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Sample records for adp-glucose pyrophosphorylase agpase

  1. Characterization of an autonomously activated plant ADP-glucose pyrophosphorylase.

    PubMed

    Boehlein, Susan K; Shaw, Janine R; Stewart, Jon D; Hannah, L Curtis

    2009-01-01

    ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step in starch biosynthesis in plants and changes in its catalytic and/or allosteric properties can lead to increased starch production. Recently, a maize (Zea mays)/potato (Solanum tuberosum) small subunit mosaic, MP [Mos(1-198)], containing the first 198 amino acids of the small subunit of the maize endosperm enzyme and the last 277 amino acids from the potato tuber enzyme, was expressed with the maize endosperm large subunit and was reported to have favorable kinetic and allosteric properties. Here, we show that this mosaic, in the absence of activator, performs like a wild-type AGPase that is partially activated with 3-phosphoglyceric acid (3-PGA). In the presence of 3-PGA, enzyme properties of Mos(1-198)/SH2 are quite similar to those of the wild-type maize enzyme. In the absence of 3-PGA, however, the mosaic enzyme exhibits greater activity, higher affinity for the substrates, and partial inactivation by inorganic phosphate. The Mos(1-198)/SH2 enzyme is also more stable to heat inactivation. The different properties of this protein were mapped using various mosaics containing smaller portions of the potato small subunit. Enhanced heat stability of Mos(1-198) was shown to originate from five potato-derived amino acids between 322 and 377. These amino acids were shown previously to be important in small subunit/large subunit interactions. These five potato-derived amino acids plus other potato-derived amino acids distributed throughout the carboxyl-terminal portion of the protein are required for the enhanced catalytic and allosteric properties exhibited by Mos(1-198)/SH2. PMID:18715954

  2. Investigation of the interaction between the large and small subunits of potato ADP-glucose pyrophosphorylase.

    PubMed

    Baris, Ibrahim; Tuncel, Aytug; Ozber, Natali; Keskin, Ozlem; Kavakli, Ibrahim Halil

    2009-10-01

    ADP-glucose pyrophosphorylase (AGPase), a key allosteric enzyme involved in higher plant starch biosynthesis, is composed of pairs of large (LS) and small subunits (SS). Current evidence indicates that the two subunit types play distinct roles in enzyme function. Recently the heterotetrameric structure of potato AGPase has been modeled. In the current study, we have applied the molecular mechanics generalized born surface area (MM-GBSA) method and identified critical amino acids of the potato AGPase LS and SS subunits that interact with each other during the native heterotetrameric structure formation. We have further shown the role of the LS amino acids in subunit-subunit interaction by yeast two-hybrid, bacterial complementation assay and native gel. Comparison of the computational results with the experiments has indicated that the backbone energy contribution (rather than the side chain energies) of the interface residues is more important in identifying critical residues. We have found that lateral interaction of the LS-SS is much stronger than the longitudinal one, and it is mainly mediated by hydrophobic interactions. This study will not only enhance our understanding of the interaction between the SS and the LS of AGPase, but will also enable us to engineer proteins to obtain better assembled variants of AGPase which can be used for the improvement of plant yield. PMID:19876371

  3. Both subunits of ADP-glucose pyrophosphorylase are regulatory.

    PubMed

    Cross, Joanna M; Clancy, Maureen; Shaw, Janine R; Greene, Thomas W; Schmidt, Robert R; Okita, Thomas W; Hannah, L Curtis

    2004-05-01

    The allosteric enzyme ADP-Glc pyrophosphorylase (AGPase) catalyzes the synthesis of ADP-Glc, a rate-limiting step in starch synthesis. Plant AGPases are heterotetramers, most of which are activated by 3-phosphoglyceric acid (3-PGA) and inhibited by phosphate. The objectives of these studies were to test a hypothesis concerning the relative roles of the two subunits and to identify regions in the subunits important in allosteric regulation. We exploited an Escherichia coli expression system and mosaic AGPases composed of potato (Solanum tuberosum) tuber and maize (Zea mays) endosperm subunit fragments to pursue this objective. Whereas potato and maize subunits have long been separated by speciation and evolution, they are sufficiently similar to form active mosaic enzymes. Potato tuber and maize endosperm AGPases exhibit radically different allosteric properties. Hence, comparing the kinetic properties of the mosaics to those of the maize endosperm and potato tuber AGPases has enabled us to identify regions important in regulation. The data herein conclusively show that both subunits are involved in the allosteric regulation of AGPase. Alterations in the small subunit condition drastically different allosteric properties. In addition, extent of 3-PGA activation and extent of 3-PGA affinity were found to be separate entities, mapping to different regions in both subunits. PMID:15122037

  4. ADP-glucose pyrophosphorylase gene plays a key role in the quality of corm and yield of cormels in gladiolus.

    PubMed

    Seng, Shanshan; Wu, Jian; Sui, Juanjuan; Wu, Chenyu; Zhong, Xionghui; Liu, Chen; Liu, Chao; Gong, Benhe; Zhang, Fengqin; He, Junna; Yi, Mingfang

    2016-05-20

    Starch is the main storage compound in underground organs like corms. ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in storage organs and is likely one of the most important determinant of sink strength. Here, we identify an AGPase gene (GhAGPS1) from gladiolus. The highest transcriptional levels of GhAGPS1 were observed in cormels and corms. Transformation of GhAGPS1 into Arabidopsis rescued the phenotype of aps1 mutant. Silencing GhAGPS1 in gladiolus corms by virus-induced gene silencing (VIGS) decreased the transcriptional levels of two genes and starch content. Transmission electron microscopy analyses of leaf and corm sections confirmed that starch biosynthesis was inhibited. Corm weight and cormel number reduced significantly in the silenced plants. Taken together, these results indicate that inhibiting the expression of AGPase gene could impair starch synthesis, which results in the lowered corm quality and cormel yield in gladiolus. PMID:27107698

  5. Probing allosteric binding sites of the maize endosperm ADP-glucose pyrophosphorylase.

    PubMed

    Boehlein, Susan K; Shaw, Janine R; Hannah, L Curtis; Stewart, Jon D

    2010-01-01

    Maize (Zea mays) endosperm ADP-glucose pyrophosphorylase (AGPase) is a highly regulated enzyme that catalyzes the rate-limiting step in starch biosynthesis. Although the structure of the heterotetrameric maize endosperm AGPase remains unsolved, structures of a nonnative, low-activity form of the potato tuber (Solanum tuberosum) AGPase (small subunit homotetramer) reported previously by others revealed that several sulfate ions bind to each enzyme. These sites are also believed to interact with allosteric regulators such as inorganic phosphate and 3-phosphoglycerate (3-PGA). Several arginine (Arg) side chains contact the bound sulfate ions in the potato structure and likely play important roles in allosteric effector binding. Alanine-scanning mutagenesis was applied to the corresponding Arg residues in both the small and large subunits of maize endosperm AGPase to determine their roles in allosteric regulation and thermal stability. Steady-state kinetic and regulatory parameters were measured for each mutant. All of the Arg mutants examined--in both the small and large subunits--bound 3-PGA more weakly than the wild type (A(50) increased by 3.5- to 20-fold). By contrast, the binding of two other maize AGPase allosteric activators (fructose-6-phosphate and glucose-6-phosphate) did not always mimic the changes observed for 3-PGA. In fact, compared to 3-PGA, fructose-6-phosphate is a more efficient activator in two of the Arg mutants. Phosphate binding was also affected by Arg substitutions. The combined data support a model for the binding interactions associated with 3-PGA in which allosteric activators and inorganic phosphate compete directly. PMID:19889875

  6. ADP-glucose pyrophosphorylase large subunit 2 is essential for storage substance accumulation and subunit interactions in rice endosperm.

    PubMed

    Tang, Xiao-Jie; Peng, Cheng; Zhang, Jie; Cai, Yue; You, Xiao-Man; Kong, Fei; Yan, Hai-Gang; Wang, Guo-Xiang; Wang, Liang; Jin, Jie; Chen, Wei-Wei; Chen, Xin-Gang; Ma, Jing; Wang, Peng; Jiang, Ling; Zhang, Wen-Wei; Wan, Jian-Min

    2016-08-01

    ADP-glucose pyrophosphorylase (AGPase) controls a rate-limiting step in the starch biosynthetic pathway in higher plants. Here we isolated a shrunken rice mutant w24. Map-based cloning identified OsAGPL2, a large subunit of the cytosolic AGPase in rice endosperm, as the gene responsible for the w24 mutation. In addition to severe inhibition of starch synthesis and significant accumulation of sugar, the w24 endosperm showed obvious defects in compound granule formation and storage protein synthesis. The defect in OsAGPL2 enhanced the expression levels of the AGPase family. Meanwhile, the elevated activities of starch phosphorylase 1 and sucrose synthase in the w24 endosperm might possibly partly account for the residual starch content in the mutant seeds. Moreover, the expression of OsAGPL2 and its counterpart, OsAGPS2b, was highly coordinated in rice endosperm. Yeast two-hybrid and BiFC assays verified direct interactions between OsAGPL2 and OsAGPS2b as well as OsAGPL1 and OsAGPS1, supporting the model for spatiotemporal complex formation of AGPase isoforms in rice endosperm. Besides, our data provided no evidence for the self-binding of OsAGPS2b, implying that OsAGPS2b might not interact to form higher molecular mass aggregates in the absence of OsAGPL2. Therefore, the molecular mechanism of rice AGPase assembly might differ from that of Arabidopsis. PMID:27297991

  7. Enhanced activity of ADP glucose pyrophosphorylase and formation of starch induced by Azospirillum brasilense in Chlorella vulgaris.

    PubMed

    Choix, Francisco J; Bashan, Yoav; Mendoza, Alberto; de-Bashan, Luz E

    2014-05-10

    ADP-glucose pyrophosphorylase (AGPase) regulates starch biosynthesis in higher plants and microalgae. This study measured the effect of the bacterium Azospirillum brasilense on AGPase activity in the freshwater microalga Chlorella vulgaris and formation of starch. This was done by immobilizing both microorganisms in alginate beads, either replete with or deprived of nitrogen or phosphorus and all under heterotrophic conditions, using d-glucose or Na-acetate as the carbon source. AGPase activity during the first 72h of incubation was higher in C. vulgaris when immobilized with A. brasilense. This happened simultaneously with higher starch accumulation and higher carbon uptake by the microalgae. Either carbon source had similar effects on enzyme activity and starch accumulation. Starvation either by N or P had the same pattern on AGPase activity and starch accumulation. Under replete conditions, the population of C. vulgaris immobilized alone was higher than when immobilized together, but under starvation conditions A. brasilense induced a larger population of C. vulgaris. In summary, adding A. brasilense enhanced AGPase activity, starch formation, and mitigation of stress in C. vulgaris. PMID:24576433

  8. Multiple forms of ADP-glucose pyrophosphorylase from tomato fruit

    NASA Technical Reports Server (NTRS)

    Chen, B. Y.; Janes, H. W.

    1997-01-01

    ADP-glucose pyrophosphorylase (AGP) was purified from tomato (Lycopersicon esculentum Mill.) fruit to apparent homogeneity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis the enzyme migrated as two close bands with molecular weights of 50,000 and 51,000. Two-dimensional polyacrylamide gel electrophoresis analysis of the purified enzyme, however, revealed at least five major protein spots that could be distinguished by their slight differences in net charge and molecular weight. Whereas all of the spots were recognized by the antiserum raised against tomato fruit AGP holoenzyme, only three of them reacted strongly with antiserum raised against the potato tuber AGP large subunit, and the other two spots (with lower molecular weights) reacted specifically with antisera raised against spinach leaf AGP holoenzyme and the potato tuber AGP small subunit. The results suggest the existence of at least three isoforms of the AGP large subunit and two isoforms of the small subunit in tomato fruit in vivo. The native molecular mass of the enzyme determined by gel filtration was 220 +/- 10 kD, indicating a tetrameric structure for AGP from tomato fruit. The purified enzyme is very sensitive to 3-phosphoglycerate/inorganic phosphate regulation.

  9. Relative turnover numbers of maize endosperm and potato tuber ADP-glucose pyrophosphorylases in the absence and presence of 3-phosphoglyceric acid.

    PubMed

    Burger, Brian T; Cross, Joanna M; Shaw, Janine R; Caren, Joel R; Greene, Thomas W; Okita, Thomas W; Hannah, L Curtis

    2003-07-01

    Adenosine diphosphate glucose pyrophosphorylase (AGPase; EC 2.7.7.27) synthesizes the starch precursor, ADP-glucose. It is a rate-limiting enzyme in starch biosynthesis and its activation by 3-phosphoglyceric acid (3PGA) and/or inhibition by inorganic phosphate (Pi) are believed to be physiologically important. Leaf, tuber and cereal embryo AGPases are highly sensitive to these effectors, whereas endosperm AGPases are much less responsive. Two hypotheses can explain the 3PGA activation differences. Compared to leaf AGPases, endosperm AGPases (i) lack the marked ability to be activated by 3PGA or (ii) they are less dependent on 3PGA for activity. The absence of purified preparations has heretofore negated answering this question. To resolve this issue, heterotetrameric maize ( Zea mays L.) endosperm and potato ( Solanum tuberosum L.) tuber AGPases expressed in Escherichia coli were isolated and the relative amounts of enzyme protein were measured by reaction to antibodies against a motif resident in both small subunits. Resulting reaction rates of both AGPases are comparable in the presence but not in the absence of 3PGA when expressed on an active-protein basis. We also placed the potato tuber UpReg1 mutation into the maize AGPase. This mutation greatly enhances 3PGA sensitivity of the potato AGPase but it has little effect on the maize AGPase. Thirdly, lysines known to bind 3PGA in potato tuber AGPase, but missing from the maize endosperm AGPase, were introduced into the maize enzyme. These had minimal effect on maize endosperm activity. In conclusion, the maize endosperm AGPase is not nearly as dependent on 3PGA for activity as is the potato tuber AGPase. PMID:14520572

  10. Sucrose regulation of ADP-glucose pyrophosphorylase subunit genes transcript levels in leaves and fruits

    NASA Technical Reports Server (NTRS)

    Li, Xiangyang; Xing, Jinpeng; Gianfagna, Thomas J.; Janes, Harry W.

    2002-01-01

    ADP-glucose pyrophosphorylase (AGPase, EC2.7.7.27) is a key regulatory enzyme in starch biosynthesis. The enzyme is a heterotetramer with two S and two B subunits. In tomato, there are three multiple forms of the S subunit gene. Agp S1, S2 and B are highly expressed in fruit from 10 to 25 days after anthesis. Agp S3 is only weakly expressed in fruit. Sucrose significantly elevates expression of Agp S1, S2 and B in both leaves and fruits. Agp S1 exhibits the highest degree of regulation by sucrose. In fact, sucrose may be required for Agp S1 expression. For excised leaves incubated in water, no transcripts for Agp S1 could be detected in the absence of sucrose, whereas it took up to 16 h in water before transcripts were no longer detectable for Agp S2 and B. Neither Agp S3 nor the tubulin gene is affected by sucrose, demonstrating that this response is specifically regulated by a carbohydrate metabolic signal, and is not due to a general increase in metabolism caused by sucrose treatment. Truncated versions of the promoter for Agp S1 indicate that a specific region 1.3-3.0 kb upstream from the transcription site is responsible for sucrose sensitivity. This region of the S1 promoter contains several cis-acting elements present in the promoters of other genes that are also regulated by sucrose. c2002 Elsevier Science Ireland Ltd. All rights reserved.

  11. Heat stability of maize endosperm ADP-glucose pyrophosphorylase is enhanced by insertion of a cysteine in the N terminus of the small subunit.

    PubMed

    Linebarger, Carla R Lyerly; Boehlein, Susan K; Sewell, Aileen K; Shaw, Janine; Hannah, L Curtis

    2005-12-01

    ADP-glucose pyrophosphorylase (AGPase) is a key regulatory enzyme in starch biosynthesis. However, plant AGPases differ in several parameters, including spatial and temporal expression, allosteric regulation, and heat stability. AGPases of cereal endosperms are heat labile, while those in other tissues, such as the potato (Solanum tuberosum) tuber, are heat stable. Sequence comparisons of heat-stable and heat-labile AGPases identified an N-terminal motif unique to heat-stable enzymes. Insertion of this motif into recombinant maize (Zea mays) endosperm AGPase increased the half-life at 58 degrees C more than 70-fold. Km values for physiological substrates were unaffected, although Kcat was doubled. A cysteine within the inserted motif gives rise to small subunit homodimers not found in the wild-type maize enzyme. Placement of this N-terminal motif into a mosaic small subunit containing the N terminus from maize endosperm and the C terminus from potato tuber AGPase increases heat stability more than 300-fold. PMID:16299180

  12. Sugars and light/dark exposure trigger differential regulation of ADP-glucose pyrophosphorylase genes in Arabidopsis thaliana (thale cress).

    PubMed

    Sokolov, L N; Déjardin, A; Kleczkowski, L A

    1998-12-15

    Expression of four Arabidopsis (thale cress) genes corresponding to the small (ApS) and large subunits (ApL1, ApL2, ApL3) of ADP-glucose pyrophosphorylase (AGPase), a key enzyme of starch biosynthesis, was found to be profoundly and differentially regulated by sugar and light/dark exposures. Transcript levels of both ApL2 and ApL3, and to a lesser extent ApS, increased severalfold upon feeding sucrose or glucose to the detached leaves in the dark, whereas the mRNA content for ApL1 decreased under the same conditions. Glucose was, in general, less effective than sucrose in inducing regulation of AGPase genes, possibly due to observed limitations in its uptake when compared with sucrose uptake by detached leaves. Osmotic agents [sorbitol, poly(ethylene glycol)] had no effect on ApS, ApL2 and ApL3 transcript level, but they did mimic the effect of sucrose on ApL1 gene, suggesting that the latter is regulated by osmotic pressure rather than any particular sugar. For all the genes the sugar effect was closely mimicked by an exposure of the dark-pre-adapted leaves to the light. Under both dark and light conditions, sucrose fed to the detached leaves was found to be rapidly metabolized to hexoses and, to some extent, starch. Starch production reflected most probably an increase in substrate availability for AGPase reaction rather than being due to changes in AGPase protein content, since both the sugar feeding and light exposure had little or no effect on the activity of AGPase or on the levels of its small and large subunit proteins in leaf extracts. The data suggest tight translational or post-translational control, but they may also reflect spatial control of AGPase gene expression within a leaf. The sugar/light-dependent regulation of AGPase gene expression may represent a part of a general cellular response to the availability/allocation of carbohydrates during photosynthesis. PMID:9841881

  13. Isolation and characterization of cDNAs and genomic DNAs encoding ADP-glucose pyrophosphorylase large and small subunits from sweet potato.

    PubMed

    Zhou, Yu-Xi; Chen, Yu-Xiang; Tao, Xiang; Cheng, Xiao-Jie; Wang, Hai-Yan

    2016-04-01

    Sweet potato [Ipomoea batatas (L.) Lam.], the world's seventh most important food crop, is also a major industrial raw material for starch and ethanol production. In the plant starch biosynthesis pathway, ADP-glucose pyrophosphorylase (AGPase) catalyzes the first, rate-limiting step and plays a pivotal role in regulating this process. In spite of the importance of sweet potato as a starch source, only a few studies have focused on the molecular aspects of starch biosynthesis in sweet potato and almost no intensive research has been carried out on the AGPase gene family in this species. In this study, cDNAs encoding two small subunits (SSs) and four large subunits (LSs) of AGPase isoforms were cloned from sweet potato and the genomic organizations of the corresponding AGPase genes were elucidated. Expression pattern analysis revealed that the two SSs were constitutively expressed, whereas the four LSs displayed differential expression patterns in various tissues and at different developmental stages. Co-expression of SSs with different LSs in Escherichia coli yielded eight heterotetramers showing different catalytic activities. Interactions between different SSs and LSs were confirmed by a yeast two-hybrid experiment. Our findings provide comprehensive information about AGPase gene sequences, structures, expression profiles, and subunit interactions in sweet potato. The results can serve as a foundation for elucidation of molecular mechanisms of starch synthesis in tuberous roots, and should contribute to future regulation of starch biosynthesis to improve sweet potato starch yield. PMID:26499957

  14. Phylogenetic analysis of ADP-glucose pyrophosphorylase subunits reveals a role of subunit interfaces in the allosteric properties of the enzyme.

    PubMed

    Georgelis, Nikolaos; Shaw, Janine R; Hannah, L Curtis

    2009-09-01

    ADP-glucose pyrophosphorylase (AGPase) catalyzes a rate-limiting step in glycogen and starch synthesis in bacteria and plants, respectively. Plant AGPase consists of two large and two small subunits that were derived by gene duplication. AGPase large subunits have functionally diverged, leading to different kinetic and allosteric properties. Amino acid changes that could account for these differences were identified previously by evolutionary analysis. In this study, these large subunit residues were mapped onto a modeled structure of the maize (Zea mays) endosperm enzyme. Surprisingly, of 29 amino acids identified via evolutionary considerations, 17 were located at subunit interfaces. Fourteen of the 29 amino acids were mutagenized in the maize endosperm large subunit (SHRUNKEN-2 [SH2]), and resulting variants were expressed in Escherichia coli with the maize endosperm small subunit (BT2). Comparisons of the amount of glycogen produced in E. coli, and the kinetic and allosteric properties of the variants with wild-type SH2/BT2, indicate that 11 variants differ from the wild type in enzyme properties or in vivo glycogen level. More interestingly, six of nine residues located at subunit interfaces exhibit altered allosteric properties. These results indicate that the interfaces between the large and small subunits are important for the allosteric properties of AGPase, and changes at these interfaces contribute to AGPase functional specialization. Our results also demonstrate that evolutionary analysis can greatly facilitate enzyme structure-function analyses. PMID:19625637

  15. The potato tuber, maize endosperm and a chimeric maize-potato ADP-glucose pyrophosphorylase exhibit fundamental differences in Pi inhibition.

