Characterization of the heterooligomeric red-type rubisco activase from red algae

PubMed Central

Loganathan, Nitin; Tsai, Yi-Chin Candace; Mueller-Cajar, Oliver

2016-01-01

The photosynthetic CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) is inhibited by nonproductive binding of its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates. Reactivation requires ATP-hydrolysis–powered remodeling of the inhibited complexes by diverse molecular chaperones known as rubisco activases (Rcas). Eukaryotic phytoplankton of the red plastid lineage contain so-called red-type rubiscos, some of which have been shown to possess superior kinetic properties to green-type rubiscos found in higher plants. These organisms are known to encode multiple homologs of CbbX, the α-proteobacterial red-type activase. Here we show that the gene products of two cbbX genes encoded by the nuclear and plastid genomes of the red algae Cyanidioschyzon merolae are nonfunctional in isolation, but together form a thermostable heterooligomeric Rca that can use both α-proteobacterial and red algal-inhibited rubisco complexes as a substrate. The mechanism of rubisco activation appears conserved between the bacterial and the algal systems and involves threading of the rubisco large subunit C terminus. Whereas binding of the allosteric regulator RuBP induces oligomeric transitions to the bacterial activase, it merely enhances the kinetics of ATP hydrolysis in the algal enzyme. Mutational analysis of nuclear and plastid isoforms demonstrates strong coordination between the subunits and implicates the nuclear-encoded subunit as being functionally dominant. The plastid-encoded subunit may be catalytically inert. Efforts to enhance crop photosynthesis by transplanting red algal rubiscos with enhanced kinetics will need to take into account the requirement for a compatible Rca. PMID:27872295

Characterization of the heterooligomeric red-type rubisco activase from red algae.

PubMed

Loganathan, Nitin; Tsai, Yi-Chin Candace; Mueller-Cajar, Oliver

2016-12-06

The photosynthetic CO 2 -fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) is inhibited by nonproductive binding of its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates. Reactivation requires ATP-hydrolysis-powered remodeling of the inhibited complexes by diverse molecular chaperones known as rubisco activases (Rcas). Eukaryotic phytoplankton of the red plastid lineage contain so-called red-type rubiscos, some of which have been shown to possess superior kinetic properties to green-type rubiscos found in higher plants. These organisms are known to encode multiple homologs of CbbX, the α-proteobacterial red-type activase. Here we show that the gene products of two cbbX genes encoded by the nuclear and plastid genomes of the red algae Cyanidioschyzon merolae are nonfunctional in isolation, but together form a thermostable heterooligomeric Rca that can use both α-proteobacterial and red algal-inhibited rubisco complexes as a substrate. The mechanism of rubisco activation appears conserved between the bacterial and the algal systems and involves threading of the rubisco large subunit C terminus. Whereas binding of the allosteric regulator RuBP induces oligomeric transitions to the bacterial activase, it merely enhances the kinetics of ATP hydrolysis in the algal enzyme. Mutational analysis of nuclear and plastid isoforms demonstrates strong coordination between the subunits and implicates the nuclear-encoded subunit as being functionally dominant. The plastid-encoded subunit may be catalytically inert. Efforts to enhance crop photosynthesis by transplanting red algal rubiscos with enhanced kinetics will need to take into account the requirement for a compatible Rca.

A unique structural domain in Methanococcoides burtonii ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) acts as a small subunit mimic

PubMed Central

2017-01-01

The catalytic inefficiencies of the CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) often limit plant productivity. Strategies to engineer more efficient plant Rubiscos have been hampered by evolutionary constraints, prompting interest in Rubisco isoforms from non-photosynthetic organisms. The methanogenic archaeon Methanococcoides burtonii contains a Rubisco isoform that functions to scavenge the ribulose-1,5-bisphosphate (RuBP) by-product of purine/pyrimidine metabolism. The crystal structure of M. burtonii Rubisco (MbR) presented here at 2.6 Å resolution is composed of catalytic large subunits (LSu) assembled into pentamers of dimers, (L2)5, and differs from Rubiscos from higher plants where LSus are glued together by small subunits (SSu) into hexadecameric L8S8 enzymes. MbR contains a unique 29-amino acid insertion near the C terminus, which folds as a separate domain in the structure. This domain, which is visualized for the first time in this study, is located in a similar position to SSus in L8S8 enzymes between LSus of adjacent L2 dimers, where negatively charged residues coordinate around a Mg2+ ion in a fashion that suggests this domain may be important for the assembly process. The Rubisco assembly domain is thus an inbuilt SSu mimic that concentrates L2 dimers. MbR assembly is ligand-stimulated, and we show that only 6-carbon molecules with a particular stereochemistry at the C3 carbon can induce oligomerization. Based on MbR structure, subunit arrangement, sequence, phylogenetic distribution, and function, MbR and a subset of Rubiscos from the Methanosarcinales order are proposed to belong to a new Rubisco subgroup, named form IIIB. PMID:28154188

A unique structural domain in Methanococcoides burtonii ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) acts as a small subunit mimic.

PubMed

Gunn, Laura H; Valegård, Karin; Andersson, Inger

2017-04-21

The catalytic inefficiencies of the CO 2 -fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) often limit plant productivity. Strategies to engineer more efficient plant Rubiscos have been hampered by evolutionary constraints, prompting interest in Rubisco isoforms from non-photosynthetic organisms. The methanogenic archaeon Methanococcoides burtonii contains a Rubisco isoform that functions to scavenge the ribulose-1,5-bisphosphate (RuBP) by-product of purine/pyrimidine metabolism. The crystal structure of M. burtonii Rubisco (MbR) presented here at 2.6 Å resolution is composed of catalytic large subunits (LSu) assembled into pentamers of dimers, (L 2 ) 5 , and differs from Rubiscos from higher plants where LSus are glued together by small subunits (SSu) into hexadecameric L 8 S 8 enzymes. MbR contains a unique 29-amino acid insertion near the C terminus, which folds as a separate domain in the structure. This domain, which is visualized for the first time in this study, is located in a similar position to SSus in L 8 S 8 enzymes between LSus of adjacent L 2 dimers, where negatively charged residues coordinate around a Mg 2+ ion in a fashion that suggests this domain may be important for the assembly process. The Rubisco assembly domain is thus an inbuilt SSu mimic that concentrates L 2 dimers. MbR assembly is ligand-stimulated, and we show that only 6-carbon molecules with a particular stereochemistry at the C 3 carbon can induce oligomerization. Based on MbR structure, subunit arrangement, sequence, phylogenetic distribution, and function, MbR and a subset of Rubiscos from the Methanosarcinales order are proposed to belong to a new Rubisco subgroup, named form IIIB. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Subunit interface dynamics in hexadecameric rubisco.

PubMed

van Lun, Michiel; van der Spoel, David; Andersson, Inger

2011-09-02

Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) plays an important role in the global carbon cycle as a hub for biomass. Rubisco catalyzes not only the carboxylation of RuBP with carbon dioxide but also a competing oxygenation reaction of RuBP with a negative impact on photosynthetic yield. The functional active site is built from two large (L) subunits that form a dimer. The octameric core of four L(2) dimers is held at each end by a cluster of four small (S) subunits, forming a hexadecamer. Each large subunit contacts more than one S subunit. These interactions exploit the dynamic flexibility of Rubisco, which we address in this study. Here, we describe seven different types of interfaces of hexadecameric Rubisco. We have analyzed these interfaces with respect to the size of the interface area and the number of polar interactions, including salt bridges and hydrogen bonds in a variety of Rubisco enzymes from different organisms and different kingdoms of life, including the Rubisco-like proteins. We have also performed molecular dynamics simulations of Rubisco from Chlamydomonas reinhardtii and mutants thereof. From our computational analyses, we propose structural checkpoints of the S subunit to ensure the functionality and/or assembly of the Rubisco holoenzyme. These checkpoints appear to fine-tune the dynamics of the enzyme in a way that could influence enzyme performance. Copyright © 2011 Elsevier Ltd. All rights reserved.

Glucose Synthesis in a Protein-Based Artificial Photosynthesis System.

PubMed

Lu, Hao; Yuan, Wenqiao; Zhou, Jack; Chong, Parkson Lee-Gau

2015-09-01

The objective of this study was to understand glucose synthesis of a protein-based artificial photosynthesis system affected by operating conditions, including the concentrations of reactants, reaction temperature, and illumination. Results from non-vesicle-based glyceraldehyde-3-phosphate (GAP) and glucose synthesis showed that the initial concentrations of ribulose-1,5-bisphosphate (RuBP) and adenosine triphosphate (ATP), lighting source, and temperature significantly affected glucose synthesis. Higher initial concentrations of RuBP and ATP significantly enhanced GAP synthesis, which was linearly correlated to glucose synthesis, confirming the proper functions of all catalyzing enzymes in the system. White fluorescent light inhibited artificial photosynthesis and reduced glucose synthesis by 79.2 % compared to in the dark. The reaction temperature of 40 °C was optimum, whereas lower or higher temperature reduced glucose synthesis. Glucose synthesis in the vesicle-based artificial photosynthesis system reconstituted with bacteriorhodopsin, F 0 F 1 ATP synthase, and polydimethylsiloxane-methyloxazoline-polydimethylsiloxane triblock copolymer was successfully demonstrated. This system efficiently utilized light-induced ATP to drive glucose synthesis, and 5.2 μg ml(-1) glucose was synthesized in 0.78-ml reaction buffer in 7 h. Light-dependent reactions were found to be the bottleneck of the studied artificial photosynthesis system.

In Vivo Studies in Rhodospirillum rubrum Indicate That Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Catalyzes Two Obligatorily Required and Physiologically Significant Reactions for Distinct Carbon and Sulfur Metabolic Pathways*♦

PubMed Central

Dey, Swati; North, Justin A.; Sriram, Jaya; Evans, Bradley S.; Tabita, F. Robert

2015-01-01

All organisms possess fundamental metabolic pathways to ensure that needed carbon and sulfur compounds are provided to the cell in the proper chemical form and oxidation state. For most organisms capable of using CO2 as sole source of carbon, ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) catalyzes primary carbon dioxide assimilation. In addition, sulfur salvage pathways are necessary to ensure that key sulfur-containing compounds are both available and, where necessary, detoxified in the cell. Using knock-out mutations and metabolomics in the bacterium Rhodospirillum rubrum, we show here that Rubisco concurrently catalyzes key and essential reactions for seemingly unrelated but physiologically essential central carbon and sulfur salvage metabolic pathways of the cell. In this study, complementation and mutagenesis studies indicated that representatives of all known extant functional Rubisco forms found in nature are capable of simultaneously catalyzing reactions required for both CO2-dependent growth as well as growth using 5-methylthioadenosine as sole sulfur source under anaerobic photosynthetic conditions. Moreover, specific inactivation of the CO2 fixation reaction did not affect the ability of Rubisco to support anaerobic 5-methylthioadenosine metabolism, suggesting that the active site of Rubisco has evolved to ensure that this enzyme maintains both key functions. Thus, despite the coevolution of both functions, the active site of this protein may be differentially modified to affect only one of its key functions. PMID:26511314

Downregulation of Rubisco Activity by Non-enzymatic Acetylation of RbcL.

PubMed

Gao, Xiang; Hong, Hui; Li, Wei-Chao; Yang, Lili; Huang, Jirong; Xiao, You-Li; Chen, Xiao-Ya; Chen, Gen-Yun

2016-07-06

Atmospheric carbon dioxide (CO2) is assimilated by the most abundant but sluggish enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Here we show that acetylation of lysine residues of the Rubisco large subunit (RbcL), including Lys201 and Lys334 in the active sites, may be an important mechanism in the regulation of Rubisco activities. It is well known that Lys201 reacts with CO2 for carbamylation, a prerequisite for both carboxylase and oxygenase activities of Rubisco, and Lys334 contacts with ribulose-1,5-bisphosphate (RuBP). The acetylation level of RbcL in plants is lower during the day and higher at night, inversely correlating with the Rubisco carboxylation activity. A search of the chloroplast proteome database did not reveal a canonical acetyltransferase; instead, we found that a plant-derived metabolite, 7-acetoxy-4-methylcoumarin (AMC), can non-enzymatically acetylate both native Rubisco and synthesized RbcL peptides spanning Lys334 or Lys201. Furthermore, lysine residues were modified by synthesized 4-methylumbelliferone esters with different electro- and stereo-substitutes, resulting in varied Rubisco activities. 1-Chloroethyl 4-methylcoumarin-7-yl carbonate (ClMC) could transfer the chloroethyl carbamate group to lysine residues of RbcL and completely inactivate Rubisco, whereas bis(4-methylcoumarin-7-yl) carbonate (BMC) improved Rubisco activity through increasing the level of Lys201 carbamylation. Our findings indicate that RbcL acetylation negatively regulates Rubisco activity, and metabolic derivatives can be designed to dissect and improve CO2 fixation efficiency of plants through lysine modification. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

In Vivo Studies in Rhodospirillum rubrum Indicate That Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Catalyzes Two Obligatorily Required and Physiologically Significant Reactions for Distinct Carbon and Sulfur Metabolic Pathways.

PubMed

Dey, Swati; North, Justin A; Sriram, Jaya; Evans, Bradley S; Tabita, F Robert

2015-12-25

All organisms possess fundamental metabolic pathways to ensure that needed carbon and sulfur compounds are provided to the cell in the proper chemical form and oxidation state. For most organisms capable of using CO2 as sole source of carbon, ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) catalyzes primary carbon dioxide assimilation. In addition, sulfur salvage pathways are necessary to ensure that key sulfur-containing compounds are both available and, where necessary, detoxified in the cell. Using knock-out mutations and metabolomics in the bacterium Rhodospirillum rubrum, we show here that Rubisco concurrently catalyzes key and essential reactions for seemingly unrelated but physiologically essential central carbon and sulfur salvage metabolic pathways of the cell. In this study, complementation and mutagenesis studies indicated that representatives of all known extant functional Rubisco forms found in nature are capable of simultaneously catalyzing reactions required for both CO2-dependent growth as well as growth using 5-methylthioadenosine as sole sulfur source under anaerobic photosynthetic conditions. Moreover, specific inactivation of the CO2 fixation reaction did not affect the ability of Rubisco to support anaerobic 5-methylthioadenosine metabolism, suggesting that the active site of Rubisco has evolved to ensure that this enzyme maintains both key functions. Thus, despite the coevolution of both functions, the active site of this protein may be differentially modified to affect only one of its key functions. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Optimizing Rubisco and its regulation for greater resource use efficiency.

PubMed

Carmo-Silva, Elizabete; Scales, Joanna C; Madgwick, Pippa J; Parry, Martin A J

2015-09-01

Rubisco catalyses the carboxylation of ribulose-1,5-bisphosphate (RuBP), enabling net CO2 assimilation in photosynthesis. The properties and regulation of Rubisco are not optimal for biomass production in current and projected future environments. Rubisco is relatively inefficient, and large amounts of the enzyme are needed to support photosynthesis, requiring large investments in nitrogen. The competing oxygenation of RuBP by Rubisco decreases photosynthetic efficiency. Additionally, Rubisco is inhibited by some sugar phosphates and depends upon interaction with Rubisco activase (Rca) to be reactivated. Rca activity is modulated by the chloroplast redox status and ADP/ATP ratios, thereby mediating Rubisco activation and photosynthetic induction in response to irradiance. The extreme thermal sensitivity of Rca compromises net CO2 assimilation at moderately high temperatures. Given its central role in carbon assimilation, the improvement of Rubisco function and regulation is tightly linked with irradiance, nitrogen and water use efficiencies. Although past attempts have had limited success, novel technologies and an expanding knowledge base make the challenge of improving Rubisco activity in crops an achievable goal. Strategies to optimize Rubisco and its regulation are addressed in relation to their potential to improve crop resource use efficiency and climate resilience of photosynthesis. © 2014 Rothamsted Research Ltd. Plant, Cell & Environment published by John Wiley & Sons Ltd.

Synthetic CO2-fixation enzyme cascades immobilized on self-assembled nanostructures that enhance CO2/O2 selectivity of RubisCO.

PubMed

Satagopan, Sriram; Sun, Yuan; Parquette, Jon R; Tabita, F Robert

2017-01-01

With increasing concerns over global warming and depletion of fossil-fuel reserves, it is attractive to develop innovative strategies to assimilate CO 2 , a greenhouse gas, into usable organic carbon. Cell-free systems can be designed to operate as catalytic platforms with enzymes that offer exceptional selectivity and efficiency, without the need to support ancillary reactions of metabolic pathways operating in intact cells. Such systems are yet to be exploited for applications involving CO 2 utilization and subsequent conversion to valuable products, including biofuels. The Calvin-Benson-Bassham (CBB) cycle and the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) play a pivotal role in global CO 2 fixation. We hereby demonstrate the co-assembly of two RubisCO-associated multienzyme cascades with self-assembled synthetic amphiphilic peptide nanostructures. The immobilized enzyme cascades sequentially convert either ribose-5-phosphate (R-5-P) or glucose, a simpler substrate, to ribulose 1,5-bisphosphate (RuBP), the acceptor for incoming CO 2 in the carboxylation reaction catalyzed by RubisCO. Protection from proteolytic degradation was observed in nanostructures associated with the small dimeric form of RubisCO and ancillary enzymes. Furthermore, nanostructures associated with a larger variant of RubisCO resulted in a significant enhancement of the enzyme's selectivity towards CO 2 , without adversely affecting the catalytic activity. The ability to assemble a cascade of enzymes for CO 2 capture using self-assembling nanostructure scaffolds with functional enhancements show promise for potentially engineering entire pathways (with RubisCO or other CO 2 -fixing enzymes) to redirect carbon from industrial effluents into useful bioproducts.

Albibacter methylovorans gen. nov., sp. nov., a novel aerobic, facultatively autotrophic and methylotrophic bacterium that utilizes dichloromethane.

PubMed

Doronina, N V; Trotsenko, Y A; Tourova, T P; Kuznetsov, B B; Leisinger, T

2001-05-01

A novel genus, Albibacter, with one species, Albibacter methylovorans sp. nov., is proposed for a facultatively chemolithotrophic and methylotrophic bacterium (strain DM10T) with the ribulose bisphosphate (RuBP) pathway of C1 assimilation. The bacterium is a Gram-negative, aerobic, asporogenous, nonmotile, colourless rod that multiplies by binary fission. The organism utilizes dichloromethane, methanol, methylamine, formate and CO2/H2, as well as a variety of polycarbon compounds, as carbon and energy sources. It is neutrophilic and mesophilic. The major cellular fatty acids are straight-chain unsaturated C18:1, saturated C16:0 and cyclopropane C19:0 acids. The main ubiquinone is Q-10. The dominant phospholipids are phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl choline and cardiolipin. The DNA G+C content is 66.7 mol%. Strain DM10T has a very low degree of DNA-DNA hybridization (4-7%) with the type species of the genera Paracoccus, Xanthobacter, Blastobacter, Angulomicrobium, Ancylobacter and Ralstonia of RuBP pathway methylobacteria. Another approach, involving comparative 16S rDNA analysis, has shown that the novel isolate represents a separate branch within the alpha-2 subgroup of the Proteobacteria. The type species of the new genus is Albibacter methylovorans sp. nov.; the type strain is DM10T (= VKM B-2236T = DSM 13819T).

Structural Changes Associated with the Acute Thermal Instability of Rubisco Activase

USDA-ARS?s Scientific Manuscript database

The inhibition of photosynthesis at moderately high temperatures has been linked to a decrease in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation. This decrease is thought to be a consequence of the thermal instability of Rubisco’s chaperone, ribulose-1,5-bisphosphate carboxyla...

Isolation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase from Leaves

USDA-ARS?s Scientific Manuscript database

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a multi-functional enzyme that catalyzes the fixation of CO2 and O2 in photosynthesis and photorespiration, respectively. As the rate-limiting step in photosynthesis, improving the catalytic properties of Rubisco has long been viewed as a...

Rubisco activity and regulation as targets for crop improvement.

PubMed

Parry, Martin A J; Andralojc, P John; Scales, Joanna C; Salvucci, Michael E; Carmo-Silva, A Elizabete; Alonso, Hernan; Whitney, Spencer M

2013-01-01

Rubisco (ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase) enables net carbon fixation through the carboxylation of RuBP. However, some characteristics of Rubisco make it surprisingly inefficient and compromise photosynthetic productivity. For example, Rubisco catalyses a wasteful reaction with oxygen that leads to the release of previously fixed CO(2) and NH(3) and the consumption of energy during photorespiration. Furthermore, Rubisco is slow and large amounts are needed to support adequate photosynthetic rates. Consequently, Rubisco has been studied intensively as a prime target for manipulations to 'supercharge' photosynthesis and improve both productivity and resource use efficiency. The catalytic properties of Rubiscos from diverse sources vary considerably, suggesting that changes in turnover rate, affinity, or specificity for CO(2) can be introduced to improve Rubisco performance in specific crops and environments. While attempts to manipulate plant Rubisco by nuclear transformation have had limited success, modifying its catalysis by targeted changes to its catalytic large subunit via chloroplast transformation have been much more successful. However, this technique is still in need of development for most major food crops including maize, wheat, and rice. Other bioengineering approaches for improving Rubisco performance include improving the activity of its ancillary protein, Rubisco activase, in addition to modulating the synthesis and degradation of Rubisco's inhibitory sugar phosphate ligands. As the rate-limiting step in carbon assimilation, even modest improvements in the overall performance of Rubisco pose a viable pathway for obtaining significant gains in plant yield, particularly under stressful environmental conditions.

Improved analysis of C4 and C3 photosynthesis via refined in vitro assays of their carbon fixation biochemistry

PubMed Central

Sharwood, Robert E.; Sonawane, Balasaheb V.; Ghannoum, Oula; Whitney, Spencer M.

2016-01-01

Plants operating C3 and C4 photosynthetic pathways exhibit differences in leaf anatomy and photosynthetic carbon fixation biochemistry. Fully understanding this underpinning biochemical variation is requisite to identifying solutions for improving photosynthetic efficiency and growth. Here we refine assay methods for accurately measuring the carboxylase and decarboxylase activities in C3 and C4 plant soluble protein. We show that differences in plant extract preparation and assay conditions are required to measure NADP-malic enzyme and phosphoenolpyruvate carboxylase (pH 8, Mg2+, 22 °C) and phosphoenolpyruvate carboxykinase (pH 7, >2mM Mn2+, no Mg2+) maximal activities accurately. We validate how the omission of MgCl2 during leaf protein extraction, lengthy (>1min) centrifugation times, and the use of non-pure ribulose-1,5-bisphosphate (RuBP) significantly underestimate Rubisco activation status. We show how Rubisco activation status varies with leaf ontogeny and is generally lower in mature C4 monocot leaves (45–60% activation) relative to C3 monocots (55–90% activation). Consistent with their >3-fold lower Rubisco contents, full Rubisco activation in soluble protein from C4 leaves (<5min) was faster than in C3 plant samples (<10min), with addition of Rubisco activase not required for full activation. We conclude that Rubisco inactivation in illuminated leaves primarily stems from RuBP binding to non-carbamylated enzyme, a state readily reversible by dilution during cellular protein extraction. PMID:27122573

Composition, quaternary structure, and catalytic properties of D-ribulose-1, 5-bisphosphate carboxylase from Euglena gracilis.

PubMed

McFadden, B A; Lord, J M; Rowe, A; Dilks, S

1975-05-01

D-Ribulose-1,5-bisphosphate carboxylase has been purified in one step by sedimenting extracts of autotrophically-grown Euglena gracilis into a linear 0.2-0.8 M sucrose density gradient. The resultant product was pure by the criteria of disc electrophoresis in gels polymerized from 5 or 7.5% acrylamide and sedimentation. The molecular weight of the enzyme estimated by density gradient centrifugation and electrophoresis in gels polymerized from various concentrations of acrylamide was 5.25 X 10(5). The S20,W was 16.4 S. Dissociation and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate established that the enzyme was composed of two types of subunits (mr 50,000 and 15,000). The oligomeric structure was visualized through negative staining and transmission electron microscopy leading to a model for the quaternary structure. Although the enzyme was moderately unstable, the estimated maximal specific activity was 1.6 mumol CO2 fixed min-1 mg protien-1 at 30 degrees C and pH 8.0 Km values were 2.2 m M, 15. 1 MUM and 0.63 mM for Mg2+, ribulose 1,5-bisphosphate, and CO2, respectively, when measured under air. 6-Phospho-D-gluconate was a noncompetitive inhibitor with respect to ribulose 1,5-bisphosphate (Ki = 0.04 mM). Oxygen was a competitive inhibitor with respect to CO2 suggesting that the enzyme was also an oxygenase. The latter was confirmed by experiments showing a molar equivalence between ribulose-1,5-bisphosphate-dependent oxygen consumption and phosphoglycerate production.

CO2 Enhancement of Growth and Photosynthesis in Rice (Oryza sativa) 1

PubMed Central

Ziska, Lewis H.; Teramura, Alan H.

1992-01-01

Two cultivars of rice (Oryza sativa L.) IR-36 and Fujiyama-5 were grown at ambient (360 microbars) and elevated CO2 (660 microbars) from germination through reproduction in unshaded greenhouses at the Duke University Phytotron. Growth at elevated CO2 resulted in significant decreases in nighttime respiration and increases in photosynthesis, total biomass, and yield for both cultivars. However, in plants exposed to simultaneous increases in CO2 and ultraviolet-B (UV-B) radiation, CO2 enhancement effects on respiration, photosynthesis, and biomass were eliminated in IR-36 and significantly reduced in Fujiyama-5. UV-B radiation simulated a 25% depletion in stratospheric ozone at Durham, North Carolina. Analysis of the response of CO2 uptake to internal CO2 concentration at light saturation suggested that, for IR-36, the predominant limitation to photosynthesis with increased UV-B radiation was the capacity for regeneration of ribulose bisphosphate (RuBP), whereas for Fujiyama-5 the primary photosynthetic decrease appeared to be related to a decline in apparent carboxylation efficiency. Changes in the RuBP regeneration limitation in IR-36 were consistent with damage to the photochemical efficiency of photosystem II as estimated from the ratio of variable to maximum chlorophyll fluorescence. Little change in RuBP regeneration and photochemistry was evident in cultivar Fujiyama-5, however. The degree of sensitivity of photochemical reactions with increased UV-B radiation appeared to be related to leaf production of UV-B-absorbing compounds. Fujiyama-5 had a higher concentration of these compounds than IR-36 in all environments, and the production of these compounds in Fujiyama-5 was stimulated by UV-B fluence. Results from this study suggest that in rice alterations in growth or photosynthesis as a result of enhanced CO2 may be eliminated or reduced if UV-B radiation continues to increase. PMID:16668910

Slow induction of photosynthesis on shade to sun transitions in wheat may cost at least 21% of productivity.

PubMed

Taylor, Samuel H; Long, Stephen P

2017-09-26

Wheat is the second most important direct source of food calories in the world. After considerable improvement during the Green Revolution, increase in genetic yield potential appears to have stalled. Improvement of photosynthetic efficiency now appears a major opportunity in addressing the sustainable yield increases needed to meet future food demand. Effort, however, has focused on increasing efficiency under steady-state conditions. In the field, the light environment at the level of individual leaves is constantly changing. The speed of adjustment of photosynthetic efficiency can have a profound effect on crop carbon gain and yield. Flag leaves of wheat are the major photosynthetic organs supplying the grain of wheat, and will be intermittently shaded throughout a typical day. Here, the speed of adjustment to a shade to sun transition in these leaves was analysed. On transfer to sun conditions, the leaf required about 15 min to regain maximum photosynthetic efficiency. In vivo analysis based on the responses of leaf CO 2 assimilation ( A ) to intercellular CO 2 concentration ( c i ) implied that the major limitation throughout this induction was activation of the primary carboxylase of C3 photosynthesis, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This was followed in importance by stomata, which accounted for about 20% of the limitation. Except during the first few seconds, photosynthetic electron transport and regeneration of the CO 2 acceptor molecule, ribulose-1,5-bisphosphate (RubP), did not affect the speed of induction. The measured kinetics of Rubisco activation in the sun and de-activation in the shade were predicted from the measurements. These were combined with a canopy ray tracing model that predicted intermittent shading of flag leaves over the course of a June day. This indicated that the slow adjustment in shade to sun transitions could cost 21% of potential assimilation.This article is part of the themed issue 'Enhancing photosynthesis in crop plants: targets for improvement'. © 2017 The Authors.

Isolated spinach ribulose-1,5-bisphosphate carboxylase/oxgenase large subunit .epsilon. n-methyltransferase and method of inactivating ribulose-1,5-bishosphatase .epsilon. n-methyltransferase activity

DOEpatents

Houtz, Robert L.

2001-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltansferase (protein methylase III or Rubisco LSMT) from a plant which has a des(methyl) lysyl residue in the LS is disclosed. In addition, the full-length cDNA clones for Rubisco LSMT are disclosed. Transgenic plants and methods of producing same which have the Rubisco LSMT gene inserted into the DNA are also provided. Further, methods of inactivating the enzymatic activity of Rubisco LSMT are also disclosed.

Biogeochemical Cycling of Manganese at Hydrothermal Vents

DTIC Science & Technology

1990-01-01

from an anoxic basin) contain the gene for the large subunit of Ribulose- 1,5-bisphosphate Carboxylase Oxygenase ( RubisCO ) suggestive of autotrophy... RubisCO gene probing on the bacterial isolates obtained from the hydrothermal vent environments as part of an ongoing ONR contract. In addition, we have...to test the feasibility of using gene probes for Ribulose-l,5- bisphosphate Carboxylase Oxygenase ( RubisCO ) for identifying autotrophic Mn(II

Mechanistic Models of Light-limited and Light-saturated Rates of Photosynthesis

DTIC Science & Technology

1997-09-30

concentrations of the rate-limiting enzyme ribulose-1,5-bisphosphate carboxylase ( Rubisco ) that catalyzes carbon fixation. This work is supported by...saturated photosynthesis as a function of the activity of the key photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase ( Rubisco ) for a Report...concentration and its specific rate. The Rubisco activities were determined at the same temperature as the photosynthetic measurements, and hence did not

Rubisco activity is associated with photosynthetic thermotolerance in a wild rice (Oryza meridionalis).

PubMed

Scafaro, Andrew P; Yamori, Wataru; Carmo-Silva, A Elizabete; Salvucci, Michael E; von Caemmerer, Susanne; Atwell, Brian J

2012-09-01

Oryza meridionalis is a wild species of rice, endemic to tropical Australia. It shares a significant genome homology with the common domesticated rice Oryza sativa. Exploiting the fact that the two species are highly related but O. meridionalis has superior heat tolerance, experiments were undertaken to identify the impact of temperature on key events in photosynthesis. At an ambient CO(2) partial pressure of 38 Pa and irradiance of 1500 µmol quanta m(-2) s(-1), the temperature optimum of photosynthesis was 33.7 ± 0.8°C for O. meridionalis, significantly higher than the 30.6 ± 0.7°C temperature optimum of O. sativa. To understand the basis for this difference, we measured gas exchange and rubisco activation state between 20 and 42°C and modeled the response to determine the rate-limiting steps of photosynthesis. The temperature response of light respiration (R(light)) and the CO(2) compensation point in the absence of respiration (Γ(*)) were determined and found to be similar for the two species. C3 photosynthesis modeling showed that despite the difference in susceptibility to high temperature, both species had a similar temperature-dependent limitation to photosynthesis. Both rice species were limited by ribulose-1,5-bisphosphate (RuBP) regeneration at temperatures of 25 and 30°C but became RuBP carboxylation limited at 35 and 40°C. The activation state of rubisco in O. meridionalis was more stable at higher temperatures, explaining its greater heat tolerance compared with O. sativa. Copyright © Physiologia Plantarum 2012.

Isolated spinach ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase and method of inactivating ribulose-1,5-bisphosphatase carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase activity

DOEpatents

Houtz, Robert L.

1999-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) from a plant which has a des(methyl) lysyl residue in the LS is disclosed. In addition, the full-length cDNA clones for Rubisco LSMT are disclosed. Transgenic plants and methods of producing same which have the Rubisco LSMT gene inserted into the DNA are also provided. Further, methods of inactivating the enzymatic activity of Rubisco LSMT are also disclosed.

The single-process biochemical reaction of Rubisco: a unified theory and model with the effects of irradiance, CO₂ and rate-limiting step on the kinetics of C₃ and C₄ photosynthesis from gas exchange.

PubMed

Farazdaghi, Hadi

2011-02-01

Photosynthesis is the origin of oxygenic life on the planet, and its models are the core of all models of plant biology, agriculture, environmental quality and global climate change. A theory is presented here, based on single process biochemical reactions of Rubisco, recognizing that: In the light, Rubisco activase helps separate Rubisco from the stored ribulose-1,5-bisphosphate (RuBP), activates Rubisco with carbamylation and addition of Mg²(+), and then produces two products, in two steps: (Step 1) Reaction of Rubisco with RuBP produces a Rubisco-enediol complex, which is the carboxylase-oxygenase enzyme (Enco) and (Step 2) Enco captures CO₂ and/or O₂ and produces intermediate products leading to production and release of 3-phosphoglycerate (PGA) and Rubisco. PGA interactively controls (1) the carboxylation-oxygenation, (2) electron transport, and (3) triosephosphate pathway of the Calvin-Benson cycle that leads to the release of glucose and regeneration of RuBP. Initially, the total enzyme participates in the two steps of the reaction transitionally and its rate follows Michaelis-Menten kinetics. But, for a continuous steady state, Rubisco must be divided into two concurrently active segments for the two steps. This causes a deviation of the steady state from the transitional rate. Kinetic models are developed that integrate the transitional and the steady state reactions. They are tested and successfully validated with verifiable experimental data. The single-process theory is compared to the widely used two-process theory of Farquhar et al. (1980. Planta 149, 78-90), which assumes that the carboxylation rate is either Rubisco-limited at low CO₂ levels such as CO₂ compensation point, or RuBP regeneration-limited at high CO₂. Since the photosynthesis rate cannot increase beyond the two-process theory's Rubisco limit at the CO₂ compensation point, net photosynthesis cannot increase above zero in daylight, and since there is always respiration at night, it leads to progressively negative daily CO₂ fixation with no possibility of oxygenic life on the planet. The Rubisco-limited theory at low CO₂ also contradicts all experimental evidence for low substrate reactions, and for all known enzymes, Rubisco included. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Photosynthetic Fractionation of the Stable Isotopes of Oxygen and Carbon.

PubMed Central

Guy, R. D.; Fogel, M. L.; Berry, J. A.

1993-01-01

Isotope discrimination during photosynthetic exchange of O2 and CO2 was measured using enzyme, thylakoid, and whole cell preparations. Evolved oxygen from isolated spinach thylakoids was isotopically identical (within analytical error) to its source water. Similar results were obtained with Anacystis nidulans Richter and Phaeodactylum tricornutum Bohlin cultures purged with helium. For consumptive reactions, discrimination ([delta], where 1 + [delta]/1000 equals the isotope effect, k16/k18 or k12/k13) was determined by analysis of residual substrate (O2 or CO2). The [delta] for the Mehler reaction, mediated by ferredoxin or methylviologen, was 15.3[per mille (thousand) sign]. Oxygen isotope discrimination during oxygenation of ribulose-1,5-bisphosphate (RuBP) catalyzed by RuBP carboxylase/oxygenase (Rubisco) was 21.3[per mille (thousand) sign] and independent of enzyme source, unlike carbon isotope discrimination: 30.3[per mille (thousand) sign] for spinach enzyme and 19.6 to 23[per mille (thousand) sign] for Rhodospirillum rubrum and A. nidulans enzymes, depending on reaction conditions. The [delta] for O2 consumption catalyzed by glycolate oxidase was 22.7[per mille (thousand) sign]. The expected overall [delta] for photorespiration is about 21.7[per mille (thousand) sign]. Consistent with this, when Asparagus sprengeri Regel mesophyll cells approached the compensation point within a sealed vessel, the [delta]18O of dissolved O2 came to a steady-state value of about 21.5[per mille (thousand) sign] relative to the source water. The results provide improved estimates of discrimination factors in several reactions prominent in the global O cycle and indicate that photorespiration plays a significant part in determining the isotopic composition of atmospheric oxygen. PMID:12231663

Crystal structure of the unactivated ribulose 1,5-bisphosphate carboxylase/oxygenase complexed with a transition state analog, 2-carboxy-D-arabinitol 1,5-bisphosphate.

PubMed Central

Zhang, K. Y.; Cascio, D.; Eisenberg, D.

1994-01-01

The crystal structure of unactivated ribulose 1,5-bisphosphate carboxylase/oxygenase from Nicotiana tabacum complexed with a transition state analog, 2-carboxy-D-arabinitol 1,5-bisphosphate, was determined to 2.7 A resolution by X-ray crystallography. The transition state analog binds at the active site in an extended conformation. As compared to the binding of the same analog in the activated enzyme, the analog binds in a reverse orientation. The active site Lys 201 is within hydrogen bonding distance of the carboxyl oxygen of the analog. Loop 6 (residues 330-339) remains open and flexible upon binding of the analog in the unactivated enzyme, in contrast to the closed and ordered loop 6 in the activated enzyme complex. The transition state analog is exposed to solvent due to the open conformation of loop 6. PMID:8142899

Combined Inoculation with Multiple Arbuscular Mycorrhizal Fungi Improves Growth, Nutrient Uptake and Photosynthesis in Cucumber Seedlings.

PubMed

Chen, Shuangchen; Zhao, Hongjiao; Zou, Chenchen; Li, Yongsheng; Chen, Yifei; Wang, Zhonghong; Jiang, Yan; Liu, Airong; Zhao, Puyan; Wang, Mengmeng; Ahammed, Golam J

2017-01-01

Mycorrhizal inoculation stimulates growth, photosynthesis and nutrient uptake in a wide range of host plants. However, the ultimate effects of arbuscular mycorrhyzal (AM) symbiosis vary with the plants and fungal species involved in the association. Therefore, identification of the appropriate combinations of AM fungi (AMF) that interact synergistically to improve their benefits is of high significance. Here, three AM fungal compositions namely VT ( Claroideoglomus sp., Funneliformis sp., Diversispora sp., Glomus sp., and Rhizophagus sp.) and BF ( Glomus intraradices , G. microageregatum BEG and G. Claroideum BEG 210), and Funneliformis mosseae (Fm) were investigated with respect to the growth, gas exchange parameters, enzymes activities in Calvin cycles and related gene expression in cucumber seedlings. The results showed that VT, BF and Fm could successfully colonize cucumber root to a different degree with the colonization rates 82.38, 74.65, and 70.32% at 46 days post inoculation, respectively. The plant height, stem diameter, dry weight, root to shoot ratio of cucumber seedlings inoculated with AMF increased significantly compared with the non-inoculated control. Moreover, AMF colonization greatly increased the root activity, chlorophyll content, net photosynthetic rate, light saturated rate of the CO 2 assimilation ( A sat), maximum carboxylation rate ( V cmax ) and maximum ribulose-1,5-bis-phosphate (RuBP) regeneration rate ( J max), which were increased by 52.81, 30.75, 58.76, 47.00, 69.15, and 65.53% when inoculated with VT, respectively. The activities of some key enzymes such RuBP carboxylase/oxygenase (RuBisCO), D-fructose-1,6-bisphosphatase (FBPase), D-fructose-6-phosphatase (F6P) and ribulose-5-phosphate kinase (Ru5PK), and related gene expression involved in the Calvin cycle including RCA , FBPase , FBPA , SBPase , rbcS and rbcL were upregulated by AMF colonization. AMF inoculation also improved macro- and micro nutrient contents such as N, P, K, S, Ca, Cu, Fe, Mn, Mg, and Zn in roots. Further analysis revealed that inoculation with VT had relatively better effect on growth of cucumber seedling followed by BF and Fm, indicating that AMF composition consisting of distant AMF species may have a better effect than a single or closely related AMF spp. This study advances the understanding of plant responses to different AM fungi toward development of strategies on AMF-promoted vegetable production.

Ribulose bisphosphate carboxylase activity and a Calvin cycle gene cluster in Sulfobacillus species.

PubMed

Caldwell, Paul E; MacLean, Martin R; Norris, Paul R

2007-07-01

The Calvin-Benson-Bassham (CBB) cycle has been extensively studied in proteobacteria, cyanobacteria, algae and plants, but hardly at all in Gram-positive bacteria. Some characteristics of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) and a cluster of potential CBB cycle genes in a Gram-positive bacterium are described in this study with two species of Sulfobacillus (Gram-positive, facultatively autotrophic, mineral sulfide-oxidizing acidophiles). In contrast to the Gram-negative, iron-oxidizing acidophile Acidithiobacillus ferrooxidans, Sulfobacillus thermosulfidooxidans grew poorly autotrophically unless the CO(2) concentration was enhanced over that in air. However, the RuBisCO of each organism showed similar affinities for CO(2) and for ribulose 1,5-bisphosphate, and similar apparent derepression of activity under CO(2) limitation. The red-type, form I RuBisCO of Sulfobacillus acidophilus was confirmed as closely related to that of the anoxygenic phototroph Oscillochloris trichoides. Eight genes potentially involved in the CBB cycle in S. acidophilus were clustered in the order cbbA, cbbP, cbbE, cbbL, cbbS, cbbX, cbbG and cbbT.

Regulation of 2-carboxyarabinitol 1-phosphatase.

PubMed

Holbrook, G P; Galasinski, S C; Salvucci, M E

1991-11-01

The regulation of 2-carboxyarabinitol 1-phosphatase (CA 1-Pase) by phosphorylated effectors was studied with enzyme purified from tobacco (Nicotiana tabacum) leaves. CA 1-Pase activity was most stimulated by fructose 1,6-bisphosphate, exhibiting an A(0.5) value of 1.9 millimolar and a 10-fold enhancement of catalysis. With ribulose-1,5-bisphosphate, the A(0.5) was 0.6 millimolar, and maximal stimulation of activity was 5.3-fold. Among the monophosphates, 3-phosphoglycerate and phosphoglycolate were more potent positive effectors than glyceraldehyde 3-phosphate, glucose 1-phosphate, glucose 6-phosphate, and dihydroxyacetone phosphate. Stimulation of CA 1-Pase by ribulose-1,5-bisphosphate and fructose 1,6-bisphosphate increased V(max) but did not appreciably alter K(m) (2-carboxyarabinitol 1-phosphate) values. Inorganic phosphate appeared to inhibit CA 1-Pase noncompetitively with respect to 2-carboxyarabinitol 1-phosphate, exhibiting a K(i) of 0.3 millimolar. The results suggest that these positive and negative effectors bind to a regulatory site on CA 1-Pase and may have a physiologial role in the light regulation of this enzyme. Related experiments with CA 1-Pase inactivated by dialysis in the absence of dithiothreitol show that partial reactivation can be achieved in the presence of a range of reducing reagents, including dithiothreitol, cysteine, and reduced glutathione. This could imply an ancillary involvement of sulfhydryl reduction during light activation of CA 1-Pase in vivo. The enzyme was thermally stable up to 35 degrees C, in contrast to ribulose-1,5-bisphosphate carboxylase/oxygenase activase which lost activity above 30 degrees C. The activation energy for CA 1-Pase was calculated to be 56.14 kilojoules per mole.

Regulation of 2-Carboxyarabinitol 1-Phosphatase 1

PubMed Central

Holbrook, Gabriel P.; Galasinski, Scott C.; Salvucci, Michael E.

1991-01-01

The regulation of 2-carboxyarabinitol 1-phosphatase (CA 1-Pase) by phosphorylated effectors was studied with enzyme purified from tobacco (Nicotiana tabacum) leaves. CA 1-Pase activity was most stimulated by fructose 1,6-bisphosphate, exhibiting an A0.5 value of 1.9 millimolar and a 10-fold enhancement of catalysis. With ribulose-1,5-bisphosphate, the A0.5 was 0.6 millimolar, and maximal stimulation of activity was 5.3-fold. Among the monophosphates, 3-phosphoglycerate and phosphoglycolate were more potent positive effectors than glyceraldehyde 3-phosphate, glucose 1-phosphate, glucose 6-phosphate, and dihydroxyacetone phosphate. Stimulation of CA 1-Pase by ribulose-1,5-bisphosphate and fructose 1,6-bisphosphate increased Vmax but did not appreciably alter Km (2-carboxyarabinitol 1-phosphate) values. Inorganic phosphate appeared to inhibit CA 1-Pase noncompetitively with respect to 2-carboxyarabinitol 1-phosphate, exhibiting a Ki of 0.3 millimolar. The results suggest that these positive and negative effectors bind to a regulatory site on CA 1-Pase and may have a physiologial role in the light regulation of this enzyme. Related experiments with CA 1-Pase inactivated by dialysis in the absence of dithiothreitol show that partial reactivation can be achieved in the presence of a range of reducing reagents, including dithiothreitol, cysteine, and reduced glutathione. This could imply an ancillary involvement of sulfhydryl reduction during light activation of CA 1-Pase in vivo. The enzyme was thermally stable up to 35°C, in contrast to ribulose-1,5-bisphosphate carboxylase/oxygenase activase which lost activity above 30°C. The activation energy for CA 1-Pase was calculated to be 56.14 kilojoules per mole. PMID:16668528

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase

DOEpatents

Houtz, Robert L.

1998-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .epsilon.N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase

DOEpatents

Houtz, Robert L.

1999-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase

DOEpatents

Houtz, R.L.

1998-03-03

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) {epsilon}N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the {epsilon}-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed. 5 figs.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase

DOEpatents

Houtz, R.L.

1999-02-02

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS){sup {epsilon}}N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the {epsilon}-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed. 8 figs.

Biology of Symbioses between Marine Invertebrates and Intracellular Bacteria

DTIC Science & Technology

1990-01-30

bisphosphate carboxylase We designed from published sequence information oligonucleotide primers which are complementary to conserved regions on RubisCO ...large and small subunit genes. These primers were used successfully to amplify using polymerase chain reaction (PCR) specific regions of RubisCO ...for the large subunit of ribulose bisphosphate carboxylase/oxygenase ( RubisCO ) to symbiont DNA shows that the symbionts from both deep-sea and shallow

Elucidating the biosynthesis of 2-carboxyarabinitol 1-phosphate through reduced expression of chloroplastic fructose 1,6-bisphosphate phosphatase and radiotracer studies with 14CO2

PubMed Central

Andralojc, P. John; Keys, Alfred J.; Kossmann, Jens; Parry, Martin A. J.

2002-01-01

2-Carboxyarabinitol 1-phosphate limits photosynthetic CO2 assimilation at low light because it is a potent, naturally occurring inhibitor of ribulose 1,5-bisphosphate carboxylase/oxygenase. Evidence is presented that this inhibitor is derived from chloroplastic fructose 1,6-bisphosphate. First, transgenic plants containing decreased amounts of chloroplastic fructose 1,6-bisphosphate phosphatase contained increased amounts of fructose 1,6-bisphosphate and 2-carboxyarabinitol 1-phosphate and greatly increased amounts of the putative intermediates hamamelose and 2-carboxyarabinitol, which in some cases were as abundant as sucrose. Second, French bean leaves in the light were shown to incorporate 14C from 14CO2 sequentially into fructose 1,6-bisphosphate, hamamelose bisphosphate, hamamelose monophosphate, hamamelose, and 2-carboxyarabinitol. As shown previously, 14C assimilated by photosynthesis was also incorporated into 2-carboxyarabinitol 1-phosphate during subsequent darkness. PMID:11917127

Isolation and characterization of cbbL and cbbS genes encoding form I ribulose-1,5-bisphosphate carboxylase/oxygenase large and small subunits in Nitrosomonas sp. strain ENI-11.

PubMed

Hirota, Ryuichi; Kato, Junichi; Morita, Hiromu; Kuroda, Akio; Ikeda, Tsukasa; Takiguchi, Noboru; Ohtake, Hisao

2002-03-01

The cbbL and cbbS genes encoding form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large and small subunits in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11 were cloned and sequenced. The deduced gene products, CbbL and CbbS, had 93 and 87% identity with Thiobacillus intermedius CbbL and Nitrobacter winogradskyi CbbS, respectively. Expression of cbbL and cbbS in Escherichia coli led to the detection of RubisCO activity in the presence of 0.1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). To our knowledge, this is the first paper to report the genes involved in the carbon fixation reaction in chemolithotrophic ammonia-oxidizing bacteria.

A non-radioactive method for measuring Rubisco activase activity in the presence of variable ATP: ADP ratios, including modifications for measuring the activity and activation state of Rubisco.

PubMed

Scales, Joanna C; Parry, Martin A J; Salvucci, Michael E

2014-03-01

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes carboxylation of ribulose-1,5-bisphosphate, the first in a series of reactions leading to the incorporation of atmospheric CO₂ into biomass. Rubisco requires Rubisco activase (RCA), an AAA+ ATPase that reactivates Rubisco by remodelling the conformation of inhibitor-bound sites. RCA is regulated by the ratio of ADP:ATP, with the precise response potentiated by redox regulation of the alpha-isoform. Measuring the effects of ADP on the activation of Rubisco by RCA using the well-established photometric assay is problematic because of the adenine nucleotide requirement of 3-phosphoglycerate (3-PGA) kinase. Described here is a novel assay for measuring RCA activity in the presence of variable ratios of ADP:ATP. The assay couples the formation of 3-PGA from ribulose 1,5-bisphosphate and CO₂ to NADH oxidation through cofactor-dependent phosphoglycerate mutase, enolase, PEP carboxylase and malate dehydrogenase. The assay was used to determine the effects of Rubisco and RCA concentration and ADP:ATP ratio on RCA activity, and to measure the activation of a modified Rubisco by RCA. Variations of the basic assay were used to measure the activation state of Rubisco in leaf extracts and the activity of purified Rubisco. The assay can be automated for high-throughput processing by conducting the reactions in two stages.

Comparison of the A-Cc curve fitting methods in determining maximum ribulose 1.5-bisphosphate carboxylase/oxygenase carboxylation rate, potential light saturated electron transport rate and leaf dark respiration.

PubMed

Miao, Zewei; Xu, Ming; Lathrop, Richard G; Wang, Yufei

2009-02-01

A review of the literature revealed that a variety of methods are currently used for fitting net assimilation of CO2-chloroplastic CO2 concentration (A-Cc) curves, resulting in considerable differences in estimating the A-Cc parameters [including maximum ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco) carboxylation rate (Vcmax), potential light saturated electron transport rate (Jmax), leaf dark respiration in the light (Rd), mesophyll conductance (gm) and triose-phosphate utilization (TPU)]. In this paper, we examined the impacts of fitting methods on the estimations of Vcmax, Jmax, TPU, Rd and gm using grid search and non-linear fitting techniques. Our results suggested that the fitting methods significantly affected the predictions of Rubisco-limited (Ac), ribulose 1,5-bisphosphate-limited (Aj) and TPU-limited (Ap) curves and leaf photosynthesis velocities because of the inconsistent estimate of Vcmax, Jmax, TPU, Rd and gm, but they barely influenced the Jmax : Vcmax, Vcmax : Rd and Jmax : TPU ratio. In terms of fitting accuracy, simplicity of fitting procedures and sample size requirement, we recommend to combine grid search and non-linear techniques to directly and simultaneously fit Vcmax, Jmax, TPU, Rd and gm with the whole A-Cc curve in contrast to the conventional method, which fits Vcmax, Rd or gm first and then solves for Vcmax, Jmax and/or TPU with V(cmax), Rd and/or gm held as constants.

Requirement of carbon dioxide for initial growth of facultative methylotroph, Acidomonas methanolica MB58.

PubMed

Mitsui, Ryoji; Katayama, Hiroko; Tanaka, Mitsuo

2015-07-01

The facultative methylotrophic bacterium Acidomonas methanolica MB58 can utilize C1 compounds via the ribulose monophosphate pathway. A large gene cluster comprising three components related to C1 metabolism was found in the genome. From upstream, the first was an mxa cluster encoding proteins for oxidation of methanol to formaldehyde; the second was the rmp cluster encoding enzymes for formaldehyde fixation; and the third was the cbb gene cluster encoding proteins for carbon dioxide (CO2) fixation. Examination of CO2 requirements for growth of A. methanolica MB58 cells demonstrated that it did not grow on any carbon source under CO2-free conditions. Measurement of ribulose-1,5-bisphosphate carboxylase activity and RT-PCR analysis demonstrated enzymatic activity was detected in A. methanolica MB58 at growth phase, regardless of carbon sources. However, methanol dehydrogenase and 3-hexlose-6-phosphate synthase expression was regulated by methanol or formaldehyde; it were detected during growth and apparently differed from ribulose-1,5-bisphosphate carboxylase expression. These results suggested that A. methanolica MB58 may be initially dependent on autotrophic growth and that carbon assimilation was subsequently coupled with the ribulose monophosphate pathway at early- to mid-log phases during methylotrophic growth. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Does long-term cultivation of saplings under elevated CO2 concentration influence their photosynthetic response to temperature?

PubMed Central

Šigut, Ladislav; Holišová, Petra; Klem, Karel; Šprtová, Mirka; Calfapietra, Carlo; Marek, Michal V.; Špunda, Vladimír; Urban, Otmar

2015-01-01

Background and Aims Plants growing under elevated atmospheric CO2 concentrations often have reduced stomatal conductance and subsequently increased leaf temperature. This study therefore tested the hypothesis that under long-term elevated CO2 the temperature optima of photosynthetic processes will shift towards higher temperatures and the thermostability of the photosynthetic apparatus will increase. Methods The hypothesis was tested for saplings of broadleaved Fagus sylvatica and coniferous Picea abies exposed for 4–5 years to either ambient (AC; 385 µmol mol−1) or elevated (EC; 700 µmol mol−1) CO2 concentrations. Temperature response curves of photosynthetic processes were determined by gas-exchange and chlorophyll fluorescence techniques. Key Results Initial assumptions of reduced light-saturated stomatal conductance and increased leaf temperatures for EC plants were confirmed. Temperature response curves revealed stimulation of light-saturated rates of CO2 assimilation (Amax) and a decline in photorespiration (RL) as a result of EC within a wide temperature range. However, these effects were negligible or reduced at low and high temperatures. Higher temperature optima (Topt) of Amax, Rubisco carboxylation rates (VCmax) and RL were found for EC saplings compared with AC saplings. However, the shifts in Topt of Amax were instantaneous, and disappeared when measured at identical CO2 concentrations. Higher values of Topt at elevated CO2 were attributed particularly to reduced photorespiration and prevailing limitation of photosynthesis by ribulose-1,5-bisphosphate (RuBP) regeneration. Temperature response curves of fluorescence parameters suggested a negligible effect of EC on enhancement of thermostability of photosystem II photochemistry. Conclusions Elevated CO2 instantaneously increases temperature optima of Amax due to reduced photorespiration and limitation of photosynthesis by RuBP regeneration. However, this increase disappears when plants are exposed to identical CO2 concentrations. In addition, increased heat-stress tolerance of primary photochemistry in plants grown at elevated CO2 is unlikely. The hypothesis that long-term cultivation at elevated CO2 leads to acclimation of photosynthesis to higher temperatures is therefore rejected. Nevertheless, incorporating acclimation mechanisms into models simulating carbon flux between the atmosphere and vegetation is necessary. PMID:25851132

Immunochemical localization of ribulose-1,5-bisphosphate carboxylase in the symbiont-containing gills of Solemya velum (Bivalvia: Mollusca).

PubMed

Cavanaugh, C M; Abbott, M S; Veenhuis, M

1988-10-01

The distribution of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase (RbuP(2)Case; EC 4.1.1.39) was examined by using two immunological methods in tissues of Solemya velum, an Atlantic coast bivalve containing putative chemoautotrophic symbionts. Antibodies elicited by the purified large subunit of RbuP(2)Case from tobacco (Nicotiana tabacum) cross-reacted on immunoblots with a protein of similar molecular mass occurring in extracts of the symbiont-containing gill tissue of S. velum. No cross-reactivity was detected in symbiont-free tissue extracts. The antiserum also cross-reacted in immunoblots with proteins of Thiobacillus neapolitanus, a free-living sulfuroxidizing chemoautotroph whose RbuP(2)Case has been well characterized. In protein A-gold immunoelectron microscopy studies, this antiserum consistently labeled the symbionts but not surrounding host gill tissue, indicating that the symbionts are responsible for the RbuP(2)Case activity.

Species Variation in the Predawn Inhibition of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase 1

PubMed Central

Servaites, Jerome C.; Parry, Martin A. J.; Gutteridge, Steven; Keys, Alfred J.

1986-01-01

The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase was measured in extracts of leaves collected before dawn (predawn activity, pa) and at midday (midday activity, ma). Twenty-three of the 37 species examined showed a pa/ma ratio (≤0.75, while only Capsicum frutescens, Cucumis sativa, Glycine max, Nicotiana tabacum, Vigna unguiculata, and 3 Solanum species showed a pa/ma ratio ≤0.5. Phaseolus vulgaris consistently showed a pa/ma ratio of ≤0.1. Activities and pa/ma ratios of the same species grown in the United States and the United Kingdom were very similar. Gel filtration of extracts before assay had no effect on the observed activities and the pa/ma ratios. These data are consistent with the hypothesis that in a number of species the enzyme is partially inhibited following the night period by the presence of a tight-binding inhibitor. PMID:16665155

Photocontrol of the expression of genes encoding chlorophyll a/b binding proteins and small subunit of ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (L. ) and Nicotiana tabacum (L. )

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wehmeyer, B.; Cashmore, A.R.; Schaefer, E.

Phytochrome and the blue ultraviolet-A photoreceptor control light-induced expression of genes encoding the chlorophyll a/b binding protein of photosystem II and photosystem I and the genes for the small subunit of the ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (tomato) and Nicotiana tabacum (tobacco). A high irradiance response also controls the induction of these genes. Genes encoding photosystem II- and I-associated chlorophyll a/b binding proteins both exhibit a transient rapid increase in expression in response to light pulse or to continuous irradiation. In contrast, genes encoding the small subunit exhibit a continuous increase in expression in response to light.more » These distinct expression characteristics are shown to reflect differences at the level of transcription.« less

Ribulose-1,5-bisphosphate Carboxylase/Oxygenase and Polyphenol Oxidase in the Tobacco Mutant Su/su and Three Green Revertant Plants 1

PubMed Central

Koivuniemi, Paul J.; Tolbert, N. E.; Carlson, Peter S.

1980-01-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) was crystallized from a heterozygous tobacco (Nicotiana tabacum L.) aurea mutant (Su/su), its wild-type sibling (su/su), and green revertant plants regenerated from green spots found on leaves of haploid Su plants. No differences were found in the specific activity or kinetic parameters of this enzyme, when comparing Su/su and su/su plants of the same age, which had been grown under identical conditions. The enzyme crystallized from revertant plants was also identical to the enzyme from wild-type plants with the exception of one clone, designated R2. R2 has a chromosome number approximately double that of the wild-type (87.0 ± 11.1 versus 48). The enzyme from R2 had a lower Vmax for CO2, although the Km values were identical to those for the enzyme from the wild-type plant. The enzyme from all mutant plants had identical isoelectric points, identical molecular weight as demonstrated by migration on native and sodium dodecyl sulfate (SDS)-polyacrylamide gels, and the same ratio of large to small subunits as the enzyme from the wild-type. The large subunit of the enzyme from tobacco leaves exhibited a different electrophoretic pattern than did the large subunit from spinach; there were two to three bands on SDS-polyacrylamide gels for the tobacco enzyme whereas the enzyme from spinach had only one species of large subunit. Total polyphenol oxidase activity was the same in leaves from the heterozygous mutant (Su/su) and wild-type (su/su) plants when correlated with developmental age as represented by morphology rather than by the chronological age of the plants. There was a marked increase in the soluble activity of this enzyme with increasing age of both plant types and also as a result of varying environmental conditions. Ribulose-1,5-bisphosphate carboxylase/oxygenase activity correlated inversely with increases in the soluble activity of polyphenol oxidase in crude homogenates from which the carboxylase/oxygenase was crystallized over a generation of Su/su and su/su plants. Criteria are outlined for determining if differences in activity of ribulose-1,5-bisphosphate carboxylase/oxygenase are caused by an effect of polyphenol oxidase activity and/or by some other extrinsic parameter. PMID:16661290

Rubisco Activase Activity Assays

USDA-ARS?s Scientific Manuscript database

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase functions as a mechano-chemical motor protein using the energy from ATP hydrolysis to contort the structure of its target protein, Rubisco. This action modulates the activation state of Rubisco by removing tightly-bound inhibitory s...

Biophysical characterization of higher plant Rubisco activase

USDA-ARS?s Scientific Manuscript database

Rubisco activase (Rca) is a chaperone-like protein of the AAA+ family, which uses mechanochemical energy derived from ATP hydrolysis to release tightly bound inhibitors from the active site of the primary carbon fixing enzyme ribulose 1,5-bisphosphate oxygenase/carboxylase (Rubisco). Mechanistic and...

Comparative modeling and molecular dynamics suggest high carboxylase activity of the Cyanobium sp. CACIAM14 RbcL protein.

PubMed

Siqueira, Andrei Santos; Lima, Alex Ranieri Jerônimo; Dall'Agnol, Leonardo Teixeira; de Azevedo, Juliana Simão Nina; da Silva Gonçalves Vianez, João Lídio; Gonçalves, Evonnildo Costa

2016-03-01

Rubisco catalyzes the first step reaction in the carbon fixation pathway, bonding atmospheric CO2/O2 to ribulose 1,5-bisphosphate; it is therefore considered one of the most important enzymes in the biosphere. Genetic modifications to increase the carboxylase activity of rubisco are a subject of great interest to agronomy and biotechnology, since this could increase the productivity of biomass in plants, algae and cyanobacteria and give better yields in crops and biofuel production. Thus, the aim of this study was to characterize in silico the catalytic domain of the rubisco large subunit (rbcL gene) of Cyanobium sp. CACIAM14, and identify target sites to improve enzyme affinity for ribulose 1,5-bisphosphate. A three-dimensional model was built using MODELLER 9.14, molecular dynamics was used to generate a 100 ns trajectory by AMBER12, and the binding free energy was calculated using MM-PBSA, MM-GBSA and SIE methods with alanine scanning. The model obtained showed characteristics of form-I rubisco, with 15 beta sheets and 19 alpha helices, and maintained the highly conserved catalytic site encompassing residues Lys175, Lys177, Lys201, Asp203, and Glu204. The binding free energy of the enzyme-substrate complexation of Cyanobium sp. CACIAM14 showed values around -10 kcal mol(-1) using the SIE method. The most important residues for the interaction with ribulose 1,5-bisphosphate were Arg295 followed by Lys334. The generated model was successfully validated, remaining stable during the whole simulation, and demonstrated characteristics of enzymes with high carboxylase activity. The binding analysis revealed candidates for directed mutagenesis sites to improve rubisco's affinity.

Characterization of Fructose 1,6-Bisphosphatase and Sedoheptulose 1,7-Bisphosphatase from the Facultative Ribulose Monophosphate Cycle Methylotroph Bacillus methanolicus

PubMed Central

Stolzenberger, Jessica; Lindner, Steffen N.; Persicke, Marcus; Brautaset, Trygve

2013-01-01

The genome of the facultative ribulose monophosphate (RuMP) cycle methylotroph Bacillus methanolicus encodes two bisphosphatases (GlpX), one on the chromosome (GlpXC) and one on plasmid pBM19 (GlpXP), which is required for methylotrophy. Both enzymes were purified from recombinant Escherichia coli and were shown to be active as fructose 1,6-bisphosphatases (FBPases). The FBPase-negative Corynebacterium glutamicum Δfbp mutant could be phenotypically complemented with glpXC and glpXP from B. methanolicus. GlpXP and GlpXC share similar functional properties, as they were found here to be active as homotetramers in vitro, activated by Mn2+ ions and inhibited by Li+, but differed in terms of the kinetic parameters. GlpXC showed a much higher catalytic efficiency and a lower Km for fructose 1,6-bisphosphate (86.3 s−1 mM−1 and 14 ± 0.5 μM, respectively) than GlpXP (8.8 s−1 mM−1 and 440 ± 7.6 μM, respectively), indicating that GlpXC is the major FBPase of B. methanolicus. Both enzymes were tested for activity as sedoheptulose 1,7-bisphosphatase (SBPase), since a SBPase variant of the ribulose monophosphate cycle has been proposed for B. methanolicus. The substrate for the SBPase reaction, sedoheptulose 1,7-bisphosphate, could be synthesized in vitro by using both fructose 1,6-bisphosphate aldolase proteins from B. methanolicus. Evidence for activity as an SBPase could be obtained for GlpXP but not for GlpXC. Based on these in vitro data, GlpXP is a promiscuous SBPase/FBPase and might function in the RuMP cycle of B. methanolicus. PMID:24013630

Characterization of fructose 1,6-bisphosphatase and sedoheptulose 1,7-bisphosphatase from the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus.

PubMed

Stolzenberger, Jessica; Lindner, Steffen N; Persicke, Marcus; Brautaset, Trygve; Wendisch, Volker F

2013-11-01

The genome of the facultative ribulose monophosphate (RuMP) cycle methylotroph Bacillus methanolicus encodes two bisphosphatases (GlpX), one on the chromosome (GlpX(C)) and one on plasmid pBM19 (GlpX(P)), which is required for methylotrophy. Both enzymes were purified from recombinant Escherichia coli and were shown to be active as fructose 1,6-bisphosphatases (FBPases). The FBPase-negative Corynebacterium glutamicum Δfbp mutant could be phenotypically complemented with glpX(C) and glpX(P) from B. methanolicus. GlpX(P) and GlpX(C) share similar functional properties, as they were found here to be active as homotetramers in vitro, activated by Mn(2+) ions and inhibited by Li(+), but differed in terms of the kinetic parameters. GlpX(C) showed a much higher catalytic efficiency and a lower Km for fructose 1,6-bisphosphate (86.3 s(-1) mM(-1) and 14 ± 0.5 μM, respectively) than GlpX(P) (8.8 s(-1) mM(-1) and 440 ± 7.6 μM, respectively), indicating that GlpX(C) is the major FBPase of B. methanolicus. Both enzymes were tested for activity as sedoheptulose 1,7-bisphosphatase (SBPase), since a SBPase variant of the ribulose monophosphate cycle has been proposed for B. methanolicus. The substrate for the SBPase reaction, sedoheptulose 1,7-bisphosphate, could be synthesized in vitro by using both fructose 1,6-bisphosphate aldolase proteins from B. methanolicus. Evidence for activity as an SBPase could be obtained for GlpX(P) but not for GlpX(C). Based on these in vitro data, GlpX(P) is a promiscuous SBPase/FBPase and might function in the RuMP cycle of B. methanolicus.

Expression of endogenous and foreign ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) genes in a RubisCO deletion mutant of Rhodobacter sphaeroides.

PubMed Central

Falcone, D L; Tabita, F R

1991-01-01

A Rhodobacter sphaeroides ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) deletion strain was constructed that was complemented by plasmids containing either the form I or form II CO2 fixation gene cluster. This strain was also complemented by genes encoding foreign RubisCO enzymes expressed from a Rhodospirillum rubrum RubisCO promoter. In R. sphaeroides, the R. rubrum promoter was regulated, resulting in variable levels of disparate RubisCO molecules under different growth conditions. Photosynthetic growth of the R. sphaeroides deletion strain complemented with cyanobacterial RubisCO revealed physiological properties reflective of the unique cellular environment of the cyanobacterial enzyme. The R. sphaeroides RubisCO deletion strain and R. rubrum promoter system may be used to assess the properties of mutagenized proteins in vivo, as well as provide a potential means to select for altered RubisCO molecules after random mutagenesis of entire genes or gene regions encoding RubisCO enzymes. Images PMID:1900508

Small oligomers of ribulose-bisphosphate carboxylase/oxygenase (Rubisco) activase are required for biological activity.

PubMed

Keown, Jeremy R; Griffin, Michael D W; Mertens, Haydyn D T; Pearce, F Grant

2013-07-12

Ribulose-bisphosphate carboxylase/oxygenase (Rubisco) activase uses the energy from ATP hydrolysis to remove tight binding inhibitors from Rubisco, thus playing a key role in regulating photosynthesis in plants. Although several structures have recently added much needed structural information for different Rubisco activase enzymes, the arrangement of these subunits in solution remains unclear. In this study, we use a variety of techniques to show that Rubisco activase forms a wide range of structures in solution, ranging from monomers to much higher order species, and that the distribution of these species is highly dependent on protein concentration. The data support a model in which Rubisco activase forms an open spiraling structure rather than a closed hexameric structure. At protein concentrations of 1 μM, corresponding to the maximal activity of the enzyme, Rubisco activase has an oligomeric state of 2-4 subunits. We propose a model in which Rubisco activase requires at least 1 neighboring subunit for hydrolysis of ATP.

Small Oligomers of Ribulose-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Are Required for Biological Activity

PubMed Central

Keown, Jeremy R.; Griffin, Michael D. W.; Mertens, Haydyn D. T.; Pearce, F. Grant

2013-01-01

Ribulose-bisphosphate carboxylase/oxygenase (Rubisco) activase uses the energy from ATP hydrolysis to remove tight binding inhibitors from Rubisco, thus playing a key role in regulating photosynthesis in plants. Although several structures have recently added much needed structural information for different Rubisco activase enzymes, the arrangement of these subunits in solution remains unclear. In this study, we use a variety of techniques to show that Rubisco activase forms a wide range of structures in solution, ranging from monomers to much higher order species, and that the distribution of these species is highly dependent on protein concentration. The data support a model in which Rubisco activase forms an open spiraling structure rather than a closed hexameric structure. At protein concentrations of 1 μm, corresponding to the maximal activity of the enzyme, Rubisco activase has an oligomeric state of 2–4 subunits. We propose a model in which Rubisco activase requires at least 1 neighboring subunit for hydrolysis of ATP. PMID:23720775

Differential expression of the eight genes of the petunia ribulose bisphosphate carboxylase small subunit multi-gene family

PubMed Central

Dean, Caroline; Elzen, Peter van den; Tamaki, Stanley; Dunsmuir, Pamela; Bedbrook, John

1985-01-01

Of the eight nuclear genes in the plant multi-gene family which encodes the small subunit (rbcS) of Petunia (Mitchell) ribulose bisphosphate carboxylase, one rbcS gene accounts for 47% of the total rbcS gene expression in petunia leaf tissue. Expression of each of five other rbcS genes is detected at levels between 2 and 23% of the total rbcS expression in leaf tissue, while expression of the remaining two rbcS genes is not detected. There is considerable variation (500-fold) in the levels of total rbcS mRNA in six organs of petunia (leaves, sepals, petals, stems, roots and stigmas/anthers). One gene, SSU301, showed the highest levels of steady-state mRNA in each of the organs examined. We discuss the differences in the steady-state mRNA levels of the individual rbcS genes in relation to their gene structure, nucleotide sequence and genomic linkage. ImagesFig. 2.Fig. 3. PMID:16453647

Carbon isotope fractionation by thermophilic phototrophic sulfur bacteria: evidence for autotrophic growth in natural populations

NASA Technical Reports Server (NTRS)

Madigan, M. T.; Takigiku, R.; Lee, R. G.; Gest, H.; Hayes, J. M.

1989-01-01

Purple phototrophic bacteria of the genus Chromatium can grow as either photoautotrophs or photoheterotrophs. To determine the growth mode of the thermophilic Chromatium species, Chromatium tepidum, under in situ conditions, we have examined the carbon isotope fractionation patterns in laboratory cultures of this organism and in mats of C. tepidum which develop in sulfide thermal springs in Yellowstone National Park. Isotopic analysis (13C/12C) of total carbon, carotenoid pigments, and bacteriochlorophyll from photoautotrophically grown cultures of C. tepidum yielded 13C fractionation factors near -20%. Cells of C. tepidum grown on excess acetate, wherein synthesis of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase ribulose bisphosphate carboxylase) was greatly repressed, were isotopically heavier, fractionation factors of ca. -7% being observed. Fractionation factors determined by isotopic analyses of cells and pigment fractions of natural populations of C. tepidum growing in three different sulfide thermal springs in Yellowstone National Park were approximately -20%, indicating that this purple sulfur bacterium grows as a photoautotroph in nature.

Mechanistic diversity in the RuBisCO superfamily: RuBisCO from Rhodospirillum rubrum is not promiscuous for reactions catalyzed by RuBisCO-like proteins.

PubMed

Warlick, Benjamin P E; Imker, Heidi J; Sriram, Jaya; Tabita, F Robert; Gerlt, John A

2012-11-27

d-Ribulose 1,5-bisphosphate carboxylase/oxygenases (RuBisCOs) are promiscuous, catalyzing not only carboxylation and oxygenation of d-ribulose 1,5-bisphosphate but also other promiscuous, presumably nonphysiological, reactions initiated by abstraction of the 3-proton of d-ribulose 1,5-bisphosphate. Also, RuBisCO has homologues that do not catalyze carboxylation; these are designated RuBisCO-like proteins or RLPs. Members of the two families of RLPs catalyze reactions in the recycling of 5'-methylthioadenosine (MTA) generated by polyamine synthesis: (1) the 2,3-diketo-5-methylthiopentane 1-phosphate (DK-MTP 1-P) "enolase" reaction in the well-known "methionine salvage" pathway in Bacillus sp. and (2) the 5-methylthio-d-ribulose 1-phosphate (MTRu 1-P) 1,3-isomerase reaction in the recently discovered "MTA-isoprenoid shunt" that generates 1-deoxy-d-xylulose 5-phosphate for nonmevalonate isoprene synthesis in Rhodospirillum rubrum. We first studied the structure and reactivity of DK-MTP 1-P that was reported to decompose rapidly [Ashida, H., Saito, Y., Kojima, C., and Yokota, A. (2008) Biosci., Biotechnol., Biochem. 72, 959-967]. The 2-carbonyl group of DK-MTP 1-P is rapidly hydrated and can undergo enolization both nonenzymatically and enzymatically via the small amount of unhydrated material that is present. We then examined the ability of RuBisCO from R. rubrum to catalyze both of the RLP-catalyzed reactions. Contrary to a previous report [Ashida, H., Saito, Y., Kojima, C., Kobayashi, K., Ogasawara, N., and Yokota, A. (2003) Science 302, 286-290], we were unable to confirm that this RuBisCO catalyzes the DK-MTP 1-P "enolase" reaction either in vitro or in vivo. We also determined that this RuBisCO does not catalyze the MTRu 1-P 1,3-isomerase reaction in vitro. Thus, although RuBisCOs can be functionally promiscuous, RuBisCO from R. rubrum is not promiscuous for either of the known RLP-catalyzed reactions.

Induction of Heat-Shock Proteins in Coxiella burnetii

DTIC Science & Technology

1990-06-26

the groEL protein in Escherichia coli is believed to aid the * assembly of ribulose bisphosphate carboxylase/oxygenase ( RuBisCO ) within chloroplasts...responsible for the assembly of RuBisCo within chloroplasts must also be translocated prior to serving this function.4 It is therefore not surprising that its

Recycling carbon dioxide during xylose fermentation by engineered Saccharomyces cerevisiae

USDA-ARS?s Scientific Manuscript database

In this study, we introduced the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase (PRK) into an engineered S. cerevisiae (SR8) harboring the XR/XDH pathway and up-regulated PPP 10, to enable CO2 recycling through a synthetic rPPP during xylose fermentation (Fig. 1). ...

Linkage and homology analysis divides the eight genes for the small subunit of petunia ribulose 1,5-bisphosphate carboxylase into three gene families

PubMed Central

Dean, Caroline; van den Elzen, Peter; Tamaki, Stanley; Dunsmuir, Pamela; Bedbrook, John

1985-01-01

Twenty-six λ phage clones with homology to coding sequences of the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase have been isolated from an EMBL3 λ phage bank of Petunia (Mitchell) DNA. Restriction mapping of the phage inserts shows that the clones were obtained from five nonoverlapping regions of petunia DNA that carry seven SSU genes. Comparison of the HindIII genomic fragments of petunia DNA with the HindIII restriction fragments of the isolated phage indicates that petunia nuclear DNA encodes eight SSU genes, seven of which are present in the phage clones. Two incomplete genes, which contain only the 3′ end of an SSU gene, were also found in the phage clones. We demonstrate that the eight SSU genes of petunia can be divided into three gene families based on homology to three petunia cDNA clones. Two gene families contain single SSU genes and the third contains six genes, four of which are closely linked within petunia nuclear DNA. Images PMID:16593584

Polysome Immunoselection Combined with cDNA Cloning to Obtain Specific Genes from Chlamydomonas reinhardtii

DTIC Science & Technology

1989-02-05

speclficlty of an avallable antitody (IgG) aginst the ribulose bisphosphate carboxylase sma"Ll subunit ( RUBISCO SSU). First, polysomal poly A+) RNA was...Lnlunopreclpitated by the antibody is the same size as the precursor of the RUBISCO S&U ,Fig. ,. When intact polysones were added to the wheat germ

Older Thinopyrum intermedium (Poaceae) plants exhibit superior photosynthetic tolerance to cold stress and greater increases in two photosynthetic enzymes under freezing stress compared with young plants

PubMed Central

Jaikumar, Nikhil S.; Snapp, Sieglinde S.; Sharkey, Thomas D.

2016-01-01

Effects of plant age on resource acquisition and stress tolerance processes is a largely unstudied subject in herbaceous perennials. In a field experiment, we compared rates of photosynthesis (A), ribulose-1,5-bisphosphate (RuBP) carboxylation capacity (V Cmax), maximum electron transport rate (J max), and triose phosphate utilization (TPU), as well as concentrations of Rubisco and sucrose-phosphate synthase (SPS) in 5-year-old and 2-year-old intermediate wheatgrass (Thinopyrum intermedium) under both optimal growing conditions and cold stress in early spring and autumn. This species is a relative of wheat undergoing domestication. An additional experiment compared photosynthetic rates in different cohorts at mid-season and under colder conditions. We hypothesized that photosynthetic capacity in older plants would be lower under favorable conditions but higher under cold stress. Our hypothesis was generally supported. Under cold stress, 5-year-old plants exhibited higher A, TPU, and temperature-adjusted V Cmax than younger plants, as well as 50% more SPS and 37% more Rubisco. In contrast, at mid-season, photosynthetic capacities in older plants were lower than in younger plants in one experiment, and similar in the other, independent of differences in water status. Both cohorts increased A, temperature-adjusted TPU and J max, [Rubisco], and [SPS] under cold stress, but changes were greater in older plants. Photosynthetic differences were largest at 1.2 ºC in very early spring, where older plants had 200% higher A and maintained up to 17% of their peak photosynthetic capacity. We find evidence of increased cold tolerance in older cohorts of wheatgrass, consistent with a growing body of research in woody perennials. PMID:27401911

Expression of the ribulose-1,5-bisphosphate carboxylase large subunit gene and three small subunit genes in two cell types of maize leaves

PubMed Central

Sheen, Jenq-Yunn; Bogorad, Lawrence

1986-01-01

Transcripts of three distinct ribulose-1,5-bisphosphate carboxylase (RuBPC) small subunit (SS) genes account for ∼90% of the mRNA for this protein in maize leaves. Transcripts of two of them constitute >80% of the SS mRNA in 24-h greening maize leaves. The third gene contribute ∼10%. Transcripts of all three nuclear-encoded SS genes are detectable in bundle sheath (BSC) and mesophyll cells (MC) of etiolated maize leaves. The level of mRNA for each gene is different in etioplasts of MC but all drop during photoregulated development of chloroplasts in MC and follow a pattern of transitory rise and fall in BSC. The amounts of LS and SS proteins continue to increase steadily well after the mRNA levels reach their peaks in BSC. The molar ratio of mRNA for chloroplast-encoded RuBPC large subunit (LS) to the nuclear genome encoded SS is about 10:1 although LS and SS proteins are present in about equimolar amounts. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4.Fig. 5.Fig. 6. PMID:16453739

Silencing ribulose-1,5-bisphosphate carboxylase/oxygenase expression does not disrupt nitrogen allocation to defense after simulated herbivory in Nicotiana attenuata.

PubMed

Stanton, Mariana A; Ullmann-Zeunert, Lynn; Wielsch, Natalie; Bartram, Stefan; Svatoš, Aleš; Baldwin, Ian T; Groten, Karin

2013-01-01

Ribulose-1,5-bisphosphate carboxylase/ oxygenase (RuBisCO) is the most abundant protein on the planet and in addition to its central role in photosynthesis it is thought to function as a nitrogen (N)-storage protein and a potential source of N for defense biosynthesis in plants. In a recent study in the wild tobacco Nicotiana attenuata, we showed that the decrease in absolute N invested in soluble proteins and RuBisCO elicited by simulated herbivory was much larger than the N-requirements of nicotine and phenolamide biosynthesis; (15)N flux studies revealed that N for defensive phenolamide synthesis originates from recently assimilated N rather than from RuBisCO turnover. Here we show that a transgenic line of N. attenuata silenced in the expression of RuBisCO (asRUB) invests similar or even larger amounts of N into phenolamide biosynthesis compared with wild type plants, consistent with our previous conclusion that recently assimilated N is channeled into phenolamide synthesis after elicitation. We suggest that the decrease in leaf proteins after simulated herbivory is a tolerance mechanism, rather than a consequence of N-demand for defense biosynthesis.

Engineering Microorganisms for Energy Production

DTIC Science & Technology

2006-06-01

the oxygen sensi- tivity of fuel-forming catalysts in biological systems. Hydrogenases, nitrogenases, and rubisco in C3 plants are all oxygen...sensitive. Indeed, C4 plants are more efficient because they developed an independent mechanism to isolate the rubisco from oxygen. Photodamage is a key...single CO 2 to sugars requires 8 photons. The reactions converting CO 2 to sugars are catalyzed by the enzyme rubisco (ribulose 1,5-bisphosphate 33

Ribulose-1,5-bis-phosphate carboxylase/oxygenase accumulation factor1 is required for holoenzyme assembly in maize.

PubMed

Feiz, Leila; Williams-Carrier, Rosalind; Wostrikoff, Katia; Belcher, Susan; Barkan, Alice; Stern, David B

2012-08-01

Most life is ultimately sustained by photosynthesis and its rate-limiting carbon fixing enzyme, ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco). Although the structurally comparable cyanobacterial Rubisco is amenable to in vitro assembly, the higher plant enzyme has been refractory to such manipulation due to poor understanding of its assembly pathway. Here, we report the identification of a chloroplast protein required for Rubisco accumulation in maize (Zea mays), RUBISCO ACCUMULATION FACTOR1 (RAF1), which lacks any characterized functional domains. Maize lines lacking RAF1 due to Mutator transposon insertions are Rubisco deficient and seedling lethal. Analysis of transcripts and proteins showed that Rubisco large subunit synthesis in raf1 plants is not compromised; however, newly synthesized Rubisco large subunit appears in a high molecular weight form whose accumulation requires a specific chaperonin 60 isoform. Gel filtration analysis and blue native gels showed that endogenous and recombinant RAF1 are trimeric; however, following in vivo cross-linking, RAF1 copurifies with Rubisco large subunit, suggesting that they interact weakly or transiently. RAF1 is predominantly expressed in bundle sheath chloroplasts, consistent with a Rubisco accumulation function. Our results support the hypothesis that RAF1 acts during Rubisco assembly by releasing and/or sequestering the large subunit from chaperonins early in the assembly process.

Examination of Chemical Adsorption and Marine Biofouling on Metal Surfaces Using Raman Scattering Techniques and Electrochemical Impedance Spectroscopy

DTIC Science & Technology

1991-03-14

analyses of labeled model compounds, such as the protein, RuBisCO (Ribulose Bisphosphate CarboxylaselOxygenase). (3) Examine adsorption of...coverages were determined radiometrically using tritiated RuBisCO . Although natural thin films were detectable using Raman scattering techniques...optical, electrochemical, and radiometric techniques and the protein RuBisCO as a model adsorbate on titanium, copper, and iron, we have been able to

A gene duplication/loss event in the ribulose-1,5-bisphosphate-carboxylase/oxygenase (rubisco) small subunit gene family among accessions of Arabidopsis thaliana.

PubMed

Schwarte, Sandra; Tiedemann, Ralph

2011-06-01

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39), the most abundant protein in nature, catalyzes the assimilation of CO(2) (worldwide about 10(11) t each year) by carboxylation of ribulose-1,5-bisphosphate. It is a hexadecamer consisting of eight large and eight small subunits. Although the Rubisco large subunit (rbcL) is encoded by a single gene on the multicopy chloroplast genome, the Rubisco small subunits (rbcS) are encoded by a family of nuclear genes. In Arabidopsis thaliana, the rbcS gene family comprises four members, that is, rbcS-1a, rbcS-1b, rbcS-2b, and rbcS-3b. We sequenced all Rubisco genes in 26 worldwide distributed A. thaliana accessions. In three of these accessions, we detected a gene duplication/loss event, where rbcS-1b was lost and substituted by a duplicate of rbcS-2b (called rbcS-2b*). By screening 74 additional accessions using a specific polymerase chain reaction assay, we detected five additional accessions with this duplication/loss event. In summary, we found the gene duplication/loss in 8 of 100 A. thaliana accessions, namely, Bch, Bu, Bur, Cvi, Fei, Lm, Sha, and Sorbo. We sequenced an about 1-kb promoter region for all Rubisco genes as well. This analysis revealed that the gene duplication/loss event was associated with promoter alterations (two insertions of 450 and 850 bp, one deletion of 730 bp) in rbcS-2b and a promoter deletion (2.3 kb) in rbcS-2b* in all eight affected accessions. The substitution of rbcS-1b by a duplicate of rbcS-2b (i.e., rbcS-2b*) might be caused by gene conversion. All four Rubisco genes evolve under purifying selection, as expected for central genes of the highly conserved photosystem of green plants. We inferred a single positive selected site, a tyrosine to aspartic acid substitution at position 72 in rbcS-1b. Exactly the same substitution compromises carboxylase activity in the cyanobacterium Anacystis nidulans. In A. thaliana, this substitution is associated with an inferred recombination. Functional implications of the substitution remain to be evaluated.

Constraining the timing of the Great Oxidation Event within the Rubisco phylogenetic tree.

PubMed

Kacar, B; Hanson-Smith, V; Adam, Z R; Boekelheide, N

2017-09-01

Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO, or Rubisco) catalyzes a key reaction by which inorganic carbon is converted into organic carbon in the metabolism of many aerobic and anaerobic organisms. Across the broader Rubisco protein family, homologs exhibit diverse biochemical characteristics and metabolic functions, but the evolutionary origins of this diversity are unclear. Evidence of the timing of Rubisco family emergence and diversification of its different forms has been obscured by a meager paleontological record of early Earth biota, their subcellular physiology and metabolic components. Here, we use computational models to reconstruct a Rubisco family phylogenetic tree, ancestral amino acid sequences at branching points on the tree, and protein structures for several key ancestors. Analysis of historic substitutions with respect to their structural locations shows that there were distinct periods of amino acid substitution enrichment above background levels near and within its oxygen-sensitive active site and subunit interfaces over the divergence between Form III (associated with anoxia) and Form I (associated with oxia) groups in its evolutionary history. One possible interpretation is that these periods of substitutional enrichment are coincident with oxidative stress exerted by the rise of oxygenic photosynthesis in the Precambrian era. Our interpretation implies that the periods of Rubisco substitutional enrichment inferred near the transition from anaerobic Form III to aerobic Form I ancestral sequences predate the acquisition of Rubisco by fully derived cyanobacterial (i.e., dual photosystem-bearing, oxygen-evolving) clades. The partitioning of extant lineages at high clade levels within our Rubisco phylogeny indicates that horizontal transfer of Rubisco is a relatively infrequent event. Therefore, it is possible that the mutational enrichment periods between the Form III and Form I common ancestral sequences correspond to the adaptation of key oxygen-sensitive components of Rubisco prior to, or coincident with, the Great Oxidation Event. © 2017 The Authors. Geobiology Published by John Wiley & Sons Ltd.

Contrasting root and photosynthesis traits in a large-acreage Canadian durum variety and its distant parent of Algerian origin for assembling drought/heat tolerance attributes

NASA Astrophysics Data System (ADS)

Ashe, Paula; Shaterian, Hamid; Akhov, Leonid; Kulkarni, Manoj; Selvaraj, Gopalan

2017-12-01

In Canada, the world’s top exporter of high-protein durum, varietal development over its nearly six-decade history has been driven by a quest for yield improvement without compromise on grain protein content and other quality aspects. Pelissier, a landrace selection from Algeria that was introduced into North America more than a century ago and the variety Strongfield that was released in 2004 are notable. Pelissier, known to elaborate more roots and considered as drought tolerant, has been cultivated commercially and thus deemed adapted. Strongfield has Pelissier in its pedigree, and it remains a high-acreage variety. Strongfield was found to elaborate only about half of the root biomass of Pelissier at maturity in greenhouse trials under well-watered conditions. Extended drought stress caused a significant reduction in the root biomass of both lines. However, Pelissier under drought maintained at least as much root biomass as that of Strongfield under well-watered conditions. In comparison to Pelissier, it had a superior photosynthesis rate (27.16 µmol CO2 m-2 s-1), capacity for carboxylation (Vcmax: 132.83 µmol CO2 m-2 s-1) and electron transport/ribulose-1,5-bisphosphate (RuBP) regeneration (Jmax: 265.40 µmol CO2 m-2 s-1); the corresponding values for Pelissier were 19.62 µmol CO2 m-2 s-1, 91.87 µmol CO2 m-2 s-1, and 163.83 µmol CO2 m-2 s-1, respectively, under well-watered conditions. Under short-term/mild drought conditions, the carbon assimilation rate remained stable in Pelissier while it declined in Strongfield to the Pelissier level. However, Strongfield succumbed to extended drought sooner than Pelissier. Photosynthesis in Strongfield but not Pelissier was found to be sensitive to high temperature stress. These results provide encouraging prospects for further exploitation of beneficial physiological traits from Pelissier in constructing climate-resilient, agronomically favourable wheat ideotypes.