    PubMed

    Boehlein, Susan K; Shaw, Janine R; McCarty, Donald R; Hwang, Seon-Kap; Stewart, Jon D; Hannah, L Curtis

    2013-09-15

    ADP-glucose pyrophosphorylase (AGPase) is highly regulated by allosteric effectors acting both positively and negatively. Enzymes from various sources differ, however, in the mechanism of allosteric regulation. Here, we determined how the effector, inorganic phosphate (Pi), functions in the presence and absence of saturating amounts of the activator, 3-phosphoglyceric acid (3-PGA). This regulation was examined in the maize endosperm enzyme, the oxidized and reduced forms of the potato tuber enzyme as well as a small subunit chimeric AGPase (MP), which contains both maize endosperm and potato tuber sequences paired with a wild-type maize large subunit. These data, combined with our previous kinetic studies of these enzymes led to a model of Pi inhibition for the various enzymes. The Pi inhibition data suggest that while the maize enzyme contains a single effector site that binds both 3-PGA and Pi, the other enzymes exhibit more complex behavior and most likely have at least two separate interacting binding sites for Pi. The possible physiological implications of the differences in Pi inhibition distinguishing the maize endosperm and potato tuber AGPases are discussed. PMID:23906662

  16. Comparison of Starch and ADP-Glucose Pyrophosphorylase Levels in Nonembryogenic Cells and Developing Embryos from Induced Carrot Cultures

    PubMed Central

    Keller, Gregory L.; Nikolau, Basil J.; Ulrich, Thomas H.; Wurtele, Eve Syrkin

    1988-01-01

    Cultures of carrot (Daucus carota L.) in a medium without added 2,4-dichlorophenoxyacetic acid were separated into fractions of embryos at different stages of development (large globular and heart, torpedo, and germinating) and nonembryogenic cells. The average starch content per cell in these fractions was similar. However, due to the smaller sizes of the cells of the embryos relative to the nonembryogenic cells, starch content per weight of tissue was higher in the embryos. The ADP-glucose pyrophosphorylase activity per cell in the nonembryogenic cells was double that of the embryo cells. Furthermore, the ratio of ADP-glucose pyrophosphorylase to starch was over 2-fold higher in the nonembryogenic cells, indicating that starch content is not simply determined by ADP-glucose pyrophosphorylase levels. ADP-glucose pyrophosphorylase activity of all culture fractions was directly proportional to the level of a single 50 kilodalton polypeptide detected by immunoblot analysis, using antiserum raised to the purified spinach leaf enzyme. In the same immunoblot analysis, novel polypeptides of 63 and 100 kilodalton were detected in embryos but were absent from nonembryogenic cells. This is one of the few reported examples of specific proteins which differentially accumulate in embryos and nonembryogenic cells. Images Fig. 2 PMID:16665929

  17. Characterization of an ADP-glucose Pyrophosphorylase Small Subunit Gene Expressed in Developing Cotton (Gossypium hirsutum) Fibers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ADP-glucose pyrophosphorylase (ADPGp) plays a rate limiting role in the biosynthesis of starch and has been shown to be involved in cell expansion of tobacco sepals. A cotton gene encoding ADPGp small subunit was isolated and sequenced. The gene contains 8 introns similar to other ADPGp genes. The o...

  18. Deciphering the kinetic mechanisms controlling selected plant ADP-glucose pyrophosphorylases.

    PubMed

    Boehlein, Susan K; Shaw, Janine R; Hwang, Seon K; Stewart, Jon D; Curtis Hannah, L

    2013-07-15

    ADP-Glc pyrophosphorylase (AGPase), a rate-limiting enzyme in starch biosynthesis, is controlled by thermostability and allosteric regulation. Previous studies suggested that redox affects turnover number and heat stability of AGPases. Here, we investigated how allostery and redox state affect kinetic mechanisms of the reduced, heat labile and the oxidized, heat stable potato tuber enzymes; the heat labile maize endosperm enzyme and a chimeric maize/potato heat stable enzyme that lacks the cysteine responsible for redox changes. With 3-PGA, all AGPases followed a Theorell-Chance Bi Bi mechanism with ATP binding first and ADP-Glc releasing last. 3-PGA increases the binding affinity for both substrates with little effect on velocity for the maize and MP isoforms. By contrast, 3-PGA increases the velocity and the affinity for G-1-P for the potato enzymes. Redox state does not affect kcat of the two potato isoforms. Without 3-PGA the oxidized potato enzyme exhibits a rapid equilibrium random Bi Bi mechanism with a dead end ternary complex. This fundamental change from rapid, ordered binding with little buildup of intermediates to a mechanism featuring relatively slow, random binding is unique to the oxidized potato tuber enzyme. Finally, ADP-Glc the physiologically relevant product of this enzyme has complex, isoform-specific effects on catalysis. PMID:23603314

  19. PCR cloning and characterization of multiple ADP-glucose pyrophosphorylase cDNAs from tomato

    NASA Technical Reports Server (NTRS)

    Chen, B. Y.; Janes, H. W.; Gianfagna, T.

    1998-01-01

    Four ADP-glucose pyrophosphorylase (AGP) cDNAs were cloned from tomato fruit and leaves by the PCR techniques. Three of them (agp S1, agp S2, and agp S3) encode the large subunit of AGP, the fourth one (agp B) encodes the small subunit. The deduced amino acid sequences of the cDNAs show very high identities (96-98%) to the corresponding potato AGP isoforms, although there are major differences in tissue expression profiles. All four tomato AGP transcripts were detected in fruit and leaves; the predominant ones in fruit are agp B and agp S1, whereas in leaves they are agp B and agp S3. Genomic southern analysis suggests that the four AGP transcripts are encoded by distinct genes.

  20. Substrate binding properties of potato tuber ADP-glucose pyrophosphorylase as determined by isothermal titration calorimetry.

    PubMed

    Cakir, Bilal; Tuncel, Aytug; Green, Abigail R; Koper, Kaan; Hwang, Seon-Kap; Okita, Thomas W; Kang, ChulHee

    2015-06-01

    Substrate binding properties of the large (LS) and small (SS) subunits of potato tuber ADP-glucose pyrophosphorylase were investigated by using isothermal titration calorimetry. Our results clearly show that the wild type heterotetramer (S(WT)L(WT)) possesses two distinct types of ATP binding sites, whereas the homotetrameric LS and SS variant forms only exhibited properties of one of the two binding sites. The wild type enzyme also exhibited significantly increased affinity to this substrate compared to the homotetrameric enzyme forms. No stable binding was evident for the second substrate, glucose-1-phosphate, in the presence or absence of ATPγS suggesting that interaction of glucose-1-phosphate is dependent on hydrolysis of ATP and supports the Theorell-Chance bi bi reaction mechanism. PMID:25953126

  1. Regulatory properties of potato-Arabidopsis hybrid ADP-glucose pyrophosphorylase.

    PubMed

    Ventriglia, Tiziana; Ballicora, Miguel A; Crevillén, Pedro; Preiss, Jack; Romero, José M

    2007-06-01

    In higher plants, ADP-glucose pyrophosphorylase (ADPGlc-PPase) is a heterotetrameric enzyme comprised of two small and two large subunits. Potato-Arabidopsis hybrid ADPGlc-PPases were generated and their regulatory properties analyzed. We show that ADPGlc-PPase subunits from two different species can interact, producing active enzymes with new regulatory properties. Depending on the subunit combinations, hybrid heterotetramers showed responses to allosteric effectors [3-phosphoglycerate (3-PGA) and Pi] in the micromolar or millimolar range. While hybrid potato small subunit (PSS) and the Arabidopsis large subunit APL1 showed an extremely sensitive response to 3-PGA and Pi, hybrid PSS/Arabidopsis APL2 was very insensitive to them. Intermediate responses were determined for other subunit combinations. PMID:17452341

  2. Ostreococcus tauri ADP-glucose Pyrophosphorylase Reveals Alternative Paths for the Evolution of Subunit Roles*

    PubMed Central

    Kuhn, Misty L.; Falaschetti, Christine A.; Ballicora, Miguel A.

    2009-01-01

    ADP-glucose pyrophosphorylase controls starch synthesis in plants and is an interesting case to study the evolution and differentiation of roles in heteromeric enzymes. It includes two homologous subunits, small (S) and large (L), that originated from a common photosynthetic eukaryotic ancestor. In present day organisms, these subunits became complementary after loss of certain roles in a process described as subfunctionalization. For instance, the potato tuber enzyme has a noncatalytic L subunit that complements an S subunit with suboptimal allosteric properties. To understand the evolution of catalysis and regulation in this family, we artificially synthesized both subunit genes from the unicellular alga Ostreococcus tauri. This is among the most ancient species in the green lineage that diverged from the ancestor of all green plants and algae. After heterologous gene expression, we purified and characterized the proteins. The O. tauri enzyme was not redox-regulated, suggesting that redox regulation of ADP-glucose pyrophosphorylases appeared later in evolution. The S subunit had a typical low apparent affinity for the activator 3-phosphoglycerate, but it was atypically defective in the catalytic efficiency (Vmax/Km) for the substrate Glc-1-P. The L subunit needed the S subunit for soluble expression. In the presence of a mutated S subunit (to avoid interference), the L subunit had a high apparent affinity for 3-phosphoglycerate and substrates suggesting a leading role in catalysis. Therefore, the subfunctionalization of the O. tauri enzyme was different from previously described cases. To the best of our knowledge, this is the first biochemical description of a system with alternative subfunctionalization paths. PMID:19737928

  3. A truncated version of an ADP-glucose pyrophosphorylase promoter from potato specifies guard cell-selective expression in transgenic plants.

    PubMed Central

    Müller-Röber, B; La Cognata, U; Sonnewald, U; Willmitzer, L

    1994-01-01

    ADP-glucose pyrophosphorylase (AGPase) is a key regulatory enzyme in starch biosynthesis in higher plants. A 3.2-kb promoter of the large subunit gene of the AGPase from potato has been isolated and its activity analyzed in transgenic potato and tobacco plants using a promoter-beta-glucuronidase fusion system. The promoter was active in various starch-containing cells, including guard cells, tuber parenchyma cells, and the starch sheath layer of stems and petioles. No expression was observed in mesophyll cells. Analysis of various promoter derivatives showed that with respect to expression in petioles and stems, essential elements must be located in the 5' distal region of the promoter, whereas elements important for expression in tuber parenchyma cells are located in an internal fragment comprising nucleotides from positions -500 to -1200. Finally, a 0.3-kb 5' proximal promoter fragment was identified that was sufficient to obtain exclusive expression in guard cells of transgenic potato and tobacco plants. The implications of our observations are discussed with respect to starch synthesis in various tissues and the use of the newly identified promoter as a tool for stomatal biology. PMID:8038601

  4. Structural Analysis of ADP-Glucose Pyrophosphorylase From the Bacterium Agrobacterium Tumefaciens

    SciTech Connect

    Cupp-Vickery, J.R.; Igarashi, R.Y.; Perez, M.; Poland, M.; Meyer, C.R.

    2009-05-14

    ADP-glucose pyrophosphorylase (ADPGlc PPase) catalyzes the conversion of glucose 1-phosphate and ATP to ADP-glucose and pyrophosphate. As a key step in glucan synthesis, the ADPGlc PPases are highly regulated by allosteric activators and inhibitors in accord with the carbon metabolism pathways of the organism. Crystals of Agrobacterium tumefaciens ADPGlc PPase were obtained using lithium sulfate as a precipitant. A complete anomalous selenomethionyl derivative X-ray diffraction data set was collected with unit cell dimensions a = 85.38 {angstrom}, b = 93.79 {angstrom}, and c = 140.29 {angstrom} ({alpha} = {beta} = {gamma} = 90{sup o}) and space group I{sub 222}. The A. tumefaciens ADPGlc PPase model was refined to 2.1 {angstrom} with an R{sub factor} = 22% and R{sub free} = 26.6%. The model consists of two domains: an N-terminal {alpha}{beta}{alpha} sandwich and a C-terminal parallel {beta}-helix. ATP and glucose 1-phosphate were successfully modeled in the proposed active site, and site-directed mutagenesis of conserved glycines in this region (G20, G21, and G23) resulted in substantial loss of activity. The interface between the N- and the C-terminal domains harbors a strong sulfate-binding site, and kinetic studies revealed that sulfate is a competitive inhibitor for the allosteric activator fructose 6-phosphate. These results suggest that the interface between the N- and C-terminal domains binds the allosteric regulator, and fructose 6-phosphate was modeled into this region. The A. tumefaciens ADPGlc PPase/fructose 6-phosphate structural model along with sequence alignment analysis was used to design mutagenesis experiments to expand the activator specificity to include fructose 1,6-bisphosphate. The H379R and H379K enzymes were found to be activated by fructose 1,6-bisphosphate.

  5. Maize endosperm ADP-glucose pyrophosphorylase SHRUNKEN2 and BRITTLE2 subunit interactions

    PubMed Central

    Greene, TW; Hannah, LC

    1998-01-01

    ADP-glucose pyrophosphorylase (AGP) represents a key regulatory step in polysaccharide synthesis in organisms ranging from bacteria to plants. Higher plant AGPs are complex in nature and are heterotetramers consisting of two similar but distinct subunits. How the subunits are assembled into enzymatically active polymers is not yet understood. Here, we address this issue by using naturally occurring null mutants of the Shrunken2 (Sh2) and Brittle2 (Bt2) loci of maize as well as the yeast two-hybrid expression system. In the absence of the maize endosperm large AGP subunit (SH2), the BT2 subunit remains as a monomer in the developing endosperm. In contrast, the SH2 protein, in the absence of BT2, is found in a complex of 100 kD. A direct interaction between SH2 and BT2 was proven when they were both expressed in yeast. Several motifs are essential for SH2:BT2 interaction because truncations removing the N or C terminus of either subunit eliminate SH2:BT2 interactions. Analysis of subunit interaction mutants (sim) also identified motifs essential for protein interactions. PMID:9707530

  6. Two Arabidopsis ADP-Glucose Pyrophosphorylase Large Subunits (APL1 and APL2) Are Catalytic1

    PubMed Central

    Ventriglia, Tiziana; Kuhn, Misty L.; Ruiz, Ma Teresa; Ribeiro-Pedro, Marina; Valverde, Federico; Ballicora, Miguel A.; Preiss, Jack; Romero, José M.

    2008-01-01

    ADP-glucose (Glc) pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in starch biosynthesis. Higher plant ADP-Glc PPase is a heterotetramer (α2β2) consisting of two small and two large subunits. There is increasing evidence that suggests that catalytic and regulatory properties of the enzyme from higher plants result from the synergy of both types of subunits. In Arabidopsis (Arabidopsis thaliana), two genes encode small subunits (APS1 and APS2) and four large subunits (APL1–APL4). Here, we show that in Arabidopsis, APL1 and APL2, besides their regulatory role, have catalytic activity. Heterotetramers formed by combinations of a noncatalytic APS1 and the four large subunits showed that APL1 and APL2 exhibited ADP-Glc PPase activity with distinctive sensitivities to the allosteric activator (3-phosphoglycerate). Mutation of the Glc-1-P binding site of Arabidopsis and potato (Solanum tuberosum) isoforms confirmed these observations. To determine the relevance of these activities in planta, a T-DNA mutant of APS1 (aps1) was characterized. aps1 is starchless, lacks ADP-Glc PPase activity, APS1 mRNA, and APS1 protein, and is late flowering in long days. Transgenic lines of the aps1 mutant, expressing an inactivated form of APS1, recovered the wild-type phenotype, indicating that APL1 and APL2 have catalytic activity and may contribute to ADP-Glc synthesis in planta. PMID:18614708

  7. Two Arabidopsis ADP-glucose pyrophosphorylase large subunits (APL1 and APL2) are catalytic.

    PubMed

    Ventriglia, Tiziana; Kuhn, Misty L; Ruiz, Ma Teresa; Ribeiro-Pedro, Marina; Valverde, Federico; Ballicora, Miguel A; Preiss, Jack; Romero, José M

    2008-09-01

    ADP-glucose (Glc) pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in starch biosynthesis. Higher plant ADP-Glc PPase is a heterotetramer (alpha(2)beta(2)) consisting of two small and two large subunits. There is increasing evidence that suggests that catalytic and regulatory properties of the enzyme from higher plants result from the synergy of both types of subunits. In Arabidopsis (Arabidopsis thaliana), two genes encode small subunits (APS1 and APS2) and four large subunits (APL1-APL4). Here, we show that in Arabidopsis, APL1 and APL2, besides their regulatory role, have catalytic activity. Heterotetramers formed by combinations of a noncatalytic APS1 and the four large subunits showed that APL1 and APL2 exhibited ADP-Glc PPase activity with distinctive sensitivities to the allosteric activator (3-phosphoglycerate). Mutation of the Glc-1-P binding site of Arabidopsis and potato (Solanum tuberosum) isoforms confirmed these observations. To determine the relevance of these activities in planta, a T-DNA mutant of APS1 (aps1) was characterized. aps1 is starchless, lacks ADP-Glc PPase activity, APS1 mRNA, and APS1 protein, and is late flowering in long days. Transgenic lines of the aps1 mutant, expressing an inactivated form of APS1, recovered the wild-type phenotype, indicating that APL1 and APL2 have catalytic activity and may contribute to ADP-Glc synthesis in planta. PMID:18614708

  8. Molecular characterization of multiple cDNA clones for ADP-glucose pyrophosphorylase from Arabidopsis thaliana.

    PubMed

    Villand, P; Olsen, O A; Kleczkowski, L A

    1993-12-01

    PCR amplification of cDNA prepared from poly(A)+ RNA from aerial parts of Arabidopsis thaliana, using degenerate nucleotide primers based on conserved regions between the large and small subunits of ADP-glucose pyrophosphorylase (AGP), yielded four different cDNAs of ca. 550 nucleotides each. Based on derived amino acid sequences, the identities between the clones varied from 49 to 69%. Sequence comparison to previously published cDNAs for AGP from various species and tissues has revealed that three of the amplified cDNAs (ApL1, ApL2 and ApL3) correspond to the large subunit of AGP, and one cDNA (ApS) encodes the small subunit of AGP. Both ApL1 and ApS were subsequently found to be present in a cDNA library made from Arabidopsis leaves. All four PCR products are encoded by single genes, as found by genomic Southern analysis. PMID:8292792

  9. The different large subunit isoforms of Arabidopsis thaliana ADP-glucose pyrophosphorylase confer distinct kinetic and regulatory properties to the heterotetrameric enzyme.

    PubMed

    Crevillén, Pedro; Ballicora, Miguel A; Mérida, Angel; Preiss, Jack; Romero, José M

    2003-08-01

    ADP-glucose pyrophosphorylase catalyzes the first and limiting step in starch biosynthesis and is allosterically regulated by the levels of 3-phosphoglycerate and phosphate in plants. ADP-glucose pyrophosphorylases from plants are heterotetramers composed of two types of subunits (small and large). In this study, the six Arabidopsis thaliana genes coding for ADP-glucose pyrophosphorylase isoforms (two small and four large subunits) have been cloned and expressed in an Escherichia coli mutant deficient in ADP-glucose pyrophosphorylase activity. The co-expression of the small subunit APS1 with the different Arabidopsis large subunits (APL1, APL2, APL3, and APL4) resulted in heterotetramers with different regulatory and kinetic properties. Heterotetramers composed of APS1 and APL1 showed the highest sensitivity to the allosteric effectors as well as the highest apparent affinity for the substrates (glucose-1-phosphate and ATP), whereas heterotetramers formed by APS1 and APL2 showed the lower response to allosteric effectors and the lower affinity for the substrates. No activity was detected for the second gene coding for a small subunit isoform (APS2) annotated in the Arabidopsis genome. This lack of activity is possibly due to the absence of essential amino acids involved in catalysis and/or in the binding of glucose-1-phosphate and 3-phosphoglycerate. Kinetic and regulatory properties of the different heterotetramers, together with sequence analysis has allowed us to make a distinction between sink and source enzymes, because the combination of different large subunits would provide a high plasticity to ADP-glucose pyrophosphorylase activity and regulation. This is the first experimental data concerning the role that all the ADP-glucose pyrophosphorylase isoforms play in a single plant species. This phenomenon could have an important role in vivo, because different large subunits would confer distinct regulatory properties to ADP-glucose pyrophosphorylase according

  10. The ancestral activation promiscuity of ADP-glucose pyrophosphorylases from oxygenic photosynthetic organisms

    PubMed Central

    2013-01-01

    Background ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in the synthesis of glycogen in bacteria and starch in algae and plants. In oxygenic photosynthetic organisms, ADP-Glc PPase is mainly activated by 3-phosphoglycerate (3-PGA) and to a lesser extent by other metabolites. In this work, we analyzed the activation promiscuity of ADP-Glc PPase subunits from the cyanobacterium Anabaena PCC 7120, the green alga Ostreococcus tauri, and potato (Solanum tuberosum) tuber by comparing a specificity constant for 3-PGA, fructose-1,6-bisphosphate (FBP), fructose-6-phosphate, and glucose-6-phosphate. Results The 3-PGA specificity constant for the enzymes from Anabaena (homotetramer), O. tauri, and potato tuber was considerably higher than for other activators. O. tauri and potato tuber enzymes were heterotetramers comprising homologous small and large subunits. Conversely, the O. tauri small subunit (OtaS) homotetramer was more promiscuous because its FBP specificity constant was similar to that for 3-PGA. To explore the role of both OtaS and OtaL (O. tauri large subunit) in determining the specificity of the heterotetramer, we knocked out the catalytic activity of each subunit individually by site-directed mutagenesis. Interestingly, the mutants OtaSD148A/OtaL and OtaS/OtaLD171A had higher specificity constants for 3-PGA than for FBP. Conclusions After gene duplication, OtaS seemed to have lost specificity for 3-PGA compared to FBP. This was physiologically and evolutionarily feasible because co-expression of both subunits restored the specificity for 3-PGA of the resulting heterotetrameric wild type enzyme. This widespread promiscuity seems to be ancestral and intrinsic to the enzyme family. Its presence could constitute an efficient evolutionary mechanism to accommodate the ADP-Glc PPase regulation to different metabolic needs. PMID:23433303

  11. Genetic Variability in Carbon Fixation, Sucrose-P-Synthase and ADP Glucose Pyrophosphorylase in Maize Plants of Differing Growth Rate

    PubMed Central

    Rocher, J. P.; Prioul, J. L.; Lecharny, A.; Reyss, A.; Joussaume, M.

    1989-01-01

    The net photosynthetic rate and the activities of ribulose 1,5 bisphosphate carboxylase (RubisCo), phosphoenolpyruvate carboxylase, sucrose-P-synthase, and ADP glucose-pyrophosphorylase, key enzymes of the leaf carbohydrate metabolism were compared in eight maize (Zea mays L.) genotypes presenting large differences in growth rate. The sucrose-P-synthase activity varied in the ratio 1 to 3 from the less active to the more active genotype and this variation was highly correlated with those in growth rate. ADP glucose pyrophosphorylase activity was not significantly different from one genotype to another whatever the basis for expression, leaf area, or soluble protein. The photosynthetic rate varied with similar amplitude (1:1) to the RubisCo activity or RubisCo quantity but the correlation with growth rate was highly significant for photosynthesis and nonsignificant for RubisCo or phosphoenolpyruvate carboxylase. So, in our series of genotypes the sucrose synthesis capacities as expressed by sucrose phosphate synthase activity seem to have a good predicting value for mean growth rate at a young stage. PMID:16666558

  12. Regulatory properties of ADP glucose pyrophosphorylase are required for adjustment of leaf starch synthesis in different photoperiods.

    PubMed

    Mugford, Sam T; Fernandez, Olivier; Brinton, Jemima; Flis, Anna; Krohn, Nicole; Encke, Beatrice; Feil, Regina; Sulpice, Ronan; Lunn, John E; Stitt, Mark; Smith, Alison M

    2014-12-01

    Arabidopsis (Arabidopsis thaliana) leaves synthesize starch faster in short days than in long days, but the mechanism that adjusts the rate of starch synthesis to daylength is unknown. To understand this mechanism, we first investigated whether adjustment occurs in mutants lacking components of the circadian clock or clock output pathways. Most mutants adjusted starch synthesis to daylength, but adjustment was compromised in plants lacking the GIGANTEA or FLAVIN-BINDING, KELCH REPEAT, F BOX1 components of the photoperiod-signaling pathway involved in flowering. We then examined whether the properties of the starch synthesis enzyme adenosine 5'-diphosphate-glucose pyrophosphorylase (AGPase) are important for adjustment of starch synthesis to daylength. Modulation of AGPase activity is known to bring about short-term adjustments of photosynthate partitioning between starch and sucrose (Suc) synthesis. We found that adjustment of starch synthesis to daylength was compromised in plants expressing a deregulated bacterial AGPase in place of the endogenous AGPase and in plants containing mutant forms of the endogenous AGPase with altered allosteric regulatory properties. We suggest that the rate of starch synthesis is in part determined by growth rate at the end of the preceding night. If growth at night is low, as in short days, there is a delay before growth recovers during the next day, leading to accumulation of Suc and stimulation of starch synthesis via activation of AGPase. If growth at night is fast, photosynthate is used for growth at the start of the day, Suc does not accumulate, and starch synthesis is not up-regulated. PMID:25293961

  13. Conserved residues of the Pro103–Arg115 loop are involved in triggering the allosteric response of the Escherichia coli ADP-glucose pyrophosphorylase

    PubMed Central

    Hill, Benjamin L; Wong, Jennifer; May, Brian M; Huerta, Fidel B; Manley, Tara E; Sullivan, Peter RF; Olsen, Kenneth W; Ballicora, Miguel A

    2015-01-01

    The synthesis of glycogen in bacteria and starch in plants is allosterically controlled by the production of ADP-glucose by ADP-glucose pyrophosphorylase. Using computational studies, site-directed mutagenesis, and kinetic characterization, we found a critical region for transmitting the allosteric signal in the Escherichia coli ADP-glucose pyrophosphorylase. Molecular dynamics simulations and structural comparisons with other ADP-glucose pyrophosphorylases provided information to hypothesize that a Pro103–Arg115 loop is part of an activation path. It had strongly correlated movements with regions of the enzyme associated with regulation and ATP binding, and a network analysis showed that the optimal network pathways linking ATP and the activator binding Lys39 mainly involved residues of this loop. This hypothesis was biochemically tested by mutagenesis. We found that several alanine mutants of the Pro103–Arg115 loop had altered activation profiles for fructose-1,6-bisphosphate. Mutants P103A, Q106A, R107A, W113A, Y114A, and R115A had the most altered kinetic profiles, primarily characterized by a lack of response to fructose-1,6-bisphosphate. This loop is a distinct insertional element present only in allosterically regulated sugar nucleotide pyrophosphorylases that could have been acquired to build a triggering mechanism to link proto-allosteric and catalytic sites. PMID:25620658

  14. Structure and expression analysis of genes encoding ADP-glucose pyrophosphorylase large subunit in wheat and its relatives.

    PubMed

    Zhang, Xiao-Wei; Li, Si-Yu; Zhang, Ling-Ling; Yang, Qiang; Jiang, Qian-Tao; Ma, Jian; Qi, Peng-Fei; Li, Wei; Chen, Guo-Yue; Lan, Xiu-Jin; Deng, Mei; Lu, Zhen-Xiang; Liu, Chunji; Wei, Yu-Ming; Zheng, You-Liang

    2016-07-01

    ADP-glucose pyrophosphorylase (AGP), which consists of two large subunits (AGP-L) and two small subunits (AGP-S), controls the rate-limiting step in the starch biosynthetic pathway. In this study, a full-length open reading frame (ORF) of AGP-L gene (named as Agp2) in wheat and a series of Agp2 gene sequences in wheat relatives were isolated. The coding region of Agp2 contained 15 exons and 14 introns including a full-length ORF of 1566 nucleotides, and the deduced protein contained 522 amino acids (57.8 kDa). Generally, the phylogenetic tree of Agp2 indicated that sequences from A- and D-genome donor species were most similar to each other and sequences from B-genome donor species contained more variation. Starch accumulation and Agp2 expression in wheat grains reached their peak at 21 and 15 days post anthesis (DPA), respectively. PMID:27299732

  15. ADP-glucose pyrophosphorylase is localized to both the cytoplasm and plastids in developing pericarp of tomato fruit

    NASA Technical Reports Server (NTRS)

    Chen, B. Y.; Wang, Y.; Janes, H. W.

    1998-01-01

    The intracellular location of ADP-glucose pyrophosphorylase (AGP) in developing pericarp of tomato (Lycopersicon esculentum Mill) has been investigated by immunolocalization. With the use of a highly specific anti-tomato fruit AGP antibody, the enzyme was localized in cytoplasm as well as plastids at both the light and electron microscope levels. The immunogold particles in plastids were localized in the stroma and at the surface of the starch granule, whereas those in the cytoplasm occurred in cluster-like patterns. Contrary to the fruit, the labeling in tomato leaf cells occurred exclusively in the chloroplasts. These data demonstrate that AGP is localized to both the cytoplasm and plastids in developing pericarp cells of tomato.

  16. On the Kinetic and Allosteric Regulatory Properties of the ADP-Glucose Pyrophosphorylase from Rhodococcus jostii: An Approach to Evaluate Glycogen Metabolism in Oleaginous Bacteria

    PubMed Central

    Cereijo, Antonela E.; Asencion Diez, Matías D.; Dávila Costa, José S.; Alvarez, Héctor M.; Iglesias, Alberto A.

    2016-01-01

    Rhodococcus spp. are oleaginous bacteria that accumulate glycogen during exponential growth. Despite the importance of these microorganisms in biotechnology, little is known about the regulation of carbon and energy storage, mainly the relationship between glycogen and triacylglycerols metabolisms. Herein, we report the molecular cloning and heterologous expression of the gene coding for ADP-glucose pyrophosphorylase (EC 2.7.7.27) of Rhodococcus jostii, strain RHA1. The recombinant enzyme was purified to electrophoretic homogeneity to accurately characterize its oligomeric, kinetic, and regulatory properties. The R. jostii ADP-glucose pyrophosphorylase is a homotetramer of 190 kDa exhibiting low basal activity to catalyze synthesis of ADP-glucose, which is markedly influenced by different allosteric effectors. Glucose-6P, mannose-6P, fructose-6P, ribose-5P, and phosphoenolpyruvate were major activators; whereas, NADPH and 6P-gluconate behaved as main inhibitors of the enzyme. The combination of glucose-6P and other effectors (activators or inhibitors) showed a cross-talk effect suggesting that the different metabolites could orchestrate a fine regulation of ADP-glucose pyrophosphorylase in R. jostii. The enzyme exhibited some degree of affinity toward ATP, GTP, CTP, and other sugar-1P substrates. Remarkably, the use of glucosamine-1P was sensitive to allosteric activation. The relevance of the fine regulation of R. jostii ADP-glucose pyrophosphorylase is further analyzed in the framework of proteomic studies already determined for the bacterium. Results support a critical role for glycogen as a temporal reserve that provides a pool of carbon able of be re-routed to produce long-term storage of lipids under certain conditions. PMID:27313571

  17. A polymorphic motif in the small subunit of ADP-glucose pyrophosphorylase modulates interactions between the small and large subunits.

    PubMed

    Cross, Joanna M; Clancy, Maureen; Shaw, Janine R; Boehlein, Susan K; Greene, Thomas W; Schmidt, Robert R; Okita, Thomas W; Hannah, L Curtis

    2005-02-01

    The heterotetrameric, allosterically regulated enzyme, adenosine-5'-diphosphoglucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step in starch synthesis. Despite vast differences in allosteric properties and a long evolutionary separation, heterotetramers of potato small subunit and maize large subunit have activity comparable to either parent in an Escherichia coli expression system. In contrast, co-expression of maize small subunit with the potato large subunit produces little activity as judged by in vivo activity stain. To pinpoint the region responsible for differential activity, we expressed chimeric maize/potato small subunits in E. coli. This identified a 55-amino acid motif of the potato small subunit that is critical for glycogen production when expressed with the potato large subunit. Potato and maize small subunit sequences differ at five amino acids in this motif. Replacement experiments revealed that at least four amino acids of maize origin were required to reduce staining. An AGPase composed of a chimeric potato small subunit containing the 55-amino acid maize motif with the potato large subunit exhibited substantially less affinity for the substrates, glucose-1-phosphate and ATP and an increased Ka for the activator, 3-phosphoglyceric acid. Placement of the potato motif into the maize small subunit restored glycogen synthesis with the potato large subunit. Hence, a small polymorphic motif within the small subunit influences both catalytic and allosteric properties by modulating subunit interactions. PMID:15686515

  18. [Enhancement of photoassimilate utilization by manipulation of ADP-glucose pyrophosphorylase gene]. Final progress report

    SciTech Connect

    Okita, T.W.

    1999-04-01

    Part 1 of this research focuses on patterns of gene expression of ADPG-pyrophosphorylase in native and transgenic potato plants. To elucidate the mechanism controlling AGP expression during plant development, the expression of the potato tuber AGP small subunit (sAGP) gene was analyzed in transgenic potato plants using a promoter-{beta}-glucuronidase expression system. Part II evaluated the structure-function relationships of AGP.

  19. The ADP-glucose pyrophosphorylase from Streptococcus mutans provides evidence for the regulation of polysaccharide biosynthesis in Firmicutes.

    PubMed

    Asención Diez, Matías D; Demonte, Ana M; Guerrero, Sergio A; Ballicora, Miguel A; Iglesias, Alberto A

    2013-12-01

    Streptococcus mutans is the leading cause of dental caries worldwide. The bacterium accumulates a glycogen-like internal polysaccharide, which mainly contributes to its carionegic capacity. S.mutans has two genes (glgC and glgD) respectively encoding putative ADP-glucose pyrophosphorylases (ADP-Glc PPase), a key enzyme for glycogen synthesis in most bacteria. Herein, we report the molecular cloning and recombinant expression of both genes (separately or together) followed by the characterization of the respective enzymes. When expressed individually GlgC had ADP-Glc PPase activity, whereas GlgD was inactive. Interestingly, the coexpressed GlgC/GlgD protein was one order of magnitude more active than GlgC alone. Kinetic characterization of GlgC and GlgC/GlgD pointed out remarkable differences between them. Fructose-1,6-bis-phosphate activated GlgC by twofold, but had no effect on GlgC/GlgD. Conversely, phospho-enol-pyruvate and inorganic salts inhibited GlgC/GlgD without affecting GlgC. However, in the presence of fructose-1,6-bis-phosphate GlgC acquired a GlgC/GlgD-like behaviour, becoming sensitive to the stated inhibitors. Results indicate that S. mutans ADP-Glc PPase is an allosteric regulatory enzyme exhibiting sensitivity to modulation by key intermediates of carbohydrates metabolism in the cell. The particular regulatory properties of the S.mutans enzyme agree with phylogenetic analysis, where GlgC and GlgD proteins found in other Firmicutes arrange in distinctive clusters. PMID:24112771

  20. Enhanced stability of maize endosperm ADP-glucose pyrophosphorylase is gained through mutants that alter subunit interactions.

    PubMed

    Greene, T W; Hannah, L C

    1998-10-27

    Temperature lability of ADP-glucose pyrophosphorylase (AGP; glucose-1-phosphate adenylyltransferase; ADP: alpha-D-glucose-1-phosphate adenylyltransferase, EC 2.7.7.27), a key starch biosynthetic enzyme, may play a significant role in the heat-induced loss in maize seed weight and yield. Here we report the isolation and characterization of heat-stable variants of maize endosperm AGP. Escherichia coli cells expressing wild type (WT) Shrunken2 (Sh2), and Brittle2 (Bt2) exhibit a reduced capacity to produce glycogen when grown at 42 degreesC. Mutagenesis of Sh2 and coexpression with WT Bt2 led to the isolation of multiple mutants capable of synthesizing copious amounts of glycogen at this temperature. An increase in AGP stability was found in each of four mutants examined. Initial characterization revealed that the BT2 protein was elevated in two of these mutants. Yeast two-hybrid studies were conducted to determine whether the mutant SH2 proteins more efficiently recruit the BT2 subunit into tetramer assembly. These experiments showed that replacement of WT SH2 with the heat-stable SH2HS33 enhanced interaction between the SH2 and BT2 subunits. In agreement, density gradient centrifugation of heated and nonheated extracts from WT and one of the mutants, Sh2hs33, identified a greater propensity for heterotetramer dissociation in WT AGP. Sequencing of Sh2hs33 and several other mutants identified a His-to-Tyr mutation at amino acid position 333. Hence, a single point mutation in Sh2 can increase the stability of maize endosperm AGP through enhanced subunit interactions. PMID:9789090

  1. Analysis of ADP-glucose pyrophosphorylase expression during turion formation induced by abscisic acid in Spirodela polyrhiza (greater duckweed)

    PubMed Central

    2012-01-01

    Background Aquatic plants differ in their development from terrestrial plants in their morphology and physiology, but little is known about the molecular basis of the major phases of their life cycle. Interestingly, in place of seeds of terrestrial plants their dormant phase is represented by turions, which circumvents sexual reproduction. However, like seeds turions provide energy storage for starting the next growing season. Results To begin a characterization of the transition from the growth to the dormant phase we used abscisic acid (ABA), a plant hormone, to induce controlled turion formation in Spirodela polyrhiza and investigated their differentiation from fronds, representing their growth phase, into turions with respect to morphological, ultra-structural characteristics, and starch content. Turions were rich in anthocyanin pigmentation and had a density that submerged them to the bottom of liquid medium. Transmission electron microscopy (TEM) of turions showed in comparison to fronds shrunken vacuoles, smaller intercellular space, and abundant starch granules surrounded by thylakoid membranes. Turions accumulated more than 60% starch in dry mass after two weeks of ABA treatment. To further understand the mechanism of the developmental switch from fronds to turions, we cloned and sequenced the genes of three large-subunit ADP-glucose pyrophosphorylases (APLs). All three putative protein and exon sequences were conserved, but the corresponding genomic sequences were extremely variable mainly due to the invasion of miniature inverted-repeat transposable elements (MITEs) into introns. A molecular three-dimensional model of the SpAPLs was consistent with their regulatory mechanism in the interaction with the substrate (ATP) and allosteric activator (3-PGA) to permit conformational changes of its structure. Gene expression analysis revealed that each gene was associated with distinct temporal expression during turion formation. APL2 and APL3 were highly

  2. Regulatory Properties of ADP Glucose Pyrophosphorylase Are Required for Adjustment of Leaf Starch Synthesis in Different Photoperiods1[W][OPEN

    PubMed Central

    Mugford, Sam T.; Fernandez, Olivier; Brinton, Jemima; Flis, Anna; Krohn, Nicole; Encke, Beatrice; Feil, Regina; Sulpice, Ronan; Lunn, John E.; Stitt, Mark; Smith, Alison M.

    2014-01-01

    Arabidopsis (Arabidopsis thaliana) leaves synthesize starch faster in short days than in long days, but the mechanism that adjusts the rate of starch synthesis to daylength is unknown. To understand this mechanism, we first investigated whether adjustment occurs in mutants lacking components of the circadian clock or clock output pathways. Most mutants adjusted starch synthesis to daylength, but adjustment was compromised in plants lacking the GIGANTEA or FLAVIN-BINDING, KELCH REPEAT, F BOX1 components of the photoperiod-signaling pathway involved in flowering. We then examined whether the properties of the starch synthesis enzyme adenosine 5′-diphosphate-glucose pyrophosphorylase (AGPase) are important for adjustment of starch synthesis to daylength. Modulation of AGPase activity is known to bring about short-term adjustments of photosynthate partitioning between starch and sucrose (Suc) synthesis. We found that adjustment of starch synthesis to daylength was compromised in plants expressing a deregulated bacterial AGPase in place of the endogenous AGPase and in plants containing mutant forms of the endogenous AGPase with altered allosteric regulatory properties. We suggest that the rate of starch synthesis is in part determined by growth rate at the end of the preceding night. If growth at night is low, as in short days, there is a delay before growth recovers during the next day, leading to accumulation of Suc and stimulation of starch synthesis via activation of AGPase. If growth at night is fast, photosynthate is used for growth at the start of the day, Suc does not accumulate, and starch synthesis is not up-regulated. PMID:25293961

  3. A sepal-expressed ADP-glucose pyrophosphorylase gene (NtAGP) is required for petal expansion growth in 'Xanthi' tobacco.

    PubMed

    Kwak, Man Sup; Min, Sung Ran; Lee, Si-Myung; Kim, Kyung-Nam; Liu, Jang Ryol; Paek, Kyung-Hee; Shin, Jeong Sheop; Bae, Jung Myung

    2007-09-01

    In this study, a tobacco (Nicotiana tabacum 'Xanthi') ADP-glucose pyrophosphorylase cDNA (NtAGP) was isolated from a flower bud cDNA library and the role of NtAGP in the growth of the floral organ was characterized. The expression of NtAGP was high in the sepal, moderate in the carpel and stamen, and low in the petal tissues. NtAGP-antisense plants produced flowers with abnormal petal limbs due to the early termination of the expansion growth of the petal limbs between the corolla lobes. Microscopic observation of the limb region revealed that cell expansion was limited in NtAGP-antisense plants but that cell numbers remained unchanged. mRNA levels of NtAGP, ADP-glucose pyrophosphorylase activity, and starch content in the sepal tissues of NtAGP-antisense plants were reduced, resulting in significantly lower levels of sugars (sucrose, glucose, and fructose) in the petal limbs. The feeding of these sugars to flower buds of the NtAGP-antisense plants restored the expansion growth in the limb area between the corolla lobes. Expansion growth of the petal limb between the corolla lobes was severely arrested in 'Xanthi' flowers from which sepals were removed, indicating that sepal carbohydrates are essential for petal limb expansion growth. These results demonstrate that NtAGP plays a crucial role in the morphogenesis of petal limbs in 'Xanthi' through the synthesis of starch, which is the main carbohydrate source for expansion growth of petal limbs, in sepal tissues. PMID:17660352

  4. Carbon Dynamics, Development and Stress Responses in Arabidopsis: Involvement of the APL4 Subunit of ADP-Glucose Pyrophosphorylase (Starch Synthesis)

    PubMed Central

    Sulmon, Cécile; Gouesbet, Gwenola; Ramel, Fanny; Cabello-Hurtado, Francisco; Penno, Christophe; Bechtold, Nicole; Couée, Ivan; Amrani, Abdelhak El

    2011-01-01

    An Arabidopsis thaliana T-DNA insertional mutant was identified and characterized for enhanced tolerance to the singlet-oxygen-generating herbicide atrazine in comparison to wild-type. This enhanced atrazine tolerance mutant was shown to be affected in the promoter structure and in the regulation of expression of the APL4 isoform of ADP-glucose pyrophosphorylase, a key enzyme of the starch biosynthesis pathway, thus resulting in decrease of APL4 mRNA levels. The impact of this regulatory mutation was confirmed by the analysis of an independent T-DNA insertional mutant also affected in the promoter of the APL4 gene. The resulting tissue-specific modifications of carbon partitioning in plantlets and the effects on plantlet growth and stress tolerance point out to specific and non-redundant roles of APL4 in root carbon dynamics, shoot-root relationships and sink regulations of photosynthesis. Given the effects of exogenous sugar treatments and of endogenous sugar levels on atrazine tolerance in wild-type Arabidopsis plantlets, atrazine tolerance of this apl4 mutant is discussed in terms of perception of carbon status and of investment of sugar allocation in xenobiotic and oxidative stress responses. PMID:22073207

  5. ADP-glucose pyrophosphorylase-deficient pea embryos reveal specific transcriptional and metabolic changes of carbon-nitrogen metabolism and stress responses.