Older Thinopyrum intermedium (Poaceae) plants exhibit superior photosynthetic tolerance to cold stress and greater increases in two photosynthetic enzymes under freezing stress compared with young plants.

PubMed

Jaikumar, Nikhil S; Snapp, Sieglinde S; Sharkey, Thomas D

2016-08-01

Effects of plant age on resource acquisition and stress tolerance processes is a largely unstudied subject in herbaceous perennials. In a field experiment, we compared rates of photosynthesis (A), ribulose-1,5-bisphosphate (RuBP) carboxylation capacity (V Cmax), maximum electron transport rate (J max), and triose phosphate utilization (TPU), as well as concentrations of Rubisco and sucrose-phosphate synthase (SPS) in 5-year-old and 2-year-old intermediate wheatgrass (Thinopyrum intermedium) under both optimal growing conditions and cold stress in early spring and autumn. This species is a relative of wheat undergoing domestication. An additional experiment compared photosynthetic rates in different cohorts at mid-season and under colder conditions. We hypothesized that photosynthetic capacity in older plants would be lower under favorable conditions but higher under cold stress. Our hypothesis was generally supported. Under cold stress, 5-year-old plants exhibited higher A, TPU, and temperature-adjusted V Cmax than younger plants, as well as 50% more SPS and 37% more Rubisco. In contrast, at mid-season, photosynthetic capacities in older plants were lower than in younger plants in one experiment, and similar in the other, independent of differences in water status. Both cohorts increased A, temperature-adjusted TPU and J max, [Rubisco], and [SPS] under cold stress, but changes were greater in older plants. Photosynthetic differences were largest at 1.2 ºC in very early spring, where older plants had 200% higher A and maintained up to 17% of their peak photosynthetic capacity. We find evidence of increased cold tolerance in older cohorts of wheatgrass, consistent with a growing body of research in woody perennials. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Biology of Symbioses between Marine Invertebrates and Intracellular Bacteria

DTIC Science & Technology

1989-01-05

number of gene probes for enzymes of CO2 (ribulose-1,5-bisphosphate carboxylase; RuBisCo ) and N2 (nitrogenase) fixation (see table 2). Using these probes... RuBisCo we could establish relationships and homologies for this enzyme among different symbionts. Table 1. Type and disposition of symblont DNA samples...st:;bution I Avail ?, I Table 2. Molecular probes available for this study. Prokaryote Type Plasrnid Probe Carbon Fixation ( RuBisCo ) Anabaena 7120

Gene Discovery and Expression Profiling in the Toxin-Producing Marine Diatom, Pseudo-nitzschia Multiseries (Hasle) Hasle

DTIC Science & Technology

2004-09-01

carboxylase/oxygenase ( rubisco ) is a another principal carbon fixation enzyme, which is alleged to represent the most abundant enzyme on earth...known to have plastid-encoded rubisco , which would account for why this gene was not identified in the P. multiseries library (Hwang and Tabita, 1989...thought to have evolved in certain plants as an adaptation to the competition of oxygen with carbon dioxide for ribulose- 15-bisphosphate ( rubisco ), a

Ribulose-1,5-Bis-Phosphate Carboxylase/Oxygenase Accumulation Factor1 Is Required for Holoenzyme Assembly in Maize[C][W

PubMed Central

Feiz, Leila; Williams-Carrier, Rosalind; Wostrikoff, Katia; Belcher, Susan; Barkan, Alice; Stern, David B.

2012-01-01

Most life is ultimately sustained by photosynthesis and its rate-limiting carbon fixing enzyme, ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco). Although the structurally comparable cyanobacterial Rubisco is amenable to in vitro assembly, the higher plant enzyme has been refractory to such manipulation due to poor understanding of its assembly pathway. Here, we report the identification of a chloroplast protein required for Rubisco accumulation in maize (Zea mays), RUBISCO ACCUMULATION FACTOR1 (RAF1), which lacks any characterized functional domains. Maize lines lacking RAF1 due to Mutator transposon insertions are Rubisco deficient and seedling lethal. Analysis of transcripts and proteins showed that Rubisco large subunit synthesis in raf1 plants is not compromised; however, newly synthesized Rubisco large subunit appears in a high molecular weight form whose accumulation requires a specific chaperonin 60 isoform. Gel filtration analysis and blue native gels showed that endogenous and recombinant RAF1 are trimeric; however, following in vivo cross-linking, RAF1 copurifies with Rubisco large subunit, suggesting that they interact weakly or transiently. RAF1 is predominantly expressed in bundle sheath chloroplasts, consistent with a Rubisco accumulation function. Our results support the hypothesis that RAF1 acts during Rubisco assembly by releasing and/or sequestering the large subunit from chaperonins early in the assembly process. PMID:22942379

Influence of Plant Growth at High CO2 Concentrations on Leaf Content of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Intracellular Distribution of Soluble Carbohydrates in Tobacco, Snapdragon, and Parsley.

PubMed Central

Moore, Bd.; Palmquist, D. E.; Seemann, J. R.

1997-01-01

We have examined the possible role of leaf cytosolic hexoses and the expression of mannitol metabolism as mechanisms that may affect the repression of photosynthetic capacity when plants are grown at 1000 versus 380 [mu]L L-1 CO2. In plants grown at high CO2, leaf ribulose-1,5-bisphosphate carboxylase/oxygenase content declined by [greater than or equal to]20% in tobacco (Nicotiana sylvestris) but was not affected in the mannitol-producing species snapdragon (Antirrhinum majus) and parsley (Petroselinum hortense). In the three species mesophyll glucose and fructose at midday occurred almost entirely in the vacuole (>99%), irrespective of growth CO2 levels. The estimated cytosolic concentrations of glucose and fructose were [less than or equal to]100 [mu]M. In the three species grown at high CO2, total leaf carbohydrates increased 60 to 100%, but mannitol metabolism did not function as an overflow mechanism for the increased accumulation of carbohydrate. In both snapdragon and parsley grown at ambient or high CO2, mannitol occurred in the chloroplast and cytosol at estimated midday concentrations of 0.1 M or more each. The compartmentation of leaf hexoses and the metabolism of alternate carbohydrates are further considered in relation to photosynthetic acclimation to high levels of CO2. PMID:12223804

Expression of glnB and a glnB-Like Gene (glnK) in a Ribulose Bisphosphate Carboxylase/Oxygenase-Deficient Mutant of Rhodobacter sphaeroides

PubMed Central

Qian, Yilei; Tabita, F. Robert

1998-01-01

In a ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO)-deficient mutant of Rhodobacter sphaeroides, strain 16PHC, nitrogenase activity was derepressed in the presence of ammonia under photoheterotrophic growth conditions. Previous studies also showed that reintroduction of a functional RubisCO and Calvin-Benson-Bassham (CBB) pathway suppressed the deregulation of nitrogenase synthesis in this strain. In this study, the derepression of nitrogenase synthesis in the presence of ammonia in strain 16PHC was further explored by using a glnB::lacZ fusion, since the product of the glnB gene is known to have a negative effect on ammonia-regulated nif control. It was found that glnB expression was repressed in strain 16PHC under photoheterotrophic growth conditions with either ammonia or glutamate as the nitrogen source; glutamine synthetase (GS) levels were also affected in this strain. However, when cells regained a functional CBB pathway by trans complementation of the deleted genes, wild-type levels of GS and glnB expression were restored. Furthermore, a glnB-like gene, glnK, was isolated from this organism, and its expression was found to be under tight nitrogen control in the wild type. Surprisingly, glnK expression was found to be derepressed in strain 16PHC under photoheterotrophic conditions in the presence of ammonia. PMID:9721307

Variation in Rubisco and other photosynthetic parameters in the life cycle of Haematococcus pluvialis

NASA Astrophysics Data System (ADS)

Chen, Zhangfan; Wang, Guangce; Niu, Jianfeng

2012-01-01

Cells of Haematococcus pluvialis Flot. et Will were collected in four different growth phases. We quantified the initial and total enzyme activity of ribulose-1,5-bisphosphate carboxylase (Rubisco) in crude extracts, and the relative expression of large-subunit ribulose-1,5-bisphosphate caboxylase / oxygenase ( rbcL) mRNA. We measured the ratio of photosynthetic rate to respiration rate (P/R), maximal effective quantum yield of photosystem II ( F v/ F m), electron transport rate (ETR), actual photochemical efficiency of PSII in the light (PSII), and non-photochemical quenching (NPQ). Green vegetative cells were found to be in the most active state, with a relatively higher P/R ratio. These cells also displayed the lowest NPQ and the highest F v/ F m, ETR, and PSII, indicating the most effective PSII. However, both Rubisco activity and rbcL mRNA expression were the lowest measured. In orange resting cysts with relatively lower P/R and NPQ, Rubisco activity and rbcL expression reached a peak, while F v/ F m, ETR, and ΦPSII were the lowest measured. Taking into account the methods of astaxanthin induction used in industry, we suggest that Rubisco may participate in astaxanthin accumulation in H. pluvialis. A continuous and sufficient supply of a carbon source such as CO2 may therefore aid the large scale production of astaxanthin.

Characterization of Thylakoid-Derived Lipid-Protein Particles Bearing the Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase.

PubMed Central

Smith, M. D.; Ghosh, S.; Dumbroff, E. B.; Thompson, J. E.

1997-01-01

Lipid-protein particles bearing the 55-kD ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (EC 4.1.1.39) large subunit (RLSU) and no detectable corresponding Rubisco small subunit (RSSU) were isolated from the stroma of intact chloroplasts by flotation centrifugation. Stromal RLSU-bearing particles appear to originate from thylakoids because they can also be generated in vitro by illumination of isolated thylakoids. Their formation in vitro is largely heat denaturable and is facilitated by light or ATP. RLSU-containing lipid-protein particles range from 0.05 to 0.10 [mu]m in radius, contain the same fatty acids as thylakoids, but have a 10- to 15-fold higher free-to-esterified fatty acid ratio than thylakoids. RLSU-bearing lipid-protein particles with no detectable RSSU were also immunopurified from the populations of both stromal lipid-protein particles and those generated in vitro from illuminated thylakoids. Protease shaving indicated that the RLSU is embedded in the lipid-protein particles and that there is also a protease-protected RLSU in thylakoids. These observations collectively indicate that the RLSU associated with thylakoids is released into the stroma by light-facilitated blebbing of lipid-protein particles. The release of RLSU-containing particles may in turn be coordinated with the assembly of Rubisco holoenzyme because chaperonin 60 is also associated with lipid-protein particles isolated from stroma. PMID:12223858

Photosynthesis in Ulva fasciata

PubMed Central

Beer, Sven; Israel, Alvaro; Drechsler, Zivia; Cohen, Yael

1990-01-01

Evidence of an inorganic carbon concentrating system in a marine macroalga is provided here. Based on an O2 technique, supported by determinations of inorganic carbon concentrations, of experimental media (as well as compensation points) using infrared gas analysis, it was found that Ulva fasciata maintained intracellular inorganic carbon levels of 2.3 to 6.0 millimolar at bulk medium concentrations ranging from 0.02 to 1.5 millimolar. Bicarbonate seemed to be the preferred carbon form taken up at all inorganic carbon levels. It was found that ribulose-1,5-bisphosphate carboxylase/oxygenase from Ulva had a Km(CO2) of 70 micromolar and saturated at about 250 micromolar CO2. Assuming a cytoplasmic pH of 7.2 (as measured for another Ulva species, P Lundberg et al. [1988] Plant Physiol 89: 1380-1387), and given the high activity of internal carbonic anhydrase (S Beer, A Israel [1990] Plant Cell Environ [in press]) and the here measured internal inorganic carbon level, it was concluded that internal CO2 in Ulva could, at ambient external inorganic carbon concentrations, be maintained at a high enough level to saturate ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation. It is suggested that this suppresses photorespiration and optimizes net photosynthetic production in an alga representing a large group of marine plants faced with limiting external CO2 concentrations in nature. PMID:16667887

Enhancing Natural Attenuation Through Bioaugmentation with Aerobic Bacteria that Degrade Cis-1,2-Dichloroethene

DTIC Science & Technology

2008-04-01

have genes that encode enzymes of the Calvin-Benson cycle, specifically for the key enzyme ribulose 1,5-bisphosphate carboxylase ( RubisCO ) which...Closer examination of the gene sequence annotated as RubisCO revealed that is more closely related to RubisCO -like (Hanson and Tabita 2001) proteins than...to RubisCO and is likely not involved in the Calvin-Benson cycle. Ionic Strength A recent paper (Müller, Walter et al. 2006) explored the

Effects of water stress on photosynthetic electron transport, photophosphorylation, and metabolite levels of Xanthium strumarium mesophyll cells.

PubMed

Sharkey, T D; Badger, M R

1982-12-01

Several component processes of photosynthesis were measured in osmotically stressed mesophyll cells of Xanthium strumarium L. The ribulose-1,5-bisphosphate regeneration capacity was reduced by water stress. Photophoshorylation was sensitive to water stress but photosynthetic electron transport was unaffected by water potentials down to-40 bar (-4 MPa). The concentrations of several intermediates of the photosynthetic carbon-reduction cycle remained relatively constant and did not indicate that ATP supply was limiting photosynthesis in the water-stressed cells.

Carbon isotope geochemistry and geobiology

NASA Technical Reports Server (NTRS)

Desmarais, D.

1985-01-01

Carbon isotope fractionation values were used to understand the history of the biosphere. For example, plankton analyses confirmed that marine extinctions at the end of the Cretaceous period were indeed severe (see Hsu's article in Sundquist and Broeker, 1984). Variations in the isotopic compositions of carbonates and evaporitic sulfates during the Paleozoic reflect the relative abundances of euxinic (anoxic) marine environments and organic deposits from terrestrial flora. The carbon isotopic composition of Precambrian sediments suggest that the enzyme ribulose bisphosphate carboxylase has existed for perhaps 3.5 billion years.

[Effect of DNA-damaging agents on the aerobic methylobacteria capable and incapable of utilizing dichloromethane].

PubMed

Firsova, Iu E; Torgonskaia, M L; Doronina, N V; Trotsenko, Iu A

2005-01-01

Methylobacterium dichloromethanicum DM4, a degrader of dichloromethane (DCM), was more tolerant to the effect of H2O2 and UV irradiation than Methylobacterium extorquens AM1, which does not consume DCM. Addition of CH2Cl2 to methylobacteria with active serine, ribulose monophosphate, and ribulose bisphosphate pathways of C1 metabolism, grown on methanol, resulted in a 1.1- to 2.5-fold increase in the incorporation of [alpha-32P]dATP into DNA Klenow fragment (exo-). As DCM dehalogenase was not induced in this process, the increase in total lengths of DNA gaps resulted from the action of DCM rather than S-chloromethylglutathione (intermediate of primary dehalogenation). The degree of DNA damage in the presence of CH2Cl2 was lower in DCM degraders than methylobacteria incapable of degrading this pollutant. This suggests that DCM degraders possess a more efficient mechanism of DNA repair.

Quantitative analyses of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large-subunit genes (cbbL) in typical paddy soils.

PubMed

Xiao, Ke-Qing; Bao, Peng; Bao, Qiong-Li; Jia, Yan; Huang, Fu-Yi; Su, Jian-Qiang; Zhu, Yong-Guan

2014-01-01

The Calvin cycle is known to be the major pathway for CO2 fixation, but our current understanding of its occurrence and importance in paddy soils is poor. In this study, the diversity of three ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes (cbbLG, cbbLR, cbbM) was investigated by clone library, T-RFLP, qPCR, and enzyme assay in five paddy soils in China. The cbbLG sequences revealed a relatively low level of diversity and were mostly related to the sequences of species from Thiobacillus. In contrast, highly diverse cbbLR and cbbM sequences were dispersed on the phylogenetic trees, and most of them were distantly related to known sequences, even forming separate clusters. Abundances of three cbbL genes ranged from 10(6) to 10(9) copies g(-1) soil, and cbbLR outnumbered cbbM and cbbLG in all soil samples, indicating that cbbLR may play a more important role than other two cbbL genes. Soil properties significantly influenced cbbL diversity in five paddy soils, of which clay content, C/N ratio, CEC, pH, and SOC correlated well with variations in microbial composition and abundance. In summary, this study provided a comparison of three cbbL genes, advancing our understanding of their role in carbon sequestration and nutrient turnover in the paddy soil. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Purification and Assay of Rubisco Activase from Leaves 1

PubMed Central

Robinson, Simon P.; Streusand, Virginia J.; Chatfield, J. Mark; Portis, Archie R.

1988-01-01

Ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) activase protein was purified from spinach leaves by ammonium sulfate precipitation and ion exchange fast protein liquid chromatography. This resulted in 48-fold purification with 70% recovery of activity and yielded up to 18 milligrams of rubisco activase protein from 100 grams of leaves. Based on these figures, the protein comprised approximately 2% by weight of soluble protein in spinach (Spinacia oleracea L.) leaves. The preparations were at least 95% pure and were stable when frozen in liquid nitrogen. Addition of ATP during purification and storage was necessary to maintain activity. Assay of rubisco activase was based on its ability to promote activation of rubisco in the presence of ribulose-1,5-bisphosphate. There was an absolute requirement for ATP which could not be replaced by other nucleoside phosphates. The initial rate of increase of rubisco activity and the final rubisco specific activity achieved were both dependent on the concentration of rubisco activase. The initial rate was directly proportional to the rubisco activase concentration and was used as the basis of activity. The rate of activation of rubisco was also dependent on the rubisco concentration, suggesting that the activation process is a second order reaction dependent on the concentrations of both rubisco and rubisco activase. It is suggested that deactivation of rubisco occurs simultaneously with rubisco activase-mediated activation, and that rubisco activation state represents a dynamic equilibrium between these two processes. Images Fig. 2 PMID:16666412

Differential Protein Composition and Gene Expression in Leaf Mesophyll Cells and Bundle Sheath Cells of the C(4) Plant Digitaria sanguinalis (L.) Scop.

PubMed

Potter, J W; Black, C C

1982-08-01

The distribution and molecular weights of cellular proteins in soluble and membrane-associated locations were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining of leaf (Digitaria sanguinalis L. Scop.) extracts and isolated cell extracts. Leaf polypeptides also were pulse-labeled, followed by isolation of the labeled leaf cell types and analysis of the newly synthesized polypeptides in each cell type by electrophoresis and fluorography.Comparison of the electrophoretic patterns of crabgrass whole leaf polypeptides with isolated cell-type polypeptides indicated a difference in protein distribution patterns for the two cell types. The mesophyll cells exhibited a greater allocation of total cellular protein into membrane-associated proteins relative to soluble proteins. In contrast, the bundle sheath cells exhibited a higher percentage of total cellular protein in soluble proteins. Phosphoenolpyruvate carboxylase was the major soluble protein in the mesophyll cell and ribulose bisphosphate carboxylase was the major soluble protein in the bundle sheath cell. The majority of in vivo(35)S-pulse-labeled proteins synthesized by the two crabgrass cell types corresponded in molecular weight to the proteins present in the cell types which were detected by conventional staining techniques. The bundle sheath cell and mesophyll cell fluorograph profiles each had 15 major (35)S-labeled proteins. The major incorporation of (35)S by bundle sheath cells was into products which co-electrophoresed with the large and small subunits of ribulose bisphosphate carboxylase. In contrast, a major (35)S-labeled product in mesophyll cell extracts co-electrophoresed with the subunit of phosphoenolpyruvate carboxylase. Both cell types exhibited equivalent in vivo labeling of a polypeptide with one- and two-dimensional electrophoretic behavior similar to the major apoprotein of the light-harvesting chlorophyll a/b protein. Results from the use of protein synthesis inhibitors during pulse-labeling experiments indicated intercellular differences in both organelle and cytoplasmic protein synthesis. A majority of the (35)S incorporation by crabgrass mesophyll cell 70S ribosomes was associated with a pair of membrane-associated polypeptides of molecular weight 32,000 and 34,500; a comparison of fluorograph and stained gel profiles suggests these products resemble the precursor and mature forms of the maize chloroplast 32,000 dalton protein reported by Grebanier et al. (1978 J. Cell Biol. 28:734-746). In contrast, crabgrass bundle sheath cell organelle translation was directed predominantly into a product which co-electrophoresed with the large subunit of ribulose bisphosphate carboxylase.

Isoleucine 309 acts as a C4 catalytic switch that increases ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) carboxylation rate in Flaveria.

PubMed

Whitney, Spencer M; Sharwood, Robert E; Orr, Douglas; White, Sarah J; Alonso, Hernan; Galmés, Jeroni

2011-08-30

Improving global yields of important agricultural crops is a complex challenge. Enhancing yield and resource use by engineering improvements to photosynthetic carbon assimilation is one potential solution. During the last 40 million years C(4) photosynthesis has evolved multiple times, enabling plants to evade the catalytic inadequacies of the CO(2)-fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). Compared with their C(3) ancestors, C(4) plants combine a faster rubisco with a biochemical CO(2)-concentrating mechanism, enabling more efficient use of water and nitrogen and enhanced yield. Here we show the versatility of plastome manipulation in tobacco for identifying sequences in C(4)-rubisco that can be transplanted into C(3)-rubisco to improve carboxylation rate (V(C)). Using transplastomic tobacco lines expressing native and mutated rubisco large subunits (L-subunits) from Flaveria pringlei (C(3)), Flaveria floridana (C(3)-C(4)), and Flaveria bidentis (C(4)), we reveal that Met-309-Ile substitutions in the L-subunit act as a catalytic switch between C(4) ((309)Ile; faster V(C), lower CO(2) affinity) and C(3) ((309)Met; slower V(C), higher CO(2) affinity) catalysis. Application of this transplastomic system permits further identification of other structural solutions selected by nature that can increase rubisco V(C) in C(3) crops. Coengineering a catalytically faster C(3) rubisco and a CO(2)-concentrating mechanism within C(3) crop species could enhance their efficiency in resource use and yield.

Characterization of Chlamydomonas Ribulose-1,5-bisphosphate carboxylase/oxygenase variants mutated at residues that are post-translationally modified.

PubMed

Rasineni, Girish Kumar; Loh, Pek Chin; Lim, Boon Hoe

2017-02-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the chloroplast enzyme that fixes CO 2 in photosynthesis, but the enzyme also fixes O 2 , which leads to the wasteful photorespiratory pathway. If we better understand the structure-function relationship of the enzyme, we might be able to engineer improvements. When the crystal structure of Chlamydomonas Rubisco was solved, four new posttranslational modifications were observed which are not present in other species. The modifications were 4-hydroxylation of the conserved Pro-104 and 151 residues, and S-methylation of the variable Cys-256 and 369 residues, which are Phe-256 and Val-369 in land plants. Because the modifications were only observed in Chlamydomonas Rubisco, they might account for the differences in kinetic properties between the algal and plant enzymes. Site-directed mutagenesis and chloroplast transformation have been used to test the essentiality of these modifications by replacing each of the residues with alanine (Ala). Biochemical analyses were done to determine the specificity factors and kinetic constants. Replacing the modified-residues in Chlamydomonas Rubisco affected the enzyme's catalytic activity. Substituting hydroxy-Pro-104 and methyl-Cys-256 with alanine influenced Rubisco catalysis. This is the first study on these posttranslationally-modified residues in Rubisco by genetic engineering. As these forms of modifications/regulation are not available in plants, the modified residues could be a means to modulate Rubisco activity. With a better understanding of Rubisco structure-function, we can define targets for improving the enzyme. Copyright © 2016 Elsevier B.V. All rights reserved.

Isoleucine 309 acts as a C4 catalytic switch that increases ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) carboxylation rate in Flaveria

PubMed Central

Whitney, Spencer M.; Sharwood, Robert E.; Orr, Douglas; White, Sarah J.; Alonso, Hernan; Galmés, Jeroni

2011-01-01

Improving global yields of important agricultural crops is a complex challenge. Enhancing yield and resource use by engineering improvements to photosynthetic carbon assimilation is one potential solution. During the last 40 million years C4 photosynthesis has evolved multiple times, enabling plants to evade the catalytic inadequacies of the CO2-fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). Compared with their C3 ancestors, C4 plants combine a faster rubisco with a biochemical CO2-concentrating mechanism, enabling more efficient use of water and nitrogen and enhanced yield. Here we show the versatility of plastome manipulation in tobacco for identifying sequences in C4-rubisco that can be transplanted into C3-rubisco to improve carboxylation rate (VC). Using transplastomic tobacco lines expressing native and mutated rubisco large subunits (L-subunits) from Flaveria pringlei (C3), Flaveria floridana (C3-C4), and Flaveria bidentis (C4), we reveal that Met-309-Ile substitutions in the L-subunit act as a catalytic switch between C4 (309Ile; faster VC, lower CO2 affinity) and C3 (309Met; slower VC, higher CO2 affinity) catalysis. Application of this transplastomic system permits further identification of other structural solutions selected by nature that can increase rubisco VC in C3 crops. Coengineering a catalytically faster C3 rubisco and a CO2-concentrating mechanism within C3 crop species could enhance their efficiency in resource use and yield. PMID:21849620

Rubisco Accumulation Factor 1 from Thermosynechococcus elongatus participates in the final stages of ribulose-1,5-bisphosphate carboxylase/oxygenase assembly in Escherichia coli cells and in vitro.

PubMed

Kolesinski, Piotr; Belusiak, Iwona; Czarnocki-Cieciura, Mariusz; Szczepaniak, Andrzej

2014-09-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) biosynthesis is a multi-step process in which specific chaperones are involved. Recently, a novel polypeptide, Rubisco Accumulation Factor 1 (RAF1), has been identified as a protein that is necessary for proper assembly of this enzyme in maize cells (Zea mays). However, neither its specific function nor its mode of action have as yet been determined. The results presented here show that the prokaryotic homolog of RAF1 from Thermosynechococcus elongatus is expressed in cyanobacterial cells and interacts with a large Rubisco subunit (RbcL). Using a heterologous expression system, it was demonstrated that this protein promotes Rubisco assembly in Escherichia coli cells. Moreover, when co-expressed with RbcL alone, a stable RbcL-RAF1 complex is formed. Molecular mass determination for this Rubisco assembly intermediate by size-exclusion chromatography coupled with multi-angle light scattering indicates that it consists of an RbcL dimer and two RAF1 molecules. A purified RbcL-RAF1 complex dissociated upon addition of a small Rubisco subunit (RbcS), leading to formation of the active holoenzyme. Moreover, titration of the octameric (RbcL8) core of Rubisco with RAF1 results in disassembly of such a stucture and creation of an RbcL-RAF1 intermediate. The results presented here are the first attempt to elucidate the role of cyanobacterial Rubisco Accumulation Factor 1 in the Rubisco biosynthesis process. © 2014 FEBS.

Distinctive Responses of Ribulose-1,5-Bisphosphate Carboxylase and Carbonic Anhydrase in Wheat Leaves to Nitrogen Nutrition and their Possible Relationships to CO2-Transfer Resistance 1

PubMed Central

Makino, Amane; Sakashita, Hiroshi; Hidema, Jun; Mae, Tadahiko; Ojima, Kunihiko; Osmond, Barry

1992-01-01

The amounts of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), total chlorophyll (Chl), and total leaf nitrogen were measured in fully expanded, young leaves of wheat (Triticum aestivum L.), rice (Oryza sativa L.), spinach (Spinacia oleracea L.), bean (Phaseolus vulgaris L.), and pea (Pisum sativum L.). In addition, the activities of whole-chain electron transport and carbonic anhydrase were measured. All plants were grown hydroponically at different nitrogen concentrations. Although a greater than proportional increase in Rubisco content relative to leaf nitrogen content and Chl was found with increasing nitrogen supply for rice, spinach, bean, and pea, the ratio of Rubisco to total leaf nitrogen or Chl in wheat was essentially independent of nitrogen treatment. In addition, the ratio of Rubisco to electron transport activities remained constant only in wheat. Nevertheless, gas-exchange analysis showed that the in vivo balance between the capacities of Rubisco and electron transport in wheat, rice, and spinach remained almost constant, irrespective of nitrogen treatment. The in vitro carbonic anhydrase activity in wheat was very low and strongly responsive to increasing nitrogen content. Such a response was not found for the other C3 plants examined, which had 10- to 30-fold higher carbonic anhydrase activity than wheat at any leaf-nitrogen content. These distinctive responses of carbonic anhydrase activity in wheat were discussed in relation to CO2-transfer resistance and the in vivo balance between the capacities of Rubisco and electron transport. PMID:16653191

Analysis of Facultative Lithotroph Distribution and Diversity on Volcanic Deposits by Use of the Large Subunit of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase†

PubMed Central

Nanba, K.; King, G. M.; Dunfield, K.

2004-01-01

A 492- to 495-bp fragment of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) (rbcL) was amplified by PCR from facultatively lithotrophic aerobic CO-oxidizing bacteria, colorless and purple sulfide-oxidizing microbial mats, and genomic DNA extracts from tephra and ash deposits from Kilauea volcano, for which atmospheric CO and hydrogen have been previously documented as important substrates. PCR products from the mats and volcanic sites were used to construct rbcL clone libraries. Phylogenetic analyses showed that the rbcL sequences from all isolates clustered with form IC rbcL sequences derived from facultative lithotrophs. In contrast, the microbial mat clone sequences clustered with sequences from obligate lithotrophs representative of form IA rbcL. Clone sequences from volcanic sites fell within the form IC clade, suggesting that these sites were dominated by facultative lithotrophs, an observation consistent with biogeochemical patterns at the sites. Based on phylogenetic and statistical analyses, clone libraries differed significantly among volcanic sites, indicating that they support distinct lithotrophic assemblages. Although some of the clone sequences were similar to known rbcL sequences, most were novel. Based on nucleotide diversity and average pairwise difference, a forested site and an 1894 lava flow were found to support the most diverse and least diverse lithotrophic populations, respectively. These indices of diversity were not correlated with rates of atmospheric CO and hydrogen uptake but were correlated with estimates of respiration and microbial biomass. PMID:15066819

Analysis of facultative lithotroph distribution and diversity on volcanic deposits by use of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase.

PubMed

Nanba, K; King, G M; Dunfield, K

2004-04-01

A 492- to 495-bp fragment of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) (rbcL) was amplified by PCR from facultatively lithotrophic aerobic CO-oxidizing bacteria, colorless and purple sulfide-oxidizing microbial mats, and genomic DNA extracts from tephra and ash deposits from Kilauea volcano, for which atmospheric CO and hydrogen have been previously documented as important substrates. PCR products from the mats and volcanic sites were used to construct rbcL clone libraries. Phylogenetic analyses showed that the rbcL sequences from all isolates clustered with form IC rbcL sequences derived from facultative lithotrophs. In contrast, the microbial mat clone sequences clustered with sequences from obligate lithotrophs representative of form IA rbcL. Clone sequences from volcanic sites fell within the form IC clade, suggesting that these sites were dominated by facultative lithotrophs, an observation consistent with biogeochemical patterns at the sites. Based on phylogenetic and statistical analyses, clone libraries differed significantly among volcanic sites, indicating that they support distinct lithotrophic assemblages. Although some of the clone sequences were similar to known rbcL sequences, most were novel. Based on nucleotide diversity and average pairwise difference, a forested site and an 1894 lava flow were found to support the most diverse and least diverse lithotrophic populations, respectively. These indices of diversity were not correlated with rates of atmospheric CO and hydrogen uptake but were correlated with estimates of respiration and microbial biomass.

Cinnamic acid-inhibited ribulose-1,5-bisphosphate carboxylase activity is mediated through decreased spermine and changes in the ratio of polyamines in cowpea.

PubMed

Huang, Xingxue; Bie, Zhilong

2010-01-01

This study investigated the effects of cinnamic acid (CA) on ribulose-1,5-bisphosphate carboxylase (RuBPC) activity and the endogenous polyamine levels of cowpea leaves. The results show that 0.1 mM CA treatment decreased photosynthetic rate (P(n)) and RuBPC activity, but it did not affect the maximal photochemical efficiency of PSII (F(v)/F(m)), the actual photochemical efficiency of PSII (PhiPSII), intercellular CO(2) concentration (C(i)), and relative chlorophyll content. These suggest that the decrease in P(n) is at least partially attributed to a lowered RuBPC activity. In addition, 0.1 mM CA treatment increased the putrescine (Put) level, but decreased spermidine (Spd) and spermine (Spm) levels, thereby reducing the (Spd+Spm)/Put (PAs) ratio in the leaves. The exogenous application of 1 mM Spd markedly reversed these CA-induced effects for polyamine and partially restored the PAs ratio and RuBPC activity in leaves. Methylglyoxal-bis (guanylhydrazone) (MGBG), which is an inhibitor of S-adenosylmethionine decarboxylase (SAMDC), results in the inability of activated cells to synthesize Spd and exacerbates the negative effects induced by CA. The exogenous application of 1 mM D-arginine (D-Arg), which is an inhibitor of Put biosynthesis, decreased the levels of Put, but increased the PAs ratio and RuBPC activity in leaves. These results suggest that 0.1 mM CA inhibits RuBPC activity by decreasing the levels of endogenous free and perchloric acid soluble (PS) conjugated Spm, as well as the PAs ratio.

Contrasting Root and Photosynthesis Traits in a Large-Acreage Canadian Durum Variety and Its Distant Parent of Algerian Origin for Assembling Drought/Heat Tolerance Attributes

PubMed Central

Ashe, Paula; Shaterian, Hamid; Akhov, Leonid; Kulkarni, Manoj; Selvaraj, Gopalan

2017-01-01

In Canada, the world's top exporter of high-protein durum, varietal development over its nearly six-decade history has been driven by a quest for yield improvement without compromise on grain protein content and other quality aspects. Pelissier, a landrace selection from Algeria that was introduced into North America more than a century ago and the variety Strongfield that was released in 2004 are notable. Pelissier, known to elaborate more roots and considered as drought tolerant, has been cultivated commercially and thus deemed adapted. Strongfield has Pelissier in its pedigree, and it remains a high-acreage variety. Strongfield was found to elaborate only about half of the root biomass of Pelissier at maturity in greenhouse trials under well-watered conditions. Extended drought stress caused a significant reduction in the root biomass of both lines. However, Pelissier under drought maintained at least as much root biomass as that of Strongfield under well-watered conditions. In comparison to Pelissier, it had a superior photosynthesis rate (27.16 μmol CO2 m−2 s−1), capacity for carboxylation (Vcmax: 132.83 μmol CO2 m−2 s−1) and electron transport/ribulose-1,5–bisphosphate (RuBP) regeneration (Jmax: 265.40 μmol CO2 m−2 s−1); the corresponding values for Pelissier were 19.62 μmol CO2 m−2 s−1, 91.87 μmol CO2 m−2 s−1, and 163.83 μmol CO2 m−2 s−1, respectively, under well-watered conditions. Under short-term/mild drought conditions, the carbon assimilation rate remained stable in Pelissier while it declined in Strongfield to the Pelissier level. However, Strongfield succumbed to extended drought sooner than Pelissier. Photosynthesis in Strongfield but not Pelissier was found to be sensitive to high temperature stress. These results provide encouraging prospects for further exploitation of beneficial physiological traits from Pelissier in constructing climate-resilient, agronomically favorable wheat ideotypes. PMID:29312927

One crop breeding cycle from starvation? How engineering crop photosynthesis for rising CO2 and temperature could be one important route to alleviation.

PubMed

Kromdijk, Johannes; Long, Stephen P

2016-03-16

Global climate change is likely to severely impact human food production. This comes at a time when predicted demand for primary foodstuffs by a growing human population and changing global diets is already outpacing a stagnating annual rate of increase in crop productivity. Additionally, the time required by crop breeding and bioengineering to release improved varieties to farmers is substantial, meaning that any crop improvements needed to mitigate food shortages in the 2040s would need to start now. In this perspective, the rationale for improvements in photosynthetic efficiency as a breeding objective for higher yields is outlined. Subsequently, using simple simulation models it is shown how predicted changes in temperature and atmospheric [CO2] affect leaf photosynthetic rates. The chloroplast accounts for the majority of leaf nitrogen in crops. Within the chloroplast about 25% of nitrogen is invested in the carboxylase, Rubisco, which catalyses the first step of CO2 assimilation. Most of the remaining nitrogen is invested in the apparatus to drive carbohydrate synthesis and regenerate ribulose-1:5-bisphosphate (RuBP), the CO2-acceptor molecule at Rubisco. At preindustrial [CO2], investment in these two aspects may have been balanced resulting in co-limitation. At today's [CO2], there appears to be over-investment in Rubisco, and despite the counter-active effects of rising temperature and [CO2], this imbalance is predicted to worsen with global climate change. By breeding or engineering restored optimality under future conditions increased productivity could be achieved in both tropical and temperate environments without additional nitrogen fertilizer. Given the magnitude of the potential shortfall, better storage conditions, improved crop management and better crop varieties will all be needed. With the short time-scale at which food demand is expected to outpace supplies, all available technologies to improve crop varieties, from classical crop breeding to crop genetic engineering should be employed. This will require vastly increased public and private investment to support translation of first discovery in laboratories to replicated field trials, and an urgent re-evaluation of regulation of crop genetic engineering. © 2016 The Authors.

Flora of Nam Kading National Protected Area I: a new species of yellow-flowered Strobilanthes (Acanthaceae), S. namkadingensis.

PubMed

Souladeth, Phetlasy; Tagane, Shuichiro; Zhang, Meng; Okabe, Norikazu; Yahara, Tetsukazu

2017-01-01

A new species of Acanthaceae, Strobilanthes namkadingensis Soulad. & Tagane from Nam Kading National Protected Area, Bolikhamxay Province, central Laos, is described and illustrated. It is characterized by long spicate inflorescences consisting of 6-32 flowers, yellow corolla, the absence of long white hairs on the bracts and 4-6 seeds per capsule. Three DNA barcode regions of the partial genes for the large sub-unit ribulose-1,5-bisphosphate carboxylase oxygenase ( rbcL ) and maturase K ( matK ) and internal transcribed spacers (ITS) are also provided.

Nitrosomonas communis strain YNSRA, an ammonia-oxidizing bacterium, isolated from the reed rhizoplane in an aquaponics plant.

PubMed

Tokuyama, Tatsuaki; Mine, Atsusi; Kamiyama, Kaoru; Yabe, Ryuichi; Satoh, Kazuo; Matsumoto, Hirotoshi; Takahashi, Reiji; Itonaga, Koji

2004-01-01

An ammonia-oxidizing bacterium (strain YNSRA) was isolated from the rhizoplane of the reed (Phragmites communis) used in an aquaponics plant which is a wastewater treatment plant. Strain YNSRA was identified as Nitrosomonas communis by taxonomic studies. The hydroxylamine-cytochrome c reductase (HCR) of strain YNSRA was found to have a higher activity (25.60 u/mg) than that of Nitrosomonas europaea ATCC25978T (8.94 u/mg). Ribulose-1,5-bisphosphate carboxylase (RubisCO) activity was detected at very low levels in strain YNSRA, whereas strain ATCC25978T had definite activity.

Interactions of Nitrate and CO2 Enrichment on Growth, Carbohydrates, and Rubisco in Arabidopsis Starch Mutants. Significance of Starch and Hexose1

PubMed Central

Sun, Jindong; Gibson, Kelly M.; Kiirats, Olavi; Okita, Thomas W.; Edwards, Gerald E.

2002-01-01

Wild-type (wt) Arabidopsis plants, the starch-deficient mutant TL46, and the near-starchless mutant TL25 were grown in hydroponics under two levels of nitrate, 0.2 versus 6 mm, and two levels of CO2, 35 versus 100 Pa. Growth (fresh weight and leaf area basis) was highest in wt plants, lower in TL46, and much lower in TL25 plants under a given treatment. It is surprising that the inability to synthesize starch restricted leaf area development under both low N (NL) and high N (NH). For each genotype, the order of greatest growth among the four treatments was high CO2/NH > low CO2/NH, > high CO2/NL, which was similar to low CO2/NL. Under high CO2/NL, wt and TL46 plants retained considerable starch in leaves at the end of the night period, and TL25 accumulated large amounts of soluble sugars, indicative of N-limited restraints on utilization of photosynthates. The lowest ribulose-1,5-bisphosphate carboxylase/oxygenase per leaf area was in plants grown under high CO2/NL. When N supply is limited, the increase in soluble sugars, particularly in the starch mutants, apparently accentuates the feedback and down-regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase, resulting in greater reduction of growth. With an adequate supply of N, growth is limited in the starch mutants due to insufficient carbohydrate reserves during the dark period. A combination of limited N and a limited capacity to synthesize starch, which restrict the capacity to use photosynthate, and high CO2, which increases the potential to produce photosynthate, provides conditions for strong down-regulation of photosynthesis. PMID:12428022

Relationships between the Efficiencies of Photosystems I and II and Stromal Redox State in CO2-Free Air 1

PubMed Central

Harbinson, Jeremy; Foyer, Christine H.

1991-01-01

The responses of the efficiencies of photosystems I and II, stromal redox state (as indicated by NADP-malate dehydrogenase activation state), and activation of the Benson-Calvin cycle enzymes ribulose 1,5-bisphosphate carboxylase and fructose 1,6-bisphosphatase to varying irradiance were measured in pea (Pisum sativum L.) leaves operating close to the CO2 compensation point. A comparison of the relationships among these parameters obtained from leaves in air was made with those obtained when the leaves were maintained in air from which the CO2 had been removed. P700 was more oxidized at any measured irradiance in CO2-free air than in air. The relationship between the quantum efficiencies of the photosystems in CO2-free air was distinctly curvilinear in contrast to the predominantly linear relationship obtained with leaves in air. This nonlinearity may be consistent with the operation of cyclic electron flow around photosystem I because the quantum efficiency of photosystem II was much more restricted than the quantum efficiency of photosystem I. In CO2-free air, measured NADP-malate dehydrogenase activities varied considerably at low irradiances. However, at high irradiance the activity of the enzyme was low, implying that the stroma was oxidized. In contrast, fructose-1,6-bisphosphatase activities tended to increase with increasing electron flux through the photosystems. Ribulose-1,5-bisphosphate carboxylase activity remained relatively constant with respect to irradiance in CO2-free air, with an activation state 50% of maximum. We conclude that, at the CO2 compensation point and high irradiance, low redox states are favored and that cyclic electron flow may be substantial. These two features may be the requirements necessary to trigger and maintain the dissipative processes in the thylakoid membrane. PMID:16668401

Insertion Mutation of the Form I cbbL Gene Encoding Ribulose Bisphosphate Carboxylase/Oxygenase (RuBisCO) in Thiobacillus neapolitanus Results in Expression of Form II RuBisCO, Loss of Carboxysomes, and an Increased CO2 Requirement for Growth

PubMed Central

Baker, Stefanie H.; Jin, Songmu; Aldrich, Henry C.; Howard, Gary T.; Shively, Jessup M.

1998-01-01

It has been previously established that Thiobacillus neapolitanus fixes CO2 by using a form I ribulose bisphosphate carboxylase/oxygenase (RuBisCO), that much of the enzyme is sequestered into carboxysomes, and that the genes for the enzyme, cbbL and cbbS, are part of a putative carboxysome operon. In the present study, cbbL and cbbS were cloned and sequenced. Analysis of RNA showed that cbbL and cbbS are cotranscribed on a message approximately 2,000 nucleotides in size. The insertion of a kanamycin resistance cartridge into cbbL resulted in a premature termination of transcription; a polar mutant was generated. The mutant is able to fix CO2, but requires a CO2 supplement for growth. Separation of cellular proteins from both the wild type and the mutant on sucrose gradients and subsequent analysis of the RuBisCO activity in the collected fractions showed that the mutant assimilates CO2 by using a form II RuBisCO. This was confirmed by immunoblot analysis using antibodies raised against form I and form II RuBisCOs. The mutant does not possess carboxysomes. Smaller, empty inclusions are present, but biochemical analysis indicates that if they are carboxysome related, they are not functional, i.e., do not contain RuBisCO. Northern analysis showed that some of the shell components of the carboxysome are produced, which may explain the presence of these inclusions in the mutant. PMID:9696760

A mechanism for intergenomic integration: abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA.

PubMed Central

Rodermel, S; Haley, J; Jiang, C Z; Tsai, C H; Bogorad, L

1996-01-01

Multimeric protein complexes in chloroplasts and mitochondria are generally composed of products of both nuclear and organelle genes of the cell. A central problem of eukaryotic cell biology is to identify and understand the molecular mechanisms for integrating the production and accumulation of the products of the two separate genomes. Ribulose bisphosphate carboxylase (Rubisco) is localized in the chloroplasts of photosynthetic eukaryotic cells and is composed of small subunits (SS) and large subunits (LS) coded for by nuclear rbcS and chloroplast rbcL genes, respectively. Transgenic tobacco plants containing antisense rbcS DNA have reduced levels of rbcS mRNA, normal levels of rbcL mRNA, and coordinately reduced LS and SS proteins. Our previous experiments indicated that the rate of translation of rbcL mRNA might be reduced in some antisense plants; direct evidence is presented here. After a short-term pulse there is less labeled LS protein in the transgenic plants than in wild-type plants, indicating that LS accumulation is controlled in the mutants at the translational and/or posttranslational levels. Consistent with a primary restriction at translation, fewer rbcL mRNAs are associated with polysomes of normal size and more are free or are associated with only a few ribosomes in the antisense plants. Effects of the rbcS antisense mutation on mRNA and protein accumulation, as well as on the distribution of mRNAs on polysomes, appear to be minimal for other chloroplast and nuclear photosynthetic genes. Our results suggest that SS protein abundance specifically contributes to the regulation of LS protein accumulation at the level of rbcL translation initiation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 6 Fig. 7 Fig. 8 PMID:8632983

Characterization of ribulose-1, 5-bisphosphate carboxylase/oxygenase and transcriptional analysis of its related genes in Saccharina japonica (Laminariales, Phaeophyta)

NASA Astrophysics Data System (ADS)

Shao, Zhanru; Liu, Fuli; Li, Qiuying; Yao, Jianting; Duan, Delin

2014-03-01

Saccharina japonica is a common macroalga in sublittoral communities of cold seawater environments, and consequently may have highly efficient ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activity for carbon assimilation. In our study, we cloned the full-length Rubisco gene from S. japonica ( SJ-rbc). It contained an open reading frame for a large subunit gene ( SJ — rbcL) of 1 467 bp, a small subunit gene ( SJ-rbcS) of 420 bp, and a SJ-rbcL/S intergenic spacer of 269 bp. The deduced peptides of SJ-rbcL and SJ-rbcS were 488 and 139 amino acids with theoretical molecular weights and isoelectric points of 53.97 kDa, 5.81 and 15.84 kDa, 4.71, respectively. After induction with 1 mmol/L isopropyl- β-D-thiogalactopyranoside for 5 h and purification by Ni2+ affinity chromatography, electrophoresis and western blot detection demonstrated successful expression of the 55 kDa SJ-rbcL protein. Real-time quantitative PCR showed that the mRNA levels of SJ-rbcL in gametophytes increased when transferred into normal growth conditions and exhibited diurnal variations: increased expression during the day but suppressed expression at night. This observation implied that Rubisco played a role in normal gametophytic growth and development. In juvenile sporophytes, mRNA levels of SJ-rbcL, carbonic anhydrase, Calvin-Benson-Bassham cycle-related enzyme, and chloroplast light-harvesting protein were remarkably increased under continuous light irradiance. Similarly, expression of these genes was up-regulated under blue light irradiance at 350 μmol/(m2·s). Our results indicate that long-term white light and short-term blue light irradiance enhances juvenile sporophytic growth by synergistic effects of various photosynthetic elements.

RubisCO selection using the vigorously aerobic and metabolically versatile bacterium Ralstonia eutropha.

PubMed

Satagopan, Sriram; Tabita, F Robert

2016-08-01

Recapturing atmospheric CO2 is key to reducing global warming and increasing biological carbon availability. Ralstonia eutropha is a biotechnologically useful aerobic bacterium that uses the Calvin-Benson-Bassham (CBB) cycle and the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) for CO2 utilization, suggesting that it may be a useful host to bioselect RubisCO molecules with improved CO2 -capture capabilities. A host strain of R. eutropha was constructed for this purpose after deleting endogenous genes encoding two related RubisCOs. This strain could be complemented for CO2 -dependent growth by introducing native or heterologous RubisCO genes. Mutagenesis and suppressor selection identified amino acid substitutions in a hydrophobic region that specifically influences RubisCO's interaction with its substrates, particularly O2 , which competes with CO2 at the active site. Unlike most RubisCOs, the R. eutropha enzyme has evolved to retain optimal CO2 -fixation rates in a fast-growing host, despite the presence of high levels of competing O2 . Yet its structure-function properties resemble those of several commonly found RubisCOs, including the higher plant enzymes, allowing strategies to engineer analogous enzymes. Because R. eutropha can be cultured rapidly under harsh environmental conditions (e.g., with toxic industrial flue gas), in the presence of near saturation levels of oxygen, artificial selection and directed evolution studies in this organism could potentially impact efforts toward improving RubisCO-dependent biological CO2 utilization in aerobic environments. d-ribulose 1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39; phosphoribulokinase, EC 2.7.1.19. © 2016 Federation of European Biochemical Societies.

ATP and magnesium promote cotton short-form ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase hexamer formation at low micromolar concentrations.

PubMed

Kuriata, Agnieszka M; Chakraborty, Manas; Henderson, J Nathan; Hazra, Suratna; Serban, Andrew J; Pham, Tuong V T; Levitus, Marcia; Wachter, Rebekka M

2014-11-25

We report a fluorescence correlation spectroscopy (FCS) study of the assembly pathway of the AAA+ protein ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca), a ring-forming ATPase responsible for activation of inhibited Rubisco complexes for biological carbon fixation. A thermodynamic characterization of simultaneously populated oligomeric states appears critical in understanding Rca structure and function. Using cotton β-Rca, we demonstrate that apparent diffusion coefficients vary as a function of concentration, nucleotide, and cation. Using manual fitting procedures, we provide estimates for the equilibrium constants for the stepwise assembly and find that in the presence of ATPγS, the Kd for hexamerization is 10-fold lower than with ADP (∼0.1 vs ∼1 μM). Hexamer fractions peak at 30 μM and dominate at 8-70 μM Rca, where they comprise 60-80% of subunits with ATPγS, compared with just 30-40% with ADP. Dimer fractions peak at 1-4 μM Rca, where they comprise 15-18% with ATPγS and 26-28% with ADP. At 30 μM Rca, large aggregates begin to form that comprise ∼10% of total protein with ATPγS and ∼25% with ADP. FCS data collected on the catalytically impaired WalkerB-D173N variant in the presence of ATP provided strong support for these results. Titration with free magnesium ions lead to the disaggregation of larger complexes in favor of hexameric forms, suggesting that a second magnesium binding site with a Kd value of 1-3 mM mediates critical subunit contacts. We propose that closed-ring toroidal hexameric forms are stabilized by binding of Mg·ATP plus Mg2+, whereas Mg·ADP promotes continuous assembly to supramolecular aggregates such as spirals.

Atomic resolution x-ray structure of the substrate recognition domain of higher plant ribulose-bisphosphate carboxylase/oxygenase (Rubisco) activase.

PubMed

Henderson, J Nathan; Kuriata, Agnieszka M; Fromme, Raimund; Salvucci, Michael E; Wachter, Rebekka M

2011-10-14

The rapid release of tight-binding inhibitors from dead-end ribulose-bisphosphate carboxylase/oxygenase (Rubisco) complexes requires the activity of Rubisco activase, an AAA+ ATPase that utilizes chemo-mechanical energy to catalyze the reactivation of Rubisco. Activase is thought to play a central role in coordinating the rate of CO(2) fixation with the light reactions of photosynthesis. Here, we present a 1.9 Å crystal structure of the C-domain core of creosote activase. The fold consists of a canonical four-helix bundle, from which a paddle-like extension protrudes that entails a nine-turn helix lined by an irregularly structured peptide strand. The residues Lys-313 and Val-316 involved in the species-specific recognition of Rubisco are located near the tip of the paddle. An ionic bond between Lys-313 and Glu-309 appears to stabilize the glycine-rich end of the helix. Structural superpositions onto the distant homolog FtsH imply that the paddles extend away from the hexameric toroid in a fan-like fashion, such that the hydrophobic sides of each blade bearing Trp-302 are facing inward and the polar sides bearing Lys-313 and Val-316 are facing outward. Therefore, we speculate that upon binding, the activase paddles embrace the Rubisco cylinder by placing their hydrophobic patches near the partner protein. This model suggests that conformational adjustments at the remote end of the paddle may relate to selectivity in recognition, rather than specific ionic contacts involving Lys-313. Additionally, the superpositions predict that the catalytically critical Arg-293 does not interact with the bound nucleotide. Hypothetical ring-ring stacking and peptide threading models for Rubisco reactivation are briefly discussed.

A reversible decrease in ribulose 1,5-bisphosphate carboxylase/oxygenase carboxylation activity caused by the aggregation of the enzyme's large subunit is triggered in response to the exposure of moderate irradiance-grown plants to low irradiance.

PubMed

Grabsztunowicz, Magda; Górski, Zbigniew; Luciński, Robert; Jackowski, Grzegorz

2015-08-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is highly regulated in response to fluctuations in the environment, including changes in irradiance. However, no complex data are available on Rubisco regulatory mechanisms triggered in plants which are submitted to moderate-low irradiance shift. Therefore, we investigated in a comprehensive way the changes at the level of amount of Rubisco protein, its structural organization and carboxylase activity of the holoenzyme as triggered by exposure of moderate irradiance-grown Arabidopsis thaliana plants to low irradiance conditions. An exposure of moderate irradiance-grown plants to low irradiance for a single photoperiod caused the exclusion of a certain pool of Rubisco under altered conditions owing to oxidative modifications resulting in the formation of protein aggregates involving Rubisco large subunit (LS). As a result, both initial and total Rubisco carboxylase activities were reduced, whereas Rubisco activation state remained largely unchanged. The results of the determination of reactive oxygen species indicated that a moderate/low irradiance transition had stimulated (1) O2 accumulation and we strongly suggest that Rubisco oxidative modifications leading to formation of aggregates encompassing Rubisco-LS were triggered by (1) O2 . When moderate irradiance regime was resumed, the majority of Rubisco-LS containing aggregates tended to be resolubilized, and this allowed Rubisco carboxylation activities to be largely recovered, without changes in the activation state of the enzyme. In the longer term, these results allow us to better understand a complexity of Rubisco regulatory mechanisms activated in response to abiotic stresses and during recovery from the stresses. © 2015 Scandinavian Plant Physiology Society.

Structure-Function Studies with the Unique Hexameric Form II Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) from Rhodopseudomonas palustris*

PubMed Central

Satagopan, Sriram; Chan, Sum; Perry, L. Jeanne; Tabita, F. Robert

2014-01-01

The first x-ray crystal structure has been solved for an activated transition-state analog-bound form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This enzyme, from Rhodopseudomonas palustris, assembles as a unique hexamer with three pairs of catalytic large subunit homodimers around a central 3-fold symmetry axis. This oligomer arrangement is unique among all known Rubisco structures, including the form II homolog from Rhodospirillum rubrum. The presence of a transition-state analog in the active site locked the activated enzyme in a “closed” conformation and revealed the positions of critical active site residues during catalysis. Functional roles of two form II-specific residues (Ile165 and Met331) near the active site were examined via site-directed mutagenesis. Substitutions at these residues affect function but not the ability of the enzyme to assemble. Random mutagenesis and suppressor selection in a Rubisco deletion strain of Rhodobacter capsulatus identified a residue in the amino terminus of one subunit (Ala47) that compensated for a negative change near the active site of a neighboring subunit. In addition, substitution of the native carboxyl-terminal sequence with the last few dissimilar residues from the related R. rubrum homolog increased the enzyme's kcat for carboxylation. However, replacement of a longer carboxyl-terminal sequence with termini from either a form III or a form I enzyme, which varied both in length and sequence, resulted in complete loss of function. From these studies, it is evident that a number of subtle interactions near the active site and the carboxyl terminus account for functional differences between the different forms of Rubiscos found in nature. PMID:24942737

The Diverse AAA+ Machines that Repair Inhibited Rubisco Active Sites

PubMed Central

Mueller-Cajar, Oliver

2017-01-01

Gaseous carbon dioxide enters the biosphere almost exclusively via the active site of the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). This highly conserved catalyst has an almost universal propensity to non-productively interact with its substrate ribulose 1,5-bisphosphate, leading to the formation of dead-end inhibited complexes. In diverse autotrophic organisms this tendency has been counteracted by the recruitment of dedicated AAA+ (ATPases associated with various cellular activities) proteins that all use the energy of ATP hydrolysis to remodel inhibited Rubisco active sites leading to release of the inhibitor. Three evolutionarily distinct classes of these Rubisco activases (Rcas) have been discovered so far. Green and red-type Rca are mostly found in photosynthetic eukaryotes of the green and red plastid lineage respectively, whereas CbbQO is associated with chemoautotrophic bacteria. Ongoing mechanistic studies are elucidating how the various motors are utilizing both similar and contrasting strategies to ultimately perform their common function of cracking the inhibited Rubisco active site. The best studied mechanism utilized by red-type Rca appears to involve transient threading of the Rubisco large subunit C-terminal peptide, reminiscent of the action performed by Clp proteases. As well as providing a fascinating example of convergent molecular evolution, Rca proteins can be considered promising crop-improvement targets. Approaches aiming to replace Rubisco in plants with improved enzymes will need to ensure the presence of a compatible Rca protein. The thermolability of the Rca protein found in crop plants provides an opportunity to fortify photosynthesis against high temperature stress. Photosynthesis also appears to be limited by Rca when light conditions are fluctuating. Synthetic biology strategies aiming to enhance the autotrophic CO2 fixation machinery will need to take into consideration the requirement for Rubisco activases as well as their properties. PMID:28580359

Structure-function studies with the unique hexameric form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodopseudomonas palustris.

PubMed

Satagopan, Sriram; Chan, Sum; Perry, L Jeanne; Tabita, F Robert

2014-08-01

The first x-ray crystal structure has been solved for an activated transition-state analog-bound form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This enzyme, from Rhodopseudomonas palustris, assembles as a unique hexamer with three pairs of catalytic large subunit homodimers around a central 3-fold symmetry axis. This oligomer arrangement is unique among all known Rubisco structures, including the form II homolog from Rhodospirillum rubrum. The presence of a transition-state analog in the active site locked the activated enzyme in a "closed" conformation and revealed the positions of critical active site residues during catalysis. Functional roles of two form II-specific residues (Ile(165) and Met(331)) near the active site were examined via site-directed mutagenesis. Substitutions at these residues affect function but not the ability of the enzyme to assemble. Random mutagenesis and suppressor selection in a Rubisco deletion strain of Rhodobacter capsulatus identified a residue in the amino terminus of one subunit (Ala(47)) that compensated for a negative change near the active site of a neighboring subunit. In addition, substitution of the native carboxyl-terminal sequence with the last few dissimilar residues from the related R. rubrum homolog increased the enzyme's kcat for carboxylation. However, replacement of a longer carboxyl-terminal sequence with termini from either a form III or a form I enzyme, which varied both in length and sequence, resulted in complete loss of function. From these studies, it is evident that a number of subtle interactions near the active site and the carboxyl terminus account for functional differences between the different forms of Rubiscos found in nature. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Biphasic Activation of Ribulose Bisphosphate Carboxylase in Spinach Leaves as Determined from Nonsteady-State CO2 Exchange 1

PubMed Central

Woodrow, Ian E.; Mott, Keith A.

1992-01-01

The activation kinetics of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) following an increase in photon flux density (PFD) were studied by analyzing CO2 assimilation time courses in spinach leaves (Spinacia oleracea). When leaves were exposed to 45 minutes of darkness before illumination at 690 micromoles per square meter per second, Rubisco activation followed apparent first-order kinetics with a relaxation time of about 3.8 minutes. But when leaves were illuminated for 45 minutes at 160 micromoles per square meter per second prior to illumination at 690 micromoles per square meter per second the relaxation time for Rubisco activation was only 2.1 minutes. The kinetics of this change in relaxation times were investigated by exposing dark-adapted leaves to 160 micromoles per square meter per second for different periods before increasing the PFD to 690 micromoles per square meter per second. It was found that the apparent relaxation time for Rubisco activation changed from 3.8 to 2.1 minutes slowly, requiring at least 8 minutes for completion. This result indicates that at least two sequential, slow processes are involved in light-mediated activation of Rubisco in spinach leaves and that the relaxation times characterizing these two processes are about 4 and 2 minutes, respectively. The kinetics of the first process in the reverse direction and the dependence of the relaxation time for the second process on the magnitude of the increase in PFD were also determined. Evidence that the first slow process is activation of the enzyme Rubisco activase and that the second slow process is the catalytic activation of Rubisco by activase is discussed. PMID:16668865

Effect of pH, Mg2+, CO2 and Mercurials on the Circular Dichroism, Thermal Stability and Light Scattering of Ribulose 1,5-Bisphosphate Carboxylases from Alfalfa, Spinach and Tobacco

PubMed Central

Tomimatsu, Yoshio; Donovan, John W.

1981-01-01

Circular dichroism, differential scanning calorimetry and light-scattering measurements of ribulose 1,5-bisphosphate carboxylase (E.C. 4.1.1.39) from alfalfa, spinach and tobacco show: a) The conformation and thermal stability of the native carboxylases are sensitive to changes in pH and to activation of the enzyme with Mg2+ and CO2. The helical content, denaturation temperature (Td) and specific enthalpy of denaturation (Δq) decreased with increase in pH. Addition of Mg2+ and CO2 at pH 9 increased Td by 4 to 5 C; at pH 7.5 the changes in Td were smaller. b) Addition of mercurials produced changes in conformation and thermal stability. The decrease in helical content of the enzymes with increase in pH was enhanced by the addition of p-chloromercuribenzoate. At pH 9, addition of p-chloromercuribenzoate or of 1-(3-(chloromercuri)-2-methoxypropyl)urea decreased Td by 11.4 to 20.2 C and Δq by 2.1 to 2.8 calories per gram. c) The spinach carboxylase undergoes the largest and the tobacco the smallest changes in conformation and thermal stability upon change in pH or treatment with mercurials. d) The calorimetric data suggest that the large and small subunits are heat denatured independently but at the same temperature. e) Light scattering measurements at pH 9 of p-chloromercuribenzoate treated tobacco enzyme showed that there is no dissociation into subunits upon heating to temperatures greater than Td. A `ball and string' model for the carboxylase molecule is proposed to reconcile independence of subunit denaturation with apparent strong interactions between subunits. PMID:16662003

Association of Carbonic Anhydrase Activity with Carboxysomes Isolated from the Cyanobacterium Synechococcus PCC7942 1

PubMed Central

Price, G. Dean; Coleman, John R.; Badger, Murray R.

1992-01-01

The development of a simple method for the isolation of purified carboxysomes from the cyanobacterium Synechococcus PCC7942 has made it possible to identify a specific and inducible, intracellular carbonic anhydrase (CA) activity that is strongly associated with carboxysomes. This was shown, in part, through enzyme recovery experiments that indicated that a clear majority of a CA activity that is sensitive to the CA inhibitor ethoxyzolamide (I50 = 4 μm) copurifies with a majority of the cell's ribulose-1,5-bisphosphate carboxylase/oxygenase activity in a highly purified pelletable fraction. Electron microscopy of this pelletable fraction revealed the presence of carboxysomes that were physically intact. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of carboxysome proteins showed that the large and small subunits of ribulose-1,5-bisphosphate carbosylase/oxygenase were clearly prominent and that several other minor proteins could be distinguished. The specific location of this carboxysomal CA activity is further reinforced by the finding that a previously isolated high CO2-requiring mutant, Type II/No. 68 (G.D. Price, M.R. Badger [1989] Plant Physiol 91: 514-525), displayed a 30-fold reduction in carboxysome-associated CA activity when tested under optimal conditions. Carboxysomal CA has the unusual property of being inactivated by dithiothreitol. The enzyme also requires 20 mm Mg2+ (as MgSO4) for near maximum activity; other divalent cations, such as Ca2+ and Mn2+, also stimulate carboxysomal CA activity, but to a lesser extent than Mg2+. Results are discussed in relation to the role of carboxysomes in the CO2-concentrating mechanism in cyanobacteria and the role that carboxysomal CA activity appears to play in this process. Images Figure 1 Figure 7 PMID:16653059

Nitrate and Ammonium Induced Photosynthetic Suppression in N-Limited Selenastrum minutum.

PubMed

Elrifi, I R; Turpin, D H

1986-05-01

Nitrate-limited chemostat cultures of Selenastrum minutum Naeg. Collins (Chlorophyta) were used to determine the effects of nitrogen addition on photosynthesis, dark respiration, and dark carbon fixation. Addition of NO(3) (-) or NH(4) (+) induced a transient suppression of photosynthetic carbon fixation (70 and 40% respectively). Intracellular ribulose bisphosphate levels decreased during suppression and recovered in parallel with photosynthesis. Photosynthetic oxygen evolution was decreased by N-pulsing under saturating light (650 microeinsteins per square meter per second). Under subsaturating light intensities (<165 microeinsteins per square meter per second) NH(4) (+) addition resulted in O(2) consumption in the light which was alleviated by the presence of the tricarboxylic acid cycle inhibitor fluoroacetate. Addition of NO(3) (-) or NH(4) (+) resulted in a large stimulation of dark respiration (67 and 129%, respectively) and dark carbon fixation (360 and 2080%, respectively). The duration of N-induced perturbations was dependent on the concentration of added N. Inhibition of glutamine 2-oxoglutarate aminotransferase by azaserine alleviated all these effects. It is proposed that suppression of photosynthetic carbon fixation in response to N pulsing was the result of a competition for metabolites between the Calvin cycle and nitrogen assimilation. Carbon skeletons required for nitrogen assimilation would be derived from tricarboxylic acid cycle intermediates. To maintain tricarboxylic acid cycle activity triose phosphates would be exported from the chloroplast. This would decrease the rate of ribulose bisphosphate regeneration and consequently decrease net photosynthetic carbon accumulation. Stoichiometric calculations indicate that the Calvin cycle is one source of triose phosphates for N assimilation; however, during transient N resupply the major demand for triose phosphates must be met by starch or sucrose breakdown. The effects of N-pulsing on O(2) evolution, dark respiration, and dark C-fixation are shown to be consistent with this model.

Enzymatic Characterization of AMP Phosphorylase and Ribose-1,5-Bisphosphate Isomerase Functioning in an Archaeal AMP Metabolic Pathway

PubMed Central

Aono, Riku; Sato, Takaaki; Yano, Ayumu; Yoshida, Shosuke; Nishitani, Yuichi; Miki, Kunio; Imanaka, Tadayuki

2012-01-01

AMP phosphorylase (AMPpase), ribose-1,5-bisphosphate (R15P) isomerase, and type III ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been proposed to constitute a novel pathway involved in AMP metabolism in the Archaea. Here we performed a biochemical examination of AMPpase and R15P isomerase from Thermococcus kodakarensis. R15P isomerase was specific for the α-anomer of R15P and did not recognize other sugar compounds. We observed that activity was extremely low with the substrate R15P alone but was dramatically activated in the presence of AMP. Using AMP-activated R15P isomerase, we reevaluated the substrate specificity of AMPpase. AMPpase exhibited phosphorylase activity toward CMP and UMP in addition to AMP. The [S]-v plot (plot of velocity versus substrate concentration) of the enzyme toward AMP was sigmoidal, with an increase in activity observed at concentrations higher than approximately 3 mM. The behavior of the two enzymes toward AMP indicates that the pathway is intrinsically designed to prevent excess degradation of intracellular AMP. We further examined the formation of 3-phosphoglycerate from AMP, CMP, and UMP in T. kodakarensis cell extracts. 3-Phosphoglycerate generation was observed from AMP alone, and from CMP or UMP in the presence of dAMP, which also activates R15P isomerase. 3-Phosphoglycerate was not formed when 2-carboxyarabinitol 1,5-bisphosphate, a Rubisco inhibitor, was added. The results strongly suggest that these enzymes are actually involved in the conversion of nucleoside monophosphates to 3-phosphoglycerate in T. kodakarensis. PMID:23065974

Photosynthetic carbon fixation characteristics of fruiting structures of Brassica campestris L

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singal, H.R.; Sheoran, I.S.; Singh, R.

1987-04-01

Activities of key enzymes of the Calvin cycle and C/sub 4/ metabolism, rates of CO/sub 2/ fixation, and the initial products of photosynthetic /sup 14/CO/sub 2/ fixation were determined in the podwall, seed coat (fruiting structures), and the subtending leaf (leaf below a receme) of Brassica campestris L. cv Toria. Compared to activities of ribulose-1,5-bisphosphate carboxylase and other Calvin cycle enzymes, e.g. NADP-glyceraldehyde-3-phosphate-dehydrogenase and ribulose-5-phosphate kinase, the activities of phosphoenol pyruvate carboxylase and other enzymes of C/sub 4/ metabolism, viz. NADP-malate dehydrogenase, NADP-malic enzyme, glutamate pyruvate transaminase, and glutamate oxaloacetate transaminase, were generally much higher in seed than in podwallmore » and leaf. Podwall and leaf were comparable to each other. Pulse-chase experiments showed that in seed the major product of /sup 14/CO/sub 2/ assimilation was malate (in short time), whereas in podwall and leaf, the label initially appeared in 3-PGA. With time, the label moved to sucrose. In contrast to legumes, Brassica pods were able to fix net CO/sub 2/ during light. However, respiratory losses were very high during the dark period.« less

Strong thermal acclimation of photosynthesis in tropical and temperate wet-forest tree species: the importance of altered Rubisco content.

PubMed

Scafaro, Andrew P; Xiang, Shuang; Long, Benedict M; Bahar, Nur H A; Weerasinghe, Lasantha K; Creek, Danielle; Evans, John R; Reich, Peter B; Atkin, Owen K

2017-07-01

Understanding of the extent of acclimation of light-saturated net photosynthesis (A n ) to temperature (T), and associated underlying mechanisms, remains limited. This is a key knowledge gap given the importance of thermal acclimation for plant functioning, both under current and future higher temperatures, limiting the accuracy and realism of Earth system model (ESM) predictions. Given this, we analysed and modelled T-dependent changes in photosynthetic capacity in 10 wet-forest tree species: six from temperate forests and four from tropical forests. Temperate and tropical species were each acclimated to three daytime growth temperatures (T growth ): temperate - 15, 20 and 25 °C; tropical - 25, 30 and 35 °C. CO 2 response curves of A n were used to model maximal rates of RuBP (ribulose-1,5-bisphosphate) carboxylation (V cmax ) and electron transport (J max ) at each treatment's respective T growth and at a common measurement T (25 °C). SDS-PAGE gels were used to determine abundance of the CO 2 -fixing enzyme, Rubisco. Leaf chlorophyll, nitrogen (N) and mass per unit leaf area (LMA) were also determined. For all species and T growth , A n at current atmospheric CO 2 partial pressure was Rubisco-limited. Across all species, LMA decreased with increasing T growth . Similarly, area-based rates of V cmax at a measurement T of 25 °C (V cmax 25 ) linearly declined with increasing T growth , linked to a concomitant decline in total leaf protein per unit leaf area and Rubisco as a percentage of leaf N. The decline in Rubisco constrained V cmax and A n for leaves developed at higher T growth and resulted in poor predictions of photosynthesis by currently widely used models that do not account for T growth -mediated changes in Rubisco abundance that underpin the thermal acclimation response of photosynthesis in wet-forest tree species. A new model is proposed that accounts for the effect of T growth -mediated declines in V cmax 25 on A n , complementing current photosynthetic thermal acclimation models that do not account for T sensitivity of V cmax 25 . © 2017 John Wiley & Sons Ltd.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spreitzer, Robert J.

CO{sub 2} and O{sub 2} are mutually competitive at the active site of ribulose-1,5-biphosphate (RuBP) carboxylase/oxygenase (Rubisco). Rubisco contains two subunits, each present in eight copies. The 15-kD small subunit is coded by a family of nuclear RbcS genes. Until now, the role of the small subunit in Rubisco structure or catalytic efficiency is not known. Because of other work in eliminating the two RbcS genes in the green algo Chlamydomonas reinhardtii, it is now possible to address questions about the structure-function relationships of the eukaryotic small subunit. There are three specific aims in this project: (1) Alanine scanning mutagenesismore » is being used to dissect the importance of the {beta}A/{beta}B loop, a feature unique to the eukaryotic small subunit. (2) Random mutagenesis is being used to identify additional residues or regions of the small subunit that are important for holoenzyme assembly and function. (3) Attempts are being made to express foreign small subunits in Chlamydomonas to examine the contribution of small subunits to holoenzyme assembly, catalytic efficiency, and CO{sub 2}/O{sub 2} specificity.« less

Carbon dioxide diffusion across stomata and mesophyll and photo-biochemical processes as affected by growth CO2 and phosphorus nutrition in cotton.

PubMed

Singh, Shardendu K; Badgujar, Girish; Reddy, Vangimalla R; Fleisher, David H; Bunce, James A

2013-06-15

Nutrients such as phosphorus may exert a major control over plant response to rising atmospheric carbon dioxide concentration (CO2), which is projected to double by the end of the 21st century. Elevated CO2 may overcome the diffusional limitations to photosynthesis posed by stomata and mesophyll and alter the photo-biochemical limitations resulting from phosphorus deficiency. To evaluate these ideas, cotton (Gossypium hirsutum) was grown in controlled environment growth chambers with three levels of phosphate (Pi) supply (0.2, 0.05 and 0.01mM) and two levels of CO2 concentration (ambient 400 and elevated 800μmolmol(-1)) under optimum temperature and irrigation. Phosphate deficiency drastically inhibited photosynthetic characteristics and decreased cotton growth for both CO2 treatments. Under Pi stress, an apparent limitation to the photosynthetic potential was evident by CO2 diffusion through stomata and mesophyll, impairment of photosystem functioning and inhibition of biochemical process including the carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase/oxyganase and the rate of ribulose-1,5-bisphosphate regeneration. The diffusional limitation posed by mesophyll was up to 58% greater than the limitation due to stomatal conductance (gs) under Pi stress. As expected, elevated CO2 reduced these diffusional limitations to photosynthesis across Pi levels; however, it failed to reduce the photo-biochemical limitations to photosynthesis in phosphorus deficient plants. Acclimation/down regulation of photosynthetic capacity was evident under elevated CO2 across Pi treatments. Despite a decrease in phosphorus, nitrogen and chlorophyll concentrations in leaf tissue and reduced stomatal conductance at elevated CO2, the rate of photosynthesis per unit leaf area when measured at the growth CO2 concentration tended to be higher for all except the lowest Pi treatment. Nevertheless, plant biomass increased at elevated CO2 across Pi nutrition with taller plants, increased leaf number and larger leaf area. Copyright © 2013 Elsevier GmbH. All rights reserved.

Relationships between the Efficiencies of Photosystems I and II and Stromal Redox State in CO(2)-Free Air : Evidence for Cyclic Electron Flow in Vivo.

PubMed

Harbinson, J; Foyer, C H

1991-09-01

The responses of the efficiencies of photosystems I and II, stromal redox state (as indicated by NADP-malate dehydrogenase activation state), and activation of the Benson-Calvin cycle enzymes ribulose 1,5-bisphosphate carboxylase and fructose 1,6-bisphosphatase to varying irradiance were measured in pea (Pisum sativum L.) leaves operating close to the CO(2) compensation point. A comparison of the relationships among these parameters obtained from leaves in air was made with those obtained when the leaves were maintained in air from which the CO(2) had been removed. P700 was more oxidized at any measured irradiance in CO(2)-free air than in air. The relationship between the quantum efficiencies of the photosystems in CO(2)-free air was distinctly curvilinear in contrast to the predominantly linear relationship obtained with leaves in air. This nonlinearity may be consistent with the operation of cyclic electron flow around photosystem I because the quantum efficiency of photosystem II was much more restricted than the quantum efficiency of photosystem I. In CO(2)-free air, measured NADP-malate dehydrogenase activities varied considerably at low irradiances. However, at high irradiance the activity of the enzyme was low, implying that the stroma was oxidized. In contrast, fructose-1,6-bisphosphatase activities tended to increase with increasing electron flux through the photosystems. Ribulose-1,5-bisphosphate carboxylase activity remained relatively constant with respect to irradiance in CO(2)-free air, with an activation state 50% of maximum. We conclude that, at the CO(2) compensation point and high irradiance, low redox states are favored and that cyclic electron flow may be substantial. These two features may be the requirements necessary to trigger and maintain the dissipative processes in the thylakoid membrane.

Induction of Reduced Photorespiratory Activity in Submersed and Amphibious Aquatic Macrophytes 1

PubMed Central

Salvucci, Michael E.; Bowes, George

1981-01-01

Incubation under water in a 30 C/14-hour or 12 C/10-hour photoperiod caused the CO2 compensation points of 10 aquatic macrophytes to decrease below 25 or increase above 50 microliters CO2 per liter, respectively. Submerged and aerial leaves of two amphibious angiosperms (Myriophyllum brasiliense and Proserpinaca palustris) maintained high compensation points when incubated in air but, when the submerged or aerial leaves of Proserpinaca were incubated under water, the compensation points dropped as low as 10. This suggests that, in addition to temperature and photoperiod, some factor associated with submergence regulates the compensation point of aquatic plants. In the high-compensation point plants, photorespiration, as a percentage of net photosynthesis, was equivalent to that in terrestrial C3 plants. For Hydrilla verticillata, the decreasing CO2 compensation points (110, 40, and 10) were associated with reduced photorespiration, as indicated by decreased O2 inhibition, decreased rates of CO2 evolution into CO2-free air, and increased net photosynthetic rates. The decrease in the CO2 compensation points of Hydrilla, Egeria densa, and Cabomba caroliniana was accompanied by an increase in the activity of phosphoenolpyruvate, but not of ribulose bisphosphate, carboxylase. In Hydrilla, several C4 enzymes also increased in activity to the following levels (micromoles per gram fresh weight per hour): pyruvate Pi dikinase (35), pyrophosphatase (716), adenylate kinase (525), NAD and NADP malate dehydrogenase (6565 and 30), NAD and NADP malic enzymes (239 and 44), and aspartate and alanine aminotransferases (357 and 85), whereas glycolate oxidase (6) and phosphoglycolate and phosphoglycerate phosphatases (76 and 32) showed no change. Glycolate dehydrogenase and phosphoenolpyruvate carboxykinase were undetectable. The reduced photorespiration in these plants may be due to increased CO2 fixation via a C4 acid pathway. However, for three Myriophyllum species, some other mechanism appears operative, as phosphoenolpyruvate carboxylase was not increased in the low compensation point state, and ribulose bisphosphate carboxylase remained the predominant carboxylation enzyme. PMID:16661670

Artificially evolved Synechococcus PCC6301 Rubisco variants exhibit improvements in folding and catalytic efficiency.

PubMed

Greene, Dina N; Whitney, Spencer M; Matsumura, Ichiro

2007-06-15

The photosynthetic CO2-fixing enzyme, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), is responsible for most of the world's biomass, but is a slow non-specific catalyst. We seek to identify and overcome the chemical and biological constraints that limit the evolutionary potential of Rubisco in Nature. Recently, the horizontal transfer of Calvin cycle genes (rbcL, rbcS and prkA) from cyanobacteria (Synechococcus PCC6301) to gamma-proteobacteria (Escherichia coli) was emulated in the laboratory. Three unique Rubisco variants containing single (M259T) and double (M259T/A8S, M259T/F342S) amino acid substitutions in the L (large) subunit were identified after three rounds of random mutagenesis and selection in E. coli. Here we show that the M259T mutation did not increase steady-state levels of rbcL mRNA or L protein. It instead improved the yield of properly folded L subunit in E. coli 4-9-fold by decreasing its natural propensity to misfold in vivo and/or by enhancing its interaction with the GroES-GroEL chaperonins. The addition of osmolites to the growth media enhanced productive folding of the M259T L subunit relative to the wild-type L subunit, while overexpression of the trigger factor and DnaK/DnaJ/GrpE chaperones impeded Rubisco assembly. The evolved enzymes showed improvement in their kinetic properties with the M259T variant showing a 12% increase in carboxylation turnover rate (k(c)cat), a 15% improvement in its K(M) for CO2 and no change in its K(M) for ribulose-1,5-bisphosphate or its CO2/O2 selectivity. The results of the present study show that the directed evolution of the Synechococcus Rubisco in E. coli can elicit improvements in folding and catalytic efficiency.

Nitrate and Ammonium Induced Photosynthetic Suppression in N-Limited Selenastrum minutum1

PubMed Central

Elrifi, Ivor R.; Turpin, David H.

1986-01-01

Nitrate-limited chemostat cultures of Selenastrum minutum Naeg. Collins (Chlorophyta) were used to determine the effects of nitrogen addition on photosynthesis, dark respiration, and dark carbon fixation. Addition of NO3− or NH4+ induced a transient suppression of photosynthetic carbon fixation (70 and 40% respectively). Intracellular ribulose bisphosphate levels decreased during suppression and recovered in parallel with photosynthesis. Photosynthetic oxygen evolution was decreased by N-pulsing under saturating light (650 microeinsteins per square meter per second). Under subsaturating light intensities (<165 microeinsteins per square meter per second) NH4+ addition resulted in O2 consumption in the light which was alleviated by the presence of the tricarboxylic acid cycle inhibitor fluoroacetate. Addition of NO3− or NH4+ resulted in a large stimulation of dark respiration (67 and 129%, respectively) and dark carbon fixation (360 and 2080%, respectively). The duration of N-induced perturbations was dependent on the concentration of added N. Inhibition of glutamine 2-oxoglutarate aminotransferase by azaserine alleviated all these effects. It is proposed that suppression of photosynthetic carbon fixation in response to N pulsing was the result of a competition for metabolites between the Calvin cycle and nitrogen assimilation. Carbon skeletons required for nitrogen assimilation would be derived from tricarboxylic acid cycle intermediates. To maintain tricarboxylic acid cycle activity triose phosphates would be exported from the chloroplast. This would decrease the rate of ribulose bisphosphate regeneration and consequently decrease net photosynthetic carbon accumulation. Stoichiometric calculations indicate that the Calvin cycle is one source of triose phosphates for N assimilation; however, during transient N resupply the major demand for triose phosphates must be met by starch or sucrose breakdown. The effects of N-pulsing on O2 evolution, dark respiration, and dark C-fixation are shown to be consistent with this model. PMID:16664788

Effects of overexpressing photosynthetic carbon flux control enzymes in the cyanobacterium Synechocystis PCC 6803.

PubMed

Liang, Feiyan; Lindblad, Peter

2016-11-01

Synechocystis PCC 6803 is a model unicellular cyanobacterium used in e.g. photosynthesis and CO 2 assimilation research. In the present study we examined the effects of overexpressing Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), sedoheptulose 1,7-biphosphatase (SBPase), fructose-bisphosphate aldolase (FBA) and transketolase (TK), confirmed carbon flux control enzymes of the Calvin-Bassham-Benson (CBB) cycle in higher plants, in Synechocystis PCC 6803. Overexpressing RuBisCO, SBPase and FBA resulted in increased in vivo oxygen evolution (maximal 115%), growth rate and biomass accumulation (maximal 52%) under 100μmolphotonsm -2 s -1 light condition. Cells overexpressing TK showed a chlorotic phenotype but increased biomass by approximately 42% under 100μmolphotonsm -2 s -1 light condition. Under 15μmolphotonsm -2 s -1 light condition, cells overexpressing TK showed enhanced in vivo oxygen evolution. This study demonstrates increased growth and biomass accumulation when overexpressing selected enzymes of the CBB cycle. RuBisCO, SBPase, FBA and TK are identified as four potential targets to improve growth and subsequently also yield of valuable products from Synechocystis PCC 6803. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Differential Expression of Rubisco in Sporophytes and Gametophytes of Some Marine Macroalgae

PubMed Central

Wang, Guangce; Niu, Jianfeng; Zhou, Baicheng

2011-01-01

Rubisco (ribulose-1, 5-bisphosphate carboxylase/oxygenase), a key enzyme of photosynthetic CO2 fixation, is one of the most abundant proteins in both higher plants and algae. In this study, the differential expression of Rubisco in sporophytes and gametophytes of four seaweed species — Porphyra yezoensis, P. haitanensis, Bangia fuscopurpurea (Rhodophyte) and Laminaria japonica (Phaeophyceae) — was studied in terms of the levels of transcription, translation and enzyme activity. Results indicated that both the Rubisco content and the initial carboxylase activity were notably higher in algal gametophytes than in the sporophytes, which suggested that the Rubisco content and the initial carboxylase activity were related to the ploidy of the generations of the four algal species. PMID:21283730

Biotechnological storage and utilization of entrapped solar energy.

PubMed

Bhattacharya, Sumana; Schiavone, Marc; Nayak, Amiya; Bhattacharya, Sanjoy K

2005-03-01

Our laboratory has recently developed a device employing immobilized F0F1 adenosine triphosphatase (ATPase) that allows synthesis of adenosine triphosphate (ATP) from adenosine 5'-diphosphate and inorganic phosphate using solar energy. We present estimates of total solar energy received by Earth's land area and demonstrate that its efficient capture may allow conversion of solar energy and storage into bonds of biochemicals using devices harboring either immobilized ATPase or NADH dehydrogenase. Capture and storage of solar energy into biochemicals may also enable fixation of CO2 emanating from polluting units. The cofactors ATP and NADH synthesized using solar energy could be used for regeneration of acceptor D-ribulose-1,5-bisphosphate from 3-phosphoglycerate formed during CO2 fixation.

Characterization of Chloroplastic Fructose 1,6-Bisphosphate Aldolases as Lysine-methylated Proteins in Plants*

PubMed Central

Mininno, Morgane; Brugière, Sabine; Pautre, Virginie; Gilgen, Annabelle; Ma, Sheng; Ferro, Myriam; Tardif, Marianne; Alban, Claude; Ravanel, Stéphane

2012-01-01

In pea (Pisum sativum), the protein-lysine methyltransferase (PsLSMT) catalyzes the trimethylation of Lys-14 in the large subunit (LS) of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the enzyme catalyzing the CO2 fixation step during photosynthesis. Homologs of PsLSMT, herein referred to as LSMT-like enzymes, are found in all plant genomes, but methylation of LS Rubisco is not universal in the plant kingdom, suggesting a species-specific protein substrate specificity of the methyltransferase. In this study, we report the biochemical characterization of the LSMT-like enzyme from Arabidopsis thaliana (AtLSMT-L), with a focus on its substrate specificity. We show that, in Arabidopsis, LS Rubisco is not naturally methylated and that the physiological substrates of AtLSMT-L are chloroplastic fructose 1,6-bisphosphate aldolase isoforms. These enzymes, which are involved in the assimilation of CO2 through the Calvin cycle and in chloroplastic glycolysis, are trimethylated at a conserved lysyl residue located close to the C terminus. Both AtLSMT-L and PsLSMT are able to methylate aldolases with similar kinetic parameters and product specificity. Thus, the divergent substrate specificity of LSMT-like enzymes from pea and Arabidopsis concerns only Rubisco. AtLSMT-L is able to interact with unmethylated Rubisco, but the complex is catalytically unproductive. Trimethylation does not modify the kinetic properties and tetrameric organization of aldolases in vitro. The identification of aldolases as methyl proteins in Arabidopsis and other species like pea suggests a role of protein lysine methylation in carbon metabolism in chloroplasts. PMID:22547063

The effect of energy substrates on PHB accumulation of Acidiphilium cryptum DX1-1.

PubMed

Xu, Ai-ling; Xia, Jin-lan; Song, Zhi-wen; Jiang, Peng; Xia, Yan; Wan, Min-xi; Zhang, Rui-yong; Yang, Yi; Liu, Ke-ke

2013-09-01

The effect of glucose and elemental sulfur on the growth and PHB accumulation of Acidiphilium cryptum DX1-1 was investigated. Meanwhile, the differential expressions of 19 genes related with PHB accumulation, sulfur metabolism and carbon fixed in heterotrophy, phytotrophy and mixotrophy were studied by RT-qPCR. The results showed that strain DX1-1 could accumulate PHB with sulfur as the energy substance and atmospheric CO2 as carbon resource. Glucose could improve the growth of strain DX1-1 cultured in medium with sulfur as the energy substance, and almost all the key enzyme-encoding genes related with PHB, sulfur metabolism and carbon fixed were basically up-regulated. PHB polymerase (Arcy_3030), ribulose-bisphosphate carboxylase (Acry_0825), ribulose-phosphate-epimerase (Acry_0022), and cysteine synthase A (Acry_2560) played important role in PHB accumulation, the modified expression of which could influence the PHB yield. With CO2 as carbon resource, the main initial substance of PHB accumulation for strain DX1-1 was acetyl-CoA, instead of acetate with the glucose as the carbon resource. Because of accumulating PHB by fixed atmospheric CO2 while independent of light, A. cryptum DX1-1 may have specifically potential in production of PHB.

Inhibition of the light-independent synthesis of chlorophyll in pine cotyledons at low temperature.

PubMed

Muramatsu, S; Kojima, K; Igasaki, T; Azumi, Y; Shinohara, K

2001-08-01

Cotyledons of Japanese black pine (Pinus thunbergii) were yellow when they developed in darkness at 8 degrees C since the light-independent synthesis of chlorophyll was almost completely inhibited in these cotyledons. The level of chlorophyll in dark-grown cotyledons was less than one-twentieth of that in light-grown cotyledons at the same temperature. In the yellow cotyledons, levels of transcripts of cab, rbcS, rbcL and psbA genes were quite high. The large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase were also detected at relatively high levels in yellow cotyledons. However, the accumulation of the two apoproteins of the light-harvesting chlorophyll a/b-binding protein of PSII was limited because of the limited supply of chlorophyll.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Landry, L.G.; Pell, E.J.

Exposing hybrid poplar (Populus maximowizii x trichocarpa) plants to ozone (O[sub 3]) resulted in an acceleration of the visual symptoms of senescence and a decrease in the activity and quantity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Whole plants, crude leaf extracts, and isolated intact chloroplasts of hybrid poplar clone 245 were used to test the hypothesis that O[sub 3]-induced structural modifications of Rubisco affect the activity of this key photosynthetic enzyme. Proteolytic activity, per se, could not account for losses in Rubisco; acidic and alkaline protease activities declined or were unaffected in foliage of O[sub 3]-treated poplar saplings. In vitro treatment ofmore » leaf extracts with O[sub 3] decreased total Rubisco activity and binding of the enzyme's transition-state analog, 2-carboxyarabinitol bisphosphate. Additionally, O[sub 3] increased the loss of Rubisco large subunit (LSU) when extracts were incubated at 37[degrees]C. Treatment of isolated intact chloroplasts with O[sub 3] accelerated both the loss of the 55-kD Rubisco LSU and the accumulation of Rubisco LSU aggregates, as visualized by immunoblotting. The time-dependent modification in rubisco structure was the primary response of the isolated organelles to O[sub 3] treatment, with little proteolytic degradation of the LSU detected. 32 refs., 5 figs., 1 tab.« less

Remodeling of chloroplast proteome under salinity affects salt tolerance of Festuca arundinacea.

PubMed

Pawłowicz, Izabela; Waśkiewicz, Agnieszka; Perlikowski, Dawid; Rapacz, Marcin; Ratajczak, Dominika; Kosmala, Arkadiusz

2018-06-07

Acclimation of photosynthetic apparatus to variable environmental conditions is an important component of tolerance to dehydration stresses, including salinity. The present study deals with the research on alterations in chloroplast proteome of the forage grasses. Based on chlorophyll fluorescence parameters, two genotypes of a model grass species-Festuca arundinacea with distinct levels of salinity tolerance: low salt tolerant (LST) and high salt tolerant (HST), were selected. Next, two-dimensional electrophoresis and mass spectrometry were applied under both control and salt stress conditions to identify proteins accumulated differentially between these two genotypes. The physiological analysis revealed that under NaCl treatment the studied plants differed in photosystem II activity, water content, and ion accumulation. The differentially accumulated proteins included ATPase B, ATP synthase, ribulose-1,5-bisphosphate carboxylase large and small subunits, cytochrome b6-f complex iron-sulfur subunit, oxygen-evolving enhancer proteins (OEE), OEE1 and OEE2, plastidic fructose-bisphosphate aldolase (pFBA), and lipocalin. A higher level of lipocalin, potentially involved in prevention of lipid peroxidation under stress, was also observed in the HST genotype. Our physiological and proteomic results performed for the first time on the species of forage grasses clearly showed that chloroplast metabolism adjustment could be a crucial factor in developing salinity tolerance.

Genetic engineering of the Calvin cycle toward enhanced photosynthetic CO2 fixation in microalgae.

PubMed

Yang, Bo; Liu, Jin; Ma, Xiaonian; Guo, Bingbing; Liu, Bin; Wu, Tao; Jiang, Yue; Chen, Feng

2017-01-01

Photosynthetic microalgae are emerging as potential biomass feedstock for sustainable production of biofuels and value-added bioproducts. CO 2 biomitigation through these organisms is considered as an eco-friendly and promising alternative to the existing carbon sequestration methods. Nonetheless, the inherent relatively low photosynthetic capacity of microalgae has hampered the practical use of this strategy for CO 2 biomitigation applications. Here, we demonstrate the feasibility of improving photosynthetic capacity by the genetic manipulation of the Calvin cycle in the typical green microalga Chlorella vulgaris . Firstly, we fused a plastid transit peptide to upstream of the enhanced green fluorescent protein (EGFP) and confirmed its expression in the chloroplast of C. vulgaris . Then we introduced the cyanobacterial fructose 1,6-bisphosphate aldolase, guided by the plastid transit peptide, into C. vulgaris chloroplast, leading to enhanced photosynthetic capacity (~ 1.2-fold) and cell growth. Molecular and physiochemical analyses suggested a possible role for aldolase overexpression in promoting the regeneration of ribulose 1,5-bisphosphate in the Calvin cycle and energy transfer in photosystems. Our work represents a proof-of-concept effort to enhance photosynthetic capacity by the engineering of the Calvin cycle in green microalgae. Our work also provides insights into targeted genetic engineering toward algal trait improvement for CO 2 biomitigation uses.

Carbonic Anhydrase Activity Associated with the Cyanobacterium Synechococcus PCC7942 1

PubMed Central

Badger, Murray R.; Price, G. Dean

1989-01-01

Intact cells and crude homogenates of high (1% CO2) and low dissolved inorganic carbon (Ci) (30-50 microliters per liter of CO2) grown Synechococcus PCC7942 have carbonic anhydrase (CA)-like activity, which enables them to catalyze the exchange of 18O from CO2 to H2O. This activity was studied using a mass spectrometer coupled to a cuvette with a membrane inlet system. Intact high and low Ci cells were found to contain CA activity, separated from the medium by a membrane which is preferentially permeable to CO2. This activity is most apparent in the light, where 18O-labeled CO2 species are being taken up by the cells but the effluxing CO2 has lost most of its label to water. In the dark, low Ci cells catalyze the depletion of the 18O enrichment of CO2 and this activity is inhibited by both ethoxyzolamide and 2-(trifluoromethoxy)carbonyl cyanide. This may occur via a common inhibition of the Ci pump and the Ci pump is proposed as a potential site for the exchange of 18O. CA activity was measurable in homogenates of both cell types but was 5- to 10-fold higher in low Ci cells. This was inhibited by ethoxyzolamide with an I50 of 50 to 100 micromolar in both low and high Ci cells. A large proportion of the internal CA activity appears to be pelletable in nature. This pelletability is increased by the presence of Mg2+ in a manner similar to that of ribulose bisphosphate carboxylase-oxygenase activity and chlorophyll (thylakoids) and may be the result of nonspecific aggregation. Separation of crude homogenates on sucrose gradients is consistent with the notion that CA and ribulose bisphosphate carboxylase-oxygenase activity may be associated with the same pelletable fraction. However, we cannot unequivocally establish that CA is located within the carboxysome. The sucrose gradients show the presence of separate soluble and pelletable CA activity. This may be due to the presence of separate forms of the enzyme or may arise from the same pelletable association which is unstable during extraction. PMID:16666546

Pathogenesis-Related Proteins of Tomato 1

PubMed Central

Vera, Pablo; Conejero, Vicente

1988-01-01

An endoproteinase induced by citrus exocortis viroid has been purified from tomato (Lycopersicon esculentum Mill, cv “Rutgers”) leaves. The proteinase corresponds to one of the major pathogenesis-related proteins of tomato plants and was designated proteinase P-69 as it has a molecular weight of 69,000 to 70,000. The proteinase was purified in four steps: (NH4)2SO4 fractionation, chromatography on Bio-Gel P-60, DEAE-Sepharose chromatography, and casein-Sepharose affinity chromatography. The proteinase had a pH optimum of 8.5 to 9.0 when assayed with either fluorescein thiocarbamoyl derivative (FTC)-casein or FTC-ribulose 1,5-bisphosphate carboxylase/oxygenase as substrates. The proteinase activity was inhibited by pCMB and strongly activated by calcium and magnesium ions as well as by DTT. When analyzed by electrofocusing, the activity showed a pI around 9.0. Images Fig. 4 Fig. 8 PMID:16666127

Plastid transformation for Rubisco engineering and protocols for assessing expression.

PubMed

Whitney, Spencer M; Sharwood, Robert E

2014-01-01

The assimilation of CO2 within chloroplasts is catalyzed by the bi-functional enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, Rubisco. Within higher plants the Rubisco large subunit gene, rbcL, is encoded in the plastid genome, while the Rubisco small subunit gene, RbcS is coded in the nucleus by a multi-gene family. Rubisco is considered a poor catalyst due to its slow turnover rate and its additional fixation of O2 that can result in wasteful loss of carbon through the energy requiring photorespiratory cycle. Improving the carboxylation efficiency and CO2/O2 selectivity of Rubisco within higher plants has been a long-term goal which has been greatly advanced in recent times using plastid transformation techniques. Here we present experimental methodologies for efficiently engineering Rubisco in the plastids of a tobacco master-line and analyzing leaf Rubisco content.

Pea amyloplast DNA is qualitatively similar to pea chloroplast DNA

NASA Technical Reports Server (NTRS)

Gaynor, J. J.

1984-01-01

Amyloplast DNA (apDNA), when subjected to digestion with restriction endonucleases, yields patterns nearly identical to that of DNA from mature pea chloroplasts (ctDNA). Southern transfers of apDNA and ctDNA, probed with the large subunit (LS) gene of ribulose-1,5-bisphosphate carboxylase (Rubisco), shows hybridization to the expected restriction fragments for both apDNA and ctDNA. However, Northern transfers of total RNA from chloroplasts and amyloplasts, probed again with the LS gene of Rubisco, shows that no detectable LS meggage is found in amyloplasts although LS expression in mature chloroplasts is high. Likewise, two dimensional polyacrylamide gel electrophoresis of etiolated gravisensitive pea tissue shows that both large and small subunits of Rubisco are conspicuously absent; however, in greening tissue these two constitute the major soluble proteins. These findings suggest that although the informational content of these two organelle types is equivalent, gene expression is quite different and is presumably under nuclear control.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wrighton, Kelly C.; Thomas, Brian C.; Sharon, I.

BD1-5, OP11, and OD1 bacteria have been widely detected in anaerobic environments, but their metabolisms remain unclear owing to lack of cultivated representatives and minimal genomic sampling. We uncovered metabolic characteristics for members of these phyla, and a new lineage, PER, via cultivation-independent recovery of 49 partial to near-complete genomes from an acetate-amended aquifer. All organisms were nonrespiring anaerobes predicted to ferment. Three augment fermentation with archaeal-like type II and III ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) that couples adenosine monophosphate salvage with CO2 fixation, a pathway previously not described in Bacteria. Members of OD1 reduce sulfur and may pump protons using archaeal-typemore » hydrogenases. For six organisms, the UGA stop codon is translated as tryptophan. All bacteria studied here may play previously unrecognized roles in hydrogen production, sulfur cycling, and fermentation of refractory sedimentary carbon.« less

Proteolytic Activity at Alkaline pH in Oat Leaves, Isolation of an Aminopeptidase 1

PubMed Central

Casano, Leonardo M.; Desimone, Marcelo; Trippi, Victorio S.

1989-01-01

Proteolytic activity in oat leaf extracts was measured with both azocasein and ribulose bisphosphate carboxylase (Rubisco) as substrates over a wide range of pH (3.0-9.2). With either azocasein or Rubisco activity peaks appeared at pH 4.8, 6.6, and 8.4. An aminopeptidase (AP) which hydrolyzes leucine-nitroanilide was partially purified. Purification consisted of a series of six steps which included ammonium sulfate precipitation, gel filtration, and two ionic exchange chromatographies. The enzyme was purified more than 100-fold. The apparent Km for leucine-nitroanilide is 0.08 millimolar at its pH optimum of 8.4. AP may be a cystein protease since it is inhibited by heavy metals and activated by 2-mercaptoethanol. Isolated chloroplasts were also able to hydrolyze leucine-nitroanilide at a pH optimum of 8.4, indicating that AP could be localized inside the photosynthetic organelles. PMID:16667194

Carboxysomes: metabolic modules for CO 2 fixation

DOE PAGES

Turmo, Aiko; Gonzalez-Esquer, Cesar Raul; Kerfeld, Cheryl A.

2017-08-14

The carboxysome is a bacterial microcompartment encapsulating the enzymes carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase. As the site of CO 2 fixation, it serves an essential role in the carbon dioxide concentrating mechanism of many chemoautotrophs and all cyanobacteria. Carboxysomes and other bacterial microcompartments self-assemble through specific protein–protein interactions that are typically mediated by conserved protein domains. In this review, we frame our current understanding of carboxysomes in the context of their component protein domains with their inherent function as the ‘building blocks’ of carboxysomes. These building blocks are organized in genetic modules (conserved chromosomal loci) that encode for carboxysomes andmore » ancillary proteins essential for the integration of the organelle with the rest of cellular metabolism. This conceptual framework provides the foundation for ‘plug-and-play’ engineering of carboxysomes as CO 2 fixation modules in a variety of biotechnological applications.« less

Nitrogen balancing and xylose addition enhances growth capacity and protein content in Chlorella minutissima cultures.

PubMed

Freitas, B C B; Esquível, M G; Matos, R G; Arraiano, C M; Morais, M G; Costa, J A V

2016-10-01

This study aimed to examine the metabolic changes in Chlorella minutissima cells grown under nitrogen-deficient conditions and with the addition of xylose. The cell density, maximum photochemical efficiency, and chlorophyll and lipid levels were measured. The expression of two photosynthetic proteins, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the beta subunit (AtpB) of adenosine triphosphate synthase, were measured. Comparison of cells grown in medium with a 50% reduction in the nitrogen concentration versus the traditional medium solution revealed that the cells grown under nitrogen-deficient conditions exhibited an increased growth rate, higher maximum cell density (12.7×10(6)cellsmL(-1)), optimal PSII efficiency (0.69) and decreased lipid level (25.08%). This study has taken the first steps toward protein detection in Chlorella minutissima, and the results can be used to optimize the culturing of other microalgae. Copyright © 2016 Elsevier Ltd. All rights reserved.

An Examination of the Plastid DNA of Hypohaploid Nicotiana plumbaginifolia Plants

PubMed Central

Cannon, Gordon C.; Van, K. Tran Thanh; Heinhorst, Sabine; Trinh, T. H.; Weissbach, Arthur

1989-01-01

DNA was extracted from different morphological types of hypohaploid Nicotiana plumbaginifolia plants. The cellular levels of chloroplast DNA (expressed as percent of total DNA) were found to be approximately two- to threefold higher in two albino hypohaploids than in a green hypohaploid. The level of chloroplast DNA in the green hypohaploid was not significantly different from either in vitro or in vivo grown haploid N. plumbaginifolia plants. Molecular hybridization with DNA probes for the large subunit of ribulose bisphosphate carboxylase from spinach and with Pvull fragments representing the entire Nicotiana tabacum chloroplast genome revealed no gross qualitative differences in the chloroplast DNAs of hypohaploid plants. Based on these observations we have concluded that the lack of chloroplast function observed in the albino forms of hypohaploid N. plumbaginifolia plants is not due to changes in the chloroplast genome. Images Figure 1 Figure 2 PMID:16666781

Serine 363 of a Hydrophobic Region of Archaeal Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from Archaeoglobus fulgidus and Thermococcus kodakaraensis Affects CO2/O2 Substrate Specificity and Oxygen Sensitivity.

PubMed

Kreel, Nathan E; Tabita, F Robert

2015-01-01

Archaeal ribulose 1, 5-bisphospate carboxylase/oxygenase (RubisCO) is differentiated from other RubisCO enzymes and is classified as a form III enzyme, as opposed to the form I and form II RubisCOs typical of chemoautotrophic bacteria and prokaryotic and eukaryotic phototrophs. The form III enzyme from archaea is particularly interesting as several of these proteins exhibit unusual and reversible sensitivity to molecular oxygen, including the enzyme from Archaeoglobus fulgidus. Previous studies with A. fulgidus RbcL2 had shown the importance of Met-295 in oxygen sensitivity and pointed towards the potential significance of another residue (Ser-363) found in a hydrophobic pocket that is conserved in all RubisCO proteins. In the current study, further structure/function studies have been performed focusing on Ser-363 of A. fulgidus RbcL2; various changes in this and other residues of the hydrophobic pocket point to and definitively establish the importance of Ser-363 with respect to interactions with oxygen. In addition, previous findings had indicated discrepant CO2/O2 specificity determinations of the Thermococcus kodakaraensis RubisCO, a close homolog of A. fulgidus RbcL2. It is shown here that the T. kodakaraensis enzyme exhibits a similar substrate specificity as the A. fulgidus enzyme and is also oxygen sensitive, with equivalent residues involved in oxygen interactions.

Serine 363 of a Hydrophobic Region of Archaeal Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from Archaeoglobus fulgidus and Thermococcus kodakaraensis Affects CO2/O2 Substrate Specificity and Oxygen Sensitivity

PubMed Central

Kreel, Nathan E.; Tabita, F. Robert

2015-01-01

Archaeal ribulose 1, 5-bisphospate carboxylase/oxygenase (RubisCO) is differentiated from other RubisCO enzymes and is classified as a form III enzyme, as opposed to the form I and form II RubisCOs typical of chemoautotrophic bacteria and prokaryotic and eukaryotic phototrophs. The form III enzyme from archaea is particularly interesting as several of these proteins exhibit unusual and reversible sensitivity to molecular oxygen, including the enzyme from Archaeoglobus fulgidus. Previous studies with A. fulgidus RbcL2 had shown the importance of Met-295 in oxygen sensitivity and pointed towards the potential significance of another residue (Ser-363) found in a hydrophobic pocket that is conserved in all RubisCO proteins. In the current study, further structure/function studies have been performed focusing on Ser-363 of A. fulgidus RbcL2; various changes in this and other residues of the hydrophobic pocket point to and definitively establish the importance of Ser-363 with respect to interactions with oxygen. In addition, previous findings had indicated discrepant CO2/O2 specificity determinations of the Thermococcus kodakaraensis RubisCO, a close homolog of A. fulgidus RbcL2. It is shown here that the T. kodakaraensis enzyme exhibits a similar substrate specificity as the A. fulgidus enzyme and is also oxygen sensitive, with equivalent residues involved in oxygen interactions. PMID:26381513

Serine 363 of a hydrophobic region of Archaeal ribulose 1,5-bisphosphate carboxylase/oxygenase from Archaeoglobus fulgidus and Thermococcus kodakaraensis affects CO 2/O 2 substrate specificity and oxygen sensitivity

DOE PAGES

Kreel, Nathan E.; Tabita, F. Robert; Berg, Ivan

2015-09-18

Archaeal ribulose 1, 5-bisphospate carboxylase/oxygenase (RubisCO) is differentiated from other RubisCO enzymes and is classified as a form III enzyme, as opposed to the form I and form II RubisCOs typical of chemoautotrophic bacteria and prokaryotic and eukaryotic phototrophs. The form III enzyme from archaea is particularly interesting as several of these proteins exhibit unusual and reversible sensitivity to molecular oxygen, including the enzyme from Archaeoglobus fulgidus. Previous studies with A. fulgidus RbcL2 had shown the importance of Met-295 in oxygen sensitivity and pointed towards the potential significance of another residue (Ser-363) found in a hydrophobic pocket that is conservedmore » in all RubisCO proteins. In the current study, further structure/function studies have been performed focusing on Ser-363 of A. fulgidus RbcL2; various changes in this and other residues of the hydrophobic pocket point to and definitively establish the importance of Ser-363 with respect to interactions with oxygen. In addition, previous findings had indicated discrepant CO 2/O 2 specificity determinations of the Thermococcus kodakaraensis RubisCO, a close homolog of A. fulgidus RbcL2. As a result, it is shown here that the T. kodakaraensis enzyme exhibits a similar substrate specificity as the A. fulgidus enzyme and is also oxygen sensitive, with equivalent residues involved in oxygen interactions.« less

Spatial variation of phytoplankton community structure in Daya Bay, China.

PubMed

Jiang, Zhao-Yu; Wang, You-Shao; Cheng, Hao; Zhang, Jian-Dong; Fei, Jiao

2015-10-01

Daya Bay is one of the largest and most important gulfs in the southern coast of China, in the northern part of the South China Sea. The phylogenetic diversity and spatial distribution of phytoplankton from the Daya Bay surface water and the relationship with the in situ water environment were investigated by the clone library of the large subunit of ribulose-1, 5-bisphosphate carboxylase (rbcL) gene. The dominant species of phytoplankton were diatoms and eustigmatophytes, which accounted for 81.9 % of all the clones of the rbcL genes. Prymnesiophytes were widely spread and wide varieties lived in Daya Bay, whereas the quantity was limited. The community structure of phytoplankton was shaped by pH and salinity and the concentration of silicate, phosphorus and nitrite. The phytoplankton biomass was significantly positively affected by phosphorus and nitrite but negatively by salinity and pH. Therefore, the phytoplankton distribution and biomass from Daya Bay were doubly affected by anthropic activities and natural factors.

[Effect of cultivation conditions on the growth and activities of sulfur metabolism enzymes and carboxylases of Sulfobacillus thermosulfidooxidans subsp. asporogenes strain 41].

PubMed

Egorova, M A; Tsaplina, I A; Zakharchuk, L M; Bogdanova, T I; Krasil'nikova, E N

2004-01-01

The moderately thermophilic acidophilic bacterium Sulfobacillus thermosulfidooxidans subsp. asporogenes strain 41 is capable of utilizing sulfides of gold-arsenic concentrate and elemental sulfur as a source of energy. The growth in the presence of S0 under auto- or mixotrophic conditions was less stable compared with the media containing iron monoxide. The enzymes involved in oxidation of sulfur inorganic compounds--thiosulfate-oxidizing enzyme, tetrathionate hydrolase, rhodonase, adenylyl sulfate reductase, sulfite oxidase, and sulfur oxygenase--were discovered in the cells of Sulfobacillus grown in the mineral medium containing 0.02% yeast extract and either sulfur or iron monoxide and thiosulfate. Cell-free extracts of the cultures grown in the medium with sulfur under auto- or mixotrophic conditions displayed activity of the key enzyme of the Calvin cycle--ribulose bisphosphate carboxylase--and several other enzymes involved in heterotrophic fixation of carbonic acid. Activities of carboxylases depended on the composition of cultivation media.

A short history of RubisCO: the rise and fall (?) of Nature's predominant CO2 fixing enzyme.

PubMed

Erb, Tobias J; Zarzycki, Jan

2018-02-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is arguably one of the most abundant proteins in the biosphere and a key enzyme in the global carbon cycle. Although RubisCO has been intensively studied, its evolutionary origins and rise as Nature's most dominant carbon dioxide (CO 2 )-fixing enzyme still remain in the dark. In this review we will bring together biochemical, structural, physiological, microbiological, as well as phylogenetic data to speculate on the evolutionary roots of the CO 2 -fixation reaction of RubisCO, the emergence of RubisCO-based autotrophic CO 2 -fixation in the context of the Calvin-Benson-Bassham cycle, and the further evolution of RubisCO into the 'RubisCOsome', a complex of various proteins assembling and interacting with the enzyme to improve its operational capacity (functionality) under different biological and environmental conditions. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Many-molecule encapsulation by an icosahedral shell

PubMed Central

Perlmutter, Jason D; Mohajerani, Farzaneh; Hagan, Michael F

2016-01-01

We computationally study how an icosahedral shell assembles around hundreds of molecules. Such a process occurs during the formation of the carboxysome, a bacterial microcompartment that assembles around many copies of the enzymes ribulose 1,5-bisphosphate carboxylase/ oxygenase and carbonic anhydrase to facilitate carbon fixation in cyanobacteria. Our simulations identify two classes of assembly pathways leading to encapsulation of many-molecule cargoes. In one, shell assembly proceeds concomitantly with cargo condensation. In the other, the cargo first forms a dense globule; then, shell proteins assemble around and bud from the condensed cargo complex. Although the model is simplified, the simulations predict intermediates and closure mechanisms not accessible in experiments, and show how assembly can be tuned between these two pathways by modulating protein interactions. In addition to elucidating assembly pathways and critical control parameters for microcompartment assembly, our results may guide the reengineering of viruses as nanoreactors that self-assemble around their reactants. DOI: http://dx.doi.org/10.7554/eLife.14078.001 PMID:27166515

Evolving Methanococcoides burtonii archaeal Rubisco for improved photosynthesis and plant growth

PubMed Central

Wilson, Robert H.; Alonso, Hernan; Whitney, Spencer M.

2016-01-01

In photosynthesis Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the often rate limiting CO2-fixation step in the Calvin cycle. This makes Rubisco both the gatekeeper for carbon entry into the biosphere and a target for functional improvement to enhance photosynthesis and plant growth. Encumbering the catalytic performance of Rubisco is its highly conserved, complex catalytic chemistry. Accordingly, traditional efforts to enhance Rubisco catalysis using protracted “trial and error” protein engineering approaches have met with limited success. Here we demonstrate the versatility of high throughput directed (laboratory) protein evolution for improving the carboxylation properties of a non-photosynthetic Rubisco from the archaea Methanococcoides burtonii. Using chloroplast transformation in the model plant Nicotiana tabacum (tobacco) we confirm the improved forms of M. burtonii Rubisco increased photosynthesis and growth relative to tobacco controls producing wild-type M. burtonii Rubisco. Our findings indicate continued directed evolution of archaeal Rubisco offers new potential for enhancing leaf photosynthesis and plant growth. PMID:26926260

Evolving Methanococcoides burtonii archaeal Rubisco for improved photosynthesis and plant growth.

PubMed

Wilson, Robert H; Alonso, Hernan; Whitney, Spencer M

2016-03-01

In photosynthesis Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the often rate limiting CO2-fixation step in the Calvin cycle. This makes Rubisco both the gatekeeper for carbon entry into the biosphere and a target for functional improvement to enhance photosynthesis and plant growth. Encumbering the catalytic performance of Rubisco is its highly conserved, complex catalytic chemistry. Accordingly, traditional efforts to enhance Rubisco catalysis using protracted "trial and error" protein engineering approaches have met with limited success. Here we demonstrate the versatility of high throughput directed (laboratory) protein evolution for improving the carboxylation properties of a non-photosynthetic Rubisco from the archaea Methanococcoides burtonii. Using chloroplast transformation in the model plant Nicotiana tabacum (tobacco) we confirm the improved forms of M. burtonii Rubisco increased photosynthesis and growth relative to tobacco controls producing wild-type M. burtonii Rubisco. Our findings indicate continued directed evolution of archaeal Rubisco offers new potential for enhancing leaf photosynthesis and plant growth.

Opposing effects of folding and assembly chaperones on evolvability of Rubisco.

PubMed

Durão, Paulo; Aigner, Harald; Nagy, Péter; Mueller-Cajar, Oliver; Hartl, F Ulrich; Hayer-Hartl, Manajit

2015-02-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the fixation of CO2 in photosynthesis. Despite its pivotal role, Rubisco is an inefficient enzyme and thus is a key target for directed evolution. Rubisco biogenesis depends on auxiliary factors, including the GroEL/ES-type chaperonin for folding and the chaperone RbcX for assembly. Here we performed directed evolution of cyanobacterial form I Rubisco using a Rubisco-dependent Escherichia coli strain. Overexpression of GroEL/ES enhanced Rubisco solubility and tended to expand the range of permissible mutations. In contrast, the specific assembly chaperone RbcX had a negative effect on evolvability by preventing a subset of mutants from forming holoenzyme. Mutation F140I in the large Rubisco subunit, isolated in the absence of RbcX, increased carboxylation efficiency approximately threefold without reducing CO2 specificity. The F140I mutant resulted in a ∼55% improved photosynthesis rate in Synechocystis PCC6803. The requirement of specific biogenesis factors downstream of chaperonin may have retarded the natural evolution of Rubisco.

A RuBisCO-mediated carbon metabolic pathway in methanogenic archaea

PubMed Central

Kono, Takunari; Mehrotra, Sandhya; Endo, Chikako; Kizu, Natsuko; Matusda, Mami; Kimura, Hiroyuki; Mizohata, Eiichi; Inoue, Tsuyoshi; Hasunuma, Tomohisa; Yokota, Akiho; Matsumura, Hiroyoshi; Ashida, Hiroki

2017-01-01

Two enzymes are considered to be unique to the photosynthetic Calvin–Benson cycle: ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), responsible for CO2 fixation, and phosphoribulokinase (PRK). Some archaea possess bona fide RuBisCOs, despite not being photosynthetic organisms, but are thought to lack PRK. Here we demonstrate the existence in methanogenic archaea of a carbon metabolic pathway involving RuBisCO and PRK, which we term ‘reductive hexulose-phosphate' (RHP) pathway. These archaea possess both RuBisCO and a catalytically active PRK whose crystal structure resembles that of photosynthetic bacterial PRK. Capillary electrophoresis-mass spectrometric analysis of metabolites reveals that the RHP pathway, which differs from the Calvin–Benson cycle only in a few steps, is active in vivo. Our work highlights evolutionary and functional links between RuBisCO-mediated carbon metabolic pathways in methanogenic archaea and photosynthetic organisms. Whether the RHP pathway allows for autotrophy (that is, growth exclusively with CO2 as carbon source) remains unknown. PMID:28082747

Rubisco small-subunit α-helices control pyrenoid formation in Chlamydomonas

PubMed Central

Meyer, Moritz T.; Genkov, Todor; Skepper, Jeremy N.; Jouhet, Juliette; Mitchell, Madeline C.; Spreitzer, Robert J.; Griffiths, Howard

2012-01-01

The pyrenoid is a subcellular microcompartment in which algae sequester the primary carboxylase, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The pyrenoid is associated with a CO2-concentrating mechanism (CCM), which improves the operating efficiency of carbon assimilation and overcomes diffusive limitations in aquatic photosynthesis. Using the model alga Chlamydomonas reinhardtii, we show that pyrenoid formation, Rubisco aggregation, and CCM activity relate to discrete regions of the Rubisco small subunit (SSU). Specifically, pyrenoid occurrence was shown to be conditioned by the amino acid composition of two surface-exposed α-helices of the SSU: higher plant-like helices knock out the pyrenoid, whereas native algal helices establish a pyrenoid. We have also established that pyrenoid integrity was essential for the operation of an active CCM. With the algal CCM being functionally analogous to the terrestrial C4 pathway in higher plants, such insights may offer a route toward transforming algal and higher plant productivity for the future. PMID:23112177

Chloroplast-cytoplasmic interrelations involved in chloroplast development in Chlamydomonas reinhardi y-1: effect of selective depletion of chloroplast translates

PubMed Central

1980-01-01

Chlamydomonas reinhardi y-1 cells grown in the dark in the presence of chloramphenicol (CD cells) are depleted of photosynthetic membranes and 70S translates. These cells were found to be unable to synthesize chlorophyll in the light until chloroplast protein synthesis was resumed. On the other hand, CD cells acquired the capacity to partially green in the presence of cycloheximide. This greening was characterized by the development of photosynthetic activity, as demonstrated by light- dependent oxygen evolution of whole cells and by measurements of ribulose-1,5-bisphosphate carboxylase and fluorescence kinetics. The chlorophyll synthesized de novo during greening in the absence of 80S ribosomal activity was organized in chlorophyll-protein complexes, as ascertained by low-temperature fluorescence-emission spectra. The morphology of these cells appeared to be normal. A model has been proposed as a working hypothesis, which could account for the phenomena described above and previously reported data pertaining to chloroplast development. PMID:7419587

Vacuolar Localization of Endoproteinases EP(1) and EP(2) in Barley Mesophyll Cells.

PubMed

Thayer, S S; Huffaker, R C

1984-05-01

The localization of two previously characterized endoproteinases (EP(1) and EP(2)) that comprise more than 95% of the protease activity in primary Hordeum vulgare L. var Numar leaves was determined. Intact vacuoles released from washed mesophyll protoplasts by gentle osmotic shock and increase in pH, were purified by flotation through a four-step Ficoll gradient. These vacuoles contained endoproteinases that rapidly degraded purified barley ribulose-1,5-bisphosphate carboxylase (RuBPCase) substrate. Breakdown products and extent of digestion of RuBPCase were determined using 12% polyacrylamide-sodium dodecyl sulfate gels. Coomassie brilliant blue- or silver-stained gels were scanned, and the peaks were integrated to provide quantitative information. The characteristics of the vacuolar endoproteinases (e.g. sensitivity to various inhibitors and activators, and the molecular weights of the breakdown products, i.e. peptide maps) closely resembled those of purified EP(1) and partially purified EP(2). It is therefore concluded that EP(1) and EP(2) are localized in the vacuoles of mesophyll cells.

Stable carbon isotopes: possible clues to early life on Mars.

PubMed

Schidlowski, M

1992-01-01

Organic and inorganic carbon in terrestrial near-surface environments are characterized by a marked difference in their 13C/12C ratios which can be traced back in the Earth's sedimentary record over almost 4 billion years. There is no doubt that the bias in favour of 12C displayed by biogenic matter derives, for the most part, from the isotope-selecting properties of the carbon-fixing enzyme (ribulose-1,5-bisphosphate carboxylase) that is operative in the principal photosynthetic pathway and promotes most of the carbon transfer from the non-living to the living realm. Postulating a universality of biological principles in analogy to the proven universality of the laws of physics and chemistry, we may expect enzymatic reactions in exobiological systems to be beset with B similar kinetic fractionation effects. Hence, the retrieval from the oldest Martian sediments of isotopic fractionations between reduced and oxidized (carbonate) carbon may substantially constrain current conjectures on the possible existence of former life on Mars.

A 3-hydroxypropionate/4-hydroxybutyrate autotrophic carbon dioxide assimilation pathway in Archaea.

PubMed

Berg, Ivan A; Kockelkorn, Daniel; Buckel, Wolfgang; Fuchs, Georg

2007-12-14

The assimilation of carbon dioxide (CO2) into organic material is quantitatively the most important biosynthetic process. We discovered that an autotrophic member of the archaeal order Sulfolobales, Metallosphaera sedula, fixed CO2 with acetyl-coenzyme A (acetyl-CoA)/propionyl-CoA carboxylase as the key carboxylating enzyme. In this system, one acetyl-CoA and two bicarbonate molecules were reductively converted via 3-hydroxypropionate to succinyl-CoA. This intermediate was reduced to 4-hydroxybutyrate and converted into two acetyl-CoA molecules via 4-hydroxybutyryl-CoA dehydratase. The key genes of this pathway were found not only in Metallosphaera but also in Sulfolobus, Archaeoglobus, and Cenarchaeum species. Moreover, the Global Ocean Sampling database contains half as many 4-hydroxybutyryl-CoA dehydratase sequences as compared with those found for another key photosynthetic CO2-fixing enzyme, ribulose-1,5-bisphosphate carboxylase-oxygenase. This indicates the importance of this enzyme in global carbon cycling.

Significance of Phosphoenolpyruvate Carboxylase during Ammonium Assimilation

PubMed Central

Guy, Robert D.; Vanlerberghe, Greg C.; Turpin, David H.

1989-01-01

The effect of N-assimilation on the partitioning of carbon fixation between phosphoenolpyruvate carboxylase (PEPcase) and ribulose bisphosphate carboxylase/oxygenase (Rubisco) was determined by measuring stable carbon isotope discrimination during photosynthesis by an N-limited green alga, Selenastrum minutum (Naeg.) Collins. This was facilitated by a two process model accounting for simultaneous CO2 fixation and respiratory CO2 release. Discrimination by control cells was consistent with the majority of carbon being fixed by Rubisco. During nitrogen assimilation however, discrimination was greatly reduced indicating an enhanced flux through PEPcase which accounted for upward of 70% of total carbon fixation. This shift toward anaplerotic metabolism supports a large increase in tricarboxylic acid cycle activity primarily between oxaloacetate and α-ketoglutarate thereby facilitating the provision of carbon skeletons for amino acid synthesis. This provides an example of a unique set of conditions under which anaplerotic carbon fixation by PEPcase exceeds photosynthetic carbon fixation by Rubisco in a C3 organism. Images Figure 6 PMID:16666678

Significance of Phosphoenolpyruvate Carboxylase during Ammonium Assimilation: Carbon Isotope Discrimination in Photosynthesis and Respiration by the N-Limited Green Alga Selenastrum minutum.

PubMed

Guy, R D; Vanlerberghe, G C; Turpin, D H

1989-04-01

The effect of N-assimilation on the partitioning of carbon fixation between phosphoenolpyruvate carboxylase (PEPcase) and ribulose bisphosphate carboxylase/oxygenase (Rubisco) was determined by measuring stable carbon isotope discrimination during photosynthesis by an N-limited green alga, Selenastrum minutum (Naeg.) Collins. This was facilitated by a two process model accounting for simultaneous CO(2) fixation and respiratory CO(2) release. Discrimination by control cells was consistent with the majority of carbon being fixed by Rubisco. During nitrogen assimilation however, discrimination was greatly reduced indicating an enhanced flux through PEPcase which accounted for upward of 70% of total carbon fixation. This shift toward anaplerotic metabolism supports a large increase in tricarboxylic acid cycle activity primarily between oxaloacetate and alpha-ketoglutarate thereby facilitating the provision of carbon skeletons for amino acid synthesis. This provides an example of a unique set of conditions under which anaplerotic carbon fixation by PEPcase exceeds photosynthetic carbon fixation by Rubisco in a C(3) organism.

Polypyrrole membranes as scaffolds for biomolecule immobilization

NASA Astrophysics Data System (ADS)

Hery, Travis M.; Satagopan, Sriram; Northcutt, Robert G.; Tabita, F. Robert; Sundaresan, Vishnu-Baba

2016-12-01

Enzymes have evolved over hundreds of years through changes in ecosystems (climate, atmosphere, hydrology, etc). The evolutionary changes driven by the need to survive has led to enzymes with diverse functionality such as reduction of carbon dioxide and methane to other forms of carbon, fixation of nitrogen, and high temperature biochemical processes. While these enzymes have useful properties, engineering a scalable cell-free system with these enzymes will be useful for stable production of desired products without involving the vagaries of cellular metabolism. This article presents various approaches to incorporate ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in a conducting polymer (polypyrrole (PPy)) doped with a bulky anion (dodecylbenzenesulfonate (DBS)) in an effort to create functional devices for the conversion of carbon dioxide into precursors for high-value chemicals. We demonstrate that the tailored device creates an environment where the enzyme can retain its function while being protected from denaturing conditions. It is envisioned that the 3-PGA produced by RuBisCO will be converted into value-added products.

Purification of nitrate reductase from Nicotiana plumbaginifolia by affinity chromatography using 5'AMP-sepharose and monoclonal antibodies.

PubMed

Moureaux, T; Leydecker, M T; Meyer, C

1989-02-15

Nitrate reductase was purified from leaves of Nicotiana plumbaginifolia using either 5'AMP-Sepharose chromatography or two steps of immunoaffinity chromatography involving monoclonal antibodies directed against nitrate reductase from maize and against ribulose-1,5-bisphosphate carboxylase from N. plumbaginifolia. Nitrate reductase obtained by the first method was purified 1000-fold to a specific activity of 9 units/mg protein. The second method produced an homogenous enzyme, purified 21,000-fold to a specific activity of 80 units/mg protein. SDS/PAGE of nitrate reductase always resulted in two bands of 107 and 99.5 kDa. The 107-kDa band was the nitrate reductase subunit of N. plumbaginifolia; the smaller one of 99.5 kDa is thought, as commonly reported, to result from proteolysis of the larger protein. The molecular mass of 107 kDa is close to the values calculated from the coding sequences of the two nitrate reductase genes recently cloned from tobacco (Nicotiana tabacum cv Xanthi).

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rejda, J.M.; Johal, S.; Chollet, R.

Homogeneous preparations of ribulose 1,5-bisphosphate carboxylase/oxygenase were isolated from several diploid and tetraploid cultivars of perennial ryegrass by three different purification protocols. The apparent K/sub m/ values for substrate CO/sub 2/ were essentially identical for the fully CO/sub 2//Mg/sup 2 +/-activated diploid and tetraploid enzymes, as were the kinetics for deactivation and activation of the CO/sub 2//Mg/sup 2 +/-activated and -depleted carboxylases, respectively. Similarly, virtually indistinguishable electrophoretic properties were observed for both the native and dissociated diploid and tetraploid ryegrass proteins, including native and subunit molecular weights and the isoelectric points of the native proteins and the large and smallmore » subunit component polypeptides. The quantity of carboxylase protein or total soluble leaf protein did not differ significantly between the diploid and tetraploid cultivars. Contrary to a previous report, these results indicate that increased ploidy level has had essentially no effect on the quantity or enzymic and physicochemical properties of ribulosebisphosphate carboxylase/oxygenase in perennial ryegrass.« less

Quantitative proteomic analysis reveals evolutionary divergence and species-specific peptides in the Alexandrium tamarense complex (Dinophyceae).

PubMed

Li, Cheng; Zhang, Yong; Xie, Zhang-Xian; He, Zhi-Ping; Lin, Lin; Wang, Da-Zhi

2013-06-28

The Alexandrium tamarense/catenella/fundyense complex is the major causative agent responsible for harmful algal blooms and paralytic shellfish poisoning around the world. However, taxonomy of the A. tamarense complex is contentious and the evolutionary relationships within the complex are unclear. This study compared protein profiles of the A. tamarense complex collected from different geographic regions using the two dimensional fluorescence difference gel electrophoresis (2-D DIGE) approach, and identified species-specific peptides using MALDI-TOF/TOF mass spectrometry. The results showed that three Alexandrium morphotypes presented significantly different protein expression patterns with about 30-40% shared proteins. However, ecotypes from different geographic regions within a species exhibited the same expression patterns, although a few proteins were altered in abundance. Several proteins, i.e. ribulose-1,5-bisphosphate carboxylase oxygenase form II, plastid protein NAP50, methionine S-adenosyltransferase, and peridinin-chlorophyll a-binding protein, were identified and presented different shift patterns in isoelectric point and/or molecular weight in the 2-D DIGE gels, indicating that amino acid mutation and/or posttranslational modification of these proteins had occurred. The species-specific peptide mass fingerprint and amino acid sequence of ribulose-1,5-bisphosphate carboxylase oxygenase were characterized in the A. tamarense complex, and amino acid substitution occurred among them. This study indicated that evolutionary divergence had occurred at the proteomic level in the A. tamarense complex, and that the species-specific peptides could be used as potential biomarkers to distinguish the three morphotypes. Scientific question: The Alexandrium tamarense/catenella/fundyense complex is the major causative agent responsible for harmful algal blooms and paralytic shellfish poisoning around the world. However, taxonomy of the A. tamarense complex is contentious and the evolutionary relationships within the complex are unclear, which has seriously impeded our understanding of Alexandrium-causing HABs and, consequently, the monitoring, mitigation and prevention. Technical significance: This study, for the first time, compared the global protein expression patterns of eight ecotypes from the A. tamarense complex and identified species-specific peptides using a quantitative proteomic approach combining 2-D DIGE and MALDI-TOF/TOF MS. This study demonstrated that the evolutionary divergence had occurred in the A. tamarense complex at the proteomic level, and the complex should be classified into three species, i.e. A. tamarense, A. catenella, and A. fundyense. Moreover, the species-specific peptide mass fingerprints could be used as potential biomarkers to distinguish the three morphotypes. Copyright © 2013 Elsevier B.V. All rights reserved.

Quantification of growth-defense trade-offs in a common currency: nitrogen required for phenolamide biosynthesis is not derived from ribulose-1,5-bisphosphate carboxylase/oxygenase turnover

PubMed Central

Wielsch, Nathalie; Bartram, Stefan; Hummert, Christian; Svatoš, Aleš; Baldwin, Ian T.; Groten, Karin

2014-01-01

Induced defenses are thought to be economical: growth and fitness-limiting resources are only invested into defenses when needed. To date, this putative growth-defense trade-off has not been quantified in a common currency at the level of individual compounds. Here, a quantification method for 15N-labeled proteins enabled a direct comparison of nitrogen (N) allocation to proteins, specifically, ribulose-1,5-bisposphate carboxylase/oxygenase (RuBisCO) as proxy for growth, with that into small N-containing defense metabolites (nicotine, phenolamides) as proxies for defense after herbivory. After repeated simulated herbivory, total N decreased in the shoots of wild type (WT) Nicotiana attenuata plants, but not in two transgenic lines impaired in jasmonate defense signaling (irLOX3) and phenolamide biosynthesis (irMYB8). N was reallocated among different compounds within elicited rosette leaves: in WT, a strong decrease in total soluble protein (TSP) and RuBisCO was accompanied by an increase in defense metabolites; irLOX3 showed a similar, albeit attenuated pattern; while irMYB8 rosette leaves were the least responsive to elicitation with overall higher levels of RuBisCO. Induced defenses were higher in the older compared to the younger rosette leaves, supporting the hypothesis that tissue developmental stage influences defense investments. We propose that MYB8, probably by regulating the production of phenolamides, indirectly mediates protein pool sizes after herbivory. Although the decrease in absolute N invested in TSP and RuBisCO elicited by simulated herbivory was much larger than the N-requirements of nicotine and phenolamide biosynthesis, 15N flux studies revealed that N for phenolamide synthesis originates from recently assimilated N rather than from RuBisCO turnover. PMID:23590461

Upregulated Transcription of Plasmid and Chromosomal Ribulose Monophosphate Pathway Genes Is Critical for Methanol Assimilation Rate and Methanol Tolerance in the Methylotrophic Bacterium Bacillus methanolicus

PubMed Central

Jakobsen, Øyvind M.; Benichou, Aline; Flickinger, Michael C.; Valla, Svein; Ellingsen, Trond E.; Brautaset, Trygve

2006-01-01

The natural plasmid pBM19 carries the key mdh gene needed for the oxidation of methanol into formaldehyde by Bacillus methanolicus. Five more genes, glpX, fba, tkt, pfk, and rpe, with deduced roles in the cell primary metabolism, are also located on this plasmid. By using real-time PCR, we show that they are transcriptionally upregulated (6- to 40-fold) in cells utilizing methanol; a similar induction was shown for two chromosomal genes, hps and phi. These seven genes are involved in the fructose bisphosphate aldolase/sedoheptulose bisphosphatase variant of the ribulose monophosphate (RuMP) pathway for formaldehyde assimilation. Curing of pBM19 causes higher methanol tolerance and reduced formaldehyde tolerance, and the methanol tolerance is reversed to wild-type levels by reintroducing mdh. Thus, the RuMP pathway is needed to detoxify the formaldehyde produced by the methanol dehydrogenase-mediated conversion of methanol, and the in vivo transcription levels of mdh and the RuMP pathway genes reflect the methanol tolerance level of the cells. The transcriptional inducer of hps and phi genes is formaldehyde, and not methanol, and introduction of multiple copies of these two genes into B. methanolicus made the cells more tolerant of growth on high methanol concentrations. The recombinant strain also had a significantly higher specific growth rate on methanol than the wild type. While pBM19 is critical for growth on methanol and important for formaldehyde detoxification, the maintenance of this plasmid represents a burden for B. methanolicus when growing on mannitol. Our data contribute to a new and fundamental understanding of the regulation of B. methanolicus methylotrophy. PMID:16585766

Upregulated transcription of plasmid and chromosomal ribulose monophosphate pathway genes is critical for methanol assimilation rate and methanol tolerance in the methylotrophic bacterium Bacillus methanolicus.

PubMed

Jakobsen, Øyvind M; Benichou, Aline; Flickinger, Michael C; Valla, Svein; Ellingsen, Trond E; Brautaset, Trygve

2006-04-01

The natural plasmid pBM19 carries the key mdh gene needed for the oxidation of methanol into formaldehyde by Bacillus methanolicus. Five more genes, glpX, fba, tkt, pfk, and rpe, with deduced roles in the cell primary metabolism, are also located on this plasmid. By using real-time PCR, we show that they are transcriptionally upregulated (6- to 40-fold) in cells utilizing methanol; a similar induction was shown for two chromosomal genes, hps and phi. These seven genes are involved in the fructose bisphosphate aldolase/sedoheptulose bisphosphatase variant of the ribulose monophosphate (RuMP) pathway for formaldehyde assimilation. Curing of pBM19 causes higher methanol tolerance and reduced formaldehyde tolerance, and the methanol tolerance is reversed to wild-type levels by reintroducing mdh. Thus, the RuMP pathway is needed to detoxify the formaldehyde produced by the methanol dehydrogenase-mediated conversion of methanol, and the in vivo transcription levels of mdh and the RuMP pathway genes reflect the methanol tolerance level of the cells. The transcriptional inducer of hps and phi genes is formaldehyde, and not methanol, and introduction of multiple copies of these two genes into B. methanolicus made the cells more tolerant of growth on high methanol concentrations. The recombinant strain also had a significantly higher specific growth rate on methanol than the wild type. While pBM19 is critical for growth on methanol and important for formaldehyde detoxification, the maintenance of this plasmid represents a burden for B. methanolicus when growing on mannitol. Our data contribute to a new and fundamental understanding of the regulation of B. methanolicus methylotrophy.

Plasmid-dependent methylotrophy in thermotolerant Bacillus methanolicus.

PubMed

Brautaset, Trygve; Jakobsen M, Øyvind M; Flickinger, Michael C; Valla, Svein; Ellingsen, Trond E

2004-03-01

Bacillus methanolicus can efficiently utilize methanol as a sole carbon source and has an optimum growth temperature of 50 degrees C. With the exception of mannitol, no sugars have been reported to support rapid growth of this organism, which is classified as a restrictive methylotroph. Here we describe the DNA sequence and characterization of a 19,167-bp circular plasmid, designated pBM19, isolated from B. methanolicus MGA3. Sequence analysis of pBM19 demonstrated the presence of the methanol dehydrogenase gene, mdh, which is crucial for methanol consumption in this bacterium. In addition, five genes (pfk, encoding phosphofructokinase; rpe, encoding ribulose-5-phosphate 3-epimerase; tkt, encoding transketolase; glpX, encoding fructose-1,6-bisphosphatase; and fba, encoding fructose-1,6-bisphosphate aldolase) with deduced roles in methanol assimilation via the ribulose monophosphate pathway are encoded by pBM19. A shuttle vector, pTB1.9, harboring the pBM19 minimal replicon (repB and ori) was constructed and used to transform MGA3. Analysis of the resulting recombinant strain demonstrated that it was cured of pBM19 and was not able to grow on methanol. A pTB1.9 derivative harboring the complete mdh gene could not restore growth on methanol when it was introduced into the pBM19-cured strain, suggesting that additional pBM19 genes are required for consumption of this carbon source. Screening of 13 thermotolerant B. methanolicus wild-type strains showed that they all harbor plasmids similar to pBM19, and this is the first report describing plasmid-linked methylotrophy in any microorganism. Our findings should have an effect on future genetic manipulations of this organism, and they contribute to a new understanding of the biology of methylotrophs.

Plasmid-Dependent Methylotrophy in Thermotolerant Bacillus methanolicus

PubMed Central

Brautaset, Trygve; Jakobsen, Øyvind M.; Flickinger, Michael C.; Valla, Svein; Ellingsen, Trond E.

2004-01-01

Bacillus methanolicus can efficiently utilize methanol as a sole carbon source and has an optimum growth temperature of 50°C. With the exception of mannitol, no sugars have been reported to support rapid growth of this organism, which is classified as a restrictive methylotroph. Here we describe the DNA sequence and characterization of a 19,167-bp circular plasmid, designated pBM19, isolated from B. methanolicus MGA3. Sequence analysis of pBM19 demonstrated the presence of the methanol dehydrogenase gene, mdh, which is crucial for methanol consumption in this bacterium. In addition, five genes (pfk, encoding phosphofructokinase; rpe, encoding ribulose-5-phosphate 3-epimerase; tkt, encoding transketolase; glpX, encoding fructose-1,6-bisphosphatase; and fba, encoding fructose-1,6-bisphosphate aldolase) with deduced roles in methanol assimilation via the ribulose monophosphate pathway are encoded by pBM19. A shuttle vector, pTB1.9, harboring the pBM19 minimal replicon (repB and ori) was constructed and used to transform MGA3. Analysis of the resulting recombinant strain demonstrated that it was cured of pBM19 and was not able to grow on methanol. A pTB1.9 derivative harboring the complete mdh gene could not restore growth on methanol when it was introduced into the pBM19-cured strain, suggesting that additional pBM19 genes are required for consumption of this carbon source. Screening of 13 thermotolerant B. methanolicus wild-type strains showed that they all harbor plasmids similar to pBM19, and this is the first report describing plasmid-linked methylotrophy in any microorganism. Our findings should have an effect on future genetic manipulations of this organism, and they contribute to a new understanding of the biology of methylotrophs. PMID:14973041

Quantitative proteome analysis of barley seeds using ruthenium(II)-tris-(bathophenanthroline-disulphonate) staining.

PubMed

Witzel, Katja; Surabhi, Giridara-Kumar; Jyothsnakumari, Gottimukkala; Sudhakar, Chinta; Matros, Andrea; Mock, Hans-Peter

2007-04-01

This paper describes the application of the recently introduced fluorescence stain Ruthenium(II)-tris-(bathophenanthroline-disulphonate) (RuBP) on a comparative proteome analysis of two phenotypically different barley lines. We carried out an analysis of protein patterns from 2-D gels of the parental lines of the Oregon Wolfe Barley mapping population DOM and REC and stained with either the conventional colloidal Coomassie Brilliant Blue (cCBB) or with the novel RuBP solution. We wished to experimentally verify the usefulness of such a stain in evaluating the complex pattern of a seed proteome, in comparison to the previously used cCBB staining technique. To validate the efficiency of visualization by both stains, we first compared the overall number of detected protein spots. On average, 790 spots were visible by cCBB staining and 1200 spots by RuBP staining. Then, the intensity of a set of spots was assessed, and changes in relative abundance were determined using image analysis software. As expected, staining with RuBP performed better in quantitation in terms of sensitivity and dynamic range. Furthermore, spots from a cultivar-specific region in the protein map were chosen for identification to asses the gain of biological information due to the staining procedure. From this particular region, eight spots were visualized exclusively by RuBP and identification was successful for all spots, proving the ability to identify even very low abundant proteins. Performance in MS analysis was comparable for both protein stains. Proteins were identified by MALDI-TOF MS peptide mass fingerprinting. This approach was not successful for all spots, due to the restricted entry number for barley in the database. Therefore, we subsequently used LC-ESI-Q-TOF MS/MS and de novo sequencing for identification. Because only an insufficient number of proteins from barley is annotated, an EST-based identification strategy was chosen for our experiment. We wished to test whether under these limitations the application of a more sensitive stain would lead to a more advanced proteome approach. In summary, we demonstrate here that the application of RuBP as an economical but reliable and sensitive fluorescence stain is highly suitable for quantitative proteome analysis of plant seeds.

Chemiluminescence reactions with cationic, neutral, and anionic ruthenium(II) complexes containing 2,2'-bipyridine and bathophenanthroline disulfonate ligands.

PubMed

Francis, Paul S; Papettas, Dimitra; Zammit, Elizabeth M; Barnett, Neil W

2010-07-15

Ruthenium complexes containing 4,7-diphenyl-1,10-phenanthroline disulfonate (bathophenanthroline disulfonate; BPS) ligands, Ru(BPS)(3)(4-), Ru(BPS)(2)(bipy)(2-) and Ru(BPS)(bipy)(2), were compared to tris(2,2'-bipyridine)ruthenium(II) (Ru(bipy)(3)(2+)), including examination of the wavelengths of maximum absorption and corrected emission intensity, photoluminescence quantum yield, stability of their oxidised ruthenium(III) form, and relative chemiluminescence intensities and signal-to-blank ratios with cerium(IV) sulfate and six analytes (codeine, morphine cocaine, potassium oxalate, furosemide and hydrochlorothiazide) in acidic aqueous solution. The presence of BPS ligands in the complex increased the photoluminescence quantum yield, but decreased the stability of the oxidised form of the reagent. In contrast to previous evidence showing much greater electrochemiluminescence intensities using Ru(BPS)(2)(bipy)(2-) and Ru(BPS)(bipy)(2), these complexes did not provide superior chemiluminescence signals than their homoleptic analogues. Copyright 2010 Elsevier B.V. All rights reserved.

Singlet oxygen-dependent translational control in the tigrina-d.12 mutant of barley.

PubMed

Khandal, Dhriti; Samol, Iga; Buhr, Frank; Pollmann, Stephan; Schmidt, Holger; Clemens, Stephan; Reinbothe, Steffen; Reinbothe, Christiane

2009-08-04

The tigrina (tig)-d.12 mutant of barley is impaired in the negative control limiting excess protochlorophyllide (Pchlide) accumulation in the dark. Upon illumination, Pchlide operates as photosensitizer and triggers singlet oxygen production and cell death. Here, we show that both Pchlide and singlet oxygen operate as signals that control gene expression and metabolite accumulation in tig-d.12 plants. In vivo labeling, Northern blotting, polysome profiling, and protein gel blot analyses revealed a selective suppression of synthesis of the small and large subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RBCSs and RBCLs), the major light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCB2), as well as other chlorophyll-binding proteins, in response to singlet oxygen. In part, these effects were caused by an arrest in translation initiation of photosynthetic transcripts at 80S cytoplasmic ribosomes. The observed changes in translation correlated with a decline in the phosphorylation level of ribosomal protein S6. At later stages, ribosome dissociation occurred. Together, our results identify translation as a major target of singlet oxygen-dependent growth control and cell death in higher plants.

The Structure of Isolated Synechococcus Strain WH8102 Carboxysomes as Revealed by Electron Cryotomography

PubMed Central

Iancu, Cristina V.; Ding, H. Jane; Morris, Dylan M.; Dias, D. Prabha; Gonzales, Arlene D.; Martino, Anthony; Jensen, Grant J.

2007-01-01

Carboxysomes are organelle-like polyhedral bodies found in cyanobacteria and many chemoautotrophic bacteria that are thought to facilitate carbon fixation. Carboxysomes are bounded by a proteinaceous outer shell and filled with ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the first enzyme in the CO2 fixation pathway, but exactly how they enhance carbon fixation is unclear. Here we report the three-dimensional structure of purified carboxysomes from Synechococcus species strain WH8102 as revealed by electron cryotomography. We found that while the sizes of individual carboxysomes in this organism varied from 114 to 137 nm, surprisingly, all were approximately icosahedral. There were on average ∼250 RuBisCOs per carboxysome, organized into 3-4 concentric layers. Some models of carboxysome function depend on specific contacts between individual RuBisCOs and the shell, but no evidence of such contacts was found: no systematic patterns of connecting densities or RuBisCO positions against the shell's presumed hexagonal lattice could be discerned, and simulations showed that packing forces alone could account for the layered organization of RuBisCOs. PMID:17669419

Modified rubisco large subunit n-methyltransferase useful for targeting molecules to the active-site vicinity of ribulose-1,5-bisphosphate

DOEpatents

Houtz, Robert L [Lexington, KY

2012-03-20

The present invention generally relates to a modified Rubisco large subunit .sup..epsilon.N-Methyltransferase (Rubisco LSMT, or RLSMT). The present invention also relates to a modified RLSMT-carbonic anhydrase (RLSMT-CA). This modified RLSMT-CA improves the efficiency of the reduction of CO.sub.2 during photosynthesis, which may increase plant growth rates. The present invention also relates to nucleic acids encoding the modified RLSMT-CA or modified RLSMT. Also, the present invention relates to cells including the modified RLSMT-CA or modified RLSMT, plants containing the modified RLSMT-CA or modified RLSMT, and methods using compositions of the present invention. In addition, the present invention relates to antibodies conjugated to CA which may bind to Rubisco, and antibodies which bind a modified RLSMT-CA. The invention also relates to modified forms of the LS and SS of Rubisco where the modified forms are fusions with CA or biologically active fragments thereof. The present invention provides methods of altering Rubisco carboxylase activity and altering plant growth.

The coupling of glycolysis and the Rubisco-based pathway through the non-oxidative pentose phosphate pathway to achieve low carbon dioxide emission fermentation.

PubMed

Li, Ya-Han; Ou-Yang, Fan-Yu; Yang, Cheng-Han; Li, Si-Yu

2015-01-01

In this study, Rubisco-based engineered Escherichia coli, containing two heterologous enzymes of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoribulokinase (PrkA), has been shown to be capable of the in situ recycling of carbon dioxide (CO2) during glycolysis. Two alternative approaches have been proposed to further enhance the carbon flow from glycolysis to a Rubisco-based pathway through the non-oxidative pentose phosphate pathway (NOPPP). The first is achieved by elevating the expression of transketolase I (TktA) and the second by blocking the native oxidation-decarboxylation reaction of E. coli by deleting the zwf gene from the chromosome (designated as JB/pTA and MZB, respectively). Decreases in the CO2 yield and the CO2 evolution per unit mole of ethanol production by at least 81% and 40% are observed. It is demonstrated in this study that the production of one mole of ethanol using E. coli strain MZB, the upper limit of CO2 emission is 0.052mol. Copyright © 2015 Elsevier Ltd. All rights reserved.

Metabolic Adaptation in Transplastomic Plants Massively Accumulating Recombinant Proteins

PubMed Central

Bally, Julia; Job, Claudette; Belghazi, Maya; Job, Dominique

2011-01-01

Background Recombinant chloroplasts are endowed with an astonishing capacity to accumulate foreign proteins. However, knowledge about the impact on resident proteins of such high levels of recombinant protein accumulation is lacking. Methodology/Principal Findings Here we used proteomics to characterize tobacco (Nicotiana tabacum) plastid transformants massively accumulating a p-hydroxyphenyl pyruvate dioxygenase (HPPD) or a green fluorescent protein (GFP). While under the conditions used no obvious modifications in plant phenotype could be observed, these proteins accumulated to even higher levels than ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the most abundant protein on the planet. This accumulation occurred at the expense of a limited number of leaf proteins including Rubisco. In particular, enzymes involved in CO2 metabolism such as nuclear-encoded plastidial Calvin cycle enzymes and mitochondrial glycine decarboxylase were found to adjust their accumulation level to these novel physiological conditions. Conclusions/Significance The results document how protein synthetic capacity is limited in plant cells. They may provide new avenues to evaluate possible bottlenecks in recombinant protein technology and to maintain plant fitness in future studies aiming at producing recombinant proteins of interest through chloroplast transformation. PMID:21966485

Understanding the molecular basis of plant growth promotional effect of Pseudomonas fluorescens on rice through protein profiling.

PubMed

Kandasamy, Saveetha; Loganathan, Karthiba; Muthuraj, Raveendran; Duraisamy, Saravanakumar; Seetharaman, Suresh; Thiruvengadam, Raguchander; Ponnusamy, Balasubramanian; Ramasamy, Samiyappan

2009-12-24

Plant Growth Promoting Rhizobacteria (PGPR), Pseudomonas fluorescens strain KH-1 was found to exhibit plant growth promotional activity in rice under both in-vitro and in-vivo conditions. But the mechanism underlying such promotional activity of P. fluorescens is not yet understood clearly. In this study, efforts were made to elucidate the molecular responses of rice plants to P. fluorescens treatment through protein profiling. Two-dimensional polyacrylamide gel electrophoresis strategy was adopted to identify the PGPR responsive proteins and the differentially expressed proteins were analyzed by mass spectrometry. Priming of P. fluorescens, 23 different proteins found to be differentially expressed in rice leaf sheaths and MS analysis revealed the differential expression of some important proteins namely putative p23 co-chaperone, Thioredoxin h- rice, Ribulose-bisphosphate carboxylase large chain precursor, Nucleotide diPhosphate kinase, Proteosome sub unit protein and putative glutathione S-transferase protein. Functional analyses of the differential proteins were reported to be directly or indirectly involved in growth promotion in plants. Thus, this study confirms the primary role of PGPR strain KH-1 in rice plant growth promotion.

Production and characterization of biodiesel from carbon dioxide concentrating chemolithotrophic bacteria, Serratia sp. ISTD04.

PubMed

Bharti, Randhir K; Srivastava, Shaili; Thakur, Indu Shekhar

2014-02-01

A chemolithotrophic bacterium, Serratia sp. ISTD04, enriched in the chemostat in presence of sodium bicarbonate as sole carbon source was evaluated for potential of carbon dioxide (CO2) sequestration and biofuel production. CO2 sequestration efficiency of the bacterium was determined by enzymatic activity of carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Further, Western blot analysis confirmed presence of RuBisCO. The bacterium produced 0.487 and 0.647mgmg(-1) per unit cell dry weight of hydrocarbons and lipids respectively. The hydrocarbons were within the range of C13-C24 making it equivalent to light oil. GC-MS analysis of lipids produced by the bacterium indicated presence of C15-C20 organic compounds that made it potential source of biodiesel after transesterification. GC-MS, FTIR and NMR spectroscopic characterization of the fatty acid methyl esters revealed the presence of 55% and 45% of unsaturated and saturated organic compounds respectively, thus making it a balanced biodiesel composition. Copyright © 2013 Elsevier Ltd. All rights reserved.

Altered invertase activities of symptomatic tissues on Beet severe curly top virus (BSCTV) infected Arabidopsis thaliana.

PubMed

Park, Jungan; Kim, Soyeon; Choi, Eunseok; Auh, Chung-Kyun; Park, Jong-Bum; Kim, Dong-Giun; Chung, Young-Jae; Lee, Taek-Kyun; Lee, Sukchan

2013-09-01

Arabidopsis thaliana infected with Beet severe curly top virus (BSCTV) exhibits systemic symptoms such as stunting of plant growth, callus induction on shoot tips, and curling of leaves and shoot tips. The regulation of sucrose metabolism is essential for obtaining the energy required for viral replication and the development of symptoms in BSCTV-infected A. thaliana. We evaluated the changed transcript level and enzyme activity of invertases in the inflorescence stems of BSCTV-infected A. thaliana. These results were consistent with the increased pattern of ribulose-1,5-bisphosphate carboxylase/oxygenase activity and photosynthetic pigment concentration in virus-infected plants to supply more energy for BSCTV multiplication. The altered gene expression of invertases during symptom development was functionally correlated with the differential expression patterns of D-type cyclins, E2F isoforms, and invertase-related genes. Taken together, our results indicate that sucrose sensing by BSCTV infection may regulate the expression of sucrose metabolism and result in the subsequent development of viral symptoms in relation with activation of cell cycle regulation.

An Arabidopsis mutant showing reduced feedback inhibition of photosynthesis.

PubMed

Van Oosten, J J; Gerbaud, A; Huijser, C; Dijkwel, P P; Chua, N H; Smeekens, S C

1997-11-01

Many plant genes are responsive to sugars but the mechanisms used by plants to sense sugars are unknown. A genetic approach has been used in Arabidopsis to identify genes involved in perception and transduction of sugar signals. For this purpose, an in vivo reporter system was established consisting of the light- and sugar-regulated plastocyanin promoter, fused to the luciferase coding sequence (PC-LUC construct). At the seedling stage, expression of the PC-LUC gene is repressed by sucrose, and a number of sucrose-uncoupled (sun) mutants were selected in which sucrose is unable to repress the activity of the PC promoter. Three mutants have been characterized in more detail. The sugar analog 2-deoxy-D-glucose (2DG) was used to repress whole plant photosynthesis, PC-LUC gene expression and total ribulose-1,5-bisphosphate activity. It was found that the sun6 mutation makes plants unresponsive to these 2DG-induced effects. Moreover, unlike wild-type plants, sun6 mutants are insensitive to elevated levels of glucose in the growth medium. These findings suggest that the SUN6 gene is active in a hexose-activated signal transduction pathway.

Effect of ethephon on protein degradation and the accumulation of pathogensis-related (PR) proteins in tomato leaf discs. [Lycopersicon esculentum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vera, P.; Conejero, V.

The effect of ethephon (2-chloroetylphosphonic acid) on the degradation of proteins and on the induction of Lycopersicon esculentum pathogenesis-related (PR) proteins was studied in tomato leaf discs. The rate of ribulose, -1,5-bisphosphate carboxylase/oxygenase (Rubisco) degradation was maximal in discs after 48 hours of incubation with 1 millimolar ethephon, leading to complete disappearance of Rubisco after 96 hours. This effect was correlated with an increase in PR protein synthesis and the induction of the previously reported alkaline proteolytic enzyme PR-P69. In vivo pulse-chase experiments demonstrated that ethephon not only affected Rubisco content but that of many other {sup 35}S-labeled proteins asmore » well, indicating that ethylene activates a general and nonspecific mechanism of protein degradation. This effect was partially inhibited in vivo by the action of pCMB, a selective inhibitor of cysteine-proteinases such as P69. These data reinforce the hypothesis that P69 and perhaps other PR proteins are involved in the mechanism of accelerated protein degradation activated by ethylene.« less

Protein changes in Lepidium sativum L. exposed to Hg during soil phytoremediation.

PubMed

Smolinska, Beata; Szczodrowska, Agnieszka; Leszczynska, Joanna

2017-08-03

Some investigations have been carried out in this study to find the best technique of soil reclamation in mercurypolluted soil. In this study, we examined Lepidium sativum L. as a plant useful for Hg phytoextraction. The simultaneous application of compost and thiosulfate was explored as a possible method of enhancing the process of phytoextraction. The results of the investigations of plant protein changes during assisted Hg phytoextraction were also provided. The results of the study show that combined use of compost and thiosulfate significantly increased both the total Hg accumulation and its translocation to aerial plant tissues. Plant protein analysis showed that L. sativum L. has the ability to respond to environmental stress condition by the activation of additional proteins. The additional proteins, like homocysteine methyltransferase, ribulose bisphosphate carboxylases (long and short chains), 14-3-3-like protein, and biosynthesis-related 40S ribosomal protein S15, were activated in plant shoots only in experiments carried out in Hg-polluted soil. There were no protein changes observed in plants exposed to compost and thiosulfate. It suggests that the combined use of compost and thiosulfate decreased Hg toxicity.

Respiration accumulates Calvin cycle intermediates for the rapid start of photosynthesis in Synechocystis sp. PCC 6803.

PubMed

Shimakawa, Ginga; Hasunuma, Tomohisa; Kondo, Akihiko; Matsuda, Mami; Makino, Amane; Miyake, Chikahiro

2014-01-01

We tested the hypothesis that inducing photosynthesis in cyanobacteria requires respiration. A mutant deficient in glycogen phosphorylase (∆GlgP) was prepared in Synechocystis sp. PCC 6803 to suppress respiration. The accumulated glycogen in ΔGlgP was 250-450% of that accumulated in wild type (WT). The rate of dark respiration in ΔGlgP was 25% of that in WT. In the dark, P700(+) reduction was suppressed in ΔGlgP, and the rate corresponded to that in (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone)-treated WT, supporting a lower respiration rate in ∆GlgP. Photosynthetic O2-evolution rate reached a steady-state value much slower in ∆GlgP than in WT. This retardation was solved by addition of d-glucose. Furthermore, we found that the contents of Calvin cycle intermediates in ∆GlgP were lower than those in WT under dark conditions. These observations indicated that respiration provided the carbon source for regeneration of ribulose 1,5-bisphosphate in order to drive the rapid start of photosynthesis.

Protein profiling of single epidermal cell types from Arabidopsis thaliana using surface-enhanced laser desorption and ionization technology.

PubMed

Ebert, Berit; Melle, Christian; Lieckfeldt, Elke; Zöller, Daniela; von Eggeling, Ferdinand; Fisahn, Joachim

2008-08-25

Here, we describe a novel approach for investigating differential protein expression within three epidermal cell types. In particular, 3000 single pavement, basal, and trichome cells from leaves of Arabidopsis thaliana were harvested by glass micro-capillaries. Subsequently, these single cell samples were joined to form pools of 100 individual cells and analyzed using the ProteinChip technology; SELDI: surface-enhanced laser desorption and ionization. As a result, numerous protein signals that were differentially expressed in the three epidermal cell types could be detected. One of these proteins was characterized by tryptical digestion and subsequent identification via tandem quadrupole-time of flight (Q-TOF) mass spectrometry. Down regulation of this sequenced small subunit precursor of ribulose-1,5 bisphosphate carboxylase(C) oxygenase(O) (RuBisCo) in trichome and basal cells indicates the sink status of these cell types that are located on the surface of A. thaliana source leaves. Based on the obtained protein profiles, we suggest a close functional relationship between basal and trichome cells at the protein level.

rbcL gene sequences provide evidence for the evolutionary lineages of leptosporangiate ferns.

PubMed

Hasebe, M; Omori, T; Nakazawa, M; Sano, T; Kato, M; Iwatsuki, K

1994-06-07

Pteriodophytes have a longer evolutionary history than any other vascular land plant and, therefore, have endured greater loss of phylogenetically informative information. This factor has resulted in substantial disagreements in evaluating characters and, thus, controversy in establishing a stable classification. To compare competing classifications, we obtained DNA sequences of a chloroplast gene. The sequence of 1206 nt of the large subunit of the ribulose-bisphosphate carboxylase gene (rbcL) was determined from 58 species, representing almost all families of leptosporangiate ferns. Phlogenetic trees were inferred by the neighbor-joining and the parsimony methods. The two methods produced almost identical phylogenetic trees that provided insights concerning major general evolutionary trends in the leptosporangiate ferns. Interesting findings were as follows: (i) two morphologically distinct heterosporous water ferns, Marsilea and Salvinia, are sister genera; (ii) the tree ferns (Cyatheaceae, Dicksoniaceae, and Metaxyaceae) are monophyletic; and (iii) polypodioids are distantly related to the gleichenioids in spite of the similarity of their exindusiate soral morphology and are close to the higher indusiate ferns. In addition, the affinities of several "problematic genera" were assessed.

Determination of proteins induced in response to jasmonic acid and salicylic acid in resistant and susceptible cultivars of tomato.

PubMed

Afroz, Amber; Khan, Muhammad Rashid; Komatsu, Setsuko

2010-07-01

Jasmonic acid (JA) and salicylic acid (SA) are signaling molecules that play key roles in the regulation of metabolic processes, reproduction, and defense against pathogens. The proteomics approach was used to identify proteins that are induced by JA and SA in the tomato cultivars Roma and Pant Bahr, which are susceptible and resistant to bacterial wilt, respectively. Threonine deaminase and leucine amino peptidase were upregulated, and ribulose-1,5-bisphosphate carboxylase/oxygenase small chain was downregulated by time-course application of JA. Translationally controlled tumor protein was upregulated by time-course application of SA. Protein disulfide isomerase was upregulated by application of either JA or SA. Proteins related to defense, energy, and protein destination/storage are suspected to be responsible for the susceptibility or resistance of the cultivars. Furthermore, in Roma, iron ABC transporter was upregulated by JA and down-regulated by SA. Iron ABC transporter plays a part in the signal transduction of both JA and SA in cultivars of tomato that are resistant to bacterial wilt.

Structure of Arabidopsis thaliana Rubisco activase.

PubMed

Hasse, Dirk; Larsson, Anna M; Andersson, Inger

2015-04-01

The CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is inactivated by the formation of dead-end complexes with inhibitory sugar phosphates. In plants and green algae, the ATP-dependent motor protein Rubisco activase restores catalytic competence by facilitating conformational changes in Rubisco that promote the release of the inhibitory compounds from the active site. Here, the crystal structure of Rubisco activase from Arabidopsis thaliana is presented at 2.9 Å resolution. The structure reveals an AAA+ two-domain structure. More than 100 residues in the protein were not visible in the electron-density map owing to conformational disorder, but were verified to be present in the crystal by mass spectrometry. Two sulfate ions were found in the structure. One was bound in the loop formed by the Walker A motif at the interface of the domains. A second sulfate ion was bound at the N-terminal end of the first helix of the C-terminal domain. The protein packs in a helical fashion in the crystal, as observed previously for Rubisco activase, but differences in the helical pitch indicate flexibility in the packing of the protein.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Imker,H.; Fedorov, A.; Fedorov, E.

D-Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant enzyme, is the paradigm member of the recently recognized mechanistically diverse RuBisCO superfamily. The RuBisCO reaction is initiated by abstraction of the proton from C3 of the D-ribulose 1,5-bisphosphate substrate by a carbamate oxygen of carboxylated Lys 201 (spinach enzyme). Heterofunctional homologues of RuBisCO found in species of Bacilli catalyze the tautomerization ('enolization') of 2,3-diketo-5-methylthiopentane 1-phosphate (DK-MTP 1-P) in the methionine salvage pathway in which 5-methylthio-D-ribose (MTR) derived from 5'-methylthioadenosine is converted to methionine [Ashida, H., Saito, Y., Kojima, C., Kobayashi, K., Ogasawara, N., and Yokota, A. (2003) A functional link between RuBisCO-likemore » protein of Bacillus and photosynthetic RuBisCO, Science 302, 286-290]. The reaction catalyzed by this 'enolase' is accomplished by abstraction of a proton from C1 of the DK-MTP 1-P substrate to form the tautomerized product, a conjugated enol. Because the RuBisCO- and 'enolase'-catalyzed reactions differ in the regiochemistry of proton abstraction but are expected to share stabilization of an enolate anion intermediate by coordination to an active site Mg{sup 2+}, we sought to establish structure-function relationships for the 'enolase' reaction so that the structural basis for the functional diversity could be established. We determined the stereochemical course of the reaction catalyzed by the 'enolases' from Bacillus subtilis and Geobacillus kaustophilus. Using stereospecifically deuterated samples of an alternate substrate derived from D-ribose (5-OH group instead of the 5-methylthio group in MTR) as well as of the natural DK-MTP 1-P substrate, we determined that the 'enolase'-catalyzed reaction involves abstraction of the 1-proS proton. We also determined the structure of the activated 'enolase' from G. kaustophilus (carboxylated on Lys 173) liganded with Mg{sup 2+} and 2,3-diketohexane 1-phosphate, a stable alternate substrate. The stereospecificity of proton abstraction restricts the location of the general base to the N-terminal {alpha}+ {beta} domain instead of the C-terminal ({beta}/{alpha}){sub 8}-barrel domain that contains the carboxylated Lys 173. Lys 98 in the N-terminal domain, conserved in all 'enolases', is positioned to abstract the 1-proS proton. Consistent with this proposed function, the K98A mutant of the G. kaustophilus 'enolase' is unable to catalyze the 'enolase' reaction. Thus, we conclude that this functionally divergent member of the RuBisCO superfamily uses the same structural strategy as RuBisCO for stabilizing the enolate anion intermediate, i.e., coordination to an essential Mg{sup 2+}, but the proton abstraction is catalyzed by a different general base.« less

Over-expressing the C3 photosynthesis cycle enzyme Sedoheptulose-1-7 Bisphosphatase improves photosynthetic carbon gain and yield under fully open air CO2 fumigation (FACE)

PubMed Central

2011-01-01

Background Biochemical models predict that photosynthesis in C3 plants is most frequently limited by the slower of two processes, the maximum capacity of the enzyme Rubisco to carboxylate RuBP (Vc,max), or the regeneration of RuBP via electron transport (J). At current atmospheric [CO2] levels Rubisco is not saturated; consequently, elevating [CO2] increases the velocity of carboxylation and inhibits the competing oxygenation reaction which is also catalyzed by Rubisco. In the future, leaf photosynthesis (A) should be increasingly limited by RuBP regeneration, as [CO2] is predicted to exceed 550 ppm by 2050. The C3 cycle enzyme sedoheptulose-1,7 bisphosphatase (SBPase, EC 3.1.3.17) has been shown to exert strong metabolic control over RuBP regeneration at light saturation. Results We tested the hypothesis that tobacco transformed to overexpressing SBPase will exhibit greater stimulation of A than wild type (WT) tobacco when grown under field conditions at elevated [CO2] (585 ppm) under fully open air fumigation. Growth under elevated [CO2] stimulated instantaneous A and the diurnal photosynthetic integral (A') more in transformants than WT. There was evidence of photosynthetic acclimation to elevated [CO2] via downregulation of Vc,max in both WT and transformants. Nevertheless, greater carbon assimilation and electron transport rates (J and Jmax) for transformants led to greater yield increases than WT at elevated [CO2] compared to ambient grown plants. Conclusion These results provide proof of concept that increasing content and activity of a single photosynthesis enzyme can enhance carbon assimilation and yield of C3 crops grown at [CO2] expected by the middle of the 21st century. PMID:21884586

Quantification of growth-defense trade-offs in a common currency: nitrogen required for phenolamide biosynthesis is not derived from ribulose-1,5-bisphosphate carboxylase/oxygenase turnover.

PubMed

Ullmann-Zeunert, Lynn; Stanton, Mariana A; Wielsch, Nathalie; Bartram, Stefan; Hummert, Christian; Svatoš, Aleš; Baldwin, Ian T; Groten, Karin

2013-08-01

Induced defenses are thought to be economical: growth and fitness-limiting resources are only invested into defenses when needed. To date, this putative growth-defense trade-off has not been quantified in a common currency at the level of individual compounds. Here, a quantification method for ¹⁵N-labeled proteins enabled a direct comparison of nitrogen (N) allocation to proteins, specifically, ribulose-1,5-bisposphate carboxylase/oxygenase (RuBisCO), as proxy for growth, with that to small N-containing defense metabolites (nicotine and phenolamides), as proxies for defense after herbivory. After repeated simulated herbivory, total N decreased in the shoots of wild-type (WT) Nicotiana attenuata plants, but not in two transgenic lines impaired in jasmonate defense signaling (irLOX3) and phenolamide biosynthesis (irMYB8). N was reallocated among different compounds within elicited rosette leaves: in the WT, a strong decrease in total soluble protein (TSP) and RuBisCO was accompanied by an increase in defense metabolites, irLOX3 showed a similar, albeit attenuated, pattern, whereas irMYB8 rosette leaves were the least responsive to elicitation, with overall higher levels of RuBisCO. Induced defenses were higher in the older compared with the younger rosette leaves, supporting the hypothesis that tissue developmental stage influences defense investments. We propose that MYB8, probably by regulating the production of phenolamides, indirectly mediates protein pool sizes after herbivory. Although the decrease in absolute N invested in TSP and RuBisCO elicited by simulated herbivory was much larger than the N-requirements of nicotine and phenolamide biosynthesis, ¹⁵N flux studies revealed that N for phenolamide synthesis originates from recently assimilated N, rather than from RuBisCO turnover. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Characterization of two transketolases encoded on the chromosome and the plasmid pBM19 of the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus

PubMed Central

2014-01-01

Background Transketolase (TKT) is a key enzyme of the pentose phosphate pathway (PPP), the Calvin cycle and the ribulose monophosphate (RuMP) cycle. Bacillus methanolicus is a facultative RuMP pathway methylotroph. B. methanolicus MGA3 harbors two genes putatively coding for TKTs; one located on the chromosome (tkt C ) and one located on the natural occurring plasmid pBM19 (tkt P ). Results Both enzymes were produced in recombinant Escherichia coli, purified and shown to share similar biochemical parameters in vitro. They were found to be active as homotetramers and require thiamine pyrophosphate for catalytic activity. The inactive apoform of the TKTs, yielded by dialysis against buffer containing 10 mM EDTA, could be reconstituted most efficiently with Mn2+ and Mg2+. Both TKTs were thermo stable at physiological temperature (up to 65°C) with the highest activity at neutral pH. Ni2+, ATP and ADP significantly inhibited activity of both TKTs. Unlike the recently characterized RuMP pathway enzymes fructose 1,6-bisphosphate aldolase (FBA) and fructose 1,6-bisphosphatase/sedoheptulose 1,7-bisphosphatase (FBPase/SBPase) from B. methanolicus MGA3, both TKTs exhibited similar kinetic parameters although they only share 76% identical amino acids. The kinetic parameters were determined for the reaction with the substrates xylulose 5-phosphate (TKTC: kcat/KM: 264 s-1 mM-1; TKTP: kcat/KM: 231 s-1 mM) and ribulose 5-phosphate (TKTC: kcat/KM: 109 s-1 mM; TKTP: kcat/KM: 84 s-1 mM) as well as for the reaction with the substrates glyceraldehyde 3-phosphate (TKTC: kcat/KM: 108 s-1 mM; TKTP: kcat/KM: 71 s-1 mM) and fructose 6-phosphate (TKTC kcat/KM: 115 s-1 mM; TKTP: kcat/KM: 448 s-1 mM). Conclusions Based on the kinetic parameters no major TKT of B. methanolicus could be determined. Increased expression of tkt P , but not of tkt C during growth with methanol [J Bacteriol 188:3063–3072, 2006] argues for TKTP being the major TKT relevant in the RuMP pathway. Neither TKT exhibited activity as dihydroxyacetone synthase, as found in methylotrophic yeast, or as the evolutionary related 1-deoxyxylulose-5-phosphate synthase. The biological significance of the two TKTs for B. methanolicus methylotrophy is discussed. PMID:24405865

Characterization of two transketolases encoded on the chromosome and the plasmid pBM19 of the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus.

PubMed

Markert, Benno; Stolzenberger, Jessica; Brautaset, Trygve; Wendisch, Volker F

2014-01-09

Transketolase (TKT) is a key enzyme of the pentose phosphate pathway (PPP), the Calvin cycle and the ribulose monophosphate (RuMP) cycle. Bacillus methanolicus is a facultative RuMP pathway methylotroph. B. methanolicus MGA3 harbors two genes putatively coding for TKTs; one located on the chromosome (tkt(C)) and one located on the natural occurring plasmid pBM19 (tkt(P)). Both enzymes were produced in recombinant Escherichia coli, purified and shown to share similar biochemical parameters in vitro. They were found to be active as homotetramers and require thiamine pyrophosphate for catalytic activity. The inactive apoform of the TKTs, yielded by dialysis against buffer containing 10 mM EDTA, could be reconstituted most efficiently with Mn(2+) and Mg(2+). Both TKTs were thermo stable at physiological temperature (up to 65°C) with the highest activity at neutral pH. Ni(2+), ATP and ADP significantly inhibited activity of both TKTs. Unlike the recently characterized RuMP pathway enzymes fructose 1,6-bisphosphate aldolase (FBA) and fructose 1,6-bisphosphatase/sedoheptulose 1,7-bisphosphatase (FBPase/SBPase) from B. methanolicus MGA3, both TKTs exhibited similar kinetic parameters although they only share 76% identical amino acids. The kinetic parameters were determined for the reaction with the substrates xylulose 5-phosphate (TKT(C): kcat/KM: 264 s(-1) mM(-1); TKT(P): kcat/KM: 231 s(-1) mM) and ribulose 5-phosphate (TKT(C): kcat/KM: 109 s(-1) mM; TKT(P): kcat/KM: 84 s(-1) mM) as well as for the reaction with the substrates glyceraldehyde 3-phosphate (TKT(C): kcat/KM: 108 s(-1) mM; TKT(P): kcat/KM: 71 s(-1) mM) and fructose 6-phosphate (TKT(C) kcat/KM: 115 s(-1) mM; TKT(P): kcat/KM: 448 s(-1) mM). Based on the kinetic parameters no major TKT of B. methanolicus could be determined. Increased expression of tkt(P), but not of tkt(C) during growth with methanol [J Bacteriol 188:3063-3072, 2006] argues for TKT(P) being the major TKT relevant in the RuMP pathway. Neither TKT exhibited activity as dihydroxyacetone synthase, as found in methylotrophic yeast, or as the evolutionary related 1-deoxyxylulose-5-phosphate synthase. The biological significance of the two TKTs for B. methanolicus methylotrophy is discussed.

Rice proteome database: a step toward functional analysis of the rice genome.

PubMed

Komatsu, Setsuko

2005-09-01

The technique of proteome analysis using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has the power to monitor global changes that occur in the protein complement of tissues and subcellular compartments. In this study, the proteins of rice were cataloged, a rice proteome database was constructed, and a functional characterization of some of the identified proteins was undertaken. Proteins extracted from various tissues and subcellular compartments in rice were separated by 2D-PAGE and an image analyzer was used to construct a display of the proteins. The Rice Proteome Database contains 23 reference maps based on 2D-PAGE of proteins from various rice tissues and subcellular compartments. These reference maps comprise 13129 identified proteins, and the amino acid sequences of 5092 proteins are entered in the database. Major proteins involved in growth or stress responses were identified using the proteome approach. Some of these proteins, including a beta-tubulin, calreticulin, and ribulose-1,5-bisphosphate carboxylase/oxygenase activase in rice, have unexpected functions. The information obtained from the Rice Proteome Database will aid in cloning the genes for and predicting the function of unknown proteins.

Metabolic traits of an uncultured archaeal lineage--MSBL1--from brine pools of the Red Sea.

PubMed

Mwirichia, Romano; Alam, Intikhab; Rashid, Mamoon; Vinu, Manikandan; Ba-Alawi, Wail; Anthony Kamau, Allan; Kamanda Ngugi, David; Göker, Markus; Klenk, Hans-Peter; Bajic, Vladimir; Stingl, Ulrich

2016-01-13

The candidate Division MSBL1 (Mediterranean Sea Brine Lakes 1) comprises a monophyletic group of uncultured archaea found in different hypersaline environments. Previous studies propose methanogenesis as the main metabolism. Here, we describe a metabolic reconstruction of MSBL1 based on 32 single-cell amplified genomes from Brine Pools of the Red Sea (Atlantis II, Discovery, Nereus, Erba and Kebrit). Phylogeny based on rRNA genes as well as conserved single copy genes delineates the group as a putative novel lineage of archaea. Our analysis shows that MSBL1 may ferment glucose via the Embden-Meyerhof-Parnas pathway. However, in the absence of organic carbon, carbon dioxide may be fixed via the ribulose bisphosphate carboxylase, Wood-Ljungdahl pathway or reductive TCA cycle. Therefore, based on the occurrence of genes for glycolysis, absence of the core genes found in genomes of all sequenced methanogens and the phylogenetic position, we hypothesize that the MSBL1 are not methanogens, but probably sugar-fermenting organisms capable of autotrophic growth. Such a mixotrophic lifestyle would confer survival advantage (or possibly provide a unique narrow niche) when glucose and other fermentable sugars are not available.

Engineering of chimeric eukaryotic/bacterial Rubisco large subunits in Escherichia coli.

PubMed

Koay, Teng Wei; Wong, Hann Ling; Lim, Boon Hoe

2016-11-26

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a rate-limiting photosynthetic enzyme that catalyzes carbon fixation in the Calvin cycle. Much interest has been devoted to engineering this ubiquitous enzyme with the goal of increasing plant growth. However, experiments that have successfully produced improved Rubisco variants, via directed evolution in Escherichia coli, are limited to bacterial Rubisco because the eukaryotic holoenzyme cannot be produced in E. coli. The present study attempts to determine the specific differences between bacterial and eukaryotic Rubisco large subunit primary structure that are responsible for preventing heterologous eukaryotic holoenzyme formation in E. coli. A series of chimeric Synechococcus Rubiscos were created in which different sections of the large subunit were swapped with those of the homologous Chlamydomonas Rubisco. Chimeric holoenzymes that can form in vivo would indicate that differences within the swapped sections do not disrupt holoenzyme formation. Large subunit residues 1-97, 198-247 and 448-472 were successfully swapped without inhibiting holoenzyme formation. In all ten chimeras, protein expression was observed for the separate subunits at a detectable level. As a first approximation, the regions that can tolerate swapping may be targets for future engineering.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Landry, L.G.; Pell, E.J.