    PubMed

    Weigelt, Kathleen; Küster, Helge; Rutten, Twan; Fait, Aaron; Fernie, Alisdair R; Miersch, Otto; Wasternack, Claus; Emery, R J Neil; Desel, Christine; Hosein, Felicia; Müller, Martin; Saalbach, Isolde; Weber, Hans

    2009-01-01

    We present a comprehensive analysis of ADP-glucose pyrophosphorylase (AGP)-repressed pea (Pisum sativum) seeds using transcript and metabolite profiling to monitor the effects that reduced carbon flow into starch has on carbon-nitrogen metabolism and related pathways. Changed patterns of transcripts and metabolites suggest that AGP repression causes sugar accumulation and stimulates carbohydrate oxidation via glycolysis, tricarboxylic acid cycle, and mitochondrial respiration. Enhanced provision of precursors such as acetyl-coenzyme A and organic acids apparently support other pathways and activate amino acid and storage protein biosynthesis as well as pathways fed by cytosolic acetyl-coenzyme A, such as cysteine biosynthesis and fatty acid elongation/metabolism. As a consequence, the resulting higher nitrogen (N) demand depletes transient N storage pools, specifically asparagine and arginine, and leads to N limitation. Moreover, increased sugar accumulation appears to stimulate cytokinin-mediated cell proliferation pathways. In addition, the deregulation of starch biosynthesis resulted in indirect changes, such as increased mitochondrial metabolism and osmotic stress. The combined effect of these changes is an enhanced generation of reactive oxygen species coupled with an up-regulation of energy-dissipating, reactive oxygen species protection, and defense genes. Transcriptional activation of mitogen-activated protein kinase pathways and oxylipin synthesis indicates an additional activation of stress signaling pathways. AGP-repressed embryos contain higher levels of jasmonate derivatives; however, this increase is preferentially in nonactive forms. The results suggest that, although metabolic/osmotic alterations in iAGP pea seeds result in multiple stress responses, pea seeds have effective mechanisms to circumvent stress signaling under conditions in which excessive stress responses and/or cellular damage could prematurely initiate senescence or apoptosis. PMID

  6. Site-directed mutagenesis of Lysine{sup 382}, the activator-binding site, of ADP-Glucose pyrophosphorylase from Anabaena PCC 6120

    SciTech Connect

    Sheng, Jun; Charng, Yee-yung; Preiss, J.

    1996-03-05

    Previous studies have shown that a highly conserved lysyl residue (Lys{sup 419}) near the C-terminus of Anabaena ADP-glucose pyrophosphorylase is involved in the binding of 3-P-glycerate, the allosteric activator. Phosphopyridoxylation of the K419R mutant enzyme modified another conserved lysyl residue (Lys{sup 382}), suggesting that this residue might be also located within the activator-binding site. Site-directed mutagenesis of Lys{sup 382} of the Anabaena enzyme was performed to determine the role of this residue. Replacing Lys{sup 382} with either arginine, alanine, or glutamine produced mutant enzymes with apparent affinities for 3-P-glycerate 10-160-fold lower than that of the wild-type enzyme. The glutamic acid mutant enzyme was inhibited by 3-P-glycerate. These mutations had lesser impact on the kinetic constants for the substrates and inhibitor, P{sub i}, and on the thermal stability. These results indicate that both the charge and size of the residue at position 382 influence the binding of 3-P-glycerate. Site-directed mutagenesis was also performed to obtain a K382R-K419R double mutant. The apparent affinity for 3-P-glycerate of this double-mutant enzyme was 104-fold lower than that of the wild-type enzyme, and the specificity for activator of this mutant enzyme was altered. The K382R-K419R enzyme could not be phosphopyridoxylated, suggesting that other lysine residues are not involved in the binding of 3-P-glycerate. 32 refs., 2 figs., 3 tabs.

  7. Structure Function Relationships of ADP-Glucose Pyrophosphorylase and Branching Enzyme: Manipulation of Their Genes for Alteration of Starch Quanlity and Quantity

    SciTech Connect

    Jack Preiss

    2006-02-16

    Conversion of the Potato tuber ADP-glucose Pyrophopshorylase Regulatory Subunit into a Catalytic Subunit. ADP-glucose synthesis, a rate-limiting reaction in starch synthesis, is catalyzed by ADP-glucose pyrophosphorylase (ADPGlc PPase). The enzyme in plants is allosterically activated by 3-phosphoglycerate (3PGA) and inhibited by inorganic phosphate (Pi) and is composed of two subunits as a heterotetramer, a2b2. Subunit a is the catalytic subunit and subunit b is designated as the regulatory subunit.The b subunit increases the affinty of the activator for the catalytic subunit. Recent results have shown that the subunits are derived from the same ancestor subunit as the regulatory subunit can be converted to a catalytically subunit via mutation of just two amino acids. Lys44 and Thr54 in the large subunit from potato tuber were converted to the homologous catalytic subunit residues, Arg33 and Lys43. The activity of the large subunit mutants cannot be readily tested with a co-expressed wild-type small (catalytic) subunit because of the intrinsic activity of the latter. We co-expressed the regulatory-subunit mutants with SmallD145N, an inactive S subunit in which the catalytic Asp145 was mutated. The activity of the small (catalytic) subunit was reduced more than three orders of magnitude. Coexpression of the L subunit double mutant LargeK44R/T54K with SmallD145N generated an enzyme with considerable activity, 10% and 18% of the wildtype enzyme, in the ADP-glucose synthetic and pyrophosphorolytic direction, respectively. Replacement of those two residues in the small subunit by the homologous amino acids in the L subunits (mutations R33K and K43T) decreased the activity one and two orders of magnitude. The wild-type enzyme and SmallD145NLargeK44R/T54K had very similar kinetic properties indicating that the substrate site has been conserved. The fact that only two mutations in the L subunit restored enzyme activity is very strong evidence that the large subunit is

  8. Structural Comparison, Substrate Specificity, and Inhibitor Binding of AGPase Small Subunit from Monocot and Dicot: Present Insight and Future Potential

    PubMed Central

    Choudhury, Manabendra D.; Modi, Mahendra K.

    2014-01-01

    ADP-glucose pyrophosphorylase (AGPase) is the first rate limiting enzyme of starch biosynthesis pathway and has been exploited as the target for greater starch yield in several plants. The structure-function analysis and substrate binding specificity of AGPase have provided enormous potential for understanding the role of specific amino acid or motifs responsible for allosteric regulation and catalytic mechanisms, which facilitate the engineering of AGPases. We report the three-dimensional structure, substrate, and inhibitor binding specificity of AGPase small subunit from different monocot and dicot crop plants. Both monocot and dicot subunits were found to exploit similar interactions with the substrate and inhibitor molecule as in the case of their closest homologue potato tuber AGPase small subunit. Comparative sequence and structural analysis followed by molecular docking and electrostatic surface potential analysis reveal that rearrangements of secondary structure elements, substrate, and inhibitor binding residues are strongly conserved and follow common folding pattern and orientation within monocot and dicot displaying a similar mode of allosteric regulation and catalytic mechanism. The results from this study along with site-directed mutagenesis complemented by molecular dynamics simulation will shed more light on increasing the starch content of crop plants to ensure the food security worldwide. PMID:25276800

  9. Purification and characterization of adenosine diphosphate glucose pyrophosphorylase from maize/potato mosaics.

    PubMed

    Boehlein, Susan K; Sewell, Aileen K; Cross, Joanna; Stewart, Jon D; Hannah, L Curtis

    2005-07-01

    Adenosine diphosphate glucose pyrophosphorylase (AGPase) catalyzes a rate-limiting step in starch biosynthesis. The reaction produces ADP-glucose and pyrophosphate from glucose-1-P and ATP. Investigations from a number of laboratories have shown that alterations in allosteric properties as well as heat stability of this enzyme have dramatic positive effects on starch synthesis in the potato (Solanum tuberosum) tuber and seeds of important cereals. Here, we report the characterization of purified recombinant mosaic AGPases derived from protein motifs normally expressed in the maize (Zea mays) endosperm and the potato tuber. These exhibit properties that should be advantageous when expressed in plants. We also present an in-depth characterization of the kinetic and allosteric properties of these purified recombinant AGPases. These data point to previously unrecognized roles for known allosteric effectors. PMID:15951484

  10. The two AGPase subunits evolve at different rates in angiosperms, yet they are equally sensitive to activity-altering amino acid changes when expressed in bacteria.

    PubMed

    Georgelis, Nikolaos; Braun, Edward L; Shaw, Janine R; Hannah, L Curtis

    2007-05-01

    The rate of protein evolution is generally thought to reflect, at least in part, the proportion of amino acids within the protein that are needed for proper function. In the case of ADP-glucose pyrophosphorylase (AGPase), this premise led to the hypothesis that, because the AGPase small subunit is more conserved compared with the large subunit, a higher proportion of the amino acids of the small subunit are required for enzyme activity compared with the large subunit. Evolutionary analysis indicates that the AGPase small subunit has been subject to more intense purifying selection than the large subunit in the angiosperms. However, random mutagenesis and expression of the maize (Zea mays) endosperm AGPase in bacteria show that the two AGPase subunits are equally predisposed to enzyme activity-altering amino acid changes when expressed in one environment with a single complementary subunit. As an alternative hypothesis, we suggest that the small subunit exhibits more evolutionary constraints in planta than does the large subunit because it is less tissue specific and thus must form functional enzyme complexes with different large subunits. Independent approaches provide data consistent with this alternative hypothesis. PMID:17496118

  11. Accelerated evolution and coevolution drove the evolutionary history of AGPase sub-units during angiosperm radiation

    PubMed Central

    Corbi, Jonathan; Dutheil, Julien Y.; Damerval, Catherine; Tenaillon, Maud I.; Manicacci, Domenica

    2012-01-01

    Background and Aims ADP-glucose pyrophosphorylase (AGPase) is a key enzyme of starch biosynthesis. In the green plant lineage, it is composed of two large (LSU) and two small (SSU) sub-units encoded by paralogous genes, as a consequence of several rounds of duplication. First, our aim was to detect specific patterns of molecular evolution following duplication events and the divergence between monocotyledons and dicotyledons. Secondly, we investigated coevolution between amino acids both within and between sub-units. Methods A phylogeny of each AGPase sub-unit was built using all gymnosperm and angiosperm sequences available in databases. Accelerated evolution along specific branches was tested using the ratio of the non-synonymous to the synonymous substitution rate. Coevolution between amino acids was investigated taking into account compensatory changes between co-substitutions. Key Results We showed that SSU paralogues evolved under high functional constraints during angiosperm radiation, with a significant level of coevolution between amino acids that participate in SSU major functions. In contrast, in the LSU paralogues, we identified residues under positive selection (1) following the first LSU duplication that gave rise to two paralogues mainly expressed in angiosperm source and sink tissues, respectively; and (2) following the emergence of grass-specific paralogues expressed in the endosperm. Finally, we found coevolution between residues that belong to the interaction domains of both sub-units. Conclusions Our results support the view that coevolution among amino acid residues, especially those lying in the interaction domain of each sub-unit, played an important role in AGPase evolution. First, within SSU, coevolution allowed compensating mutations in a highly constrained context. Secondly, the LSU paralogues probably acquired tissue-specific expression and regulatory properties via the coevolution between sub-unit interacting domains. Finally, the

  12. Cumulative effect of heterologous AtWRI1 gene expression and endogenous BjAGPase gene silencing increases seed lipid content in Indian mustard Brassica juncea.

    PubMed

    Bhattacharya, Surajit; Das, Natasha; Maiti, Mrinal K

    2016-10-01

    The production of vegetable oil in many countries of the world, including India has not been able to keep pace with the increasing requirement, leading to a very large gap in the demand-supply chain. Thus, there is an urgent need to increase the yield potential of the oilseed crops so as to enhance the storage lipid productivity. The present study describes a novel metabolic engineering ploy involving the constitutive down-regulation of endogenous ADP-glucose pyrophosphorylase (BjAGPase) enzyme and the seed-specific expression of WRINKLED1 transcription factor (AtWRI1) from Arabidopsis thaliana in Indian mustard (Brassica juncea) with an aim to divert the photosynthetically fixed carbon pool from starch to lipid synthesis in the seeds for the enhanced production of storage lipids in the seeds of transgenic mustard plants. The starch content, in both the vegetative leaf and developing seed tissues of the transgenic B. juncea lines exhibited a reduction by about 45-53% compared to the untransformed control, whereas the soluble sugar content was increased by 2.4 and 1.3-fold in the leaf and developing seed tissues, respectively. Consequently, the transgenic lines showed a significant enhancement in total seed lipid content ranging between 7.5 and 16.9%. The results indicate that the adopted metabolic engineering strategy was successful in significantly increasing the seed oil content. Therefore, findings of our research suggest that the metabolic engineering strategy adopted in this study for shifting the anabolic carbon flux from starch synthesis to lipid biosynthesis can be employed for increasing the storage lipid content of seeds in other plant species. PMID:27314514

  13. Loss of Starch Granule Initiation Has a Deleterious Effect on the Growth of Arabidopsis Plants Due to an Accumulation of ADP-Glucose1[W

    PubMed Central

    Ragel, Paula; Streb, Sebastian; Feil, Regina; Sahrawy, Mariam; Annunziata, Maria Grazia; Lunn, John E.; Zeeman, Samuel; Mérida, Ángel

    2013-01-01

    STARCH SYNTHASE4 (SS4) is required for proper starch granule initiation in Arabidopsis (Arabidopsis thaliana), although SS3 can partially replace its function. Unlike other starch-deficient mutants, ss4 and ss3/ss4 mutants grow poorly even under long-day conditions. They have less chlorophyll and carotenoids than the wild type and lower maximal rates of photosynthesis. There is evidence of photooxidative damage of the photosynthetic apparatus in the mutants from chlorophyll a fluorescence parameters and their high levels of malondialdehyde. Metabolite profiling revealed that ss3/ss4 accumulates over 170 times more ADP-glucose (Glc) than wild-type plants. Restricting ADP-Glc synthesis, by introducing mutations in the plastidial phosphoglucomutase (pgm1) or the small subunit of ADP-Glc pyrophosphorylase (aps1), largely restored photosynthetic capacity and growth in pgm1/ss3/ss4 and aps1/ss3/ss4 triple mutants. It is proposed that the accumulation of ADP-Glc in the ss3/ss4 mutant sequesters a large part of the plastidial pools of adenine nucleotides, which limits photophosphorylation, leading to photooxidative stress, causing the chlorotic and stunted growth phenotypes of the plants. PMID:23872660

  14. Loss of starch granule initiation has a deleterious effect on the growth of arabidopsis plants due to an accumulation of ADP-glucose.

    PubMed

    Ragel, Paula; Streb, Sebastian; Feil, Regina; Sahrawy, Mariam; Annunziata, Maria Grazia; Lunn, John E; Zeeman, Samuel; Mérida, Ángel

    2013-09-01

    STARCH SYNTHASE4 (SS4) is required for proper starch granule initiation in Arabidopsis (Arabidopsis thaliana), although SS3 can partially replace its function. Unlike other starch-deficient mutants, ss4 and ss3/ss4 mutants grow poorly even under long-day conditions. They have less chlorophyll and carotenoids than the wild type and lower maximal rates of photosynthesis. There is evidence of photooxidative damage of the photosynthetic apparatus in the mutants from chlorophyll a fluorescence parameters and their high levels of malondialdehyde. Metabolite profiling revealed that ss3/ss4 accumulates over 170 times more ADP-glucose (Glc) than wild-type plants. Restricting ADP-Glc synthesis, by introducing mutations in the plastidial phosphoglucomutase (pgm1) or the small subunit of ADP-Glc pyrophosphorylase (aps1), largely restored photosynthetic capacity and growth in pgm1/ss3/ss4 and aps1/ss3/ss4 triple mutants. It is proposed that the accumulation of ADP-Glc in the ss3/ss4 mutant sequesters a large part of the plastidial pools of adenine nucleotides, which limits photophosphorylation, leading to photooxidative stress, causing the chlorotic and stunted growth phenotypes of the plants. PMID:23872660

  15. Modification of carbon partitioning, photosynthetic capacity, and O{sub 2} sensitivity in Arabidopsis plants with low ADP-glucose pyrophosphorylase activity

    SciTech Connect

    Sun, J.; Okita, T.W.; Edwards, G.E.

    1999-01-01

    Wild-type Arabidopsis plants, the starch-deficient mutant TL46, and the near-starchless mutant TL25 were evaluated by noninvasive in situ methods for their capacity for net CO{sub 2} assimilation, true rates of photosynthetic O{sub 2} evolution (determined from chlorophyll fluorescence measurements of photosystem II), partitioning of photosynthate into sucrose and starch, and plant growth. Compared with wild-type plants, the starch mutants showed reduced photosynthetic capacity, with the largest reduction occurring in mutant TL25 subjected to high light and increased CO{sub 2} partial pressure. The extent of stimulation of CO{sub 2} assimilation by increasing CO{sub 2} or by reducing O{sub 2} partial pressure was significantly less for the starch mutants than for wild-type plants. Under high light and moderate to high levels of CO{sub 2}, the rates of CO{sub 2} assimilation and O{sub 2} evolution and the percentage inhibition of photosynthesis by low O{sub 2} were higher for the wild type than for the mutants. The relative rates of {sup 14}CO{sub 2} incorporation into starch under high light and high CO{sub 2} followed the patterns of photosynthetic capacity, with TL46 showing 31% to 40% of the starch-labeling rates of the wild type and TL25 showing less than 14% incorporation. Overall, there were significant correlations between the rates of starch synthesis and CO{sub 2} assimilation and between the rates of starch synthesis and cumulative leaf area. These results indicate that leaf starch plays an important role as a transient reserve, the synthesis of which can ameliorate any potential reduction in photosynthesis caused by feedback regulation.

  16. Analysis of the Rice ADP-Glucose Transporter (OsBT1) Indicates the Presence of Regulatory Processes in the Amyloplast Stroma That Control ADP-Glucose Flux into Starch1[OPEN

    PubMed Central

    Shiraishi, Shota; Matsusaka, Hiroaki; Singh, Salvinder; Hosaka, Yuko; Satoh, Hikaru

    2016-01-01

    Previous studies showed that efforts to further elevate starch synthesis in rice (Oryza sativa) seeds overproducing ADP-glucose (ADPglc) were prevented by processes downstream of ADPglc synthesis. Here, we identified the major ADPglc transporter by studying the shrunken3 locus of the EM1093 rice line, which harbors a mutation in the BRITTLE1 (BT1) adenylate transporter (OsBt1) gene. Despite containing elevated ADPglc levels (approximately 10-fold) compared with the wild-type, EM1093 grains are small and shriveled due to the reduction in the amounts and size of starch granules. Increases in ADPglc levels in EM1093 were due to their poor uptake of ADP-[14C]glc by amyloplasts. To assess the potential role of BT1 as a rate-determining step in starch biosynthesis, the maize ZmBt1 gene was overexpressed in the wild-type and the GlgC (CS8) transgenic line expressing a bacterial glgC-TM gene. ADPglc transport assays indicated that transgenic lines expressing ZmBT1 alone or combined with GlgC exhibited higher rates of transport (approximately 2-fold), with the GlgC (CS8) and GlgC/ZmBT1 (CS8/AT5) lines showing elevated ADPglc levels in amyloplasts. These increases, however, did not lead to further enhancement in seed weights even when these plant lines were grown under elevated CO2. Overall, our results indicate that rice lines with enhanced ADPglc synthesis and import into amyloplasts reveal additional barriers within the stroma that restrict maximum carbon flow into starch. PMID:26754668

  17. Improving starch yield in cereals by over-expression of ADPglucose pyrophosphorylase: expectations and unanticipated outcomes.

    PubMed

    Tuncel, Aytug; Okita, Thomas W

    2013-10-01

    Significant improvements in crop productivity are required to meet the nutritional requirements of a growing world population. This challenge is magnified by an increased demand for bioenergy as a means to mitigate carbon inputs into the environment. Starch is a major component of the harvestable organs of many crop plants, and various endeavors have been taken to improve the yields of starchy organs through the manipulation of starch synthesis. Substantial efforts have centered on the starch regulatory enzyme ADPglucose pyrophosphorylase (AGPase) due to its pivotal role in starch biosynthesis. These efforts include over-expression of this enzyme in cereal plants such as maize, rice and wheat as well as potato and cassava, as they supply the bulk of the staple food worldwide. In this perspective, we describe efforts to increase starch yields in cereal grains by first providing an introduction about the importance of source-sink relationship and the motives behind the efforts to alter starch biosynthesis and turnover in leaves. We then discuss the catalytic and regulatory properties of AGPase and the molecular approaches used to enhance starch synthesis by manipulation of this process during grain filling using seed-specific promoters. Several studies have demonstrated increases in starch content per seed using endosperm-specific promoters, but other studies have demonstrated an increase in seed number with only marginal impact on seed weight. Potential mechanisms that may be responsible for this paradoxical increase in seed number will also be discussed. Finally, we describe current efforts and future prospects to improve starch yield in cereals. These efforts include further enhancement of starch yield in rice by augmenting the process of ADPglucose transport into amyloplast as well as other enzymes involved in photoassimilate partitioning in seeds. PMID:23987811

  18. The α-Glucan Phosphorylase MalP of Corynebacterium glutamicum Is Subject to Transcriptional Regulation and Competitive Inhibition by ADP-Glucose

    PubMed Central

    Clermont, Lina; Macha, Arthur; Müller, Laura M.; Derya, Sami M.; von Zaluskowski, Philipp; Eck, Alexander; Eikmanns, Bernhard J.