Hybrid poplar clones exposed to ozone exhibit symptoms of accelerated senescence, including early decline in activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). The authors examined the hypothesis that ozone-induced reduction in rubisco occurred as a result of increased protease activity. To test this hypothesis, saplings of Populus maximowizii x trichocarpa were exposed to 0.08 {mu}l/l ozone, 4 h/day, from initiation of sample leaf expansion to foliar abscission. Periodically throughout the treatment the sample leaf was analyzed for chlorophyll content, total protein content, rubisco activity, and proteolytic activity at pH 4.5 and 7.8. At the time of peak rubisco activity, protein was subjectmore » to SDS-PAGE to quantify rubisco. Total protein content of sample leaves was unaffected by ozone treatment. Proteolysis measured under acidic conditions was lower in ozone-treated than control plants throughout the exposure. Proteolysis determined under alkaline conditions only revealed decreases in the second half of the experiment. Ozone induced a more rapid decline in rubisco activity than occurred in control tissue. Quantitative effects of rubisco reflected results of activity assays. It did not appear that enhanced proteolysis could explain the ozone-induced accelerated decline in rubisco.« less

Rubisco activity in Mediterranean species is regulated by the chloroplastic CO2 concentration under water stress

PubMed Central

Galmés, Jeroni; Ribas-Carbó, Miquel; Medrano, Hipólito; Flexas, Jaume

2011-01-01

Water stress decreases the availability of the gaseous substrate for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) by decreasing leaf conductance to CO2. In spite of limiting photosynthetic carbon assimilation, especially in those environments where drought is the predominant factor affecting plant growth and yield, the effects of water deprivation on the mechanisms that control Rubisco activity are unclear. In the present study, 11 Mediterranean species, representing different growth forms, were subject to increasing levels of drought stress, the most severe one followed by rewatering. The results confirmed species-specific patterns in the decrease in the initial activity and activation state of Rubisco as drought stress and leaf dehydration intensified. Nevertheless, all species followed roughly the same trend when Rubisco activity was related to stomatal conductance (gs) and chloroplastic CO2 concentration (Cc), suggesting that deactivation of Rubisco sites could be induced by low Cc, as a result of water stress. The threshold level of Cc that triggered Rubisco deactivation was dependent on leaf characteristics and was related to the maximum attained for each species under non-stressing conditions. Those species adapted to low Cc were more capable of maintaining active Rubisco as drought stress intensified. PMID:21115663

The mechanism of the molecular interaction between cerium (III) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco).

PubMed

Liu, Chao; Hong, Fa-shui; Tao, Ye; Liu, Tao; Xie, Ya-ning; Xu, Jian-hua; Li, Zhong-rui

2011-11-01

The mechanism of the molecular interaction between Ce3+, a member of rare earth elements, and Rubisco in vitro is investigated. The carboxylase activity of Rubisco greatly increased under low concentrations of Ce3+ and decreased under high concentrations of Ce3+. The ultraviolet absorption spectra show that the various concentrations of Ce3+ treatment do not shift the characteristic peaks of Rubisco while the characteristic peak intensity of Rubisco increases with increasing Ce3+ concentration. The Rubisco-Ce3+ interactions also do not cause any noticeable change in the λmax of Rubisco fluorescence spectra. However, the fluorescence intensity of Rubisco is found quenched by the addition of Ce3+, which strongly suggests that Ce3+ could directly bind to the Rubisco protein. and the binding sites is estimated to 1.52 per protein. The binding between Ce3+ and Rubisco is also proved by extended X-ray absorption fine-structure essay; Ce3+ coordinated with eight oxygen atoms of Rubisco in first shells and six oxygen atoms in second shells. The results implied that Ce3+ might improve the microenvironment of Rubisco and, in turn, affected the carboxylase capacity of Rubisco greatly.

Proteomic Analysis of the Salt-Responsive Leaf and Root Proteins in the Anticancer Plant Andrographis paniculata Nees

PubMed Central

Rafii, Mohd Yusop; Maziah, Mahmood

2014-01-01

Separation of proteins based on the physicochemical properties with different molecular weight and isoelectric points would be more accurate. In the current research, the 45-day-old seedlings were treated with 0 (control) and 12 dS m−1 of sodium chloride in the hydroponic system. After 15 days of salt exposure, the total protein of the fresh leaves and roots was extracted and analyzed using two-dimensional electrophoresis system (2-DE). The analysis led to the detection of 32 induced proteins (19 proteins in leaf and 13 proteins in the root) as well as 12 upregulated proteins (four proteins in leaf and eight proteins in the root) in the salt-treated plants. Of the 44 detected proteins, 12 were sequenced, and three of them matched with superoxide dismutase, ascorbate peroxidase and ribulose-1, 5-bisphosphate oxygenase whereas the rest remained unknown. The three known proteins associate with plants response to environmental stresses and could represent the general stress proteins in the present study too. In addition, the proteomic feedback of different accessions of A. paniculata to salt stress can potentially be used to breed salt-tolerant varieties of the herb. PMID:25423252

Identification and Levels of 2′-Carboxyarabinitol in Leaves 1

PubMed Central

Moore, Brandon d.; Sharkey, Thomas D.; Kobza, John; Seemann, Jeffrey R.

1992-01-01

2′-Carboxyarabinitol 1-phosphate (CA1P) is a naturally occurring inhibitor of ribulose-1,5 bisphosphate carboxylase/oxygenase activity. A chloroplast phosphatase has previously been identified that degrades CA1P in vitro to carboxyarabinitol (CA) plus phosphate, but CA has not yet been detected in plants. Here, we detail procedures to isolate and assay CA from leaves and utilize mass spectrometry to demonstrate for the first time that CA is present in plants. CA was present in leaves of all 13 species examined, including those of C3, C4, and Crassulacean acid metabolism photosynthetic subgroups. CA was present both in species with high levels of CA1P (e.g. Phaseolus vulgaris, Lycopersicon esculentum, Beta vulgaris) as well as in species with low levels of CA1P (e.g. Spinacea oleracea, Triticum aestivum). CA levels in the light were sometimes greater than those in the dark. Bean leaves had the most CA of any species tested, with levels in the light approaching 1 micromole per milligram of chlorophyll. In illuminated bean leaves, about 63% of the CA is located outside the chloroplast. CA is one of only a few branched chain sugar acids to be identified from plants. PMID:16669072

A novel method for determination of the (15) N isotopic composition of Rubisco in wheat plants exposed to elevated atmospheric carbon dioxide.

PubMed

Aranjuelo, Iker; Molero, Gemma; Avice, Jean Christophe; Bourguignon, Jacques

2015-02-01

Although ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is mostly known as a key enzyme involved in CO2 assimilation during the Calvin cycle, comparatively little is known about its role as a pool of nitrogen storage in leaves. For this purpose, we developed a protocol to purify Rubisco that enables later analysis of its (15) N isotope composition (δ(15) N) at the natural abundance and (15) N-labeled plants. In order to test the utility of this protocol, durum wheat (Triticum durum var. Sula) exposed to an elevated CO2 concentration (700 vs 400 µmol mol(-1) ) was labeled with K(15) NO3 (enriched at 2 atom %) during the ear development period. The developed protocol proves to be selective, simple, cost effective and reproducible. The study reveals that (15) N labeling was different in total organic matter, total soluble protein and the Rubisco fraction. The obtained data suggest that photosynthetic acclimation in wheat is caused by Rubisco depletion. This depletion may be linked to preferential nitrogen remobilization from Rubisco toward grain filling. © 2014 Scandinavian Plant Physiology Society.

Transcriptional response of the extremophile red alga Cyanidioschyzon merolae to changes in CO2 concentrations.

PubMed

Rademacher, Nadine; Wrobel, Thomas J; Rossoni, Alessandro W; Kurz, Samantha; Bräutigam, Andrea; Weber, Andreas P M; Eisenhut, Marion

2017-10-01

Cyanidioschyzon merolae (C. merolae) is an acidophilic red alga growing in a naturally low carbon dioxide (CO 2 ) environment. Although it uses a ribulose 1,5-bisphosphate carboxylase/oxygenase with high affinity for CO 2 , the survival of C. merolae relies on functional photorespiratory metabolism. In this study, we quantified the transcriptomic response of C. merolae to changes in CO 2 conditions. We found distinct changes upon shifts between CO 2 conditions, such as a concerted up-regulation of photorespiratory genes and responses to carbon starvation. We used the transcriptome data set to explore a hypothetical CO 2 concentrating mechanism in C. merolae, based on the assumption that photorespiratory genes and possible candidate genes involved in a CO 2 concentrating mechanism are co-expressed. A putative bicarbonate transport protein and two α-carbonic anhydrases were identified, which showed enhanced transcript levels under reduced CO 2 conditions. Genes encoding enzymes of a PEPCK-type C 4 pathway were co-regulated with the photorespiratory gene cluster. We propose a model of a hypothetical low CO 2 compensation mechanism in C. merolae integrating these low CO 2 -inducible components. Copyright © 2017 Elsevier GmbH. All rights reserved.

Understanding the molecular basis of plant growth promotional effect of Pseudomonas fluorescens on rice through protein profiling

PubMed Central

2009-01-01

Background Plant Growth Promoting Rhizobacteria (PGPR), Pseudomonas fluorescens strain KH-1 was found to exhibit plant growth promotional activity in rice under both in-vitro and in-vivo conditions. But the mechanism underlying such promotional activity of P. fluorescens is not yet understood clearly. In this study, efforts were made to elucidate the molecular responses of rice plants to P. fluorescens treatment through protein profiling. Two-dimensional polyacrylamide gel electrophoresis strategy was adopted to identify the PGPR responsive proteins and the differentially expressed proteins were analyzed by mass spectrometry. Results Priming of P. fluorescens, 23 different proteins found to be differentially expressed in rice leaf sheaths and MS analysis revealed the differential expression of some important proteins namely putative p23 co-chaperone, Thioredoxin h- rice, Ribulose-bisphosphate carboxylase large chain precursor, Nucleotide diPhosphate kinase, Proteosome sub unit protein and putative glutathione S-transferase protein. Conclusion Functional analyses of the differential proteins were reported to be directly or indirectly involved in growth promotion in plants. Thus, this study confirms the primary role of PGPR strain KH-1 in rice plant growth promotion. PMID:20034395

Protein phosphorylation differs significantly among ontogenetic phases in Malus seedlings

PubMed Central

2014-01-01

Background Although protein phosphorylation is an important post-translational modification affecting protein function and metabolism, dynamic changes in this process during ontogenesis remain unexplored in woody angiosperms. Methods Phosphorylated proteins from leaves of three apple seedlings at juvenile, adult vegetative and reproductive stages were extracted and subjected to alkaline phosphatase pre-treatment. After separating the proteins by two-dimensional gel electrophoresis and phosphoprotein-specific Pro-Q Diamond staining, differentially expressed phosphoproteins were identified by MALDI-TOF-TOF mass spectrometry. Results A total of 107 phosphorylated protein spots on nine gels (three ontogenetic phases × three seedlings) were identified by MALDI-TOF-TOF mass spectrometry. The 55 spots of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) large-chain fragments varied significantly in protein abundance and degree of phosphorylation among ontogenetic phases. Abundances of the 27 spots corresponding to Rubisco activase declined between juvenile and reproductive phases. More extensively, phosphorylated β-tubulin chain spots with lower isoelectric points were most abundant during juvenile and adult vegetative phases. Conclusions Protein phosphorylation varied significantly during vegetative phase change and floral transition in apple seedlings. Most of the observed changes were consistent among seedlings and between hybrid populations. PMID:24904238

Expression of holo and apo forms of spinach acyl carrier protein-I in leaves of transgenic tobacco plants.

PubMed Central

Post-Beittenmiller, M A; Schmid, K M; Ohlrogge, J B

1989-01-01

Acyl carrier protein (ACP) is a chloroplast-localized cofactor of fatty acid synthesis, desaturation, and acyl transfer. We have transformed tobacco with a chimeric gene consisting of the tobacco ribulose-1,5-bisphosphate carboxylase promoter and transit peptide and the sequence encoding the mature spinach ACP-I. Spinach ACP-I was expressed in the transformed plants at levels twofold to threefold higher than the endogenous tobacco ACPs as determined by protein immunoblots and assays of ACP in leaf extracts. In addition to these elevated levels of the holo form, there were high levels of apoACP-I, a form lacking the 4'-phosphopantetheine prosthetic group and not previously detected in vivo. The mature forms of both apoACP-I and holoACP-I were located in the chloroplasts, indicating that the transit peptide was cleaved and that attachment of the prosthetic group was not required for uptake into the plastid. There were also significant levels of spinach acyl-ACP-I, demonstrating that spinach ACP-I participated in tobacco fatty acid metabolism. Lipid analyses of the transformed plants indicated that the increased ACP levels caused no significant alterations in leaf lipid biosynthesis. PMID:2535529

Comparative analysis of cells and proteins of pumpkin plants for the control of fruit size.

PubMed

Nakata, Yumiko; Taniguchi, Go; Takazaki, Shinya; Oda-Ueda, Naoko; Miyahara, Kohji; Ohshima, Yasumi

2012-09-01

Common pumpkin plants (Cucurbita maxima) produce fruits of 1-2 kg size on the average, while special varieties of the same species called Atlantic Giant are known to produce a huge fruit up to several hundred kilograms. As an approach to determine the factors controlling the fruit size in C. maxima, we cultivated both AG and control common plants, and found that both the cell number and cell sizes were increased in a large fruit while DNA content of the cell did not change significantly. We also compared protein patterns in the leaves, stems, ripe and young fruits by two-dimensional (2D) gel electrophoresis, and identified those differentially expressed between them with mass spectroscopy. Based on these results, we suggest that factors in photosynthesis such as ribulose-bisphosphate carboxylase, glycolysis pathway enzymes, heat-shock proteins and ATP synthase play positive or negative roles in the growth of a pumpkin fruit. These results provide a step toward the development of plant biotechnology to control fruit size in the future. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Changes in Protein Synthesis in Rapeseed (Brassica napus) Seedlings during a Low Temperature Treatment 1

PubMed Central

Meza-Basso, Luis; Alberdi, Miren; Raynal, Monique; Ferrero-Cadinanos, Maria-Luz; Delseny, Michel

1986-01-01

Changes induced by cold treatment in young rapeseed (Brassica napus) seedlings were investigated at the molecular level. Following germination at 18°C for 48 hours, one half of the seedlings was transferred to 0°C for another 48 hour period, the other half being kept at 18°C as a control. Newly synthesized proteins were labeled for the last 6 hours of incubation with [35S]methionine. The different polypeptides were separated by two-dimensional electrophoresis in polyacrylamide gels. Newly synthesized proteins were revealed by fluorography. Protein synthesis clearly continues at 0°C and some polypeptides preferentially accumulate at this temperature. On the other hand, synthesis of several others is repressed while many are insensitive to cold treatment. Similar changes are also observed when mRNA is prepared from cold treated seedlings, translated in vitro in a reticulocyte cell free system and compared with the products of mRNA extracted from control samples. Among the genes which are repressed we identified the small subunit of ribulose 1,6-bisphosphate carboxylase. These changes are also detectable after shorter treatments. Images Fig. 1 Fig. 2 Fig. 3 PMID:16665102

Green Roots: Photosynthesis and Photoautotrophy in an Underground Plant Organ.

PubMed Central

Flores, H. E.; Dai, Yr.; Cuello, J. L.; Maldonado-Mendoza, I. E.; Loyola-Vargas, V. M.

1993-01-01

The potential for photosynthetic and photoautotrophic growth was studied in hairy root cultures of Asteraceae and Solanaceae species. Upon transfer to light, initially heterotrophic root cultures of Acmella oppositifolia and Datura innoxia greened rapidly, differentiated chloroplasts, and developed light-dependent CO2 fixation in the cortical cells. Photosynthetic potential was expressed in root cultures of all the Asteraceae genera examined (Acmella, Artemisia, Rudbeckia, Stevia, and Tagetes). Hairy roots of A. oppositifolia and D. innoxia were further adapted to photoautotrophy by growing in the presence of light and added CO2 (1-5%) and by direct or sequential transfers into media containing progressively lower sugar concentrations. The transition to photoautotrophy was accompanied by an increase in CO2 fixation and in the specific activity of 1,5-ribulose-bisphosphate carboxylase/ oxygenase (Rubisco). During the adaptation of A. oppositifolia roots to photoautotrophy, the ratio of Rubisco to phosphoenolpyruvate carboxylase increased significantly, approaching that found in the leaves. The levels and patterns of alkaloids and polyacetylenes produced by Solanaceae and Asteraceae hairy roots, respectively, were dramatically altered in photomixotrophic and photoautotrophic cultures. Photoautotrophic roots of A. oppositifolia have been mainitained in vitro for over 2 years. PMID:12231691

Application of an Improved Proteomics Method for Abundant Protein Cleanup: Molecular and Genomic Mechanisms Study in Plant Defense*

PubMed Central

Zhang, Yixiang; Gao, Peng; Xing, Zhuo; Jin, Shumei; Chen, Zhide; Liu, Lantao; Constantino, Nasie; Wang, Xinwang; Shi, Weibing; Yuan, Joshua S.; Dai, Susie Y.

2013-01-01

High abundance proteins like ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) impose a consistent challenge for the whole proteome characterization using shot-gun proteomics. To address this challenge, we developed and evaluated Polyethyleneimine Assisted Rubisco Cleanup (PARC) as a new method by combining both abundant protein removal and fractionation. The new approach was applied to a plant insect interaction study to validate the platform and investigate mechanisms for plant defense against herbivorous insects. Our results indicated that PARC can effectively remove Rubisco, improve the protein identification, and discover almost three times more differentially regulated proteins. The significantly enhanced shot-gun proteomics performance was translated into in-depth proteomic and molecular mechanisms for plant insect interaction, where carbon re-distribution was used to play an essential role. Moreover, the transcriptomic validation also confirmed the reliability of PARC analysis. Finally, functional studies were carried out for two differentially regulated genes as revealed by PARC analysis. Insect resistance was induced by over-expressing either jacalin-like or cupin-like genes in rice. The results further highlighted that PARC can serve as an effective strategy for proteomics analysis and gene discovery. PMID:23943779

Genus-Specific Real-Time PCR and HRM Assays to Distinguish Liriope from Ophiopogon Samples.

PubMed

Masiero, Eva; Banik, Dipanwita; Abson, John; Greene, Paul; Slater, Adrian; Sgamma, Tiziana

2017-10-26

Liriope and Ophiopogon species have a long history of use as traditional medicines across East Asia. They have also become widely used around the world for ornamental and landscaping purposes. The morphological similarities between Liriope and Ophiopogon taxa have made the taxonomy of the two genera problematic and caused confusion about the identification of individual specimens. Molecular approaches could be a useful tool for the discrimination of these two genera in combination with traditional methods. Seventy-five Liriope and Ophiopogon samples from the UK National Plant Collections of Ophiopogon and Liriope were analyzed. The 5' end of the DNA barcode region of the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase ( rbcLa ) was used for the discrimination of the two genera. A single nucleotide polymorphism (SNP) between the two genera allowed the development of discriminatory tests for genus-level identification based on specific PCR and high-resolution melt curve (HRM) assays. The study highlights the advantage of incorporating DNA barcoding methods into plant identification protocols and provides simple assays that could be used for the quality assurance of commercially traded plants and herbal drugs.

Limitations to soybean photosynthesis at elevated carbon dioxide in free-air enrichment and open top chamber systems.

PubMed

Bunce, James A

2014-09-01

It has been suggested that the stimulation of soybean photosynthesis by elevated CO2 was less in free-air carbon dioxide enrichment (FACE) systems than in open top chambers (OTC), which might explain smaller yield increases at elevated CO2 in FACE systems. However, this has not been tested using the same cultivars grown in the same location. I tested whether soybean photosynthesis at high light and elevated CO2 (ambient+180 μmol mol(-1)) was limited by electron transport (J) in FACE systems but by ribulose-bisphosphate carboxylation capacity (VCmax) in OTC. FACE systems with daytime and continuous CO2 enrichment were also compared. The results indicated that in both cultivars examined, midday photosynthesis at high light was always limited by VCmax, both in the FACE and in the OTC systems. Daytime only CO2 enrichment did not affect photosynthetic parameters or limitations, but did result in significantly smaller yields in both cultivars than continuous elevation. Photosynthesis measured at low photosynthetic photon flux density (PPFD) was not higher at elevated than at ambient CO2, because of an acclimation to elevated CO2 which was only evident at low measurement PPFDs. Published by Elsevier Ireland Ltd.

Two Lactarius species associated with a relict Fagus grandifolia var. mexicana population in a Mexican montane cloud forest.

PubMed

Montoya, L; Haug, I; Bandala, V M

2010-01-01

Ectomycorrhizal (EM) fleshy fungi are being monitored in a population of Fagus grandifolia var. mexicana persisting in a montane cloud forest refuge on a volcano in a subtropical region of central Veracruz (eastern Mexico). The population of Fagus studied represents one of the 10 recognized forest fragments still housing this tree genus in Mexico. This is the first attempt to document EM fungi associated with this tree species in Mexico. We present evidence of the ectomycorrhizal symbiosis for Lactarius badiopallescens and L. cinereus with this endemic tree. Species identification of Lactarius on Fagus grandifolia var. mexicana was based on the comparison of DNAsequences (ITS rDNA) of spatiotemporally co-occurring basidiomes and EM root tips. The host of the EM tips was identified by comparison of the large subunit of the ribulose-bisphosphate carboxylase gene (rbcL). The occurrence of Lactarius badiopallescens and L. cinereus populations in the area of study represent the southernmost record known to date of these two species in North America and are new for the Neotropical Lactarius mycota. Descriptions coupled with illustrations of macro- and micromorphological features of basidiomes as well as photographs of ectomycorrhizas are presented.

Singlet oxygen-dependent translational control in the tigrina-d.12 mutant of barley

PubMed Central

Khandal, Dhriti; Samol, Iga; Buhr, Frank; Pollmann, Stephan; Schmidt, Holger; Clemens, Stephan; Reinbothe, Steffen; Reinbothe, Christiane

2009-01-01

The tigrina (tig)-d.12 mutant of barley is impaired in the negative control limiting excess protochlorophyllide (Pchlide) accumulation in the dark. Upon illumination, Pchlide operates as photosensitizer and triggers singlet oxygen production and cell death. Here, we show that both Pchlide and singlet oxygen operate as signals that control gene expression and metabolite accumulation in tig-d.12 plants. In vivo labeling, Northern blotting, polysome profiling, and protein gel blot analyses revealed a selective suppression of synthesis of the small and large subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RBCSs and RBCLs), the major light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCB2), as well as other chlorophyll-binding proteins, in response to singlet oxygen. In part, these effects were caused by an arrest in translation initiation of photosynthetic transcripts at 80S cytoplasmic ribosomes. The observed changes in translation correlated with a decline in the phosphorylation level of ribosomal protein S6. At later stages, ribosome dissociation occurred. Together, our results identify translation as a major target of singlet oxygen-dependent growth control and cell death in higher plants. PMID:19620736

The importance of methane and thiosulfate in the metabolism of the bacterial symbionts of two deep-sea mussels

USGS Publications Warehouse

Fisher, C.R.; Childress, J.J.; Oremland, R.S.; Bidigare, R.R.

1987-01-01

Undescribed hydrocarbon-seep mussels were collected from the Louisiana Slope, Gulf of Mexico, during March 1986, and the ultrastructure of their gills was examined and compared to Bathymodiolus thermophilus, a mussel collected from the deep-sea hydrothermal vents on the Gala??pagos Rift in March 1985. These closely related mytilids both contain abundant symbiotic bacteria in their gills. However, the bacteria from the two species are distinctly different in both morphology and biochemistry, and are housed differently within the gills of the two mussels. The symbionts from the seep mussel are larger than the symbionts from B. thermophilus and, unlike the latter, contain stacked intracytoplasmic membranes. In the seep mussel three or fewer symbionts appear to be contained in each host-cell vacuole, while in B. thermophilus there are often more than twenty bacteria visible in a single section through a vacuole. The methanotrophic nature of the seep-mussel symbionts was confirmed in 14C-methane uptake experiments by the appearance of label in both CO2 and acid-stable, non-volatile, organic compounds after a 3 h incubation of isolated gill tissue. Furthermore, methane consumption was correlated with methanol dehydrogenase activity in isolated gill tissue. Activity of ribulose-1,5-biphosphate (RuBP) carboxylase and 14CO2 assimilation studies indicate the presence of either a second type of symbiont or contaminating bacteria on the gills of freshly captured seep mussels. A reevaluation of the nutrition of the symbionts in B. thermophilus indicates that while the major symbiont is not a methanotroph, its status as a sulfur-oxidizing chemoautotroph, as has been suggested previously, is far from proven. ?? 1987 Springer-Verlag.

Photorespiration plays an important role in the regulation of photosynthetic electron flow under fluctuating light in tobacco plants grown under full sunlight.

PubMed

Huang, Wei; Hu, Hong; Zhang, Shi-Bao

2015-01-01

Plants usually experience dynamic fluctuations of light intensities under natural conditions. However, the responses of mesophyll conductance, CO2 assimilation, and photorespiration to light fluctuation are not well understood. To address this question, we measured photosynthetic parameters of gas exchange and chlorophyll fluorescence in tobacco leaves at 2-min intervals while irradiance levels alternated between 100 and 1200 μmol photons m(-2) s(-1). Compared with leaves exposed to a constant light of 1200 μmol photons m(-2) s(-1), both stomatal and mesophyll conductances were significantly restricted in leaves treated with fluctuating light condition. Meanwhile, CO2 assimilation rate and electron flow devoted to RuBP carboxylation at 1200 μmol photons m(-2) s(-1) under fluctuating light were limited by the low chloroplast CO2 concentration. Analysis based on the C3 photosynthesis model indicated that, at 1200 μmol photons m(-2) s(-1) under fluctuating light, the CO2 assimilation rate was limited by RuBP carboxylation. Electron flow devoted to RuBP oxygenation at 1200 μmol photons m(-2) s(-1) under fluctuating light remained at nearly the maximum level throughout the experimental period. We conclude that fluctuating light restricts CO2 assimilation by decreasing both stomatal and mesophyll conductances. Under such conditions, photorespiration plays an important role in the regulation of photosynthetic electron flow.

Characterization of C₃--C₄ intermediate species in the genus Heliotropium L. (Boraginaceae): anatomy, ultrastructure and enzyme activity.

PubMed

Muhaidat, Riyadh; Sage, Tammy L; Frohlich, Michael W; Dengler, Nancy G; Sage, Rowan F

2011-10-01

Photosynthetic pathway characteristics were studied in nine species of Heliotropium (sensu lato, including Euploca), using assessments of leaf anatomy and ultrastructure, activities of PEP carboxylase and C₄ acid decarboxylases, and immunolocalization of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) and the P-subunit of glycine decarboxylase (GDC). Heliotropium europaeum, Heliotropium calcicola and Heliotropium tenellum are C₃ plants, while Heliotropium texanum and Heliotropium polyphyllum are C₄ species. Heliotropium procumbens and Heliotropium karwinskyi are functionally C₃, but exhibit 'proto-Kranz' anatomy where bundle sheath (BS) cells are enlarged and mitochondria primarily occur along the centripetal (inner) wall of the BS cells; GDC is present throughout the leaf. Heliotropium convolvulaceum and Heliotropium greggii are C₃--C₄ intermediates, with Kranz-like enlargement of the BS cells, localization of mitochondria along the inner BS wall and a loss of GDC in the mesophyll (M) tissue. These C₃--C₄ species of Heliotropium probably shuttle photorespiratory glycine from the M to the BS tissue for decarboxylation. Heliotropium represents an important new model for studying C₄ evolution. Where existing models such as Flaveria emphasize diversification of C₃--C₄ intermediates, Heliotropium has numerous C₃ species expressing proto-Kranz traits that could represent a critical initial phase in the evolutionary origin of C₄ photosynthesis. © 2011 Blackwell Publishing Ltd.

A "footprint" of plant carbon fixation cycle functions during the development of a heterotrophic fungus.

PubMed

Lyu, Xueliang; Shen, Cuicui; Xie, Jiatao; Fu, Yanping; Jiang, Daohong; Hu, Zijin; Tang, Lihua; Tang, Liguang; Ding, Feng; Li, Kunfei; Wu, Song; Hu, Yanping; Luo, Lilian; Li, Yuanhao; Wang, Qihua; Li, Guoqing; Cheng, Jiasen

2015-08-11

Carbon fixation pathway of plants (CFPP) in photosynthesis converts solar energy to biomass, bio-products and biofuel. Intriguingly, a large number of heterotrophic fungi also possess enzymes functionally associated with CFPP, raising the questions about their roles in fungal development and in evolution. Here, we report on the presence of 17 CFPP associated enzymes (ten in Calvin-Benson-Basham reductive pentose phosphate pathway and seven in C4-dicarboxylic acid cycle) in the genome of Sclerotinia sclerotiorum, a heterotrophic phytopathogenic fungus, and only two unique enzymes: ribulose-1, 5-bisphosphate carboxylase-oxygenase (Rubisco) and phosphoribulokinase (PRK) were absent. This data suggested an incomplete CFPP-like pathway (CLP) in fungi. Functional profile analysis demonstrated that the activity of the incomplete CLP was dramatically regulated during different developmental stages of S. sclerotiorum. Subsequent experiments confirmed that many of them were essential to the virulence and/or sclerotial formation. Most of the CLP associated genes are conserved in fungi. Phylogenetic analysis showed that many of them have undergone gene duplication, gene acquisition or loss and functional diversification in evolutionary history. These findings showed an evolutionary links in the carbon fixation processes of autotrophs and heterotrophs and implicated the functions of related genes were in course of continuous change in different organisms in evolution.

Evaluation of proteome alterations induced by cadmium stress in sunflower (Helianthus annuus L.) cultures.

PubMed

Lopes Júnior, Cícero Alves; Barbosa, Herbert de Sousa; Moretto Galazzi, Rodrigo; Ferreira Koolen, Hector Henrique; Gozzo, Fábio Cesar; Arruda, Marco Aurélio Zezzi

2015-09-01

The present study evaluates, at a proteomic level, changes in protein abundance in sunflower leaves in the absence or presence (at 50 or 700mg) of cadmium (as CdCl2). At the end of the cultivation period (45 days), proteins are extracted from leaves with phenol, separated by two-dimensional difference gel electrophoresis (2-D DIGE), and excised from the gels. The differential protein abundances (for proteins differing by more than 1.8 fold, which corresponds to 90% variation) are characterized using nESI-LC-MS/MS. The protein content decreases by approximately 41% in plants treated with 700mg Cd compared with control plants. By comparing all groups of plants evaluated in this study (Control vs. Cd-lower, Control vs. Cd-higher and Cd-lower vs. Cd-higher), 39 proteins are found differential and 18 accurately identified; the control vs. Cd-higher treatment is that presenting the most differential proteins. From identified proteins, those involved in energy and disease/defense (including stress), are the ribulose bisphosphate carboxylase large chain, transketolase, and heat shock proteins are the most differential abundant proteins. Thus, at the present study, photosynthesis is the main process affected by Cd in sunflowers, although these plants are highly tolerant to Cd. Copyright © 2015 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

MacIntyre, H.L.; Geider, R.J.; McKay, R.M.

Net phytoplankton (>20 {mu}m) comprised 51 {plus_minus} 9% of the total chlorophyll (Chl) in a Skeletonema costatum-dominated spring bloom in Delaware Bay. The net phytoplankton had low C:N and high protein:carbohydrate ratios, indicating that their growth was nutrient-replete. Their photosynthetic responses were characterized by low specific absorption, low light-limited and light-saturated rates of photosynthesis, and high quantum yields, indicative of acclimation to low irradiance and internal self-shading. High fucoxanthin: Chl ratios also indicated low light acclimation, but high photoprotective xanthophyll: Chl ratios suggested a high capacity for photoprotective energy dissipation. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) could be activated and deactivated in responsemore » to changes in irradiance and was fully activated at the surface of the water column and fully deactivated in aphotic deep water. Maximum Rubisco activity was correlated with Rubisco content and bulk protein content of the phytoplankton and with light-saturated rates of photosynthesis measured in short (<20-min) incubations. Long (60-min) incubations caused a decrease in the light-saturated rate of photosynthesis, possibly because of feedback limitation. While feedback limitation is unlikely to occur in the water column it should be considered when estimating productivity in well-mixed waters from fixed light-depth incubations. 90 refs., 7 figs., 2 tabs.« less

The Differences between NAD-ME and NADP-ME Subtypes of C4 Photosynthesis: More than Decarboxylating Enzymes.

PubMed

Rao, Xiaolan; Dixon, Richard A

2016-01-01

As an adaptation to changing climatic conditions that caused high rates of photorespiration, C 4 plants have evolved to display higher photosynthetic efficiency than C 3 plants under elevated temperature, high light intensities, and drought. The C 4 plants independently evolved more than 60 times in 19 families of angiosperms to establish similar but not uniform C 4 mechanisms to concentrate CO 2 around the carboxylating enzyme Rubisco (ribulose bisphosphate carboxylase oxygenase). C 4 photosynthesis is divided into at least two basic biochemical subtypes based on the primary decarboxylating enzymes, NAD-dependent malic enzyme (NAD-ME) and NADP-dependent malic enzyme (NADP-ME). The multiple polygenetic origins of these subtypes raise questions about the association of C 4 variation between biochemical subtypes and diverse lineages. This review addresses the differences in evolutionary scenario, leaf anatomy, and especially C 4 metabolic flow, C 4 transporters, and cell-specific function deduced from recently reported cell-specific transcriptomic, proteomic, and metabolic analyses of NAD-ME and NADP-ME subtypes. Current omic analysis has revealed the extent to which component abundances differ between the two biochemical subtypes, leading to a better understanding of C 4 photosynthetic mechanisms in NAD-ME and NADP-ME subtypes.

The Differences between NAD-ME and NADP-ME Subtypes of C4 Photosynthesis: More than Decarboxylating Enzymes

PubMed Central

Rao, Xiaolan; Dixon, Richard A.

2016-01-01

As an adaptation to changing climatic conditions that caused high rates of photorespiration, C4 plants have evolved to display higher photosynthetic efficiency than C3 plants under elevated temperature, high light intensities, and drought. The C4 plants independently evolved more than 60 times in 19 families of angiosperms to establish similar but not uniform C4 mechanisms to concentrate CO2 around the carboxylating enzyme Rubisco (ribulose bisphosphate carboxylase oxygenase). C4 photosynthesis is divided into at least two basic biochemical subtypes based on the primary decarboxylating enzymes, NAD-dependent malic enzyme (NAD-ME) and NADP-dependent malic enzyme (NADP-ME). The multiple polygenetic origins of these subtypes raise questions about the association of C4 variation between biochemical subtypes and diverse lineages. This review addresses the differences in evolutionary scenario, leaf anatomy, and especially C4 metabolic flow, C4 transporters, and cell-specific function deduced from recently reported cell-specific transcriptomic, proteomic, and metabolic analyses of NAD-ME and NADP-ME subtypes. Current omic analysis has revealed the extent to which component abundances differ between the two biochemical subtypes, leading to a better understanding of C4 photosynthetic mechanisms in NAD-ME and NADP-ME subtypes. PMID:27790235

Recycling Carbon Dioxide during Xylose Fermentation by Engineered Saccharomyces cerevisiae.

PubMed

Xia, Peng-Fei; Zhang, Guo-Chang; Walker, Berkley; Seo, Seung-Oh; Kwak, Suryang; Liu, Jing-Jing; Kim, Heejin; Ort, Donald R; Wang, Shu-Guang; Jin, Yong-Su

2017-02-17

Global climate change caused by the emission of anthropogenic greenhouse gases (GHGs) is a grand challenge to humanity. To alleviate the trend, the consumption of fossil fuels needs to be largely reduced and alternative energy technologies capable of controlling GHG emissions are anticipated. In this study, we introduced a synthetic reductive pentose phosphate pathway (rPPP) into a xylose-fermenting Saccharomyces cerevisiae strain SR8 to achieve simultaneous lignocellulosic bioethanol production and carbon dioxide recycling. Specifically, ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum and phosphoribulokinase from Spinacia oleracea were introduced into the SR8 strain. The resulting strain with the synthetic rPPP was able to exhibit a higher yield of ethanol and lower yields of byproducts (xylitol and glycerol) than a control strain. In addition, the reduced release of carbon dioxide by the engineered strain was observed during xylose fermentation, suggesting that the carbon dioxide generated by pyruvate decarboxylase was partially reassimilated through the synthetic rPPP. These results demonstrated that recycling of carbon dioxide from the ethanol fermentation pathway in yeast can be achieved during lignocellulosic bioethanol production through a synthetic carbon conservative metabolic pathway. This strategy has a great potential to alleviate GHG emissions during the production of second-generation ethanol.

Community genomic analysis of an extremely acidophilic sulfur-oxidizing biofilm

PubMed Central

Jones, Daniel S; Albrecht, Heidi L; Dawson, Katherine S; Schaperdoth, Irene; Freeman, Katherine H; Pi, Yundan; Pearson, Ann; Macalady, Jennifer L

2012-01-01

Highly acidic (pH 0–1) biofilms, known as ‘snottites', form on the walls and ceilings of hydrogen sulfide-rich caves. We investigated the population structure, physiology and biogeochemistry of these biofilms using metagenomics, rRNA methods and lipid geochemistry. Snottites from the Frasassi cave system (Italy) are dominated (>70% of cells) by Acidithiobacillus thiooxidans, with smaller populations including an archaeon in the uncultivated ‘G-plasma' clade of Thermoplasmatales (>15%) and a bacterium in the Acidimicrobiaceae family (>5%). Based on metagenomic evidence, the Acidithiobacillus population is autotrophic (ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), carboxysomes) and oxidizes sulfur by the sulfide–quinone reductase and sox pathways. No reads matching nitrogen fixation genes were detected in the metagenome, whereas multiple matches to nitrogen assimilation functions are present, consistent with geochemical evidence, that fixed nitrogen is available in the snottite environment to support autotrophic growth. Evidence for adaptations to extreme acidity include Acidithiobacillus sequences for cation transporters and hopanoid synthesis, and direct measurements of hopanoid membrane lipids. Based on combined metagenomic, molecular and geochemical evidence, we suggest that Acidithiobacillus is the snottite architect and main primary producer, and that snottite morphology and distributions in the cave environment are directly related to the supply of C, N and energy substrates from the cave atmosphere. PMID:21716305

Photosynthetic characteristics and mycosporine-like amino acids under UV radiation: a competitive advantage of Mastocarpus stellatus over Chondrus crispus at the Helgoland shoreline?

NASA Astrophysics Data System (ADS)

Bischof, K.; Kräbs, G.; Hanelt, D.; Wiencke, C.

2000-05-01

Chondrus crispus and Mastocarpus stellatus both inhabit the intertidal and upper sublittoral zone of Helgoland, but with C. crispus generally taking a lower position. Measurements of chlorophyll fluorescence, activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), and content and composition of UV absorbing mycosporine-like amino acids (MAAs) were conducted in the laboratory, to test whether susceptibility to UV radiation may play a role in the vertical distribution of these two species. Effective and maximal quantum yield of photochemistry as well as maximal electron transport rate (ETRmax) in C. crispus were more strongly affected by UV-B radiation than in M. stellatus. In both species, no negative effects of the respective radiation conditions were found on total activity of RubisCO. Total MAA content in M. stellatus was up to 6-fold higher than in C. crispus and the composition of MAAs in the two species was different. The results indicate that, among others, UV-B sensitivity may be a factor restricting C. crispus to the lower intertidal and upper sublittoral zone, whereas M. stellatus is better adapted to UV radiation and is therefore more competitive in the upper intertidal zone.

XAP5 CIRCADIAN TIMEKEEPER Coordinates Light Signals for Proper Timing of Photomorphogenesis and the Circadian Clock in Arabidopsis[W

PubMed Central

Martin-Tryon, Ellen L.; Harmer, Stacey L.

2008-01-01

Numerous, varied, and widespread taxa have an internal circadian clock that allows anticipation of rhythmic changes in the environment. We have identified XAP5 CIRCADIAN TIMEKEEPER (XCT), an Arabidopsis thaliana gene important for light regulation of the circadian clock and photomorphogenesis. XCT is essential for proper clock function: xct mutants display a shortened circadian period in all conditions tested. Interestingly, XCT plays opposite roles in plant responses to light depending both on trait and wavelength. The clock in xct plants is hypersensitive to red but shows normal responses to blue light. By contrast, inhibition of hypocotyl elongation in xct is hyposensitive to red light but hypersensitive to blue light. Finally, XCT is important for ribulose-1,5-bisphosphate carboxylase/oxygenase production and plant greening in response to light. This novel combination of phenotypes suggests XCT may play a global role in coordinating growth in response to the light environment. XCT contains a XAP5 domain and is well conserved across diverse taxa, suggesting it has a common function in higher eukaryotes. Downregulation of the XCT ortholog in Caenorhabditis elegans is lethal, suggesting that studies in Arabidopsis may be instrumental to understanding the biochemical activity of XCT. PMID:18515502

Comparative Physiological and Proteomic Analysis Reveals the Leaf Response to Cadmium-Induced Stress in Poplar (Populus yunnanensis)

PubMed Central

Yang, Shihai; Zhou, Yanli; Dong, Chao; Ren, Jian; Sun, Xudong; Yang, Yongping

2015-01-01

Excess amounts of heavy metals are important environmental pollutants with significant ecological and nutritional effects. Cdmium (Cd) is of particular concern because of its widespread occurrence and high toxicity. We conducted physiological and proteomic analyses to improve our understanding of the responses of Populus yunnanensis to Cd stress. The plantlets experienced two apparent stages in their response to Cd stress. During the first stage, transiently induced defense-response molecules, photosynthesis- and energy-associated proteins, antioxidant enzymes and heat shock proteins (HSPs) accumulated to enhance protein stability and establish a new cellular homeostasis. This activity explains why plant photosynthetic capability during this period barely changed. During the second stage, a decline of ribulose-1, 5-bisphosphate carboxylase (RuBisCO) and HSP levels led to imbalance of the plant photosynthetic system. Additionally, the expression of Mitogen-activated protein kinase 3 (MPK3), Mitogen-activated protein kinase 6 (MPK6) and a homeobox-leucine zipper protein was higher in the second stage. Higher expression of caffeoyl-CoA O-methyltransferase (CCoAOMT) may regulate plant cell wall synthesis for greater Cd storage. These genes may be candidates for further research and use in genetic manipulation of poplar tolerance to Cd stress. PMID:26349064

The complex character of photosynthesis in cucumber fruit

PubMed Central

Sui, Xiaolei; Shan, Nan; Hu, Liping; Yu, Changqing; Ren, Huazhong; Zhang, Zhenxian

2017-01-01

Abstract The surface area of a mature green cucumber (Cucumis sativa L.) fruit is comparable with that of a functional leaf, but the characteristics of fruit photosynthesis and its contribution to growth are poorly understood. Here, the photosynthetic properties of two genotypes of cucumber (dark green and light green fruits) were studied using a combination of electron microscopy, immunogold enzyme localization, chlorophyll fluorescence imaging, isotope tracer, and fruit darkening techniques. Chlorophyll content of the exocarp is similar to that of leaves, but there are no distinctive palisade and spongy tissues. The efficiency of PSII is similar to that in leaves, but with lower non-photochemical quenching (NPQ). Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is found mainly in the exocarp, while phosphoenolpyruvate carboxylase (PEPC) is primarily localized to vascular bundles and placenta tissue. Rubisco and PEPC expression at both transcriptional and translational levels increases concurrently during fruit growth. The contribution of fruit photosynthesis in exocarp to its own C accumulation is 9.4%, while ~88% of respiratory CO2 in fruit was captured and re-fixed. Photosynthesis by cucumber fruits, through direct fixation of atmospheric CO2 and recapture of respired CO2, as verified by 14CO2 uptake and gas exchange, makes an important contribution to fruit growth. PMID:28369547

An engineered Streptomyces hygroscopicus aph 7" gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii.

PubMed

Berthold, Peter; Schmitt, Rüdiger; Mages, Wolfgang

2002-12-01

We have developed a positively selectable marker for the green alga Chlamydomonas reinhardtii using the Streptomyces hygroscopicus aminoglycoside phosphotransferase gene (aph7"). Its expression is controlled by C. reinhardtii regulatory elements, namely, the beta2-tubulin gene promoter in combination with the first intron and the 3' untranslated region of the small subunit of ribulose bisphosphate carboxylase, rbcS2. C. reinhardtii cell-wall deficient and wild-type strains were transformed at rates up to 5 x 10(-5) with two constructs, pHyg3 and pHyg4 (intron-less). Transformants selected on plates with 10 microg/ml hygromycin B exhibited diverse levels of resistance of up to 200 microg/ml that were stably maintained for at least seven months; they contained two to five copies of the construct integrated in their genomes. Transcription of the chimeric aph7" gene, correct splicing of the rbcS2 intron, and polyadenylation of the transcripts have been verified by sequencing of RT-PCR products. Average co-transformation rates using pHyg3 and a second selectable plasmid were about 11%. This advocates the hygromycin-resistance plasmid, pHyg3, as a new versatile tool for the transformation of a broad range of C. reinhardtii strains without the sustained need for using auxotrophic mutants as recipients.

Proteomic analysis of carbon concentrating chemolithotrophic bacteria Serratia sp. for sequestration of carbon dioxide.

PubMed

Bharti, Randhir K; Srivastava, Shaili; Thakur, Indu Shekhar

2014-01-01

A chemolithotrophic bacterium enriched in the chemostat in presence of sodium bicarbonate as sole carbon source was identified as Serratia sp. by 16S rRNA sequencing. Carbon dioxide sequestering capacity of bacterium was detected by carbonic anhydrase enzyme and ribulose-1, 5- bisphosphate carboxylase/oxygenase (RuBisCO). The purified carbonic anhydrase showed molecular weight of 29 kDa. Molecular weight of RuBisCO was 550 kDa as determined by fast protein liquid chromatography (FPLC), however, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed presence of two subunits whose molecular weights were 56 and 14 kDa. The Western blot analysis of the crude protein and purified sample cross reacted with RuBisCO large-subunit polypeptides antibodies showed strong band pattern at molecular weight around 56 kDa regions. Whole cell soluble proteins of Serratia sp. grown under autotrophic and heterotrophic conditions were resolved by two-dimensional gel electrophoresis and MALDI-TOF/MS for differential expression of proteins. In proteomic analysis of 63 protein spots, 48 spots were significantly up-regulated in the autotrophically grown cells; seven enzymes showed its utilization in autotrophic carbon fixation pathways and other metabolic activities of bacterium including lipid metabolisms indicated sequestration potency of carbon dioxide and production of biomaterials.

Proteomic Analysis of Carbon Concentrating Chemolithotrophic Bacteria Serratia sp. for Sequestration of Carbon Dioxide

PubMed Central

Bharti, Randhir K.; Srivastava, Shaili; Thakur, Indu Shekhar

2014-01-01

A chemolithotrophic bacterium enriched in the chemostat in presence of sodium bicarbonate as sole carbon source was identified as Serratia sp. by 16S rRNA sequencing. Carbon dioxide sequestering capacity of bacterium was detected by carbonic anhydrase enzyme and ribulose-1, 5- bisphosphate carboxylase/oxygenase (RuBisCO). The purified carbonic anhydrase showed molecular weight of 29 kDa. Molecular weight of RuBisCO was 550 kDa as determined by fast protein liquid chromatography (FPLC), however, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed presence of two subunits whose molecular weights were 56 and 14 kDa. The Western blot analysis of the crude protein and purified sample cross reacted with RuBisCO large-subunit polypeptides antibodies showed strong band pattern at molecular weight around 56 kDa regions. Whole cell soluble proteins of Serratia sp. grown under autotrophic and heterotrophic conditions were resolved by two-dimensional gel electrophoresis and MALDI-TOF/MS for differential expression of proteins. In proteomic analysis of 63 protein spots, 48 spots were significantly up-regulated in the autotrophically grown cells; seven enzymes showed its utilization in autotrophic carbon fixation pathways and other metabolic activities of bacterium including lipid metabolisms indicated sequestration potency of carbon dioxide and production of biomaterials. PMID:24619032

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stochaj, W.R.; Grossman, A.R.

One- and two-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunological analyses were used to visualize differences in polypeptides synthesized by Symbiodinium sp. from the anemone Aiptasia pallida when grown in the cultured and endosymbiotic states (freshly isolated zooxanthellae). Surprisingly, a comparison of proteins in cultured and endosymbiotic Symbiodinium sp. revealed only four major polypeptides with similar isoelectric and molecular mass characteristics. Using monospecific antibodies, we demonstrated differences in specific proteins synthesized by the dinoflagellate in the two different growth states. The dimeric, 14 kDa form of the peripheral membrane peridinin-chlorophyll a binding protein predominates under endosymbiotic conditions, whereas the monomeric, 35more » kDa form predominates under the culture conditions used in this study. Antibodies to form II ribulose-1,5-bisphosphate carboxylase revealed 62 and 60 kDa forms of this protein in the alga grown as an endosymbiont and in culture, respectively. Differences in the integral membrane peridinin-chlorophyll a-c-binding proteins were also observed. These results demonstrate that there are major changes in the populations of proteins synthesized by Symbiodinium sp. in response to the conditions in hospite. Such changes may reflect a developmental switch that tailors the physiology of the alga to the conditions encountered in the endosymbiotic state. 77 refs., 6 figs.« less

Chloroplast and nuclear gene sequences indicate late Pennsylvanian time for the last common ancestor of extant seed plants.

PubMed Central

Savard, L; Li, P; Strauss, S H; Chase, M W; Michaud, M; Bousquet, J

1994-01-01

We have estimated the time for the last common ancestor of extant seed plants by using molecular clocks constructed from the sequences of the chloroplastic gene coding for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) and the nuclear gene coding for the small subunit of rRNA (Rrn18). Phylogenetic analyses of nucleotide sequences indicated that the earliest divergence of extant seed plants is likely represented by a split between conifer-cycad and angiosperm lineages. Relative-rate tests were used to assess homogeneity of substitution rates among lineages, and annual angiosperms were found to evolve at a faster rate than other taxa for rbcL and, thus, these sequences were excluded from construction of molecular clocks. Five distinct molecular clocks were calibrated using substitution rates for the two genes and four divergence times based on fossil and published molecular clock estimates. The five estimated times for the last common ancestor of extant seed plants were in agreement with one another, with an average of 285 million years and a range of 275-290 million years. This implies a substantially more recent ancestor of all extant seed plants than suggested by some theories of plant evolution. PMID:8197201

Occurrence of a number of enzymes involved in either gluconeogenesis or other processes in the pericarp of three cultivars of grape (Vitis vinifera L.) during development.

PubMed

Famiani, Franco; Moscatello, Stefano; Ferradini, Nicoletta; Gardi, Tiziano; Battistelli, Alberto; Walker, Robert P

2014-11-01

It is uncertain whether the enzymes pyruvate orthophosphate dikinase (PPDK) or isocitrate lyase (ICL) are present in the pericarp of grape, in which they could function in gluconeogenesis. The occurrence of these and other enzymes was investigated in the pericarp of three cultivars of grape (Vitis vinifera L.). In particular, the abundance of the enzymes aldolase, glutamine synthase (GS), acid invertase, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), phosphoenolpyruvate carboxylase (PEPC), PPDK and ICL were determined during the development of the pericarp of the cultivars Cabernet Sauvignon, Chardonnay and Zibibbo. PPDK and ICL were not detected at any stage of development. Each of the other enzymes showed different changes in abundance during development. However, for a given enzyme its changes in abundance were similar in each cultivar. In the ripe pericarp of Cabernet Sauvignon, PEPC, cytosolic GS and aldolase were equally distributed between the vasculature and parenchyma cells of the flesh and skin. The absence or very low abundance of PPDK provides strong evidence that any gluconeogenesis from malate utilises phosphoenolpyruvate carboxykinase (PEPCK). The absence or very low abundance of ICL in the pericarp precludes any gluconeogenesis from ethanol. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Fe deficiency induced changes in rice (Oryza sativa L.) thylakoids.

PubMed

Wang, Yuwen; Xu, Chao; Li, Kang; Cai, Xiaojie; Wu, Min; Chen, Guoxiang

2017-01-01

Iron deficiency is an important abiotic stress that limits productivity of crops all over the world. We selected a hybrid rice (Oryza sativa L.), LYPJ, which is super high-yield and widely cultured in China, to investigate changes in the components and structure of thylakoid membranes and photosynthetic performance in response to iron deficiency. Our results demonstrated that photosystem I (PSI) is the primary target for iron deficiency, while the changes in photosystem II (PSII) are important for rebuilding a balance in disrupted energy utilization and dissipation caused by differential degradation of photosynthetic components. The result of immunoblot analysis suggested that the core subunit PsaA declined drastically, while PsbA remained relatively stable. Furthermore, several organizational changes of the photosynthetic apparatus were found by BN-PAGE, including a marked decrease in the PSI core complexes, the Cytb 6 /f complex, and the trimeric form of the LHCII antenna, consistent with the observed unstacking grana. The fluorescence induction analysis indicated a descending PSII activity with energy dissipation enhanced markedly. In addition, we proposed that the crippled CO 2 assimilation could be compensated by the enhanced of phosphoenolpyruvate carboxylase (PEPC), which is suggested by the decreased ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and photosynthetic efficiency.

RHON1 mediates a Rho-like activity for transcription termination in plastids of Arabidopsis thaliana.

PubMed

Chi, Wei; He, Baoye; Manavski, Nikolay; Mao, Juan; Ji, Daili; Lu, Congming; Rochaix, Jean David; Meurer, Jörg; Zhang, Lixin

2014-12-01

Although transcription termination is essential to generate functional RNAs, its underlying molecular mechanisms are still poorly understood in plastids of vascular plants. Here, we show that the RNA binding protein RHON1 participates in transcriptional termination of rbcL (encoding large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase) in Arabidopsis thaliana. Inactivation of RHON1 leads to enhanced rbcL read-through transcription and to aberrant accD (encoding β-subunit of the acetyl-CoA carboxylase) transcriptional initiation, which may result from inefficient transcription termination of rbcL. RHON1 can bind to the mRNA as well as to single-stranded DNA of rbcL, displays an RNA-dependent ATPase activity, and terminates transcription of rbcL in vitro. These results suggest that RHON1 terminates rbcL transcription using an ATP-driven mechanism similar to that of Rho of Escherichia coli. This RHON1-dependent transcription termination occurs in Arabidopsis but not in rice (Oryza sativa) and appears to reflect a fundamental difference between plastomes of dicotyledonous and monocotyledonous plants. Our results point to the importance and significance of plastid transcription termination and provide insights into its machinery in an evolutionary context. © 2014 American Society of Plant Biologists. All rights reserved.

Sequences 5' to translation start regulate expression of petunia rbcS genes.

PubMed Central

Dean, C; Favreau, M; Bedbrook, J; Dunsmuir, P

1989-01-01

The promoter sequences that contribute to quantitative differences in expression of the petunia genes (rbcS) encoding the small subunit of ribulose bisphosphate carboxylase have been characterized. The promoter regions of the two most abundantly expressed petunia rbcS genes, SSU301 and SSU611, show sequence similarity not present in other rbcS genes. We investigated the significance of these and other sequences by adding specific regions from the SSU301 promoter (the most strongly expressed gene) to equivalent regions in the SSU911 promoter (the least strongly expressed gene) and assaying the expression of the fusions in transgenic tobacco plants. In this way, we characterized an SSU301 promoter region (either from -285 to -178 or -291 to -204) which, when added to SSU911, in either orientation, increased SSU911 expression 25-fold. This increase was equivalent to that caused by addition of the entire SSU301 5'-flanking region. Replacement of SSU911 promoter sequences between -198 and the start codon with sequences from the equivalent region of SSU301 did not increase SSU911 expression significantly. The -291 to -204 SSU301 promoter fragment contributes significantly to quantitative differences in expression between the petunia rbcS genes. PMID:2535543

Engineering Rubisco activase from thermophilic cyanobacteria into high-temperature sensitive plants.

PubMed

Ogbaga, Chukwuma C; Stepien, Piotr; Athar, Habib-Ur-Rehman; Ashraf, Muhammad

2018-06-01

In the past decade, various strategies to improve photosynthesis and crop yield, such as leaf morphology, light interception and use efficiency, biochemistry of light reactions, stomatal conductance, carboxylation efficiency, and source to sink regulation, have been discussed at length. Leaf morphology and physiology are tightly coupled to light capturing efficiency, gas exchange capacity, and temperature regulation. However, apart from the photoprotective mechanism of photosystem-II (PSII), i.e. non-photochemical quenching, very low genetic variation in the components of light reactions has been observed in plants. In the last decade, biochemistry-based enhancement of carboxylation efficiency that improves photosynthesis in plants was one of the potential strategies for improving plant biomass production. Enhancement of activation of the ubiquitous enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) by Rubisco activase may be another potential strategy for improving a photosynthesis-driven increase in crop yield. Rubisco activase modifies the conformation of the active center in Rubisco by removing tightly bound inhibitors, thereby contributing to enzyme activation and rapid carboxylation. Thermophilic cyanobacteria are oxygenic photosynthetic bacteria that thrive in high-temperature environments. This critical review discusses the prospects for and the potential of engineering Rubisco activase from thermophilic cyanobacteria into temperature-sensitive plants, to increase the threshold temperature and survival of these plants in arid regions.

Effects of 60 Hz sinusoidal magnetic field on in vitro establishment, multiplication, and acclimatization phases of Coffea arabica seedlings.

PubMed

Isaac Alemán, Elizabeth; Oliveira Moreira, Rafael; Almeida Lima, Andre; Chaves Silva, Samuel; González-Olmedo, Justo Lorenzo; Chalfun-Junior, Antonio

2014-09-01

The influence of extremely low frequency electromagnetic fields on net photosynthesis, transpiration, photosynthetic pigment concentration, and gene expression of ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit (RBCS1), during in vitro establishment, in vitro multiplication and acclimatization phases of coffee seedlings were investigated. Untreated coffee plants were considered as control, whereas treated plants were exposed to a 60 Hz sinusoidal magnetic field of 2 mT of magnetic induction during 3 min. This magnetic field was generated by an electromagnet, connected to a wave generator. The results revealed that magnetically treated plants showed a significant increase in net photosynthesis (85.4% and 117.9%, in multiplication and acclimatization phases, respectively), and in photosynthetic pigment concentration (66.6% for establishment phase, 79.9% for multiplication phase, and 43.8% for acclimatization phase). They also showed a differential RBCS1 gene expression (approximately twofold) and a decrease of transpiration rates in regard to their control plants. In conclusion, the findings suggest that the application of 60 Hz magnetic field to in vitro coffee plants may improve the seedlings quality by modifying some photosynthetic physiological and molecular processes, increasing their vigor, and ensuring better plant development in later stages. © 2014 Wiley Periodicals, Inc.

Metabolism of d-Arabinose: Origin of a d-Ribulokinase Activity in Escherichia coli1

PubMed Central

LeBlanc, Donald J.; Mortlock, Robert P.

1971-01-01

The kinase responsible for the phosphorylation of d-ribulose was purified 45.5-fold from a strain of Escherichia coli K-12 capable of growth on d-arabinose with no separation of d-ribulo- or l-fuculokinase activities. Throughout the purification, the ratios of activities remained essentially constant. A nonadditive effect of combining both substrates in an assay mixture; identical Km values for adenosine triphosphate with either l-fuculose or d-ribulose as substrate; and, the irreversible loss of activity on both substrates, after removal of magnesium ions from the enzyme preparation, suggest that the dual activity is due to the same enzyme. A fourfold greater affinity of the enzyme for l-fuculose than for d-ribulose, as well as a higher relative activity on l-fuculose, suggest that the natural substrate for this enzyme is l-fuculose. The product of the purified enzyme, with d-ribulose as substrate, was prepared. The ratio of total phosphorous to ribulose phosphate was 1.01:1, indicating that the product was ribulose monophosphate. The behavior of the kinase product in the cysteine-carbazole and orcinol reactions, as well as the results of periodate oxidation assays, provided evidence that it was not d-ribulose-5-phosphate. Reaction of this compound with a cell-free extract of E. coli possessing l-fuculose-l-phosphate aldolase activity resulted in the production of dihydroxyacetone phosphate and glycolaldehyde. The kinase product failed to reduce 2,3,5-triphenyltetrazolium and possessed a half-life of approximately 1.5 min in the presence of 1 n HCl at 100 C. These properties suggested that the phosphate group was attached to carbon atom 1 of d-ribulose. PMID:4323967

Properties of ribulose diphosphate carboxylase immobilized on porous glass

NASA Technical Reports Server (NTRS)

Shapira, J.; Hanson, C. L.; Lyding, J. M.; Reilly, P. J.

1974-01-01

Ribulose-1,5-diphosphate carboxylase from spinach has been bound to arylamine porous glass with a diazo linkage and to alklamine porous glass with glutaraldehyde. Stability at elevated temperatures and responses to changes of pH and ribulose-1,5-diphosphate, Mg(2+), and dithiothreitol concentrations were not significantly different from the soluble enzyme, though stability at 4 C was somewhat improved.

[Response and adaptation of photosynthesis of cucumber seedlings to high temperature stress].

PubMed

Sun, Sheng Nan; Wang, Qiang; Sun, Chen Chen; Liu, Feng Jiao; Bi, Huan Gai; Ai, Xi Zhen

2017-05-18

Cucumber seedlings (Cucumis sativus Jinyou 35) were used to study the effects of high temperature (HT: 42 ℃/32 ℃) and sub-high temperature (SHT: 35 ℃/25 ℃) on its photosynthesis and growth. The results showed that the growth of cucumber seedlings was dramatically inhibited by the high and sub-high temperature stresses. The photosynthetic rate (P n ) was gradually reduced, while intercellular CO 2 concentration (C i ) was increased as heat stress lasted. Under heat stress, stomatal conductance (g s ), transpiration rate (T r ), photorespiration rate (P r ) and dark respiration rate (D r ) showed a trend from rise to decline in cucumber seedlings, which implied that heat-induced decline of photosynthesis was mainly due to non-stomatal limitation. Maximal photochemical efficiency of PS2 in darkness (F v /F m ), actual photochemical efficiency (χ PS 2 ), photochemical quenching (q P ) and electron transport rate (ETR) were severely hampered, while initial fluorescence (F o ) and non-chemical quenching (NPQ) were increased as a result of high and sub-high temperature stresses. Under extended high temperature stress, the activities of RuBP carboxylase (RuBPCase) and Rubisco activase (RCA) as well as the mRNA abundance of Rubisco and RCA were in the trend of decrease, while they were reduced 3 days following the sub-high temperature treatment. The activities and mRNA expressions of sedoheptulose-1,7-bisphosphatase (SBPase) and fructose 1,6-bisphosphate aldolase (FBA) increased initially, but decreased afterwards under heat stress. Taken together, our data suggested that short-term sub-high temperature did not cause photoinhibition under optimal light conditions, however, high temperature led to severe damage to PS2 reaction center in cucumber seedlings. The photosynthetic enzymes were induced by high temperature stress and the induction was affected by temperature and stress duration.

Photorespiration plays an important role in the regulation of photosynthetic electron flow under fluctuating light in tobacco plants grown under full sunlight

PubMed Central

Huang, Wei; Hu, Hong; Zhang, Shi-Bao

2015-01-01

Plants usually experience dynamic fluctuations of light intensities under natural conditions. However, the responses of mesophyll conductance, CO2 assimilation, and photorespiration to light fluctuation are not well understood. To address this question, we measured photosynthetic parameters of gas exchange and chlorophyll fluorescence in tobacco leaves at 2-min intervals while irradiance levels alternated between 100 and 1200 μmol photons m−2 s−1. Compared with leaves exposed to a constant light of 1200 μmol photons m−2 s−1, both stomatal and mesophyll conductances were significantly restricted in leaves treated with fluctuating light condition. Meanwhile, CO2 assimilation rate and electron flow devoted to RuBP carboxylation at 1200 μmol photons m−2 s−1 under fluctuating light were limited by the low chloroplast CO2 concentration. Analysis based on the C3 photosynthesis model indicated that, at 1200 μmol photons m−2 s−1 under fluctuating light, the CO2 assimilation rate was limited by RuBP carboxylation. Electron flow devoted to RuBP oxygenation at 1200 μmol photons m−2 s−1 under fluctuating light remained at nearly the maximum level throughout the experimental period. We conclude that fluctuating light restricts CO2 assimilation by decreasing both stomatal and mesophyll conductances. Under such conditions, photorespiration plays an important role in the regulation of photosynthetic electron flow. PMID:26322062

Characterization of ribulose diphosphate carboxylase and phosphoribulokinase from Thiobacillus thioparus and Thiobacillus neapolitanus.

NASA Technical Reports Server (NTRS)

Johnson, E. J.; Johnson, M. K.; Macelroy, R. D.

1968-01-01

Ribulose diphosphate carboxylase and phosphoribulokinase activity in chemosynthetic autotrophs Thiobacillus thioparus and Thiobacillus neapolitanus, noting sedimentation and gel filtration characteristics

Characterization of herbaspirillum- and limnobacter-related strains isolated from young volcanic deposits in miyake-jima island, Japan.

PubMed

Lu, Hongsheng; Fujimura, Reiko; Sato, Yoshinori; Nanba, Kenji; Kamijo, Takashi; Ohta, Hiroyuki

2008-01-01

The role of microbes in the early development of ecosystems on new volcanic materials seems to be crucial to primary plant succession but is not well characterized. Here we analyzed the bacterial community colonizing 22-year-old volcanic deposits of the Miyake-jima Island (Japan) using culture-based and 16S rRNA gene clone library methods. The majority of 91 bacterial isolates were placed phylogenetically in two clusters (A and B) of the Betaproteobacteria. Cluster A (82% of isolates) was related to the genus Limnobacter and Cluster B (9%) was affiliated with the Herbaspirillum clade. The clone library analysis supported the predominance of Cluster B rather than Cluster A. Strain KP1-50 of Cluster B was able to grow on a mineral medium under an atmosphere of H(2), O(2), and CO(2) (85:5:10), and characterized by its large-subunit gene of ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcL) and nitrogenase reductase gene (nifH). In contrast, strains of Cluster A did not grow chemolithoautotrophically with H(2), O(2), and CO(2) but increased their cell biomass with the addition of thiosulfate to the succinate medium, suggesting the use of thiosulfate as an energy source. From phenotypic characterization, it was suggested that the Cluster A and B strains were novel species in the genus Limnobacter and Herbaspirillum, respectively.

Gene activity during germination of spores of the fern, Onoclea sensibilis. Cell-free translation analysis of mRNA of spores and the effect of alpha-amanitin on spore germination

NASA Technical Reports Server (NTRS)

Raghavan, V.

1992-01-01

Poly(A)-RNA fractions of dormant, dark-imbibed (non-germinating) and photoinduced (germinating) spores of Onoclea sensibilis were poor templates in the rabbit reticulocyte lysate protein synthesizing system, but the translational efficiency of poly(A)+RNA was considerably higher than that of unfractionated RNA. Poly(A)+RNA isolated from photoinduced spores had a consistently higher translational efficiency than poly(A)+RNA from dark-imbibed spores. Analysis of the translation products by one-dimensional polyacrylamide gel electrophoresis showed no qualitative differences in the mRNA populations of dormant, dark-imbibed, and photoinduced spores. However, poly(A)+RNA from dark-imbibed spores appeared to encode in vitro fewer detectable polypeptides at a reduced intensity than photoinduced spores. A DNA clone encoding the large subunit of maize ribulose bisphosphate carboxylase hybridized at strong to moderate intensity to RNA isolated from dark-imbibed spores, indicating the absence of mRNA degradation. Although alpha-amanitin did not inhibit the germination of spores, the drug prevented the elongation of the rhizoid and protonemal initial with a concomitant effect on the synthesis of poly(A)+RNA. These results are consistent with the view that some form of translational control involving stored mRNA operates during dark-imbibition and photoinduced germination of spores.

Photorespiration Is Crucial for Dynamic Response of Photosynthetic Metabolism and Stomatal Movement to Altered CO2 Availability.

PubMed

Eisenhut, Marion; Bräutigam, Andrea; Timm, Stefan; Florian, Alexandra; Tohge, Takayuki; Fernie, Alisdair R; Bauwe, Hermann; Weber, Andreas P M

2017-01-09

The photorespiratory pathway or photorespiration is an essential process in oxygenic photosynthetic organisms, which can reduce the efficiency of photosynthetic carbon assimilation and is hence frequently considered as a wasteful process. By comparing the response of the wild-type plants and mutants impaired in photorespiration to a shift in ambient CO 2 concentrations, we demonstrate that photorespiration also plays a beneficial role during short-term acclimation to reduced CO 2 availability. The wild-type plants responded with few differentially expressed genes, mostly involved in drought stress, which is likely a consequence of enhanced opening of stomata and concomitant water loss upon a shift toward low CO 2 . In contrast, mutants with impaired activity of photorespiratory enzymes were highly stressed and not able to adjust stomatal conductance to reduced external CO 2 availability. The transcriptional response of mutant plants was congruent, indicating a general reprogramming to deal with the consequences of reduced CO 2 availability, signaled by enhanced oxygenation of ribulose-1,5-bisphosphate and amplified by the artificially impaired photorespiratory metabolism. Central in this reprogramming was the pronounced reallocation of resources from growth processes to stress responses. Taken together, our results indicate that unrestricted photorespiratory metabolism is a prerequisite for rapid physiological acclimation to a reduction in CO 2 availability. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

Algal evolution in relation to atmospheric CO2: carboxylases, carbon-concentrating mechanisms and carbon oxidation cycles

PubMed Central

Raven, John A.; Giordano, Mario; Beardall, John; Maberly, Stephen C.

2012-01-01

Oxygenic photosynthesis evolved at least 2.4 Ga; all oxygenic organisms use the ribulose bisphosphate carboxylase-oxygenase (Rubisco)–photosynthetic carbon reduction cycle (PCRC) rather than one of the five other known pathways of autotrophic CO2 assimilation. The high CO2 and (initially) O2-free conditions permitted the use of a Rubisco with a high maximum specific reaction rate. As CO2 decreased and O2 increased, Rubisco oxygenase activity increased and 2-phosphoglycolate was produced, with the evolution of pathways recycling this inhibitory product to sugar phosphates. Changed atmospheric composition also selected for Rubiscos with higher CO2 affinity and CO2/O2 selectivity correlated with decreased CO2-saturated catalytic capacity and/or for CO2-concentrating mechanisms (CCMs). These changes increase the energy, nitrogen, phosphorus, iron, zinc and manganese cost of producing and operating Rubisco–PCRC, while biosphere oxygenation decreased the availability of nitrogen, phosphorus and iron. The majority of algae today have CCMs; the timing of their origins is unclear. If CCMs evolved in a low-CO2 episode followed by one or more lengthy high-CO2 episodes, CCM retention could involve a combination of environmental factors known to favour CCM retention in extant organisms that also occur in a warmer high-CO2 ocean. More investigations, including studies of genetic adaptation, are needed. PMID:22232762

The effect of silicon on the leaf proteome of rice (Oryza sativa L.) plants under cadmium-stress.

PubMed

Nwugo, Chika C; Huerta, Alfredo J

2011-02-04

The best known silicon (Si)-accumulating plant, rice (Oryza sativa L.), stores most of its Si in leaves, but the importance of Si has been limited to a mechanical role. Our initial studies showed that Si-induced cadmium (Cd) tolerance is mediated by the enhancement of instantaneous water-use-efficiency, carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO), and light-use-efficiency in leaves of rice plants. In this study, we investigated changes in the rice leaf proteome in order to identify molecular mechanisms involved in Si-induced Cd tolerance. Our study identified 60 protein spots that were differentially regulated due to Cd and/or Si treatments. Among them, 50 were significantly regulated by Si, including proteins associated with photosynthesis, redox homeostasis, regulation/protein synthesis, pathogen response and chaperone activity. Interestingly, we observed a Si-induced up-regulation of a class III peroxidase and a thaumatin-like protein irrespective of Cd treatment, in addition to a Cd-induced up-regulation of protein disulfide isomerase, a HSP70 homologue, a NADH-ubiquinone oxidoreductase, and a putative phosphogluconate dehydrogenase, especially in the presence of Si. Taken together, our study sheds light on molecular mechanisms involved in Si-induced Cd tolerance in rice leaves and suggests a more active involvement of Si in plant physiological processes than previously proposed.

S-nitrosoglutathione spraying improves stomatal conductance, Rubisco activity and antioxidant defense in both leaves and roots of sugarcane plants under water deficit.

PubMed

Silveira, Neidiquele M; Marcos, Fernanda C C; Frungillo, Lucas; Moura, Bárbara B; Seabra, Amedea B; Salgado, Ione; Machado, Eduardo C; Hancock, John T; Ribeiro, Rafael V

2017-08-01

Water deficit is a major environmental constraint on crop productivity and performance and nitric oxide (NO) is an important signaling molecule associated with many biochemical and physiological processes in plants under stressful conditions. This study aims to test the hypothesis that leaf spraying of S-nitrosoglutathione (GSNO), an NO donor, improves the antioxidant defense in both roots and leaves of sugarcane plants under water deficit, with positive consequences for photosynthesis. In addition, the roles of key photosynthetic enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC) in maintaining CO 2 assimilation of GSNO-sprayed plants under water deficit were evaluated. Sugarcane plants were sprayed with water or GSNO 100 μM and subjected to water deficit, by adding polyethylene glycol (PEG-8000) to the nutrient solution. Sugarcane plants supplied with GSNO presented increases in the activity of antioxidant enzymes such as superoxide dismutase in leaves and catalase in roots, indicating higher antioxidant capacity under water deficit. Such adjustments induced by GSNO were sufficient to prevent oxidative damage in both organs and were associated with better leaf water status. As a consequence, GSNO spraying alleviated the negative impact of water deficit on stomatal conductance and photosynthetic rates, with plants also showing increases in Rubisco activity under water deficit. © 2017 Scandinavian Plant Physiology Society.

Depletion of abundant plant RuBisCO protein using the protamine sulfate precipitation method.

PubMed

Kim, Yu Ji; Lee, Hye Min; Wang, Yiming; Wu, Jingni; Kim, Sang Gon; Kang, Kyu Young; Park, Ki Hun; Kim, Yong Chul; Choi, In Soo; Agrawal, Ganesh Kumar; Rakwal, Randeep; Kim, Sun Tae

2013-07-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant plant leaf protein, hampering deep analysis of the leaf proteome. Here, we describe a novel protamine sulfate precipitation (PSP) method for the depletion of RuBisCO. For this purpose, soybean leaf total proteins were extracted using Tris-Mg/NP-40 extraction buffer. Obtained clear supernatant was subjected to the PSP method, followed by 13% SDS-PAGE analysis of total, PS-supernatant and -precipitation derived protein samples. In a dose-dependent experiment, 0.1% w/v PS was found to be sufficient for precipitating RuBisCO large and small subunits (LSU and SSU). Western blot analysis confirmed no detection of RuBisCO LSU in the PS-supernatant proteins. Application of this method to Arabidopsis, rice, and maize leaf proteins revealed results similar to soybean. Furthermore, 2DE analyses of PS-treated soybean leaf displayed enriched protein profile for the protein sample derived from the PS-supernatant than total proteins. Some enriched 2D spots were subjected to MALDI-TOF-TOF analysis and were successfully assigned for their protein identity. Hence, the PSP method is: (i) simple, fast, economical, and reproducible for RuBisCO precipitation from the plant leaf sample; (ii) applicable to both dicot and monocot plants; and (iii) suitable for downstream proteomics analysis. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sutter, Markus; Roberts, Evan W.; Gonzalez, Raul C.

Carboxysomes are bacterial microcompartments that enhance carbon fixation by concentrating ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its substrate CO 2 within a proteinaceous shell. They are found in all cyanobacteria, some purple photoautotrophs and many chemoautotrophic bacteria. Carboxysomes consist of a protein shell that encapsulates several hundred molecules of RuBisCO, and contain carbonic anhydrase and other accessory proteins. Genes coding for carboxysome shell components and the encapsulated proteins are typically found together in an operon. The α-carboxysome operon is embedded in a cluster of additional, conserved genes that are presumably related to its function. In many chemoautotrophs, products of the expanded carboxysomemore » locus include CbbO and CbbQ, a member of the AAA+ domain superfamily. We bioinformatically identified subtypes of CbbQ proteins and show that their genes frequently co-occur with both Form IA and Form II RuBisCO. The α-carboxysome-associated ortholog, CsoCbbQ, from Halothiobacillus neapolitanus forms a hexamer in solution and hydrolyzes ATP. The crystal structure shows that CsoCbbQ is a hexamer of the typical AAA+ domain; the additional C-terminal domain, diagnostic of the CbbQ subfamily, structurally fills the inter-monomer gaps, resulting in a distinctly hexagonal shape. Finally, we show that CsoCbbQ interacts with CsoCbbO and is a component of the carboxysome shell, the first example of ATPase activity associated with a bacterial microcompartment.« less

The plant G box promoter sequence activates transcription in Saccharomyces cerevisiae and is bound in vitro by a yeast activity similar to GBF, the plant G box binding factor.

PubMed Central

Donald, R G; Schindler, U; Batschauer, A; Cashmore, A R

1990-01-01

G box and I box sequences of the Arabidopsis thaliana ribulose-bisphosphate-1,5-carboxylase small subunit (RBCS) promoter are required for expression mediated by the Arabidopsis rbcS-1A promoter in transgenic tobacco plants and are bound in vitro by factors from plant nuclear extracts termed GBF and GA-1, respectively. We show here that a -390 to -60 rbcS-1A promoter fragment containing the G box and two I boxes activates transcription from a truncated iso-1-cytochrome c (CYC1) gene promoter in Saccharomyces cerevisiae. Mutagenesis of either the rbcS-1A G box or both I box sequences eliminated the expression mediated by this fragment. When polymerized, I box oligonucleotides were also capable of enhancing expression from the truncated CYC1 promoter. Single-copy G box sequences from the Arabidopsis rbcS-1A, Arabidopsis Adh and tomato rbcS-3A promoters were more potent activators and were used in mobility shift assays to identify a DNA binding activity in yeast functionally similar to GBF. In methylation interference experiments, the binding specificity of the yeast protein was indistinguishable from that obtained with plant nuclear extracts. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:2161333

Cytonuclear Coordination Is Not Immediate upon Allopolyploid Formation in Tragopogon miscellus (Asteraceae) Allopolyploids

PubMed Central

Sehrish, Tina; Symonds, V. Vaughan; Soltis, Douglas E.; Soltis, Pamela S.; Tate, Jennifer A.

2015-01-01

Allopolyploids, formed by hybridization and chromosome doubling, face the immediate challenge of having duplicated nuclear genomes that interact with the haploid and maternally inherited cytoplasmic (plastid and mitochondrial) genomes. Most of our knowledge of the genomic consequences of allopolyploidy has focused on the fate of the duplicated nuclear genes without regard to their potential interactions with cytoplasmic genomes. As a step toward understanding the fates of nuclear-encoded subunits that are plastid-targeted, here we examine the retention and expression of the gene encoding the small subunit of Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco; rbcS) in multiple populations of allotetraploid Tragopogon miscellus (Asteraceae). These polyploids formed recently (~80 years ago) and repeatedly from T. dubius and T. pratensis in the northwestern United States. Examination of 79 T. miscellus individuals from 10 natural populations, as well as 25 synthetic allotetraploids, including reciprocally formed plants, revealed a low percentage of naturally occurring individuals that show a bias in either gene (homeolog) loss (12%) or expression (16%), usually toward maintaining the maternal nuclear copy of rbcS. For individuals showing loss, seven retained the maternally derived rbcS homeolog only, while three had the paternally derived copy. All of the synthetic polyploid individuals examined (S0 and S1 generations) retained and expressed both parental homeologs. These results demonstrate that cytonuclear coordination does not happen immediately upon polyploid formation in Tragopogon miscellus. PMID:26646761

Cellular expression of C3 and C4 photosynthetic enzymes in the amphibious sedge Eleocharis retroflexa ssp. chaetaria.

PubMed

Ueno, Osamu; Wakayama, Masataka

2004-12-01

The amphibious leafless sedge Eleocharis retroflexa ssp. chaetaria expresses C(4)-like biochemical characteristics in both the terrestrial and submerged forms. Culms of the terrestrial form have Kranz anatomy, whereas those of the submerged form have Kranz-like anatomy combined with anatomical features of aquatic plant leaves. We examined the immunolocalization of C(3) and C(4) enzymes in culms of the two forms. In both forms, phosphoenolpyruvate carboxylase; pyruvate, Pi dikinase; and NAD-malic enzyme were compartmentalized between the mesophyll (M) and Kranz cells, but their levels were somewhat reduced in the submerged form. In the terrestrial form, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) occurred mainly in the Kranz cells, and weakly in the M chloroplasts. In the submerged form, the rubisco occurred at higher levels in the M cells than in the terrestrial form. In both forms, the C(4) pattern of enzyme expression was clearer in the M cells adjacent to Kranz cells than in distant M cells. During the transition from terrestrial to submerged conditions, the enzyme expression pattern changed in submerged mature culms that had been formed in air before submergence, and matched that in culms newly developed underwater. It seems that effects of both environmental and developmental factors overlap in the C(4) pattern expression in this plant.

Unravelling the Carbon and Sulphur Metabolism in Coastal Soil Ecosystems Using Comparative Cultivation-Independent Genome-Level Characterisation of Microbial Communities

PubMed Central

Yousuf, Basit; Kumar, Raghawendra; Mishra, Avinash; Jha, Bhavanath

2014-01-01

Bacterial autotrophy contributes significantly to the overall carbon balance, which stabilises atmospheric CO2 concentration and decelerates global warming. Little attention has been paid to different modes of carbon/sulphur metabolism mediated by autotrophic bacterial communities in terrestrial soil ecosystems. We studied these pathways by analysing the distribution and abundance of the diagnostic metabolic marker genes cbbM, apsA and soxB, which encode for ribulose-1,5-bisphosphate carboxylase/oxygenase, adenosine phosphosulphate reductase and sulphate thiohydrolase, respectively, among different contrasting soil types. Additionally, the abundance of community members was assessed by quantifying the gene copy numbers for 16S rRNA, cbbL, cbbM, apsA and soxB. Distinct compositional differences were observed among the clone libraries, which revealed a dominance of phylotypes associated with carbon and sulphur cycling, such as Gammaproteobacteria (Thiohalomonas, Allochromatium, Chromatium, Thiomicrospira) and Alphaproteobacteria (Rhodopseudomonas, Rhodovulum, Paracoccus). The rhizosphere soil was devoid of sulphur metabolism, as the soxB and apsA genes were not observed in the rhizosphere metagenome, which suggests the absence or inadequate representation of sulphur-oxidising bacteria. We hypothesise that the novel Gammaproteobacteria sulphur oxidisers might be actively involved in sulphur oxidation and inorganic carbon fixation, particularly in barren saline soil ecosystems, suggesting their significant putative ecological role and contribution to the soil carbon pool. PMID:25225969

Understanding the physiological roles of polyhydroxybutyrate (PHB) in Rhodospirillum rubrum S1 under aerobic chemoheterotrophic conditions.

PubMed

Narancic, Tanja; Scollica, Elisa; Kenny, Shane T; Gibbons, Helena; Carr, Eibhlin; Brennan, Lorraine; Cagney, Gerard; Wynne, Kieran; Murphy, Cormac; Raberg, Matthias; Heinrich, Daniel; Steinbüchel, Alexander; O'Connor, Kevin E

2016-10-01

Polyhydroxybutyrate (PHB) is an important biopolymer accumulated by bacteria and associated with cell survival and stress response. Here, we make two surprising findings in the PHB-accumulating species Rhodospirillum rubrum S1. We first show that the presence of PHB promotes the increased assimilation of acetate preferentially into biomass rather than PHB. When R. rubrum is supplied with (13)C-acetate as a PHB precursor, 83.5 % of the carbon in PHB comes from acetate. However, only 15 % of the acetate ends up in PHB with the remainder assimilated as bacterial biomass. The PHB-negative mutant of R. rubrum assimilates 2-fold less acetate into biomass compared to the wild-type strain. Acetate assimilation proceeds via the ethylmalonyl-CoA pathway with (R)-3-hydroxybutyrate as a common intermediate with the PHB pathway. Secondly, we show that R. rubrum cells accumulating PHB have reduced ribulose 1,5-bisphosphate carboxylase (RuBisCO) activity. RuBisCO activity reduces 5-fold over a 36-h period after the onset of PHB. In contrast, a PHB-negative mutant maintains the same level of RuBisCO activity over the growth period. Since RuBisCO controls the redox potential in R. rubrum, PHB likely replaces RuBisCO in this role. R. rubrum is the first bacterium found to express RuBisCO under aerobic chemoheterotrophic conditions.

Manipulation of the hypocotyl sink activity by reciprocal grafting of two Raphanus sativus varieties: its effects on morphological and physiological traits of source leaves and whole-plant growth.

PubMed

Sugiura, Daisuke; Betsuyaku, Eriko; Terashima, Ichiro

2015-12-01

To reveal whether hypocotyl sink activities are regulated by the aboveground parts, and whether physiology and morphology of source leaves are affected by the hypocotyl sink activities, we conducted grafting experiments using two Raphanus sativus varieties with different hypocotyl sink activities. Comet (C) and Leafy (L) varieties with high and low hypocotyl sink activities were reciprocally grafted and resultant plants were called by their scion and stock such as CC, LC, CL and LL. Growth, leaf mass per area (LMA), total non-structural carbohydrates (TNCs) and photosynthetic characteristics were compared among them. Comet hypocotyls in CC and LC grew well regardless of the scions, whereas Leafy hypocotyls in CL and LL did not. Relative growth rate was highest in LL and lowest in CC. Photosynthetic capacity was correlated with Rubisco (ribulose 1·5-bisphosphate carboxylase/oxygenase) content but unaffected by TNC. High C/N ratio and accumulation of TNC led to high LMA and structural LMA. These results showed that the hypocotyl sink activity was autonomously regulated by hypocotyl and that the down-regulation of photosynthesis was not induced by TNC. We conclude that the change in the sink activity alters whole-plant growth through the changes in both biomass allocation and leaf morphological characteristics in R. sativus. © 2015 John Wiley & Sons Ltd.

Long-distance transport of mRNA via parenchyma cells and phloem across the host-parasite junction in Cuscuta.

PubMed

David-Schwartz, Rakefet; Runo, Steven; Townsley, Brad; Machuka, Jesse; Sinha, Neelima

2008-01-01

It has been shown that the parasitic plant dodder (Cuscuta pentagona) establishes a continuous vascular system through which water and nutrients are drawn. Along with solutes, viruses and proteins, mRNA transcripts are transported from the host to the parasite. The path of the transcripts and their stability in the parasite have yet to be revealed. To discover the route of mRNA transportation, the in situ reverse transcriptase-polymerase chain reaction (RT-PCR) technique was used to locally amplify host transcript within parasitic tissue. The stability of host mRNA molecules was also checked by monitoring specific transcripts along the growing dodder thread. Four mRNAs, alpha and beta subunits of PYROPHOSPHATE (PPi)-DEPENDENT PHOSPHOFRUCTOKINASE (LePFP), the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), and GIBBERELLIC ACID INSENSITIVE (LeGAI), were found to move from host (tomato (Solanum lycopersicum)) to dodder. LePFP mRNA was localized to the dodder parenchyma cells and to the phloem. LePFP transcripts were found in the growing dodder stem up to 30 cm from the tomato-dodder connection. These results suggest that mRNA molecules are transferred from host to parasite via symplastic connections between parenchyma cells, move towards the phloem, and are stable for a long distance in the parasite. This may allow developmental coordination between the parasite and its host.

Evidence of form II RubisCO (cbbM) in a perennially ice-covered Antarctic lake.

PubMed

Kong, Weidong; Dolhi, Jenna M; Chiuchiolo, Amy; Priscu, John; Morgan-Kiss, Rachael M

2012-11-01

The permanently ice-covered lakes of the McMurdo Dry Valleys, Antarctica, harbor microbially dominated food webs. These organisms are adapted to a variety of unusual environmental extremes, including low temperature, low light, and permanently stratified water columns with strong chemo- and oxy-clines. Owing to the low light levels during summer caused by thick ice cover as well as 6 months of darkness during the polar winter, chemolithoautotrophic microorganisms could play a key role in the production of new carbon for the lake ecosystems. We used clone library sequencing and real-time quantitative PCR of the gene encoding form II Ribulose 1, 5-bisphosphate carboxylase/oxygenase to determine spatial and seasonal changes in the chemolithoautotrophic community in Lake Bonney, a 40-m-deep lake covered by c. 4 m of permanent ice. Our results revealed that chemolithoautotrophs harboring the cbbM gene are restricted to layers just above the chemo- and oxi-cline (≤ 15 m) in the west lobe of Lake Bonney (WLB). Our data reveal that the WLB is inhabited by a unique chemolithoautotrophic community that resides in the suboxic layers of the lake where there are ample sources of alternative electron sources such as ammonium, reduced iron and reduced biogenic sulfur species. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

An Excel tool for deriving key photosynthetic parameters from combined gas exchange and chlorophyll fluorescence: theory and practice.

PubMed

Bellasio, Chandra; Beerling, David J; Griffiths, Howard

2016-06-01

Combined photosynthetic gas exchange and modulated fluorometres are widely used to evaluate physiological characteristics associated with phenotypic and genotypic variation, whether in response to genetic manipulation or resource limitation in natural vegetation or crops. After describing relatively simple experimental procedures, we present the theoretical background to the derivation of photosynthetic parameters, and provide a freely available Excel-based fitting tool (EFT) that will be of use to specialists and non-specialists alike. We use data acquired in concurrent variable fluorescence-gas exchange experiments, where A/Ci and light-response curves have been measured under ambient and low oxygen. From these data, the EFT derives light respiration, initial PSII (photosystem II) photochemical yield, initial quantum yield for CO2 fixation, fraction of incident light harvested by PSII, initial quantum yield for electron transport, electron transport rate, rate of photorespiration, stomatal limitation, Rubisco (ribulose 1·5-bisphosphate carboxylase/oxygenase) rate of carboxylation and oxygenation, Rubisco specificity factor, mesophyll conductance to CO2 diffusion, light and CO2 compensation point, Rubisco apparent Michaelis-Menten constant, and Rubisco CO2 -saturated carboxylation rate. As an example, a complete analysis of gas exchange data on tobacco plants is provided. We also discuss potential measurement problems and pitfalls, and suggest how such empirical data could subsequently be used to parameterize predictive photosynthetic models. © 2015 John Wiley & Sons Ltd.

Mesophyll cells of C4 plants have fewer chloroplasts than those of closely related C3 plants.

PubMed

Stata, Matt; Sage, Tammy L; Rennie, Troy D; Khoshravesh, Roxana; Sultmanis, Stefanie; Khaikin, Yannay; Ludwig, Martha; Sage, Rowan F

2014-11-01

The evolution of C(4) photosynthesis from C(3) ancestors eliminates ribulose bisphosphate carboxylation in the mesophyll (M) cell chloroplast while activating phosphoenolpyruvate (PEP) carboxylation in the cytosol. These changes may lead to fewer chloroplasts and different chloroplast positioning within M cells. To evaluate these possibilities, we compared chloroplast number, size and position in M cells of closely related C(3), C(3) -C(4) intermediate and C(4) species from 12 lineages of C(4) evolution. All C(3) species had more chloroplasts per M cell area than their C(4) relatives in high-light growth conditions. C(3) species also had higher chloroplast coverage of the M cell periphery than C(4) species, particularly opposite intercellular air spaces. In M cells from 10 of the 12 C(4) lineages, a greater fraction of the chloroplast envelope was pulled away from the plasmalemma in the C(4) species than their C(3) relatives. C(3) -C(4) intermediate species generally exhibited similar patterns as their C(3) relatives. We interpret these results to reflect adaptive shifts that facilitate efficient C(4) function by enhancing diffusive access to the site of primary carbon fixation in the cytosol. Fewer chloroplasts in C(4) M cells would also reduce shading of the bundle sheath chloroplasts, which also generate energy required by C(4) photosynthesis. © 2014 John Wiley & Sons Ltd.

From milk to diet: feed recognition for milk authenticity.

PubMed

Ponzoni, E; Gianì, S; Mastromauro, F; Breviario, D

2009-11-01

The presence of plastidial DNA fragments of plant origin in animal milk samples has been confirmed. An experimental plan was arranged with 4 groups of goats, each provided with a different monophytic diet: 3 fresh forages (oats, ryegrass, and X-triticosecale) and one 2-wk-old silage (X-triticosecale). Feed-derived rubisco (ribulose bisphosphate carboxylase, rbcL) DNA fragments were detected in 100% of the analyzed goat milk samples, and the nucleotide sequence of the PCR-amplified fragments was found to be 100% identical to the corresponding fragments amplified from the plant species consumed in the diet. Two additional chloroplast-based molecular markers were used to set up an assay for distinctiveness, conveniently based on a simple PCR. In one case, differences in single nucleotides occurring within the gene encoding for plant maturase K (matK) were exploited. In the other, plant species recognition was based on the difference in the length of the intron present within the transfer RNA leucine (trnL) gene. The presence of plastidial plant DNA, ascertained by the PCR-based amplification of the rbcL fragment, was also assessed in raw cow milk samples collected directly from stock farms or taken from milk sold on the commercial market. In this case, the nucleotide sequence of the amplified DNA fragments reflected the multiple forages present in the diet fed to the animals.

Functional metagenomic selection of RubisCOs from uncultivated bacteria

USGS Publications Warehouse

Varaljay, Vanessa A; Satagopan, Sriram; North, Justin A.; Witteveen, Briana; Dourado, Manuella N.; Anantharaman, Karthik; Arbing, Mark A.; McCann, Shelley; Oremland, Ronald S.; Banfield, Jillian F.; Wrighton, Kelly C.; Tabita, F. Robert

2016-01-01

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a critical yet severely inefficient enzyme that catalyses the fixation of virtually all of the carbon found on Earth. Here, we report a functional metagenomic selection that recovers physiologically active RubisCO molecules directly from uncultivated and largely unknown members of natural microbial communities. Selection is based on CO2-dependent growth in a host strain capable of expressing environmental deoxyribonucleic acid (DNA), precluding the need for pure cultures or screening of recombinant clones for enzymatic activity. Seventeen functional RubisCO-encoded sequences were selected using DNA extracted from soil and river autotrophic enrichments, a photosynthetic biofilm and a subsurface groundwater aquifer. Notably, three related form II RubisCOs were recovered which share high sequence similarity with metagenomic scaffolds from uncultivated members of theGallionellaceae family. One of the Gallionellaceae RubisCOs was purified and shown to possessCO2/O2 specificity typical of form II enzymes. X-ray crystallography determined that this enzyme is a hexamer, only the second form II multimer ever solved and the first RubisCO structure obtained from an uncultivated bacterium. Functional metagenomic selection leverages natural biological diversity and billions of years of evolution inherent in environmental communities, providing a new window into the discovery of CO2-fixing enzymes not previously characterized.

Isolation and characterization of a Treponema pallidum major 60-kilodalton protein resembling the groEL protein of Escherichia coli.

PubMed Central

Houston, L S; Cook, R G; Norris, S J

1990-01-01

A native structure containing the major 60-kilodalton common antigen polypeptide (designated TpN60) was isolated from Treponema pallidum subsp. pallidum (Nichols strain) through a combination of differential centrifugation and sucrose density gradient sedimentation. Gel filtration chromatography indicated that this structure is a high-molecular-weight homo-oligomer of TpN60. Antisera to TpN60 reacted with the groEL polypeptide of Escherichia coli, as determined by immunoperoxidase staining of two-dimensional electroblots. Electron microscopy of the isolated complex revealed a ringlike structure with a diameter of approximately 16 nm which was very similar in appearance to the groEL protein. Comparison of the N-terminal amino acid sequence of TpN60 with the deduced sequences of the E. coli groEL protein, related chaperonin proteins from mycobacteria and Coxiella burnetti, the hsp60 protein of Saccharomyces cerevisiae, the wheat ribulose bisphosphate carboxylase-oxygenase-subunit-binding protein (alpha subunit), and the human P1 mitochondrial protein indicated sequence identity at 8 of 22 to 10 of 22 residues (36 to 45% identity). We conclude that the oligomer of TpN60 is homologous to the groEL protein and related chaperonins found in a wide variety of procaryotes and eucaryotes and thus may represent a heat shock protein involved in protein folding and assembly. Images PMID:1971618

Physiological and Proteomic Adaptation of the Alpine Grass Stipa purpurea to a Drought Gradient

PubMed Central

Yang, Yunqiang; Dong, Chao; Yang, Shihai; Li, Xiong; Sun, Xudong; Yang, Yongping

2015-01-01

Stipa purpurea, an endemic forage species on the Tibetan Plateau, is highly resistant to cold and drought, but the mechanisms underlying its responses to drought stress remain elusive. An understanding of such mechanisms may be useful for developing cultivars that are adaptable to water deficit. In this study, we analyzed the physiological and proteomic responses of S. purpurea under increasing drought stress. Seedlings of S. purpurea were subjected to a drought gradient in a controlled experiment, and proteins showing changes in abundance under these conditions were identified by two-dimensional electrophoresis followed by mass spectrometry analysis. A western blotting analysis was conducted to confirm the increased abundance of a heat-shock protein, NCED2, and a dehydrin in S. purpurea seedlings under drought conditions. We detected carbonylated proteins to identify oxidation-sensitive proteins in S. purpurea seedlings, and found that ribulose-1, 5-bisphosphate carboxylase oxygenase (RuBisCO) was one of the oxidation-sensitive proteins under drought. Together, these results indicated drought stress might inhibit photosynthesis in S. purpurea by oxidizing RuBisCO, but the plants were able to maintain photosynthetic efficiency by a compensatory upregulation of unoxidized RuBisCO and other photosynthesis-related proteins. Further analyses confirmed that increased abundance of antioxidant enzymes could balance the redox status of the plants to mitigate drought-induced oxidative damage. PMID:25646623

[Advenella kashmirensis subsp. methylica PK1, a facultative methylotroph from carex rhizosphere].

PubMed

Poroshina, M N; Doronina, N V; Kaparullina, E N; Trotsenko, Iu A

2015-01-01

A strain (PK1) of facultative methylobacteria growing on methanol as a carbon and energy source was isolated from carex rhizosphere (Pamukkale National Park, Turkey). The cells were nonmotile gram-negative rods propagating by binary fission. The organism was a strict anaerobe, oxidase- and catalase-positive. Optimal growth occurred at 29°C, pH 8.0-8.5, and 0.5% NaCl; no growth occurred at 2% NaCl. The organism used the ribulose bisphosphate pathway of C1 assimilation. Predominant fatty acids were 11-octodecenoic (18:1ω7) and cis-hexadecenoic (16:1ω7c). Phosphatidylethanolamine and diphosphatidylglycerol were the dominant phospholipids. Q8 was the main ubiquinone. DNA G+C content was 55.4 mol % (mp). Sequencing of the 16S rRNA gene revealed that strain PK1 belonged to the genus Advenella with 98.8 and 99.2% similarity to the type strains A. incenata CCUG 45225T and A. kashmirensis WT001T, respectively. DNA-DNA homology of strain PK1 and A. kashmirensis WT001T was 70%. While MALDI analysis confirmed their close clusterization, RAPD analysis revealed the differences between strain PKI and other Advenella strains. Based on its geno- and phenotypic properties, the isolate PK1 was classified as A. kashmirensis subsp. methylica PK1 (VKM-B 2850 = DSM 27514), the first known methylotroph of the genus Advenella.

Protein methylation in pea chloroplasts. [Pisum sativum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Niemi, K.J.; Adler, J.; Selman, B.R.

1990-07-01

The methylation of chloroplast proteins has been investigated by incubating intact pea (Pisum sativum) chloroplasts with ({sup 3}H-methyl)-S-adenosylmethionine. Incubation in the light increases the amount of methylation in both the thylakoid and stromal fractions. Numerous thylakoid proteins serve as substrates for the methyltransfer reactions. Three of these thylakoid proteins are methylated to a significantly greater extent in the light than in the dark. The primary stromal polypeptide methylated is the large subunit of ribulose bisphosphate carboxylase/oxygenase. One other stromal polypeptide is also methylated much more in the light than in the dark. Two distinct types of protein methylation occur. Onemore » methylinkage is stable to basic conditions whereas a second type is base labile. The base-stable linkage is indicative of N-methylation of amino acid residues while base-lability is suggestive of carboxymethylation of amino acid residues. Labeling in the light increases the percentage of methylation that is base labile in the thylakoid fraction while no difference is observed in the amount of base-labile methylations in light-labeled and dark-labeled stromal proteins. Also suggestive of carboxymethylation is the detection of volatile ({sup 3}H)methyl radioactivity which increases during the labeling period and is greater in chloroplasts labeled in the light as opposed to being labeled in the dark; this implies in vivo turnover of the ({sup 3}H)methyl group.« less

Unravelling the carbon and sulphur metabolism in coastal soil ecosystems using comparative cultivation-independent genome-level characterisation of microbial communities.

PubMed

Yousuf, Basit; Kumar, Raghawendra; Mishra, Avinash; Jha, Bhavanath

2014-01-01

Bacterial autotrophy contributes significantly to the overall carbon balance, which stabilises atmospheric CO2 concentration and decelerates global warming. Little attention has been paid to different modes of carbon/sulphur metabolism mediated by autotrophic bacterial communities in terrestrial soil ecosystems. We studied these pathways by analysing the distribution and abundance of the diagnostic metabolic marker genes cbbM, apsA and soxB, which encode for ribulose-1,5-bisphosphate carboxylase/oxygenase, adenosine phosphosulphate reductase and sulphate thiohydrolase, respectively, among different contrasting soil types. Additionally, the abundance of community members was assessed by quantifying the gene copy numbers for 16S rRNA, cbbL, cbbM, apsA and soxB. Distinct compositional differences were observed among the clone libraries, which revealed a dominance of phylotypes associated with carbon and sulphur cycling, such as Gammaproteobacteria (Thiohalomonas, Allochromatium, Chromatium, Thiomicrospira) and Alphaproteobacteria (Rhodopseudomonas, Rhodovulum, Paracoccus). The rhizosphere soil was devoid of sulphur metabolism, as the soxB and apsA genes were not observed in the rhizosphere metagenome, which suggests the absence or inadequate representation of sulphur-oxidising bacteria. We hypothesise that the novel Gammaproteobacteria sulphur oxidisers might be actively involved in sulphur oxidation and inorganic carbon fixation, particularly in barren saline soil ecosystems, suggesting their significant putative ecological role and contribution to the soil carbon pool.

Cyanobacterial-based approaches to improving photosynthesis in plants.

PubMed

Zarzycki, Jan; Axen, Seth D; Kinney, James N; Kerfeld, Cheryl A

2013-01-01

Plants rely on the Calvin-Benson (CB) cycle for CO(2) fixation. The key carboxylase of the CB cycle is ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). Efforts to enhance carbon fixation in plants have traditionally focused on RubisCO or on approaches that can help to remedy RubisCO's undesirable traits: its low catalytic efficiency and photorespiration. Towards reaching the goal of improving plant photosynthesis, cyanobacteria may be instrumental. Because of their evolutionary relationship to chloroplasts, they represent ideal model organisms for photosynthesis research. Furthermore, the molecular understanding of cyanobacterial carbon fixation provides a rich source of strategies that can be exploited for the bioengineering of chloroplasts. These strategies include the cyanobacterial carbon concentrating mechanism (CCM), which consists of active and passive transporter systems for inorganic carbon and a specialized organelle, the carboxysome. The carboxysome encapsulates RubisCO together with carbonic anhydrase in a protein shell, resulting in an elevated CO(2) concentration around RubisCO. Moreover, cyanobacteria differ from plants in the isoenzymes involved in the CB cycle and the photorespiratory pathways as well as in mechanisms that can affect the activity of RubisCO. In addition, newly available cyanobacterial genome sequence data from the CyanoGEBA project, which has more than doubled the amount of genomic information available for cyanobacteria, increases our knowledge on the CCM and the occurrence and distribution of genes of interest.

Phakopsora euvitis Causes Unusual Damage to Leaves and Modifies Carbohydrate Metabolism in Grapevine

PubMed Central

Nogueira Júnior, Antonio F.; Ribeiro, Rafael V.; Appezzato-da-Glória, Beatriz; Soares, Marli K. M.; Rasera, Júlia B.; Amorim, Lilian

2017-01-01

Asian grapevine rust (Phakopsora euvitis) is a serious disease, which causes severe leaf necrosis and early plant defoliation. These symptoms are unusual for a strict biotrophic pathogen. This work was performed to quantify the effects of P. euvitis on photosynthesis, carbohydrates, and biomass accumulation of grapevine. The reduction in photosynthetic efficiency of the green leaf tissue surrounding the lesions was quantified using the virtual lesion concept (β parameter). Gas exchange and responses of CO2 assimilation to increasing intercellular CO2 concentration were analyzed. Histopathological analyses and quantification of starch were also performed on diseased leaves. Biomass and carbohydrate accumulation were quantified in different organs of diseased and healthy plants. Rust reduced the photosynthetic rate, and β was estimated at 5.78, indicating a large virtual lesion. Mesophyll conductance, maximum rubisco carboxylation rate, and regeneration of ribulose-1,5-bisphosphate dependent on electron transport rate were reduced, causing diffusive and biochemical limitations to photosynthesis. Hypertrophy, chloroplast degeneration of mesophyll cells, and starch accumulation in cells close to lesions were observed. Root carbohydrate concentration was reduced, even at low rust severity. Asian grapevine rust dramatically reduced photosynthesis and altered the dynamics of production and accumulation of carbohydrates, unlike strict biotrophic pathogens. The reduction in carbohydrate reserves in roots would support polyetic damage on grapevine, caused by a polycyclic disease. PMID:29018470

Fructose 2,6-bisphosphate and the climacteric in bananas.

PubMed

Ball, K L; ap Rees, T

1988-11-15

This work was done to test the view that there is a marked rise in the content of fructose 2,6-bisphosphate during the climacteric of the fruit of banana (Musa cavendishii Lamb ex. Paxton). Bananas were ripened in the dark in a continuous stream of air in the absence of exogenous ethylene. CO2 production and the contents of fructose 2,6-bisphosphate and sucrose were monitored over a 15-day period. A range of extraction procedures for fructose 2,6-bisphosphate were compared. Recovery of fructose 2,6-bisphosphate added to samples of unripe fruit varied from poor to unmeasurable. Recoveries from samples of ripe fruit were high. It is argued that this differential recovery of fructose 2,6-bisphosphate undermines claims that the amount of this compound increases at the climacteric. When recoveries are taken into account, our data suggest that there is no major change in fructose 2,6-bisphosphate content during the onset of the climacteric in bananas.

The metabolic enzyme fructose-1,6-bisphosphate aldolase acts as a transcriptional regulator in pathogenic Francisella.

PubMed

Ziveri, Jason; Tros, Fabiola; Guerrera, Ida Chiara; Chhuon, Cerina; Audry, Mathilde; Dupuis, Marion; Barel, Monique; Korniotis, Sarantis; Fillatreau, Simon; Gales, Lara; Cahoreau, Edern; Charbit, Alain

2017-10-11

The enzyme fructose-bisphosphate aldolase occupies a central position in glycolysis and gluconeogenesis pathways. Beyond its housekeeping role in metabolism, fructose-bisphosphate aldolase has been involved in additional functions and is considered as a potential target for drug development against pathogenic bacteria. Here, we address the role of fructose-bisphosphate aldolase in the bacterial pathogen Francisella novicida. We demonstrate that fructose-bisphosphate aldolase is important for bacterial multiplication in macrophages in the presence of gluconeogenic substrates. In addition, we unravel a direct role of this metabolic enzyme in transcription regulation of genes katG and rpoA, encoding catalase and an RNA polymerase subunit, respectively. We propose a model in which fructose-bisphosphate aldolase participates in the control of host redox homeostasis and the inflammatory immune response.The enzyme fructose-bisphosphate aldolase (FBA) plays central roles in glycolysis and gluconeogenesis. Here, Ziveri et al. show that FBA of the pathogen Francisella novicida acts, in addition, as a transcriptional regulator and is important for bacterial multiplication in macrophages.

Xylose utilization in recombinant zymomonas

DOEpatents

Caimi, Perry G; McCole, Laura; Tao, Luan; Tomb, Jean-Francois; Viitanen, Paul V

2014-03-25

Xylose-utilizing Zymomonas strains studied were found to accumulate ribulose when grown in xylose-containing media. Engineering these strains to increase ribose-5-phosphate isomerase activity led to reduced ribulose accumulation, improved growth, improved xylose utilization, and increased ethanol production.

Mineral induced formation of pentose-2,4-bisphosphates

NASA Technical Reports Server (NTRS)

Krishnamurthy, R.; Pitsch, S.; Arrhenius, G.; Bada, J. L. (Principal Investigator)

1999-01-01

Formation of rac.-pentose-2,4-bisphosphates is demonstrated, starting from glycolaldehyde phosphate and glyceraldehyde-2-phosphate, and induced by mixed valence double layer metal hydroxide minerals. The reactions proceed from dilute aqueous reactant solutions (1.5 mM) at near neutral pH. Conditions have been established, where ribose-2,4-bisphosphate is the major product (approximately 48%) among the pentose-2,4-bisphosphates, which are formed with up to 25% yield.

Leaf-age effects on temperature responses of photosynthesis and respiration of an alpine oak, Quercus aquifolioides, in southwestern China.

PubMed

Zhou, Haoran; Xu, Ming; Pan, Hongli; Yu, Xiubo

2015-11-01

Temperature responses and sensitivity of photosynthesis (A(n_)T) and respiration for leaves at different ages are crucial to modeling ecosystem carbon (C) cycles and productivity of evergreen forests. Understanding the mechanisms and processes of temperature sensitivity may further shed lights on temperature acclimation of photosynthesis and respiration with leaf aging. The current study examined temperature responses of photosynthesis and respiration of young leaves (YLs) (fully expanded in current growth season) and old leaves (OLs) (fully expanded in last growth season) of Quercus aquifolioides Rehder and E.H. Wilson in an alpine oak forest, southwestern China. Temperature responses of dark respiration (R(dark)), net assimilation (A(n)), maximal velocity of carboxylation (V(cmax)) and maximum rate of electron transport (J(max)) were significantly different between the two leaf ages. Those differences implied different temperature response parameters should be used for leaves of different ages in modeling vegetation productivity and ecosystem C cycles in Q. aquifolioides forests and other evergreen forests. We found that RuBP carboxylation determined the downward shift of A(n_)T in OLs, while RuBP regeneration and the balance between Rubisco carboxylation and RuBP regeneration made little contribution. Sensitivity of stomatal conductance to vapor pressure deficit changed in OLs and compensated part of the downward shift. We also found that OLs of Q. aquifolioides had lower An due to lower stomatal conductance, higher stomatal conductance limitation and deactivation of the biochemical processes. In addition, the balance between R(dark) and A(n) changed between OLs and YLs, which was represented by a higher R(dark)/A(n) ratio for OLs. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Construction of genetically engineered Candida tropicalis for conversion of l-arabinose to l-ribulose.

PubMed

Yeo, In-Seok; Shim, Woo-Yong; Kim, Jung Hoe

2018-05-20

For the biological production of l-ribulose, conversion by enzymes or resting cells has been investigated. However, expensive or concentrated substrates, an additional purification step to remove borate and the requirement for cell cultivation and harvest steps before utilization of resting cells make the production process complex and unfavorable. Microbial fermentation may help overcome these limitations. In this study, we constructed a genetically engineered Candida tropicalis strain to produce l-ribulose by fermentation with a glucose/l-arabinose mixture. For the uptake of l-arabinose as a substrate and conversion of l-arabinose to l-ribulose, two heterologous genes coding for l-arabinose transporter and l-arabinose isomerase, were constitutively expressed in C. tropicalis under the GAPDH promoter. The Arabidopsis thaliana-originated l-arabinose transporter gene (STP2)-expressing strain exhibited a high l-arabinose uptake rate of 0.103 g/g cell/h and the expression of l-arabinose isomerase from Lactobacillus sakei 23 K showed 30% of conversion (9 g/L) from 30 g/L of l-arabinose. This genetically engineered strain can be used for l-ribulose production by fermentation using mixed sugars of glucose and l-arabinose. Copyright © 2018 Elsevier B.V. All rights reserved.

DNA Barcoding Green Microalgae Isolated from Neotropical Inland Waters

PubMed Central

Hadi, Sámed I. I. A.; Santana, Hugo; Brunale, Patrícia P. M.; Gomes, Taísa G.; Oliveira, Márcia D.; Matthiensen, Alexandre; Oliveira, Marcos E. C.; Silva, Flávia C. P.; Brasil, Bruno S. A. F.

2016-01-01

This study evaluated the feasibility of using the Ribulose Bisphosphate Carboxylase Large subunit gene (rbcL) and the Internal Transcribed Spacers 1 and 2 of the nuclear rDNA (nuITS1 and nuITS2) markers for identifying a very diverse, albeit poorly known group, of green microalgae from neotropical inland waters. Fifty-one freshwater green microalgae strains isolated from Brazil, the largest biodiversity reservoir in the neotropics, were submitted to DNA barcoding. Currently available universal primers for ITS1-5.8S-ITS2 region amplification were sufficient to successfully amplify and sequence 47 (92%) of the samples. On the other hand, new sets of primers had to be designed for rbcL, which allowed 96% of the samples to be sequenced. Thirty-five percent of the strains could be unambiguously identified to the species level based either on nuITS1 or nuITS2 sequences’ using barcode gap calculations. nuITS2 Compensatory Base Change (CBC) and ITS1-5.8S-ITS2 region phylogenetic analysis, together with morphological inspection, confirmed the identification accuracy. In contrast, only 6% of the strains could be assigned to the correct species based solely on rbcL sequences. In conclusion, the data presented here indicates that either nuITS1 or nuITS2 are useful markers for DNA barcoding of freshwater green microalgae, with advantage for nuITS2 due to the larger availability of analytical tools and reference barcodes deposited at databases for this marker. PMID:26900844

Bioinformatic analysis of the distribution of inorganic carbon transporters and prospective targets for bioengineering to increase Ci uptake by cyanobacteria.

PubMed

Gaudana, Sandeep B; Zarzycki, Jan; Moparthi, Vamsi K; Kerfeld, Cheryl A

2015-10-01

Cyanobacteria have evolved a carbon-concentrating mechanism (CCM) which has enabled them to inhabit diverse environments encompassing a range of inorganic carbon (Ci: [Formula: see text] and CO2) concentrations. Several uptake systems facilitate inorganic carbon accumulation in the cell, which can in turn be fixed by ribulose 1,5-bisphosphate carboxylase/oxygenase. Here we survey the distribution of genes encoding known Ci uptake systems in cyanobacterial genomes and, using a pfam- and gene context-based approach, identify in the marine (alpha) cyanobacteria a heretofore unrecognized number of putative counterparts to the well-known Ci transporters of beta cyanobacteria. In addition, our analysis shows that there is a huge repertoire of transport systems in cyanobacteria of unknown function, many with homology to characterized Ci transporters. These can be viewed as prospective targets for conversion into ancillary Ci transporters through bioengineering. Increasing intracellular Ci concentration coupled with efforts to increase carbon fixation will be beneficial for the downstream conversion of fixed carbon into value-added products including biofuels. In addition to CCM transporter homologs, we also survey the occurrence of rhodopsin homologs in cyanobacteria, including bacteriorhodopsin, a class of retinal-binding, light-activated proton pumps. Because they are light driven and because of the apparent ease of altering their ion selectivity, we use this as an example of re-purposing an endogenous transporter for the augmentation of Ci uptake by cyanobacteria and potentially chloroplasts.

RubisCO Gene Clusters Found in a Metagenome Microarray from Acid Mine Drainage

PubMed Central

Guo, Xue; Yin, Huaqun; Cong, Jing; Dai, Zhimin; Liang, Yili

2013-01-01

The enzyme responsible for carbon dioxide fixation in the Calvin cycle, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), is always detected as a phylogenetic marker to analyze the distribution and activity of autotrophic bacteria. However, such an approach provides no indication as to the significance of genomic content and organization. Horizontal transfers of RubisCO genes occurring in eubacteria and plastids may seriously affect the credibility of this approach. Here, we presented a new method to analyze the diversity and genomic content of RubisCO genes in acid mine drainage (AMD). A metagenome microarray containing 7,776 large-insertion fosmids was constructed to quickly screen genome fragments containing RubisCO form I large-subunit genes (cbbL). Forty-six cbbL-containing fosmids were detected, and six fosmids were fully sequenced. To evaluate the reliability of the metagenome microarray and understand the microbial community in AMD, the diversities of cbbL and the 16S rRNA gene were analyzed. Fosmid sequences revealed that the form I RubisCO gene cluster could be subdivided into form IA and IB RubisCO gene clusters in AMD, because of significant divergences in molecular phylogenetics and conservative genomic organization. Interestingly, the form I RubisCO gene cluster coexisted with the form II RubisCO gene cluster in one fosmid genomic fragment. Phylogenetic analyses revealed that horizontal transfers of RubisCO genes may occur widely in AMD, which makes the evolutionary history of RubisCO difficult to reconcile with organismal phylogeny. PMID:23335778

A protein with an inactive pterin-4a-carbinolamine dehydratase domain is required for Rubisco biogenesis in plants.

PubMed

Feiz, Leila; Williams-Carrier, Rosalind; Belcher, Susan; Montano, Monica; Barkan, Alice; Stern, David B

2014-12-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a critical role in sustaining life by catalysis of carbon fixation in the Calvin-Benson pathway. Incomplete knowledge of the assembly pathway of chloroplast Rubisco has hampered efforts to fully delineate the enzyme's properties, or seek improved catalytic characteristics via directed evolution. Here we report that a Mu transposon insertion in the Zea mays (maize) gene encoding a chloroplast dimerization co-factor of hepatocyte nuclear factor 1 (DCoH)/pterin-4α-carbinolamine dehydratases (PCD)-like protein is the causative mutation in a seedling-lethal, Rubisco-deficient mutant named Rubisco accumulation factor 2 (raf2-1). In raf2 mutants newly synthesized Rubisco large subunit accumulates in a high-molecular weight complex, the formation of which requires a specific chaperonin 60-kDa isoform. Analogous observations had been made previously with maize mutants lacking the Rubisco biogenesis proteins RAF1 and BSD2. Chemical cross-linking of maize leaves followed by immunoprecipitation with antibodies to RAF2, RAF1 or BSD2 demonstrated co-immunoprecipitation of each with Rubisco small subunit, and to a lesser extent, co-immunoprecipitation with Rubisco large subunit. We propose that RAF2, RAF1 and BSD2 form transient complexes with the Rubisco small subunit, which in turn assembles with the large subunit as it is released from chaperonins. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eckardt, N.A.; Pell, E.J.

Leaf senescence is characterized by loss of the major photosynthetic enzyme, Ribulose bisphosphate carboxylase (Rubisco). Exposure to ozone (O[sub 3]) is often associated with a premature decline in the quantity of this enzyme. Declines in Rubisco quantity could arise through inhibition of synthesis or enhancement of degradation. Several experiments were conducted to investigate the effect of O[sub 3] on these events in immature and mature leaves of potato. The effect of O[sub 3] on Rubisco synthesis was investigated indirectly by measuring the relative quantities of mRNA for the rubisco large (rbcL) and small (rbcS) subunits following a 5 hour exposuremore » to 0.309 [mu]L L[sup [minus]1] O[sup 3] or charcoal-filtered air. O[sup 3] treatment was associated with a significant loss in rbcS mRNA in immature and mature potato leaves sampled immediately following the exposure. After the O[sup 3] exposure, a set of plants was placed in the dark at 30 C for two days. Levels of rbcS mRNA declined rapidly during the first twelve hours of dark incubation, thus declines in Rubisco quantity following two days of dark incubation were ascribed to degradation. Enhanced degradation due to O[sub 3] during the dark incubation was observed in the mature leaves, but not in the immature leaves. We conclude that O[sub 3] can cause both inhibited synthesis and enhanced degradation of Rubisco, and the response in dependent on leaf age.« less

Development of an activity-directed selection system enabled significant improvement of the carboxylation efficiency of Rubisco.

PubMed

Cai, Zhen; Liu, Guoxia; Zhang, Junli; Li, Yin

2014-07-01

Photosynthetic CO(2) fixation is the ultimate source of organic carbon on earth and thus is essential for crop production and carbon sequestration. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the first step of photosynthetic CO(2) fixation. However, the extreme low carboxylation efficiency of Rubisco makes it the most attractive target for improving photosynthetic efficiency. Extensive studies have focused on re-engineering a more efficient enzyme, but the effort has been impeded by the limited understanding of its structure-function relationships and the lack of an efficient selection system towards its activity. To address the unsuccessful molecular engineering of Rubisco, we developed an Escherichia coli-based activity-directed selection system which links the growth of host cell solely to the Rubisco activity therein. A Synechococcus sp. PCC7002 Rubisco mutant with E49V and D82G substitutions in the small subunit was selected from a total of 15,000 mutants by one round of evolution. This mutant showed an 85% increase in specific carboxylation activity and a 45% improvement in catalytic efficiency towards CO(2). The small-subunit E49V mutation was speculated to influence holoenzyme catalysis through interaction with the large-subunit Q225. This interaction is conserved among various Rubisco from higher plants and Chlamydomonas reinhardtii. Knowledge of these might provide clues for engineering Rubisco from higher plants, with the potential of increasing the crop yield.

A tomato chloroplast-targeted DnaJ protein protects Rubisco activity under heat stress.

PubMed

Wang, Guodong; Kong, Fanying; Zhang, Song; Meng, Xia; Wang, Yong; Meng, Qingwei

2015-06-01

Photosynthesis is one of the biological processes most sensitive to heat stress in plants. Carbon assimilation, which depends on ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is one of the major sites sensitive to heat stress in photosynthesis. In this study, the roles of a tomato (Solanum lycopersicum) chloroplast-targeted DnaJ protein (SlCDJ2) in resisting heat using sense and antisense transgenic tomatoes were examined. SlCDJ2 was found to be uniformly distributed in the thylakoids and stroma of the chloroplasts. Under heat stress, sense plants exhibited higher chlorophyll contents and fresh weights, and lower accumulation of reactive oxygen species (ROS) and membrane damage. Moreover, Rubisco activity, Rubisco large subunit (RbcL) content, and CO2 assimilation capacity were all higher in sense plants and lower in antisense plants compared with wild-type plants. Thus, SlCDJ2 contributes to maintenance of CO2 assimilation capacity mainly by protecting Rubisco activity under heat stress. SlCDJ2 probably achieves this by keeping the levels of proteolytic enzymes low, which prevents accelerated degradation of Rubisco under heat stress. Furthermore, a chloroplast heat-shock protein 70 was identified as a binding partner of SlCDJ2 in yeast two-hybrid assays. Taken together, these findings establish a role for SlCDJ2 in maintaining Rubisco activity in plants under heat stress. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Environmentally driven evolution of Rubisco and improved photosynthesis and growth within the C3 genus Limonium (Plumbaginaceae).

PubMed

Galmés, Jeroni; Andralojc, P John; Kapralov, Maxim V; Flexas, Jaume; Keys, Alfred J; Molins, Arántzazu; Parry, Martin A J; Conesa, Miquel À

2014-08-01

Carbon assimilation by most ecosystems requires ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Its kinetic parameters are likely to have evolved in parallel with intracellular CO2 availability, with the result that faster forms of Rubisco occur in species with CO2 -concentrating mechanisms. The Rubisco catalytic properties were determined and evaluated in relation to growth and carbon assimilation capacity in Mediterranean Limonium species, inhabiting severe stress environments. Significant kinetic differences between closely related species depended on two amino acid substitutions at functionally important residues 309 and 328 within the Rubisco large subunit. The Rubisco of species facing the largest CO2 restrictions during drought had relatively high affinity for CO2 (low Michaelis-Menten constant for CO2 Kc) but low maximum rates of carboxylation (kcatc), while the opposite was found for species that maintained higher CO2 concentrations under similar conditions. Rubisco kinetic characteristics were correlated with photosynthetic rate in both well-watered and drought-stressed plants. Moreover, the drought-mediated decrease in plant biomass accumulation was consistently lower in species with higher Rubisco carboxylase catalytic efficiency (kcatc/Kc). The present study is the first demonstration of Rubisco adaptation during species diversification within closely related C3 plants, revealing a direct relationship between Rubisco molecular evolution and the biomass accumulation of closely related species subjected to unfavourable conditions. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

Thermal responses of Symbiodinium photosynthetic carbon assimilation

NASA Astrophysics Data System (ADS)

Oakley, Clinton A.; Schmidt, Gregory W.; Hopkinson, Brian M.

2014-06-01

The symbiosis between hermatypic corals and their dinoflagellate endosymbionts, genus Symbiodinium, is based on carbon exchange. This symbiosis is disrupted by thermally induced coral bleaching, a stress response in which the coral host expels its algal symbionts as they become physiologically impaired. The disruption of the dissolved inorganic carbon (DIC) supply or the thermal inactivation of Rubisco have been proposed as sites of initial thermal damage that leads to the bleaching response. Symbiodinium possesses a highly unusual Form II ribulose bisphosphate carboxylase/oxygenase (Rubisco), which exhibits a lower CO2:O2 specificity and may be more thermally unstable than the Form I Rubiscos of other algae and land plants. Components of the CO2 concentrating mechanism (CCM), which supplies inorganic carbon for photosynthesis, may also be temperature sensitive. Here, we examine the ability of four cultured Symbiodinium strains to acquire and fix DIC across a temperature gradient. Surprisingly, the half-saturation constant of photosynthesis with respect to DIC concentration ( K P), an index of CCM function, declined with increasing temperature in three of the four strains, indicating a greater potential for photosynthetic carbon acquisition at elevated temperatures. In the fourth strain, there was no effect of temperature on K P. Finding no evidence for thermal inhibition of the CCM, we conclude that CCM components are not likely to be the primary sites of thermal damage. Reduced photosynthetic quantum yields, a hallmark of thermal bleaching, were observed at low DIC concentrations, leaving open the possibility that reduced inorganic carbon availability is involved in bleaching.

Is RAF1 protein from Synechocystis sp. PCC 6803 really needed in the cyanobacterial Rubisco assembly process?

PubMed

Kolesinski, Piotr; Rydzy, Malgorzata; Szczepaniak, Andrzej

2017-05-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is responsible for carbon dioxide conversion during photosynthesis and, therefore, is the most important protein in biomass generation. Modifications of this biocatalyst toward improvements in its properties are hindered by the complicated and not yet fully understood assembly process required for the formation of active holoenzymes. An entire set of auxiliary factors, including chaperonin GroEL/GroES and assembly chaperones RbcX or Rubisco accumulation factor 1 (RAF1), is involved in the folding and subsequent assembly of Rubisco subunits. Recently, it has been shown that cyanobacterial RAF1 acts during the formation of the large Rubisco subunit (RbcL) dimer. However, both its physiological function and its necessity in the prokaryotic Rubisco formation process remain elusive. Here, we demonstrate that the Synechocystis sp. PCC 6803 strain with raf1 gene disruption shows the same growth rate as wild-type cells under standard conditions. Moreover, the Rubisco biosynthesis process seems to be unperturbed in mutant cells despite the absence of RbcL-RAF1 complexes. However, in the tested environmental conditions, sulfur starvation triggers the degradation of RbcL and subsequent proteolysis of other polypeptides in wild-type but not Δraf1 strains. Pull-down experiments also indicate that, apart from Rubisco, RAF1 co-purifies with phycocyanins. We postulate that RAF1 is not an obligatory factor in cyanobacterial Rubisco assembly, but rather participates in environmentally regulated Rubisco homeostasis.

Diversity and expression of different forms of RubisCO genes in polluted groundwater under different redox conditions.

PubMed

Alfreider, Albin; Schirmer, Mario; Vogt, Carsten

2012-03-01

Groundwater polluted with methyl-tert-butyl ether (MTBE) and ammonium was investigated for chemolithoautotrophic CO(2) fixation capabilities based on detailed analyses of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large subunit genes. Samples retrieved from a groundwater conditioning unit, characterized by different redox conditions, were examined for the presence of form IA, form IC (cbbL) and form II (cbbM) RubisCO genes and transcripts obtained from DNA- and RNA-extracts. Form IA RubisCO sequences, which revealed a complex and distinct variety in different sampling stations, were expressed in the original groundwater and in samples amended with oxygen, but not in the aquifer groundwater enriched with nitrate. Form IC RubisCO genes were exclusively detected in groundwater supplied with oxygen and sequences were affiliated with cbbL genes in nitrifying bacteria. cbbM genes were not expressed in the oxygen-amended groundwater, probably due to the low CO(2) /O(2) substrate specificity of this enzyme. Most form II RubisCO transcripts were affiliated with RubisCO genes of denitrifiers, which are important residents in the groundwater supplied with nitrate. The distinct distribution pattern and diversity of RubisCO genes and transcripts obtained in this study suggest that the induction of different RubisCO enzymes is highly regulated and closely linked to the actual environmental conditions. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

The Arabidopsis ppi1 Mutant Is Specifically Defective in the Expression, Chloroplast Import, and Accumulation of Photosynthetic ProteinsW⃞

PubMed Central

Kubis, Sybille; Baldwin, Amy; Patel, Ramesh; Razzaq, Azam; Dupree, Paul; Lilley, Kathryn; Kurth, Joachim; Leister, Dario; Jarvis, Paul

2003-01-01

The import of nucleus-encoded proteins into chloroplasts is mediated by translocon complexes in the envelope membranes. A component of the translocon in the outer envelope membrane, Toc34, is encoded in Arabidopsis by two homologous genes, atTOC33 and atTOC34. Whereas atTOC34 displays relatively uniform expression throughout development, atTOC33 is strongly upregulated in rapidly growing, photosynthetic tissues. To understand the reason for the existence of these two related genes, we characterized the atTOC33 knockout mutant ppi1. Immunoblotting and proteomics revealed that components of the photosynthetic apparatus are deficient in ppi1 chloroplasts and that nonphotosynthetic chloroplast proteins are unchanged or enriched slightly. Furthermore, DNA array analysis of 3292 transcripts revealed that photosynthetic genes are moderately, but specifically, downregulated in ppi1. Proteome differences in ppi1 could be correlated with protein import rates: ppi1 chloroplasts imported the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit and 33-kD oxygen-evolving complex precursors at significantly reduced rates, but the import of a 50S ribosomal subunit precursor was largely unaffected. The ppi1 import defect occurred at the level of preprotein binding, which is consistent with a role for atToc33 during preprotein recognition. The data suggest that atToc33 is involved preferentially in the import of photosynthetic proteins and, by extension, that atToc34 is involved in the import of nonphotosynthetic chloroplast proteins. PMID:12897258

The LysR-type transcription factor PacR is a global regulator of photosynthetic carbon assimilation in Anabaena.

PubMed

Picossi, Silvia; Flores, Enrique; Herrero, Antonia

2015-09-01

Cyanobacteria perform water-splitting photosynthesis and are important primary producers impacting the carbon and nitrogen cycles at global scale. They fix CO2 through ribulose-bisphosphate carboxylase/oxygenase (RuBisCo) and have evolved a distinct CO2 concentrating mechanism (CCM) that builds high CO2 concentrations in the vicinity of RuBisCo favouring its carboxylase activity. Filamentous cyanobacteria such as Anabaena fix CO2 in photosynthetic vegetative cells, which donate photosynthate to heterocysts that rely on a heterotrophic metabolism to fix N2 . CCM elements are induced in response to inorganic carbon limitation, a cue that exposes the photosynthetic apparatus to photodamage by over-reduction. An Anabaena mutant lacking the LysR-type transcription factor All3953 grew poorly and dies under high light. The rbcL operon encoding RuBisCo was induced upon carbon limitation in the wild type but not in the mutant. ChIP-Seq analysis was used to globally identify All3953 targets under carbon limitation. Targets include, besides rbcL, genes encoding CCM elements, photorespiratory pathway- photosystem- and electron transport-related components, and factors, including flavodiiron proteins, with a demonstrated or putative function in photoprotection. Quantitative reverse transcription polymerase chain reaction analysis of selected All3953 targets showed regulation in the wild type but not in the mutant. All3953 (PacR) is a global regulator of carbon assimilation in an oxygenic photoautotroph. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Cochliobolus lunatus down-regulates proteome at late stage of colonization and transiently alters StNPR1 expression in Solanum tuberosum L.

PubMed

Louis, Bengyella; Waikhom, Sayanika D; Jose, Robinson C; Goyari, Sailendra; Bhardwaj, Pardeep Kumar; Talukdar, Narayan C; Roy, Pranab

2017-03-01

Cochliobolus lunatus abundantly produces four-celled conidia at high temperatures (>30 °C) and under suitable conditions; the fungus colonizes potato (Solanum tuberosum L.) cultivars by adopting different invasion strategies at the microscopic level. Long-lasting defence during infection requires an upsurge in proteome changes particularly pathogenesis-related proteins chiefly under the control of nonexpresser of pathogenesis-related proteins. In order to gain molecular insights, we profiled the changes in proteome and potato nonexpresser of pathogenesis-related proteins (StNPR1) during the infection process. It is found that C. lunatus significantly (P < 0.05) suppressed the host functional proteome by 96 h after infection (hai), principally, affecting the expression of ribulose bisphosphate carboxylase enzyme, plastidic aldolase enzyme, alcohol dehydrogenase 2 and photosystem II protein prior to the formation of brown-to-black leaf spot disease. Strongest host response was observed at 24 hai hallmarked by 307 differentially expressed peptide spots concurring with the active phase of production of penetrating hyphae. Additionally, C. lunatus differentially down-regulated StNPR1 transcript by 8.19 fold by 24 hai. This study is the first to elucidate that C. lunatus transiently down-regulates the expression of StNPR1 at the onset of infection, and as a whole, infection negatively affects the expression of proteome components involved in photosynthesis, carbon fixation and light assimilation. This study contributes towards better understanding of the mechanism underlining the invasion strategies of C. lunatus.

Determination of ¹⁵N-incorporation into plant proteins and their absolute quantitation: a new tool to study nitrogen flux dynamics and protein pool sizes elicited by plant-herbivore interactions.

PubMed

Ullmann-Zeunert, Lynn; Muck, Alexander; Wielsch, Natalie; Hufsky, Franziska; Stanton, Mariana A; Bartram, Stefan; Böcker, Sebastian; Baldwin, Ian T; Groten, Karin; Svatoš, Aleš

2012-10-05

Herbivory leads to changes in the allocation of nitrogen among different pools and tissues; however, a detailed quantitative analysis of these changes has been lacking. Here, we demonstrate that a mass spectrometric data-independent acquisition approach known as LC-MS(E), combined with a novel algorithm to quantify heavy atom enrichment in peptides, is able to quantify elicited changes in protein amounts and (15)N flux in a high throughput manner. The reliable identification/quantitation of rabbit phosphorylase b protein spiked into leaf protein extract was achieved. The linear dynamic range, reproducibility of technical and biological replicates, and differences between measured and expected (15)N-incorporation into the small (SSU) and large (LSU) subunits of ribulose-1,5-bisphosphate-carboxylase/oxygenase (RuBisCO) and RuBisCO activase 2 (RCA2) of Nicotiana attenuata plants grown in hydroponic culture at different known concentrations of (15)N-labeled nitrate were used to further evaluate the procedure. The utility of the method for whole-plant studies in ecologically realistic contexts was demonstrated by using (15)N-pulse protocols on plants growing in soil under unknown (15)N-incorporation levels. Additionally, we quantified the amounts of lipoxygenase 2 (LOX2) protein, an enzyme important in antiherbivore defense responses, demonstrating that the approach allows for in-depth quantitative proteomics and (15)N flux analyses of the metabolic dynamics elicited during plant-herbivore interactions.

Contrasting Diversity and Host Association of Ectomycorrhizal Basidiomycetes versus Root-Associated Ascomycetes in a Dipterocarp Rainforest

PubMed Central

Sato, Hirotoshi; Tanabe, Akifumi S.; Toju, Hirokazu

2015-01-01

Root-associated fungi, including ectomycorrhizal and root-endophytic fungi, are among the most diverse and important belowground plant symbionts in dipterocarp rainforests. Our study aimed to reveal the biodiversity, host association, and community structure of ectomycorrhizal Basidiomycota and root-associated Ascomycota (including root-endophytic Ascomycota) in a lowland dipterocarp rainforest in Southeast Asia. The host plant chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) region and fungal internal transcribed spacer 2 (ITS2) region were sequenced using tag-encoded, massively parallel 454 pyrosequencing to identify host plant and root-associated fungal taxa in root samples. In total, 1245 ascomycetous and 127 putative ectomycorrhizal basidiomycetous taxa were detected from 442 root samples. The putative ectomycorrhizal Basidiomycota were likely to be associated with closely related dipterocarp taxa to greater or lesser extents, whereas host association patterns of the root-associated Ascomycota were much less distinct. The community structure of the putative ectomycorrhizal Basidiomycota was possibly more influenced by host genetic distances than was that of the root-associated Ascomycota. This study also indicated that in dipterocarp rainforests, root-associated Ascomycota were characterized by high biodiversity and indistinct host association patterns, whereas ectomycorrhizal Basidiomycota showed less biodiversity and a strong host phylogenetic preference for dipterocarp trees. Our findings lead to the working hypothesis that root-associated Ascomycota, which might be mainly represented by root-endophytic fungi, have biodiversity hotspots in the tropics, whereas biodiversity of ectomycorrhizal Basidiomycota increases with host genetic diversity. PMID:25884708

Benefit of pulsation in soft corals

PubMed Central

Kremien, Maya; Shavit, Uri; Mass, Tali; Genin, Amatzia

2013-01-01

Soft corals of the family Xeniidae exhibit a unique, rhythmic pulsation of their tentacles (Movie S1), first noted by Lamarck nearly 200 y ago. However, the adaptive benefit of this perpetual, energetically costly motion is poorly understood. Using in situ underwater particle image velocimetry, we found that the pulsation motions thrust water upward and enhance mixing across the coral–water boundary layer. The induced upward motion effectively prevents refiltration of water by neighboring polyps, while the intensification of mixing, together with the upward flow, greatly enhances the coral’s photosynthesis. A series of controlled laboratory experiments with the common xeniid coral Heteroxenia fuscescens showed that the net photosynthesis rate during pulsation was up to an order of magnitude higher than during the coral’s resting, nonpulsating state. This enhancement diminished when the concentration of oxygen in the ambient water was artificially raised, indicating that the enhancement of photosynthesis was due to a greater efflux of oxygen from the coral tissues. By lowering the internal oxygen concentration, pulsation alleviates the problem of reduced affinity of ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) to CO2 under conditions of high oxygen concentrations. The photosynthesis–respiration ratio of the pulsating H. fuscescens was markedly higher than the ratios reported for nonpulsating soft and stony corals. Although pulsation is commonly used for locomotion and filtration in marine mobile animals, its occurrence in sessile (bottom-attached) species is limited to members of the ancient phylum Cnidaria, where it is used to accelerate water and enhance physiological processes. PMID:23610420

Impact of Raw and Bioaugmented Olive-Mill Wastewater and Olive-Mill Solid Waste on the Content of Photosynthetic Molecules in Tobacco Plants.

PubMed

Parrotta, Luigi; Campani, Tommaso; Casini, Silvia; Romi, Marco; Cai, Giampiero

2016-08-03

Disposal and reuse of olive-mill wastes are both an economic and environmental problem, especially in countries where the cultivation of olive trees is extensive. Microorganism-based bioaugmentation can be used to reduce the pollutant capacity of wastes. In this work, bioaugmentation was used to reduce the polyphenolic content of both liquid and solid wastes. After processing, bioaugmented wastes were tested on the root development of maize seeds and on photosynthesis-related molecules of tobacco plants. In maize, we found that bioaugmentation made olive-mill wastes harmless for seed germination. In tobacco, we analyzed the content of RuBisCO (ribulose-1,5-bisphosphate carboxylase oxygenase) and of the photosynthetic pigments lutein, chlorophylls, and β-carotene. Levels of RuBisCO were negatively affected by untreated wastewater but increased if plants were treated with bioaugmented wastewater. On the contrary, levels of RuBisCO increased in the case of plants treated with raw olive-mill solid waste. Pigment levels showed dissimilar behavior because their concentration increased if plants were irrigated with raw wastewater or treated with raw olive-mill solid waste. Treatment with bioaugmented wastes restored pigment content. Findings show that untreated wastes are potentially toxic at the commencement of treatment, but plants can eventually adapt after an initial stress period. Bioaugmented wastes do not induce immediate damages, and plants rapidly recover optimal levels of photosynthetic molecules.

BioDry: An Inexpensive, Low-Power Method to Preserve Aquatic Microbial Biomass at Room Temperature.

PubMed

Tuorto, Steven J; Brown, Chris M; Bidle, Kay D; McGuinness, Lora R; Kerkhof, Lee J

2015-01-01

This report describes BioDry (patent pending), a method for reliably preserving the biomolecules associated with aquatic microbial biomass samples, without the need of hazardous materials (e.g. liquid nitrogen, preservatives, etc.), freezing, or bulky storage/sampling equipment. Gel electrophoresis analysis of nucleic acid extracts from samples treated in the lab with the BioDry method indicated that molecular integrity was protected in samples stored at room temperature for up to 30 days. Analysis of 16S/18S rRNA genes for presence/absence and relative abundance of microorganisms using both 454-pyrosequencing and TRFLP profiling revealed statistically indistinguishable communities from control samples that were frozen in liquid nitrogen immediately after collection. Seawater and river water biomass samples collected with a portable BioDry "field unit", constructed from off-the-shelf materials and a battery-operated pumping system, also displayed high levels of community rRNA preservation, despite a slight decrease in nucleic acid recovery over the course of storage for 30 days. Functional mRNA and protein pools from the field samples were also effectively conserved with BioDry, as assessed by respective RT-PCR amplification and western blot of ribulose-1-5-bisphosphate carboxylase/oxygenase. Collectively, these results demonstrate that BioDry can adequately preserve a suite of biomolecules from aquatic biomass at ambient temperatures for up to a month, giving it great potential for high resolution sampling in remote locations or on autonomous platforms where space and power are limited.

Analysis of early bacterial communities on volcanic deposits on the island of Miyake (Miyake-jima), Japan: a 6-year study at a fixed site.

PubMed

Fujimura, Reiko; Sato, Yoshinori; Nishizawa, Tomoyasu; Nanba, Kenji; Oshima, Kenshiro; Hattori, Masahira; Kamijo, Takashi; Ohta, Hiroyuki

2012-01-01

Microbial colonization on new terrestrial substrates represents the initiation of new soil ecosystem formation. In this study, we analyzed early bacterial communities growing on volcanic ash deposits derived from the 2000 Mount Oyama eruption on the island of Miyake (Miyake-jima), Japan. A site was established in an unvegetated area near the summit and investigated over a 6-year period from 2003 to 2009. Collected samples were acidic (pH 3.0-3.6), did not utilize any organic substrates in ECO microplate assays (Biolog), and harbored around 106 cells (g dry weight)(-1) of autotrophic Fe(II) oxidizers by most-probable-number (MPN) counts. Acidithiobacillus ferrooxidans, Acidithiobacillus ferrivorans, and the Leptospirillum groups I, II and III were found to be abundant in the deposits by clone library analysis of bacterial 16S rRNA genes. The numerical dominance of Acidithiobacillus ferrooxidans was also supported by analysis of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Comparing the 16S rRNA gene clone libraries from samples differing in age, shifts in Fe(II)-oxidizing populations seemed to occur with deposit aging. The detection of known 16S rRNA gene sequences from Fe(III)-reducing acidophiles promoted us to propose the acidity-driven iron cycle for the early microbial ecosystem on the deposit.

Analysis of Early Bacterial Communities on Volcanic Deposits on the Island of Miyake (Miyake-jima), Japan: a 6-year Study at a Fixed Site

PubMed Central

Fujimura, Reiko; Sato, Yoshinori; Nishizawa, Tomoyasu; Nanba, Kenji; Oshima, Kenshiro; Hattori, Masahira; Kamijo, Takashi; Ohta, Hiroyuki

2012-01-01

Microbial colonization on new terrestrial substrates represents the initiation of new soil ecosystem formation. In this study, we analyzed early bacterial communities growing on volcanic ash deposits derived from the 2000 Mount Oyama eruption on the island of Miyake (Miyake-jima), Japan. A site was established in an unvegetated area near the summit and investigated over a 6-year period from 2003 to 2009. Collected samples were acidic (pH 3.0–3.6), did not utilize any organic substrates in ECO microplate assays (Biolog), and harbored around 106 cells (g dry weight)−1 of autotrophic Fe(II) oxidizers by most-probable-number (MPN) counts. Acidithiobacillus ferrooxidans, Acidithiobacillus ferrivorans, and the Leptospirillum groups I, II and III were found to be abundant in the deposits by clone library analysis of bacterial 16S rRNA genes. The numerical dominance of Acidithiobacillus ferrooxidans was also supported by analysis of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Comparing the 16S rRNA gene clone libraries from samples differing in age, shifts in Fe(II)-oxidizing populations seemed to occur with deposit aging. The detection of known 16S rRNA gene sequences from Fe(III)-reducing acidophiles promoted us to propose the acidity-driven iron cycle for the early microbial ecosystem on the deposit. PMID:22075623

Benefit of pulsation in soft corals.

PubMed

Kremien, Maya; Shavit, Uri; Mass, Tali; Genin, Amatzia

2013-05-28

Soft corals of the family Xeniidae exhibit a unique, rhythmic pulsation of their tentacles (Movie S1), first noted by Lamarck nearly 200 y ago. However, the adaptive benefit of this perpetual, energetically costly motion is poorly understood. Using in situ underwater particle image velocimetry, we found that the pulsation motions thrust water upward and enhance mixing across the coral-water boundary layer. The induced upward motion effectively prevents refiltration of water by neighboring polyps, while the intensification of mixing, together with the upward flow, greatly enhances the coral's photosynthesis. A series of controlled laboratory experiments with the common xeniid coral Heteroxenia fuscescens showed that the net photosynthesis rate during pulsation was up to an order of magnitude higher than during the coral's resting, nonpulsating state. This enhancement diminished when the concentration of oxygen in the ambient water was artificially raised, indicating that the enhancement of photosynthesis was due to a greater efflux of oxygen from the coral tissues. By lowering the internal oxygen concentration, pulsation alleviates the problem of reduced affinity of ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) to CO2 under conditions of high oxygen concentrations. The photosynthesis-respiration ratio of the pulsating H. fuscescens was markedly higher than the ratios reported for nonpulsating soft and stony corals. Although pulsation is commonly used for locomotion and filtration in marine mobile animals, its occurrence in sessile (bottom-attached) species is limited to members of the ancient phylum Cnidaria, where it is used to accelerate water and enhance physiological processes.

Structure and immunocytochemical localization of photosynthetic enzymes in the lamina joint and sheath pulvinus of the C4 grass Arundinella hirta.

PubMed

Wakayama, Masataka; Ohnishi, Jun-ichi; Ueno, Osamu

2013-03-01

The C(4) grass Arundinella hirta exhibits a unique C(4) anatomy, with isolated Kranz cells (distinctive cells) and C(4)-type expression of photosynthetic enzymes in the leaf sheath and stem as well as in the leaf blade. The border zones between these organs are pale green. Those between the leaf blade and sheath and between the sheath and stem are called the lamina joint and sheath pulvinus, respectively, and are involved in gravity sensing. We investigated the structure and localization of C(3) and C(4) photosynthetic enzymes in these tissues. In both zones the epidermis lacked stomata. The inner tissue was composed of parenchyma cells and vascular bundles. The parenchyma cells were densely packed with small intercellular spaces and contained granal chloroplasts with large starch grains. No C(4)-type cellular differentiation was recognized. Western blot analysis showed that the lamina joint and pulvinus accumulated substantial amounts of phosphoenolpyruvate carboxylase (PEPC), pyruvate,Pi dikinase (PPDK), and ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco). Immunogold electron microscopy revealed PEPC in the cytosol and both PPDK and rubisco in the chloroplasts of parenchyma cells, suggesting the occurrence of C(3) and C(4) enzymes within a single type of chlorenchyma cell. These data indicate that the lamina joint and pulvinus have unique expression patterns of C(3) and C(4) enzymes, unlike those in C(4)-type anatomy.

Characterization of Cannabis sativa allergens.

PubMed

Nayak, Ajay P; Green, Brett J; Sussman, Gordon; Berlin, Noam; Lata, Hemant; Chandra, Suman; ElSohly, Mahmoud A; Hettick, Justin M; Beezhold, Donald H

2013-07-01

Allergic sensitization to Cannabis sativa is rarely reported, but the increasing consumption of marijuana has resulted in an increase in the number of individuals who become sensitized. To date, little is known about the causal allergens associated with C sativa. To characterize marijuana allergens in different components of the C sativa plant using serum IgE from marijuana sensitized patients. Serum samples from 23 patients with a positive skin prick test result to a crude C sativa extract were evaluated. IgE reactivity was variable between patients and C sativa extracts. IgE reactivity to C sativa proteins in Western blots was heterogeneous and ranged from 10 to 70 kDa. Putative allergens derived from 2-dimensional gels were identified. Prominent IgE reactive bands included a 23-kDa oxygen-evolving enhancer protein 2 and a 50-kDa protein identified to be the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. Additional proteins were identified in the proteomic analysis, including those from adenosine triphosphate synthase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and luminal binding protein (heat shock protein 70), suggesting these proteins are potential allergens. Deglycosylation studies helped refine protein allergen identification and demonstrated significant IgE antibodies against plant oligosaccharides that could help explain cross-reactivity. Identification and characterization of allergens from C sativa may be helpful in further understanding allergic sensitization to this plant species. Copyright © 2013 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Mendel's green cotyledon gene encodes a positive regulator of the chlorophyll-degrading pathway

PubMed Central

Sato, Yutaka; Morita, Ryouhei; Nishimura, Minoru; Yamaguchi, Hiroyasu; Kusaba, Makoto

2007-01-01

Mutants that retain greenness of leaves during senescence are known as “stay-green” mutants. The most famous stay-green mutant is Mendel's green cotyledon pea, one of the mutants used in determining the law of genetics. Pea plants homozygous for this recessive mutation (known as i at present) retain greenness of the cotyledon during seed maturation and of leaves during senescence. We found tight linkage between the I locus and stay-green gene originally found in rice, SGR. Molecular analysis of three i alleles including one with no SGR expression confirmed that the I gene encodes SGR in pea. Functional analysis of sgr mutants in pea and rice further revealed that leaf functionality is lowered despite a high chlorophyll a (Chl a) and chlorophyll b (Chl b) content in the late stage of senescence, suggesting that SGR is primarily involved in Chl degradation. Consistent with this observation, a wide range of Chl–protein complexes, but not the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit, were shown to be more stable in sgr than wild-type plants. The expression of OsCHL and NYC1, which encode the first enzymes in the degrading pathways of Chl a and Chl b, respectively, was not affected by sgr in rice. The results suggest that SGR might be involved in activation of the Chl-degrading pathway during leaf senescence through translational or posttranslational regulation of Chl-degrading enzymes. PMID:17709752

Mendel's green cotyledon gene encodes a positive regulator of the chlorophyll-degrading pathway.

PubMed

Sato, Yutaka; Morita, Ryouhei; Nishimura, Minoru; Yamaguchi, Hiroyasu; Kusaba, Makoto

2007-08-28

Mutants that retain greenness of leaves during senescence are known as "stay-green" mutants. The most famous stay-green mutant is Mendel's green cotyledon pea, one of the mutants used in determining the law of genetics. Pea plants homozygous for this recessive mutation (known as i at present) retain greenness of the cotyledon during seed maturation and of leaves during senescence. We found tight linkage between the I locus and stay-green gene originally found in rice, SGR. Molecular analysis of three i alleles including one with no SGR expression confirmed that the I gene encodes SGR in pea. Functional analysis of sgr mutants in pea and rice further revealed that leaf functionality is lowered despite a high chlorophyll a (Chl a) and chlorophyll b (Chl b) content in the late stage of senescence, suggesting that SGR is primarily involved in Chl degradation. Consistent with this observation, a wide range of Chl-protein complexes, but not the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit, were shown to be more stable in sgr than wild-type plants. The expression of OsCHL and NYC1, which encode the first enzymes in the degrading pathways of Chl a and Chl b, respectively, was not affected by sgr in rice. The results suggest that SGR might be involved in activation of the Chl-degrading pathway during leaf senescence through translational or posttranslational regulation of Chl-degrading enzymes.

BioDry: An Inexpensive, Low-Power Method to Preserve Aquatic Microbial Biomass at Room Temperature

PubMed Central

Tuorto, Steven J.; Brown, Chris M.; Bidle, Kay D.; McGuinness, Lora R.; Kerkhof, Lee J.

2015-01-01

This report describes BioDry (patent pending), a method for reliably preserving the biomolecules associated with aquatic microbial biomass samples, without the need of hazardous materials (e.g. liquid nitrogen, preservatives, etc.), freezing, or bulky storage/sampling equipment. Gel electrophoresis analysis of nucleic acid extracts from samples treated in the lab with the BioDry method indicated that molecular integrity was protected in samples stored at room temperature for up to 30 days. Analysis of 16S/18S rRNA genes for presence/absence and relative abundance of microorganisms using both 454-pyrosequencing and TRFLP profiling revealed statistically indistinguishable communities from control samples that were frozen in liquid nitrogen immediately after collection. Seawater and river water biomass samples collected with a portable BioDry “field unit", constructed from off-the-shelf materials and a battery-operated pumping system, also displayed high levels of community rRNA preservation, despite a slight decrease in nucleic acid recovery over the course of storage for 30 days. Functional mRNA and protein pools from the field samples were also effectively conserved with BioDry, as assessed by respective RT-PCR amplification and western blot of ribulose-1-5-bisphosphate carboxylase/oxygenase. Collectively, these results demonstrate that BioDry can adequately preserve a suite of biomolecules from aquatic biomass at ambient temperatures for up to a month, giving it great potential for high resolution sampling in remote locations or on autonomous platforms where space and power are limited. PMID:26710122

Application of HB17, an Arabidopsis class II homeodomain-leucine zipper transcription factor, to regulate chloroplast number and photosynthetic capacity.

PubMed

Hymus, Graham J; Cai, Suqin; Kohl, Elizabeth A; Holtan, Hans E; Marion, Colleen M; Tiwari, Shiv; Maszle, Don R; Lundgren, Marjorie R; Hong, Melissa C; Channa, Namitha; Loida, Paul; Thompson, Rebecca; Taylor, J Philip; Rice, Elena; Repetti, Peter P; Ratcliffe, Oliver J; Reuber, T Lynne; Creelman, Robert A

2013-11-01

Transcription factors are proposed as suitable targets for the control of traits such as yield or food quality in plants. This study reports the results of a functional genomics research effort that identified ATHB17, a transcription factor from the homeodomain-leucine zipper class II family, as a novel target for the enhancement of photosynthetic capacity. It was shown that ATHB17 is expressed natively in the root quiescent centre (QC) from Arabidopsis embryos and seedlings. Analysis of the functional composition of genes differentially expressed in the QC from a knockout mutant (athb17-1) compared with its wild-type sibling revealed the over-representation of genes involved in auxin stimulus, embryo development, axis polarity specification, and plastid-related processes. While no other phenotypes were observed in athb17-1 plants, overexpression of ATHB17 produced a number of phenotypes in Arabidopsis including enhanced chlorophyll content. Image analysis of isolated mesophyll cells of 35S::ATHB17 lines revealed an increase in the number of chloroplasts per unit cell size, which is probably due to an increase in the number of proplastids per meristematic cell. Leaf physiological measurements provided evidence of improved photosynthetic capacity in 35S::ATHB17 lines on a per unit leaf area basis. Estimates of the capacity for ribulose-1,5-bisphosphate-saturated and -limited photosynthesis were significantly higher in 35S::ATHB17 lines.

Application of HB17, an Arabidopsis class II homeodomain-leucine zipper transcription factor, to regulate chloroplast number and photosynthetic capacity

PubMed Central

Kohl, Elizabeth A.; Tiwari, Shiv; Lundgren, Marjorie R.; Channa, Namitha; Creelman, Robert A.

2013-01-01

Transcription factors are proposed as suitable targets for the control of traits such as yield or food quality in plants. This study reports the results of a functional genomics research effort that identified ATHB17, a transcription factor from the homeodomain-leucine zipper class II family, as a novel target for the enhancement of photosynthetic capacity. It was shown that ATHB17 is expressed natively in the root quiescent centre (QC) from Arabidopsis embryos and seedlings. Analysis of the functional composition of genes differentially expressed in the QC from a knockout mutant (athb17-1) compared with its wild-type sibling revealed the over-representation of genes involved in auxin stimulus, embryo development, axis polarity specification, and plastid-related processes. While no other phenotypes were observed in athb17-1 plants, overexpression of ATHB17 produced a number of phenotypes in Arabidopsis including enhanced chlorophyll content. Image analysis of isolated mesophyll cells of 35S::ATHB17 lines revealed an increase in the number of chloroplasts per unit cell size, which is probably due to an increase in the number of proplastids per meristematic cell. Leaf physiological measurements provided evidence of improved photosynthetic capacity in 35S::ATHB17 lines on a per unit leaf area basis. Estimates of the capacity for ribulose-1,5-bisphosphate-saturated and -limited photosynthesis were significantly higher in 35S::ATHB17 lines. PMID:24006420

Non-native, N-terminal Hsp70 Molecular Motor Recognition Elements in Transit Peptides Support Plastid Protein Translocation*

PubMed Central

Chotewutmontri, Prakitchai; Bruce, Barry D.

2015-01-01

Previously, we identified the N-terminal domain of transit peptides (TPs) as a major determinant for the translocation step in plastid protein import. Analysis of Arabidopsis TP dataset revealed that this domain has two overlapping characteristics, highly uncharged and Hsp70-interacting. To investigate these two properties, we replaced the N-terminal domains of the TP of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and its reverse peptide with a series of unrelated peptides whose affinities to the chloroplast stromal Hsp70 have been determined. Bioinformatic analysis indicated that eight out of nine peptides in this series are not similar to the TP N terminus. Using in vivo and in vitro protein import assays, the majority of the precursors containing Hsp70-binding elements were targeted to plastids, whereas none of the chimeric precursors lacking an N-terminal Hsp70-binding element were targeted to the plastids. Moreover, a pulse-chase assay showed that two chimeric precursors with the most uncharged peptides failed to translocate into the stroma. The ability of multiple unrelated Hsp70-binding elements to support protein import verified that the majority of TPs utilize an N-terminal Hsp70-binding domain during translocation and expand the mechanistic view of the import process. This work also indicates that synthetic biology may be utilized to create de novo TPs that exceed the targeting activity of naturally occurring sequences. PMID:25645915

Structural Characterization of a Newly Identified Component of α-Carboxysomes: The AAA+ Domain Protein CsoCbbQ

DOE PAGES

Sutter, Markus; Roberts, Evan W.; Gonzalez, Raul C.; ...

2015-11-05

Carboxysomes are bacterial microcompartments that enhance carbon fixation by concentrating ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its substrate CO 2 within a proteinaceous shell. They are found in all cyanobacteria, some purple photoautotrophs and many chemoautotrophic bacteria. Carboxysomes consist of a protein shell that encapsulates several hundred molecules of RuBisCO, and contain carbonic anhydrase and other accessory proteins. Genes coding for carboxysome shell components and the encapsulated proteins are typically found together in an operon. The α-carboxysome operon is embedded in a cluster of additional, conserved genes that are presumably related to its function. In many chemoautotrophs, products of the expanded carboxysomemore » locus include CbbO and CbbQ, a member of the AAA+ domain superfamily. We bioinformatically identified subtypes of CbbQ proteins and show that their genes frequently co-occur with both Form IA and Form II RuBisCO. The α-carboxysome-associated ortholog, CsoCbbQ, from Halothiobacillus neapolitanus forms a hexamer in solution and hydrolyzes ATP. The crystal structure shows that CsoCbbQ is a hexamer of the typical AAA+ domain; the additional C-terminal domain, diagnostic of the CbbQ subfamily, structurally fills the inter-monomer gaps, resulting in a distinctly hexagonal shape. Finally, we show that CsoCbbQ interacts with CsoCbbO and is a component of the carboxysome shell, the first example of ATPase activity associated with a bacterial microcompartment.« less

Proteomic Analysis Reveals Differences in Tolerance to Acid Rain in Two Broad-Leaf Tree Species, Liquidambar formosana and Schima superba

PubMed Central

Wang, Chao; Liu, Ting-Wu; Chalifour, Annie; Chen, Juan; Shen, Zhi-Jun; Liu, Xiang; Wang, Wen-Hua; Zheng, Hai-Lei

2014-01-01

Acid rain (AR) is a serious environmental issue inducing harmful impacts on plant growth and development. It has been reported that Liquidambar formosana, considered as an AR-sensitive tree species, was largely injured by AR, compared with Schima superba, an AR-tolerant tree species. To clarify the different responses of these two species to AR, a comparative proteomic analysis was conducted in this study. More than 1000 protein spots were reproducibly detected on two-dimensional electrophoresis gels. Among them, 74 protein spots from L. formosana gels and 34 protein spots from S. superba gels showed significant changes in their abundances under AR stress. In both L. formosana and S. superba, the majority proteins with more than 2 fold changes were involved in photosynthesis and energy production, followed by material metabolism, stress and defense, transcription, post-translational and modification, and signal transduction. In contrast with L. formosana, no hormone response-related protein was found in S. superba. Moreover, the changes of proteins involved in photosynthesis, starch synthesis, and translation were distinctly different between L. formosana and S. superba. Protein expression analysis of three proteins (ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, ascorbate peroxidase and glutathione-S-transferase) by Western blot was well correlated with the results of proteomics. In conclusion, our study provides new insights into AR stress responses in woody plants and clarifies the differences in strategies to cope with AR between L. formosana and S. superba. PMID:25025692

Acclimation of the summer annual species, lolium temulentum, to CO(2) enrichment

PubMed

Lewis; Peratoner; Cairns; Causton; Foyer

1999-11-01

Lolium temulentum L. Ba 3081 was grown hydroponically in air (350 &mgr;mol mol(-1) CO(2)) and elevated CO(2) (700 &mgr;mol mol(-1) CO(2)) at two irradiances (150 and 500 &mgr;mol m(-2) s(-1)) for 35 days at which point the plants were harvested. Elevated CO(2) did not modify relative growth rate or biomass at either irradiance. Foliar carbon-to-nitrogen ratios were decreased at elevated CO(2) and plants had a greater number of shorter tillers, particularly at the lower growth irradiance. Both light-limited and light-saturated rates of photosynthesis were stimulated. The amount of ribulose-1, 5-bisphosphate carboxylase-oxygenase (Rubisco) protein was increased at elevated CO(2), but maximum extractable Rubisco activities were not significantly increased. A pronounced decrease in the Rubisco activation state was found with CO(2) enrichment, particularly at the higher growth irradiance. Elevated-CO(2)-induced changes in leaf carbohydrate composition were small in comparison to those caused by changes in irradiance. No CO(2)-dependent effects on fructan biosynthesis were observed. Leaf respiration rates were increased by 68% in plants grown with CO(2) enrichment and low light. We conclude that high CO(2) will only result in increased biomass if total light input favourably increases the photosynthesis-to-respiration ratio. At low irradiances, biomass is more limited by increased rates of respiration than by CO(2)-induced enhancement of photosynthesis.

Phosphatidylinositol(4,5)bisphosphate and phosphatidylinositol(4)phosphate in plant tissues. [Pisum sativum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Irvine, R.F.; Letcher, A.J.; Lander, D.J.

1989-03-01

Pea (Pisum sativum) leaf discs or swimming suspensions of Chlamydomonas eugametos were radiolabeled with ({sup 3}H)myo-inositol or ({sup 32}P)Pi and the lipids were extracted, deacylated, and their glycerol moieties removed. The resulting inositol trisphosphate and bisphosphate fractions were examined by periodate degradation, reduction and dephosphorylation, or by incubation with human red cell membranes. Their likely structures were identified as D-myo-inositol(1,4,5)trisphosphate and D-myo-inositol(1,4,)-bisphosphate. It is concluded that plants contain phosphatidylinositol(4)phosphate and phosphatidylinositol(4,5)bisphosphate; no other polyphosphoinositides were detected.

Identification and expression of fructose-1,6-bisphosphate aldolase genes and their relations to oil content in developing seeds of tea oil tree (Camellia oleifera)

USDA-ARS?s Scientific Manuscript database

Tea oil tree (Camellia oleifera, Co) provides a fine edible oil source in China. Tea oil from the seeds is very beneficial to human health. Fructose-1,6-bisphosphate aldolase (FBA) hydrolyzes fructose-1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, two critical metab...

Structural insight into mechanism and diverse substrate selection strategy of L-ribulokinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agarwal R.; Swaminathan S.; Burley, S. K.

2012-01-01

The araBAD operon encodes three different enzymes required for catabolism of L-arabinose, which is one of the most abundant monosaccharides in nature. L-ribulokinase, encoded by the araB gene, catalyzes conversion of L-ribulose to L-ribulose-5-phosphate, the second step in the catabolic pathway. Unlike other kinases, ribulokinase exhibits diversity in substrate selectivity and catalyzes phosphorylation of all four 2-ketopentose sugars with comparable k{sub cat} values. To understand ribulokinase recognition and phosphorylation of a diverse set of substrates, we have determined the X-ray structure of ribulokinase from Bacillus halodurans bound to L-ribulose and investigated its substrate and ATP co-factor binding properties. The polypeptidemore » chain is folded into two domains, one small and the other large, with a deep cleft in between. By analogy with related sugar kinases, we identified {sup 447}{und GG}LPQ{und K}{sup 452} as the ATP-binding motif within the smaller domain. L-ribulose binds in the cleft between the two domains via hydrogen bonds with the side chains of highly conserved Trp126, Lys208, Asp274, and Glu329 and the main chain nitrogen of Ala96. The interaction of L-ribulokinase with L-ribulose reveals versatile structural features that help explain recognition of various 2-ketopentose substrates and competitive inhibition by L-erythrulose. Comparison of our structure to that of the structures of other sugar kinases revealed conformational variations that suggest domain-domain closure movements are responsible for establishing the observed active site environment.« less

Metal sites in 3,4-dihydroxy-2-butanone 4-phosphate synthase from Methanococcus jannaschii in complex with the substrate ribulose 5-phosphate.

PubMed

Steinbacher, Stefan; Schiffmann, Susanne; Bacher, Adelbert; Fischer, Markus

2004-07-01

The crystal structure of Methanococcus jannaschii 3,4-dihydroxy-2-butanone 4-phosphate synthase in complex with the substrate ribulose 5-phosphate at a dimetal centre has recently been determined at 1.7 A resolution. The enzyme converts ribulose 5-phosphate into 3,4-dihydroxy-2-butanone 4-phosphate, while its C4 atom is released as formate. The resulting four-carbon body supplies all eight C atoms for the xylene moiety of riboflavin. Three of the four hydroxyl groups of ribulose 5-phosphate were coordinated by the metal ions. Based on crystallographic refinement, the metals were assigned as zinc and calcium, which were present in the crystallization buffer. Neither metal supports the enzymatic reaction. In the present study, the correctness of this assignment is assessed using anomalous diffraction data collected at the high-energy side of the zinc absorption edge (lambda = 1.2823 A). Only the three tentative zinc ions give strong peaks in an anomalous difference Fourier map (>20sigma), whereas the four tentative calcium ions do not show anomalous signals above the noise level. These results confirm the initial assignment. In addition, the resolution was improved to 1.55 A.

Fructose 2,6-bisphosphate and 6-phosphofructo-2-kinase during liver regeneration.

PubMed Central

Rosa, J L; Ventura, F; Carreras, J; Bartrons, R

1990-01-01

Glycogen and fructose 2,6-bisphosphate levels in rat liver decreased quickly after partial hepatectomy. After 7 days the glycogen level was normalized and fructose 2,6-bisphosphate concentration still remained low. The 'active' (non-phosphorylated) form of 6-phosphofructo-2-kinase varied in parallel with fructose 2,6-bisphosphate levels, whereas the 'total' activity of the enzyme decreased only after 24 h, similarly to glucokinase. The response of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from hepatectomized rats (96 h) to sn-glycerol 3-phosphate and to cyclic AMP-dependent protein kinase was different from that of the enzyme from control animals and similar to that of the foetal isoenzyme. PMID:2173548

Exploring substrate binding and discrimination in fructose1, 6-bisphosphate and tagatose 1,6-bisphosphate aldolases.

PubMed

Zgiby, S M; Thomson, G J; Qamar, S; Berry, A

2000-03-01

Fructose 1,6-bisphosphate aldolase catalyses the reversible condensation of glycerone-P and glyceraldehyde 3-phosphate into fructose 1,6-bisphosphate. A recent structure of the Escherichia coli Class II fructose 1,6-bisphosphate aldolase [Hall, D.R., Leonard, G.A., Reed, C.D., Watt, C.I., Berry, A. & Hunter, W.N. (1999) J. Mol. Biol. 287, 383-394] in the presence of the transition state analogue phosphoglycolohydroxamate delineated the roles of individual amino acids in binding glycerone-P and in the initial proton abstraction steps of the mechanism. The X-ray structure has now been used, together with sequence alignments, site-directed mutagenesis and steady-state enzyme kinetics to extend these studies to map important residues in the binding of glyceraldehyde 3-phosphate. From these studies three residues (Asn35, Ser61 and Lys325) have been identified as important in catalysis. We show that mutation of Ser61 to alanine increases the Km value for fructose 1, 6-bisphosphate 16-fold and product inhibition studies indicate that this effect is manifested most strongly in the glyceraldehyde 3-phosphate binding pocket of the active site, demonstrating that Ser61 is involved in binding glyceraldehyde 3-phosphate. In contrast a S61T mutant had no effect on catalysis emphasizing the importance of an hydroxyl group for this role. Mutation of Asn35 (N35A) resulted in an enzyme with only 1.5% of the activity of the wild-type enzyme and different partial reactions indicate that this residue effects the binding of both triose substrates. Finally, mutation of Lys325 has a greater effect on catalysis than on binding, however, given the magnitude of the effects it is likely that it plays an indirect role in maintaining other critical residues in a catalytically competent conformation. Interestingly, despite its proximity to the active site and high sequence conservation, replacement of a fourth residue, Gln59 (Q59A) had no significant effect on the function of the enzyme. In a separate study to characterize the molecular basis of aldolase specificity, the agaY-encoded tagatose 1,6-bisphosphate aldolase of E. coli was cloned, expressed and kinetically characterized. Our studies showed that the two aldolases are highly discriminating between the diastereoisomers fructose bisphosphate and tagatose bisphosphate, each enzyme preferring its cognate substrate by a factor of 300-1500-fold. This produces an overall discrimination factor of almost 5 x 105 between the two enzymes. Using the X-ray structure of the fructose 1,6-bisphosphate aldolase and multiple sequence alignments, several residues were identified, which are highly conserved and are in the vicinity of the active site. These residues might potentially be important in substrate recognition. As a consequence, nine mutations were made in attempts to switch the specificity of the fructose 1,6-bisphosphate aldolase to that of the tagatose 1,6-bisphosphate aldolase and the effect on substrate discrimination was evaluated. Surprisingly, despite making multiple changes in the active site, many of which abolished fructose 1, 6-bisphosphate aldolase activity, no switch in specificity was observed. This highlights the complexity of enzyme catalysis in this family of enzymes, and points to the need for further structural studies before we fully understand the subtleties of the shaping of the active site for complementarity to the cognate substrate.

Over Expression of Sedoheptulose 1,7, bisphosphatase (SBPase) Significantly Improves Carbon Assimilation at Elevated CO2

USDA-ARS?s Scientific Manuscript database

Biochemical models of photosynthesis (A) show that it is most frequently limited by the slowest of two processes, maximum carboxylation capacity of the enzyme Rubisco (Vc,max) or the regeneration of RuBP via electron transport (J). At current CO2 levels Rubisco is not saturated by its substrate, the...

The characteristics of pyrophosphate: D-fructose-6-phosphate 1-phosphotransferases from Sansevieria trifasciata leaves and Phaseolus coccineus stems.

PubMed

Kowalczyk, S

1987-01-01

Three different molecular forms of pyrophosphate-dependent phosphofructokinase have been isolated: one from Sansevieria trifasciata leaves and two from Phaseolus coccineus stems. The form isolated from S. trifasciata has the molecular weight of about 115,000. The apparent molecular weights for the two forms from mung bean were approximately 220,000 and 450,000. All three forms have the same pH optima, an absolute requirement for Mg2+ ions both in the forward and reverse reaction, but differ in their sensitivity toward fructose 2,6-bisphosphate. Kinetic properties of the partially purified enzymes have been investigated in the presence and absence of fructose 2,6-bisphosphate. Pyrophosphate-dependent phosphofructokinase from S. trifasciata exhibited hyperbolic kinetics with all substrates tested. The saturation curves of the enzyme (form A) from mung bean for pyrophosphate, fructose 6-phosphate and fructose 1,6-bisphosphate were sigmoidal in the absence of fructose 2,6-bisphosphate. In the presence of fructose 2,6-bisphosphate these kinetics became hyperbolic.

Regulatory components of carbon concentrating mechanisms in aquatic unicellular photosynthetic organisms.

PubMed

Tomar, Vandana; Sidhu, Gurpreet Kaur; Nogia, Panchsheela; Mehrotra, Rajesh; Mehrotra, Sandhya

2017-11-01

This review provides an insight into the regulation of the carbon concentrating mechanisms (CCMs) in lower organisms like cyanobacteria, proteobacteria, and algae. CCMs evolved as a mechanism to concentrate CO 2 at the site of primary carboxylating enzyme Ribulose-1, 5-bisphosphate carboxylase oxygenase (Rubisco), so that the enzyme could overcome its affinity towards O 2 which leads to wasteful processes like photorespiration. A diverse set of CCMs exist in nature, i.e., carboxysomes in cyanobacteria and proteobacteria; pyrenoids in algae and diatoms, the C 4 system, and Crassulacean acid metabolism in higher plants. Prime regulators of CCM in most of the photosynthetic autotrophs belong to the LysR family of transcriptional regulators, which regulate the activity of the components of CCM depending upon the ambient CO 2 concentrations. Major targets of these regulators are carbonic anhydrase and inorganic carbon uptake systems (CO 2 and HCO 3 - transporters) whose activities are modulated either at transcriptional level or by changes in the levels of their co-regulatory metabolites. The article provides information on the localization of the CCM components as well as their function and participation in the development of an efficient CCM. Signal transduction cascades leading to activation/inactivation of inducible CCM components on perception of low/high CO 2 stimuli have also been brought into picture. A detailed study of the regulatory components can aid in identifying the unraveled aspects of these mechanisms and hence provide information on key molecules that need to be explored to further provide a clear understanding of the mechanism under study.

A tribute to Ulrich Heber (1930-2016) for his contribution to photosynthesis research: understanding the interplay between photosynthetic primary reactions, metabolism and the environment.

PubMed

Dietz, Karl-Josef; Krause, G Heinrich; Siebke, Katharina; Krieger-Liszkay, Anja

2018-07-01

The dynamic and efficient coordination of primary photosynthetic reactions with leaf energization and metabolism under a wide range of environmental conditions is a fundamental property of plants involving processes at all functional levels. The present historical perspective covers 60 years of research aiming to understand the underlying mechanisms, linking major breakthroughs to current progress. It centers on the contributions of Ulrich Heber who had pioneered novel concepts, fundamental methods, and mechanistic understanding of photosynthesis. An important first step was the development of non-aqueous preparation of chloroplasts allowing the investigation of chloroplast metabolites ex vivo (meaning that the obtained results reflect the in vivo situation). Later on, intact chloroplasts, retaining their functional envelope membranes, were isolated in aqueous media to investigate compartmentation and exchange of metabolites between chloroplasts and external medium. These studies elucidated metabolic interaction between chloroplasts and cytoplasm during photosynthesis. Experiments with isolated intact chloroplasts clarified that oxygenation of ribulose-1.5-bisphosphate generates glycolate in photorespiration. The development of non-invasive optical methods enabled researchers identifying mechanisms that balance electron flow in the photosynthetic electron transport system avoiding its over-reduction. Recording chlorophyll a (Chl a) fluorescence allowed one to monitor, among other parameters, thermal energy dissipation by means of 'nonphotochemical quenching' of the excited state of Chl a. Furthermore, studies both in vivo and in vitro led to basic understanding of the biochemical mechanisms of freezing damage and frost tolerance of plant leaves, to SO 2 tolerance of tree leaves and dehydrating lichens and mosses.

Niche specialization of novel Thaumarchaeota to oxic and hypoxic acidic geothermal springs of Yellowstone National Park

PubMed Central

Beam, Jacob P; Jay, Zackary J; Kozubal, Mark A; Inskeep, William P

2014-01-01

Novel lineages of the phylum Thaumarchaeota are endemic to thermal habitats, and may exhibit physiological capabilities that are not yet observed in members of this phylum. The primary goals of this study were to conduct detailed phylogenetic and functional analyses of metagenome sequence assemblies of two different thaumarchaeal populations found in high-temperature (65–72 °C), acidic (pH∼3) iron oxide and sulfur sediment environments of Yellowstone National Park (YNP). Metabolic reconstruction was coupled with detailed geochemical measurements of each geothermal habitat and reverse-transcriptase PCR to confirm the in situ activity of these populations. Phylogenetic analyses of ribosomal and housekeeping proteins place these archaea near the root of the thaumarchaeal branch. Metabolic reconstruction suggests that these populations are chemoorganotrophic and couple growth with the reduction of oxygen or nitrate in iron oxide habitats, or sulfur in hypoxic sulfur sediments. The iron oxide population has the potential for growth via the oxidation of sulfide to sulfate using a novel reverse sulfate reduction pathway. Possible carbon sources include aromatic compounds (for example, 4-hydroxyphenylacetate), complex carbohydrates (for example, starch), oligopeptides and amino acids. Both populations contain a type III ribulose bisphosphate carboxylase/oxygenase used for carbon dioxide fixation or adenosine monophosphate salvage. No evidence for the oxidation of ammonia was obtained from de novo sequence assemblies. Our results show that thermoacidophilic Thaumarchaeota from oxic iron mats and hypoxic sulfur sediments exhibit different respiratory machinery depending on the presence of oxygen versus sulfide, represent deeply rooted lineages within the phylum Thaumarchaeota and are endemic to numerous sites in YNP. PMID:24196321

Overexpression of the rubisco activase gene improves growth and low temperature and weak light tolerance in Cucumis sativus.