    2015-01-01

    ABSTRACT α-Glucan phosphorylases contribute to degradation of glycogen and maltodextrins formed in the course of maltose metabolism in bacteria. Accordingly, bacterial α-glucan phosphorylases are classified as either glycogen or maltodextrin phosphorylase, GlgP or MalP, respectively. GlgP and MalP enzymes follow the same catalytic mechanism, and thus their substrate spectra overlap; however, they differ in their regulation: GlgP genes are constitutively expressed and the enzymes are controlled on the activity level, whereas expression of MalP genes are transcriptionally controlled in response to the carbon source used for cultivation. We characterize here the modes of control of the α-glucan phosphorylase MalP of the Gram-positive Corynebacterium glutamicum. In accordance to the proposed function of the malP gene product as MalP, we found transcription of malP to be regulated in response to the carbon source. Moreover, malP transcription is shown to depend on the growth phase and to occur independently of the cell glycogen content. Surprisingly, we also found MalP activity to be tightly regulated competitively by the presence of ADP-glucose, an intermediate of glycogen synthesis. Since the latter is considered a typical feature of GlgPs, we propose that C. glutamicum MalP acts as both maltodextrin and glycogen phosphorylase and, based on these findings, we question the current system for classification of bacterial α-glucan phosphorylases. IMPORTANCE Bacterial α-glucan phosphorylases have been classified conferring to their purpose as either glycogen or maltodextrin phosphorylases. We found transcription of malP in C. glutamicum to be regulated in response to the carbon source, which is recognized as typical for maltodextrin phosphorylases. Surprisingly, we also found MalP activity to be tightly regulated competitively by the presence of ADP-glucose, an intermediate of glycogen synthesis. The latter is considered a typical feature of GlgPs. These findings

  19. Arabidopsis UDP-Sugar Pyrophosphorylase: Evidence for Two Isoforms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arabidopsis UDP-sugar pyrophosphorylase (AtUSP, EC 2.7.7.64) is a broad substrate pyrophosphorylase that exhibits activity with GlcA-1-P, Gal-1-P, and Glc-1-P. AtUSP, a single gene in Arabidopsis, is widely expressed in tissues. Although USP exhibits activity with GlcA-1-P, it is not clear whether U...

  20. Arabidopsis UDP-sugar pyrophosphorylase: evidence for two isoforms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arabidopsis UDP-sugar pyrophosphorylase (AtUSP, EC 2.7.7.64) is a broad substrate pyrophosphorylase that exhibits activity with GlcA-1-P, Gal-1-P, and Glc-1-P. Immunoblots using polyclonal antibodies raised to recombinant AtUSP demonstrated the presence of two USP isoforms of approximately 70 kDa (U...

  1. Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADPglucose pyrophosphorylase and higher rates of starch synthesis in Arabidopsis thaliana

    PubMed Central

    Lunn, John E.; Feil, Regina; Hendriks, Janneke H. M.; Gibon, Yves; Morcuende, Rosa; Osuna, Daniel; Scheible, Wolf-Rüdiger; Carillo, Petronia; Hajirezaei, Mohammad-Reza; Stitt, Mark

    2006-01-01

    Tre6P (trehalose 6-phosphate) is implicated in sugar-signalling pathways in plants, but its exact functions in vivo are uncertain. One of the main obstacles to discovering these functions is the difficulty of measuring the amount of Tre6P in plant tissues. We have developed a highly specific assay, using liquid chromatography coupled to MS-Q3 (triple quadrupole MS), to measure Tre6P in the femto-picomole range. The Tre6P content of sucrose-starved Arabidopsis thaliana seedlings in axenic culture increased from 18 to 482 pmol·g−1FW (fresh weight) after adding sucrose. Leaves from soil-grown plants contained 67 pmol·g−1FW at the end of the night, which rose to 108 pmol·g−1FW after 4 h of illumination. Even greater changes in Tre6P content were seen after a 6 h extension of the dark period, and in the starchless mutant, pgm. The intracellular concentration of Tre6P in wild-type leaves was estimated to range from 1 to 15 μM. It has recently been reported that the addition of Tre6P to isolated chloroplasts leads to redox activation of AGPase (ADPglucose pyrophosphorylase) [Kolbe, Tiessen, Schluepmann, Paul, Ulrich and Geigenberger (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 11118–11123]. Using the new assay for Tre6P, we found that rising sugar levels in plants are accompanied by increases in the level of Tre6P, redox activation of AGPase and the stimulation of starch synthesis in vivo. These results indicate that Tre6P acts as a signalling metabolite of sugar status in plants, and support the proposal that Tre6P mediates sucrose-induced changes in the rate of starch synthesis. PMID:16551270

  2. Genetic modification of cassava for enhanced starch production.

    PubMed

    Ihemere, Uzoma; Arias-Garzon, Diana; Lawrence, Susan; Sayre, Richard

    2006-07-01

    To date, transgenic approaches to biofortify subsistence crops have been rather limited. This is particularly true for the starchy root crop cassava (Manihot esculenta Crantz). Cassava has one of the highest rates of CO(2) fixation and sucrose synthesis for any C3 plant, but rarely reaches its yield potentials in the field. It was our hypothesis that starch production in cassava tuberous roots could be increased substantially by increasing the sink strength for carbohydrate. To test this hypothesis, we generated transgenic plants with enhanced tuberous root ADP-glucose pyrophosphorylase (AGPase) activity. This was achieved by expressing a modified form of the bacterial glgC gene under the control of a Class I patatin promoter. AGPase catalyses the rate-limiting step in starch biosynthesis, and therefore the expression of a more active bacterial form of the enzyme was expected to lead to increased starch production. To facilitate maximal AGPase activity, we modified the Escherichia coli glgC gene (encoding AGPase) by site-directed mutagenesis (G336D) to reduce allosteric feedback regulation by fructose-1,6-bisphosphate. Transgenic plants (three) expressing the glgC gene had up to 70% higher AGPase activity than control plants when assayed under conditions optimal for plant and not bacterial AGPase activity. Plants having the highest AGPase activities had up to a 2.6-fold increase in total tuberous root biomass when grown under glasshouse conditions. In addition, plants with the highest tuberous root AGPase activity had significant increases in above-ground biomass, consistent with a possible reduction in feedback inhibition on photosynthetic carbon fixation. These results demonstrate that targeted modification of enzymes regulating source-sink relationships in crop plants having high carbohydrate source strengths is an effective strategy for increasing carbohydrate yields in sink tissues. PMID:17177810

  3. Immunocytochemical localization of ADPglucose pyrophosphorylase in developing potato tuber cells

    SciTech Connect

    Kim, Woo Taek; Franceschi, V.R.; Okita, T.W. ); Robinson, N.L.; Morell, M.; Preiss, J. )

    1989-09-01

    The subcellular localization of ADPglucose pyrophosphorylase, a key regulatory enzyme in starch biosynthesis, was determined in developing potato tuber cells by immunocytochemical localization techniques at the light microscopy level. Specific labeling of ADPglucose pyrophosphorylase by either immunofluorescence or immunogold followed by silver enhancement was detected only in the amyloplasts and indicates that this enzyme is located exclusively in the amyloplasts in developing potato tuber cells. Labeling occurred on the starch grains and, in some instances, specific labeling patterns were evident which may be related to sites active in starch deposition.

  4. Structural Basis of Glycogen Biosynthesis Regulation in Bacteria.

    PubMed

    Cifuente, Javier O; Comino, Natalia; Madariaga-Marcos, Julene; López-Fernández, Sonia; García-Alija, Mikel; Agirre, Jon; Albesa-Jové, David; Guerin, Marcelo E

    2016-09-01

    ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step of bacterial glycogen and plant starch biosynthesis, the most common carbon storage polysaccharides in nature. A major challenge is to understand how AGPase activity is regulated by metabolites in the energetic flux within the cell. Here we report crystal structures of the homotetrameric AGPase from Escherichia coli in complex with its physiological positive and negative allosteric regulators, fructose-1,6-bisphosphate (FBP) and AMP, and sucrose in the active site. FBP and AMP bind to partially overlapping sites located in a deep cleft between glycosyltransferase A-like and left-handed β helix domains of neighboring protomers, accounting for the fact that sensitivity to inhibition by AMP is modulated by the concentration of the activator FBP. We propose a model in which the energy reporters regulate EcAGPase catalytic activity by intra-protomer interactions and inter-protomer crosstalk, with a sensory motif and two regulatory loops playing a prominent role. PMID:27545622

  5. Biosynthesis of starch in chloroplasts.

    PubMed

    Nomura, T; Nakayama, N; Murata, T; Akazawa, T

    1967-03-01

    The enzymic synthesis of ADP-glucose and UDP-glucose by chloroplastic pyrophosphorylase of bean and rice leaves has been demonstrated by paper chromatographic techniques. In both tissues, the activity of UDP-glucose-pyrophosphorylase was much higher than ADP-glucose-pyrophosphorylase. Glycerate-3-phosphate, phosphoenolpyruvate and fructose-1,6-diphosphate did not stimulate ADP-glucose formation by a pyrophosphorylation reaction. The major metabolic pathway for UDP-glucose utilization appears to be the synthesis of either sucrose or sucrose-P. On the other hand, a specific precursor role of ADP-glucose for synthesizing chloroplast starch by the ADP-glucose-starch transglucosylase reaction is supported by the coupled enzyme system of ADP-glucose-pyrophosphorylase and transglucosylase, isolated from chloroplasts. None of the glycolytic intermediates stimulated the glucose transfer in the enzyme sequence of reaction system employed. PMID:4292567

  6. Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

    DOEpatents

    Jarvis, Eric E.; Roessler, Paul G.

    1999-01-01

    The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities.

  7. Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

    DOEpatents

    Jarvis, E.E.; Roessler, P.G.

    1999-07-27

    The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities. 8 figs.

  8. Both UDP N-acetylglucosamine pyrophosphorylases of Tribolium castaneum are critical for molting, survival, and fecundity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A bioinformatics search of the genome of the red flour beetle, Tribolium castaneum, resulted in the identification of two genes encoding proteins closely related to UDP-N-acetylglucosamine pyrophosphorylases (UAP), which provide the activated precursor, UDP-N-acetylglucosamine, for the synthesis of ...

  9. The subunit structure of potato tuber ADPglucose pyrophosphorylase. [Solanum tuberosum L

    SciTech Connect

    Okita, T.W.; Nakata, P.A.; Anderson, J.M. ); Sowokinos, J. ); Morell, M.; Preiss, J. )

    1990-06-01

    ADPglucose pyrophosphorylase has been extensively purified from potato (Solanum tuberosum L.) tuber tissue to study its structure. By employing a modified published procedure together with Mono Q chromatography, a near homogeneous enzyme preparation was obtained with substantial improvement in enzyme yield and specific activity. In single dimensional sodium dodecyl sulfate polyacrylamide gels, the enzyme migrated as a single polypeptide band with a mobility of about 50,000 daltons. Analysis by two-dimensional polyacrylamide gel electrophoresis, however, revealed the presence of two types of subunits which could be distinguished by their slight differences in net charge and molecular weight. The smaller potato tuber subunit was recognized by antiserum prepared against the smaller spinach leaf 51 kilodalton ADPglucose pyrophosphorylase subunit. In contrast, the anti-54 kilodalton raised against the spinach leaf subunit did not significantly react to the tuber enzyme subunits. The results are consistent with the hypothesis that the potato tuber ADPglucose pyrophosphorylase is not composed of a simple homotetramer as previously suggested, but is a product of two separate and distinct subunits as observed for the spinach leaf and maize enzymes.

  10. Identification of a GDP-mannose pyrophosphorylase gene from Sulfolobus solfataricus.

    PubMed

    Sacchetti, Silvana; Bartolucci, Simonetta; Rossi, Mosè; Cannio, Raffaele

    2004-05-12

    An open reading frame (ORF) encoding a putative GDP-mannose pyrophosphorylase (SsoGMPP) was identified on the genome sequence of Sulfolobus solfataricus P2, the predicted gene product showing high amino acid sequence homology to several archaeal, bacterial, and eukaryal GDP-mannose pyrophosphorylases such as guanidine diphosphomannose pyrophosphorylases (GMPPs) from Saccharomyces cerevisiae and Arabidopsis thaliana. The sequence was PCR amplified from genomic DNA of S. solfataricus P2 and heterologous gene expression obtained as a fusion to glutathione S-transferase in Escherichia coli, under conditions suitable to reduce the formation of inclusion bodies. Specific assays performed at 60 degrees C revealed the presence of the archaeal synthesizing GDP-mannose enzyme activity in the cell extracts of the transformed E. coli. As a positive control, the same assays were performed at the mesophilic enzyme optimum temperature on the already characterized yeast recombinant GMPP. The recombinant protein was purified to homogeneity by glutathione sepharose affinity chromatography and its thermophilic nature could be verified. The enzyme was definitively identified by demonstrating its capability to catalyze also the reverse reaction of pyrophosphorolysis and, most interestingly, its high specificity for synthesizing GDP-mannose. PMID:15145064

  11. [Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase gene]. Progress report, [March 15, 1989--April 14, 1990

    SciTech Connect

    Okita, T.W.

    1990-12-31

    The long term aim of this project is to assess the feasibility of increasing the conversion of photosynthate into starch via manipulation of the gene that encodes for ADPglucose pyrophosphorylase, a key regulatory enzyme of starch biosynthesis. In developing storage tissues such as cereal seeds and tubers, starch biosynthesis is regulated by the gene activation and expression of ADPglucose pyrophosphorylase, starch synthase, branching enzyme and other ancillary starch modifying enzymes, as well as the allosteric-controlled behavior of ADPglucose pyrophosphorylase activity. During the last two years we have obtained information on the structure of this enzyme from both potato tuber and rice endosperm, using a combination of biochemical and molecular biological approaches. Moreover, we present evidence that this enzyme may be localized at discrete regions of the starch grain within the amyloplast, and plays a role in controlling overall starch biosynthesis in potato tubers.

  12. On the Ancestral UDP-Glucose Pyrophosphorylase Activity of GalF from Escherichia coli

    PubMed Central

    Ebrecht, Ana C.; Orlof, Agnieszka M.; Sasoni, Natalia; Figueroa, Carlos M.; Iglesias, Alberto A.; Ballicora, Miguel A.

    2015-01-01

    In bacteria, UDP-glucose is a central intermediate in carbohydrate metabolism. The enzyme responsible for its synthesis is encoded by the galU gene and its deletion generates cells unable to ferment galactose. In some bacteria, there is a second gene, galF, encoding for a protein with high sequence identity to GalU. However, the role of GalF has been contradictory regarding its catalytic capability and not well understood. In this work we show that GalF derives from a catalytic (UDP-glucose pyrophosphorylase) ancestor, but its activity is very low compared to GalU. We demonstrated that GalF has some residual UDP-glucose pyrophosphorylase activity by in vitro and in vivo experiments in which the phenotype of a galU- strain was reverted by the over-expression of GalF and its mutant. To demonstrate its evolutionary path of “enzyme inactivation” we enhanced the catalysis by mutagenesis and showed the importance of the quaternary structure. This study provides important information to understand the structural and functional evolutionary origin of the protein GalF in enteric bacteria. PMID:26617591

  13. [Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase gene]. Summary of progress, [April 15, 1991--April 14, 1992

    SciTech Connect

    Okita, T.W.

    1992-12-31

    The long term aim of this project is to assess the feasibility of increasing the conversion of photosynthate into starch via manipulation of genes encoding enzymes that may be rate-limiting in starch biosynthesis. In developing storage tissues such as tubers, starch biosynthesis is regulated by the gene activation and expression of ADPglucose pyrophosphorylase, starch synthase, branching enzyme and other ancillary starch modifying enzymes, as well as the allosteric-controlled behavior of ADPglucose pyrophosphorylase activity. In view of the regulatory role of ADPglucose pyrophosphorylase in starch biosynthesis at both the genetic and biochemical level, we have focused our attention on the genes that encode for this enzyme in potato tubers. The proposed objectives of the grant were to (1) analyze the structure of the tuber enzyme, (2) isolate and characterize the structure of its genes, and (3) identify the regulatory elements controlling ADPglucose pyrophosphorylase during plant development. During the last two and 1/2 years we have met or have made considerable progress in achieving these objectives as discussed in more detail below.

  14. Purification and Properties of Adenosine Diphosphoglucose Pyrophosphorylase from Sweet Corn 1

    PubMed Central

    Amir, Jacob; Cherry, Joe H.

    1972-01-01

    A 40-fold purification of adenosine diphosphoglucose pyrophosphorylase from sweet corn (Zea mays var. Golden Beauty) revealed the enzyme to be specific for adenosine triphosphate. The enzyme has an absolute requirement for Mg2+ and is activated by 3-phosphoglycerate and to a lesser extent by ribose-5-phosphate and fructose-6-phosphate. The apparent Km values of the enzyme for glucose-1-phosphate, adenosine triphosphate, pyrophosphate, and adenosine diphosphoglucose are 1.9 × 10−4, 3.2 × 10−5, 3.3 × 10−5, and 6.2 × 10−4m, respectively. Pyrophosphate inhibits adenosine diphosphoglucose synthesis competitively (Ki = 3.8 × 10−7m), while orthophosphate and sulfate appear to inhibit the reacion noncompetitively. These results show that the production of this sugar nucleotide can be controlled by the concentration of pyrophosphate. PMID:16658078

  15. Leishmania major UDP-sugar pyrophosphorylase salvages galactose for glycoconjugate biosynthesis

    PubMed Central

    Damerow, Sebastian; Hoppe, Carolin; Bandini, Giulia; Zarnovican, Patricia; Buettner, Falk F.R.; Ferguson, Michael A.J.; Routier, Françoise H.

    2015-01-01

    Leishmaniases are a set of tropical and sub-tropical diseases caused by protozoan parasites of the genus Leishmania whose severity ranges from self-healing cutaneous lesions to fatal visceral infections. Leishmania parasites synthesise a wide array of cell surface and secreted glycoconjugates that play important roles in infection. These glycoconjugates are particularly abundant in the promastigote form and known to be essential for establishment of infection in the insect midgut and effective transmission to the mammalian host. Since they are rich in galactose, their biosynthesis requires an ample supply of UDP-galactose. This nucleotide-sugar arises from epimerisation of UDP-glucose but also from an uncharacterised galactose salvage pathway. In this study, we evaluated the role of the newly characterised UDP-sugar pyrophosphorylase (USP) of Leishmania major in UDP-galactose biosynthesis. Upon deletion of the USP encoding gene, L. major lost the ability to synthesise UDP-galactose from galactose-1-phosphate but its ability to convert glucose-1-phosphate into UDP-glucose was fully maintained. Thus USP plays a role in UDP-galactose activation but does not significantly contribute to the de novo synthesis of UDP-glucose. Accordingly, USP was shown to be dispensable for growth and glycoconjugate biosynthesis under standard growth conditions. However, in a mutant seriously impaired in the de novo synthesis of UDP-galactose (due to deficiency of the UDP-glucose pyrophosphorylase) addition of extracellular galactose increased biosynthesis of the cell surface lipophosphoglycan. Thus under restrictive conditions, such as those encountered by Leishmania in its natural habitat, galactose salvage by USP may play a substantial role in biosynthesis of the UDP-galactose pool. We hypothesise that USP recycles galactose from the blood meal within the midgut of the insect for synthesis of the promastigote glycocalyx and thereby contributes to successful vector infection. PMID

  16. Allosteric regulation of the partitioning of glucose-1-phosphate between glycogen and trehalose biosynthesis in Mycobacterium tuberculosis

    PubMed Central

    Asención Diez, Matías D.; Demonte, Ana M.; Syson, Karl; Arias, Diego G.; Gorelik, Andrii; Guerrero, Sergio A.; Bornemann, Stephen; Iglesias, Alberto A.

    2015-01-01

    Background Mycobacterium tuberculosis is a pathogenic prokaryote adapted to survive in hostile environments. In this organism and other Gram-positive actinobacteria, the metabolic pathways of glycogen and trehalose are interconnected. Results In this work we show the production, purification and characterization of recombinant enzymes involved in the partitioning of glucose-1-phosphate between glycogen and trehalose in M. tuberculosis H37Rv, namely: ADP-glucose pyrophosphorylase, glycogen synthase, UDP-glucose pyrophosphorylase and trehalose-6-phosphate synthase. The substrate specificity, kinetic parameters and allosteric regulation of each enzyme were determined. ADP-glucose pyrophosphorylase was highly specific for ADP-glucose while trehalose-6-phosphate synthase used not only ADP-glucose but also UDP-glucose, albeit to a lesser extent. ADP-glucose pyrophosphorylase was allosterically activated primarily by phosphoenolpyruvate and glucose-6-phosphate, while the activity of trehalose-6-phosphate synthase was increased up to 2-fold by fructose-6-phosphate. None of the other two enzymes tested exhibited allosteric regulation. Conclusions Results give information about how the glucose-1-phosphate/ADP-glucose node is controlled after kinetic and regulatory properties of key enzymes for mycobacteria metabolism. General significance This work increases our understanding of oligo and polysaccharides metabolism in M. tuberculosis and reinforces the importance of the interconnection between glycogen and trehalose biosynthesis in this human pathogen. PMID:25277548

  17. Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase gene. Progress report, [April 15, 1988--April 14, 1989

    SciTech Connect

    Okita, T.W.

    1989-12-31

    During this period researchers have been successful in determining the structure of the rice pyrophosphorylase gene. Potato tuber ADPglucose pyrophosphorylse purification and structure studies were carried out as well as recombinant DNA studies. Evidence suggests that the tuber form is made up of subunits with similar molecular weights and immunological relatedness. In contrast, the spinach leaf enzyme and presumably the maize endosperm species is composed of two dissimilar sununits encoded by different genes.

  18. Proteomic Analysis Reveals Key Proteins and Phosphoproteins upon Seed Germination of Wheat (Triticum aestivum L.).

    PubMed

    Dong, Kun; Zhen, Shoumin; Cheng, Zhiwei; Cao, Hui; Ge, Pei; Yan, Yueming

    2015-01-01

    Wheat (Triticum aestivum L.) is one of the oldest cultivated crops and the second most important food crop in the world. Seed germination is the key developmental process in plant growth and development, and poor germination directly affects plant growth and subsequent grain yield. In this study, we performed the first dynamic proteome analysis of wheat seed germination using a two-dimensional differential gel electrophoresis (2D-DIGE)-based proteomic approach. A total of 166 differentially expressed protein (DEP) spots representing 73 unique proteins were identified, which are mainly involved in storage, stress/defense/detoxification, carbohydrate metabolism, photosynthesis, cell metabolism, and transcription/translation/transposition. The identified DEPs and their dynamic expression profiles generally correspond to three distinct seed germination phases after imbibition: storage degradation, physiological processes/morphogenesis, and photosynthesis. Some key DEPs involved in storage substance degradation and plant defense mechanisms, such as globulin 3, sucrose synthase type I, serpin, beta-amylase, and plastid ADP-glucose pyrophosphorylase (AGPase) small subunit, were found to be phosphorylated during seed germination. Particularly, the phosphorylation site Ser(355) was found to be located in the enzyme active region of beta-amylase, which promotes substrate binding. Phosphorylated modification of several proteins could promote storage substance degradation and environmental stress defense during seed germination. The central metabolic pathways involved in wheat seed germination are proposed herein, providing new insights into the molecular mechanisms of cereal seed germination. PMID:26635843

  19. Influence of tryptophan and indole-3-acetic acid on starch accumulation in the synthetic mutualistic Chlorella sorokiniana-Azospirillum brasilense system under heterotrophic conditions.

    PubMed

    Palacios, Oskar A; Choix, Francisco J; Bashan, Yoav; de-Bashan, Luz E

    2016-06-01

    This study measured the relations between tryptophan production, the phytohormone indole-3-acetic acid (IAA) and the metabolism and accumulation of starch during synthetic mutualism between the microalgae Chlorella sorokiniana and the microalgae growth-promoting bacteria Azospirillum brasilense, created by co-immobilization in alginate beads. Experiments used two wild-type A. brasilense strains (Cd and Sp6) and an IAA-attenuated mutant (SpM7918) grown under nitrogen-replete and nitrogen-starved conditions tested under dark, heterotrophic and aerobic growth conditions. Under all incubating conditions, C. sorokiniana, but not A. brasilense, produced tryptophan. A significant correlation between IAA-production by A. brasilense and starch accumulation in C. sorokiniana was found, since the IAA-attenuated mutant was not producing increased starch levels. The highest ADP-glucose pyrophosphorylase (AGPase) activity, starch content and glucose uptake were found during the interaction of A. brasilense wild type strains with the microalgae. When the microalgae were grown alone, they produced only small amounts of starch. Supplementation with synthetic IAA to C. sorokiniana grown alone enhanced the above parameters, but only transiently. Activity of α-amylase decreased under nitrogen-replete conditions, but increased under nitrogen-starved conditions. In summary, this study demonstrated that, during synthetic mutualism, the exchange of tryptophan and IAA between the partners is a mechanism that governs several changes in starch metabolism of C. sorokiniana, yielding an increase in starch content. PMID:26924113

  20. Systematic Analysis of Pericarp Starch Accumulation and Degradation during Wheat Caryopsis Development.

    PubMed

    Yu, Xurun; Li, Bo; Wang, Leilei; Chen, Xinyu; Wang, Wenjun; Wang, Zhong; Xiong, Fei

    2015-01-01

    Although wheat (Triticum aestivum L.) pericarp starch granule (PSG) has been well-studied, our knowledge of its features and mechanism of accumulation and degradation during pericarp growth is poor. In the present study, developing wheat caryopses were collected and starch granules were extracted from their pericarp to investigate the morphological and structural characteristics of PSGs using microscopy, X-ray diffraction and Fourier transform infrared spectroscopy techniques. Relative gene expression levels of ADP-glucose pyrophosphorylase (APGase), granule-bound starch synthase II (GBSS II), and α-amylase (AMY) were quantified by quantitative real-time polymerase chain reaction. PSGs presented as single or multiple starch granules and were synthesized both in the amyloplast and chloroplast in the pericarp. PSG degradation occurred in the mesocarp, beginning at 6 days after anthesis. Amylose contents in PSGs were lower and relative degrees of crystallinity were higher at later stages of development than at earlier stages. Short-range ordered structures in the external regions of PSGs showed no differences in the developing pericarp. When hydrolyzed by α-amylase, PSGs at various developmental stages showed high degrees of enzymolysis. Expression levels of AGPase, GBSS II, and AMY were closely related to starch synthesis and degradation. These results help elucidate the mechanisms of accumulation and degradation as well as the functions of PSG during wheat caryopsis development. PMID:26394305

  1. Raman spectroscopy provides a rapid, non-invasive method for quantitation of starch in live, unicellular microalgae.

    PubMed

    Ji, Yuetong; He, Yuehui; Cui, Yanbin; Wang, Tingting; Wang, Yun; Li, Yuanguang; Huang, Wei E; Xu, Jian

    2014-12-01

    Conventional methods for quantitation of starch content in cells generally involve starch extraction steps and are usually labor intensive, thus a rapid and non-invasive method will be valuable. Using the starch-producing unicellular microalga Chlamydomonas reinhardtii as a model, we employed a customized Raman spectrometer to capture the Raman spectra of individual single cells under distinct culture conditions and along various growth stages. The results revealed a nearly linear correlation (R(2) = 0.9893) between the signal intensity at 478 cm(-1) and the starch content of the cells. We validated the specific correlation by showing that the starch-associated Raman peaks were eliminated in a mutant strain where the AGPase (ADP-glucose pyrophosphorylase) gene was disrupted and consequentially the biosynthesis of starch blocked. Furthermore, the method was validated in an industrial algal strain of Chlorella pyrenoidosa. This is the first demonstration of starch quantitation in individual live cells. Compared to existing cellular starch quantitation methods, this single-cell Raman spectra-based approach is rapid, label-free, non-invasive, culture-independent, low-cost, and potentially able to simultaneously track multiple metabolites in individual live cells, therefore should enable many new applications. PMID:24906189

  2. Proteomic Analysis Reveals Key Proteins and Phosphoproteins upon Seed Germination of Wheat (Triticum aestivum L.)

    PubMed Central

    Dong, Kun; Zhen, Shoumin; Cheng, Zhiwei; Cao, Hui; Ge, Pei; Yan, Yueming

    2015-01-01

    Wheat (Triticum aestivum L.) is one of the oldest cultivated crops and the second most important food crop in the world. Seed germination is the key developmental process in plant growth and development, and poor germination directly affects plant growth and subsequent grain yield. In this study, we performed the first dynamic proteome analysis of wheat seed germination using a two-dimensional differential gel electrophoresis (2D-DIGE)-based proteomic approach. A total of 166 differentially expressed protein (DEP) spots representing 73 unique proteins were identified, which are mainly involved in storage, stress/defense/detoxification, carbohydrate metabolism, photosynthesis, cell metabolism, and transcription/translation/transposition. The identified DEPs and their dynamic expression profiles generally correspond to three distinct seed germination phases after imbibition: storage degradation, physiological processes/morphogenesis, and photosynthesis. Some key DEPs involved in storage substance degradation and plant defense mechanisms, such as globulin 3, sucrose synthase type I, serpin, beta-amylase, and plastid ADP-glucose pyrophosphorylase (AGPase) small subunit, were found to be phosphorylated during seed germination. Particularly, the phosphorylation site Ser355 was found to be located in the enzyme active region of beta-amylase, which promotes substrate binding. Phosphorylated modification of several proteins could promote storage substance degradation and environmental stress defense during seed germination. The central metabolic pathways involved in wheat seed germination are proposed herein, providing new insights into the molecular mechanisms of cereal seed germination. PMID:26635843

  3. Systematic Analysis of Pericarp Starch Accumulation and Degradation during Wheat Caryopsis Development

    PubMed Central

    Yu, Xurun; Li, Bo; Wang, Leilei; Chen, Xinyu; Wang, Wenjun; Wang, Zhong; Xiong, Fei

    2015-01-01

    Although wheat (Triticum aestivum L.) pericarp starch granule (PSG) has been well-studied, our knowledge of its features and mechanism of accumulation and degradation during pericarp growth is poor. In the present study, developing wheat caryopses were collected and starch granules were extracted from their pericarp to investigate the morphological and structural characteristics of PSGs using microscopy, X-ray diffraction and Fourier transform infrared spectroscopy techniques. Relative gene expression levels of ADP-glucose pyrophosphorylase (APGase), granule-bound starch synthase II (GBSS II), and α-amylase (AMY) were quantified by quantitative real-time polymerase chain reaction. PSGs presented as single or multiple starch granules and were synthesized both in the amyloplast and chloroplast in the pericarp. PSG degradation occurred in the mesocarp, beginning at 6 days after anthesis. Amylose contents in PSGs were lower and relative degrees of crystallinity were higher at later stages of development than at earlier stages. Short-range ordered structures in the external regions of PSGs showed no differences in the developing pericarp. When hydrolyzed by α-amylase, PSGs at various developmental stages showed high degrees of enzymolysis. Expression levels of AGPase, GBSS II, and AMY were closely related to starch synthesis and degradation. These results help elucidate the mechanisms of accumulation and degradation as well as the functions of PSG during wheat caryopsis development. PMID:26394305

  4. Changes in carbohydrate metabolism in Plasmopara viticola-infected grapevine leaves.

    PubMed

    Gamm, Magdalena; Héloir, Marie-Claire; Bligny, Richard; Vaillant-Gaveau, Nathalie; Trouvelot, Sophie; Alcaraz, Gérard; Frettinger, Patrick; Clément, Christophe; Pugin, Alain; Wendehenne, David; Adrian, Marielle

    2011-09-01

    The oomycete Plasmopara viticola is responsible for downy mildew, a severe grapevine disease. In infected grapevine leaves, we have observed an abnormal starch accumulation at the end of the dark period, suggesting modifications in starch metabolism. Therefore, several complementary approaches, including transcriptomic analyses, measurements of enzyme activities, and sugar quantification, were performed in order to investigate and to understand the effects of P. viticola infection on leaf starch and-to a larger extent-carbohydrate metabolism. Our results indicate that starch accumulation is associated with an increase in ADP-glucose pyrophosphorylase (AGPase) activity and modifications in the starch degradation pathway, especially an increased α-amylase activity. Together with these alterations in starch metabolism, we have observed an accumulation of hexoses, an increase in invertase activity, and a reduction of photosynthesis, indicating a source-to-sink transition in infected leaf tissue. Additionally, we have measured an accumulation of the disaccharide trehalose correlated to an increased trehalase gene expression and enzyme activity. Altogether, these results highlight a dramatic alteration of carbohydrate metabolism correlated with later stages of P. viticola development in leaves. PMID:21649510

  5. Can fast-growing plantation trees escape biochemical down-regulation of photosynthesis when grown throughout their complete production cycle in the open air under elevated carbon dioxide?

    PubMed

    Davey, P A; Olcer, H; Zakhleniuk, O; Bernacchi, C J; Calfapietra, C; Long, S P; Raines, C A

    2006-07-01

    Poplar trees sustain close to the predicted increase in leaf photosynthesis when grown under long-term elevated CO2 concentration ([CO2]). To investigate the mechanisms underlying this response, carbohydrate accumulation and protein expression were determined over four seasons of growth. No increase in the levels of soluble carbohydrates was observed in the young expanding or mature sun leaves of the three poplar genotypes during this period. However, substantial increases in starch levels were observed in the mature leaves of all three poplar genotypes grown in elevated [CO2]. Despite the very high starch levels, no changes in the expression of photosynthetic Calvin cycle proteins, or in the starch biosynthetic enzyme ADP-glucose pyrophosphorylase (AGPase), were observed. This suggested that no long-term photosynthetic acclimation to CO2 occurred in these plants. Our data indicate that poplar trees are able to 'escape' from long-term, acclimatory down-regulation of photosynthesis through a high capacity for starch synthesis and carbon export. These findings show that these poplar genotypes are well suited to the elevated [CO2] conditions forecast for the middle of this century and may be particularly suited for planting for the long-term carbon sequestration into wood. PMID:17080946

  6. Distinct metabolic changes between wheat embryo and endosperm during grain development revealed by 2D-DIGE-based integrative proteome analysis.

    PubMed

    Cao, Hui; He, Miao; Zhu, Chong; Yuan, Linlin; Dong, Liwei; Bian, Yanwei; Zhang, Wenying; Yan, Yueming

    2016-05-01

    Two Chinese bread wheat cultivars, Jinghua 9 and Zhongmai 175, distinct in grain weight and dough quality, were used to study proteome changes in the embryo and endosperm during grain development using a two-dimensional difference gel electrophoresis (2D-DIGE)-based proteomics approach. In total, 138 and 127 differentially expressed protein (DEP) spots representing 116 and 113 unique DEPs were identified in the embryo and endosperm, respectively. Among them, 54 (31%) DEPs were commonly present in both organs while 62 (35%) and 59 (34%) DEPs occurred only in the embryo and endosperm, respectively. Embryonic DEPs are primarily stress-related proteins and involved in carbohydrate and lipid metabolism, while those from the endosperm are related primarily to carbohydrate metabolism and storage. Principal component analysis (PCA) indicated that the proteome differences in the endosperm caused by different cultivars were greater than those by development stages, while the differences in the embryo showed the opposite pattern. Protein-protein interaction (PPI) analysis revealed a complex network centered primarily on enzymes involved in carbohydrate and protein metabolism. The transcriptional levels of fourteen important DEPs encoding genes showed high similarity between organs and cultivars. In particular, some key DEPs of the endosperm, such as phosphoglucomutase, ADP-glucose pyrophosphorylase (AGPase), and sucrose synthase (SUS), showed significantly upregulated expression, indicating their key roles in starch biosynthesis and grain yield. Moreover, upregulated expression of some storage proteins in the endosperm could improve wheat bread-making quality. PMID:26968330

  7. Salinity induces carbohydrate accumulation and sugar-regulated starch biosynthetic genes in tomato (Solanum lycopersicum L. cv. ‘Micro-Tom’) fruits in an ABA- and osmotic stress-independent manner

    PubMed Central

    Yin, Yong-Gen; Kobayashi, Yoshie; Sanuki, Atsuko; Kondo, Satoru; Fukuda, Naoya; Ezura, Hiroshi; Sugaya, Sumiko; Matsukura, Chiaki

    2010-01-01

    Salinity stress enhances sugar accumulation in tomato (Solanum lycopersicum) fruits. To elucidate the mechanisms underlying this phenomenon, the transport of carbohydrates into tomato fruits and the regulation of starch synthesis during fruit development in tomato plants cv. ‘Micro-Tom’ exposed to high levels of salinity stress were examined. Growth with 160 mM NaCl doubled starch accumulation in tomato fruits compared to control plants during the early stages of development, and soluble sugars increased as the fruit matured. Tracer analysis with 13C confirmed that elevated carbohydrate accumulation in fruits exposed to salinity stress was confined to the early development stages and did not occur after ripening. Salinity stress also up-regulated sucrose transporter expression in source leaves and increased activity of ADP-glucose pyrophosphorylase (AGPase) in fruits during the early development stages. The results indicate that salinity stress enhanced carbohydrate accumulation as starch during the early development stages and it is responsible for the increase in soluble sugars in ripe fruit. Quantitative RT-PCR analyses of salinity-stressed plants showed that the AGPase-encoding genes, AgpL1 and AgpS1 were up-regulated in developing fruits, and AgpL1 was obviously up-regulated by sugar at the transcriptional level but not by abscisic acid and osmotic stress. These results indicate AgpL1 and AgpS1 are involved in the promotion of starch biosynthesis under the salinity stress in ABA- and osmotic stress-independent manners. These two genes are differentially regulated at the transcriptional level, and AgpL1 is suggested to play a regulatory role in this event. PMID:19995825

  8. Lauric Acid Production in a Glycogen-Less Strain of Synechococcus sp. PCC 7002

    PubMed Central

    Work, Victoria H.; Melnicki, Matthew R.; Hill, Eric A.; Davies, Fiona K.; Kucek, Leo A.; Beliaev, Alexander S.; Posewitz, Matthew C.

    2015-01-01

    The cyanobacterium Synechococcus sp. Pasteur culture collection 7002 was genetically engineered to synthesize biofuel-compatible medium-chain fatty acids (FAs) during photoautotrophic growth. Expression of a heterologous lauroyl-acyl carrier protein (C12:0-ACP) thioesterase with concurrent deletion of the endogenous putative acyl-ACP synthetase led to secretion of transesterifiable C12:0 FA in CO2-supplemented batch cultures. When grown at steady state over a range of light intensities in a light-emitting diode turbidostat photobioreactor, the C12-secreting mutant exhibited a modest reduction in growth rate and increased O2 evolution relative to the wild-type (WT). Inhibition of (i) glycogen synthesis by deletion of the glgC-encoded ADP-glucose pyrophosphorylase (AGPase) and (ii) protein synthesis by nitrogen deprivation were investigated as potential mechanisms for metabolite redistribution to increase FA synthesis. Deletion of AGPase led to a 10-fold decrease in reducing carbohydrates and secretion of organic acids during nitrogen deprivation consistent with an energy spilling phenotype. When the carbohydrate-deficient background (ΔglgC) was modified for C12 secretion, no increase in C12 was achieved during nutrient replete growth, and no C12 was recovered from any strain upon nitrogen deprivation under the conditions used. At steady state, the growth rate of the ΔglgC strain saturated at a lower light intensity than the WT, but O2 evolution was not compromised and became increasingly decoupled from growth rate with rising irradiance. Photophysiological properties of the ΔglgC strain suggest energy dissipation from photosystem II and reconfiguration of electron flow at the level of the plastoquinone pool. PMID:25964950

  9. MUMMY, A UDP-N-ACETYLGLUCOSAMINE PYROPHOSPHORYLASE, MODULATES DPP SIGNALING IN THE EMBRYONIC EPIDERMIS OF DROSOPHILA

    PubMed Central

    HUMPHREYS, GREGORY B.; JUD, MOLLY C.; MONROE, KATHRYN M.; KIMBALL, SUZANNE S.; HIGLEY, MATTHEW; SHIPLEY, DANIELLE; VRABLIK, MARIE CLOUGHERTY; BATES, KATHERINE L.; LETSOU, ANTHEA

    2013-01-01

    The evolutionarily conserved JNK/AP-1 (Jun N-terminal kinase/activator protein 1) and BMP (Bone Morphogenetic Protein) signaling cascades are deployed hierarchically to regulate dorsal closure in the fruit fly Drosophila melanogaster. In this developmental context, the JNK/AP-1 signaling cascade transcriptionally activates BMP signaling in leading edge epidermal cells. Here we show that the mummy (mmy) gene product, which is required for dorsal closure, functions as a BMP signaling antagonist. Genetic and biochemical tests of Mmy’s role as a BMP-antagonist indicate that its function is independent of AP-1, the transcriptional trigger of BMP signal transduction in leading edge cells. pMAD (phosphorylated Mothers Against Dpp) activity data show the mmy gene product to be a new type of epidermal BMP regulator – one which transforms a BMP ligand from a long- to a short-range signal. mmy codes for the single UDP-N-acetylglucosamine pyrophosphorylase in Drosophila, and its requirement for attenuating epidermal BMP signaling during dorsal closure points to a new role for glycosylation in defining a highly restricted BMP activity field in the fly. These findings add a new dimension to our understanding of mechanisms modulating the BMP signaling gradient. PMID:23796903

  10. UDP-glucose pyrophosphorylase is a novel plant cell death regulator.

    PubMed

    Chivasa, Stephen; Tomé, Daniel F A; Slabas, Antoni R

    2013-04-01

    Programmed cell death (PCD) is an essential process that functions in plant organ sculpture, tissue differentiation, nutrient recycling, and defense against pathogen attack. A full understanding of the mechanism of PCD in plants is hindered by the limited identification of protein components of the complex signaling circuitry that underpins this important physiological process. Here we have used Arabidopsis thaliana and fumonisin B1 (FB1) to identify proteins that constitute part of the PCD signaling network. We made an inadvertent, but important observation that exogenous sucrose modulates FB1-induced cell death and identified sucrose-induced genes from publicly available transcriptomic data sets for reverse genetic analyses. Using transfer-DNA gene knockout plants, UDP-glucose pyrophosphorylase 1 (UGP1), a sucrose-induced gene, was demonstrated to be a critical factor that regulates FB1-induced PCD. We employed 2D-DiGE to identify proteomic changes preceding PCD after exposure of Arabidopsis to FB1 and used UGP1 knockout plants to refine the analysis and isolate downstream candidate proteins with a putative PCD regulatory function. Our results reveal chloroplasts as the predominantly essential organelles in FB1-induced PCD. Overall, this study reveals a novel function of UGP1 as a cell death regulator and provides candidate proteins likely recruited downstream in the activation of plant PCD. PMID:23438466

  11. Genetic and structural validation of Aspergillus fumigatus UDP-N-acetylglucosamine pyrophosphorylase as an antifungal target

    PubMed Central

    Fang, Wenxia; Du, Ting; Raimi, Olawale G; Hurtado-Guerrero, Ramon; Urbaniak, Michael D; Ibrahim, Adel F M; Ferguson, Michael A J; Jin, Cheng; Aalten, Daan M F

    2013-01-01

    The sugar nucleotide UDP-N-acetylglucosamine (UDP-GlcNAc) is an essential metabolite in both prokaryotes and eukaryotes. In fungi, it is the precursor for the synthesis of chitin, an essential component of the fungal cell wall. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is the final enzyme in eukaryotic UDP-GlcNAc biosynthesis, converting UTP and N-acetylglucosamine-1-phosphate (GlcNAc-1P) to UDP-GlcNAc. As such, this enzyme may provide an attractive target against pathogenic fungi. Here, we demonstrate that the fungal pathogen Aspergillus fumigatus possesses an active UAP (AfUAP1) that shows selectivity for GlcNAc-1P as the phosphosugar substrate. A conditional mutant, constructed by replacing the native promoter of the A. fumigatus uap1 gene with the Aspergillus nidulans alcA promoter, revealed that uap1 is essential for cell survival and important for cell wall synthesis and morphogenesis. The crystal structure of AfUAP1 was determined and revealed exploitable differences in the active site compared with the human enzyme. Thus AfUAP1 could represent a novel antifungal target and this work will assist the future discovery of small molecule inhibitors against this enzyme. PMID:23750903

  12. Purification, crystallization and preliminary X-ray characterization of the human GTP fucose pyrophosphorylase

    SciTech Connect

    Quirk, Stephen; Seley-Radtke, Katherine L.