PubMed

Bi, Huangai; Liu, Peipei; Jiang, Zhensheng; Ai, Xizhen

2017-10-01

Rubisco activase (RCA) is an important enzyme that can catalyze the carboxylation and oxygenation activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which is involved in the photosynthetic carbon reduction cycle. Here, we studied the effects of changes in RCA activity on photosynthesis, growth and development, as well as the low temperature and weak light tolerance of RCA overexpressing transgenic cucumber (Cucumis sativus) plants. CsRCA overexpression increased the plant height, leaf area and dry matter, and decreased the root/top ratio in transgenic cucumber plants compared with the wild-type (WT) plants. Low temperature and low light stress led to decreases in the CsRCA expression and protein levels, the photosynthetic rate (Pn) and the stomatal conductance (Gs), but an increase in the intercellular CO 2 (Ci) concentration in cucumber leaves. The actual photochemical efficiency and maximal photochemical efficiency of photosystem II in cucumber seedlings also declined, but the initial fluorescence increased during low temperature and weak light stress. Transgenic plants showed a lower decrease in the CsRCA expression level and actual and maximal photochemical efficiencies, as well as increases in the Ci and initial fluorescence relative to the WT plants. Low temperature and low light stress resulted in a significant increase in the malondialdehyde (MDA) content; however, this increase was reduced in transgenic plants compared with that in WT plants. Thus, the overexpression of CsRCA may promote the growth and low temperature and low light tolerance of cucumber plants in solar greenhouses. © 2017 Scandinavian Plant Physiology Society.

Direct characterization of the native structure and mechanics of cyanobacterial carboxysomes.

PubMed

Faulkner, Matthew; Rodriguez-Ramos, Jorge; Dykes, Gregory F; Owen, Siân V; Casella, Selene; Simpson, Deborah M; Beynon, Robert J; Liu, Lu-Ning

2017-08-03

Carboxysomes are proteinaceous organelles that play essential roles in enhancing carbon fixation in cyanobacteria and some proteobacteria. These self-assembling organelles encapsulate Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase using a protein shell structurally resembling an icosahedral viral capsid. The protein shell serves as a physical barrier to protect enzymes from the cytosol and a selectively permeable membrane to mediate transport of enzyme substrates and products. The structural and mechanical nature of native carboxysomes remain unclear. Here, we isolate functional β-carboxysomes from the cyanobacterium Synechococcus elongatus PCC7942 and perform the first characterization of the macromolecular architecture and inherent physical mechanics of single β-carboxysomes using electron microscopy, atomic force microscopy (AFM) and proteomics. Our results illustrate that the intact β-carboxysome comprises three structural domains, a single-layered icosahedral shell, an inner layer and paracrystalline arrays of interior Rubisco. We also observe the protein organization of the shell and partial β-carboxysomes that likely serve as the β-carboxysome assembly intermediates. Furthermore, the topography and intrinsic mechanics of functional β-carboxysomes are determined in native conditions using AFM and AFM-based nanoindentation, revealing the flexible organization and soft mechanical properties of β-carboxysomes compared to rigid viruses. Our study provides new insights into the natural characteristics of β-carboxysome organization and nanomechanics, which can be extended to diverse bacterial microcompartments and are important considerations for the design and engineering of functional carboxysomes in other organisms to supercharge photosynthesis. It offers an approach for inspecting the structural and mechanical features of synthetic metabolic organelles and protein scaffolds in bioengineering.

Leaf nitrogen assimilation and partitioning differ among subtropical forest plants in response to canopy addition of nitrogen treatments.

PubMed

Liu, Nan; Wu, Shuhua; Guo, Qinfeng; Wang, Jiaxin; Cao, Ce; Wang, Jun

2018-05-12

Global increases in nitrogen deposition may alter forest structure and function by interfering with plant nitrogen metabolism (e.g., assimilation and partitioning) and subsequent carbon assimilation, but it is unclear how these responses to nitrogen deposition differ among species. In this study, we conducted a 2-year experiment to investigate the effects of canopy addition of nitrogen (CAN) on leaf nitrogen assimilation and partitioning in three subtropical forest plants (Castanea henryi, Ardisia quinquegona, and Blastus cochinchinensis). We hypothesized that responses of leaf nitrogen assimilation and partitioning to CAN differ among subtropical forest plants. CAN increased leaf nitrate reductase (NR) activity, and leaf nitrogen and chlorophyll contents but reduced leaf maximum photosynthetic rate (A max ), photosynthetic nitrogen use efficiency (PNUE), ribulose-1,5-bisphosphate carboxylase (Rubisco) activity, and metabolic protein content of an overstory tree species C. henryi. In an understory tree A. quinquegona, CAN increased NR activity and glutamine synthetase activity and therefore increased metabolic protein synthesis (e.g., Rubisco) in leaves. In the shrub B. cochinchinensis, CAN increased A max , PNUE, Rubisco content, metabolic protein content, and Rubisco activity in leaves. Leaf nitrogen assimilation and partitioning results indicated that A. quinquegona and B. cochinchinensis may better acclimate to CAN than C. henryi and that the acclimation mechanism differs among the species. Results from this study suggest that long-term elevated atmospheric nitrogen deposition has contributed to the ongoing transformation of subtropical forests into communities dominated by small trees and shrubs. Copyright © 2018 Elsevier B.V. All rights reserved.

The fixABCX genes in Rhodospirillum rubrum encode a putative membrane complex participating in electron transfer to nitrogenase.

PubMed

Edgren, Tomas; Nordlund, Stefan

2004-04-01

In our efforts to identify the components participating in electron transport to nitrogenase in Rhodospirillum rubrum, we used mini-Tn5 mutagenesis followed by metronidazole selection. One of the mutants isolated, SNT-1, exhibited a decreased growth rate and about 25% of the in vivo nitrogenase activity compared to the wild-type values. The in vitro nitrogenase activity was essentially wild type, indicating that the mutation affects electron transport to nitrogenase. Sequencing showed that the Tn5 insertion is located in a region with a high level of similarity to fixC, and extended sequencing revealed additional putative fix genes, in the order fixABCX. Complementation of SNT-1 with the whole fix gene cluster in trans restored wild-type nitrogenase activity and growth. Using Western blotting, we demonstrated that expression of fixA and fixB occurs only under conditions under which nitrogenase also is expressed. SNT-1 was further shown to produce larger amounts of both ribulose 1,5-bisphosphate carboxylase/oxygenase and polyhydroxy alkanoates than the wild type, indicating that the redox status is affected in this mutant. Using Western blotting, we found that FixA and FixB are soluble proteins, whereas FixC most likely is a transmembrane protein. We propose that the fixABCX genes encode a membrane protein complex that plays a central role in electron transfer to nitrogenase in R. rubrum. Furthermore, we suggest that FixC is the link between nitrogen fixation and the proton motive force generated in the photosynthetic reactions.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hay, J.; Schwender, J.

Plant oils are an important renewable resource, and seed oil content is a key agronomical trait that is in part controlled by the metabolic processes within developing seeds. A large-scale model of cellular metabolism in developing embryos of Brassica napus (bna572) was used to predict biomass formation and to analyze metabolic steady states by flux variability analysis under different physiological conditions. Predicted flux patterns are highly correlated with results from prior 13C metabolic flux analysis of B. napus developing embryos. Minor differences from the experimental results arose because bna572 always selected only one sugar and one nitrogen source from themore » available alternatives, and failed to predict the use of the oxidative pentose phosphate pathway. Flux variability, indicative of alternative optimal solutions, revealed alternative pathways that can provide pyruvate and NADPH to plastidic fatty acid synthesis. The nutritional values of different medium substrates were compared based on the overall carbon conversion efficiency (CCE) for the biosynthesis of biomass. Although bna572 has a functional nitrogen assimilation pathway via glutamate synthase, the simulations predict an unexpected role of glycine decarboxylase operating in the direction of NH4+ assimilation. Analysis of the light-dependent improvement of carbon economy predicted two metabolic phases. At very low light levels small reductions in CO2 efflux can be attributed to enzymes of the tricarboxylic acid cycle (oxoglutarate dehydrogenase, isocitrate dehydrogenase) and glycine decarboxylase. At higher light levels relevant to the 13C flux studies, ribulose-1,5-bisphosphate carboxylase activity is predicted to account fully for the light-dependent changes in carbon balance.« less

Role of Small Subunit in Mediating Assembly of Red-type Form I Rubisco

PubMed Central

Joshi, Jidnyasa; Mueller-Cajar, Oliver; Tsai, Yi-Chin C.; Hartl, F. Ulrich; Hayer-Hartl, Manajit

2015-01-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme involved in photosynthetic carbon fixation, converting atmospheric CO2 to organic compounds. Form I Rubisco is a cylindrical complex composed of eight large (RbcL) subunits that are capped by four small subunits (RbcS) at the top and four at the bottom. Form I Rubiscos are phylogenetically divided into green- and red-type. Some red-type enzymes have catalytically superior properties. Thus, understanding their folding and assembly is of considerable biotechnological interest. Folding of the green-type RbcL subunits in cyanobacteria is mediated by the GroEL/ES chaperonin system, and assembly to holoenzyme requires specialized chaperones such as RbcX and RAF1. Here, we show that the red-type RbcL subunits in the proteobacterium Rhodobacter sphaeroides also fold with GroEL/ES. However, assembly proceeds in a chaperone-independent manner. We find that the C-terminal β-hairpin extension of red-type RbcS, which is absent in green-type RbcS, is critical for efficient assembly. The β-hairpins of four RbcS subunits form an eight-stranded β-barrel that protrudes into the central solvent channel of the RbcL core complex. The two β-barrels stabilize the complex through multiple interactions with the RbcL subunits. A chimeric green-type RbcS carrying the C-terminal β-hairpin renders the assembly of a cyanobacterial Rubisco independent of RbcX. Our results may facilitate the engineering of crop plants with improved growth properties expressing red-type Rubisco. PMID:25371207

A function-based screen for seeking RubisCO active clones from metagenomes: novel enzymes influencing RubisCO activity.

PubMed

Böhnke, Stefanie; Perner, Mirjam

2015-03-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a key enzyme of the Calvin cycle, which is responsible for most of Earth's primary production. Although research on RubisCO genes and enzymes in plants, cyanobacteria and bacteria has been ongoing for years, still little is understood about its regulation and activation in bacteria. Even more so, hardly any information exists about the function of metagenomic RubisCOs and the role of the enzymes encoded on the flanking DNA owing to the lack of available function-based screens for seeking active RubisCOs from the environment. Here we present the first solely activity-based approach for identifying RubisCO active fosmid clones from a metagenomic library. We constructed a metagenomic library from hydrothermal vent fluids and screened 1056 fosmid clones. Twelve clones exhibited RubisCO activity and the metagenomic fragments resembled genes from Thiomicrospira crunogena. One of these clones was further analyzed. It contained a 35.2 kb metagenomic insert carrying the RubisCO gene cluster and flanking DNA regions. Knockouts of twelve genes and two intergenic regions on this metagenomic fragment demonstrated that the RubisCO activity was significantly impaired and was attributed to deletions in genes encoding putative transcriptional regulators and those believed to be vital for RubisCO activation. Our new technique revealed a novel link between a poorly characterized gene and RubisCO activity. This screen opens the door to directly investigating RubisCO genes and respective enzymes from environmental samples.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reckmann, U.; Scheibe, R.; Raschke, K.

We investigated whether the reductive pentose phosphate path in guard cells of Pisum sativum had the capacity to contribute significantly to the production of osmotica during stomatal opening in the light. Amounts of ribulose 1,5-bisphophate carboxylase/oxygenase (Rubisco) were determined by the ({sup 14}C) carboxyarabinitol bisphosphate assay. A guard cell contained about 1.2 and a mesophyll cell about 324 picograms of the enzyme; the ratio was 1:270. The specific activities of Rubisco in guard cells and in mesophyll cells were equal; there was no indication of a specific inhibitor of Rubisco in guard cells. Rubisco activity was 115 femtomol per guard-cellmore » protoplast and hour. This value was different from zero with a probability of 0.99. After exposure of guard-cell protoplasts to {sup 14}CO{sub 2} for 2 seconds in the light, about one-half of the radioactivity was in phosphorylated compounds and <10% in malate. Guard cells in epidermal strips produced a different labelling pattern; in the light, <10% of the label was in phosphorylated compounds and about 60% in malate. The rate of solute accumulation in intact guard cells was estimated to have been 900 femto-osmol per cell and hour. If Rubisco operated at full capacity in guard cells, and hexoses were produced as osmotica, solutes could be supplied at a rate of 19femto-osmol per cell and hour, which would constitute 2% of the estimated requirement. The capacity of guard-cell Rubisco to meet the solute requirement for stomatal opening in leaves of Pisum sativum is insignificant.« less

Stability-activity tradeoffs constrain the adaptive evolution of RubisCO.

PubMed

Studer, Romain A; Christin, Pascal-Antoine; Williams, Mark A; Orengo, Christine A

2014-02-11

A well-known case of evolutionary adaptation is that of ribulose-1,5-bisphosphate carboxylase (RubisCO), the enzyme responsible for fixation of CO2 during photosynthesis. Although the majority of plants use the ancestral C3 photosynthetic pathway, many flowering plants have evolved a derived pathway named C4 photosynthesis. The latter concentrates CO2, and C4 RubisCOs consequently have lower specificity for, and faster turnover of, CO2. The C4 forms result from convergent evolution in multiple clades, with substitutions at a small number of sites under positive selection. To understand the physical constraints on these evolutionary changes, we reconstructed in silico ancestral sequences and 3D structures of RubisCO from a large group of related C3 and C4 species. We were able to precisely track their past evolutionary trajectories, identify mutations on each branch of the phylogeny, and evaluate their stability effect. We show that RubisCO evolution has been constrained by stability-activity tradeoffs similar in character to those previously identified in laboratory-based experiments. The C4 properties require a subset of several ancestral destabilizing mutations, which from their location in the structure are inferred to mainly be involved in enhancing conformational flexibility of the open-closed transition in the catalytic cycle. These mutations are near, but not in, the active site or at intersubunit interfaces. The C3 to C4 transition is preceded by a sustained period in which stability of the enzyme is increased, creating the capacity to accept the functionally necessary destabilizing mutations, and is immediately followed by compensatory mutations that restore global stability.

Effects of CO2 Concentration on Rubisco Activity, Amount, and Photosynthesis in Soybean Leaves 1

PubMed Central

Campbell, William J.; Allen, L. H.; Bowes, George

1988-01-01

Growth at an elevated CO2 concentration resulted in an enhanced capacity for soybean (Glycine max L. Merr. cv Bragg) leaflet photosynthesis. Plants were grown from seed in outdoor controlled-environment chambers under natural solar irradiance. Photosynthetic rates, measured during the seed filling stage, were up to 150% greater with leaflets grown at 660 compared to 330 microliters of CO2 per liter when measured across a range of intercellular CO2 concentrations and irradiance. Soybean plants grown at elevated CO2 concentrations had heavier pod weights per plant, 44% heavier with 660 compared to 330 microliters of CO2 per liter grown plants, and also greater specific leaf weights. Ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) activity showed no response (mean activity of 96 micromoles of CO2 per square meter per second expressed on a leaflet area basis) to short-term (∼1 hour) exposures to a range of CO2 concentrations (110-880 microliters per liter), nor was a response of activity (mean activity of 1.01 micromoles of CO2 per minute per milligram of protein) to growth CO2 concentration (160-990 microliters per liter) observed. The amount of rubisco protein was constant, as growth CO2 concentration was varied, and averaged 55% of the total leaflet soluble protein. Although CO2 is required for activation of rubisco, results indicated that within the range of CO2 concentrations used (110-990 microliters per liter), rubisco activity in soybean leaflets, in the light, was not regulated by CO2. PMID:16666460

Effects of carbohydrate accumulation on photosynthesis differ between sink and source leaves of Phaseolus vulgaris L.

PubMed

Araya, Takao; Noguchi, Ko; Terashima, Ichiro

2006-05-01

Accumulation of non-structural carbohydrate in leaves represses photosynthesis. However, the extent of repression should be different between sink leaves (sugar consumers) and source leaves (sugar exporters). We investigated the effects of carbohydrate accumulation on photosynthesis in the primary leaves of bean (Phaseolus vulgaris L.) during leaf expansion. To increase the carbohydrate content of the leaves, we supplied 20 mM sucrose solution to the roots for 5 d (sugar treatment). Plants supplied only with water and nutrients were used as controls. The carbohydrate contents, which are the sum of glucose, sucrose and starch, of the sugar-treated leaves were 1.5-3 times of those of the control leaves at all developmental stages. In the young sink leaves, the photosynthetic rate at saturating light and at an ambient CO2 concentration (A360) did not differ between the sugar-treated and control leaves. The A360 of sugar-treated source leaves gradually decreased relative to the control source leaves with leaf expansion. The initial slope of the A-Ci (CO2 concentration in the intercellular space) curve, and the Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) content per leaf area showed trends similar to that of A360. Differences in Amax between the treatments were slightly smaller than those in A360. These results indicate that the effect of carbohydrate accumulation on photosynthesis is significant in the source leaves, but not in the young sink leaves, and that the decrease in Rubisco content was the main cause of the carbohydrate repression of photosynthesis.

Proteomic Analysis of the Relationship between Metabolism and Nonhost Resistance in Soybean Exposed to Bipolaris maydis.

PubMed

Dong, Yumei; Su, Yuan; Yu, Ping; Yang, Min; Zhu, Shusheng; Mei, Xinyue; He, Xiahong; Pan, Manhua; Zhu, Youyong; Li, Chengyun

2015-01-01

Nonhost resistance (NHR) pertains to the most common form of plant resistance against pathogenic microorganisms of other species. Bipolaris maydis is a non-adapted pathogen affecting soybeans, particularly of maize/soybean intercropping systems. However, no experimental evidence has described the immune response of soybeans against B. maydis. To elucidate the molecular mechanism underlying NHR in soybeans, proteomics analysis based on two-dimensional polyacrylamide gel electrophoresis (2-DE) was performed to identify proteins involved in the soybean response to B. maydis. The spread of B. maydis spores across soybean leaves induced NHR throughout the plant, which mobilized almost all organelles and various metabolic processes in response to B. maydis. Some enzymes, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), mitochondrial processing peptidase (MPP), oxygen evolving enhancer (OEE), and nucleoside diphosphate kinase (NDKs), were found to be related to NHR in soybeans. These enzymes have been identified in previous studies, and STRING analysis showed that most of the protein functions related to major metabolic processes were induced as a response to B. maydis, which suggested an array of complex interactions between soybeans and B. maydis. These findings suggest a systematic NHR against non-adapted pathogens in soybeans. This response was characterized by an overlap between metabolic processes and response to stimulus. Several metabolic processes provide the soybean with innate immunity to the non-adapted pathogen, B. maydis. This research investigation on NHR in soybeans may foster a better understanding of plant innate immunity, as well as the interactions between plant and non-adapted pathogens in intercropping systems.

Proteomic Analysis of the Relationship between Metabolism and Nonhost Resistance in Soybean Exposed to Bipolaris maydis

PubMed Central

Dong, Yumei; Su, Yuan; Yu, Ping; Yang, Min; Zhu, Shusheng; Mei, Xinyue; He, Xiahong; Pan, Manhua; Zhu, Youyong; Li, Chengyun

2015-01-01

Nonhost resistance (NHR) pertains to the most common form of plant resistance against pathogenic microorganisms of other species. Bipolaris maydis is a non-adapted pathogen affecting soybeans, particularly of maize/soybean intercropping systems. However, no experimental evidence has described the immune response of soybeans against B. maydis. To elucidate the molecular mechanism underlying NHR in soybeans, proteomics analysis based on two-dimensional polyacrylamide gel electrophoresis (2-DE) was performed to identify proteins involved in the soybean response to B. maydis. The spread of B. maydis spores across soybean leaves induced NHR throughout the plant, which mobilized almost all organelles and various metabolic processes in response to B. maydis. Some enzymes, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), mitochondrial processing peptidase (MPP), oxygen evolving enhancer (OEE), and nucleoside diphosphate kinase (NDKs), were found to be related to NHR in soybeans. These enzymes have been identified in previous studies, and STRING analysis showed that most of the protein functions related to major metabolic processes were induced as a response to B. maydis, which suggested an array of complex interactions between soybeans and B. maydis. These findings suggest a systematic NHR against non-adapted pathogens in soybeans. This response was characterized by an overlap between metabolic processes and response to stimulus. Several metabolic processes provide the soybean with innate immunity to the non-adapted pathogen, B. maydis. This research investigation on NHR in soybeans may foster a better understanding of plant innate immunity, as well as the interactions between plant and non-adapted pathogens in intercropping systems. PMID:26513657

Photosynthetic characteristics of an amphibious plant, Eleocharis vivipara: Expression of C4 and C3 modes in contrasting environments

PubMed Central

Ueno, Osamu; Samejima, Muneaki; Muto, Shoshi; Miyachi, Shigetoh

1988-01-01

Eleocharis vivipara Link, a freshwater amphibious leafless plant belonging to the Cyperaceae can grow in both terrestrial and submersed aquatic conditions. Two forms of E. vivipara obtained from these contrasting environments were examined for the characteristics associated with C4 and C3 photosynthesis. In the terrestrial form (δ 13C values = -13.5 to -15.4‰, where ‰ is parts per thousand), the culms, which are photosynthetic organs, possess a Kranz-type anatomy typical of C4 plants, and well-developed bundle-sheath cells contain numerous large chloroplasts. In the submersed form (δ 13C value = -25.9‰), the culms possess anatomical features characteristic of submersed aquatic plants, and the reduced bundle-sheath cells contain only a few small chloroplasts. 14C pulse-12C chase experiments showed that the terrestrial form and the submersed form fix carbon by way of the C4 pathway, with aspartate (40%) and malate (35%) as the main primary products, and by way of the C3 pathway, with 3-phosphoglyceric acid (53%) and sugar phosphates (14%) as the main primary products, respectively. The terrestrial form showed photosynthetic enzyme activities typical of the NAD-malic enzyme-C4 subtype, whereas the submersed form showed decreased activities of key C4 enzymes and an increased ribulose 1,5-bisphosphate carboxylase (EC 4.1.1.39) activity. These data suggest that this species can differentiate into the C4 mode under terrestrial conditions and into the C3 mode under submersed conditions. Images PMID:16593980

Photosynthetic activity during olive (Olea europaea) leaf development correlates with plastid biogenesis and Rubisco levels.

PubMed

Maayan, Inbar; Shaya, Felix; Ratner, Kira; Mani, Yair; Lavee, Shimon; Avidan, Benjamin; Shahak, Yosepha; Ostersetzer-Biran, Oren

2008-11-01

Olive leaves are known to mature slowly, reaching their maximum photosynthetic activity only after full leaf expansion. Poor assimilation rates, typical to young olive leaves, were previously associated with low stomata conductance. Yet, very little is known about chloroplast biogenesis throughout olive leaf development. Here, the photosynthetic activity and plastids development throughout leaf maturation is characterized by biochemical and ultrastructural analyses. Although demonstrated only low photosynthetic activity, the plastids found in young leaves accumulated both photosynthetic pigments and proteins required for photophosphorylation and carbon fixation. However, Rubisco (ribulose-1,5-bisphosphate carboxylase-oxygenase), which catalyzes the first major step of carbon fixation and one of the most abundant proteins in plants, could not be detected in the young leaves and only slowly accumulated throughout development. In fact, Rubisco levels seemed tightly correlated with the observed photosynthetic activities. Unlike Rubisco, numerous proteins accumulated in the young olive leaves. These included the early light induced proteins, which may be required to reduce the risk of photodamage, because of light absorption by photosynthetic pigments. Also, high levels of ribosomal L11 subunit, transcription factor elF-5A, Histones H2B and H4 were observed in the apical leaves, and in particular a plastidic-like aldolase, which accounted for approximately 30% of the total proteins. These proteins may upregulate in their levels to accommodate the high demand for metabolic energy in the young developing plant tissue, further demonstrating the complex sink-to-source relationship between young and photosynthetically active mature leaves.

Physiological responses of the CAM epiphyte Tillandsia usneoides L. (Bromeliaceae) to variations in light and water supply.

PubMed

Haslam, Richard; Borland, Anne; Maxwell, Kate; Griffiths, Howard

2003-06-01

In an effort to understand the mechanisms that sustain rootless atmospheric plants, the modulation of Crassulacean acid metabolism (CAM) in response to variations in irradiance and water supply was investigated in the epiphyte Tillandsia usneoides. Plants were acclimated to three light regimes, i.e. high, intermediate and low, with integrated photon flux densities (PFD) of 14.40, 8.64 and 4.32 mol m-2 d-1 equivalent to an instantaneous PFD of 200, 100, and 50 mumol m-2 s-1, respectively. Daily watering was then withdrawn from half of the plants at each PFD for 7 d prior to sampling. In response to the three PFD treatments, chlorophyll content increased in plants acclimated to lower irradiances. Light response curves using non-invasive measurements of chlorophyll fluorescence demonstrated that photosystem II efficiency (phi PSII) was maintained in high PFD acclimated plants, as they exhibited a larger capacity for non-photochemical dissipation (NPQ) of excess light energy than low PFD acclimated plants. Net CO2 uptake increased in response to higher PFD, reflecting enhanced carboxylation capacity in terms of phosphoenolpyruvate carboxylase (PEPc) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activities. After water was withdrawn, nocturnal net CO2 uptake and accumulated levels of acidity declined in all PFD treatments, concomitant with increased respiratory recycling of malate. Examining the strategies employed by epiphytes such as T. usneodies to tolerate extreme light and water regimes has demonstrated the importance of physiological mechanisms that allow flexible carboxylation capacity and continued carbon cycling to maintain photosynthetic integrity.

A Proteomic Analysis of the Upper and Lower Flanks of the Base of Rice Shoot in the Gravitropism

NASA Astrophysics Data System (ADS)

Hu, Liwei; Chen, Haiying; Dou, Xianying; Jin, Jing; Sun, Weining; Cai, Weiming

2015-11-01

Due to gravitational stimulation, the lower part of a shoot base grows faster than the upper part, leading the shoot to curve upward. Though much research has been done on the mechanism of plant gravitropism, it still requires extensive elucidation. Recently, functional genomic strategies have been applied to study this mechanism in plants. The present study carried out a proteomic analysis to gain a better understanding of gravity stimulation in rice. Three-week-old rice seedlings were gravitropically stimulated and samples were harvested at 4 different time points: 0.5, 3, 6, and 9 h. Then, the total crude proteins were extracted from the lower and upper parts of the shoot base, separated by 2-DE, and silver stained. At each time point, proteins in the lower and upper parts were compared, and the differently expressed proteins were identified using MALDI TOF or ESI-MS/MS. After gravity stimulation, proteins involved in nine different functional categories were either up-regulated or down-regulated. Sugar metabolism, glycolysis, the tricarboxylic acid (TCA/citric) cycle, pyruvate metabolism, and transcription regulation-related proteins were regulated. Although the initiation of defense reactions mainly occurred in roots, some different defense mechanisms were also evoked in the aerial tissues. Interestingly, the abundance of some proteins changed drastically at only 0.5 h after reorientation: inosine monophosphate dehydrogenase (up to 6.49-fold higher in lower flanks at 0.5 h), ATP synthase D (4.25-fold), and ribulose-1,5 -bisphosphate carboxylase oxygenase (3.62-fold). These findings may aid in understanding the mechanism of the gravitropism.

Alterations in leaf nitrogen metabolism indicated the structural changes of subtropical forest by canopy addition of nitrogen.

PubMed

Liu, Nan; Wang, Jiaxin; Guo, Qinfeng; Wu, Shuhua; Rao, Xingquan; Cai, Xi'an; Lin, Zhifang

2018-09-30

Globally, nitrogen deposition increment has caused forest structural changes due to imbalanced plant nitrogen metabolism and subsequent carbon assimilation. Here, a 2 consecutive-year experiment was conducted to reveal the effects of canopy addition of nitrogen (CAN) on nitrogen absorption, assimilation, and allocation in leaves of three subtropical forest woody species (Castanea henryi, Ardisia quinquegona, and Blastus cochinchinensis). We hypothesized that CAN altered leaf nitrogen absorption, assimilation and partitioning of different plants in different ways in subtropical forest. It shows that CAN increased maximum photosynthetic rate (A max ), photosynthetic nitrogen use efficiency (PNUE), and metabolic protein content of the two understory species A. quinquegona and B. cochinchinensis. By contrary, for the overstory species, C. henryi, A max , PNUE, and metabolic protein content were significantly reduced in response to CAN. We found that changes in leaf nitrogen metabolism were mainly due to the differences in enzyme (e.g. Ribulose-1,5-bisphosphate carboxylase, nitrate reductase, nitrite reductase and glutamine synthetase) activities under CAN treatment. Our results indicated that C. henryi may be more susceptible to CAN treatment, and both A. quinquegona and B. cochinchinensis could better adapt to CAN treatment but in different ways. Our findings may partially explain the ongoing degradation of subtropical forest into a community dominated by small trees and shrubs in recent decades. It is possible that persistent high levels of atmospheric nitrogen deposition will lead to the steady replacement of dominant woody species in this subtropical forest. Copyright © 2018 Elsevier Inc. All rights reserved.

Novel magnetite-producing magnetotactic bacteria belonging to the Gammaproteobacteria.

PubMed

Lefèvre, Christopher T; Viloria, Nathan; Schmidt, Marian L; Pósfai, Mihály; Frankel, Richard B; Bazylinski, Dennis A

2012-02-01

Two novel magnetotactic bacteria (MTB) were isolated from sediment and water collected from the Badwater Basin, Death Valley National Park and southeastern shore of the Salton Sea, respectively, and were designated as strains BW-2 and SS-5, respectively. Both organisms are rod-shaped, biomineralize magnetite, and are motile by means of flagella. The strains grow chemolithoautotrophically oxidizing thiosulfate and sulfide microaerobically as electron donors, with thiosulfate oxidized stoichiometrically to sulfate. They appear to utilize the Calvin-Benson-Bassham cycle for autotrophy based on ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) activity and the presence of partial sequences of RubisCO genes. Strains BW-2 and SS-5 biomineralize chains of octahedral magnetite crystals, although the crystals of SS-5 are elongated. Based on 16S rRNA gene sequences, both strains are phylogenetically affiliated with the Gammaproteobacteria class. Strain SS-5 belongs to the order Chromatiales; the cultured bacterium with the highest 16S rRNA gene sequence identity to SS-5 is Thiohalocapsa marina (93.0%). Strain BW-2 clearly belongs to the Thiotrichales; interestingly, the organism with the highest 16S rRNA gene sequence identity to this strain is Thiohalospira alkaliphila (90.2%), which belongs to the Chromatiales. Each strain represents a new genus. This is the first report of magnetite-producing MTB phylogenetically associated with the Gammaproteobacteria. This finding is important in that it significantly expands the phylogenetic diversity of the MTB. Physiology of these strains is similar to other MTB and continues to demonstrate their potential in nitrogen, iron, carbon and sulfur cycling in natural environments.

Hidden diversity revealed by genome-resolved metagenomics of iron-oxidizing microbial mats from Lō'ihi Seamount, Hawai'i.

PubMed

Fullerton, Heather; Hager, Kevin W; McAllister, Sean M; Moyer, Craig L

2017-08-01

The Zetaproteobacteria are ubiquitous in marine environments, yet this class of Proteobacteria is only represented by a few closely-related cultured isolates. In high-iron environments, such as diffuse hydrothermal vents, the Zetaproteobacteria are important members of the community driving its structure. Biogeography of Zetaproteobacteria has shown two ubiquitous operational taxonomic units (OTUs), yet much is unknown about their genomic diversity. Genome-resolved metagenomics allows for the specific binning of microbial genomes based on genomic signatures present in composite metagenome assemblies. This resulted in the recovery of 93 genome bins, of which 34 were classified as Zetaproteobacteria. Form II ribulose 1,5-bisphosphate carboxylase genes were recovered from nearly all the Zetaproteobacteria genome bins. In addition, the Zetaproteobacteria genome bins contain genes for uptake and utilization of bioavailable nitrogen, detoxification of arsenic, and a terminal electron acceptor adapted for low oxygen concentration. Our results also support the hypothesis of a Cyc2-like protein as the site for iron oxidation, now detected across a majority of the Zetaproteobacteria genome bins. Whole genome comparisons showed a high genomic diversity across the Zetaproteobacteria OTUs and genome bins that were previously unidentified by SSU rRNA gene analysis. A single lineage of cosmopolitan Zetaproteobacteria (zOTU 2) was found to be monophyletic, based on cluster analysis of average nucleotide identity and average amino acid identity comparisons. From these data, we can begin to pinpoint genomic adaptations of the more ecologically ubiquitous Zetaproteobacteria, and further understand their environmental constraints and metabolic potential.

Mixotrophic and autotrophic growth of Thiobacillus acidophilus on glucose and thiosulfate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pronk, J.T.; Meulenberg, R.; van den Berg, D.J.C.

1990-11-01

Mixotrophic growth of the facultatively autotrophic acidophile Thiobacillus acidophilus on mixtures of glucose and thiosulfate or tetrathionate was studied in substrate-limited chemostat cultures. Growth yields in mixotrophic cultures were higher than the sum of the heterotrophic and autographic growth yields. Pulse experiments with thiosulfate indicated that tetrathionate is an intermediate during thiosulfate oxidation by cell suspensions of T. acidophilus. From mixotrophic growth studied, the energetic value of thiosulfate and tetrathionate redox equivalents was estimated to be 50% of that of redox equivalents derived from glucose oxidation. Ribulose 1,5-bisphosphate carboxylase (RuBPCase) activities in cell extracts and rates of sulfur compound oxidationmore » by cell suspensions increased with increasing thiosulfate/glucose ratios in the influent medium of the mixotrophic cultures. Significant RuBPCase and sulfur compound-oxidizing activities were detected in heterotrophically grown T. acidophilus. Polyhedral inclusion bodies (carboxysomes) could be observed at low frequencies in thin sections of cells grown in heterotrophic, glucose-limited chemostat cultures. Highest RuBPCase activities and carboxysome abundancy were observed in cells from autotrophic, CO{sub 2}-limited chemostat cultures. The maximum growth rate at which thiosulfate was still completely oxidized was increased when glucose was utilized simultaneously. This, together with the fact that even during heterotrophic growth the organism exhibited significant activities of enzymes involved in autotrophic metabolism, indicates that T. acidophilus is well adapted to a mixotrophic lifestyle. In this respect, T. acidophilus may have a competitive advantage over autotrophic acidophiles with respect to the sulfur compound oxidation in environments in which organic compounds are present.« less

Antioxidative enzymes and expression of rbcL gene as tools to monitor heavy metal-related stress in plants.

PubMed

Jaskulak, Marta; Rorat, Agnieszka; Grobelak, Anna; Kacprzak, Małgorzata

2018-04-14

The aim of the study was to evaluate sensitivity and potential applications of selected biomarkers in phytoremediation under complex heavy metal contamination in Sinapis alba L., Robinia pseudoacacia L. and Lupinus luteus L as a potential tools in effective phytoremediation management. The toxicity assessment was conducted using selected measurement endpoints, both classical and advanced, i.e., germination index, roots length, guaiacol peroxidase activity (GPX), chlorophyll and protein content, the amount of total phenolic compounds (TPC) and level of expression of one of the ribulose-bisphosphate carboxylase genes (rbcL). Moreover, the influence of organic additives: cattle, horse manure, and vermicompost on lowering plant abiotic stress caused by complex heavy metal contamination was studied to assess the possible applications of selected stress markers in large scale phytoremediation planning. The results demonstrated the beneficial effects of selected soil additives on plant development. The 5% difference in the quantity of applied amendment caused statistically significant differences in GPX, TPC, chlorophyll content and expression level of rbcL. Among all endpoints, GPX activity, chlorophyll, and phenolic compounds content, as well as the expression of rbcL, turned out to be the most reliable assays for determination of the type and dosage of selected soil amendments (fertilizers) in the assisted phytoremediation process. Selected markers can be used to achieve the desired level of plant abiotic stress and consequently photosynthesis efficiency and CO 2 sequestration. The results showed, that presented assays can be used in different taxonomical groups such as Fabaceae for planning effective phytoremediation process. Copyright © 2018 Elsevier Ltd. All rights reserved.

Photorespiratory Bypasses Lead to Increased Growth in Arabidopsis thaliana: Are Predictions Consistent with Experimental Evidence?

PubMed Central

Basler, Georg; Küken, Anika; Fernie, Alisdair R.; Nikoloski, Zoran

2016-01-01

Arguably, the biggest challenge of modern plant systems biology lies in predicting the performance of plant species, and crops in particular, upon different intracellular and external perturbations. Recently, an increased growth of Arabidopsis thaliana plants was achieved by introducing two different photorespiratory bypasses via metabolic engineering. Here, we investigate the extent to which these findings match the predictions from constraint-based modeling. To determine the effect of the employed metabolic network model on the predictions, we perform a comparative analysis involving three state-of-the-art metabolic reconstructions of A. thaliana. In addition, we investigate three scenarios with respect to experimental findings on the ratios of the carboxylation and oxygenation reactions of Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). We demonstrate that the condition-dependent growth phenotypes of one of the engineered bypasses can be qualitatively reproduced by each reconstruction, particularly upon considering the additional constraints with respect to the ratio of fluxes for the RuBisCO reactions. Moreover, our results lend support for the hypothesis of a reduced photorespiration in the engineered plants, and indicate that specific changes in CO2 exchange as well as in the proxies for co-factor turnover are associated with the predicted growth increase in the engineered plants. We discuss our findings with respect to the structure of the used models, the modeling approaches taken, and the available experimental evidence. Our study sets the ground for investigating other strategies for increase of plant biomass by insertion of synthetic reactions. PMID:27092301

Carbon Acquisition by Cyanobacteria: Mechanisms, Comparative Genomics, and Evolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaplan, Aaron; Hagemann, Martin; Bauwe, Hermann

2008-01-01

In this chapter we mainly focus on the mechanisms of inorganic carbon uptake, photorespiration, and the regulation between the metabolic fluxes involved in photoautotrophic, photomixotrophic and heterotrophic growth. We identify the genes involved, their regulation and phylogeny. Living in an environment where the CO₂ concentration is considerably lower than required to saturate their carboxylating enzyme, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), cyanobacteria acquired the CO₂ concentrating mechanism (CCM) that enables them to accumulate CO₂ at the carboxylation site. All the cyanobacteria examined to date are able to fix CO₂ into carbohydrates. However, in addition to variance in the range of physical growthmore » conditions, cyanobacteria also vary substantially in their ability to consume organic carbon from their surroundings. Many strains are obligate photoautotrophs where the sole carbon source is CO₂, while others are able to perform photomixotrophic or even heterotrophic growth using a wide variety of organic substances (c.f. Rippka et al., 1979; Stal and Moezelaar, 1997b). Cyanobacteria constitute a unique case where the anabolic and catabolic carbohydrate metabolisms function in the same cellular compartment. In addition, the photosynthetic and respiratory electron transport pathways share components in the thylakoid membranes. Despite its importance to our understanding of cyanobacterial metabolism, little is known about the mechanisms involved in the shifts between photoautotrophic, heterotrophic and photomixotrophic modes of growth, and their regulation; between the different pathways of carbohydrate breakdown- glycolysis, fermentation, the oxidative pentose phosphate, the Krebs cycle and the photorespiratory pathways. In this chapter we shall briefly focus on recent advances in our understanding of the CCM and carbon metabolism in cyanobacteria.« less

Carbon acquisition by Cyanobacteria: Mechanisms, Comparative Genomics and Evolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaplan, Aaron; Hagemann, Martin; Bauwe, Hermann

2008-01-01

In this chapter we mainly focus on the mechanisms of inorganic carbon uptake, photorespiration, and the regulation between the metabolic fluxes involved in photoautotrophic, photomixotrophic and heterotrophic growth. We identify the genes involved, their regulation and phylogeny. Living in an environment where the CO₂ concentration is considerably lower than required to saturate their carboxylating enzyme, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), cyanobacteria acquired the CO₂ concentrating mechanism (CCM) that enables them to accumulate CO₂ at the carboxylation site. All the cyanobacteria examined to date are able to fix CO₂ into carbohydrates. However, in addition to variance in the range of physical growthmore » conditions, cyanobacteria also vary substantially in their ability to consume organic carbon from their surroundings. Many strains are obligate photoautotrophs where the sole carbon source is CO₂, while others are able to perform photomixotrophic or even heterotrophic growth using a wide variety of organic substances (c.f. Rippka et al., 1979; Stal and Moezelaar, 1997b). Cyanobacteria constitute a unique case where the anabolic and catabolic carbohydrate metabolisms function in the same cellular compartment. In addition, the photosynthetic and respiratory electron transport pathways share components in the thylakoid membranes. Despite its importance to our understanding of cyanobacterial metabolism, little is known about the mechanisms involved in the shifts between photoautotrophic, heterotrophic and photomixotrophic modes of growth, and their regulation; between the different pathways of carbohydrate breakdown- glycolysis, fermentation, the oxidative pentose phosphate, the Krebs cycle and the photorespiratory pathways. In this chapter we shall briefly focus on recent advances in our understanding of the CCM and carbon metabolism in cyanobacteria.« less

Over-expression of LeNCED1 in tomato (Solanum lycopersicum L.) with the rbcS3C promoter allows recovery of lines that accumulate very high levels of abscisic acid and exhibit severe phenotypes.

PubMed

Tung, Swee Ang; Smeeton, Rachel; White, Charlotte A; Black, Colin R; Taylor, Ian B; Hilton, Howard W; Thompson, Andrew J

2008-07-01

Previous work where 9-cis-epoxycarotenoid dioxygenase (NCED) was over-expressed using the constitutive Gelvin Superpromoter resulted in mild increases in abscisic acid (ABA) accumulation, accompanied by stomatal closure and increased water-use efficiency (WUE), but with apparently little impact on long-term biomass production. However, one of the negative effects of the over-expression of NCED using constitutive promoters in tomato was increased seed dormancy. Here we report the use of the rbcS3C promoter, from a gene encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), to drive LeNCED1 transgene expression in tomato in a light-responsive and circadian manner. In comparison to the constitutive promoter, the rbcS3C promoter allowed the generation of transgenic plants with much higher levels of ABA accumulation in leaves and sap, but the effect on seed dormancy was diminished. These plants displayed the expected reductions in stomatal conductance and CO(2) assimilation, but they also exhibited a severe set of symptoms that included perturbed cotyledon release from the testa, increased photobleaching in young seedlings, substantially reduced chlorophyll and carotenoid content, interveinal leaf flooding, and greatly reduced growth. These symptoms illustrate adverse consequences of long-term, very high ABA accumulation. Only more moderate increases in ABA biosynthesis are likely to be useful in the context of agriculture. Implications are discussed for the design of transgenic 'high ABA' plants that exhibit increased WUE but have minimal negative phenotypic effects.

Maternal control of seed oil content in Brassica napus: the role of silique wall photosynthesis.

PubMed

Hua, Wei; Li, Rong-Jun; Zhan, Gao-Miao; Liu, Jing; Li, Jun; Wang, Xin-Fa; Liu, Gui-Hua; Wang, Han-Zhong

2012-02-01

Seed oil content is an important agronomic trait in rapeseed. However, our understanding of the regulatory processes controlling oil accumulation is still limited. Using two rapeseed lines (zy036 and 51070) with contrasting oil content, we found that maternal genotype greatly affects seed oil content. Genetic and physiological evidence indicated that difference in the local and tissue-specific photosynthetic activity in the silique wall (a maternal tissue) was responsible for the different seed oil contents. This effect was mimicked by in planta manipulation of silique wall photosynthesis. Furthermore, the starch content and expression of the important lipid synthesis regulatory gene WRINKLED1 in developing seeds were linked with silique wall photosynthetic activity. 454 pyrosequencing was performed to explore the possible molecular mechanism for the difference in silique wall photosynthesis between zy036 and 51070. Interestingly, the results suggested that photosynthesis-related genes were over-represented in both total silique wall expressed genes and genes that were differentially expressed between genotypes. A potential regulatory mechanism for elevated photosynthesis in the zy036 silique wall is proposed on the basis of knowledge from Arabidopsis. Differentially expressed ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)-related genes were used for further investigations. Oil content correlated closely with BnRBCS1A expression levels and Rubisco activities in the silique wall, but not in the leaf. Taken together, our results highlight an important role of silique wall photosynthesis in the regulation of seed oil content in terms of maternal effects. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Structure of a class I tagatose-1,6-bisphosphate aldolase: investigation into an apparent loss of stereospecificity.

PubMed

LowKam, Clotilde; Liotard, Brigitte; Sygusch, Jurgen

2010-07-02

Tagatose-1,6-bisphosphate aldolase from Streptococcus pyogenes is a class I aldolase that exhibits a remarkable lack of chiral discrimination with respect to the configuration of hydroxyl groups at both C3 and C4 positions. The enzyme catalyzes the reversible cleavage of four diastereoisomers (fructose 1,6-bisphosphate (FBP), psicose 1,6-bisphosphate, sorbose 1,6-bisphosphate, and tagatose 1,6-bisphosphate) to dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate with high catalytic efficiency. To investigate its enzymatic mechanism, high resolution crystal structures were determined of both native enzyme and native enzyme in complex with dihydroxyacetone-P. The electron density map revealed a (alpha/beta)(8) fold in each dimeric subunit. Flash-cooled crystals of native enzyme soaked with dihydroxyacetone phosphate trapped a covalent intermediate with carbanionic character at Lys(205), different from the enamine mesomer bound in stereospecific class I FBP aldolase. Structural analysis indicates extensive active site conservation with respect to class I FBP aldolases, including conserved conformational responses to DHAP binding and conserved stereospecific proton transfer at the DHAP C3 carbon mediated by a proximal water molecule. Exchange reactions with tritiated water and tritium-labeled DHAP at C3 hydrogen were carried out in both solution and crystalline state to assess stereochemical control at C3. The kinetic studies show labeling at both pro-R and pro-S C3 positions of DHAP yet detritiation only at the C3 pro-S-labeled position. Detritiation of the C3 pro-R label was not detected and is consistent with preferential cis-trans isomerism about the C2-C3 bond in the carbanion as the mechanism responsible for C3 epimerization in tagatose-1,6-bisphosphate aldolase.

Structure of a Class I Tagatose-1,6-bisphosphate Aldolase

PubMed Central

LowKam, Clotilde; Liotard, Brigitte; Sygusch, Jurgen

2010-01-01

Tagatose-1,6-bisphosphate aldolase from Streptococcus pyogenes is a class I aldolase that exhibits a remarkable lack of chiral discrimination with respect to the configuration of hydroxyl groups at both C3 and C4 positions. The enzyme catalyzes the reversible cleavage of four diastereoisomers (fructose 1,6-bisphosphate (FBP), psicose 1,6-bisphosphate, sorbose 1,6-bisphosphate, and tagatose 1,6-bisphosphate) to dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate with high catalytic efficiency. To investigate its enzymatic mechanism, high resolution crystal structures were determined of both native enzyme and native enzyme in complex with dihydroxyacetone-P. The electron density map revealed a (α/β)8 fold in each dimeric subunit. Flash-cooled crystals of native enzyme soaked with dihydroxyacetone phosphate trapped a covalent intermediate with carbanionic character at Lys205, different from the enamine mesomer bound in stereospecific class I FBP aldolase. Structural analysis indicates extensive active site conservation with respect to class I FBP aldolases, including conserved conformational responses to DHAP binding and conserved stereospecific proton transfer at the DHAP C3 carbon mediated by a proximal water molecule. Exchange reactions with tritiated water and tritium-labeled DHAP at C3 hydrogen were carried out in both solution and crystalline state to assess stereochemical control at C3. The kinetic studies show labeling at both pro-R and pro-S C3 positions of DHAP yet detritiation only at the C3 pro-S-labeled position. Detritiation of the C3 pro-R label was not detected and is consistent with preferential cis-trans isomerism about the C2–C3 bond in the carbanion as the mechanism responsible for C3 epimerization in tagatose-1,6-bisphosphate aldolase. PMID:20427286

Structure of Toxoplasma gondii fructose-1,6-bisphosphate aldolase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boucher, Lauren E.; Bosch, Jürgen, E-mail: jbosch@jhu.edu; Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205

The structure of T. gondii fructose-1,6-bisphosphate aldolase, a glycolytic enzyme and structural component of the invasion machinery, was determined to a resolution of 2.0 Å. The apicomplexan parasite Toxoplasma gondii must invade host cells to continue its lifecycle. It invades different cell types using an actomyosin motor that is connected to extracellular adhesins via the bridging protein fructose-1,6-@@bisphosphate aldolase. During invasion, aldolase serves in the role of a structural bridging protein, as opposed to its normal enzymatic role in the glycolysis pathway. Crystal structures of the homologous Plasmodium falciparum fructose-1,6-bisphosphate aldolase have been described previously. Here, T. gondii fructose-1,6-bisphosphate aldolasemore » has been crystallized in space group P22{sub 1}2{sub 1}, with the biologically relevant tetramer in the asymmetric unit, and the structure has been determined via molecular replacement to a resolution of 2.0 Å. An analysis of the quality of the model and of the differences between the four chains in the asymmetric unit and a comparison between the T. gondii and P. falciparum aldolase structures is presented.« less

Structure of fructose bisphosphate aldolase from Bartonella henselae bound to fructose 1,6-bisphosphate.

PubMed

Gardberg, Anna; Abendroth, Jan; Bhandari, Janhavi; Sankaran, Banumathi; Staker, Bart

2011-09-01

Fructose bisphosphate aldolase (FBPA) enzymes have been found in a broad range of eukaryotic and prokaryotic organisms. FBPA catalyses the cleavage of fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. The SSGCID has reported several FBPA structures from pathogenic sources, including the bacterium Brucella melitensis and the protozoan Babesia bovis. Bioinformatic analysis of the Bartonella henselae genome revealed an FBPA homolog. The B. henselae FBPA enzyme was recombinantly expressed and purified for X-ray crystallographic studies. The purified enzyme crystallized in the apo form but failed to diffract; however, well diffracting crystals could be obtained by cocrystallization in the presence of the native substrate fructose 1,6-bisphosphate. A data set to 2.35 Å resolution was collected from a single crystal at 100 K. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=72.39, b=127.71, c=157.63 Å. The structure was refined to a final free R factor of 22.2%. The structure shares the typical barrel tertiary structure and tetrameric quaternary structure reported for previous FBPA structures and exhibits the same Schiff base in the active site.

Structure of 3,4-dihydroxy-2-butanone 4-phosphate synthase from Methanococcus jannaschii in complex with divalent metal ions and the substrate ribulose 5-phosphate: implications for the catalytic mechanism.

PubMed

Steinbacher, Stefan; Schiffmann, Susanne; Richter, Gerald; Huber, Robert; Bacher, Adelbert; Fischer, Markus

2003-10-24

Skeletal rearrangements of carbohydrates are crucial for many biosynthetic pathways. In riboflavin biosynthesis ribulose 5-phosphate is converted into 3,4-dihydroxy-2-butanone 4-phosphate while its C4 atom is released as formate in a sequence of metal-dependent reactions. Here, we present the crystal structure of Methanococcus jannaschii 3,4-dihydroxy-2-butanone 4-phosphate synthase in complex with the substrate ribulose 5-phosphate at a dimetal center presumably consisting of non-catalytic zinc and calcium ions at 1.7-A resolution. The carbonyl group (O2) and two out of three free hydroxyl groups (OH3 and OH4) of the substrate are metal-coordinated. We correlate previous mutational studies on this enzyme with the present structural results. Residues of the first coordination sphere involved in metal binding are indispensable for catalytic activity. Only Glu-185 of the second coordination sphere cannot be replaced without complete loss of activity. It contacts the C3 hydrogen atom directly and probably initiates enediol formation in concert with both metal ions to start the reaction sequence. Mechanistic similarities to Rubisco acting on the similar substrate ribulose 1,5-diphosphate in carbon dioxide fixation as well as other carbohydrate (reducto-) isomerases are discussed.

Rational Design Synthesis and Evaluation of New Selective Inhibitors of Microbial Class II (Zinc Dependent) Fructose Bis-phosphate Aldolases

DOE Office of Scientific and Technical Information (OSTI.GOV)

R Daher; M Coincon; M Fonvielle

2011-12-31

We report the synthesis and biochemical evaluation of several selective inhibitors of class II (zinc dependent) fructose bis-phosphate aldolases (Fba). The products were designed as transition-state analogues of the catalyzed reaction, structurally related to the substrate fructose bis-phosphate (or sedoheptulose bis-phosphate) and based on an N-substituted hydroxamic acid, as a chelator of the zinc ion present in active site. The compounds synthesized were tested on class II Fbas from various pathogenic microorganisms and, by comparison, on a mammalian class I Fba. The best inhibitor shows Ki against class II Fbas from various pathogens in the nM range, with very highmore » selectivity (up to 105). Structural analyses of inhibitors in complex with aldolases rationalize and corroborate the enzymatic kinetics results. These inhibitors represent lead compounds for the preparation of new synthetic antibiotics, notably for tuberculosis prophylaxis.« less

Structure of Toxoplasma gondii fructose-1,6-bisphosphate aldolase.

PubMed

Boucher, Lauren E; Bosch, Jürgen

2014-09-01

The apicomplexan parasite Toxoplasma gondii must invade host cells to continue its lifecycle. It invades different cell types using an actomyosin motor that is connected to extracellular adhesins via the bridging protein fructose-1,6-bisphosphate aldolase. During invasion, aldolase serves in the role of a structural bridging protein, as opposed to its normal enzymatic role in the glycolysis pathway. Crystal structures of the homologous Plasmodium falciparum fructose-1,6-bisphosphate aldolase have been described previously. Here, T. gondii fructose-1,6-bisphosphate aldolase has been crystallized in space group P22121, with the biologically relevant tetramer in the asymmetric unit, and the structure has been determined via molecular replacement to a resolution of 2.0 Å. An analysis of the quality of the model and of the differences between the four chains in the asymmetric unit and a comparison between the T. gondii and P. falciparum aldolase structures is presented.

6-Phosphofructokinase and ribulose-5-phosphate 3-epimerase in methylotrophic Bacillus methanolicus ribulose monophosphate cycle.

PubMed

Le, Simone Balzer; Heggeset, Tonje Marita Bjerkan; Haugen, Tone; Nærdal, Ingemar; Brautaset, Trygve

2017-05-01

D-Ribulose-5-phosphate-3-epimerase (RPE) and 6-phosphofructokinase (PFK) catalyse two reactions in the ribulose monophosphate (RuMP) cycle in Bacillus methanolicus. The B. methanolicus wild-type strain MGA3 possesses two putative rpe and pfk genes encoded on plasmid pBM19 (rpe1-MGA3 and pfk1-MGA3) and on the chromosome (rpe2-MGA3 and pfk2-MGA3). The wild-type strain PB1 also encodes putative rpe and pfk genes on plasmid pBM20 (rpe1-PB1 and pfk1-PB1*); however, it only harbours a chromosomal pfk gene (pfk2-PB1). Transcription of the plasmid-encoded genes was 10-fold to 15-fold upregulated in cells growing on methanol compared to mannitol, while the chromosomal genes were transcribed at similar levels under both conditions in both strains. All seven gene products were recombinantly produced in Escherichia coli, purified and biochemically characterized. All three RPEs were active as hexamers, catalytically stimulated by Mg 2+ and Mn 2+ and displayed similar K' values (56-75 μM) for ribulose 5-phosphate. Rpe2-MGA3 showed displayed 2-fold lower V max (49 U/mg) and a significantly reduced thermostability compared to the two Rpe1 proteins. Pfk1-PB1* was shown to be non-functional. The PFKs were active both as octamers and as tetramers, were catalytically stimulated by Mg 2+ and Mn 2+ , and displayed similar thermostabilities. The PFKs have similar K m values for fructose 6-phosphate (0.61-0.94 μM) and for ATP (0.38-0.82 μM), while Pfk1-MGA3 had a 2-fold lower V max (6.3 U/mg) compared to the two Pfk2 proteins. Our results demonstrate that MGA3 and PB1 exert alternative solutions to plasmid-dependent methylotrophy, including genetic organization, regulation, and biochemistry of RuMP cycle enzymes.

Examination of Chemical Adsorption and Marine Biofouling on Metal Surfaces Using Raman Scattering Techniques and Electrochemical Impedance Spectroscopy

DTIC Science & Technology

1989-01-13

aromatic amino acids adsorbed on conditioned Ag electrodes in 0.1 N KCl. A. tryptophan. B. phenylalanine. Ribulose biphosphate carboxylase ( RuBisCo ) has...Preliminary runs with a silver electrode, UV-oxidized seawater, and a putative fouling protein, Ribulose 1,5 biphosphate carboxylase ( RuBisCo ) have been...completed. Prior to adding RuBisCo to the system, a series of runs were made to establish functionality of the cell and system parameters. The system

pH determines the energetic efficiency of the cyanobacterial CO2 concentrating mechanism.

PubMed

Mangan, Niall M; Flamholz, Avi; Hood, Rachel D; Milo, Ron; Savage, David F

2016-09-06

Many carbon-fixing bacteria rely on a CO2 concentrating mechanism (CCM) to elevate the CO2 concentration around the carboxylating enzyme ribulose bisphosphate carboxylase/oxygenase (RuBisCO). The CCM is postulated to simultaneously enhance the rate of carboxylation and minimize oxygenation, a competitive reaction with O2 also catalyzed by RuBisCO. To achieve this effect, the CCM combines two features: active transport of inorganic carbon into the cell and colocalization of carbonic anhydrase and RuBisCO inside proteinaceous microcompartments called carboxysomes. Understanding the significance of the various CCM components requires reconciling biochemical intuition with a quantitative description of the system. To this end, we have developed a mathematical model of the CCM to analyze its energetic costs and the inherent intertwining of physiology and pH. We find that intracellular pH greatly affects the cost of inorganic carbon accumulation. At low pH the inorganic carbon pool contains more of the highly cell-permeable H2CO3, necessitating a substantial expenditure of energy on transport to maintain internal inorganic carbon levels. An intracellular pH ≈8 reduces leakage, making the CCM significantly more energetically efficient. This pH prediction coincides well with our measurement of intracellular pH in a model cyanobacterium. We also demonstrate that CO2 retention in the carboxysome is necessary, whereas selective uptake of HCO3 (-) into the carboxysome would not appreciably enhance energetic efficiency. Altogether, integration of pH produces a model that is quantitatively consistent with cyanobacterial physiology, emphasizing that pH cannot be neglected when describing biological systems interacting with inorganic carbon pools.

Acclimation of CO2 Assimilation in Cotton Leaves to Water Stress and Salinity 1

PubMed Central

Plaut, Zvi; Federman, Evelyn

1991-01-01

Cotton (Gossypium hirsutum L. cv Acala SJ2) plants were exposed to three levels of osmotic or matric potentials. The first was obtained by salt and the latter by withholding irrigation water. Plants were acclimated to the two stress types by reducing the rate of stress development by a factor of 4 to 7. CO2 assimilation was then determined on acclimated and nonacclimated plants. The decrease of CO2 assimilation in salinity-exposed plants was significantly less in acclimated as compared with nonacclimated plants. Such a difference was not found under water stress at ambient CO2 partial pressure. The slopes of net CO2 assimilation versus intercellular CO2 partial pressure, for the initial linear portion of this relationship, were increased in plants acclimated to salinity of −0.3 and −0.6 megapascal but not in nonacclimated plants. In plants acclimated to water stress, this change in slopes was not significant. Leaf osmotic potential was reduced much more in acclimated than in nonacclimated plants, resulting in turgor maintenance even at −0.9 megapascal. In nonacclimated plants, turgor pressure reached zero at approximately −0.5 megapascal. The accumulation of Cl− and Na+ in the salinity-acclimated plants fully accounted for the decrease in leaf osmotic potential. The rise in concentration of organic solutes comprised only 5% of the total increase in solutes in salinity-acclimated and 10 to 20% in water-stress-acclimated plants. This acclimation was interpreted in light of the higher protein content per unit leaf area and the enhanced ribulose bisphosphate carboxylase activity. At saturating CO2 partial pressure, the declined inhibition in CO2 assimilation of stress-acclimated plants was found for both salinity and water stress. ImagesFigure 2 PMID:16668429

Comparison of three protein extraction procedures from toxic and non-toxic dinoflagellates for proteomics analysis.

PubMed

Jiang, Xi-Wen; Wang, Jing; Chan, Leo Lai; Lam, Paul Kwan Sing; Gu, Ji-Dong

2015-08-01

Three methods for extraction and preparation of high-quality proteins from both toxic and non-toxic dinoflagellates for proteomics analysis, including Trizol method, Lysis method and Tris method, were compared with the subsequent protein separation profiles using 2-D differential gel electrophoresis (2-D DIGE), Coomassie Blue and silver staining. These methods showed suitability for proteins with different pIs and molecular weights. Tris method was better for low molecular weight and low pI protein isolation; whereas both Lysis and Trizol method were better for high-molecular weight and high pI protein purification. Trizol method showed good results with Alexandrium species and Gynodinium species, and the background in gel was much clearer than the other two methods. At the same time, only Lysis method caused breaking down of the target proteins. On the other hand, Trizol method obtained higher concentration of ribulose-1,5-bisphosphate carboxylase/oxygenase proteins by Western-blotting, while Tris method was the best for peridinin-chlorophyll-protein complexes protein and T1 protein preparation. DIGE was better than Coomassie Blue and silver staining, except for some limitations, such as the high cost of the dyes, relatively short shelf life and the requirements for extensive and special image capturing equipment. Some proteins related to PSTs synthesis in dinoflagellates are hydrophobic with high molecular weight or binding on membranes and Trizol method performed better than Tris method for these proteins. The Trizol method and 2-D DIGE were effective combination for proteomics investigations of dinoflagellates. This procedure allows reliable and high recovery efficiency of proteins from dinoflagellates for better understanding on their occurrence and toxin-production for physiological and biochemical information.

Do the rich always become richer? Characterizing the leaf physiological response of the high-yielding rice cultivar Takanari to free-air CO2 enrichment.

PubMed

Chen, Charles P; Sakai, Hidemitsu; Tokida, Takeshi; Usui, Yasuhiro; Nakamura, Hirofumi; Hasegawa, Toshihiro

2014-02-01

The development of crops which are well suited to growth under future environmental conditions such as higher atmospheric CO2 concentrations ([CO2]) is essential to meeting the challenge of ensuring food security in the face of the growing human population and changing climate. A high-yielding indica rice variety (Oryza sativa L. cv. Takanari) has been recently identified as a potential candidate for such breeding, due to its high productivity in present [CO2]. To test if it could further increase its productivity under elevated [CO2] (eCO2), Takanari was grown in the paddy field under season-long free-air CO2 enrichment (FACE, approximately 200 µmol mol(-1) above ambient [CO2]) and its leaf physiology was compared with the representative japonica variety 'Koshihikari'. Takanari showed consistently higher midday photosynthesis and stomatal conductance than Koshihikari under both ambient and FACE growth conditions over 2 years. Maximum ribulose-1,5-bisphosphate carboxylation and electron transport rates were higher for Takanari at the mid-grain filling stage in both years. Mesophyll conductance was higher in Takanari than in Koshihikari at the late grain-filling stage. In contrast to Koshihikari, Takanari grown under FACE conditions showed no decrease in total leaf nitrogen on an area basis relative to ambient-grown plants. Chl content was higher in Takanari than in Koshihikari at the same leaf nitrogen level. These results indicate that Takanari maintains its superiority over Koshihikari in regards to its leaf-level productivity when grown in elevated [CO2] and it may be a valuable resource for rice breeding programs which seek to increase crop productivity under current and future [CO2].

Comparing the in Vivo Function of α-Carboxysomes and β-Carboxysomes in Two Model Cyanobacteria1[W][OPEN

PubMed Central

Whitehead, Lynne; Long, Benedict M.; Price, G. Dean; Badger, Murray R.

2014-01-01

The carbon dioxide (CO2)-concentrating mechanism of cyanobacteria is characterized by the occurrence of Rubisco-containing microcompartments called carboxysomes within cells. The encapsulation of Rubisco allows for high-CO2 concentrations at the site of fixation, providing an advantage in low-CO2 environments. Cyanobacteria with Form-IA Rubisco contain α-carboxysomes, and cyanobacteria with Form-IB Rubisco contain β-carboxysomes. The two carboxysome types have arisen through convergent evolution, and α-cyanobacteria and β-cyanobacteria occupy different ecological niches. Here, we present, to our knowledge, the first direct comparison of the carboxysome function from α-cyanobacteria (Cyanobium spp. PCC7001) and β-cyanobacteria (Synechococcus spp. PCC7942) with similar inorganic carbon (Ci; as CO2 and HCO3−) transporter systems. Despite evolutionary and structural differences between α-carboxysomes and β-carboxysomes, we found that the two strains are remarkably similar in many physiological parameters, particularly the response of photosynthesis to light and external Ci and their modulation of internal ribulose-1,5-bisphosphate, phosphoglycerate, and Ci pools when grown under comparable conditions. In addition, the different Rubisco forms present in each carboxysome had almost identical kinetic parameters. The conclusions indicate that the possession of different carboxysome types does not significantly influence the physiological function of these species and that similar carboxysome function may be possessed by each carboxysome type. Interestingly, both carboxysome types showed a response to cytosolic Ci, which is of higher affinity than predicted by current models, being saturated by 5 to 15 mm Ci. This finding has bearing on the viability of transplanting functional carboxysomes into the C3 chloroplast. PMID:24642960

Functional indicators of response mechanisms to nitrogen deposition, ozone, and their interaction in two Mediterranean tree species

PubMed Central

Palma, Adriano; Salvatori, Elisabetta; Basile, Adriana; Maresca, Viviana; Asadi Karam, Elham; Manes, Fausto

2017-01-01

The effects of nitrogen (N) deposition, tropospheric ozone (O3) and their interaction were investigated in two Mediterranean tree species, Fraxinus ornus L. (deciduous) and Quercus ilex L. (evergreen), having different leaf habits and resource use strategies. An experiment was conducted under controlled condition to analyse how nitrogen deposition affects the ecophysiological and biochemical traits, and to explore how the nitrogen-induced changes influence the response to O3. For both factors we selected realistic exposures (20 kg N ha-1 yr-1 and 80 ppb h for nitrogen and O3, respectively), in order to elucidate the mechanisms implemented by the plants. Nitrogen addition resulted in higher nitrogen concentration at the leaf level in F. ornus, whereas a slight increase was detected in Q. ilex. Nitrogen enhanced the maximum rate of assimilation and ribulose 1,5-bisphosphate regeneration in both species, whereas it influenced the light harvesting complex only in the deciduous F. ornus that was also affected by O3 (reduced assimilation rate and accelerated senescence-related processes). Conversely, Q. ilex developed an avoidance mechanism to cope with O3, confirming a substantial O3 tolerance of this species. Nitrogen seemed to ameliorate the harmful effects of O3 in F. ornus: the hypothesized mechanism of action involved the production of nitrogen oxide as the first antioxidant barrier, followed by enzymatic antioxidant response. In Q. ilex, the interaction was not detected on gas exchange and photosystem functionality; however, in this species, nitrogen might stimulate an alternative antioxidant response such as the emission of volatile organic compounds. Antioxidant enzyme activity was lower in plants treated with both O3 and nitrogen even though reactive oxygen species production did not differ between the treatments. PMID:28973038

Kranz and single-cell forms of C4 plants in the subfamily Suaedoideae show kinetic C4 convergence for PEPC and Rubisco with divergent amino acid substitutions

PubMed Central

Rosnow, Josh J.; Evans, Marc A.; Kapralov, Maxim V.; Cousins, Asaph B.; Edwards, Gerald E.; Roalson, Eric H.

2015-01-01

The two carboxylation reactions performed by phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) are vital in the fixation of inorganic carbon for C4 plants. The abundance of PEPC is substantially elevated in C4 leaves, while the location of Rubisco is restricted to one of two chloroplast types. These differences compared with C3 leaves have been shown to result in convergent enzyme optimization in some C4 species. Investigation into the kinetic properties of PEPC and Rubisco from Kranz C4, single cell C4, and C3 species in Chenopodiaceae s. s. subfamily Suaedoideae showed that these major carboxylases in C4 Suaedoideae species lack the same mutations found in other C4 systems which have been examined; but still have similar convergent kinetic properties. Positive selection analysis on the N-terminus of PEPC identified residues 364 and 368 to be under positive selection with a posterior probability >0.99 using Bayes empirical Bayes. Compared with previous analyses on other C4 species, PEPC from C4 Suaedoideae species have different convergent amino acids that result in a higher K m for PEP and malate tolerance compared with C3 species. Kinetic analysis of Rubisco showed that C4 species have a higher catalytic efficiency of Rubisco (k catc in mol CO2 mol–1 Rubisco active sites s–1), despite lacking convergent substitutions in the rbcL gene. The importance of kinetic changes to the two-carboxylation reactions in C4 leaves related to amino acid selection is discussed. PMID:26417023

Protein accumulation in leaves and roots associated with improved drought tolerance in creeping bentgrass expressing an ipt gene for cytokinin synthesis

PubMed Central

Merewitz, Emily B.; Gianfagna, Thomas; Huang, Bingru

2011-01-01

Cytokinins (CKs) may be involved in the regulation of plant adaptation to drought stress. The objectives of the study were to identify proteomic changes in leaves and roots in relation to improved drought tolerance in transgenic creeping bentgrass (Agrostis stolonifera) containing a senescence-activated promoter (SAG12) and the isopentyl transferase (ipt) transgene that increases endogenous CK content. Leaves of SAG12-ipt bentgrass exhibited less severe senescence under water stress, as demonstrated by maintaining lower electrolyte leakage and lipid peroxidation, and higher photochemical efficiency (Fv/Fm), compared with the null transformant (NT) plants. SAG12-ipt plants had higher root/shoot ratios and lower lipid peroxidation in leaves under water stress than the NT plants. The suppression of drought-induced leaf senescence and root dieback in the transgenic plants was associated with the maintenance of greater antioxidant enzyme activities (superoxide dismutase, peroxidase, and catalase). The SAG12-ipt and NT plants exhibited differential protein expression patterns under well-watered and drought conditions in both leaves and roots. Under equivalent leaf water deficit (47% relative water content), SAG12-ipt plants maintained higher abundance of proteins involved in (i) energy production within both photosynthesis and respiration [ribulose 1,5-bisphosphate carboxylase (RuBisCO) and glyceraldehyde phosphate dehydrogenase (GAPDH)]; (ii) amino acid synthesis (methionine and glutamine); (iii) protein synthesis and destination [chloroplastic elongation factor (EF-Tu) and protein disulphide isomerases (PDIs)]; and (iv) antioxidant defence system (catalase and peroxidase) than the NT plants. These results suggest that increased endogenous CKs under drought stress may directly or indirectly regulate protein abundance and enzymatic activities involved in the above-mentioned metabolic processes, thereby enhancing plant drought tolerance. PMID:21831843

RuBisCO depletion improved proteome coverage of cold responsive S-nitrosylated targets in Brassica juncea

PubMed Central

Sehrawat, Ankita; Abat, Jasmeet K.; Deswal, Renu

2013-01-01

Although in the last few years good number of S-nitrosylated proteins are identified but information on endogenous targets is still limiting. Therefore, an attempt is made to decipher NO signaling in cold treated Brassica juncea seedlings. Treatment of seedlings with substrate, cofactor and inhibitor of Nitric-oxide synthase and nitrate reductase (NR), indicated NR mediated NO biosynthesis in cold. Analysis of the in vivo thiols showed depletion of low molecular weight thiols and enhancement of available protein thiols, suggesting redox changes. To have a detailed view, S-nitrosylation analysis was done using biotin switch technique (BST) and avidin-affinity chromatography. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is S-nitrosylated and therefore, is identified as target repeatedly due to its abundance. It also competes out low abundant proteins which are important NO signaling components. Therefore, RuBisCO was removed (over 80%) using immunoaffinity purification. Purified S-nitrosylated RuBisCO depleted proteins were resolved on 2-D gel as 110 spots, including 13 new, which were absent in the crude S-nitrosoproteome. These were identified by nLC-MS/MS as thioredoxin, fructose biphosphate aldolase class I, myrosinase, salt responsive proteins, peptidyl-prolyl cis-trans isomerase and malate dehydrogenase. Cold showed differential S-nitrosylation of 15 spots, enhanced superoxide dismutase activity (via S-nitrosylation) and promoted the detoxification of superoxide radicals. Increased S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase sedoheptulose-biphosphatase, and fructose biphosphate aldolase, indicated regulation of Calvin cycle by S-nitrosylation. The results showed that RuBisCO depletion improved proteome coverage and provided clues for NO signaling in cold. PMID:24032038

Structure and Identification of a Pterin Dehydratase-like Protein as a Ribulose-bisphosphate Carboxylase/Oxygenase (RuBisCO) Assembly Factor in the α-Carboxysome*

PubMed Central

Wheatley, Nicole M.; Sundberg, Christopher D.; Gidaniyan, Soheil D.; Cascio, Duilio; Yeates, Todd O.

2014-01-01

Carboxysomes are proteinaceous bacterial microcompartments that increase the efficiency of the rate-limiting step in carbon fixation by sequestering reaction substrates. Typically, α-carboxysomes are genetically encoded as a single operon expressing the structural proteins and the encapsulated enzymes of the microcompartment. In addition, depending on phylogeny, as many as 13 other genes are found to co-occur near or within α-carboxysome operons. One of these genes codes for a protein with distant homology to pterin-4α-carbinolamine dehydratase (PCD) enzymes. It is present in all α-carboxysome containing bacteria and has homologs in algae and higher plants. Canonical PCDs play an important role in amino acid hydroxylation, a reaction not associated with carbon fixation. We determined the crystal structure of an α-carboxysome PCD-like protein from the chemoautotrophic bacterium Thiomonas intermedia K12, at 1.3-Å resolution. The protein retains a three-dimensional fold similar to canonical PCDs, although the prominent active site cleft present in PCD enzymes is disrupted in the α-carboxysome PCD-like protein. Using a cell-based complementation assay, we tested the PCD-like proteins from T. intermedia and two additional bacteria, and found no evidence for PCD enzymatic activity. However, we discovered that heterologous co-expression of the PCD-like protein from Halothiobacillus neapolitanus with RuBisCO and GroELS in Escherichia coli increased the amount of soluble, assembled RuBisCO recovered from cell lysates compared with co-expression of RuBisCO with GroELS alone. We conclude that this conserved PCD-like protein, renamed here α-carboxysome RuBisCO assembly factor (or acRAF), is a novel RuBisCO chaperone integral to α-carboxysome function. PMID:24459150

Response of Phaseolus vulgaris L. plants to low-let ionizing radiation: Growth and oxidative stress

NASA Astrophysics Data System (ADS)

Arena, C.; De Micco, V.; Aronne, G.; Pugliese, M.; Virzo De Santo, A.; De Maio, A.

2013-10-01

The scenarios for the long-term habitation of space platforms and planetary stations involve plants as fundamental part of Bioregenerative Life Support Systems (BLSS) to support the crew needs. Several constraints may limit plant growth in space: among them ionizing radiation is recognized to severely affect plant cell at morphological, physiological and biochemical level. In this work, plants of Phaseolus vulgaris L. were subjected to four different doses of X-rays (0.3, 10, 50 and 100 Gy) in order to assess the effects of ionizing radiation on this species and to analyze possible mechanisms carried out to overcome the radiation injuries. The effects of X-rays on plant growth were assessed by measuring stem elongation, number of internodes and leaf dry weight. The integrity of photosynthetic apparatus was evaluated by photosynthetic pigment composition and ribulose 1,5-bisphosphate carboxylase (Rubisco) activity, whereas changes in total antioxidant pool and glutathione S transferase activity (GST) were utilized as markers of oxidative stress. The distribution of phenolic compounds in leaf tissues as natural shielding against radiation was also determined. Irradiation of plants at 0.3 and 10 Gy did not determine differences in all considered parameters as compared to control. On the contrary, at 50 and 100 Gy a reduction of plant growth and a decrease in photosynthetic pigment content, as well as an increase in phenolic compounds and a decrease in total antioxidant content and GST activity were found. Only a slight reduction of Rubisco activity in leaves irradiated at 50 and 100 Gy was found. The overall results indicate P. vulgaris as a species with a good potential to face ionizing radiation and suggest its suitability for utilization in BLSSs.

pH determines the energetic efficiency of the cyanobacterial CO2 concentrating mechanism

PubMed Central

Flamholz, Avi; Hood, Rachel D.; Milo, Ron

2016-01-01

Many carbon-fixing bacteria rely on a CO2 concentrating mechanism (CCM) to elevate the CO2 concentration around the carboxylating enzyme ribulose bisphosphate carboxylase/oxygenase (RuBisCO). The CCM is postulated to simultaneously enhance the rate of carboxylation and minimize oxygenation, a competitive reaction with O2 also catalyzed by RuBisCO. To achieve this effect, the CCM combines two features: active transport of inorganic carbon into the cell and colocalization of carbonic anhydrase and RuBisCO inside proteinaceous microcompartments called carboxysomes. Understanding the significance of the various CCM components requires reconciling biochemical intuition with a quantitative description of the system. To this end, we have developed a mathematical model of the CCM to analyze its energetic costs and the inherent intertwining of physiology and pH. We find that intracellular pH greatly affects the cost of inorganic carbon accumulation. At low pH the inorganic carbon pool contains more of the highly cell-permeable H2CO3, necessitating a substantial expenditure of energy on transport to maintain internal inorganic carbon levels. An intracellular pH ≈8 reduces leakage, making the CCM significantly more energetically efficient. This pH prediction coincides well with our measurement of intracellular pH in a model cyanobacterium. We also demonstrate that CO2 retention in the carboxysome is necessary, whereas selective uptake of HCO3− into the carboxysome would not appreciably enhance energetic efficiency. Altogether, integration of pH produces a model that is quantitatively consistent with cyanobacterial physiology, emphasizing that pH cannot be neglected when describing biological systems interacting with inorganic carbon pools. PMID:27551079

Structure and identification of a pterin dehydratase-like protein as a ribulose-bisphosphate carboxylase/oxygenase (RuBisCO) assembly factor in the α-carboxysome.

PubMed

Wheatley, Nicole M; Sundberg, Christopher D; Gidaniyan, Soheil D; Cascio, Duilio; Yeates, Todd O

2014-03-14

Carboxysomes are proteinaceous bacterial microcompartments that increase the efficiency of the rate-limiting step in carbon fixation by sequestering reaction substrates. Typically, α-carboxysomes are genetically encoded as a single operon expressing the structural proteins and the encapsulated enzymes of the microcompartment. In addition, depending on phylogeny, as many as 13 other genes are found to co-occur near or within α-carboxysome operons. One of these genes codes for a protein with distant homology to pterin-4α-carbinolamine dehydratase (PCD) enzymes. It is present in all α-carboxysome containing bacteria and has homologs in algae and higher plants. Canonical PCDs play an important role in amino acid hydroxylation, a reaction not associated with carbon fixation. We determined the crystal structure of an α-carboxysome PCD-like protein from the chemoautotrophic bacterium Thiomonas intermedia K12, at 1.3-Å resolution. The protein retains a three-dimensional fold similar to canonical PCDs, although the prominent active site cleft present in PCD enzymes is disrupted in the α-carboxysome PCD-like protein. Using a cell-based complementation assay, we tested the PCD-like proteins from T. intermedia and two additional bacteria, and found no evidence for PCD enzymatic activity. However, we discovered that heterologous co-expression of the PCD-like protein from Halothiobacillus neapolitanus with RuBisCO and GroELS in Escherichia coli increased the amount of soluble, assembled RuBisCO recovered from cell lysates compared with co-expression of RuBisCO with GroELS alone. We conclude that this conserved PCD-like protein, renamed here α-carboxysome RuBisCO assembly factor (or acRAF), is a novel RuBisCO chaperone integral to α-carboxysome function.

Changes in PEP carboxylase, rubisco and rubisco activase mRNA levels from maize (Zea mays) exposed to a chronic ozone stress.

PubMed

Leitao, Louis; Maoret, Jean-José; Biolley, Jean-Philippe

2007-01-01

We quantified the ozone impact on levels of Zea mays L. cv. Chambord mRNAs encoding C4-phosphoenolpyruvate carboxylase (C4-PEPc), ribulose-l,5-bisphosphate carboxylase/oxygenase small and large subunits (Rubisco-SSU and Rubisco-LSU, respectively) and Rubisco activase (RCA) using real-time RT-PCR. Foliar pigment content, PEPc and Rubisco protein amounts were simultaneously determined. Two experiments were performed to study the ozone response of the 5th and the 10th leaf. For each experiment, three ozone concentrations were tested in open-top chambers: non-filtered air (NF, control) and non-filtered air containing 40 (+40) and 80 nL L-1 (+80) ozone. Regarding the 5th leaf, +40 atmosphere induced a loss in pigmentation, PEPc and Rubisco activase mRNAs. However, it was unable to notably depress carboxylase protein amounts and mRNAs encoding Rubisco. Except for Rubisco mRNAs, all other measured parameters from 5th leaf were depressed by +80 atmosphere. Regarding the 10th leaf, +40 atmosphere increased photosynthetic pigments and transcripts encoding Rubisco and Rubisco activase. Rubisco and PEPc protein amounts were not drastically changed, even if they tended to be increased. Level of C4-PEPc mRNA remained almost stable. In response to +80 atmosphere, pigments and transcripts encoding PEPc were notably decreased. Rubisco and PEPc protein amounts also declined to a lesser extent. Conversely, the level of transcripts encoding both Rubisco subunits and Rubisco activase that were not consistently disturbed tended to be slightly augmented. So, the present study suggests that maize leaves can respond differentially to a similar ozone stress.

Substitutions at the opening of the Rubisco central solvent channel affect holoenzyme stability and CO2/O 2 specificity but not activation by Rubisco activase.

PubMed

Esquivel, M Gloria; Genkov, Todor; Nogueira, Ana S; Salvucci, Michael E; Spreitzer, Robert J

2013-12-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the initial step of carbon metabolism in photosynthesis. The holoenzyme comprises eight large subunits, arranged as a tetramer of dimers around a central solvent channel that defines a fourfold axis of symmetry, and eight small subunits, arranged as two tetramers at the poles of the axis. The phylogenetically divergent small-subunit loops between β-strands A and B form the entrance to the solvent channel. In the green alga Chlamydomonas reinhardtii, Ile-58 from each of the four small-subunit βA-βB loops defines the minimal diameter of the channel opening. To understand the role of the central solvent channel in Rubisco function, directed mutagenesis and transformation of Chlamydomonas were employed to replace Ile-58 with Ala, Lys, Glu, Trp, or three Trp residues (I58W3) to close the entrance to the channel. The I58E, I58K, and I58W substitutions caused only small decreases in photosynthetic growth at 25 and 35 °C, whereas I58W3 had a substantial effect at both temperatures. The mutant enzymes had decreased carboxylation rates, but the I58W3 enzyme had decreases in both carboxylation and CO2/O2 specificity. The I58E, I58W, and I58W3 enzymes were inactivated at lower temperatures than wild-type Rubisco, and were degraded at slower rates under oxidative stress. However, these mutant enzymes were activated by Rubisco activase at normal rates, indicating that the structural transition required for carboxylation is not affected by altering the solvent channel opening. Structural dynamics alone may not be responsible for these distant effects on the Rubisco active site.

Regulation of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase

PubMed Central

Hazra, Suratna; Henderson, J. Nathan; Liles, Kevin; Hilton, Matthew T.; Wachter, Rebekka M.

2015-01-01

In many photosynthetic organisms, tight-binding Rubisco inhibitors are released by the motor protein Rubisco activase (Rca). In higher plants, Rca plays a pivotal role in regulating CO2 fixation. Here, the ATPase activity of 0.005 mm tobacco Rca was monitored under steady-state conditions, and global curve fitting was utilized to extract kinetic constants. The kcat was best fit by 22.3 ± 4.9 min−1, the Km for ATP by 0.104 ± 0.024 mm, and the Ki for ADP by 0.037 ± 0.007 mm. Without ADP, the Hill coefficient for ATP hydrolysis was extracted to be 1.0 ± 0.1, indicating noncooperative behavior of homo-oligomeric Rca assemblies. However, the addition of ADP was shown to introduce positive cooperativity between two or more subunits (Hill coefficient 1.9 ± 0.2), allowing for regulation via the prevailing ATP/ADP ratio. ADP-mediated activation was not observed, although larger amounts led to competitive product inhibition of hydrolytic activity. The catalytic efficiency increased 8.4-fold upon cooperative binding of a second magnesium ion (Hill coefficient 2.5 ± 0.5), suggesting at least three conformational states (ATP-bound, ADP-bound, and empty) within assemblies containing an average of about six subunits. The addition of excess Rubisco (24:1, L8S8/Rca6) and crowding agents did not modify catalytic rates. However, high magnesium provided for thermal Rca stabilization. We propose that magnesium mediates the formation of closed hexameric toroids capable of high turnover rates and amenable to allosteric regulation. We suggest that in vivo, the Rca hydrolytic activity is tuned by fluctuating [Mg2+] in response to changes in available light. PMID:26283786

A faster Rubisco with potential to increase photosynthesis in crops.

PubMed

Lin, Myat T; Occhialini, Alessandro; Andralojc, P John; Parry, Martin A J; Hanson, Maureen R

2014-09-25

In photosynthetic organisms, D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major enzyme assimilating atmospheric CO2 into the biosphere. Owing to the wasteful oxygenase activity and slow turnover of Rubisco, the enzyme is among the most important targets for improving the photosynthetic efficiency of vascular plants. It has been anticipated that introducing the CO2-concentrating mechanism (CCM) from cyanobacteria into plants could enhance crop yield. However, the complex nature of Rubisco's assembly has made manipulation of the enzyme extremely challenging, and attempts to replace it in plants with the enzymes from cyanobacteria and red algae have not been successful. Here we report two transplastomic tobacco lines with functional Rubisco from the cyanobacterium Synechococcus elongatus PCC7942 (Se7942). We knocked out the native tobacco gene encoding the large subunit of Rubisco by inserting the large and small subunit genes of the Se7942 enzyme, in combination with either the corresponding Se7942 assembly chaperone, RbcX, or an internal carboxysomal protein, CcmM35, which incorporates three small subunit-like domains. Se7942 Rubisco and CcmM35 formed macromolecular complexes within the chloroplast stroma, mirroring an early step in the biogenesis of cyanobacterial β-carboxysomes. Both transformed lines were photosynthetically competent, supporting autotrophic growth, and their respective forms of Rubisco had higher rates of CO2 fixation per unit of enzyme than the tobacco control. These transplastomic tobacco lines represent an important step towards improved photosynthesis in plants and will be valuable hosts for future addition of the remaining components of the cyanobacterial CCM, such as inorganic carbon transporters and the β-carboxysome shell proteins.

Temperature responses of the Rubisco maximum carboxylase activity across domains of life: phylogenetic signals, trade-offs, and importance for carbon gain.

PubMed

Galmés, J; Kapralov, M V; Copolovici, L O; Hermida-Carrera, C; Niinemets, Ü

2015-02-01

Temperature response of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalytic properties directly determines the CO2 assimilation capacity of photosynthetic organisms as well as their survival in environments with different thermal conditions. Despite unquestionable importance of Rubisco, the comprehensive analysis summarizing temperature responses of Rubisco traits across lineages of carbon-fixing organisms is lacking. Here, we present a review of the temperature responses of Rubisco carboxylase specific activity (c(cat)(c)) within and across domains of life. In particular, we consider the variability of temperature responses, and their ecological, physiological, and evolutionary controls. We observed over two-fold differences in the energy of activation (ΔH(a)) among different groups of photosynthetic organisms, and found significant differences between C3 plants from cool habitats, C3 plants from warm habitats and C4 plants. According to phylogenetically independent contrast analysis, ΔH(a) was not related to the species optimum growth temperature (T growth), but was positively correlated with Rubisco specificity factor (S(c/o)) across all organisms. However, when only land plants were analyzed, ΔH(a) was positively correlated with both T(growth) and S(c/o), indicating different trends for these traits in plants versus unicellular aquatic organisms, such as algae and bacteria. The optimum temperature (T(opt)) for k(cat)(c) correlated with S(c/o) for land plants and for all organisms pooled, but the effect of T growth on T(opt) was driven by species phylogeny. The overall phylogenetic signal was significant for all analyzed parameters, stressing the importance of considering the evolutionary framework and accounting for shared ancestry when deciphering relationships between Rubisco kinetic parameters. We argue that these findings have important implications for improving global photosynthesis models.

Mutation design of a thermophilic Rubisco based on three-dimensional structure enhances its activity at ambient temperature.

PubMed

Fujihashi, Masahiro; Nishitani, Yuichi; Kiriyama, Tomohiro; Aono, Riku; Sato, Takaaki; Takai, Tomoyuki; Tagashira, Kenta; Fukuda, Wakao; Atomi, Haruyuki; Imanaka, Tadayuki; Miki, Kunio

2016-10-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a central role in carbon dioxide fixation on our planet. Rubisco from a hyperthermophilic archaeon Thermococcus kodakarensis (Tk-Rubisco) shows approximately twenty times the activity of spinach Rubisco at high temperature, but only one-eighth the activity at ambient temperature. We have tried to improve the activity of Tk-Rubisco at ambient temperature, and have successfully constructed several mutants which showed higher activities than the wild-type enzyme both in vitro and in vivo. Here, we designed new Tk-Rubisco mutants based on its three-dimensional structure and a sequence comparison of thermophilic and mesophilic plant Rubiscos. Four mutations were introduced to generate new mutants based on this strategy, and one of the four mutants, T289D, showed significantly improved activity compared to that of the wild-type enzyme. The crystal structure of the Tk-Rubisco T289D mutant suggested that the increase in activity was due to mechanisms distinct from those involved in the improvement in activity of Tk-Rubisco SP8, a mutant protein previously reported to show the highest activity at ambient temperature. Combining the mutations of T289D and SP8 successfully generated a mutant protein (SP8-T289D) with the highest activity to date both in vitro and in vivo. The improvement was particularly pronounced for the in vivo activity of SP8-T289D when introduced into the mesophilic, photosynthetic bacterium Rhodopseudomonas palustris, which resulted in a strain with nearly two-fold higher specific growth rates compared to that of a strain harboring the wild-type enzyme at ambient temperature. Proteins 2016; 84:1339-1346. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase: product inhibition, cooperativity, and magnesium activation.

PubMed

Hazra, Suratna; Henderson, J Nathan; Liles, Kevin; Hilton, Matthew T; Wachter, Rebekka M

2015-10-02

In many photosynthetic organisms, tight-binding Rubisco inhibitors are released by the motor protein Rubisco activase (Rca). In higher plants, Rca plays a pivotal role in regulating CO2 fixation. Here, the ATPase activity of 0.005 mm tobacco Rca was monitored under steady-state conditions, and global curve fitting was utilized to extract kinetic constants. The kcat was best fit by 22.3 ± 4.9 min(-1), the Km for ATP by 0.104 ± 0.024 mm, and the Ki for ADP by 0.037 ± 0.007 mm. Without ADP, the Hill coefficient for ATP hydrolysis was extracted to be 1.0 ± 0.1, indicating noncooperative behavior of homo-oligomeric Rca assemblies. However, the addition of ADP was shown to introduce positive cooperativity between two or more subunits (Hill coefficient 1.9 ± 0.2), allowing for regulation via the prevailing ATP/ADP ratio. ADP-mediated activation was not observed, although larger amounts led to competitive product inhibition of hydrolytic activity. The catalytic efficiency increased 8.4-fold upon cooperative binding of a second magnesium ion (Hill coefficient 2.5 ± 0.5), suggesting at least three conformational states (ATP-bound, ADP-bound, and empty) within assemblies containing an average of about six subunits. The addition of excess Rubisco (24:1, L8S8/Rca6) and crowding agents did not modify catalytic rates. However, high magnesium provided for thermal Rca stabilization. We propose that magnesium mediates the formation of closed hexameric toroids capable of high turnover rates and amenable to allosteric regulation. We suggest that in vivo, the Rca hydrolytic activity is tuned by fluctuating [Mg(2+)] in response to changes in available light. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Functions, Compositions, and Evolution of the Two Types of Carboxysomes: Polyhedral Microcompartments That Facilitate CO2 Fixation in Cyanobacteria and Some Proteobacteria

PubMed Central

Rae, Benjamin D.; Long, Benedict M.; Badger, Murray R.

2013-01-01

SUMMARY Cyanobacteria are the globally dominant photoautotrophic lineage. Their success is dependent on a set of adaptations collectively termed the CO2-concentrating mechanism (CCM). The purpose of the CCM is to support effective CO2 fixation by enhancing the chemical conditions in the vicinity of the primary CO2-fixing enzyme, d-ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), to promote the carboxylase reaction and suppress the oxygenase reaction. In cyanobacteria and some proteobacteria, this is achieved by encapsulation of RubisCO within carboxysomes, which are examples of a group of proteinaceous bodies called bacterial microcompartments. Carboxysomes encapsulate the CO2-fixing enzyme within the selectively permeable protein shell and simultaneously encapsulate a carbonic anhydrase enzyme for CO2 supply from a cytoplasmic bicarbonate pool. These bodies appear to have arisen twice and undergone a process of convergent evolution. While the gross structures of all known carboxysomes are ostensibly very similar, with shared gross features such as a selectively permeable shell layer, each type of carboxysome encapsulates a phyletically distinct form of RubisCO enzyme. Furthermore, the specific proteins forming structures such as the protein shell or the inner RubisCO matrix are not identical between carboxysome types. Each type has evolutionarily distinct forms of the same proteins, as well as proteins that are entirely unrelated to one another. In light of recent developments in the study of carboxysome structure and function, we present this review to summarize the knowledge of the structure and function of both types of carboxysome. We also endeavor to cast light on differing evolutionary trajectories which may have led to the differences observed in extant carboxysomes. PMID:24006469

Activation of interspecies-hybrid Rubisco enzymes to assess different models for the Rubisco-Rubisco activase interaction.

PubMed

Wachter, Rebekka M; Salvucci, Michael E; Carmo-Silva, A Elizabete; Barta, Csengele; Genkov, Todor; Spreitzer, Robert J

2013-11-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is prone to inactivation from non-productive binding of sugar-phosphates. Reactivation of Rubisco requires conformational remodeling by a specific chaperone, Rubisco activase. Rubisco activase from tobacco and other plants in the family Solanaceae is an inefficient activator of Rubisco from non-Solanaceae plants and from the green alga Chlamydomonas reinhardtii. To determine if the Rubisco small subunit plays a role in the interaction with Rubisco activase, a hybrid Rubisco (SSNT) composed of tobacco small subunits and Chlamydomonas large subunits was constructed. The SSNT hybrid, like other hybrid Rubiscos containing plant small subunits, supported photoautotrophic growth in Chlamydomonas, but growth in air was much slower than for cells containing wild-type Rubisco. The kinetic properties of the SSNT hybrid Rubisco were similar to the wild-type enzyme, indicating that the poor growth in air was probably caused by disruption of pyrenoid formation and the consequent impairment of the CO2concentrating mechanism. Recombinant Rubisco activase from Arabidopsis activated the SSNT hybrid Rubisco and hybrid Rubiscos containing spinach and Arabidopsis small subunits at rates similar to the rates with wild-type Rubisco. However, none of the hybrid Rubiscos was activated by tobacco Rubisco activase. That replacement of Chlamydomonas small subunits with plant small subunits does not affect the species-specific interaction between Rubisco and Rubisco activase suggests that the association is not dominated by the small subunits that surround the Rubisco central solvent channel. Therefore, the geometry of a side-on binding mode is more consistent with the data than a top-on or ring-stacking binding mode.

Kranz and single-cell forms of C4 plants in the subfamily Suaedoideae show kinetic C4 convergence for PEPC and Rubisco with divergent amino acid substitutions.

PubMed

Rosnow, Josh J; Evans, Marc A; Kapralov, Maxim V; Cousins, Asaph B; Edwards, Gerald E; Roalson, Eric H

2015-12-01

The two carboxylation reactions performed by phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) are vital in the fixation of inorganic carbon for C4 plants. The abundance of PEPC is substantially elevated in C4 leaves, while the location of Rubisco is restricted to one of two chloroplast types. These differences compared with C3 leaves have been shown to result in convergent enzyme optimization in some C4 species. Investigation into the kinetic properties of PEPC and Rubisco from Kranz C4, single cell C4, and C3 species in Chenopodiaceae s. s. subfamily Suaedoideae showed that these major carboxylases in C4 Suaedoideae species lack the same mutations found in other C4 systems which have been examined; but still have similar convergent kinetic properties. Positive selection analysis on the N-terminus of PEPC identified residues 364 and 368 to be under positive selection with a posterior probability >0.99 using Bayes empirical Bayes. Compared with previous analyses on other C4 species, PEPC from C4 Suaedoideae species have different convergent amino acids that result in a higher K m for PEP and malate tolerance compared with C3 species. Kinetic analysis of Rubisco showed that C4 species have a higher catalytic efficiency of Rubisco (k catc in mol CO2 mol(-1) Rubisco active sites s(-1)), despite lacking convergent substitutions in the rbcL gene. The importance of kinetic changes to the two-carboxylation reactions in C4 leaves related to amino acid selection is discussed. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Role of small subunit in mediating assembly of red-type form I Rubisco.

PubMed

Joshi, Jidnyasa; Mueller-Cajar, Oliver; Tsai, Yi-Chin C; Hartl, F Ulrich; Hayer-Hartl, Manajit

2015-01-09

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme involved in photosynthetic carbon fixation, converting atmospheric CO2 to organic compounds. Form I Rubisco is a cylindrical complex composed of eight large (RbcL) subunits that are capped by four small subunits (RbcS) at the top and four at the bottom. Form I Rubiscos are phylogenetically divided into green- and red-type. Some red-type enzymes have catalytically superior properties. Thus, understanding their folding and assembly is of considerable biotechnological interest. Folding of the green-type RbcL subunits in cyanobacteria is mediated by the GroEL/ES chaperonin system, and assembly to holoenzyme requires specialized chaperones such as RbcX and RAF1. Here, we show that the red-type RbcL subunits in the proteobacterium Rhodobacter sphaeroides also fold with GroEL/ES. However, assembly proceeds in a chaperone-independent manner. We find that the C-terminal β-hairpin extension of red-type RbcS, which is absent in green-type RbcS, is critical for efficient assembly. The β-hairpins of four RbcS subunits form an eight-stranded β-barrel that protrudes into the central solvent channel of the RbcL core complex. The two β-barrels stabilize the complex through multiple interactions with the RbcL subunits. A chimeric green-type RbcS carrying the C-terminal β-hairpin renders the assembly of a cyanobacterial Rubisco independent of RbcX. Our results may facilitate the engineering of crop plants with improved growth properties expressing red-type Rubisco. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Effects of elevated pCO2 on physiological performance of marine microalgae Dunaliella salina (Chlorophyta, Chlorophyceae

NASA Astrophysics Data System (ADS)

Hu, Shunxin; Wang, You; Wang, Ying; Zhao, Yan; Zhang, Xinxin; Zhang, Yongsheng; Jiang, Ming; Tang, Xuexi

2018-03-01

The present study was conducted to determine the effects of elevated pCO2 on growth, photosynthesis, dark respiration and inorganic carbon acquisition in the marine microalga Dunaliella salina. To accomplish this, D. salina was incubated in semi-continuous cultures under present-day CO2 levels (390 μatm, pHNBS: 8.10), predicted year 2100 CO2 levels (1 000 μatm, pHNBS: 7.78) and predicted year 2300 CO2 levels (2 000 μatm, pHNBS: 7.49). Elevated pCO2 significantly enhanced photosynthesis (in terms of gross photosynthetic O2 evolution, effective quantum yield (Δ F/ F' m ), photosynthetic efficiency ( α), maximum relative electron transport rate (rETRmax) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity) and dark respiration of D. salina, but had insignificant effects on growth. The photosynthetic O2 evolution of D. salina was significantly inhibited by the inhibitors acetazolamide (AZ), ethoxyzolamide (EZ) and 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), indicating that D. salina is capable of acquiring HCOˉ 3 via extracellular carbonic anhydrase and anion-exchange proteins. Furthermore, the lower inhibition of the photosynthetic O2 evolution at high pCO2 levels by AZ, EZ and DIDS and the decreased carbonic anhydrase showed that carbon concentrating mechanisms were down-regulated at high pCO2. In conclusion, our results show that photosynthesis, dark respiration and CCMs will be affected by the increased pCO2/low pH conditions predicted for the future, but that the responses of D. salina to high pCO2/low pH might be modulated by other environmental factors such as light, nutrients and temperature. Therefore, further studies are needed to determine the interactive effects of pCO2, temperature, light and nutrients on marine microalgae.

Identification and In Silico Analysis of Major Redox Modulated Proteins from Brassica juncea Seedlings Using 2D Redox SDS PAGE (2-Dimensional Diagonal Redox Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis).

PubMed

Chaurasia, Satya Prakash; Deswal, Renu

2017-02-01

The thiol-disulphide exchange regulates the activity of proteins by redox modulation. Many studies to analyze reactive oxygen species (ROS), particularly, hydrogen peroxide (H 2 O 2 ) induced changes in the gene expression have been reported, but efforts to detect H 2 O 2 modified proteins are comparatively few. Two-dimensional diagonal redox sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was used to detect polypeptides which undergo thiol-disulphide exchange in Brassica juncea seedlings following H 2 O 2 (10 mM) treatment for 30 min. Eleven redox responsive polypeptides were identified which included cruciferin, NLI [Nuclear LIM (Lin11, Isl-1 & Mec-3 domains)] interacting protein phosphatase, RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) large subunit, and myrosinase. Redox modulation of RuBisCO large subunit was further confirmed by western blotting. However, the small subunit of RuBisCO was not affected by these redox changes. All redox modulated targets except NLI interacting protein (although it contains two cysteines) showed oxidation sensitive cysteines by in silico analysis. Interestingly, interactome of cruciferin and myrosinase indicated that they may have additional function(s) beside their well-known roles in the seedling development and abiotic stress respectively. Cruciferin showed interactions with stress associated proteins like defensing-like protein 192 and 2-cys peroxiredoxin. Similarly, myrosinase showed interactions with nitrilase and cytochrome p450 which are involved in nitrogen metabolism and/or hormone biosynthesis. This simple procedure can be used to detect major stress mediated redox changes in other plants.

Effects of an inhibitor of phosphoenolpyruvate carboxylase on photosynthesis of the terrestrial forms of amphibious Eleocharis species.

PubMed

Ueno, Osamu; Ishimaru, Ken

2002-01-01

The leafless amphibious sedge Eleocharis vivipara develops culms with C(4) traits and Kranz anatomy under terrestrial conditions, but develops culms with C(3) traits and non-Kranz anatomy under submerged conditions. The culms of the terrestrial form have high C(4) enzyme activities, while those of the submerged form have decreased C(4) enzyme activities. The culms accumulate ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the mesophyll cells (MC) and the bundle sheath cells. The Rubisco in the MC may be responsible for the operation of the C(3) pathway in the submerged form. To verify the presence of the C(3) cycle in the MC, we examined the effects of 3,3-dichloro-2-(dihydroxyphosphinoylmethyl) -propenoate (DCDP), an inhibitor of phosphoenolpyruvate carboxylase (PEPC), on photosynthesis in culms of the terrestrial forms of E. vivipara and related amphibious species, E. baldwinii and E. retroflexa ssp. chaetaria. When 1 mM DCDP was fed via the transpiration stream to excised leaves, photosynthesis was inhibited completely in Fimbristylis dichotoma (C(4) control), but by only 20% in potato (C(3) control). In the terrestrial Eleocharis plants, the degree of inhibition of photosynthesis by DCDP was intermediate between those of the C(4) and C(3) plants, at 58-81%. These results suggest that photosynthesis under DCDP treatment in the terrestrial Eleocharis plants is due mainly to fixation of atmospheric CO(2) by Rubisco and probably the C(3) cycle in the MC. These features are reminiscent of those in C(4)-like plants. Differential effects of DCDP on photosynthesis of the 3 Eleocharis species are discussed in relation to differences in the degree of Rubisco accumulation and C(3) activity in the MC.

Callitriche cophocarpa (water starwort) proteome under chromate stress: evidence for induction of a quinone reductase.

PubMed

Kaszycki, Paweł; Dubicka-Lisowska, Aleksandra; Augustynowicz, Joanna; Piwowarczyk, Barbara; Wesołowski, Wojciech

2018-03-01

Chromate-induced physiological stress in a water-submerged macrophyte Callitriche cophocarpa Sendtn. (water starwort) was tested at the proteomic level. The oxidative stress status of the plant treated with 1 mM Cr(VI) for 3 days revealed stimulation of peroxidases whereas catalase and superoxide dismutase activities were similar to the control levels. Employing two-dimensional electrophoresis, comparative proteomics enabled to detect five differentiating proteins subjected to identification with mass spectrometry followed by an NCBI database search. Cr(VI) incubation led to induction of light harvesting chlorophyll a/b binding protein with a concomitant decrease of accumulation of ribulose bisphosphate carboxylase (RuBisCO). The main finding was, however, the identification of an NAD(P)H-dependent dehydrogenase FQR1, detectable only in Cr(VI)-treated plants. The FQR1 flavoenzyme is known to be responsive to oxidative stress and to act as a detoxification protein by protecting the cells against oxidative damage. It exhibits the in vitro quinone reductase activity and is capable of catalyzing two-electron transfer from NAD(P)H to several substrates, presumably including Cr(VI). The enhanced accumulation of FQR1 was chromate-specific since other stressful conditions, such as salt, temperature, and oxidative stresses, all failed to induce the protein. Zymographic analysis of chromate-treated Callitriche shoots showed a novel enzymatic protein band whose activity was attributed to the newly identified enzyme. We suggest that Cr(VI) phytoremediation with C. cophocarpa can be promoted by chromate reductase activity produced by the induced quinone oxidoreductase which might take part in Cr(VI) → Cr(III) bioreduction process and thus enable the plant to cope with the chromate-generated oxidative stress.

Exposure of Lycopersicon Esculentum to Microcystin-LR: Effects in the Leaf Proteome and Toxin Translocation from Water to Leaves and Fruits

PubMed Central

Gutiérrez-Praena, Daniel; Campos, Alexandre; Azevedo, Joana; Neves, Joana; Freitas, Marisa; Guzmán-Guillén, Remédios; Cameán, Ana María; Renaut, Jenny; Vasconcelos, Vitor

2014-01-01

Natural toxins such as those produced by freshwater cyanobacteria have been regarded as an emergent environmental threat. However, the impact of these water contaminants in agriculture is not yet fully understood. The aim of this work was to investigate microcystin-LR (MC-LR) toxicity in Lycopersicon esculentum and the toxin accumulation in this horticultural crop. Adult plants (2 month-old) grown in a greenhouse environment were exposed for 2 weeks to either pure MC-LR (100 μg/L) or Microcystis aeruginosa crude extracts containing 100 μg/L MC-LR. Chlorophyll fluorescence was measured, leaf proteome investigated with two-dimensional gel electrophoresis and Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF)/TOF, and toxin bioaccumulation assessed by liquid chromatography-mass spectrometry (LC-MS)/MS. Variations in several protein markers (ATP synthase subunits, Cytochrome b6-f complex iron-sulfur, oxygen-evolving enhancer proteins) highlight the decrease of the capacity of plants to synthesize ATP and to perform photosynthesis, whereas variations in other proteins (ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and ribose-5-phosphate isomerase) suggest an increase of carbon fixation and decrease of carbohydrate metabolism reactions in plants exposed to pure MC-LR and cyanobacterial extracts, respectively. MC-LR was found in roots (1635.21 μg/kg fw), green tomatoes (5.15–5.41 μg/kg fw), mature tomatoes (10.52–10.83 μg/kg fw), and leaves (12,298.18 μg/kg fw). The results raise concerns relative to food safety and point to the necessity of monitoring the bioaccumulation of water toxins in agricultural systems affected by cyanotoxin contamination. PMID:24921194

Exposure of Lycopersicon esculentum to microcystin-LR: effects in the leaf proteome and toxin translocation from water to leaves and fruits.

PubMed

Gutiérrez-Praena, Daniel; Campos, Alexandre; Azevedo, Joana; Neves, Joana; Freitas, Marisa; Guzmán-Guillén, Remédios; Cameán, Ana María; Renaut, Jenny; Vasconcelos, Vitor

2014-06-11

Natural toxins such as those produced by freshwater cyanobacteria have been regarded as an emergent environmental threat. However, the impact of these water contaminants in agriculture is not yet fully understood. The aim of this work was to investigate microcystin-LR (MC-LR) toxicity in Lycopersicon esculentum and the toxin accumulation in this horticultural crop. Adult plants (2 month-old) grown in a greenhouse environment were exposed for 2 weeks to either pure MC-LR (100 μg/L) or Microcystis aeruginosa crude extracts containing 100 μg/L MC-LR. Chlorophyll fluorescence was measured, leaf proteome investigated with two-dimensional gel electrophoresis and Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF)/TOF, and toxin bioaccumulation assessed by liquid chromatography-mass spectrometry (LC-MS)/MS. Variations in several protein markers (ATP synthase subunits, Cytochrome b6-f complex iron-sulfur, oxygen-evolving enhancer proteins) highlight the decrease of the capacity of plants to synthesize ATP and to perform photosynthesis, whereas variations in other proteins (ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and ribose-5-phosphate isomerase) suggest an increase of carbon fixation and decrease of carbohydrate metabolism reactions in plants exposed to pure MC-LR and cyanobacterial extracts, respectively. MC-LR was found in roots (1635.21 μg/kg fw), green tomatoes (5.15-5.41 μg/kg fw), mature tomatoes (10.52-10.83 μg/kg fw), and leaves (12,298.18 μg/kg fw). The results raise concerns relative to food safety and point to the necessity of monitoring the bioaccumulation of water toxins in agricultural systems affected by cyanotoxin contamination.

Effects of elevated pCO2 on physiological performance of marine microalgae Dunaliella salina (Chlorophyta, Chlorophyceae)

NASA Astrophysics Data System (ADS)

Hu, Shunxin; Wang, You; Wang, Ying; Zhao, Yan; Zhang, Xinxin; Zhang, Yongsheng; Jiang, Ming; Tang, Xuexi

2017-06-01

The present study was conducted to determine the effects of elevated pCO2 on growth, photosynthesis, dark respiration and inorganic carbon acquisition in the marine microalga Dunaliella salina. To accomplish this, D. salina was incubated in semi-continuous cultures under present-day CO2 levels (390 μatm, pHNBS: 8.10), predicted year 2100 CO2 levels (1 000 μatm, pHNBS: 7.78) and predicted year 2300 CO2 levels (2 000 μatm, pHNBS: 7.49). Elevated pCO2 significantly enhanced photosynthesis (in terms of gross photosynthetic O2 evolution, effective quantum yield (ΔF/F' m ), photosynthetic efficiency (α), maximum relative electron transport rate (rETRmax) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity) and dark respiration of D. salina, but had insignificant effects on growth. The photosynthetic O2 evolution of D. salina was significantly inhibited by the inhibitors acetazolamide (AZ), ethoxyzolamide (EZ) and 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), indicating that D. salina is capable of acquiring HCO3 - via extracellular carbonic anhydrase and anion-exchange proteins. Furthermore, the lower inhibition of the photosynthetic O2 evolution at high pCO2 levels by AZ, EZ and DIDS and the decreased carbonic anhydrase showed that carbon concentrating mechanisms were down-regulated at high pCO2. In conclusion, our results show that photosynthesis, dark respiration and CCMs will be affected by the increased pCO2/low pH conditions predicted for the future, but that the responses of D. salina to high pCO2/low pH might be modulated by other environmental factors such as light, nutrients and temperature. Therefore, further studies are needed to determine the interactive effects of pCO2, temperature, light and nutrients on marine microalgae.

[Influence of over expression of CsRCA on photosynthesis of cucumber seedlings under high temperature stress.

PubMed

Bi, Huan Gai; Dong, Xu Bing; Liu, Pei Pei; Li, Qing Ming; Ai, Xi Zhen

2016-07-01

In the present work, transgenic cucumber seedlings over expressing CsRCA and wild-type cucumber seedlings '08-1'at three-leaf stage exposed to high temperature (40 ℃, PFD 600 μmol· m -2 · s -1 ) were used to study the regulatory mechanism of photosynthesis by CsRCA. The results showed that the mRNA abundance of rbcL and rbcS as well as the activities of ribulose bisphosphate carboxylic enzyme (Rubisco) and Rubisco activase (RCA) were significantly higher in CsRCA over-expressing cucumber seedlings than in wild type (WT). Following 2-h exposure to high temperature, a notable decrease was observed in photosynthetic rate (P n ), photochemical perfor-mance index based on the absorption of light energy (PI ABS ), activities of Rubisco and RCA as well as the relative expression of rbcL, rbcS and CsRCA in both wild-type cucumber seedlings and transgenic cucumber seedlings. It was found that high temperature stress led to higher W k , a parameter of chlorophyll (Chl) a fluorescence OJIP curve. Furthermore, high temperature greatly reduced the efficiency of electron transfer along the electron transport chain beyond Q A (ψ 0 ) and the quantum yield for electron transport (φ E0 ), indicating that PSII oxygen complexes (OEC) and electron transport chain downstream Q A were inhibited by high temperature. However, the inhibition could be alleviated by over expressing CsRCA in cucumber seedlings. Taken together, our data suggested that over expressing CsRCA improves photosynthesis in cucumber seedlings under high temperature stress by enhancing activities of the Rubisco and RCA, and maintaining the number of active reaction centers.

Marker-free transgenic rice expressing the vegetative insecticidal protein (Vip) of Bacillus thuringiensis shows broad insecticidal properties.

PubMed

Pradhan, Subrata; Chakraborty, Anirban; Sikdar, Narattam; Chakraborty, Saikat; Bhattacharyya, Jagannath; Mitra, Joy; Manna, Anulina; Dutta Gupta, Snehasish; Sen, Soumitra Kumar

2016-10-01

Genetically engineered rice lines with broad insecticidal properties against major lepidopteran pests were generated using a synthetic, truncated form of vegetative insecticidal protein (Syn vip3BR) from Bacillus thuringiensis. The selectable marker gene and the redundant transgene(s) were eliminated through Cre/ lox mediated recombination and genetic segregation to make consumer friendly Bt -rice. For sustainable resistance against lepidopteran insect pests, chloroplast targeted synthetic version of bioactive core component of a vegetative insecticidal protein (Syn vip3BR) of Bacillus thuringiensis was expressed in rice under the control of green-tissue specific ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene promoter. The transgenic plants (in Oryza sativa indica Swarna cultivar) showed high insect mortality rate in vitro against major rice pests, yellow stem borer (Scirpophaga incertulas), rice leaf folder (Cnaphalocrocis medinalis) and rice horn caterpillar (Melanitis leda ismene) in T1 generation, indicating insecticidal potency of Syn vip3BR. Under field conditions, the T1 plants showed considerable resistance against leaf folders and stem borers. The expression cassette (vip-lox-hpt-lox) as well as another vector with chimeric cre recombinase gene under constitutive rice ubiquitin1 gene promoter was designed for the elimination of selectable marker hygromycin phosphotransferase (hptII) gene. Crossing experiments were performed between T1 plants with single insertion site of vip-lox-hpt-lox T-DNA and one T1 plant with moderate expression of cre recombinase with linked bialaphos resistance (syn bar) gene. Marker gene excision was achieved in hybrids with up to 41.18 % recombination efficiency. Insect resistant transgenic lines, devoid of selectable marker and redundant transgene(s) (hptII + cre-syn bar), were established in subsequent generation through genetic segregation.

Evidence for a Role for NAD(P)H Dehydrogenase in Concentration of CO2 in the Bundle Sheath Cell of Zea mays.

PubMed

Peterson, Richard B; Schultes, Neil P; McHale, Neil A; Zelitch, Israel

2016-05-01

Prior studies with Nicotiana and Arabidopsis described failed assembly of the chloroplastic NDH [NAD(P)H dehydrogenase] supercomplex by serial mutation of several subunit genes. We examined the properties of Zea mays leaves containing Mu and Ds insertions into nuclear gene exons encoding the critical o- and n-subunits of NDH, respectively. In vivo reduction of plastoquinone in the dark was sharply diminished in maize homozygous mutant compared to normal leaves but not to the extreme degree observed for the corresponding lesions in Arabidopsis. The net carbon assimilation rate (A) at high irradiance and saturating CO2 levels was reduced by one-half due to NDH mutation in maize although no genotypic effect was evident at very low CO2 levels. Simultaneous assessment of chlorophyll fluorescence and A in maize at low (2% by volume) and high (21%) O2 levels indicated the presence of a small, yet detectable, O2-dependent component of total linear photosynthetic electron transport in 21% O2 This O2-dependent component decreased with increasing CO2 level indicative of photorespiration. Photorespiration was generally elevated in maize mutant compared to normal leaves. Quantification of the proportion of total electron transport supporting photorespiration enabled estimation of the bundle sheath cell CO2 concentration (Cb) using a simple kinetic model of ribulose bisphosphate carboxylase/oxygenase function. The A versus Cb relationships overlapped for normal and mutant lines consistent with occurrence of strictly CO2-limited photosynthesis in the mutant bundle sheath cell. The results are discussed in terms of a previously reported CO2 concentration model [Laisk A, Edwards GE (2000) Photosynth Res 66: 199-224]. © 2016 American Society of Plant Biologists. All Rights Reserved.

Sensitivity of Photosynthesis in a C4 Plant, Maize, to Heat Stress

PubMed Central

Crafts-Brandner, Steven J.; Salvucci, Michael E.

2002-01-01

Our objective was to determine the sensitivity of components of the photosynthetic apparatus of maize (Zea mays), a C4 plant, to high temperature stress. Net photosynthesis (Pn) was inhibited at leaf temperatures above 38°C, and the inhibition was much more severe when the temperature was increased rapidly rather than gradually. Transpiration rate increased progressively with leaf temperature, indicating that inhibition was not associated with stomatal closure. Nonphotochemical fluorescence quenching (qN) increased at leaf temperatures above 30°C, indicating increased thylakoid energization even at temperatures that did not inhibit Pn. Compared with CO2 assimilation, the maximum quantum yield of photosystem II (Fv/Fm) was relatively insensitive to leaf temperatures up to 45°C. The activation state of phosphoenolpyruvate carboxylase decreased marginally at leaf temperatures above 40°C, and the activity of pyruvate phosphate dikinase was insensitive to temperature up to 45°C. The activation state of Rubisco decreased at temperatures exceeding 32.5°C, with nearly complete inactivation at 45°C. Levels of 3-phosphoglyceric acid and ribulose-1,5-bisphosphate decreased and increased, respectively, as leaf temperature increased, consistent with the decrease in Rubisco activation. When leaf temperature was increased gradually, Rubisco activation acclimated in a similar manner as Pn, and acclimation was associated with the expression of a new activase polypeptide. Rates of Pn calculated solely from the kinetics of Rubisco were remarkably similar to measured rates if the calculation included adjustment for temperature effects on Rubisco activation. We conclude that inactivation of Rubisco was the primary constraint on the rate of Pn of maize leaves as leaf temperature increased above 30°C. PMID:12177490

Functional indicators of response mechanisms to nitrogen deposition, ozone, and their interaction in two Mediterranean tree species.

PubMed

Fusaro, Lina; Palma, Adriano; Salvatori, Elisabetta; Basile, Adriana; Maresca, Viviana; Asadi Karam, Elham; Manes, Fausto

2017-01-01

The effects of nitrogen (N) deposition, tropospheric ozone (O3) and their interaction were investigated in two Mediterranean tree species, Fraxinus ornus L. (deciduous) and Quercus ilex L. (evergreen), having different leaf habits and resource use strategies. An experiment was conducted under controlled condition to analyse how nitrogen deposition affects the ecophysiological and biochemical traits, and to explore how the nitrogen-induced changes influence the response to O3. For both factors we selected realistic exposures (20 kg N ha-1 yr-1 and 80 ppb h for nitrogen and O3, respectively), in order to elucidate the mechanisms implemented by the plants. Nitrogen addition resulted in higher nitrogen concentration at the leaf level in F. ornus, whereas a slight increase was detected in Q. ilex. Nitrogen enhanced the maximum rate of assimilation and ribulose 1,5-bisphosphate regeneration in both species, whereas it influenced the light harvesting complex only in the deciduous F. ornus that was also affected by O3 (reduced assimilation rate and accelerated senescence-related processes). Conversely, Q. ilex developed an avoidance mechanism to cope with O3, confirming a substantial O3 tolerance of this species. Nitrogen seemed to ameliorate the harmful effects of O3 in F. ornus: the hypothesized mechanism of action involved the production of nitrogen oxide as the first antioxidant barrier, followed by enzymatic antioxidant response. In Q. ilex, the interaction was not detected on gas exchange and photosystem functionality; however, in this species, nitrogen might stimulate an alternative antioxidant response such as the emission of volatile organic compounds. Antioxidant enzyme activity was lower in plants treated with both O3 and nitrogen even though reactive oxygen species production did not differ between the treatments.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Endow, Joshua K.; Rocha, Agostinho Gomes; Baldwin, Amy J.

PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here in this study we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins,more » the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope.« less

Structure and enzyme expression in photosynthetic organs of the atypical C4 grass Arundinella hirta.

PubMed

Wakayama, Masataka; Ohnishi, Jun-ichi; Ueno, Osamu

2006-05-01

In its leaf blade, Arundinella hirta has unusual Kranz cells that lie distant from the veins (distinctive cells; DCs), in addition to the usual Kranz units composed of concentric layers of mesophyll cells (MCs) and bundle sheath cells (BSCs; usual Kranz cells) surrounding the veins. We examined whether chlorophyllous organs other than leaf blades--namely, the leaf sheath, stem, scale leaf, and constituents of the spike--also have this unique anatomy and the C4 pattern of expression of photosynthetic enzymes. All the organs developed DCs to varying degrees, as well as BSCs. The stem, rachilla, and pedicel had C4-type anatomy with frequent occurrence of DCs, as in the leaf blade. The leaf sheath, glume, and scale leaf had a modified C4 anatomy with MCs more than two cells distant from the Kranz cells; DCs were relatively rare. An immunocytochemical study of C3 and C4 enzymes revealed that all the organs exhibited essentially the same C4 pattern of expression as in the leaf blade. In the scale leaf, however, intense expression of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) occurred in the MCs as well as in the BSCs and DCs. In the leaf sheath, the distant MCs also expressed Rubisco. In Arundinella hirta, it seems that the ratio of MC to Kranz cell volumes, and the distance from the Kranz cells, but not from the veins, affects the cellular expression of photosynthetic enzymes. We suggest that the main role of DCs is to keep a constant quantitative balance between the MCs and Kranz cells, which is a prerequisite for effective C4 pathway operation.

Do the Rich Always Become Richer? Characterizing the Leaf Physiological Response of the High-Yielding Rice Cultivar Takanari to Free-Air CO2 Enrichment

PubMed Central

Chen, Charles P.; Sakai, Hidemitsu; Tokida, Takeshi; Usui, Yasuhiro; Nakamura, Hirofumi; Hasegawa, Toshihiro

2014-01-01

The development of crops which are well suited to growth under future environmental conditions such as higher atmospheric CO2 concentrations ([CO2]) is essential to meeting the challenge of ensuring food security in the face of the growing human population and changing climate. A high-yielding indica rice variety (Oryza sativa L. cv. Takanari) has been recently identified as a potential candidate for such breeding, due to its high productivity in present [CO2]. To test if it could further increase its productivity under elevated [CO2] (eCO2), Takanari was grown in the paddy field under season-long free-air CO2 enrichment (FACE, approximately 200 µmol mol−1 above ambient [CO2]) and its leaf physiology was compared with the representative japonica variety ‘Koshihikari’. Takanari showed consistently higher midday photosynthesis and stomatal conductance than Koshihikari under both ambient and FACE growth conditions over 2 years. Maximum ribulose-1,5-bisphosphate carboxylation and electron transport rates were higher for Takanari at the mid-grain filling stage in both years. Mesophyll conductance was higher in Takanari than in Koshihikari at the late grain-filling stage. In contrast to Koshihikari, Takanari grown under FACE conditions showed no decrease in total leaf nitrogen on an area basis relative to ambient-grown plants. Chl content was higher in Takanari than in Koshihikari at the same leaf nitrogen level. These results indicate that Takanari maintains its superiority over Koshihikari in regards to its leaf-level productivity when grown in elevated [CO2] and it may be a valuable resource for rice breeding programs which seek to increase crop productivity under current and future [CO2]. PMID:24443497

Characteristics of photosynthesis and functions of the water-water cycle in rice (Oryza sativa) leaves in response to potassium deficiency.

PubMed

Weng, Xiao-Yan; Zheng, Chen-Juan; Xu, Hong-Xia; Sun, Jian-Yi

2007-12-01

The mechanisms of photoprotection of photosynthesis and dissipation of excitation energy in rice leaves in response to potassium (K) deficiency were investigated. Net photosynthetic rate and the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase decreased under K deficiency. Compared with the control, non-photochemical quenching of Chl fluorescence increased in K-deficient plant, whereas the efficiency of excitation transfer (F'(v)/F'(m)) and the photochemical quenching coefficient (q(P)) decreased. Thus, thermal dissipation of excitation energy increased as more excess electrons were accumulated in the photosynthetic chain. The electron transport rate through PSII (J(f)) was more sensitive to O2 concentration, and the fraction of electron transport rate required to sustain CO2 assimilation and photorespiration (J(g)/J(f)) was significantly decreased under K deficiency compared with the control. Furthermore, the alternative electron transport (J(a)/J(f)) was increased, indicating that a considerable amount of electrons had been transported to O2 during the water-water cycle in the K-deficient leaves. Although the fraction of electron transport to photorespiration (J(o)/J(f)) was also increased in the K-deficient leaves, it was less sensitive than that of the water-water cycle. With the generation of reactive oxygen species level, the activities of superoxide dismutase and ascorbate peroxidase, two of the key enzymes involved in scavenging of active oxygen species in the water-water cycle, also increased in K-deficient rice. Therefore, it is likely that a series of photoprotective mechanisms were initiated in rice plants in response to K deficiency and the water-water cycle might be critical for protecting photosynthetic apparatus under K deficiency in rice.

Large Fractions of CO2-Fixing Microorganisms in Pristine Limestone Aquifers Appear To Be Involved in the Oxidation of Reduced Sulfur and Nitrogen Compounds

PubMed Central

Herrmann, Martina; Rusznyák, Anna; Akob, Denise M.; Schulze, Isabel; Opitz, Sebastian; Totsche, Kai Uwe

2015-01-01

The traditional view of the dependency of subsurface environments on surface-derived allochthonous carbon inputs is challenged by increasing evidence for the role of lithoautotrophy in aquifer carbon flow. We linked information on autotrophy (Calvin-Benson-Bassham cycle) with that from total microbial community analysis in groundwater at two superimposed—upper and lower—limestone groundwater reservoirs (aquifers). Quantitative PCR revealed that up to 17% of the microbial population had the genetic potential to fix CO2 via the Calvin cycle, with abundances of cbbM and cbbL genes, encoding RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) forms I and II, ranging from 1.14 × 103 to 6 × 106 genes liter−1 over a 2-year period. The structure of the active microbial communities based on 16S rRNA transcripts differed between the two aquifers, with a larger fraction of heterotrophic, facultative anaerobic, soil-related groups in the oxygen-deficient upper aquifer. Most identified CO2-assimilating phylogenetic groups appeared to be involved in the oxidation of sulfur or nitrogen compounds and harbored both RubisCO forms I and II, allowing efficient CO2 fixation in environments with strong oxygen and CO2 fluctuations. The genera Sulfuricella and Nitrosomonas were represented by read fractions of up to 78 and 33%, respectively, within the cbbM and cbbL transcript pool and accounted for 5.6 and 3.8% of 16S rRNA sequence reads, respectively, in the lower aquifer. Our results indicate that a large fraction of bacteria in pristine limestone aquifers has the genetic potential for autotrophic CO2 fixation, with energy most likely provided by the oxidation of reduced sulfur and nitrogen compounds. PMID:25616797

Soil Carbon-Fixation Rates and Associated Bacterial Diversity and Abundance in Three Natural Ecosystems.

PubMed

Lynn, Tin Mar; Ge, Tida; Yuan, Hongzhao; Wei, Xiaomeng; Wu, Xiaohong; Xiao, Keqing; Kumaresan, Deepak; Yu, San San; Wu, Jinshui; Whiteley, Andrew S

2017-04-01

CO 2 assimilation by autotrophic microbes is an important process in soil carbon cycling, and our understanding of the community composition of autotrophs in natural soils and their role in carbon sequestration of these soils is still limited. Here, we investigated the autotrophic C incorporation in soils from three natural ecosystems, i.e., wetland (WL), grassland (GR), and forest (FO) based on the incorporation of labeled C into the microbial biomass. Microbial assimilation of 14 C ( 14 C-MBC) differed among the soils from three ecosystems, accounting for 14.2-20.2% of 14 C-labeled soil organic carbon ( 14 C-SOC). We observed a positive correlation between the cbbL (ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large-subunit gene) abundance, 14 C-SOC level, and 14 C-MBC concentration confirming the role of autotrophic bacteria in soil carbon sequestration. Distinct cbbL-bearing bacterial communities were present in each soil type; form IA and form IC RubisCO-bearing bacteria were most abundant in WL, followed by GR soils, with sequences from FO soils exclusively derived from the form IC clade. Phylogenetically, the diversity of CO 2 -fixing autotrophs and CO oxidizers differed significantly with soil type, whereas cbbL-bearing bacterial communities were similar when assessed using coxL. We demonstrate that local edaphic factors such as pH and salinity affect the C-fixation rate as well as cbbL and coxL gene abundance and diversity. Such insights into the effect of soil type on the autotrophic bacterial capacity and subsequent carbon cycling of natural ecosystems will provide information to enhance the sustainable management of these important natural ecosystems.

Interaction of Sulfate Assimilation with Carbon and Nitrogen Metabolism in Lemna minor1

PubMed Central

Kopriva, Stanislav; Suter, Marianne; von Ballmoos, Peter; Hesse, Holger; Krähenbühl, Urs; Rennenberg, Heinz; Brunold, Christian

2002-01-01

Cysteine synthesis from sulfide and O-acetyl-l-serine (OAS) is a reaction interconnecting sulfate, nitrogen, and carbon assimilation. Using Lemna minor, we analyzed the effects of omission of CO2 from the atmosphere and simultaneous application of alternative carbon sources on adenosine 5′-phosphosulfate reductase (APR) and nitrate reductase (NR), the key enzymes of sulfate and nitrate assimilation, respectively. Incubation in air without CO2 led to severe decrease in APR and NR activities and mRNA levels, but ribulose-1,5-bisphosphate carboxylase/oxygenase was not considerably affected. Simultaneous addition of sucrose (Suc) prevented the reduction in enzyme activities, but not in mRNA levels. OAS, a known regulator of sulfate assimilation, could also attenuate the effect of missing CO2 on APR, but did not affect NR. When the plants were subjected to normal air after a 24-h pretreatment in air without CO2, APR and NR activities and mRNA levels recovered within the next 24 h. The addition of Suc and glucose in air without CO2 also recovered both enzyme activities, with OAS again influenced only APR. 35SO42− feeding showed that treatment in air without CO2 severely inhibited sulfate uptake and the flux through sulfate assimilation. After a resupply of normal air or the addition of Suc, incorporation of 35S into proteins and glutathione greatly increased. OAS treatment resulted in high labeling of cysteine; the incorporation of 35S in proteins and glutathione was much less increased compared with treatment with normal air or Suc. These results corroborate the tight interconnection of sulfate, nitrate, and carbon assimilation. PMID:12428005

Compound-specific Isotope Analysis of Cyanobacterial Pure cultures and Microbial Mats: Effects of Photorespiration?

NASA Technical Reports Server (NTRS)

Jahnke, L. L.; Summons, R. E.

2006-01-01

Microbial mats are considered modern homologs of Precambrian stromatolites. The carbon isotopic compositions of organic matter and biomarker lipids provide clues to the depositional environments of ancient mat ecosystems. As the source of primary carbon fixation for over two billion years, an understanding of cyanobacterial lipid biosynthesis, associated isotopic discriminations, and the influence of physiological factors on growth and isotope expression is essential to help us compare modern microbial ecosystems to their ancient counterparts. Here, we report on the effects of photorespiration (PR) on the isotopic composition of cyanobacteria and biomarker lipids, and on potential PR effects associated with the composition of various microbial mats. The high light, high O2 and limiting CO2 conditions often present at the surface of microbial mats are known to support PR in cyanobacteria. The oxygenase function of ribulose bisphosphate carboxylase/oxygenase can result in photoexcretion of glycolate and subsequent degration by heterotrophic bacteria. We have found evidence which supports an isotopic depletion (increased apparent E) scaled to O2 level associated with growth of Phormidium luridum at low CO2 concentrations (less than 0.04%). Similar to previous studies, isotopic differences between biomass and lipid biomarkers, and between lipid classes were positively correlated with overall fractionation, and should provide a means of estimating the influence of PR on overall isotopic composition of microbial mats. Several examples of microbial mats growing in the hydrothermal waters of Yellowstone National Park and the hypersaline marine evaporation ponds at Guerrero Negro, Baja Sur Mexico will be compared with a view to PR as a possible explanation of the relatively heavy C-isotope composition of hypersaline mats.

Lipoic acid mitigates oxidative stress and recovers metabolic distortions in salt-stressed wheat seedlings by modulating ion homeostasis, the osmo-regulator level and antioxidant system.

PubMed

Gorcek, Zeynep; Erdal, Serkan

2015-11-01

Soil salinity is one of the most detrimental environmental factors affecting the growth of plants and limiting their agricultural productivity. This study investigated whether exogenous lipoic acid (LA) pretreatment plays a role in promoting salt tolerance in wheat seedlings. The seedlings were treated with LA (1.75 mmol L(-1)) and salt (100 mmol L(-1) NaCl) separately and a combination of them. Salt stress significantly reduced relative water content, leaf surface area, ribulose bisphosphate carboxylase expression, and chlorophyll content but increased the content of osmo-regulator protein, carbohydrates and proline. In addition, salinity led to an imbalance in the inorganic composition of wheat leaves. While it elevated Na(+) content compared to control, Ca content and K(+)/Na(+) ratio were reduced. Under saline conditions, despite increases in antioxidant enzyme activity and levels of antioxidant compounds (ascorbate and glutathione), the content of reactive oxygen species (superoxide anion, hydrogen peroxide) and malondialdehyde were higher than in control seedlings. LA significantly promoted osmo-regulator level and antioxidant enzyme activities compared to stressed seedlings alone. Also, it both increased levels of ascorbate and glutathione and regenerated their oxidised forms, thus contributing to maintaining cellular redox status. Similarly, LA prevented excessive accumulation of Na(+) and promoted K(+)/Na(+) ratio and Ca content. Reactive oxygen species content was significantly reduced, and the inhibitions in the above parameters markedly recovered. LA reduced salinity-induced oxidative damage and thus contributed to the growth and development of plants in saline soils by modulating ion homeostasis between plant and soil as well as in osmo-regulator content and antioxidant system. © 2014 Society of Chemical Industry.

A Non-photosynthetic Diatom Reveals Early Steps of Reductive Evolution in Plastids.

PubMed

Kamikawa, Ryoma; Moog, Daniel; Zauner, Stefan; Tanifuji, Goro; Ishida, Ken-Ichiro; Miyashita, Hideaki; Mayama, Shigeki; Hashimoto, Tetsuo; Maier, Uwe G; Archibald, John M; Inagaki, Yuji

2017-09-01

Nonphotosynthetic plastids retain important biological functions and are indispensable for cell viability. However, the detailed processes underlying the loss of plastidal functions other than photosynthesis remain to be fully understood. In this study, we used transcriptomics, subcellular localization, and phylogenetic analyses to characterize the biochemical complexity of the nonphotosynthetic plastids of the apochlorotic diatom Nitzschia sp. NIES-3581. We found that these plastids have lost isopentenyl pyrophosphate biosynthesis and ribulose-1,5-bisphosphate carboxylase/oxygenase-based carbon fixation but have retained various proteins for other metabolic pathways, including amino acid biosynthesis, and a portion of the Calvin-Benson cycle comprised only of glycolysis/gluconeogenesis and the reductive pentose phosphate pathway (rPPP). While most genes for plastid proteins involved in these reactions appear to be phylogenetically related to plastid-targeted proteins found in photosynthetic relatives, we also identified a gene that most likely originated from a cytosolic protein gene. Based on organellar metabolic reconstructions of Nitzschia sp. NIES-3581 and the presence/absence of plastid sugar phosphate transporters, we propose that plastid proteins for glycolysis, gluconeogenesis, and rPPP are retained even after the loss of photosynthesis because they feed indispensable substrates to the amino acid biosynthesis pathways of the plastid. Given the correlated retention of the enzymes for plastid glycolysis, gluconeogenesis, and rPPP and those for plastid amino acid biosynthesis pathways in distantly related nonphotosynthetic plastids and cyanobacteria, we suggest that this substrate-level link with plastid amino acid biosynthesis is a key constraint against loss of the plastid glycolysis/gluconeogenesis and rPPP proteins in multiple independent lineages of nonphotosynthetic algae/plants. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Improved tolerance to post-anthesis drought stress by pre-drought priming at vegetative stages in drought-tolerant and -sensitive wheat cultivars.

PubMed

Abid, Muhammad; Tian, Zhongwei; Ata-Ul-Karim, Syed Tahir; Liu, Yang; Cui, Yakun; Zahoor, Rizwan; Jiang, Dong; Dai, Tingbo

2016-09-01

Wheat crop endures a considerable penalty of yield reduction to escape the drought events during post-anthesis period. Drought priming under a pre-drought stress can enhance the crop potential to tolerate the subsequent drought stress by triggering a faster and stronger defense mechanism. Towards these understandings, a set of controlled moderate drought stress at 55-60% field capacity (FC) was developed to prime the plants of two wheat cultivars namely Luhan-7 (drought tolerant) and Yangmai-16 (drought sensitive) during tillering (Feekes 2 stage) and jointing (Feekes 6 stage), respectively. The comparative response of primed and non-primed plants, cultivars and priming stages was evaluated by applying a subsequent severe drought stress at 7 days after anthesis. The results showed that primed plants of both cultivars showed higher potential to tolerate the post-anthesis drought stress through improved leaf water potential, more chlorophyll, and ribulose-1, 5-bisphosphate carboxylase/oxygenase contents, enhanced photosynthesis, better photoprotection and efficient enzymatic antioxidant system leading to less yield reductions. The primed plants of Luhan-7 showed higher capability to adapt the drought stress events than Yangmai-16. The positive effects of drought priming to sustain higher grain yield were pronounced in plants primed at tillering than those primed at jointing. In consequence, upregulated functioning of photosynthetic apparatus and efficient enzymatic antioxidant activities in primed plants indicated their superior potential to alleviate a subsequently occurring drought stress, which contributed to lower yield reductions than non-primed plants. However, genotypic and priming stages differences in response to drought stress also contributed to affect the capability of primed plants to tolerate the post-anthesis drought stress conditions in wheat. Copyright © 2016. Published by Elsevier Masson SAS.

Metabolic flux of the oxidative pentose phosphate pathway under low light conditions in Synechocystis sp. PCC 6803.

PubMed

Ueda, Kentaro; Nakajima, Tsubasa; Yoshikawa, Katsunori; Toya, Yoshihiro; Matsuda, Fumio; Shimizu, Hiroshi

2018-02-27

The role of the oxidative pentose phosphate pathway (oxPPP) in Synechocystis sp. PCC 6803 under mixotrophic conditions was investigated by 13 C metabolic flux analysis. Cells were cultured under low (10 μmol m -2  s -1 ) and high light intensities (100 μmol m -2  s -1 ) in the presence of glucose. The flux of CO 2 fixation by ribulose bisphosphate carboxylase/oxygenase under the high light condition was approximately 3-fold higher than that under the low light condition. Although no flux of the oxPPP was observed under the high light condition, flux of 0.08-0.19 mmol gDCW -1  h -1 in the oxPPP was observed under the low light condition. The balance between the consumption and production of NADPH suggested that approximately 10% of the total NADPH production was generated by the oxPPP under the low light condition. The growth phenotype of a mutant with deleted zwf, which encodes glucose-6-phosphate dehydrogenase in the oxPPP, was compared to that of the parental strain under low and high light conditions. Growth of the Δzwf mutant nearly stopped during the late growth phase under the low light condition, whereas the growth rates of the two strains were identical under the high light condition. These results indicate that NADPH production in the oxPPP is essential for anabolism under low light conditions. The oxPPP appears to play an important role in producing NADPH from glucose and ATP to compensate for NADPH shortage under low light conditions. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Hydrogen sulphide improves adaptation of Zea mays seedlings to iron deficiency

PubMed Central

Chen, Juan; Wu, Fei-Hua; Shang, Yu-Ting; Wang, Wen-Hua; Hu, Wen-Jun; Simon, Martin; Liu, Xiang; Shangguan, Zhou-Ping; Zheng, Hai-Lei

2015-01-01

Hydrogen sulphide (H2S) is emerging as a potential molecule involved in physiological regulation in plants. However, whether H2S regulates iron-shortage responses in plants is largely unknown. Here, the role of H2S in modulating iron availability in maize (Zea mays L. cv Canner) seedlings grown in iron-deficient culture solution is reported. The main results are as follows: Firstly, NaHS, a donor of H2S, completely prevented leaf interveinal chlorosis in maize seedlings grown in iron-deficient culture solution. Secondly, electron micrographs of mesophyll cells from iron-deficient maize seedlings revealed plastids with few photosynthetic lamellae and rudimentary grana. On the contrary, mesophyll chloroplasts appeared completely developed in H2S-treated maize seedlings. Thirdly, H2S treatment increased iron accumulation in maize seedlings by changing the expression levels of iron homeostasis- and sulphur metabolism-related genes. Fourthly, phytosiderophore (PS) accumulation and secretion were enhanced by H2S treatment in seedlings grown in iron-deficient solution. Indeed, the gene expression of ferric-phytosiderophore transporter (ZmYS1) was specifically induced by iron deficiency in maize leaves and roots, whereas their abundance was decreased by NaHS treatment. Lastly, H2S significantly enhanced photosynthesis through promoting the protein expression of ribulose-1,5-bisphosphate carboxylase large subunit (RuBISCO LSU) and phosphoenolpyruvate carboxylase (PEPC) and the expression of genes encoding RuBISCO large subunit (RBCL), small subunit (RBCS), D1 protein (psbA), and PEPC in maize seedlings grown in iron-deficient solution. These results indicate that H2S is closely related to iron uptake, transport, and accumulation, and consequently increases chlorophyll biosynthesis, chloroplast development, and photosynthesis in plants. PMID:26208645

Hydrogen sulphide improves adaptation of Zea mays seedlings to iron deficiency.

PubMed

Chen, Juan; Wu, Fei-Hua; Shang, Yu-Ting; Wang, Wen-Hua; Hu, Wen-Jun; Simon, Martin; Liu, Xiang; Shangguan, Zhou-Ping; Zheng, Hai-Lei

2015-11-01

Hydrogen sulphide (H2S) is emerging as a potential molecule involved in physiological regulation in plants. However, whether H2S regulates iron-shortage responses in plants is largely unknown. Here, the role of H2S in modulating iron availability in maize (Zea mays L. cv Canner) seedlings grown in iron-deficient culture solution is reported. The main results are as follows: Firstly, NaHS, a donor of H2S, completely prevented leaf interveinal chlorosis in maize seedlings grown in iron-deficient culture solution. Secondly, electron micrographs of mesophyll cells from iron-deficient maize seedlings revealed plastids with few photosynthetic lamellae and rudimentary grana. On the contrary, mesophyll chloroplasts appeared completely developed in H2S-treated maize seedlings. Thirdly, H2S treatment increased iron accumulation in maize seedlings by changing the expression levels of iron homeostasis- and sulphur metabolism-related genes. Fourthly, phytosiderophore (PS) accumulation and secretion were enhanced by H2S treatment in seedlings grown in iron-deficient solution. Indeed, the gene expression of ferric-phytosiderophore transporter (ZmYS1) was specifically induced by iron deficiency in maize leaves and roots, whereas their abundance was decreased by NaHS treatment. Lastly, H2S significantly enhanced photosynthesis through promoting the protein expression of ribulose-1,5-bisphosphate carboxylase large subunit (RuBISCO LSU) and phosphoenolpyruvate carboxylase (PEPC) and the expression of genes encoding RuBISCO large subunit (RBCL), small subunit (RBCS), D1 protein (psbA), and PEPC in maize seedlings grown in iron-deficient solution. These results indicate that H2S is closely related to iron uptake, transport, and accumulation, and consequently increases chlorophyll biosynthesis, chloroplast development, and photosynthesis in plants. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Structure of fructose bisphosphate aldolase from Bartonella henselae bound to fructose 1,6-bisphosphate

PubMed Central

Gardberg, Anna; Abendroth, Jan; Bhandari, Janhavi; Sankaran, Banumathi; Staker, Bart

2011-01-01

Fructose bisphosphate aldolase (FBPA) enzymes have been found in a broad range of eukaryotic and prokaryotic organisms. FBPA catalyses the cleavage of fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxy­acetone phosphate. The SSGCID has reported several FBPA structures from pathogenic sources, including the bacterium Brucella melitensis and the protozoan Babesia bovis. Bioinformatic analysis of the Bartonella henselae genome revealed an FBPA homolog. The B. henselae FBPA enzyme was recombinantly expressed and purified for X-ray crystallographic studies. The purified enzyme crystallized in the apo form but failed to diffract; however, well diffracting crystals could be obtained by cocrystallization in the presence of the native substrate fructose 1,6-bisphosphate. A data set to 2.35 Å resolution was collected from a single crystal at 100 K. The crystal belonged to the orthorhombic space group P212121, with unit-cell parameters a = 72.39, b = 127.71, c = 157.63 Å. The structure was refined to a final free R factor of 22.2%. The structure shares the typical barrel tertiary structure and tetrameric quaternary structure reported for previous FBPA structures and exhibits the same Schiff base in the active site. PMID:21904049

Synthesis of 4-thiouridine, 6-thioinosine, and 6-thioguanosine 3',5'-O-bisphosphates as donor molecules for RNA ligation and their application to the synthesis of photoactivatable TMG-capped U1 snRNA fragments.

PubMed

Kadokura, M; Wada, T; Seio, K; Sekine, M

2000-08-25

4-Thiouridine, 6-thioguanosine, and 6-thioinosine 3',5'-bisphosphates (9, 20, and 28) were synthesized in good yields by considerably improved methods. In the former two compounds, uridine and 2-N-phenylacetylguanosine were converted via transient O-trimethylsilylation to the corresponding 4- and 6-O-benzenesulfonyl intermediates (2 and 13), which, in turn, were allowed to react with 2-cyanoethanethiol in the presence of N-methylpyrrolidine to give 4-thiouridine (3) and 2-N-phenylacetyl-6-thioguanosine derivatives (14), respectively. In situ dimethoxytritylation of these thionucleoside derivatives gave the 5'-masked products 4 and 15 in high overall yields from 1 and 11. 6-S-(2-Cyanoethyl)-5'-O-(4,4'-dimethoxytrityl)-6-thioinosine (23) was synthesized via substitution of the 5'-O-tritylated 6-chloropurine riboside derivative 22 with 2-cyanoethanethiol. These S-(2-cyanoethyl)thionucleosides were converted to the 2'-O-(tert-butyldimethylsilyl)ribonucleoside 3'-phosphoramidite derivatives 7, 18, and 26 or 3',5'-bisphosphate derivatives 8, 19, and 27. Treatment of 8, 19, and 27 with DBU gave thionucleoside 3',5'-bisphosphate derivatives 9, 20, and 28, which were found to be substrates of T4 RNA ligase. These thionucleoside 3',5'-bisphosphates were examined as donors for ligation with m3(2,2,7) G5'pppAmUmA, i.e., the 5'-terminal tetranucleotide fragment of U1 snRNA, The 4-thiouridine 3',5'-bisphosphate derivative 9 was found to serve as the most active substrate of T4 RNA ligase with a reaction efficiency of 96%.

The genes encoding fructose bisphosphate aldolase in Trypanosoma brucei are interspersed with unrelated genes.

PubMed Central

Vijayasarathy, S; Ernest, I; Itzhaki, J E; Sherman, D; Mowatt, M R; Michels, P A; Clayton, C E

1990-01-01

The fructose bisphosphate aldolase genes of Trypanosoma brucei are interspersed with unrelated genes whose transcript levels show no developmental modulation. Transcription appears approximately constant across the entire locus, suggesting that aldolase mRNA abundance is regulated post-transcriptionally. Images PMID:2349093

Synthesis of bis-Phosphate Iminoaltritol Enantiomers and Structural Characterization with Adenine Phosphoribosyltransferase.

PubMed

Harris, Lawrence D; Harijan, Rajesh K; Ducati, Rodrigo G; Evans, Gary B; Hirsch, Brett M; Schramm, Vern L

2018-01-19

Phosphoribosyl transferases (PRTs) are essential in nucleotide synthesis and salvage, amino acid, and vitamin synthesis. Transition state analysis of several PRTs has demonstrated ribocation-like transition states with a partial positive charge residing on the pentose ring. Core chemistry for synthesis of transition state analogues related to the 5-phospho-α-d-ribosyl 1-pyrophosphate (PRPP) reactant of these enzymes could be developed by stereospecific placement of bis-phosphate groups on an iminoaltritol ring. Cationic character is provided by the imino group and the bis-phosphates anchor both the 1- and 5-phosphate binding sites. We provide a facile synthetic path to these molecules. Cyclic-nitrone redox methodology was applied to the stereocontrolled synthesis of three stereoisomers of a selectively monoprotected diol relevant to the synthesis of transition-state analogue inhibitors. These polyhydroxylated pyrrolidine natural product analogues were bis-phosphorylated to generate analogues of the ribocationic form of 5-phosphoribosyl 1-phosphate. A safe, high yielding synthesis of the key intermediate represents a new route to these transition state mimics. An enantiomeric pair of iminoaltritol bis-phosphates (L-DIAB and D-DIAB) was prepared and shown to display inhibition of Plasmodium falciparum orotate phosphoribosyltransferase and Saccharomyces cerevisiae adenine phosphoribosyltransferase (ScAPRT). Crystallographic inhibitor binding analysis of L- and D-DIAB bound to the catalytic sites of ScAPRT demonstrates accommodation of both enantiomers by altered ring geometry and bis-phosphate catalytic site contacts.

Advantages of estimating parameters of photosynthesis model by fitting A-Ci curves at multiple subsaturating light intensities

NASA Astrophysics Data System (ADS)

Fu, W.; Gu, L.; Hoffman, F. M.

2013-12-01

The photosynthesis model of Farquhar, von Caemmerer & Berry (1980) is an important tool for predicting the response of plants to climate change. So far, the critical parameters required by the model have been obtained from the leaf-level measurements of gas exchange, namely the net assimilation of CO2 against intercellular CO2 concentration (A-Ci) curves, made at saturating light conditions. With such measurements, most points are likely in the Rubisco-limited state for which the model is structurally overparameterized (the model is also overparameterized in the TPU-limited state). In order to reliably estimate photosynthetic parameters, there must be sufficient number of points in the RuBP regeneration-limited state, which has no structural over-parameterization. To improve the accuracy of A-Ci data analysis, we investigate the potential of using multiple A-Ci curves at subsaturating light intensities to generate some important parameter estimates more accurately. Using subsaturating light intensities allow more RuBp regeneration-limited points to be obtained. In this study, simulated examples are used to demonstrate how this method can eliminate the errors of conventional A-Ci curve fitting methods. Some fitted parameters like the photocompensation point and day respiration impose a significant limitation on modeling leaf CO2 exchange. The multiple A-Ci curves fitting can also improve over the so-called Laisk (1977) method, which was shown by some recent publication to produce incorrect estimates of photocompensation point and day respiration. We also test the approach with actual measurements, along with suggested measurement conditions to constrain measured A-Ci points to maximize the occurrence of RuBP regeneration-limited photosynthesis. Finally, we use our measured gas exchange datasets to quantify the magnitude of resistance of chloroplast and cell wall-plasmalemma and explore the effect of variable mesophyll conductance. The variable mesophyll conductance takes into account the influence of CO2 from mitochondria, comparing to the commonly used constant value of mesophyll conductance. We show that after considering this effect the other parameters of the photosynthesis model can be re-estimated. Our results indicate that variable mesophyll conductance has most effect on the estimation of the parameter of the maximum electron transport rate (Jmax), but has a negligible impact on the estimated day respiration (Rd) and photocompensation point (<2%).

Conversion of D-ribulose 5-phosphate to D-xylulose 5-phosphate : new insights from structural and biochemical studies on human RPE.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, W.; Ouyang, S.; Shaw, N.

2011-02-01

The pentose phosphate pathway (PPP) confers protection against oxidative stress by supplying NADPH necessary for the regeneration of glutathione, which detoxifies H{sub 2}O{sub 2} into H{sub 2}O and O{sub 2}. RPE functions in the PPP, catalyzing the reversible conversion of D-ribulose 5-phosphate to D-xylulose 5-phosphate and is an important enzyme for cellular response against oxidative stress. Here, using structural, biochemical, and functional studies, we show that human D-ribulose 5-phosphate 3-epimerase (hRPE) uses Fe{sup 2+} for catalysis. Structures of the binary complexes of hRPE with D-ribulose 5-phosphate and D-xylulose 5-phosphate provide the first detailed molecular insights into the binding mode ofmore » physiological ligands and reveal an octahedrally coordinated Fe{sup 2+} ion buried deep inside the active site. Human RPE folds into a typical ({beta}/{alpha}){sub 8} triosephosphate isomerase (TIM) barrel with a loop regulating access to the active site. Two aspartic acids are well positioned to carry out the proton transfers in an acid-base type of reaction mechanism. Interestingly, mutating Ser-10 to alanine almost abolished the enzymatic activity, while L12A and M72A mutations resulted in an almost 50% decrease in the activity. The binary complexes of hRPE reported here will aid in the design of small molecules for modulating the activity of the enzyme and altering flux through the PPP.« less

Pcal_0111, a highly thermostable bifunctional fructose-1,6-bisphosphate aldolase/phosphatase from Pyrobaculum calidifontis.

PubMed

Aziz, Iram; Rashid, Naeem; Ashraf, Raza; Bashir, Qamar; Imanaka, Tadayuki; Akhtar, Muhammad

2017-05-01

Pyrobaculum calidifontis genome harbors an open reading frame Pcal_0111 annotated as fructose bisphosphate aldolase. Although the gene is annotated as fructose bisphosphate aldolase, it exhibits a high homology with previously reported fructose-1,6-bisphosphate aldolase/phosphatase from Thermoproteus neutrophilus. To examine the biochemical properties of Pcal_0111, we have cloned and expressed the gene in Escherichia coli. Purified recombinant Pcal_0111 catalyzed both phosphatase and aldolase reactions with specific activity values of 4 U and 1.3 U, respectively. These values are highest among the fructose 1,6-bisphosphatases/aldolases characterized from archaea. The enzyme activity increased linearly with the increase in temperature until 100 °C. Recombinant Pcal_0111 is highly stable with a half-life of 120 min at 100 °C. There was no significant change in the circular dichroism spectra of the protein up to 90 °C. The enzyme activity was not affected by AMP but strongly inhibited by ATP with an IC 50 value of 0.75 mM and mildly by ADP. High thermostability and inhibition by ATP make Pcal_0111 a unique fructose 1,6-bisphosphatase/aldolase.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Audigier, S.M.P.; Wang, J.K.T.; Greengard, P.

Synaptosomes, purified from rat cerebral cortex, were prelabeled with (/sup 3/H)inositol to study phosphatidylinositol turnover in nerve terminals. Labeled synaptosomes were either depolarized with 40 mM K/sup +/ or exposed to carbamoylcholine (carbachol). K/sup +/ depolarization increased the level of inositol phosphates in a time-dependent manner. The inositol bisphosphate level also increased rapidly, but its elevated level was sustained during continued depolarization. The elevated level of inositol bisphosphate was reversed upon repolarization of the synaptosomes. The level of inositol monophosphate increased slowly to 120-130% of control. These effects of K/sup +/ depolarization depended on the presence of Ca/sup 2 +/more » in the incubation medium. Carbachol stimulated the turnover of phosphatidylinositol in a dose- and time-dependent manner. The level of inositol bisphosphate increased to 210% of control, and this maximal response was seen from 15 to 60 min. Accumulation of inositol monophosphate was larger than that of inositol bisphosphate, but its time course was slower. Atropine and pirenzepine inhibited the carbachol effect with high affinities. These data show that both Ca/sup 2 +/ influx and M/sub 1/ muscarinic receptor activation stimulate phospholipase C activity in synaptosomes, suggesting that phosphatidylinositol turnover may be involved in regulating neurotransmitter release from nerve terminals.« less

Fructose-bisphosphate aldolase a is a potential metastasis-associated marker of lung squamous cell carcinoma and promotes lung cell tumorigenesis and migration.

PubMed

Du, Sha; Guan, Zhuzhu; Hao, Lihong; Song, Yang; Wang, Lan; Gong, Linlin; Liu, Lu; Qi, Xiaoyu; Hou, Zhaoyuan; Shao, Shujuan

2014-01-01

Fructose-bisphosphate aldolase A (ALDOA) is a key enzyme in glycolysis and is responsible for catalyzing the reversible conversion of fructose-1,6-bisphosphate to glyceraldehydes-3-phosphate and dihydroxyacetone phosphate. ALDOA contributes to various cellular functions such as muscle maintenance, regulation of cell shape and mobility, striated muscle contraction, actin filament organization and ATP biosynthetic process. Here, we reported that ALDOA is a highly expressed in lung squamous cell carcinoma (LSCC) and its expression level is correlated with LSCC metastasis, grades, differentiation status and poor prognosis. Depletion of ALDOA expression in the lung squamous carcinoma NCI-H520 cells reduces the capabilities of cell motility and tumorigenesis. These data suggest that ALDOA could be a potential marker for LSCC metastasis and a therapeutic target for drug development.

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of fructose-1,6-bisphosphate aldolase from Escherichia coli.

PubMed

Zhang, Li; Guo, Zheng; Huang, Jing; Liu, Meiruo; Wang, Yuandong; Ji, Chaoneng

2014-10-01

Fructose-1,6-bisphosphate aldolase is one of the most important enzymes in the glycolytic pathway and catalyzes the reversible cleavage of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The full-length fbaB gene encoding fructose-1,6-bisphosphate aldolase class I (FBPA I) was cloned from Escherichia coli strain BL21. FBPA I was overexpressed in E. coli and purified. Biochemical analysis found that the optimum reaction temperature of FBPA I is 330.5 K and that the enzyme has a high temperature tolerance. Crystals of recombinant FBPA I were obtained by the sitting-drop vapour-diffusion technique in a condition consisting of 19 mg ml(-1) FBPA I in 0.1 M Tris pH 9.0, 10%(w/v) polyethylene glycol 8000 and diffracted to 2.0 Å resolution. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 217.7, b = 114.9, c = 183.9 Å, β = 124.6°. The asymmetric unit of these crystals may contain ten molecules, giving a Matthews coefficient of 2.48 Å(3) Da(-1) and a solvent content of 50.5%.

Large fractions of CO2-fixing microorganisms in pristine limestone aquifers appear to be involved in the oxidation of reduced sulfur and nitrogen compounds

USGS Publications Warehouse

Herrmann, Martina; Rusznyák, Anna; Akob, Denise M.; Schulze, Isabel; Opitz, Sebastian; Totsche, Kai Uwe; Küsel, Kirsten

2015-01-01

The traditional view of the dependency of subsurface environments on surface-derived allochthonous carbon inputs is challenged by increasing evidence for the role of lithoautotrophy in aquifer carbon flow. We linked information on autotrophy (Calvin-Benson-Bassham cycle) with that from total microbial community analysis in groundwater at two superimposed—upper and lower—limestone groundwater reservoirs (aquifers). Quantitative PCR revealed that up to 17% of the microbial population had the genetic potential to fix CO2 via the Calvin cycle, with abundances of cbbM and cbbL genes, encoding RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) forms I and II, ranging from 1.14 × 103 to 6 × 106 genes liter−1 over a 2-year period. The structure of the active microbial communities based on 16S rRNA transcripts differed between the two aquifers, with a larger fraction of heterotrophic, facultative anaerobic, soil-related groups in the oxygen-deficient upper aquifer. Most identified CO2-assimilating phylogenetic groups appeared to be involved in the oxidation of sulfur or nitrogen compounds and harbored both RubisCO forms I and II, allowing efficient CO2 fixation in environments with strong oxygen and CO2 fluctuations. The genera Sulfuricellaand Nitrosomonas were represented by read fractions of up to 78 and 33%, respectively, within the cbbM and cbbL transcript pool and accounted for 5.6 and 3.8% of 16S rRNA sequence reads, respectively, in the lower aquifer. Our results indicate that a large fraction of bacteria in pristine limestone aquifers has the genetic potential for autotrophic CO2 fixation, with energy most likely provided by the oxidation of reduced sulfur and nitrogen compounds.

Highly conserved small subunit residues influence rubisco large subunit catalysis.

PubMed

Genkov, Todor; Spreitzer, Robert J

2009-10-30

The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.

NDH-Mediated Cyclic Electron Flow Around Photosystem I is Crucial for C4 Photosynthesis.

PubMed

Ishikawa, Noriko; Takabayashi, Atsushi; Noguchi, Ko; Tazoe, Youshi; Yamamoto, Hiroshi; von Caemmerer, Susanne; Sato, Fumihiko; Endo, Tsuyoshi

2016-10-01

C 4 photosynthesis exhibits efficient CO 2 assimilation in ambient air by concentrating CO 2 around ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) through a metabolic pathway called the C 4 cycle. It has been suggested that cyclic electron flow (CEF) around PSI mediated by chloroplast NADH dehydrogenase-like complex (NDH), an alternative pathway of photosynthetic electron transport (PET), plays a crucial role in C 4 photosynthesis, although the contribution of NDH-mediated CEF is small in C 3 photosynthesis. Here, we generated NDH-suppressed transformants of a C 4 plant, Flaveria bidentis, and showed that the NDH-suppressed plants grow poorly, especially under low-light conditions. CO 2 assimilation rates were consistently decreased in the NDH-suppressed plants under low and medium light intensities. Measurements of non-photochemical quenching (NPQ) of Chl fluorescence, the oxidation state of the reaction center of PSI (P700) and the electrochromic shift (ECS) of pigment absorbance indicated that proton translocation across the thylakoid membrane is impaired in the NDH-suppressed plants. Since proton translocation across the thylakoid membrane induces ATP production, these results suggest that NDH-mediated CEF plays a role in the supply of ATP which is required for C 4 photosynthesis. Such a role is more crucial when the light that is available for photosynthesis is limited and the energy production by PET becomes rate-determining for C 4 photosynthesis. Our results demonstrate that the physiological contribution of NDH-mediated CEF is greater in C 4 photosynthesis than in C 3 photosynthesis, suggesting that the mechanism of PET in C 4 photosynthesis has changed from that in C 3 photosynthesis accompanying the changes in the mechanism of CO 2 assimilation. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Arabidopsis thaliana ggt1 photorespiratory mutants maintain leaf carbon/nitrogen balance by reducing RuBisCO content and plant growth.

PubMed

Dellero, Younès; Lamothe-Sibold, Marlène; Jossier, Mathieu; Hodges, Michael

2015-09-01

Metabolic and physiological analyses of glutamate:glyoxylate aminotransferase 1 (GGT1) mutants were performed at the global leaf scale to elucidate the mechanisms involved in their photorespiratory growth phenotype. Air-grown ggt1 mutants showed retarded growth and development, that was not observed at high CO2 (3000 μL L(-1) ). When compared to wild-type (WT) plants, air-grown ggt1 plants exhibited glyoxylate accumulation, global changes in amino acid amounts including a decrease in serine content, lower organic acid levels, and modified ATP/ADP and NADP(+) /NADPH ratios. When compared to WT plants, their net CO2 assimilation rates (An ) were 50% lower and this mirrored decreases in ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) contents. High CO2 -grown ggt1 plants transferred to air revealed a rapid decrease of An and photosynthetic electron transfer rate while maintaining a high energetic state. Short-term (a night period and 4 h of light) transferred ggt1 leaves accumulated glyoxylate and exhibited low serine contents, while other amino acid levels were not modified. RuBisCO content, activity and activation state were not altered after a short-term transfer while the ATP/ADP ratio was lowered in ggt1 rosettes. However, plant growth and RuBisCO levels were both reduced in ggt1 leaves after a long-term (12 days) acclimation to air from high CO2 when compared to WT plants. The data are discussed with respect to a reduced photorespiratory carbon recycling in the mutants. It is proposed that the low An limits nitrogen-assimilation, this decreases leaf RuBisCO content until plants attain a new homeostatic state that maintains a constant C/N balance and leads to smaller, slower growing plants. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Soil and water warming accelerates phenology and down-regulation of leaf photosynthesis of rice plants grown under free-air CO2 enrichment (FACE).

PubMed

Adachi, Minaco; Hasegawa, Toshihiro; Fukayama, Hiroshi; Tokida, Takeshi; Sakai, Hidemitsu; Matsunami, Toshinori; Nakamura, Hirofumi; Sameshima, Ryoji; Okada, Masumi

2014-02-01

To enable prediction of future rice production in a changing climate, we need to understand the interactive effects of temperature and elevated [CO2] (E[CO2]). We therefore examined if the effect of E[CO2] on the light-saturated leaf photosynthetic rate (Asat) was affected by soil and water temperature (NT, normal; ET, elevated) under open-field conditions at the rice free-air CO2 enrichment (FACE) facility in Shizukuishi, Japan, in 2007 and 2008. Season-long E[CO2] (+200 µmol mol(-1)) increased Asat by 26%, when averaged over two years, temperature regimes and growth stages. The effect of ET (+2°C) on Asat was not significant at active tillering and heading, but became negative and significant at mid-grain filling; Asat in E[CO2]-ET was higher than in ambient [CO2] (A[CO2])-NT by only 4%. Photosynthetic down-regulation at E[CO2] also became apparent at mid-grain filling; Asat compared at the same [CO2] in the leaf cuvette was significantly lower in plants grown in E[CO2] than in those grown in A[CO2]. The additive effects of E[CO2] and ET decreased Asat by 23% compared with that of A[CO2]-NT plants. Although total crop nitrogen (N) uptake was increased by ET, N allocation to the leaves and to Rubisco was reduced under ET and E[CO2] at mid-grain filling, which resulted in a significant decrease (32%) in the maximum rate of ribulose-1,5-bisphosphate carboxylation on a leaf area basis. Because the change in N allocation was associated with the accelerated phenology in E[CO2]-ET plants, we conclude that soil and water warming accelerates photosynthetic down-regulation at E[CO2].

Appearance and accumulation of C/sub 4/ carbon pathway enzymes in developing maize leaves and differentiating maize A188 callus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aoyagi, K.; Bassham, J.A.

1986-02-01

Regenerating maize A188 tissue cultures were examined for the presence of enzymes involved in C/sub 4/ photosynthesis, for cell morphology, and for /sup 14/C labeling kinetics to study the implementation of this pathway during plant development. For comparison, sections of maize seedling leaves were examined. Protein blot analysis using antibodies to leaf enzymes showed a different profile of these enzymes during the early stages of shoot regeneration from callus from the closely-coordinated profile observed in seedling leaves. Pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1) and phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.31) were found in nonchlorophyllous callus while ribulose 1,5-bisphosphate carboxylase (RuBPC, ECmore » 4.1.1.39) and malic enzyme, NADP-specific (ME-NADP) (EC 1.3.1.37) were not detectable until later. Enzyme activity assays showed the presence of ME-NADP as well as PEPC and PPDK in nonchlorophyllous callus. However, the activities of ME-NADP and PEPC had properties similar to those of the enzymes from C/sub 3/ leaves and from etiolated C/sub 4/ leaf tissues, but differing from the corresponding enzymes in the mature leaf. Immunoprecipitation of in vitro translation products of poly(A)RNA extracted from embryoid-forming callus showed both the 110 kilodalton precursor to chloroplast PPDK and the 94 kilodalton polypeptide. Therefore, the chloroplast tye of PPDK mRNA is present prior to the appearance of leaf morphology. Analysis of the labeled products of /sup 14/CO/sub 2/ fixation by nonchlorophyllous calli indicated ..beta..-carboxylation to give acids of the tricarboxylic acid cycle, but no incorporation into phosphoglycerate. With greening of the callus, some incorporation into phosphoglycerate and sugar phosphates occurred, and this increased in shoots as they developed, although with older shoots the increase in ..beta..-carboxylation products was even greater.« less

The BSD2 ortholog in Chlamydomonas reinhardtii is a polysome-associated chaperone that co-migrates on sucrose gradients with the rbcL transcript encoding the Rubisco large subunit.

PubMed

Doron, Lior; Segal, Na'ama; Gibori, Hadas; Shapira, Michal

2014-10-01

The expression of the CO2 -fixation enzyme ribulose-bisphosphate carboxylase/oxygenase (Rubisco), which is affected by light, involves the cysteine-rich protein bundle-sheath defective-2 (BSD2) that was originally identified in maize bundle-sheath cells. We identified the BSD2 ortholog in Chlamydomonas reinhardtii as a small protein (17 kDa) localized to the chloroplast. The algal BSD2-ortholog contains four CXXCXGXG DnaJ-like elements, but lacks the other conserved domains of DnaJ. BSD2 co-migrated with the rbcL transcript on heavy polysomes, and both BSD2 and rbcL mRNA shifted to the lighter fractions under oxidizing conditions that repress the translation of the Rubisco large subunit (RbcL). This profile of co-migration supports the possibility that BSD2 is required for the de novo synthesis of RbcL. Furthermore, BSD2 co-migrated with the rbcL transcript in a C. reinhardtii premature-termination mutant that encodes the first 60 amino acids of RbcL. In both strains, BSD2 shared its migration profile with the rbcL transcript but not with psbA mRNA. The chaperone activity of BSD2 was exemplified by its ability to prevent the aggregation of both citrate synthase (CS) and RbcL in vitro following their chemical denaturation. This activity did not depend on the presence of the thiol groups on BSD2. In contrast, the activity of BSD2 in preventing the precipitation of reduced β-chains in vitro in the insulin turbidity assay was thiol-dependent. We conclude that BSD2 combines a chaperone 'holdase' function with the ability to interact with free thiols, with both activities being required to protect newly synthesized RbcL chains. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Whole-plant growth and N utilization in transgenic rice plants with increased or decreased Rubisco content under different CO2 partial pressures.

PubMed

Sudo, Emi; Suzuki, Yuji; Makino, Amane

2014-11-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) strongly limits photosynthesis at lower CO2 concentration [CO2] whereas [corrected] Rubisco limitation is cancelled by elevated [CO2]. Therefore, increase or reduction in Rubisco content by transformation with a sense or an antisense RBCS construct are expected to alter the biomass production under different CO2 levels. RBCS-sense (125% Rubisco of wild-type) and -antisense (35% Rubisco of wild-type) rice (Oryza sativa L.) plants were grown for 63 days at three different CO2 levels: low [CO2] (28 Pa), normal [CO2] (40 Pa) and elevated [CO2] (120 Pa). The biomass of RBCS-sense plants was 32% and 15% greater at low [CO2] and normal [CO2] than that of the wild-type plants, respectively, but did not differ at elevated [CO2]. Conversely, the biomass of RBCS-antisense plants was the smallest at low [CO2]. Thus, overproduction of Rubisco was effective for biomass production at low [CO2]. Greater biomass production at low [CO2] in RBCS-sense plants was caused by an increase in the net assimilation rate, and associated with an increase in the amount of N uptake. Furthermore, Rubisco overproduction in RBCS-sense plants was also promoted at low [CO2]. Although it seems that low [CO2]-growth additionally stimulates the effect of RBCS overexpression, such a phenomenon observed at low [CO2] was mediated through an increase in total leaf N content. Thus, the dependence of the growth improvement in RBCS-sense rice on growth [CO2] was closely related to the degree of Rubisco overproduction which was accompanied not only by leaf N content but also by whole plant N content. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Effects of long-term exposure to ammonium sulfate particles on growth and gas exchange rates of Fagus crenata, Castanopsis sieboldii, Larix kaempferi and Cryptomeria japonica seedlings

NASA Astrophysics Data System (ADS)

Yamaguchi, Masahiro; Otani, Yoko; Li, Peiran; Nagao, Hiroshi; Lenggoro, I. Wuled; Ishida, Atsushi; Yazaki, Kenichi; Noguchi, Kyotaro; Nakaba, Satoshi; Yamane, Kenichi; Kuroda, Katsushi; Sano, Yuzou; Funada, Ryo; Izuta, Takeshi

2014-11-01

To clarify the effects of long-term exposure to ammonium sulfate (AS) particles on growth and physiological functions of forest tree species, seedlings of Fagus crenata, Castanopsis sieboldii, Larix kaempferi and Cryptomeria japonica were exposed to submicron-size AS particles during two growing seasons from 3 June 2011 to 8 October 2012. The mean sulfate concentration in PM2.5 increased during the exposure inside the chamber in 2011 and 2012 by 2.73 and 4.32 μg SO42- m-3, respectively. No significant effects of exposure to AS particles were detected on the whole-plant dry mass of the seedlings. These results indicate that the exposure to submicrometer AS particles at the ambient level for two growing seasons did not significantly affect the growth of the seedlings. No significant effects of exposure to AS particles were found on the net photosynthetic rate in the leaves or needles of F. crenata, C. sieboldii and L. kaempferi seedlings. Also, in the previous-year needles of C. japonica seedlings, exposure to AS particles significantly reduced the net photosynthetic rate, which may be caused by the reduction in the concentration of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco). On the contrary, in current-year needles of C. japonica seedlings, net photosynthetic rate significantly increased with exposure to AS particles, which may be the result of increases in stomatal conductance and concentrations of Rubisco and chlorophyll. Furthermore, exposure to AS particles correlated with an increase in concentrations of NH4+, free amino acid and total soluble protein, suggesting that AS particles may be deliquesced, absorbed into the leaves and metabolized into amino acid and protein. These results suggest that net photosynthesis in the needles of C. japonica is relatively sensitive to submicron-size AS particles as compared with the other three tree species.

DOE Office of Scientific and Technical Information (OSTI.GOV)

John, David E.; Wang, Zhaohui A.; Liu, Xuewu

River plumes deliver large quantities of nutrients to oligotrophic oceans, often resulting in significant CO 2 drawdown. To determine the relationship between expression of the major gene in carbon fixation (large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, RuBisCO) and CO 2 dynamics, we evaluated rbcL mRNA abundance using novel quantitative PCR assays, phytoplankton cell analyses, photophysiological parameters, and pCO 2 in and around the Mississippi River plume (MRP) in the Gulf of Mexico. Lower salinity (30–32) stations were dominated by rbcL mRNA concentrations from heterokonts, such as diatoms and pelagophytes, which were at least an order of magnitude greater than haptophytes, alpha-Synechococcusmore » or high-light Prochlorococcus. However, rbcL transcript abundances were similar among these groups at oligotrophic stations (salinity 34–36). Diatom cell counts and heterokont rbcL RNA showed a strong negative correlation to seawater pCO 2. While Prochlorococcus cells did not exhibit a large difference between low and high pCO 2 water, Prochlorococcus rbcL RNA concentrations had a strong positive correlation to pCO 2, suggesting a very low level of RuBisCO RNA transcription among Prochlorococcus in the plume waters, possibly due to their relatively poor carbon concentrating mechanisms (CCMs). These results provide molecular evidence that diatom/pelagophyte productivity is largely responsible for the large CO 2 drawdown occurring in the MRP, based on the co-occurrence of elevated RuBisCO gene transcript concentrations from this group and reduced seawater pCO 2 levels. This may partly be due to efficient CCMs that enable heterokont eukaryotes such as diatoms to continue fixing CO 2 in the face of strong CO 2 drawdown. Finally, our work represents the first attempt to relate in situ microbial gene expression to contemporaneous CO 2 flux measurements in the ocean.« less

Rubisco mutagenesis provides new insight into limitations on photosynthesis and growth in Synechocystis PCC6803

PubMed Central

Marcus, Yehouda; Altman-Gueta, Hagit; Wolff, Yael; Gurevitz, Michael

2011-01-01

Orthophosphate (Pi) stimulates the activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) while paradoxically inhibiting its catalysis. Of three Pi-binding sites, the roles of the 5P- and latch sites have been documented, whereas that of the 1P-site remained unclear. Conserved residues at the 1P-site of Rubisco from the cyanobacterium Synechocystis PCC6803 were substituted and the kinetic properties of the enzyme derivatives and effects on cell photosynthesis and growth were examined. While Pi-stimulated Rubisco activation diminished for enzyme mutants T65A/S and G404A, inhibition of catalysis by Pi remained unchanged. Together with previous studies, the results suggest that all three Pi-binding sites are involved in stimulation of Rubisco activation, whereas only the 5P-site is involved in inhibition of catalysis. While all the mutations reduced the catalytic turnover of Rubisco (Kcat) between 6- and 20-fold, the photosynthesis and growth rates under saturating irradiance and inorganic carbon (Ci) concentrations were only reduced 40–50% (in the T65A/S mutants) or not at all (G404A mutant). Analysis of the mutant cells revealed a 3-fold increase in Rubisco content that partially compensated for the reduced Kcat so that the carboxylation rate per chlorophyll was one-third of that in the wild type. Correlation between the kinetic properties of Rubisco and the photosynthetic rate (Pmax) under saturating irradiance and Ci concentrations indicate that a >60% reduction in Kcat can be tolerated before Pmax in Synechocystsis PCC6803 is affected. These results indicate that the limitation of Rubisco activity on the rate of photosynthesis in Synechocystis is low. Determination of Calvin cycle metabolites revealed that unlike in higher plants, cyanobacterial photosynthesis is constrained by phosphoglycerate reduction probably due to limitation of ATP or NADPH. PMID:21551078

Statistical epistasis between candidate gene alleles for complex tuber traits in an association mapping population of tetraploid potato

PubMed Central

Li, Li; Paulo, Maria-João; van Eeuwijk, Fred

2010-01-01

Association mapping using DNA-based markers is a novel tool in plant genetics for the analysis of complex traits. Potato tuber yield, starch content, starch yield and chip color are complex traits of agronomic relevance, for which carbohydrate metabolism plays an important role. At the functional level, the genes and biochemical pathways involved in carbohydrate metabolism are among the best studied in plants. Quantitative traits such as tuber starch and sugar content are therefore models for association genetics in potato based on candidate genes. In an association mapping experiment conducted with a population of 243 tetraploid potato varieties and breeding clones, we previously identified associations between individual candidate gene alleles and tuber starch content, starch yield and chip quality. In the present paper, we tested 190 DNA markers at 36 loci scored in the same association mapping population for pairwise statistical epistatic interactions. Fifty marker pairs were associated mainly with tuber starch content and/or starch yield, at a cut-off value of q ≤ 0.20 for the experiment-wide false discovery rate (FDR). Thirteen marker pairs had an FDR of q ≤ 0.10. Alleles at loci encoding ribulose-bisphosphate carboxylase/oxygenase activase (Rca), sucrose phosphate synthase (Sps) and vacuolar invertase (Pain1) were most frequently involved in statistical epistatic interactions. The largest effect on tuber starch content and starch yield was observed for the paired alleles Pain1-8c and Rca-1a, explaining 9 and 10% of the total variance, respectively. The combination of these two alleles increased the means of tuber starch content and starch yield. Biological models to explain the observed statistical epistatic interactions are discussed. Electronic supplementary material The online version of this article (doi:10.1007/s00122-010-1389-3) contains supplementary material, which is available to authorized users. PMID:20603706

Novel Insights into the Influence of Seed Sarcotesta Photosynthesis on Accumulation of Seed Dry Matter and Oil Content in Torreya grandis cv. “Merrillii”

PubMed Central

Hu, Yuanyuan; Zhang, Yongling; Yu, Weiwu; Hänninen, Heikki; Song, Lili; Du, Xuhua; Zhang, Rui; Wu, Jiasheng

2018-01-01

Seed oil content is an important trait of nut seeds, and it is affected by the import of carbon from photosynthetic sources. Although green leaves are the main photosynthetic organs, seed sarcotesta photosynthesis also supplies assimilates to seed development. Understanding the relationship between seed photosynthesis and seed development has theoretical and practical significance in the cultivation of Torreya grandis cv. “Merrillii.” To assess the role of seed sarcotesta photosynthesis on the seed development, anatomical and physiological traits of sarcotesta were measured during two growing seasons in the field. Compared with the attached current-year leaves, the sarcotesta had higher gross photosynthetic rate at the first stage of seed development. At the late second stage of seed development, sarcotesta showed down-regulation of PSII activity, as indicated by significant decrease in the following chlorophyll fluorescence parameters: the maximum PSII efficiency (Fv/Fm), the PSII quantum yield (ΦPSII), and the photosynthetic quenching coefficient (qP). The ribulose 1, 5—bisphosphate carboxylase (Rubisco) activity, the total chlorophyll content (Chl(a+b)) and nitrogen content in the sarcotesta were also significantly decreased during that period. Treatment with DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] preventing seed photosynthesis decreased the seed dry weight and the oil content by 25.4 and 25.5%, respectively. We conclude that seed photosynthesis plays an important role in the dry matter accumulation at the first growth stage. Our results also suggest that down-regulation of seed photosynthesis is a plant response to re-balance the source-sink ratio at the second growth stage. These results suggest that seed photosynthesis is important for biomass accumulation and oil synthesis of the Torreya seeds. The results will facilitate achieving higher yields and oil contents in nut trees by selection for higher seed photosynthesis cultivars. PMID:29375592

Polyglycine Acts as a Rejection Signal for Protein Transport at the Chloroplast Envelope.