    2006-04-01

    The human GTP fucose pyrophosphohydrolase protein has been crystallized via the hanging-drop technique over a reservoir of polyethylene glycol (MW 8000) and ethylene glycol. The orthorhombic crystals diffract to 2.8 Å resolution. The human nucleotide-sugar metabolizing enzyme GTP fucose pyrophosphorylase (GFPP) has been purified to homogeneity by an affinity chromatographic procedure that utilizes a novel nucleoside analog. This new purification regime results in a protein preparation that produces significantly better crystals than traditional purification methods. The purified 66.6 kDa monomeric protein has been crystallized via hanging-drop vapor diffusion at 293 K. Crystals of the native enzyme diffract to 2.8 Å and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}. There is a single GFPP monomer in the asymmetric unit, giving a Matthews coefficient of 2.38 Å{sup 3} Da{sup −1} and a solvent content of 48.2%. A complete native data set has been collected as a first step in determining the three-dimensional structure of this enzyme.

  13. Overexpression of UDP-glucose pyrophosphorylase gene could increase cellulose content in Jute (Corchorus capsularis L.).

    PubMed

    Zhang, Gaoyang; Qi, Jianmin; Xu, Jiantang; Niu, Xiaoping; Zhang, Yujia; Tao, Aifen; Zhang, Liwu; Fang, Pingping; Lin, Lihui

    2013-12-13

    In this study, the full-length cDNA of the UDP-glucose pyrophosphorylase gene was isolated from jute by homologous cloning (primers were designed according to the sequence of UGPase gene of other plants) and modified RACE techniques; the cloned gene was designated CcUGPase. Using bioinformatic analysis, the gene was identified as a member of the UGPase gene family. Real-time PCR analysis revealed differential spatial and temporal expression of the CcUGPase gene, with the highest expression levels at 40 and 120d. PCR and Southern hybridization results indicate that the gene was integrated into the jute genome. Overexpression of CcUGPase gene in jute revealed increased height and cellulose content compared with control lines, although the lignin content remained unchanged. The results indicate that the jute UGPase gene participates in cellulose biosynthesis. These data provide an important basis for the application of the CcUGPase gene in the improvement of jute fiber quality. PMID:24269810

  14. GDP-D-mannose pyrophosphorylase from Pogonatherum paniceum enhances salinity and drought tolerance of transgenic tobacco.

    PubMed

    Ai, Taobo; Liao, Xuehong; Li, Rui; Fan, Linhong; Luo, Fengxue; Xu, Ying; Wang, Shenghua

    2016-01-01

    Pogonatherum paniceum is a highly drought- and salt-tolerant plant species that is typically used for ecological restoration and the conservation of soil and water in many countries. Understanding the molecular mechanisms underlying plant abiotic stress responses, especially to salinity and drought stresses, in species such as P. paniceum could be important to broader crop improvement efforts. GDP-D-mannose pyrophosphorylase (GMPase) is the limiting enzyme in the synthesis of L-ascorbic acid (AsA), which plays a crucial role in the detoxification of reactive oxygen species (ROS). We have cloned and characterized the cDNA of the PpGMP gene of P. paniceum encoding a GMPase. The full-length cDNA sequence contains 1411 nucleotides encoding a putative protein with 361 amino acid residues and an approximate molecular mass of 39.68 kDa. The GMPase transcript was up-regulated in P. paniceum plants subjected to salinity and drought stress, respectively. Transgenic tobacco expressing PpGMPase exhibited enhanced salinity and drought resistance, a higher seed germination rate, better growth performance, a higher AsA content, a more stable redox state, higher superoxide dismutase (SOD) activity, and lower levels of malonaldehyde (MDA) and H2O2 under drought and salinity stress. Taken together, expression of PpGMPase in tobacco conferred salinity and drought stress tolerance by increasing the content of AsA, thereby enhancing ROS-detoxifying functions. Thus, PpGMP is a potential candidate gene for crop improvement. PMID:27442366

  15. Purification, crystallization and preliminary X-ray diffraction studies of UDP-N-acetylglucosamine pyrophosphorylase from Candida albicans

    SciTech Connect

    Maruyama, Daisuke; Nishitani, Yuichi; Nonaka, Tsuyoshi; Kita, Akiko; Fukami, Takaaki A.; Mio, Toshiyuki; Yamada-Okabe, Hisafumi; Yamada-Okabe, Toshiko; Miki, Kunio

    2006-12-01

    UDP-N-acetylglucosamine pyrophosphorylase was purified and crystallized and X-ray diffraction data were collected to 2.3 Å resolution. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is an essential enzyme in the synthesis of UDP-N-acetylglucosamine. UAP from Candida albicans was purified and crystallized by the sitting-drop vapour-diffusion method. The crystals of the substrate and product complexes both diffract X-rays to beyond 2.3 Å resolution using synchrotron radiation. The crystals of the substrate complex belong to the triclinic space group P1, with unit-cell parameters a = 47.77, b = 62.89, c = 90.60 Å, α = 90.01, β = 97.72, γ = 92.88°, whereas those of the product complex belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 61.95, b = 90.87, c = 94.88 Å.

  16. Structure and Dynamics of UDP-Glucose Pyrophosphorylase from Arabidopsis thaliana with Bound UDP-Glucose and UTP

    PubMed Central

    McCoy, Jason G.; Bitto, Eduard; Bingman, Craig A.; Wesenberg, Gary E.; Bannen, Ryan M.; Kondrashov, Dmitry A.; Phillips, George N.

    2007-01-01

    The structure of the UDP-glucose pyrophosphorylase encoded by Arabidopsis thaliana gene At3g03250 has been solved to a nominal resolution of 1.86 Å. In addition, the structure has been solved in the presence of the substrates/products UTP and UDP-glucose to nominal resolutions of 1.64 Å and 1.85 Å. The three structures revealed a catalytic domain similar to that of other nucleotidyl-glucose pyrophosphorylases with a carboxy-terminal β-helix domain in a unique orientation. Conformational changes are observed between the native and substrate-bound complexes. The nucleotide binding loop and the carboxy-terminal domain, including the suspected catalytically important Lys360, move in and out of the active site in a concerted fashion. TLS refinement was employed to initially model conformational heterogeneity in the UDP-glucose complex followed by the use of multiconformer refinement for the entire molecule. Normal mode analysis generated atomic displacement predictions in good agreement in magnitude and direction with the observed conformational changes and anisotropic displacement parameters generated by TLS refinement. The structures and the observed dynamic changes provide insight into the ordered mechanism of this enzyme and previously described oligomerization effects on catalytic activity. PMID:17178129

  17. Molecular and functional analysis of UDP-N-acetylglucosamine Pyrophosphorylases from the Migratory Locust, Locusta migratoria.

    PubMed

    Liu, Xiaojian; Li, Feng; Li, Daqi; Ma, Enbo; Zhang, Wenqing; Zhu, Kun Yan; Zhang, Jianzhen

    2013-01-01

    UDP-N-acetylglucosamine pyrophosphorylases (UAP) function in the formation of extracellular matrix by producing N-acetylglucosamine (GlcNAc) residues needed for chitin biosynthesis and protein glycosylation. Herein, we report two UAP cDNA's derived from two different genes (LmUAP1 and LmUAP2) in the migratory locust Locusta migratoria. Both the cDNA and their deduced amino acid sequences showed about 70% identities between the two genes. Phylogenetic analysis suggests that LmUAP1 and LmUAP2 derive from a relatively recent gene duplication event. Both LmUAP1 and LmUAP2 were widely expressed in all the major tissues besides chitin-containing tissues. However, the two genes exhibited different developmental expression patterns. High expression of LmUAP1 was detected during early embryogenesis, then decreased greatly, and slowly increased before egg hatch. During nymphal development, the highest expression of LmUAP1 appeared just after molting but declined in each inter-molting period and then increased before molting to the next stage, whereas LmUAP2 was more consistently expressed throughout all these stages. When the early second- and fifth-instar nymphs (1-day-old) were injected with LmUAP1 double-stranded RNA (dsRNA), 100% mortality was observed 2 days after the injection. When the middle second- and fifth-instar nymphs (3- to 4-day-old) were injected with LmUAP1 dsRNA, 100% mortality was observed during their next molting process. In contrast, when the insects at the same stages were injected with LmUAP2 dsRNA, these insects were able to develop normally and molt to the next stage successfully. It is presumed that the lethality caused by RNAi of LmUAP1 is due to reduced chitin biosynthesis of the integument and midgut, whereas LmUAP2 is not essential for locust development at least in nymph stage. This study is expected to help better understand different functions of UAP1 and UAP2 in the locust and other insect species. PMID:23977188

  18. Molecular and Functional Analysis of UDP-N-Acetylglucosamine Pyrophosphorylases from the Migratory Locust, Locusta migratoria

    PubMed Central

    Li, Daqi; Ma, Enbo; Zhang, Wenqing; Zhu, Kun Yan; Zhang, Jianzhen

    2013-01-01

    UDP-N-acetylglucosamine pyrophosphorylases (UAP) function in the formation of extracellular matrix by producing N-acetylglucosamine (GlcNAc) residues needed for chitin biosynthesis and protein glycosylation. Herein, we report two UAP cDNA’s derived from two different genes (LmUAP1 and LmUAP2) in the migratory locust Locusta migratoria. Both the cDNA and their deduced amino acid sequences showed about 70% identities between the two genes. Phylogenetic analysis suggests that LmUAP1 and LmUAP2 derive from a relatively recent gene duplication event. Both LmUAP1 and LmUAP2 were widely expressed in all the major tissues besides chitin-containing tissues. However, the two genes exhibited different developmental expression patterns. High expression of LmUAP1 was detected during early embryogenesis, then decreased greatly, and slowly increased before egg hatch. During nymphal development, the highest expression of LmUAP1 appeared just after molting but declined in each inter-molting period and then increased before molting to the next stage, whereas LmUAP2 was more consistently expressed throughout all these stages. When the early second- and fifth-instar nymphs (1-day-old) were injected with LmUAP1 double-stranded RNA (dsRNA), 100% mortality was observed 2 days after the injection. When the middle second- and fifth-instar nymphs (3- to 4-day-old) were injected with LmUAP1 dsRNA, 100% mortality was observed during their next molting process. In contrast, when the insects at the same stages were injected with LmUAP2 dsRNA, these insects were able to develop normally and molt to the next stage successfully. It is presumed that the lethality caused by RNAi of LmUAP1 is due to reduced chitin biosynthesis of the integument and midgut, whereas LmUAP2 is not essential for locust development at least in nymph stage. This study is expected to help better understand different functions of UAP1 and UAP2 in the locust and other insect species. PMID:23977188

  19. Effect of nitrogen and phosphorus deficiency on transcriptional regulation of genes encoding key enzymes of starch metabolism in duckweed (Landoltia punctata).

    PubMed

    Zhao, Zhao; Shi, Hui-juan; Wang, Mao-lin; Cui, Long; Zhao, Hai; Zhao, Yun

    2015-01-01

    The production of starch by plants influences their use as biofuels. Nitrogen (N) and phosphorus (P) regulate starch gene expression during plant growth and development, yet the role of key enzymes such as ADP-glucose pyrophosphorylase (E.C. 2.7.7.27 AGPase) in starch metabolism during N- and P-deficiency remains unknown. We investigated the effect of N- and P-deficiency on the expression of large (LeAPL1, LeAPL2, and LeAPL3) and small (LeAPS) subunits of AGPase in duckweed (Landoltia punctata) and their correlation with starch content. We first isolated the full-length cDNA encoding LeAPL1 (GenBank Accession No. KJ603244) and LeAPS (GenBank Accession No. KJ603243); they contained open reading frames of 1554 bp (57.7-kDa polypeptide of 517 amino acids) and 1578 bp (57.0 kDa polypeptide of 525 amino acids), respectively. Real-time PCR analysis revealed that LeAPL1 and LeAPL3 were highly expressed during early stages of N-deficiency, while LeAPL2 was only expressed during late stage. However, in response to P-deficiency, LeAPL1 and LeAPL2 were upregulated during early stages and LeAPL3 was primarily expressed in the late stage. Interestingly, LeAPS was highly expressed following N-deficiency during both stages, but was only upregulated in the early stage after P-deficiency. The activities of AGPase and soluble starch synthesis enzyme (SSS EC 2.4.1.21) were positively correlated with changes in starch content. Furthermore, LeAPL3 and LeSSS (SSS gene) were positively correlated with changes in starch content during N-deficiency, while LeAPS and LeSSS were correlated with starch content in response to P-deficiency. These results elevate current knowledge of the molecular mechanisms underlying starch synthesis. PMID:25438139

  20. Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase gene. Progress report, [April 15, 1987--April 14, 1988

    SciTech Connect

    Okita, T.W.

    1988-12-31

    Many agronomically important crops are viewed as significant resources of renewable energy. Overall crop productivity could be increased if the efficiency of photoassimilate conversion into dry matter such as starch were improved in storage tissues. Starch production is controlled by the catalytic activity of ADPglucose pyrophosphorylase in the first step of starch biosynthesis. This research focuses on the genetic structure and molecular mechanisms by which it is controlled during plant development and how it is affected by environmental and hormonal conditions. The current goal is to isolate the genes for this enzyme present in both cereal endosperm and potato tuber tissues, and to elucidate its structure and the controlling sequences responsible for gene expression. The long term goal is the improvement of starch production in storage organs by manipulating this gene so that it encodes an enzyme refractive to inorganic phosphate inhibition.

  1. Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase genes. Progress report, [April 15, 1990--April 14, 1991

    SciTech Connect

    Okita, T.W.

    1990-12-31

    The long term goal of this project is to assess the feasibility of increasing the conversion of photosynthate a key regulatory enzyme in starch biosynthesis. In developing storage tissues such as cereal seeds and tubers, starch biosynthesis is primarily regulated by the gene activation, expression, and allosteric regulation of ADPglucose pyrophosphorylase, as well as starch synthase, and branching enzyme. During the last year we have elucidated the structure of both subunits which compose this tetrameric enzyme and determined the temporal and spatial expression of the genes encoding each subunit as well as their correlation to starch biosynthesis. Genomic clones to both subunits have also been isolated and the gene structure of the small subunit determined. Transgenic potato plants have been produced containing deletions of the small subunit promoter. Currently, cis acting elements and their involvement in spatial and temporal expression are under investigation.

  2. In silico analysis of a therapeutic target in Leishmania infantum: the guanosine-diphospho-D-mannose pyrophosphorylase.

    PubMed

    Pomel, S; Rodrigo, J; Hendra, F; Cavé, C; Loiseau, P M

    2012-02-01

    Leishmaniases are tropical and sub-tropical diseases for which classical drugs (i.e. antimonials) exhibit toxicity and drug resistance. Such a situation requires to find new chemical series with antileishmanial activity. This work consists in analyzing the structure of a validated target in Leishmania: the GDP-mannose pyrophosphorylase (GDP-MP), an enzyme involved in glycosylation and essential for amastigote survival. By comparing both human and L. infantum GDP-MP 3D homology models, we identified (i) a common motif of amino acids that binds to the mannose moiety of the substrate and, interestingly, (ii) a motif that is specific to the catalytic site of the parasite enzyme. This motif could then be used to design compounds that specifically inhibit the leishmanial GDP-MP, without any effect on the human homolog. PMID:22314241

  3. Mutagenesis of the potato ADPglucose pyrophosphorylase and characterization of an allosteric mutant defective in 3-phosphoglycerate activation

    SciTech Connect

    Greene, T.W.; Chantler, S.E.; Kahn, M.L.

    1996-02-20

    ADPglucose pyrophosphorylase (glucose-1-phosphate adenylytransferase; AD P:{alpha}-D-glucose-1-phosphate adenylyltransferase, EC 2.7.7.27) catalyzes a key regulatory step in {alpha}-glucan synthesis in bacteria and higher plants. We have previously shown that the expression of the cDNA sequences of the potato tuber large (LS) and small (SS) subunits yielded a functional heterotetrameric enzyme capable of complementing a mutation in the single AGP (glgC) structural gene of Escherichia coli. This heterologous complementation provides a powerful genetic approach to obtain biochemical information on the specific roles of LS and SS in enzyme function. By mutagenizing the LS cDNA with hydroxylamine and then coexpressing with wild-type SS in an E. coli glgC{sup {minus}} strain, >350 mutant colonies were identified that were impaired in glycogen production. One mutant exhibited enzymatic and antigen levels comparable to the wild-type recombinant enzyme but required 45-fold greater levels of the activator 3-phosphoglycerate for maximum activity. Sequence analysis identified a single nucleotide change that resulted in the change of Pro-52 to Leu. This heterologous genetic system provides and efficient means to identify residues important for catalysis and allosteric functioning and should lead to novel approaches to increase plant productivity. 31 refs., 4 figs., 1 tab.

  4. Identification, subcellular localization, biochemical properties, and high-resolution crystal structure of Trypanosoma brucei UDP-glucose pyrophosphorylase

    PubMed Central

    Mariño, Karina; Güther, Maria Lucia Sampaio; Wernimont, Amy K; Amani, Mernhaz; Hui, Raymond; Ferguson, Michael AJ

    2010-01-01

    The protozoan parasite Trypanosoma brucei is the causative agent of the cattle disease Nagana and human African sleeping sickness. Glycoproteins play key roles in the parasite’s survival and infectivity, and the de novo biosyntheses of the sugar nucleotides UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine, and GDP-fucose have been shown to be essential for their growth. The only route to UDP-Gal in T. brucei is through the epimerization of UDP-glucose (UDP-Glc) by UDP-Glc 4′-epimerase. UDP-Glc is also the glucosyl donor for the unfolded glycoprotein glucosyltransferase (UGGT) involved in glycoprotein quality control in the endoplasmic reticulum and is the presumed donor for the synthesis of base J (β-d-glucosylhydroxymethyluracil), a rare deoxynucleotide found in telomere-proximal DNA in the bloodstream form of T. brucei. Considering that UDP-Glc plays such a central role in carbohydrate metabolism, we decided to characterize UDP-Glc biosynthesis in T. brucei. We identified and characterized the parasite UDP-glucose pyrophosphorylase (TbUGP), responsible for the formation of UDP-Glc from glucose-1-phosphate and UTP, and localized the enzyme to the peroxisome-like glycosome organelles of the parasite. Recombinant TbUGP was shown to be enzymatically active and specific for glucose-1-phosphate. The high-resolution crystal structure was also solved, providing a framework for the design of potential inhibitors against the parasite enzyme. PMID:20724435

  5. Functional inactivation of UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1) induces early leaf senescence and defence responses in rice.

    PubMed

    Wang, Zhaohai; Wang, Ya; Hong, Xiao; Hu, Daoheng; Liu, Caixiang; Yang, Jing; Li, Yang; Huang, Yunqing; Feng, Yuqi; Gong, Hanyu; Li, Yang; Fang, Gen; Tang, Huiru; Li, Yangsheng

    2015-02-01

    Plant leaf senescence and defence responses are important biological processes, but the molecular mechanisms involved are not well understood. This study identified a new rice mutant, spotted leaf 29 (spl29). The SPL29 gene was identified by map-based cloning, and SPL29 was confirmed as UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1) by enzymatic analysis. The mutant spl29 lacks UAP activity. The biological phenotypes for which UAP is responsible have not previously been reported in plants. The spl29 mutant displayed early leaf senescence, confirmed by chlorophyll loss and photosystem II decline as physiological indicators, chloroplast degradation as a cellular characteristic, and both upregulation of senescence transcription factors and senescence-associated genes, and downregulation of photosynthesis-related genes, as molecular evidence. Defence responses were induced in the spl29 mutant, shown by enhanced resistance to bacterial blight inoculation and upregulation of defence response genes. Reactive oxygen species, including O2 (-) and H2O2, accumulated in spl29 plants; there was also increased malondialdehyde content. Enhanced superoxide dismutase activity combined with normal catalase activity in spl29 could be responsible for H2O2 accumulation. The plant hormones jasmonic acid and abscisic acid also accumulated in spl29 plants. ROS and plant hormones probably play important roles in early leaf senescence and defence responses in the spl29 mutant. Based on these findings, it is suggested that UAP1 is involved in regulating leaf senescence and defence responses in rice. PMID:25399020

  6. Enhanced UDP-glucose and UDP-galactose by homologous overexpression of UDP-glucose pyrophosphorylase in Lactobacillus casei.