PubMed

Endow, Joshua K; Rocha, Agostinho Gomes; Baldwin, Amy J; Roston, Rebecca L; Yamaguchi, Toshio; Kamikubo, Hironari; Inoue, Kentaro

2016-01-01

PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins, the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope.

Photosynthesis-related characteristics of the midrib and the interveinal lamina in leaves of the C3–CAM intermediate plant Mesembryanthemum crystallinum

PubMed Central

Kuźniak, Elżbieta; Kornas, Andrzej; Kaźmierczak, Andrzej; Rozpądek, Piotr; Nosek, Michał; Kocurek, Maciej; Zellnig, Günther; Müller, Maria; Miszalski, Zbigniew

2016-01-01

Background and Aims Leaf veins are usually encircled by specialized bundle sheath cells. In C4 plants, they play an important role in CO2 assimilation, and the photosynthetic activity is compartmentalized between the mesophyll and the bundle sheath. In C3 and CAM (Crassulacean acid metabolism) plants, the photosynthetic activity is generally attributed to the leaf mesophyll cells, and the vascular parenchymal cells are rarely considered for their role in photosynthesis. Recent studies demonstrate that enzymes required for C4 photosynthesis are also active in the veins of C3 plants, and their vascular system contains photosynthetically competent parenchyma cells. However, our understanding of photosynthesis in veins of C3 and CAM plants still remains insufficient. Here spatial analysis of photosynthesis-related properties were applied to the midrib and the interveinal lamina cells in leaves of Mesembryanthemum crystallinum, a C3–CAM intermediate plant. Methods The midrib anatomy as well as chloroplast structure and chlorophyll fluorescence, diurnal gas exchange profiles, the immunoblot patterns of PEPC (phosphoenolpyruvate carboxylase) and RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase), H2O2 localization and antioxidant enzyme activities were compared in the midrib and in the interveinal mesophyll cells in leaves of C3 and CAM plants. Key Results Leaf midribs were structurally competent to perform photosynthesis in C3 and CAM plants. The midrib chloroplasts resembled those in the bundle sheath cells of C4 plants and were characterized by limited photosynthetic activity. Conclusions The metabolic roles of midrib chloroplasts differ in C3 and CAM plants. It is suggested that in leaves of C3 plants the midrib chloroplasts could be involved in the supply of CO2 for carboxylation, and in CAM plants they could provide malate to different metabolic processes and mediate H2O2 signalling. PMID:27091507

Microbial communities at the borehole observatory on the Costa Rica Rift flank (Ocean Drilling Program Hole 896A).

PubMed

Nigro, Lisa M; Harris, Kate; Orcutt, Beth N; Hyde, Andrew; Clayton-Luce, Samuel; Becker, Keir; Teske, Andreas

2012-01-01

The microbiology of subsurface, hydrothermally influenced basaltic crust flanking mid-ocean ridges has remained understudied, due to the difficulty in accessing the subsurface environment. The instrumented boreholes resulting from scientific ocean drilling offer access to samples of the formation fluids circulating through oceanic crust. We analyzed the phylogenetic diversity of bacterial communities of fluid and microbial mat samples collected in situ from the observatory at Ocean Drilling Program Hole 896A, drilled into ~6.5 million-year-old basaltic crust on the flank of the Costa Rica Rift in the equatorial Pacific Ocean. Bacterial 16S rRNA gene sequences recovered from borehole fluid and from a microbial mat coating the outer surface of the fluid port revealed both unique and shared phylotypes. The dominant bacterial clones from both samples were related to the autotrophic, sulfur-oxidizing genus Thiomicrospira. Both samples yielded diverse gamma- and alphaproteobacterial phylotypes, as well as members of the Bacteroidetes, Planctomycetes, and Verrucomicrobia. Analysis of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes (cbbL and cbbM) from the sampling port mat and from the borehole fluid demonstrated autotrophic carbon assimilation potential for in situ microbial communities; most cbbL genes were related to those of the sulfur-oxidizing genera Thioalkalivibrio and Thiomicrospira, and cbbM genes were affiliated with uncultured phylotypes from hydrothermal vent plumes and marine sediments. Several 16S rRNA gene phylotypes from the 896A observatory grouped with phylotypes recovered from seawater-exposed basalts and sulfide deposits at inactive hydrothermal vents, but there is little overlap with hydrothermally influenced basaltic boreholes 1026B and U1301A on the Juan de Fuca Ridge flank, suggesting that site-specific characteristics of Hole 896A (i.e., seawater mixing into borehole fluids) affect the microbial community composition.

Land plant biochemistry.

PubMed Central

Raven, J A

2000-01-01

Biochemical studies have complemented ultrastructural and, subsequently molecular genetic evidence consistent with the Charophyceae being the closest extant algal relatives of the embryophytes. Among the genes used in such molecular phylogenetic studies is that rbcL) for the large subunit of ribulose bisphosphate carboxylase-oxygenase (RUBISCO). The RUBISCO of the embryophytes is derived, via the Chlorophyta. from that of the cyanobacteria. This clade of the molecular phylogeny of RUBISCO shows a range of kinetic characteristics, especially of CO2 affinities and of CO2/O2 selectivities. The range of these kinetic values within the bryophytes is no greater than in the rest of the embryophytes; this has implications for the evolution of the embryophytes in the high atmospheric CO2 environment of the late Lower Palaeozoic. The differences in biochemistry between charophycean algae and embryophytes can to some extent be related functionally to the structure and physiology of embryophytes. Examples of components of embryophytes, which are qualitatively or quantitatively different from those of charophytes, are the water repellent/water resistant extracellular lipids, the rigid phenolic polymers functional in water-conducting elements and mechanical support in air, and in UV-B absorption, flavonoid phenolics involved in UV-B absorption and in interactions with other organisms, and the greater emphasis on low Mr organic acids. retained in the plant as free acids or salts, or secreted to the rhizosphere. The roles of these components are discussed in relation to the environmental conditions at the time of evolution of the terrestrial embryophytes. A significant point about embryophytes is the predominance of nitrogen-free extracellular structural material (a trait shared by most algae) and UV-B screening components, by contrast with analogous components in many other organisms. An important question, which has thus far been incompletely addressed, is the extent to which the absence from bryophytes of the biochemical pathways which produce components found only in tracheophytes is the result of evolutionary loss of these functions. PMID:10905612

Adaptation to high temperature mitigates the impact of water deficit during combined heat and drought stress in C3 sunflower and C4 maize varieties with contrasting drought tolerance.

PubMed

Killi, Dilek; Bussotti, Filippo; Raschi, Antonio; Haworth, Matthew

2017-02-01

Heat and drought stress frequently occur together, however, their impact on plant growth and photosynthesis (P N ) is unclear. The frequency, duration and severity of heat and drought stress events are predicted to increase in the future, having severe implications for agricultural productivity and food security. To assess the impact on plant gas exchange, physiology and morphology we grew drought tolerant and sensitive varieties of C3 sunflower (Helianthus annuus) and C4 maize (Zea mays) under conditions of elevated temperature for 4 weeks prior to the imposition of water deficit. The negative impact of temperature on P N was most apparent in sunflower. The drought tolerant sunflower retained ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) activity under heat stress to a greater extent than its drought sensitive counterpart. Maize exhibited no varietal difference in response to increased temperature. In contrast to previous studies, where a sudden rise in temperature induced an increase in stomatal conductance (G s ), we observed no change or a reduction in G s with elevated temperature, which alongside lower leaf area mitigated the impact of drought at the higher temperature. The drought tolerant sunflower and maize varieties exhibited greater investment in root-systems, allowing greater uptake of the available soil water. Elevated temperatures associated with heat-waves will have profound negative impacts on crop growth in both sunflower and maize, but the deleterious effect on P N was less apparent in the drought tolerant sunflower and both maize varieties. As C4 plants generally exhibit water use efficiency (WUE) and resistance to heat stress, selection on the basis of tolerance to heat and drought stress would be more beneficial to the yields of C3 crops cultivated in drought prone semi-arid regions. © 2016 Scandinavian Plant Physiology Society.

Bundle Sheath Diffusive Resistance to CO2 and Effectiveness of C4 Photosynthesis and Refixation of Photorespired CO2 in a C4 Cycle Mutant and Wild-Type Amaranthus edulis1

PubMed Central

Kiirats, Olavi; Lea, Peter J.; Franceschi, Vincent R.; Edwards, Gerald E.

2002-01-01

A mutant of the NAD-malic enzyme-type C4 plant, Amaranthus edulis, which lacks phosphoenolpyruvate carboxylase (PEPC) in the mesophyll cells was studied. Analysis of CO2 response curves of photosynthesis of the mutant, which has normal Kranz anatomy but lacks a functional C4 cycle, provided a direct means of determining the liquid phase-diffusive resistance of atmospheric CO2 to sites of ribulose 1,5-bisphosphate carboxylation inside bundle sheath (BS) chloroplasts (rbs) within intact plants. Comparisons were made with excised shoots of wild-type plants fed 3,3-dichloro-2-(dihydroxyphosphinoyl-methyl)-propenoate, an inhibitor of PEPC. Values of rbs in A. edulis were 70 to 180 m2 s−1 mol−1, increasing as the leaf matured. This is about 70-fold higher than the liquid phase resistance for diffusion of CO2 to Rubisco in mesophyll cells of C3 plants. The values of rbs in A. edulis are sufficient for C4 photosynthesis to elevate CO2 in BS cells and to minimize photorespiration. The calculated CO2 concentration in BS cells, which is dependent on input of rbs, was about 2,000 μbar under maximum rates of CO2 fixation, which is about six times the ambient level of CO2. High re-assimilation of photorespired CO2 was demonstrated in both mutant and wild-type plants at limiting CO2 concentrations, which can be explained by high rbs. Increasing O2 from near zero up to ambient levels under low CO2, resulted in an increase in the gross rate of O2 evolution measured by chlorophyll fluorescence analysis in the PEPC mutant; this increase was simulated from a Rubisco kinetic model, which indicates effective refixation of photorespired CO2 in BS cells. PMID:12376660

Trends in leaf photosynthesis in historical rice varieties developed in the Philippines since 1966.

PubMed

Hubbart, S; Peng, S; Horton, P; Chen, Y; Murchie, E H

2007-01-01

Crop improvement in terms of yield is rarely linked to leaf photosynthesis. However, in certain crop plants such as rice, it is predicted that an increase in photosynthetic rate will be required to support future grain yield potential. In order to understand the relationships between yield improvement and leaf photosynthesis, controlled environment conditions were used to grow 10 varieties which were released from the International Rice Research Institute (IRRI) between 1966 and 1995 and one newly developed line. Two growth light intensities were used: high light (1500 micromol m(-2) s(-1)) and low light (300 micromol m(-2) s(-1)). Gas exchange, leaf protein, chlorophyll, and leaf morphology were measured in the ninth leaf on the main stem. A high level of variation was observed among high light-grown plants for light-saturated photosynthetic rate per unit leaf area (P(max)), stomatal conductance (g), content of ribulose bisphosphate carboxylase-oxygenase (Rubisco), and total leaf protein content. Notably, between 1966 and 1980 there was a decline in P(max), g, leaf protein, chlorophyll, and Rubisco content. Values recovered in those varieties released after 1980. This striking trend coincides with a previous published observation that grain yield in IRRI varieties released prior to 1980 correlated with harvest index whereas that for those released after 1980 correlated with biomass. P(max) showed significant correlations with both g and Rubisco content. Large differences were observed between high light- and low light-grown plants (photoacclimation). The photoacclimation 'range' for P(max) correlated with P(max) in high light-grown plants. It is concluded that (i) leaf photosynthesis may be systematically affected by breeding strategy; (ii) P(max) is a useful target for yield improvements where yield is limited by biomass production rather than partitioning; and (iii) the capacity for photoacclimation is related to high P(max) values.

Polyglycine Acts as a Rejection Signal for Protein Transport at the Chloroplast Envelope

DOE PAGES

Endow, Joshua K.; Rocha, Agostinho Gomes; Baldwin, Amy J.; ...

2016-12-09

PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here in this study we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins,more » the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope.« less

Reduced Osmotic Potential Inhibition of Photosynthesis 1

PubMed Central

Berkowitz, Gerald A.; Gibbs, Martin

1983-01-01

The effects of reduced reaction medium osmotic potential (0.67 molar sorbitol as compared to a control treatment with 0.33 molar sorbitol) on the enzymic steps of the photosynthetic carbon reduction cycle were investigated using isolated spinach (Spinacia oleracea L. var Longstanding Bloomsdale) chloroplasts. Reversal of reduced osmotic potential inhibition of photosynthetic rates by a stromal alkalating agent (NH4Cl) was associated with specific steps of the cycle. Low osmotic potential induced stromal acidification was found to be facilitated by osmotically induced chloroplast shrinkage. However, the action of the alkalating agent was found not to be associated with reversal of osmotically induced morphological changes of the stromal compartment. Labeled metabolite analyses indicated that the osmotic stress treatment caused the substrate for fructose 1,6-bisphosphatase (FBPase) to build up in the absence of NH4Cl, and the substrate for phosphoribulokinase to increase in the presence of NH4Cl. These data were interpreted as indicating that the most severe effect of osmotic stress on photosynthesis is at the site of FBPase, and that this inhibition is mediated by osmotically induced stromal acidification. Phosphoribulokinase activity inhibition at the low osmotic potential treatment was apparently less severe and not mediated by stromal acidification. A third site of osmotic inhibition, which was reversed by NH4Cl, and therefore was assumed to be mediated by stromal acidification, was at the step of ribulose 1,5-bisphosphate carboxylase. Additions of NH4Cl also enhanced the activity of the pH-insensitive phase of the photosynthetic carbon reduction cycle, 3-phosphoglyceric acid reduction, at the stress treatment. This effect was thought to be mediated by the removal of the block at FBPase. A model was proposed to outline the relative severity of osmotic stress effects at various sites of the photosynthetic carbon reduction cycle. Images Fig. 1 PMID:16663127

LCE: leaf carbon exchange data set for tropical, temperate, and boreal species of North and Central America.

PubMed

Smith, Nicholas G; Dukes, Jeffrey S

2017-11-01

Leaf canopy carbon exchange processes, such as photosynthesis and respiration, are substantial components of the global carbon cycle. Climate models base their simulations of photosynthesis and respiration on an empirical understanding of the underlying biochemical processes, and the responses of those processes to environmental drivers. As such, data spanning large spatial scales are needed to evaluate and parameterize these models. Here, we present data on four important biochemical parameters defining leaf carbon exchange processes from 626 individuals of 98 species at 12 North and Central American sites spanning ~53° of latitude. The four parameters are the maximum rate of Rubisco carboxylation (V cmax ), the maximum rate of electron transport for the regeneration of Ribulose-1,5,-bisphosphate (J max ), the maximum rate of phosphoenolpyruvate carboxylase carboxylation (V pmax ), and leaf dark respiration (R d ). The raw net photosynthesis by intercellular CO 2 (A/C i ) data used to calculate V cmax , J max , and V pmax rates are also presented. Data were gathered on the same leaf of each individual (one leaf per individual), allowing for the examination of each parameter relative to others. Additionally, the data set contains a number of covariates for the plants measured. Covariate data include (1) leaf-level traits (leaf mass, leaf area, leaf nitrogen and carbon content, predawn leaf water potential), (2) plant-level traits (plant height for herbaceous individuals and diameter at breast height for trees), (3) soil moisture at the time of measurement, (4) air temperature from nearby weather stations for the day of measurement and each of the 90 d prior to measurement, and (5) climate data (growing season mean temperature, precipitation, photosynthetically active radiation, vapor pressure deficit, and aridity index). We hope that the data will be useful for obtaining greater understanding of the abiotic and biotic determinants of these important biochemical parameters and for evaluating and improving large-scale models of leaf carbon exchange. © 2017 by the Ecological Society of America.

Abiotic and biotic determinants of leaf carbon exchange capacity from tropical to high boreal biomes

NASA Astrophysics Data System (ADS)

Smith, N. G.; Dukes, J. S.

2016-12-01

Photosynthesis and respiration on land represent the two largest fluxes of carbon dioxide between the atmosphere and the Earth's surface. As such, the Earth System Models that are used to project climate change are high sensitive to these processes. Studies have found that much of this uncertainty is due to the formulation and parameterization of plant photosynthetic and respiratory capacity. Here, we quantified the abiotic and biotic factors that determine photosynthetic and respiratory capacity at large spatial scales. Specifically, we measured the maximum rate of Rubisco carboxylation (Vcmax), the maximum rate of Ribulose-1,5-bisphosphate regeneration (Jmax), and leaf dark respiration (Rd) in >600 individuals of 98 plant species from the tropical to high boreal biomes of Northern and Central America. We also measured a bevy of covariates including plant functional type, leaf nitrogen content, short- and long-term climate, leaf water potential, plant size, and leaf mass per area. We found that plant functional type and leaf nitrogen content were the primary determinants of Vcmax, Jmax, and Rd. Mean annual temperature and mean annual precipitation were not significant predictors of these rates. However, short-term climatic variables, specifically soil moisture and air temperature over the previous 25 days, were significant predictors and indicated that heat and soil moisture deficits combine to reduce photosynthetic capacity and increase respiratory capacity. Finally, these data were used as a model benchmarking tool for the Community Land Model version 4.5 (CLM 4.5). The benchmarking analyses determined errors in the leaf nitrogen allocation scheme of CLM 4.5. Under high leaf nitrogen levels within a plant type the model overestimated Vcmax and Jmax. This result suggested that plants were altering their nitrogen allocation patterns when leaf nitrogen levels were high, an effect that was not being captured by the model. These data, taken with models in mind, provide paths forward for improving model structure and parameterization of leaf carbon exchange at large spatial scales.

Paclobutrazol affects the resistance of black spruce to high light and thermal stress.

PubMed

Mahoney, Sean R.; Ghosh, Sibdas; Peirson, David; Dumbroff, Erwin B.

1998-02-01

Detached needles from 20-week-old black spruce (Picea mariana (Mill.) B.S.P.) seedlings root-drenched with 60 mg of paclobutrazol were exposed to two temperatures (22 and 50 degrees C) and two light treatments (100 and 1900 &mgr;mol m(-2) s(-1) PAR) in a factorial combination for 4 h in vitro. Mean dry weights of individual needles from paclobutrazol-treated plants were approximately 1.9 times heavier than that of needles from untreated controls at 22 degrees C, but no differences were observed following incubation at 50 degrees C. Numbers of cells per needle remained constant in all treatments. Chlorophyll and carotenoid contents per needle were higher in seedlings treated with paclobutrazol than in untreated control seedlings, and the differences were most pronounced in the high temperature plus high light treatment. In low light at 50 degrees C, quantum efficiency of photosystem II was 45% higher in needles of paclobutrazol-treated seedlings than in needles of untreated control seedlings, but quantum efficiency of needles from treated seedlings declined when needles were exposed to high light at either temperature. Peroxidase and superoxide dismutase activities were up-regulated by paclobutrazol, whereas catalase activities were depressed and no significant differences were observed between treated and control needles at 50 degrees C in either light treatment. Paclobutrazol treatment did not moderate the depressive effects of high temperature on total soluble protein or on the activity of ribulose-1,5-bisphosphate carboxylase. In contrast, high activities of phosphoenolpyruvate carboxylase were maintained in paclobutrazol-treated needles under all stress conditions, whereas large losses in activity were recorded in untreated needles at 50 degrees C. Collectively, these observations suggest that paclobutrazol treatment may convey resistance to excessive light and high temperatures by increasing the potential of conifers to limit damage caused by oxidative stress.

Photosynthesis-related characteristics of the midrib and the interveinal lamina in leaves of the C3-CAM intermediate plant Mesembryanthemum crystallinum.

PubMed

Kuźniak, Elżbieta; Kornas, Andrzej; Kaźmierczak, Andrzej; Rozpądek, Piotr; Nosek, Michał; Kocurek, Maciej; Zellnig, Günther; Müller, Maria; Miszalski, Zbigniew

2016-06-01

Leaf veins are usually encircled by specialized bundle sheath cells. In C4 plants, they play an important role in CO2 assimilation, and the photosynthetic activity is compartmentalized between the mesophyll and the bundle sheath. In C3 and CAM (Crassulacean acid metabolism) plants, the photosynthetic activity is generally attributed to the leaf mesophyll cells, and the vascular parenchymal cells are rarely considered for their role in photosynthesis. Recent studies demonstrate that enzymes required for C4 photosynthesis are also active in the veins of C3 plants, and their vascular system contains photosynthetically competent parenchyma cells. However, our understanding of photosynthesis in veins of C3 and CAM plants still remains insufficient. Here spatial analysis of photosynthesis-related properties were applied to the midrib and the interveinal lamina cells in leaves of Mesembryanthemum crystallinum, a C3-CAM intermediate plant. The midrib anatomy as well as chloroplast structure and chlorophyll fluorescence, diurnal gas exchange profiles, the immunoblot patterns of PEPC (phosphoenolpyruvate carboxylase) and RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase), H2O2 localization and antioxidant enzyme activities were compared in the midrib and in the interveinal mesophyll cells in leaves of C3 and CAM plants. Leaf midribs were structurally competent to perform photosynthesis in C3 and CAM plants. The midrib chloroplasts resembled those in the bundle sheath cells of C4 plants and were characterized by limited photosynthetic activity. The metabolic roles of midrib chloroplasts differ in C3 and CAM plants. It is suggested that in leaves of C3 plants the midrib chloroplasts could be involved in the supply of CO2 for carboxylation, and in CAM plants they could provide malate to different metabolic processes and mediate H2O2 signalling. © The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Effect of CO2-induced seawater acidification on growth, photosynthesis and inorganic carbon acquisition of the harmful bloom-forming marine microalga, Karenia mikimotoi.

PubMed

Hu, Shunxin; Zhou, Bin; Wang, You; Wang, Ying; Zhang, Xinxin; Zhao, Yan; Zhao, Xinyu; Tang, Xuexi

2017-01-01

Karenia mikimotoi is a widespread, toxic and non-calcifying dinoflagellate, which can release and produce ichthyotoxins and hemolytic toxins affecting the food web within the area of its bloom. Shifts in the physiological characteristics of K. mikimotoi due to CO2-induced seawater acidification could alter the occurrence, severity and impacts of harmful algal blooms (HABs). Here, we investigated the effects of elevated pCO2 on the physiology of K. mikimotoi. Using semi-continuous cultures under controlled laboratory conditions, growth, photosynthesis and inorganic carbon acquisition were determined over 4-6 week incubations at ambient (390ppmv) and elevated pCO2 levels (1000 ppmv and 2000 ppmv). pH-drift and inhibitor-experiments suggested that K. mikimotoi was capable of acquiring HCO3-, and that the utilization of HCO3- was predominantly mediated by anion-exchange proteins, but that HCO3- dehydration catalyzed by external carbonic anhydrase (CAext) only played a minor role in K. mikimotoi. Even though down-regulated CO2 concentrating mechanisms (CCMs) and enhanced gross photosynthetic O2 evolution were observed under 1000 ppmv CO2 conditions, the saved energy did not stimulate growth of K. mikimotoi under 1000 ppmv CO2, probably due to the increased dark respiration. However, significantly higher growth and photosynthesis [in terms of photosynthetic oxygen evolution, effective quantum Yield (Yield), photosynthetic efficiency (α), light saturation point (Ek) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity] were observed under 2000 ppmv CO2 conditions. Furthermore, elevated pCO2 increased the photo-inhibition rate of photosystem II (β) and non-photochemical quenching (NPQ) at high light. We suggest that the energy saved through the down-regulation of CCMs might lead to the additional light stress and photo-damage. Therefore, the response of this species to elevated CO2 conditions will be determined by more than regulation and efficiency of CCMs.

Significant Role for Microbial Autotrophy in the Sequestration of Soil Carbon

PubMed Central

Yuan, Hongzhao; Ge, Tida; Chen, Caiyan; O'Donnell, Anthony G.

2012-01-01

Soils were incubated for 80 days in a continuously labeled 14CO2 atmosphere to measure the amount of labeled C incorporated into the microbial biomass. Microbial assimilation of 14C differed between soils and accounted for 0.12% to 0.59% of soil organic carbon (SOC). Assuming a terrestrial area of 1.4 × 108 km2, this represents a potential global sequestration of 0.6 to 4.9 Pg C year−1. Estimated global C sequestration rates suggest a “missing sink” for carbon of between 2 and 3 Pg C year−1. To determine whether 14CO2 incorporation was mediated by autotrophic microorganisms, the diversity and abundance of CO2-fixing bacteria and algae were investigated using clone library sequencing, terminal restriction fragment length polymorphism (T-RFLP), and quantitative PCR (qPCR) of the ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) gene (cbbL). Phylogenetic analysis showed that the dominant cbbL-containing bacteria were Azospirillum lipoferum, Rhodopseudomonas palustris, Bradyrhizobium japonicum, Ralstonia eutropha, and cbbL-containing chromophytic algae of the genera Xanthophyta and Bacillariophyta. Multivariate analyses of T-RFLP profiles revealed significant differences in cbbL-containing microbial communities between soils. Differences in cbbL gene diversity were shown to be correlated with differences in SOC content. Bacterial and algal cbbL gene abundances were between 106 and 108 and 103 to 105 copies g−1 soil, respectively. Bacterial cbbL abundance was shown to be positively correlated with RubisCO activity (r = 0.853; P < 0.05), and both cbbL abundance and RubisCO activity were significantly related to the synthesis rates of [14C]SOC (r = 0.967 and 0.946, respectively; P < 0.01). These data offer new insights into the importance of microbial autotrophy in terrestrial C cycling. PMID:22286999

D-ribulose-5-phosphate 3-epimerase: Cloning and heterologous expression of the spinach gene, and purification and characterization of the recombinant enzyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Y.R.; Hartman, F.C.; Lu, T.Y.S.

The authors have achieved, to their knowledge, the first high-level heterologous expression of the gene encoding D-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by DL-{alpha}-glycerophosphate or ethanol and destabilized by D-ribulose-5-phosphate or 2-mercaptoethanol. Despite this lability, the unprecedentedly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation. Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis. As deducedmore » from the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric.« less

Fructose-Bisphosphate Aldolase A Is a Potential Metastasis-Associated Marker of Lung Squamous Cell Carcinoma and Promotes Lung Cell Tumorigenesis and Migration

PubMed Central

Hao, Lihong; Song, Yang; Wang, Lan; Gong, Linlin; Liu, Lu; Qi, Xiaoyu; Hou, Zhaoyuan; Shao, Shujuan

2014-01-01

Fructose-bisphosphate aldolase A (ALDOA) is a key enzyme in glycolysis and is responsible for catalyzing the reversible conversion of fructose-1,6-bisphosphate to glyceraldehydes-3-phosphate and dihydroxyacetone phosphate. ALDOA contributes to various cellular functions such as muscle maintenance, regulation of cell shape and mobility, striated muscle contraction, actin filament organization and ATP biosynthetic process. Here, we reported that ALDOA is a highly expressed in lung squamous cell carcinoma (LSCC) and its expression level is correlated with LSCC metastasis, grades, differentiation status and poor prognosis. Depletion of ALDOA expression in the lung squamous carcinoma NCI-H520 cells reduces the capabilities of cell motility and tumorigenesis. These data suggest that ALDOA could be a potential marker for LSCC metastasis and a therapeutic target for drug development. PMID:24465716

The Path of Carbon in Photosynthesis XXI. The Cyclic Regeneration of Carbon Dioxide Acceptor

DOE R&D Accomplishments Database

Bassham, J. A.; Benson, A. A.; Kay, Lorel D.; Harris, Anne Z.; Wilson, A. T.; Calvin, M.

1953-10-01

Photosynthesizing plants have been exposed to C{sup 14}O{sub 2} for short periods of time (0.4 to 15 sec.) and the products of carbon dioxide reduction analyzed by paper chromatography and radio autography. Methods have been developed for the degradation of ribulose and sedoheptulose. These sugars, obtained as their phosphate esters from the above C{sup 14}O{sub 2} exposures and from other experiments, have been degraded and their distribution of radiocarbon determined. The distribution of radiocarbon in these sugars, and other data, indicate that sedoheptulose phosphate and ribulose diphosphates are formed during photosynthesis from triose and hexose phosphates, the latter being synthesized, in turn, by the reduction of 3-phosphoglyceric acid.

Glucose Induces Protein Targeting to Glycogen in Hepatocytes by Fructose 2,6-Bisphosphate-Mediated Recruitment of MondoA to the Promoter

PubMed Central

Petrie, John L.; Al-Oanzi, Ziad H.; Arden, Catherine; Tudhope, Susan J.; Mann, Jelena; Kieswich, Julius; Yaqoob, Muhammad M.; Towle, Howard C.

2013-01-01

In the liver, a high glucose concentration activates transcription of genes encoding glucose 6-phosphatase and enzymes for glycolysis and lipogenesis by elevation in phosphorylated intermediates and recruitment of the transcription factor ChREBP (carbohydrate response element binding protein) and its partner, Mlx, to gene promoters. A proposed function for this mechanism is intracellular phosphate homeostasis. In extrahepatic tissues, MondoA, the paralog of ChREBP, partners with Mlx in transcriptional induction by glucose. We tested for glucose induction of regulatory proteins of the glycogenic pathway in hepatocytes and identified the glycogen-targeting proteins, GL and PTG (protein targeting to glycogen), as being encoded by Mlx-dependent glucose-inducible genes. PTG induction by glucose was MondoA dependent but ChREBP independent and was enhanced by forced elevation of fructose 2,6-bisphosphate and by additional xylitol-derived metabolites. It was counteracted by selective depletion of fructose 2,6-bisphosphate with a bisphosphatase-active kinase-deficient variant of phosphofructokinase 2/fructosebisphosphatase 2, which prevented translocation of MondoA to the nucleus and recruitment to the PTG promoter. We identify a novel role for MondoA in the liver and demonstrate that elevated fructose 2,6-bisphosphate is essential for recruitment of MondoA to the PTG promoter. Phosphometabolite activation of MondoA and ChREBP and their recruitment to target genes is consistent with a mechanism for gene regulation to maintain intracellular phosphate homeostasis. PMID:23207906

Ion-exchange chromatography separates activities synthesizing and degrading fructose 2,6-bisphosphate from C3 and C4 leaves but not from rat liver

NASA Technical Reports Server (NTRS)

Macdonald, F. D.; Chou, Q.; Buchanan, B. B.

1987-01-01

Fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase were separated on the basis of charge from leaves of C3 (spinach, lettuce, and pea) and C4 (sorghum and amaranthus) plants but not from rat liver--a tissue known to contain a bifunctional enzyme with both activities. [2-32P]Fructose 2,6-bisphosphate binding experiments also suggest that the major forms of these activities reside on different proteins in leaves.

Import of fructose bisphosphate aldolase into the glycosomes of Trypanosoma brucei

PubMed Central

1987-01-01

The glycolytic enzymes of Trypanosomatids are compartmentalized within peroxisome-like microbodies called glycosomes. Fructose bisphosphate aldolase is synthesized on free polysomes and imported into glycosomes within 5 min. Peptide mapping reveals no primary structural differences between the in vivo-synthesized protein and that made in vitro from a synthetic template. However, native aldolase from glycosomes is partially protease resistant, whereas the in vitro translation product is not. Pulse-chase results indicate that aldolase in bloodstream trypanosomes has a much longer half-life than in the procyclic tsetse fly form. PMID:3320052

Reaction of the isosteric methylenephosphonate analog of alpha-D-glucose 1-phosphate with phosphoglucomutase. Induced-fit specificity revisited.

PubMed

Ray, W J; Post, C B; Puvathingal, J M

1993-01-12

The phospho form of phosphoglucomutase reacts with the isosteric methylenephosphonate analog of alpha-D-glucose 1-phosphate to produce the corresponding analog of alpha-D-glucose 1,6-bisphosphate plus the dephosphoenzyme. In a coupled reaction, kcat/Km = 1.7 x 10(3) M-1 s-1, which is about 2 x 10(-5) times that for the corresponding reaction with alpha-D-glucose 1-phosphate. The decrease in kcat/Km is divided more or less evenly between less efficient PO3- transfer and decreased binding, although smaller phosphates and phosphonates bind approximately equally. There is a much smaller difference in the binding of glucose 1-methylenephosphonate 6-phosphate and glucose 1,6-bisphosphate to the dephosphoenzyme: the binding ratio is < 1:35 when the glucose ring is oriented similarly. Preferred binding patterns for a number of substrates/inhibitors, studied by 31P NMR and UV-difference spectroscopy, suggest that in the ground state the phosphonate group is tolerated to a much greater extent at the catalytic subsite than at the phosphate-binding subsite, where binding specificity appears to be directed toward a tetrahedral-PO3(2-) group attached to a bridging atom that can act as a hydrogen-bond acceptor. Binding specificity at the catalytic subsite apparently is directed toward a different array, possibly (-O...PO3...O-)2-. Some of these results are considered in terms of a modified version of the "induced fit" concept of enzymic specificity, which is reexamined in view of implied thermodynamic restrictions. The internal rearrangement whereby the positions of the anionic groups of the phosphate/phosphonate are exchanged is compared with the analogous rearrangements involving glucose 1,6-bisphosphate and 1,4-butanediol bisphosphate. The supplementary material describes a three-step synthesis of 1-deoxy-alpha-D-glucose 1-methylenephosphonate together with a procedure for phosphorylating the phosphonate to produce an analog of alpha-D-glucose 1,6-bisphosphate and also describes a facile procedure for the qualitative conversion of organic phosphonates to inorganic phosphate.

Purification, properties and immunological relationship of L (+)-lactate dehydrogenase from Lactobacillus casei.

PubMed

Gordon, G L; Doelle, H W

1976-08-16

The fructose-1,6-bisphosphate-activated L-lactate dehydrogenase (EC 1.1.1.27) from Lactobacillus casei ATCC 393 has been purified to homogenity by including affinity chromatography (cibacronblue-Sephadex-G-200) and preparative polyacrylamide gel electrophoresis into the purification procedures. The enzyme has an Mr of 132000-135000 with a subunit Mr of 34000. The pH optimum was found to be 5.4 insodium acetate buffer. Tris/maleate and citrate/phosphate buffers inhibited enzyme activity at this pH. The enzyme was completely inactivated by a temperature increase from 60 degrees C to 70 degrees C. Pyruvate saturation curves were sigmoidal in the absence of fructose 1,6-bisphosphate. In the presence of 20 muM fructose 1,6-bisphosphate a Km of 1.0 mM for pyruvate was obtained, whereas fructose 1,6-bisphosphate had no effect on the Km of 0.01 mM for NADH. The use of pyruvate analogues revealed two types of pyruvate binding sites, a catalytic and an effector site. The enzyme from L. casei appears to be subject to strict metabolic control, since ADP, ATP, dihydroxyacetone phosphate and 6-phosphogluconate are strong inhibitors. Immunodiffusion experiments with a rabbit antiserum to L. casei lactate dehydrogenase revealed that L. casei ATCC 393 L (+)-lactate dehydrogenase is probably not immunologically related to group D and group N streptococci. Of 24 lactic acid bacterial strains tested only 5 strains did cross-react: L. casei ATCC 393 = L. casei var. rhamnosus ATCC 7469 - L. casei var. alactosus NCDO 680 greater than L. casei UQM 95 greater than L. plantarum ATCC 14917.

ARF1·GTP, Tyrosine-based Signals, and Phosphatidylinositol 4,5-Bisphosphate Constitute a Minimal Machinery to Recruit the AP-1 Clathrin Adaptor to Membranes

PubMed Central

Crottet, Pascal; Meyer, Daniel M.; Rohrer, Jack; Spiess, Martin

2002-01-01

At the trans-Golgi network, clathrin coats containing AP-1 adaptor complexes are formed in an ARF1-dependent manner, generating vesicles transporting cargo proteins to endosomes. The mechanism of site-specific targeting of AP-1 and the role of cargo are poorly understood. We have developed an in vitro assay to study the recruitment of purified AP-1 adaptors to chemically defined liposomes presenting peptides corresponding to tyrosine-based sorting motifs. AP-1 recruitment was found to be dependent on myristoylated ARF1, GTP or nonhydrolyzable GTP-analogs, tyrosine signals, and small amounts of phosphoinositides, most prominently phosphatidylinositol 4,5-bisphosphate, in the absence of any additional cytosolic or membrane bound proteins. AP-1 from cytosol could be recruited to a tyrosine signal independently of the lipid composition, but the rate of recruitment was increased by phosphatidylinositol 4,5-bisphosphate. The results thus indicate that cargo proteins are involved in coat recruitment and that the local lipid composition contributes to specifying the site of vesicle formation. PMID:12388765

Purification, crystallization and preliminary X-ray analysis of native and selenomethionine class I tagatose-1,6-bisphosphate aldolase from Streptococcus pyogenes.

PubMed

Liotard, Brigitte; Sygusch, Jurgen

2004-03-01

Tagatose-1,6-bisphosphate aldolase (EC 4.1.2.40) is situated at the branching of the tagatose-6-phosphate and Embden-Meyerhof-Parnas (glycolysis) metabolic pathways, where it catalyzes the reversible cleavage of tagatose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The recombinant protein from Streptococcus pyogenes was overexpressed in Escherichia coli in its native and selenomethionine-derivative forms and purified using ion-exchange and hydrophobic interaction chromatography. Orthorhombic crystals suitable for structural analysis were obtained by the hanging-drop vapour-diffusion method for both isoforms. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 63.7, b = 108.1, c = 238.7 A for the native form and a = 64.1, b = 108.3, c = 239.8 A for the selenomethionine derivative. The asymmetric unit contains four protomers, corresponding to a crystal volume per protein weight (V(M)) of 2.8 A(3) Da(-1) and a solvent content of 56% by volume.

Nonenzymatic gluconeogenesis-like formation of fructose 1,6-bisphosphate in ice.

PubMed

Messner, Christoph B; Driscoll, Paul C; Piedrafita, Gabriel; De Volder, Michael F L; Ralser, Markus

2017-07-11

The evolutionary origins of metabolism, in particular the emergence of the sugar phosphates that constitute glycolysis, the pentose phosphate pathway, and the RNA and DNA backbone, are largely unknown. In cells, a major source of glucose and the large sugar phosphates is gluconeogenesis. This ancient anabolic pathway (re-)builds carbon bonds as cleaved in glycolysis in an aldol condensation of the unstable catabolites glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, forming the much more stable fructose 1,6-bisphosphate. We here report the discovery of a nonenzymatic counterpart to this reaction. The in-ice nonenzymatic aldol addition leads to the continuous accumulation of fructose 1,6-bisphosphate in a permanently frozen solution as followed over months. Moreover, the in-ice reaction is accelerated by simple amino acids, in particular glycine and lysine. Revealing that gluconeogenesis may be of nonenzymatic origin, our results shed light on how glucose anabolism could have emerged in early life forms. Furthermore, the amino acid acceleration of a key cellular anabolic reaction may indicate a link between prebiotic chemistry and the nature of the first metabolic enzymes.

Photorespiration.

ERIC Educational Resources Information Center

Rao, K. K.; Hall, D. O.

1982-01-01

Topics in this discussion of photorespiration (light-dependent oxygen consumption and carbon dioxide evolution from leaves) include: (1) the biochemistry of photorespiration; (2) ribulose biphosphate carboxylase and glycollate synthesis; (3) metabolism of glycollate; (4) plants lacking photorespiratory systems; and (5) advantages of…

The pentose phosphate pathway of glucose metabolism. Hormonal and dietary control of the oxidative and non-oxidative reactions of the cycle in liver

PubMed Central

Novello, F.; Gumaa, J. A.; McLean, Patricia

1969-01-01

1. Measurements were made of the non-oxidative reactions of the pentose phosphate cycle in liver (transketolase, transaldolase, ribulose 5-phosphate epimerase and ribose 5-phosphate isomerase activities) in a variety of hormonal and nutritional conditions. In addition, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were measured for comparison with the oxidative reactions of the cycle; hexokinase, glucokinase and phosphoglucose isomerase activities were also included. Starvation for 2 days caused significant lowering of activity of all the enzymes of the pentose phosphate cycle based on activity in the whole liver. Re-feeding with a high-carbohydrate diet restored all the enzyme activities to the range of the control values with the exception of that of glucose 6-phosphate dehydrogenase, which showed the well-known `overshoot' effect. Re-feeding with a high-fat diet also restored the activities of all the enzymes of the pentose phosphate cycle and of hexokinase; glucokinase activity alone remained unchanged. Expressed as units/g. of liver or units/mg. of protein hexokinase, glucose 6-phosphate dehydrogenase, transketolase and pentose phosphate isomerase activities were unchanged by starvation; both 6-phosphogluconate dehydrogenase and ribulose 5-phosphate epimerase activities decreased faster than the liver weight or protein content. 2. Alloxan-diabetes resulted in a decrease of approx. 30–40% in the activities of 6-phosphogluconate dehydrogenase, ribose 5-phosphate isomerase, ribulose 5-phosphate epimerase and transketolase; in contrast with this glucose 6-phosphate dehydrogenase, transaldolase and phosphoglucose isomerase activities were unchanged. Treatment of alloxan-diabetic rats with protamine–zinc–insulin for 3 days caused a very marked increase to above normal levels of activity in all the enzymes of the pentose phosphate pathway except ribulose 5-phosphate epimerase, which was restored to the control value. Hexokinase activity was also raised by this treatment. After 7 days treatment of alloxan-diabetic rats with protamine–zinc–insulin the enzyme activities returned towards the control values. 3. In adrenalectomized rats the two most important changes were the rise in hexokinase activity and the fall in transketolase activity; in addition, ribulose 5-phosphate epimerase activity was also decreased. These effects were reversed by cortisone treatment. In addition, in cortisone-treated adrenalectomized rats glucokinase activity was significantly lower than the control value. 4. In thyroidectomized rats both ribose 5-phosphate isomerase and transketolase activities were decreased; in contrast with this transaldolase activity did not change significantly. Hypophysectomy caused a 50% fall in transketolase activity that was partially reversed by treatment with thyroxine and almost fully reversed by treatment with growth hormone for 8 days. 5. The results are discussed in relation to the hormonal control of the non-oxidative reactions of the pentose phosphate cycle, the marked changes in transketolase activity being particularly outstanding. PMID:5791534

Method for producing redox shuttles

DOEpatents

Pupek, Krzysztof Z.; Dzwiniel, Trevor L.; Krumdick, Gregory K.

2015-03-03

A single step method for producing a redox shuttle having the formula 2,5-di-tert-butyl-1,4-phenylene tetraethyl bis(phosphate) is provided, the method comprising phosphorylating tert butyl hydroquinone with a phosphate-containing reagent. Also provided is method for producing 2,5-di-tert-butyl-1,4-phenylene tetraethyl bis(phosphate), the method comprising solubilizing tert-butyl hydroquinone and tetrabutylammonium bromide with methyltetrahydrofuran to create a mixture; heating the mixture while adding base to the mixture in an amount to turn the mixture orange; and adding diethyl chlorophosphate to the orange mixture in an amount to phosphorylate the hydroquinone.

Fructose-1,6-bisphosphate aldolase (FBA)-a conserved glycolytic enzyme with virulence functions in bacteria: 'ill met by moonlight'.

PubMed

Shams, Fariza; Oldfield, Neil J; Wooldridge, Karl G; Turner, David P J

2014-12-01

Moonlighting proteins constitute an intriguing class of multifunctional proteins. Metabolic enzymes and chaperones, which are often highly conserved proteins in bacteria, archaea and eukaryotic organisms, are among the most commonly recognized examples of moonlighting proteins. Fructose-1,6-bisphosphate aldolase (FBA) is an enzyme involved in the Embden-Meyerhof-Parnas (EMP) glycolytic pathway and in gluconeogenesis. Increasingly, it is also recognized that FBA has additional functions beyond its housekeeping role in central metabolism. In the present review, we summarize the current knowledge of the moonlighting functions of FBA in bacteria.

Improving formaldehyde consumption drives methanol assimilation in engineered E. coli.

PubMed

Woolston, Benjamin M; King, Jason R; Reiter, Michael; Van Hove, Bob; Stephanopoulos, Gregory

2018-06-19

Due to volatile sugar prices, the food vs fuel debate, and recent increases in the supply of natural gas, methanol has emerged as a promising feedstock for the bio-based economy. However, attempts to engineer Escherichia coli to metabolize methanol have achieved limited success. Here, we provide a rigorous systematic analysis of several potential pathway bottlenecks. We show that regeneration of ribulose 5-phosphate in E. coli is insufficient to sustain methanol assimilation, and overcome this by activating the sedoheptulose bisphosphatase variant of the ribulose monophosphate pathway. By leveraging the kinetic isotope effect associated with deuterated methanol as a chemical probe, we further demonstrate that under these conditions overall pathway flux is kinetically limited by methanol dehydrogenase. Finally, we identify NADH as a potent kinetic inhibitor of this enzyme. These results provide direction for future engineering strategies to improve methanol utilization, and underscore the value of chemical biology methodologies in metabolic engineering.

Analysis of methylated patterns and quality-related genes in tobacco (Nicotiana tabacum) cultivars.

PubMed

Jiao, Junna; Jia, Yanlong; Lv, Zhuangwei; Sun, Chuanfei; Gao, Lijie; Yan, Xiaoxiao; Cui, Liusu; Tang, Zongxiang; Yan, Benju

2014-08-01

Methylation-sensitive amplified polymorphism was used in this study to investigate epigenetic information of four tobacco cultivars: Yunyan 85, NC89, K326, and Yunyan 87. The DNA fragments with methylated information were cloned by reamplified PCR and sequenced. The results of Blast alignments showed that the genes with methylation information included chitinase, nitrate reductase, chloroplast DNA, mitochondrial DNA, ornithine decarboxylase, ribulose carboxylase, and promoter sequences. Homologous comparison in three cloned gene sequences (nitrate reductase, ornithine decarboxylase, and ribulose decarboxylase) indicated that geographic factors had significant influence on the whole genome methylation. Introns also contained different information in different tobacco cultivars. These findings suggest that synthetic mechanisms for tobacco aromatic components could be affected by different environmental factors leading to variation of noncoding regions in the genome, which finally results in different fragrance and taste in different tobacco cultivars.

Two different Bacillus thuringiensis toxin genes confer resistance to beet armyworm (Spodoptera exigua Hübner) in transgenic Bt-shallots (Allium cepa L.).

PubMed

Zheng, Si-Jun; Henken, Betty; de Maagd, Ruud A; Purwito, Agus; Krens, Frans A; Kik, Chris

2005-06-01

Agrobacterium-mediated genetic transformation was applied to produce beet armyworm (Spodoptera exigua Hübner) resistant tropical shallots (Allium cepa L. group Aggregatum). A cry1Ca or a H04 hybrid gene from Bacillus thuringiensis, driven by the chrysanthemum ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (Rubisco SSU) promoter, along with the hygromycin phosphotransferase gene (hpt) driven by the CaMV 35S promoter, was employed for genetic transformation. An average transformation frequency of 3.68% was obtained from two shallot cultivars, Tropix and Kuning. After transfer of the in vitro plants to the greenhouse 69% of the cry1Ca and 39% of the H04 transgenic shallots survived the first half year. After one year of cultivation in the greenhouse the remaining cry1Ca and H04 transgenic plants grew vigorously and had a normal bulb formation, although the cry1Ca transgenic plants (and controls) had darker green leaves compared to their H04 counterparts. Standard PCR, adaptor ligation PCR and Southern analyses confirmed the integration of T-DNA into the shallot genome. Northern blot and ELISA analyses revealed expression of the cry1Ca or H04 gene in the transgenic plants. The amount of Cry1Ca expressed in transgenic plants was higher than the expression levels of H04 (0.39 vs. 0.16% of the total soluble leaf proteins, respectively). There was a good correlation between protein expression and beet armyworm resistance. Cry1Ca or H04 gene expression of at least 0.22 or 0.08% of the total soluble protein in shallot leaves was sufficient to give a complete resistance against beet armyworm. This confirms earlier observations that the H04 toxin is more toxic to S. exigua than the Cry1Ca toxin. The results from this study suggest that the cry1Ca and H04 transgenic shallots developed could be used for introducing resistance to beet armyworm in (sub) tropical shallot.

Growth response of Douglas-fir seedlings to nitrogen fertilization: importance of Rubisco activation state and respiration rates.

PubMed

Manter, Daniel K; Kavanagh, Kathleen L; Rose, Cathy L

2005-08-01

High foliar nitrogen concentration ([N]) is associated with high rates of photosynthesis and thus high tree productivity; however, at excessive [N], tree productivity is reduced. Reports of excessive [N] in the Douglas-fir forests of the Oregon Coast Range prompted this investigation of growth and needle physiological responses to increasing foliar N concentrations in 1-year-old Douglas-fir seedlings. After 1 year of N fertilization, total seedling biomass increased with each successive increase in N fertilizer concentration, except in the highest N fertilization treatment. Of the many physiological responses that were analyzed, only photosynthetic capacity (i.e., Vcmax), respiration rates and leaf specific conductance (KL) differed significantly between N treatments. Photosynthetic capacity showed a curvilinear relationship with foliar [N], reaching an apparent maximum rate when needle N concentrations exceeded about 12 mg g(-1). In vitro measurements of ribulose-1,5-bisphosphate carboxylase (Rubisco) activity suggested that photosynthetic capacity was best related to activated, not total, Rubisco content. Rubisco activation state declined as foliar [N] increased, and based on its significant correlation (r2= 0.63) with foliar Mn:Mg ratios, it may be related to Mn inactivation of Rubisco. Respiration rates increased linearly as foliar N concentration increased (r2= 0.84). The value of K(L) also increased as foliar [N] increased, reaching a maximum when foliar [N] exceeded about 10 mg g(-1). Changes in K(L) were unrelated to changes in leaf area or sapwood area because leaf area to sapwood area ratios remained constant. Cumulative effects of the observed physiological responses to N fertilization were analyzed by modeling annual net CO2 assimilation (Anet) based on treatment specific values of Vcmax, dark respiration (Rdark) and KL. Estimates of Anet were highly correlated with measured total seedling biomass (r2= 0.992), suggesting that long-term, cumulative effects of maximum Rubisco carboxylation, Rdark and KL responses to N fertilization may limit seedling production when foliar N exceeds about 13 mg g(-1) or is reduced to less than about 11 mg g(-1).

Identification and characterization of a carboxysomal γ-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120.

PubMed

de Araujo, Charlotte; Arefeen, Dewan; Tadesse, Yohannes; Long, Benedict M; Price, G Dean; Rowlett, Roger S; Kimber, Matthew S; Espie, George S

2014-09-01

Carboxysomes are proteinaceous microcompartments that encapsulate carbonic anhydrase (CA) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco); carboxysomes, therefore, catalyze reversible HCO3 (-) dehydration and the subsequent fixation of CO2. The N- and C-terminal domains of the β-carboxysome scaffold protein CcmM participate in a network of protein-protein interactions that are essential for carboxysome biogenesis, organization, and function. The N-terminal domain of CcmM in the thermophile Thermosynechococcus elongatus BP-1 is also a catalytically active, redox regulated γ-CA. To experimentally determine if CcmM from a mesophilic cyanobacterium is active, we cloned, expressed and purified recombinant, full-length CcmM from Nostoc sp. PCC 7120 as well as the N-terminal 209 amino acid γ-CA-like domain. Both recombinant proteins displayed ethoxyzolamide-sensitive CA activity in mass spectrometric assays, as did the carboxysome-enriched TP fraction. NstCcmM209 was characterized as a moderately active and efficient γ-CA with a k cat of 2.0 × 10(4) s(-1) and k cat/K m of 4.1 × 10(6) M(-1) s(-1) at 25 °C and pH 8, a pH optimum between 8 and 9.5 and a temperature optimum spanning 25-35 °C. NstCcmM209 also catalyzed the hydrolysis of the CO2 analog carbonyl sulfide. Circular dichroism and intrinsic tryptophan fluorescence analysis demonstrated that NstCcmM209 was progressively and irreversibly denatured above 50 °C. NstCcmM209 activity was inhibited by the reducing agent tris(hydroxymethyl)phosphine, an effect that was fully reversed by a molar excess of diamide, a thiol oxidizing agent, consistent with oxidative activation being a universal regulatory mechanism of CcmM orthologs. Immunogold electron microscopy and Western blot analysis of TP pellets indicated that Rubisco and CcmM co-localize and are concentrated in Nostoc sp. PCC 7120 carboxysomes.

Assessing the Likelihood of Gene Flow From Sugarcane (Saccharum Hybrids) to Wild Relatives in South Africa.

PubMed

Snyman, Sandy J; Komape, Dennis M; Khanyi, Hlobisile; van den Berg, Johnnie; Cilliers, Dirk; Lloyd Evans, Dyfed; Barnard, Sandra; Siebert, Stefan J

2018-01-01

Pre-commercialization studies on environmental biosafety of genetically modified (GM) crops are necessary to evaluate the potential for sexual hybridization with related plant species that occur in the release area. The aim of the study was a preliminary assessment of factors that may contribute to gene flow from sugarcane ( Saccharum hybrids) to indigenous relatives in the sugarcane production regions of Mpumalanga and KwaZulu-Natal provinces, South Africa. In the first instance, an assessment of Saccharum wild relatives was conducted based on existing phylogenies and literature surveys. The prevalence, spatial overlap, proximity, distribution potential, and flowering times of wild relatives in sugarcane production regions based on the above, and on herbaria records and field surveys were conducted for Imperata, Sorghum, Cleistachne , and Miscanthidium species. Eleven species were selected for spatial analyses based on their presence within the sugarcane cultivation region: four species in the Saccharinae and seven in the Sorghinae. Secondly, fragments of the nuclear internal transcribed spacer (ITS) regions of the 5.8s ribosomal gene and two chloroplast genes, ribulose-bisphosphate carboxylase ( rbcL ), and maturase K ( matK ) were sequenced or assembled from short read data to confirm relatedness between Saccharum hybrids and its wild relatives. Phylogenetic analyses of the ITS cassette showed that the closest wild relative species to commercial sugarcane were Miscanthidium capense, Miscanthidium junceum , and Narenga porphyrocoma. Sorghum was found to be more distantly related to Saccharum than previously described. Based on the phylogeny described in our study, the only species to highlight in terms of evolutionary divergence times from Saccharum are those within the genus Miscanthidium , most especially M. capense , and M. junceum which are only 3 million years divergent from Saccharum . Field assessment of pollen viability of 13 commercial sugarcane cultivars using two stains, iodine potassium iodide (IKI) and triphenyl tetrazolium chloride, showed decreasing pollen viability (from 85 to 0%) from the north to the south eastern regions of the study area. Future work will include other aspects influencing gene flow such as cytological compatibility and introgression between sugarcane and Miscanthidium species.

RBCS1A and RBCS3B, two major members within the Arabidopsis RBCS multigene family, function to yield sufficient Rubisco content for leaf photosynthetic capacity

PubMed Central

Tsunoda, Honami; Suzuki, Yuji; Makino, Amane; Ishida, Hiroyuki

2012-01-01

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (RBCS) is encoded by a nuclear RBCS multigene family in many plant species. The contribution of the RBCS multigenes to accumulation of Rubisco holoenzyme and photosynthetic characteristics remains unclear. T-DNA insertion mutants of RBCS1A (rbcs1a-1) and RBCS3B (rbcs3b-1) were isolated among the four Arabidopsis RBCS genes, and a double mutant (rbcs1a3b-1) was generated. RBCS1A mRNA was not detected in rbcs1a-1 and rbcs1a3b-1, while the RBCS3B mRNA level was suppressed to ∼20% of the wild-type level in rbcs3b-1 and rbcs1a3b-1 leaves. As a result, total RBCS mRNA levels declined to 52, 79, and 23% of the wild-type level in rbcs1a-1, rbcs3b-1, and rbcs1a3b-1, respectively. Rubisco contents showed declines similar to total RBCS mRNA levels, and the ratio of Rubisco-nitrogen to total nitrogen was 62, 78, and 40% of the wild-type level in rbcs1a-1, rbcs3b-1, and rbcs1a3b-1, respectively. The effects of RBCS1A and RBCS3B mutations in rbcs1a3b-1 were clearly additive. The rates of CO2 assimilation at ambient CO2 of 40 Pa were reduced with decreased Rubisco contents in the respective mutant leaves. Although the RBCS composition in the Rubisco holoenzyme changed, the CO2 assimilation rates per unit of Rubisco content were the same irrespective of the genotype. These results clearly indicate that RBCS1A and RBCS3B contribute to accumulation of Rubisco in Arabidopsis leaves and that these genes work additively to yield sufficient Rubisco for photosynthetic capacity. It is also suggested that the RBCS composition in the Rubisco holoenzyme does not affect photosynthesis under the present ambient [CO2] conditions. PMID:22223809

Accumulation and processing of a recombinant protein designed as a cleavable fusion to the endogenous Rubisco LSU protein in Chlamydomonas chloroplast

PubMed Central

Muto, Machiko; Henry, Ryan E; Mayfield, Stephen P

2009-01-01

Background Expression of recombinant proteins in green algal chloroplast holds substantial promise as a platform for the production of human therapeutic proteins. A number of proteins have been expressed in the chloroplast of Chlamydomonas reinhardtii, including complex mammalian proteins, but many of these proteins accumulate to significantly lower levels than do endogenous chloroplast proteins. We examined if recombinant protein accumulation could be enhanced by genetically fusing the recombinant reporter protein, luciferase, to the carboxy-terminal end of an abundant endogenous protein, the large subunit of ribulose bisphosphate carboxylase (Rubisco LSU). Additionally, as recombinant proteins fused to endogenous proteins are of little clinical or commercial value, we explored the possibility of engineering our recombinant protein to be cleavable from the endogenous protein in vivo. This strategy would obviate the need for further in vitro processing steps in order to produce the desired recombinant protein. To achieve this, a native protein-processing site from preferredoxin (preFd) was placed between the Rubisco LSU and luciferase coding regions in the fusion protein construct. Results The luciferase from the fusion protein accumulated to significantly higher levels than luciferase expressed alone. By eliminating the endogenous Rubisco large subunit gene (rbcL), we achieved a further increase in luciferase accumulation with respect to luciferase expression in the WT background. Importantly, near-wild type levels of functional Rubisco holoenzyme were generated following the proteolytic removal of the fused luciferase, while luciferase activity for the fusion protein was almost ~33 times greater than luciferase expressed alone. These data demonstrate the utility of using fusion proteins to enhance recombinant protein accumulation in algal chloroplasts, and also show that engineered proteolytic processing sites can be used to liberate the exogenous protein from the endogenous fusion partner, allowing for the purification of the intended mature protein. Conclusion These results demonstrate the utility of fusion proteins in algal chloroplast as a method to increase accumulation of recombinant proteins that are difficult to express. Since Rubisco is ubiquitous to land plants and green algae, this strategy may also be applied to higher plant transgenic expression systems. PMID:19323825

RBCS1A and RBCS3B, two major members within the Arabidopsis RBCS multigene family, function to yield sufficient Rubisco content for leaf photosynthetic capacity.

PubMed

Izumi, Masanori; Tsunoda, Honami; Suzuki, Yuji; Makino, Amane; Ishida, Hiroyuki

2012-03-01

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (RBCS) is encoded by a nuclear RBCS multigene family in many plant species. The contribution of the RBCS multigenes to accumulation of Rubisco holoenzyme and photosynthetic characteristics remains unclear. T-DNA insertion mutants of RBCS1A (rbcs1a-1) and RBCS3B (rbcs3b-1) were isolated among the four Arabidopsis RBCS genes, and a double mutant (rbcs1a3b-1) was generated. RBCS1A mRNA was not detected in rbcs1a-1 and rbcs1a3b-1, while the RBCS3B mRNA level was suppressed to ∼20% of the wild-type level in rbcs3b-1 and rbcs1a3b-1 leaves. As a result, total RBCS mRNA levels declined to 52, 79, and 23% of the wild-type level in rbcs1a-1, rbcs3b-1, and rbcs1a3b-1, respectively. Rubisco contents showed declines similar to total RBCS mRNA levels, and the ratio of Rubisco-nitrogen to total nitrogen was 62, 78, and 40% of the wild-type level in rbcs1a-1, rbcs3b-1, and rbcs1a3b-1, respectively. The effects of RBCS1A and RBCS3B mutations in rbcs1a3b-1 were clearly additive. The rates of CO(2) assimilation at ambient CO(2) of 40 Pa were reduced with decreased Rubisco contents in the respective mutant leaves. Although the RBCS composition in the Rubisco holoenzyme changed, the CO(2) assimilation rates per unit of Rubisco content were the same irrespective of the genotype. These results clearly indicate that RBCS1A and RBCS3B contribute to accumulation of Rubisco in Arabidopsis leaves and that these genes work additively to yield sufficient Rubisco for photosynthetic capacity. It is also suggested that the RBCS composition in the Rubisco holoenzyme does not affect photosynthesis under the present ambient [CO(2)] conditions.

Preservation and detection of microstructural and taxonomic correlations in the carbon isotopic compositions of individual Precambrian microfossils

NASA Astrophysics Data System (ADS)

Williford, Kenneth H.; Ushikubo, Takayuki; Schopf, J. William; Lepot, Kevin; Kitajima, Kouki; Valley, John W.

2013-03-01

Here we present techniques for, and new data from, in situ carbon isotope (δ13C) analysis of Precambrian permineralized microscopic fossils with a reproducibility of 1-2‰ using secondary ion mass spectrometry (SIMS). Individual microfossils, selected for their excellent preservation, were analyzed in petrographic thin sections of stromatolitic cherts from the Proterozoic Gunflint (˜1880 Ma), Bitter Springs (˜830 Ma), Min'yar (˜740 Ma), and Chichkan (˜775 Ma) Formations. The range of δ13C values (-34.6‰ to -22.1‰ VPDB) among the 46 individuals analyzed falls within that expected for photoautotrophic carbon fixation by ribulose bisphosphate carboxylase (RuBisCO), consistent with morphology-based taxonomic assignments for these specimens. Microfossils classified as cyanobacteria from the Gunflint, Bitter Springs, and Min'yar Formations (for which published carbonate carbon isotope data can be used to estimate the δ13C of the original dissolved inorganic carbon substrate) exhibit a consistent ˜19‰ total fractionation (δ13C of dissolved inorganic carbon - δ13C of biomass) similar to that observed in living cyanobacteria, over a wide range of δ13Ccarb values (-2.9‰ to 3.4‰). In stromatolitic chert of the Min'yar Formation, morphologically diverse microfossils preserved in a ˜1 mm2 part of a microbial mat exhibit systematic isotopic differences among and within taxa that correlate with their morphologically inferred biological affinities and suggest that isotopic signatures of their original biosynthetic processes (e.g., lipid and peptidoglycan synthesis) are preserved. Isotopic offsets consistent with the different RuBisCO-based fractionations typical of cyanobacteria and photosynthetic eukaryotes are documented by the differing δ13C values of a colonial cyanobacterium (-22.6 ± 0.5‰) and a phytoplanktonic protistan acritarch (-28.9 ± 1.0‰) situated <1 cm apart in the stromatolitic Chichkan chert. These findings show for the first time the possibility of using in situ isotopic microanalysis of fossil microbial mats and ancient sediments in order to distinguish metabolic fingerprints within complex microbial ecosystems and consortia.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Joshi, Gauri S; Romagnoli, Simona; Verberkmoes, Nathan C

Rhodopseudomonas palustris is unique among characterized nonsulfur purple bacteria because of its capacity for anaerobic photoheterotrophic growth using aromatic acids. Like growth with other reduced electron donors, this growth typically requires the presence of bicarbonate/CO{sub 2} or some other added electron acceptor in the growth medium. Proteomic studies indicated that there was specific accumulation of form I ribulose 1, 5-bisphosphate carboxylase/oxygenase (RubisCO) subunit proteins (CbbL and CbbS), as well as the CbbX protein, in cells grown on benzoate without added bicarbonate; such cells used the small amounts of dissolved CO{sub 2} in the medium to support growth. These proteins weremore » not observed in extracts from cells grown in the presence of high levels (10 mM) of added bicarbonate. To confirm the results of the proteomics studies, it was shown that the total RubisCO activity levels were significantly higher (five- to sevenfold higher) in wild-type (CGA010) cells grown on benzoate with a low level (0.5 mM) of added bicarbonate. Immunoblots indicated that the increase in RubisCO activity levels was due to a specific increase in the amount of form I RubisCO (CbbLS) and not in the amount of form II RubisCO (CbbM), which was constitutively expressed. Deletion of the main transcriptional regulator gene, cbbR, resulted in impaired growth on benzoate-containing low-bicarbonate media, and it was established that form I RubisCO synthesis was absolutely and specifically dependent on CbbR. To understand the regulatory role of the CbbRRS two-component system, strains with nonpolar deletions of the cbbRRS genes were grown on benzoate. Distinct from the results obtained with photoautotrophic growth conditions, the results of studies with various CbbRRS mutant strains indicated that this two-component system did not affect the observed enhanced synthesis of form I RubisCO under benzoate growth conditions. These studies indicate that diverse growth conditions differentially affect the ability of the CbbRRS two-component system to influence cbb transcription.« less

Directions for Optimization of Photosynthetic Carbon Fixation: RuBisCO's Efficiency May Not Be So Constrained After All.

PubMed

Cummins, Peter L; Kannappan, Babu; Gready, Jill E

2018-01-01

The ubiquitous enzyme Ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) fixes atmospheric carbon dioxide within the Calvin-Benson cycle that is utilized by most photosynthetic organisms. Despite this central role, RuBisCO's efficiency surprisingly struggles, with both a very slow turnover rate to products and also impaired substrate specificity, features that have long been an enigma as it would be assumed that its efficiency was under strong evolutionary pressure. RuBisCO's substrate specificity is compromised as it catalyzes a side-fixation reaction with atmospheric oxygen; empirical kinetic results show a trend to tradeoff between relative specificity and low catalytic turnover rate. Although the dominant hypothesis has been that the active-site chemistry constrains the enzyme's evolution, a more recent study on RuBisCO stability and adaptability has implicated competing selection pressures. Elucidating these constraints is crucial for directing future research on improving photosynthesis, as the current literature casts doubt on the potential effectiveness of site-directed mutagenesis to improve RuBisCO's efficiency. Here we use regression analysis to quantify the relationships between kinetic parameters obtained from empirical data sets spanning a wide evolutionary range of RuBisCOs. Most significantly we found that the rate constant for dissociation of CO 2 from the enzyme complex was much higher than previous estimates and comparable with the corresponding catalytic rate constant. Observed trends between relative specificity and turnover rate can be expressed as the product of negative and positive correlation factors. This provides an explanation in simple kinetic terms of both the natural variation of relative specificity as well as that obtained by reported site-directed mutagenesis results. We demonstrate that the kinetic behaviour shows a lesser rather than more constrained RuBisCO, consistent with growing empirical evidence of higher variability in relative specificity. In summary our analysis supports an explanation for the origin of the tradeoff between specificity and turnover as due to competition between protein stability and activity, rather than constraints between rate constants imposed by the underlying chemistry. Our analysis suggests that simultaneous improvement in both specificity and turnover rate of RuBisCO is possible.

R-Syst::diatom: an open-access and curated barcode database for diatoms and freshwater monitoring.

PubMed

Rimet, Frédéric; Chaumeil, Philippe; Keck, François; Kermarrec, Lenaïg; Vasselon, Valentin; Kahlert, Maria; Franc, Alain; Bouchez, Agnès

2016-01-01

Diatoms are micro-algal indicators of freshwater pollution. Current standardized methodologies are based on microscopic determinations, which is time consuming and prone to identification uncertainties. The use of DNA-barcoding has been proposed as a way to avoid these flaws. Combining barcoding with next-generation sequencing enables collection of a large quantity of barcodes from natural samples. These barcodes are identified as certain diatom taxa by comparing the sequences to a reference barcoding library using algorithms. Proof of concept was recently demonstrated for synthetic and natural communities and underlined the importance of the quality of this reference library. We present an open-access and curated reference barcoding database for diatoms, called R-Syst::diatom, developed in the framework of R-Syst, the network of systematic supported by INRA (French National Institute for Agricultural Research), see http://www.rsyst.inra.fr/en. R-Syst::diatom links DNA-barcodes to their taxonomical identifications, and is dedicated to identify barcodes from natural samples. The data come from two sources, a culture collection of freshwater algae maintained in INRA in which new strains are regularly deposited and barcoded and from the NCBI (National Center for Biotechnology Information) nucleotide database. Two kinds of barcodes were chosen to support the database: 18S (18S ribosomal RNA) and rbcL (Ribulose-1,5-bisphosphate carboxylase/oxygenase), because of their efficiency. Data are curated using innovative (Declic) and classical bioinformatic tools (Blast, classical phylogenies) and up-to-date taxonomy (Catalogues and peer reviewed papers). Every 6 months R-Syst::diatom is updated. The database is available through the R-Syst microalgae website (http://www.rsyst.inra.fr/) and a platform dedicated to next-generation sequencing data analysis, virtual_BiodiversityL@b (https://galaxy-pgtp.pierroton.inra.fr/). We present here the content of the library regarding the number of barcodes and diatom taxa. In addition to these information, morphological features (e.g. biovolumes, chloroplasts…), life-forms (mobility, colony-type) or ecological features (taxa preferenda to pollution) are indicated in R-Syst::diatom. Database URL: http://www.rsyst.inra.fr/. © The Author(s) 2016. Published by Oxford University Press.

Mechanisms of carbon dioxide acquisition and CO2 sensing in marine diatoms: a gateway to carbon metabolism.

PubMed

Matsuda, Yusuke; Hopkinson, Brian M; Nakajima, Kensuke; Dupont, Christopher L; Tsuji, Yoshinori

2017-09-05

Diatoms are one of the most successful marine eukaryotic algal groups, responsible for up to 20% of the annual global CO 2 fixation. The evolution of a CO 2 -concentrating mechanism (CCM) allowed diatoms to overcome a number of serious constraints on photosynthesis in the marine environment, particularly low [CO 2 ] aq in seawater relative to concentrations required by the CO 2 fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), which is partly due to the slow diffusion rate of CO 2 in water and a limited CO 2 formation rate from [Formula: see text] in seawater. Diatoms use two alternative strategies to take up dissolved inorganic carbon (DIC) from the environment: one primarily relies on the direct uptake of [Formula: see text] through plasma-membrane type solute carrier (SLC) 4 family [Formula: see text] transporters and the other is more reliant on passive diffusion of CO 2 formed by an external carbonic anhydrase (CA). Bicarbonate taken up into the cytoplasm is most likely then actively transported into the chloroplast stroma by SLC4-type transporters on the chloroplast membrane system. Bicarbonate in the stroma is converted into CO 2 only in close proximity to RubisCO preventing unnecessary CO 2 leakage. CAs play significant roles in mobilizing DIC as it is progressively moved towards the site of fixation. However, the evolutionary types and subcellular locations of CAs are not conserved between different diatoms, strongly suggesting that this DIC mobilization strategy likely evolved multiple times with different origins. By contrast, the recent discovery of the thylakoid luminal θ-CA indicates that the strategy to supply CO 2 to RubisCO in the pyrenoid may be very similar to that of green algae, and strongly suggests convergent coevolution in CCM function of the thylakoid lumen not only among diatoms but among eukaryotic algae in general. In this review, both experimental and corresponding theoretical models of the diatom CCMs are discussed.This article is part of the themed issue 'The peculiar carbon metabolism in diatoms'. © 2017 The Author(s).

Photosynthesis light-independent reactions are sensitive biomarkers to monitor lead phytotoxicity in a Pb-tolerant Pisum sativum cultivar.

PubMed

Rodriguez, Eleazar; da Conceição Santos, Maria; Azevedo, Raquel; Correia, Carlos; Moutinho-Pereira, José; Ferreira de Oliveira, José Miguel Pimenta; Dias, Maria Celeste

2015-01-01

Lead (Pb) environmental contamination remains prevalent. Pisum sativum L. plants have been used in ecotoxicological studies, but some cultivars showed to tolerate and accumulate some levels of Pb, opening new perspectives to their use in phytoremediation approaches. However, the putative use of pea plants in phytoremediation requires reliable toxicity endpoints. Here, we evaluated the sensitivity of a large number of photosynthesis-related biomarkers in Pb-exposed pea plants. Plants (cv. "Corne de Bélier") were exposed to Pb concentrations up to 1,000 mg kg(-1) soil during 28 days. The photosynthetic potential biomarkers that were analyzed included pigments, chlorophyll (Chl) a fluorescence, gas exchange, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) activity, and carbohydrates. Flow cytometry (FCM) was also used to assess the morpho-functional status of chloroplasts. Finally, Pb-induced nutrient disorders were also evaluated. Net CO2 assimilation rate (A) and RuBisCO activity decreased strongly in Pb-exposed plants. Plant dry mass (DM) accumulation, however, was only reduced in the higher Pb concentrations tested (500 and 1,000 mg kg(-1) soil). Pigment contents increased solely in plants exposed to the largest Pb concentration, and in addition, the parameters related to the light-dependent reactions of photosynthesis, Fv/Fm and ΦPSII, were not affected by Pb exposure. In contrast to this, carbohydrates showed an overall tendency to increase in Pb-exposed plants. The morphological status of chloroplasts was affected by Pb exposure, with a general trend of volume decrease and granularity increase. These results point the endpoints related to the light-independent reactions of photosynthesis as more sensitive predictors of Pb-toxicity than the light-dependent reactions ones. Among the endpoints related to the light-independent photosynthesis reactions, RuBisCO activity and A were found to be the most sensitive. We discuss here the advantages of using these parameters as biomarkers for Pb toxicity in plants. Finally, we report that, despite showing physiological disorders, these cultivar plants survived and accumulated high doses of Pb, and their use in environmental/decontamination studies is open to debate.

Proteomics analysis reveals novel host molecular mechanisms associated with thermotherapy of 'Ca. Liberibacter asiaticus'-infected citrus plants.

PubMed

Nwugo, Chika C; Doud, Melissa S; Duan, Yong-Ping; Lin, Hong

2016-11-14

Citrus Huanglongbing (HLB), which is linked to the bacterial pathogen 'Ca. Liberibacter asiaticus' (Las), is the most devastating disease of citrus plants, and longer-term control measures via breeding or genetic engineering have been unwieldy because all cultivated citrus species are susceptible to the disease. However, the degree of susceptibility varies among citrus species, which has prompted efforts to identify potential Las resistance/tolerance-related genes in citrus plants for application in breeding or genetic engineering programs. Plant exposure to one form of stress has been shown to serendipitously induce innate resistance to other forms of stress and a recent study showed that continuous heat treatment (40 to 42 °C) reduced Las titer and HLB-associated symptoms in citrus seedlings. The goal of the present study was to apply comparative proteomics analysis via 2-DE and mass spectrometry to elucidate the molecular processes associated with heat-induced mitigation of HLB in citrus plants. Healthy or Las-infected citrus grapefruit plants were exposed to room temperature or to continuous heat treatment of 40 °C for 6 days. An exhaustive total protein extraction process facilitated the identification of 107 differentially-expressed proteins in response to Las and/or heat treatment, which included a strong up-regulation of chaperones including small (23.6, 18.5 and 17.9 kDa) heat shock proteins, a HSP70-like protein and a ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO)-binding 60 kDa chaperonin, particularly in response to heat treatment. Other proteins that were generally down-regulated due to Las infection but up-regulated in response to heat treatment include RuBisCO activase, chlorophyll a/b binding protein, glucosidase II beta subunit-like protein, a putative lipoxygenase protein, a ferritin-like protein, and a glutathione S-transferase. The differentially-expressed proteins identified in this study highlights a premier characterization of the molecular mechanisms potentially involved in the reversal of Las-induced pathogenicity processes in citrus plants and are hence proposed targets for application towards the development of cisgenic Las-resistant/tolerant citrus plants.

Influence of temperature on measurements of the CO2 compensation point: differences between the Laisk and O2-exchange methods.

PubMed

Walker, Berkley J; Cousins, Asaph B

2013-04-01

The CO2 compensation point in the absence of day respiration (Γ*) is a key parameter for modelling leaf CO2 exchange. Γ* links the kinetics of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) with the stoichiometry of CO2 released per Rubisco oxygenation from photorespiration (α), two essential components of biochemical models of photosynthesis. There are two main gas-exchange methods for measuring Γ*: (i) the Laisk method, which requires estimates of mesophyll conductance to CO2 (g m) and (ii) measurements of O2 isotope exchange, which assume constant values of α and a fixed stoichiometry between O2 uptake and Rubisco oxygenation. In this study, the temperature response of Γ* measured using the Laisk and O2-exchange methods was compared under ambient (25 °C) and elevated (35 °C) temperatures to determine whether both methods yielded similar results. Previously published temperature responses of Γ* estimated with the Laisk and O2-exchange methods in Nicotiana tabacum demonstrated that the Laisk-derived model of Γ* was more sensitive to temperature compared with the O2-exchange model. Measurements in Arabidopsis thaliana indicated that the Laisk and O2-exchange methods produced similar Γ* at 25 °C; however, Γ* values from O2 exchange were lower at 35 °C compared with the Laisk method. Compared with a photorespiratory mutant (pmdh1pmdh2hpr) with increased α, wild-type (WT) plants had lower Laisk values of Γ* at 25 °C but were not significantly different at 35 °C. These differences between Laisk and O2 exchange values of Γ* at 35 °C could be explained by temperature sensitivity of α in WT and/or errors in the assumptions of O2 exchange. The differences between Γ* measured using the Laisk and O2-exchange method with temperature demonstrate that assumptions used to measure Γ*, and possibly the species-specific validity of these assumptions, need to be considered when modelling the temperature response of photosynthesis.

Enhancement of growth, photosynthetic performance and yield by exclusion of ambient UV components in C3 and C4 plants.

PubMed

Kataria, Sunita; Guruprasad, K N; Ahuja, Sumedha; Singh, Bupinder

2013-10-05

A field experiment was conducted under tropical climate for assessing the effect of ambient UV-B and UV-A by exclusion of UV components on the growth, photosynthetic performance and yield of C3 (cotton, wheat) and C4 (amaranthus, sorghum) plants. The plants were grown in specially designed UV exclusion chambers, wrapped with filters that excluded UV-B (<315nm), UV-A+B (<400nm), transmitted all the UV (280-400nm) or without filters. All the four plant species responded to UV exclusion by a significant increase in plant height, leaf area, leaf biomass, total biomass accumulation and yield. Measurements of the chlorophyll, chlorophyll fluorescence parameters, gas exchange parameters and the activity of Ribulose-1,5-bisphosphate carboxylase (Rubisco) by fixation of (14)CO2 indicated a direct relationship between enhanced rate of photosynthesis and yield of the plants. Quantum yield of electron transport was enhanced by the exclusion of UV indicating better utilization of PAR assimilation and enhancement in reducing power in all the four plant species. Exclusion of UV-B in particular significantly enhanced the net photosynthetic rate, stomatal conductance and activity of Rubisco. Additional fixation of carbon due to exclusion of ambient UV-B was channeled towards yield as there was a decrease in the level of UV-B absorbing substances and an increase in soluble proteins in all the four plant species. The magnitude of the promotion in all the parameters studied was higher in dicots (cotton, amaranthus) compared to monocots (wheat, sorghum) after UV exclusion. The results indicated a suppressive action of ambient UV-B on growth and photosynthesis; dicots were more sensitive than monocots in this suppression while no great difference in sensitivity was found between C3 and C4 plants. Experiments indicated the suppressive action of ambient UV on carbon fixation and yield of C3 and C4 plants. Exclusion of solar UV-B will have agricultural benefits in both C3 and C4 plants under tropical climate. Copyright © 2013 Elsevier B.V. All rights reserved.

Does Long-Term Elevation of CO2 Concentration Increase Photosynthesis in Forest Floor Vegetation? (Indiana Strawberry in a Maryland Forest).

PubMed

Osborne, C. P.; Drake, B. G.; LaRoche, J.; Long, S. P.

1997-05-01

As the partial pressure of CO2 (pCO2) in the atmosphere rises, photorespiratory loss of carbon in C3 photosynthesis will diminish and the net efficiency of light-limited photosynthetic carbon uptake should rise. We tested this expectation for Indiana strawberry (Duchesnea indica) growing on a Maryland forest floor. Open-top chambers were used to elevate the pCO2 of a forest floor habitat to 67 Pa and were paired with control chambers providing an ambient pCO2 of 38 Pa. After 3.5 years, D. indica leaves grown and measured in the elevated pCO2 showed a significantly greater maximum quantum efficiency of net photosynthesis (by 22%) and a lower light compensation point (by 42%) than leaves grown and measured in the control chambers. The quantum efficiency to minimize photorespiration, measured in 1% O2, was the same for controls and plants grown at elevated pCO2. This showed that the maximum efficiency of light-energy transduction into assimilated carbon was not altered by acclimation and that the increase in light-limited photosynthesis at elevated pCO2 was simply a function of the decrease in photorespiration. Acclimation did decrease the ribulose-1,5-bisphosphate carboxylase/oxygenase and light-harvesting chlorophyll protein content of the leaf by more than 30%. These changes were associated with a decreased capacity for light-saturated, but not light-limited, photosynthesis. Even so, leaves of D. indica grown and measured at elevated pCO2 showed greater light-saturated photosynthetic rates than leaves grown and measured at the current atmospheric pCO2. In situ measurements under natural forest floor lighting showed large increases in leaf photosynthesis at elevated pCO2, relative to controls, in both summer and fall. The increase in efficiency of light-limited photosynthesis with elevated pCO2 allowed positive net photosynthetic carbon uptake on days and at locations on the forest floor that light fluxes were insufficient for positive net photosynthesis in the current atmospheric pCO2.

Assessing the Likelihood of Gene Flow From Sugarcane (Saccharum Hybrids) to Wild Relatives in South Africa

PubMed Central

Snyman, Sandy J.; Komape, Dennis M.; Khanyi, Hlobisile; van den Berg, Johnnie; Cilliers, Dirk; Lloyd Evans, Dyfed; Barnard, Sandra; Siebert, Stefan J.

2018-01-01

Pre-commercialization studies on environmental biosafety of genetically modified (GM) crops are necessary to evaluate the potential for sexual hybridization with related plant species that occur in the release area. The aim of the study was a preliminary assessment of factors that may contribute to gene flow from sugarcane (Saccharum hybrids) to indigenous relatives in the sugarcane production regions of Mpumalanga and KwaZulu-Natal provinces, South Africa. In the first instance, an assessment of Saccharum wild relatives was conducted based on existing phylogenies and literature surveys. The prevalence, spatial overlap, proximity, distribution potential, and flowering times of wild relatives in sugarcane production regions based on the above, and on herbaria records and field surveys were conducted for Imperata, Sorghum, Cleistachne, and Miscanthidium species. Eleven species were selected for spatial analyses based on their presence within the sugarcane cultivation region: four species in the Saccharinae and seven in the Sorghinae. Secondly, fragments of the nuclear internal transcribed spacer (ITS) regions of the 5.8s ribosomal gene and two chloroplast genes, ribulose-bisphosphate carboxylase (rbcL), and maturase K (matK) were sequenced or assembled from short read data to confirm relatedness between Saccharum hybrids and its wild relatives. Phylogenetic analyses of the ITS cassette showed that the closest wild relative species to commercial sugarcane were Miscanthidium capense, Miscanthidium junceum, and Narenga porphyrocoma. Sorghum was found to be more distantly related to Saccharum than previously described. Based on the phylogeny described in our study, the only species to highlight in terms of evolutionary divergence times from Saccharum are those within the genus Miscanthidium, most especially M. capense, and M. junceum which are only 3 million years divergent from Saccharum. Field assessment of pollen viability of 13 commercial sugarcane cultivars using two stains, iodine potassium iodide (IKI) and triphenyl tetrazolium chloride, showed decreasing pollen viability (from 85 to 0%) from the north to the south eastern regions of the study area. Future work will include other aspects influencing gene flow such as cytological compatibility and introgression between sugarcane and Miscanthidium species. PMID:29930938

R-Syst::diatom: an open-access and curated barcode database for diatoms and freshwater monitoring

PubMed Central

Rimet, Frédéric; Chaumeil, Philippe; Keck, François; Kermarrec, Lenaïg; Vasselon, Valentin; Kahlert, Maria; Franc, Alain; Bouchez, Agnès

2016-01-01

Diatoms are micro-algal indicators of freshwater pollution. Current standardized methodologies are based on microscopic determinations, which is time consuming and prone to identification uncertainties. The use of DNA-barcoding has been proposed as a way to avoid these flaws. Combining barcoding with next-generation sequencing enables collection of a large quantity of barcodes from natural samples. These barcodes are identified as certain diatom taxa by comparing the sequences to a reference barcoding library using algorithms. Proof of concept was recently demonstrated for synthetic and natural communities and underlined the importance of the quality of this reference library. We present an open-access and curated reference barcoding database for diatoms, called R-Syst::diatom, developed in the framework of R-Syst, the network of systematic supported by INRA (French National Institute for Agricultural Research), see http://www.rsyst.inra.fr/en. R-Syst::diatom links DNA-barcodes to their taxonomical identifications, and is dedicated to identify barcodes from natural samples. The data come from two sources, a culture collection of freshwater algae maintained in INRA in which new strains are regularly deposited and barcoded and from the NCBI (National Center for Biotechnology Information) nucleotide database. Two kinds of barcodes were chosen to support the database: 18S (18S ribosomal RNA) and rbcL (Ribulose-1,5-bisphosphate carboxylase/oxygenase), because of their efficiency. Data are curated using innovative (Declic) and classical bioinformatic tools (Blast, classical phylogenies) and up-to-date taxonomy (Catalogues and peer reviewed papers). Every 6 months R-Syst::diatom is updated. The database is available through the R-Syst microalgae website (http://www.rsyst.inra.fr/) and a platform dedicated to next-generation sequencing data analysis, virtual_BiodiversityL@b (https://galaxy-pgtp.pierroton.inra.fr/). We present here the content of the library regarding the number of barcodes and diatom taxa. In addition to these information, morphological features (e.g. biovolumes, chloroplasts…), life-forms (mobility, colony-type) or ecological features (taxa preferenda to pollution) are indicated in R-Syst::diatom. Database URL: http://www.rsyst.inra.fr/ PMID:26989149

Does long-term elevation of CO{sub 2} concentration increase photosynthesis in forest floor vegetation? Indiana strawberry in a Maryland forest

DOE Office of Scientific and Technical Information (OSTI.GOV)

Osborne, C.P.; Long, S.P.; Drake, B.G.

1997-05-01

As the partial pressure of CO{sub 2} (pCO{sub 2}) in the atmosphere rises, photorespiratory loss of carbon in C, photosynthesis will diminish and the net efficiency of light-limited photosynthetic carbon uptake should rise. Indiana strawberry (Duchesnea indica) growing on a Maryland forest floor was tested. Open-top chambers were used to elevate the pCO{sub 2} of a forest floor habitat to 67 Pa and were paired with control chambers with an ambient pCO{sub 2} of 38 Pa. After 3.5 years, D. indica leaves in the elevated pCO{sub 2} showed a significantly greater maximum quantum efficiency of net photosynthesis (by 22%) andmore » a lower light compensation point (by 42%) than leaves in the control chambers. The quantum efficiency to minimize photorespiration was the same for controls and plants grown at elevated pCO{sub 2}, showing the maximum efficiency of light-energy transduction into assimilated carbon was not altered by acclimation and the increase in light-limited photosynthesis at elevated pCO{sub 2} was a function of the decrease in photorespiration. Acclimation did decrease the ribulose-1,5-bisphosphate carboxylase/oxygenase and light-harvesting chlorophyll protein content of the leaf by more than 30%. These changes were associated with a decreased capacity for light-saturated, but not light-limited, photosynthesis. Leaves of D. indica grown and measured at elevated pCO{sub 2} showed greater light-saturated photosynthetic rates than leaves grown and measured at the current atmospheric pCO{sub 2}. In situ measurements under natural lighting showed large increases in leaf photosynthesis at elevated pCO{sub 2}, relative to controls, in both summer and fall. The increase in efficiency of light-limited photosynthesis with elevated pCO{sub 2} allowed positive net photosynthetic carbon uptake on days and at locations on the forest floor that light fluxes were insufficient for positive net photosynthesis in the current atmospheric pCO{sub 2}. 33 refs., 3 figs., 3 tabs.« less