    PubMed

    Rodríguez-Díaz, Jesús; Yebra, María J

    2011-07-20

    UDP-sugars are widely used as substrates in the synthesis of oligosaccharides catalyzed by glycosyltransferases. In the present work a metabolic engineering strategy aimed to direct the carbon flux towards UDP-glucose and UDP-galactose biosynthesis was successfully applied in Lactobacillus casei. The galU gene coding for UDP-glucose pyrophosphorylase (GalU) enzyme in L. casei BL23 was cloned under control of the inducible nisA promoter and it was shown to be functional by homologous overexpression. Notably, about an 80-fold increase in GalU activity resulted in approximately a 9-fold increase of UDP-glucose and a 4-fold increase of UDP-galactose. This suggested that the endogenous UDP-galactose 4-epimerase (GalE) activity, which inter-converts both UDP-sugars, is not sufficient to maintain the UDP-glucose/UDP-galactose ratio. The L. casei galE gene coding for GalE was cloned downstream of galU and the resulting plasmid was transformed in L. casei. The new recombinant strain showed about a 4-fold increase of GalE activity, however this increment did not affect that ratio, suggesting that GalE has higher affinity for UDP-galactose than for UDP-glucose. The L. casei strains constructed here that accumulate high intracellular levels of UDP-sugars would be adequate hosts for the production of oligosaccharides. PMID:21663774

  7. Deletion of UDP-glucose pyrophosphorylase reveals a UDP-glucose independent UDP-galactose salvage pathway in Leishmania major

    PubMed Central

    Lamerz, Anne-Christin; Damerow, Sebastian; Kleczka, Barbara; Wiese, Martin; van Zandbergen, Ger; Lamerz, Jens; Wenzel, Alexander; Hsu, Fong-Fu; Turk, John; Beverley, Stephen M.; Routier, Françoise H.

    2010-01-01

    The nucleotide sugar UDP-galactose (UDP-Gal) is essential for the biosynthesis of several abundant glycoconjugates forming the surface glycocalyx of the protozoan parasite Leishmania major. Current data suggest that UDP-Gal could arise de novo by epimerization of UDP-glucose (UDP-Glc) or by a salvage pathway involving phosphorylation of Gal and the action of UDP-glucose:α-d-galactose-1-phosphate uridylyltransferase as described by Leloir. Since both pathways require UDP-Glc, inactivation of the UDP-glucose pyrophosphorylase (UGP) catalyzing activation of glucose-1 phosphate to UDP-Glc was expected to deprive parasites of UDP-Gal required for Leishmania glycocalyx formation. Targeted deletion of the gene encoding UGP, however, only partially affected the synthesis of the Gal-rich phosphoglycans. Moreover, no alteration in the abundant Gal-containing glycoinositolphospholipids was found in the deletion mutant. Consistent with these findings, the virulence of the UGP-deficient mutant was only modestly affected. These data suggest that Leishmania elaborates a UDP-Glc independent salvage pathway for UDP-Gal biosynthesis. PMID:20335578

  8. Cloning, expression, and mapping of GDP-D-mannose pyrophosphorylase cDNA from tomato (Lycopersicon esculentum).

    PubMed

    Zou, Li-Ping; Li, Han-Xia; Ouyang, Bo; Zhang, Jun-Hong; Ye, Zhi-Biao

    2006-08-01

    GDP-D-mannose pyrophosphorylase (GMP, EC 2.7.7.22) catalyzes the synthesis of GDP-D-mannose and represents the first committed step in plant ascorbic acid biosynthesis. Using potato GMP cDNA sequence as a querying probe, 65 highly homologous tomato ESTs were obtained from dbEST of GenBank and the putative cDNA sequence of tomato GMP was assembled. The full-length GMP cDNA of tomato was cloned by RACE-PCR with primers designed according to the assembled cDNA sequence. The full-length cDNA sequence contained a complete open reading frame (ORF) of 1,086 bp, which encoded 361 amino acid residues. This gene was designated as LeGMP (GenBank accession No. AY605668). Homology analysis of LeGMP showed a 96% identity with potato GMP and the deduced amino acid showed 99%, 97%, 91% and 89% homology with GMP from potato, tobacco, alfalfa and Arabidopsis thaliana, respectively. Northern blot analysis showed that LeGMP was constitutively expressed in roots, stems, leaves, flowers and fruits of tomato; but the expression levels varied. LeGMP was mapped to 3-D using 75 tomato introgression lines (ILs), each containing a single homozygous RFLP-defined chromosome segment from the green-fruited species Lycopersicon pennellii. PMID:16939010

  9. Underlying Mechanisms of Zymographic Diversity in Starch Synthase I and Pullulanase in Rice-Developing Endosperm.

    PubMed

    Chen, Yaling; Bao, Jinsong

    2016-03-01

    Amylopectin is synthesized by the coordinated actions of many (iso)enzymes, including ADP-glucose pyrophosphorylase (AGPase), starch synthases (SSs), branching enzymes (BEs), and debranching enzymes (DBEs). Here, two polymorphic forms of starch synthase I (SSI) and pullulanase (PUL) in rice-developing seeds, designated as SSI-1/SSI-2 and PUL-1/PUL-2, were discovered for the first time by zymographic analysis. The SSI and PUL polymorphisms were strongly associated with the SSI microsatellite marker (p = 3.6 × 10(-37)) and PUL insertion/deletion (InDel) markers (p < 3.6 × 10(-51)). Western blotting and mass spectrometric analysis confirmed that the polymorphic bands were truly the SSI and PUL enzymes. Only one non-synonymous variation in SSI DNA sequence (the SNP A/G) causing the change of the amino acid K438 to E438 was observed, which coincided well with the polymorphic forms of SSI. Nine non-synonymous variations were found between PUL-1 and PUL-2. Two non-synonymous variations of PUL (F316L and D770E) were identified by mass spectrometric analysis, but all of the variations did not change the structure of PUL. The co-immunoprecipitation results revealed the differences in protein-protein interaction patterns, i.e., strong or weaker signals of SSI-BEI and SSI-BEIIb, between the two forms of SSI. The results will enhance our understanding of SSI and PUL properties and provide helpful information to understand their functions in starch biosynthesis in rice endosperm. PMID:26860852

  10. Metabolic engineering of sugarcane to accumulate energy-dense triacylglycerols in vegetative biomass.

    PubMed

    Zale, Janice; Jung, Je Hyeong; Kim, Jae Yoon; Pathak, Bhuvan; Karan, Ratna; Liu, Hui; Chen, Xiuhua; Wu, Hao; Candreva, Jason; Zhai, Zhiyang; Shanklin, John; Altpeter, Fredy

    2016-02-01

    Elevating the lipid content in vegetative tissues has emerged as a new strategy for increasing energy density and biofuel yield of crops. Storage lipids in contrast to structural and signaling lipids are mainly composed of glycerol esters of fatty acids, also known as triacylglycerol (TAG). TAGs are one of the most energy-rich and abundant forms of reduced carbon available in nature. Therefore, altering the carbon-partitioning balance in favour of TAG in vegetative tissues of sugarcane, one of the highest yielding biomass crops, is expected to drastically increase energy yields. Here we report metabolic engineering to elevate TAG accumulation in vegetative tissues of sugarcane. Constitutive co-expression of WRINKLED1 (WRI1), diacylglycerol acyltransferase1-2 (DGAT1-2) and oleosin1 (OLE1) and simultaneous cosuppression of ADP-glucose pyrophosphorylase (AGPase) and a subunit of the peroxisomal ABC transporter1 (PXA1) in transgenic sugarcane elevated TAG accumulation in leaves or stems by 95- or 43-fold to 1.9% or 0.9% of dry weight (DW), respectively, while expression or suppression of one to three of the target genes increased TAG levels by 1.5- to 9.5-fold. Accumulation of TAG in vegetative progeny plants was consistent with the results from primary transgenics and contributed to a total fatty acid content of up to 4.7% or 1.7% of DW in mature leaves or stems, respectively. Lipid droplets were visible within mesophyll cells of transgenic leaves by confocal fluorescence microscopy. These results provide the basis for optimizations of TAG accumulation in sugarcane and other high yielding biomass grasses and will open new prospects for biofuel applications. PMID:26058948

  11. Defect in cell wall integrity of the yeast saccharomyces cerevisiae caused by a mutation of the GDP-mannose pyrophosphorylase gene VIG9.

    PubMed

    Yoda, K; Kawada, T; Kaibara, C; Fujie, A; Abe, M; Hitoshi; Hashimoto; Shimizu, J; Tomishige, N; Noda, Y; Yamasaki, M

    2000-09-01

    The Saccharomyces cerevisiae VIG9 gene encodes GDP-mannose pyrophosphorylase, which synthesizes GDP-mannose from GTP and mannose-1-phosphate. Although the null mutant was lethal, the vig9 mutants so far obtained showed no growth defect but immature protein glycosylation and drug hypersensitivity. During our search for cell-wall mutants, we found a novel temperature-sensitive mutant, JS30, which required an osmotic stabilizer for viability. JS30 excreted cell surface proteins in the medium without any indication of cell lysis. Although conventional genetic analysis using mating was impossible, by detailed characterization of JS30 including an in vitro enzyme assay and nucleotide sequencing, we found the defect of JS30 was due to a mutation in the VIG9 gene. These results indicated a critical role of GDP-mannose in maintenance of cell-wall integrity. PMID:11055399

  12. Soybean cotyledon starch metabolism is sensitive to altered gravity conditions

    NASA Technical Reports Server (NTRS)

    Brown, C. S.; Piastuch, W. C.; Knott, W. M.

    1994-01-01

    We have demonstrated that etiolated soybean seedlings grown under the altered gravity conditions of clinorotation (1 rpm) and centrifugation (5xg) exhibit changes in starch metabolism. Cotyledon starch concentration was lower (-28%) in clinorotated plants and higher (+24%) in centrifuged plants than in vertical control plants. The activity of ADP-glucose pyrophosphorylase in the cotyledons was affected in a similar way, i.e. lower (-37%) in the clinorotated plants and higher (+22%) in the centrifuged plants. Other starch metabolic enzyme activities, starch synthase, starch phosphorylase and total hydrolase were not affected by the altered gravity treatments. We conclude that the observed changes in starch concentrations were primarily due to gravity-mediated differences in ADP-glucose pyrophosphorylase activity.

  13. Correlation of Activities of the Enzymes α-Phosphoglucomutase, UDP-Galactose 4-Epimerase, and UDP-Glucose Pyrophosphorylase with Exopolysaccharide Biosynthesis by Streptococcus thermophilus LY03

    PubMed Central

    Degeest, Bart; De Vuyst, Luc

    2000-01-01

    The effects of different carbohydrates or mixtures of carbohydrates as substrates on bacterial growth and exopolysaccharide (EPS) production were studied for the yoghurt starter culture Streptococcus thermophilus LY03. This strain produces two heteropolysaccharides with the same monomeric composition (galactose and glucose in the ratio 4:1) but with different molecular masses. Lactose and glucose were fermented by S. thermophilus LY03 only when they were used as sole energy and carbohydrate sources. Fructose was also fermented when it was applied in combination with lactose or glucose. Both the amount of EPS produced and the carbohydrate source consumption rates were clearly influenced by the type of energy and carbohydrate source used, while the EPS monomeric composition remained constant (galactose-glucose, 4:1) under all circumstances. A combination of lactose and glucose resulted in the largest amounts of EPS. Measurements of the activities of enzymes involved in EPS biosynthesis, and of those involved in sugar nucleotide biosynthesis and the Embden-Meyerhof-Parnas pathway, demonstrated that the levels of activity of α-phosphoglucomutase, UDP-galactose 4-epimerase, and UDP-glucose pyrophosphorylase are highly correlated with the amount of EPS produced. Furthermore, a weaker relationship or no relationship between the amounts of EPS and the enzymes involved in either the rhamnose nucleotide synthetic branch of the EPS biosynthesis or the pathway leading to glycolysis was observed for S. thermophilus LY03. PMID:10919816

  14. Rice UDP-Glucose Pyrophosphorylase1 Is Essential for Pollen Callose Deposition and Its Cosuppression Results in a New Type of Thermosensitive Genic Male Sterility[W][OA

    PubMed Central

    Chen, Rongzhi; Zhao, Xiao; Shao, Zhe; Wei, Zhe; Wang, Yuanyuan; Zhu, Lili; Zhao, Jie; Sun, Mengxiang; He, Ruifeng; He, Guangcun

    2007-01-01

    UDP-glucose pyrophosphorylase (UGPase) catalyzes the reversible production of glucose-1-phosphate and UTP to UDP-glucose and pyrophosphate. The rice (Oryza sativa) genome contains two homologous UGPase genes, Ugp1 and Ugp2. We report a functional characterization of rice Ugp1, which is expressed throughout the plant, with highest expression in florets, especially in pollen during anther development. Ugp1 silencing by RNA interference or cosuppression results in male sterility. Expressing a double-stranded RNA interference construct in Ugp1-RI plants resulted in complete suppression of both Ugp1 and Ugp2, together with various pleiotropic developmental abnormalities, suggesting that UGPase plays critical roles in plant growth and development. More importantly, Ugp1-cosuppressing plants contained unprocessed intron-containing primary transcripts derived from transcription of the overexpression construct. These aberrant transcripts undergo temperature-sensitive splicing in florets, leading to a novel thermosensitive genic male sterility. Pollen mother cells (PMCs) of Ugp1-silenced plants appeared normal before meiosis, but during meiosis, normal callose deposition was disrupted. Consequently, the PMCs began to degenerate at the early meiosis stage, eventually resulting in complete pollen collapse. In addition, the degeneration of the tapetum and middle layer was inhibited. These results demonstrate that rice Ugp1 is required for callose deposition during PMC meiosis and bridges the apoplastic unloading pathway and pollen development. PMID:17400897

  15. Mutations in GDP-Mannose Pyrophosphorylase B Cause Congenital and Limb-Girdle Muscular Dystrophies Associated with Hypoglycosylation of α-Dystroglycan

    PubMed Central

    Carss, Keren J.; Stevens, Elizabeth; Foley, A. Reghan; Cirak, Sebahattin; Riemersma, Moniek; Torelli, Silvia; Hoischen, Alexander; Willer, Tobias; van Scherpenzeel, Monique; Moore, Steven A.; Messina, Sonia; Bertini, Enrico; Bönnemann, Carsten G.; Abdenur, Jose E.; Grosmann, Carla M.; Kesari, Akanchha; Punetha, Jaya; Quinlivan, Ros; Waddell, Leigh B.; Young, Helen K.; Wraige, Elizabeth; Yau, Shu; Brodd, Lina; Feng, Lucy; Sewry, Caroline; MacArthur, Daniel G.; North, Kathryn N.; Hoffman, Eric; Stemple, Derek L.; Hurles, Matthew E.; van Bokhoven, Hans; Campbell, Kevin P.; Lefeber, Dirk J.; Lin, Yung-Yao; Muntoni, Francesco

    2013-01-01

    Congenital muscular dystrophies with hypoglycosylation of α-dystroglycan (α-DG) are a heterogeneous group of disorders often associated with brain and eye defects in addition to muscular dystrophy. Causative variants in 14 genes thought to be involved in the glycosylation of α-DG have been identified thus far. Allelic mutations in these genes might also cause milder limb-girdle muscular dystrophy phenotypes. Using a combination of exome and Sanger sequencing in eight unrelated individuals, we present evidence that mutations in guanosine diphosphate mannose (GDP-mannose) pyrophosphorylase B (GMPPB) can result in muscular dystrophy variants with hypoglycosylated α-DG. GMPPB catalyzes the formation of GDP-mannose from GTP and mannose-1-phosphate. GDP-mannose is required for O-mannosylation of proteins, including α-DG, and it is the substrate of cytosolic mannosyltransferases. We found reduced α-DG glycosylation in the muscle biopsies of affected individuals and in available fibroblasts. Overexpression of wild-type GMPPB in fibroblasts from an affected individual partially restored glycosylation of α-DG. Whereas wild-type GMPPB localized to the cytoplasm, five of the identified missense mutations caused formation of aggregates in the cytoplasm or near membrane protrusions. Additionally, knockdown of the GMPPB ortholog in zebrafish caused structural muscle defects with decreased motility, eye abnormalities, and reduced glycosylation of α-DG. Together, these data indicate that GMPPB mutations are responsible for congenital and limb-girdle muscular dystrophies with hypoglycosylation of α-DG. PMID:23768512

  16. UDP-glucose pyrophosphorylase Ugp1 is involved in oxidative stress response and long-term survival during stationary phase in Saccharomyces cerevisiae.

    PubMed

    Yi, Dae-Gwan; Huh, Won-Ki

    2015-11-27

    Ugp1, UDP-glucose pyrophosphorylase, plays an important role in carbohydrate metabolism because it provides UDP-glucose that is a pivotal metabolite in several metabolic pathways in Saccharomyces cerevisiae. In this study, we show that a considerable reduction of glycogen and trehalose content in ugp1 knockdown cells is rescued by complementing the expression of Ugp1, indicating that Ugp1 is required for the production of storage carbohydrates. Because of the specific function of trehalose as a stress protectant, Ugp1 expression contributed to oxidative stress response and long-term cell survival during stationary phase. Furthermore, the modulation of Ugp1 level readjusted glycogen and trehalose accumulation in the protein kinase A (PKA)-related gene mutants. The PKA-dependent phenotypes of oxidative stress resistance and long-term cell survival were also alleviated via adjustment of Ugp1 level. Collectively, our data suggest that the regulation of UPG1 influences several PKA-dependent processes by adjusting the levels of various carbohydrates. PMID:26498530

  17. Cloning and sequencing of the Candida albicans homologue of SRB1/PSA1/VIG9, the essential gene encoding GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae.

    PubMed

    Warit, S; Walmsley, R M; Stateva, L I

    1998-09-01

    Two genomic fragments have been isolated from Candida albicans which strongly hybridize to SRB1/PSA1/VIG9, an essential gene which encodes GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae. A common 2.5 kb Xbal-Pstl fragment has been identified, which Southern analysis suggests is most likely unique in the C. albicans genome. The fragment contains an ORF, which is 82% identical and 90% homologous to the Srb1p/Psa1p/Vig9p from S. cerevisiae, contains one additional amino acid at position 254 and is able to functionally complement the major phenotypic characteristics of S. cerevisiae srb1 null and conditional mutations. The authors therefore conclude that they have cloned and sequenced from C. albicans the bona fide homologue of SRB1/PSA1/VIG9, named hereafter CaSRB1. Northern analysis data indicate that the gene is expressed in C. albicans under conditions of growth in the yeast and hyphal form and suggest that its expression might be regulated. PMID:9782489

  18. Analysis of GDP-D-mannose pyrophosphorylase gene promoter from acerola (Malpighia glabra) and increase in ascorbate content of transgenic tobacco expressing the acerola gene.

    PubMed

    Badejo, Adebanjo A; Tanaka, Nobukazu; Esaka, Muneharu

    2008-01-01

    GDP-D-mannose pyrophosphorylase (GMP) is an important enzyme in the Smirnoff-Wheeler's pathway for the biosynthesis of ascorbic acid (AsA) in plants. We have reported recently that the expression of the acerola (Malpighia glabra) GMP gene, designated MgGMP, correlates with the AsA content of the plant. The acerola plant has very high levels of AsA relative to better studied model plants such as Arabidopsis. Here we found that the GMP mRNA levels in acerola are higher than those from Arabidopsis and tomato. Also, the transient expression of the uidA reporter gene in the protoplasts of Nicotiana tabacum cultures showed the MgGMP gene promoter to have higher activity than the cauliflower mosaic virus 35S and Arabidopsis GMP promoters. The AsA content of transgenic tobacco plants expressing the MgGMP gene including its promoter was about 2-fold higher than that of the wild type. PMID:18037674

  19. Sequence differences between human muscle and liver cDNAs for UDPglucose pyrophosphorylase and kinetic properties of the recombinant enzymes expressed in Escherichia coli.

    PubMed

    Duggleby, R G; Chao, Y C; Huang, J G; Peng, H L; Chang, H Y

    1996-01-15

    UDP-Glc pyrophosphorylase (EC 2.7.7.9) catalyses the interconversion of MgUTP plus Glc1P and UDP-Glc plus MgPPi. Complementation of an Escherichia coli strain lacking this activity has allowed isolation of cDNA encoding this enzyme from a human muscle library. Two forms were identified and the nucleotide sequence of each was determined; they were found to differ only in the 5' region and we suggest that these arise from the use of a different first exon in the two transcripts. These nucleotide sequences are different from that of the cDNA which was isolated previously from a human liver library [Peng, H.-L. & Chang, H.-Y. (1993) FEBS Lett. 329, 153-158] and it is proposed that these liver and muscle forms are derived from different genes. The cDNA for muscle form I, muscle form II, the liver form, and the liver form fused to part of the lacZ gene were expressed in Escherichia coli and the kinetic properties of each enzyme were characterised. Muscle form I and the LacZ/liver fusion enzyme exhibit Michaelis-Menten kinetics towards all substrates while muscle form II has a sigmoidal dependence of rate upon the concentration of MgPPi. The liver form shows Michaelis-Menten kinetics towards MgUTP. For the remaining three substrates, complex kinetics were observed involving a combination of sigmoidicity at low substrate concentration and partial inhibition at high substrate concentration. PMID:8631325

  20. Cloning and expression of GDP-D-mannose pyrophosphorylase gene and ascorbic acid content of acerola (Malpighia glabra L.) fruit at ripening stages.

    PubMed

    Badejo, Adebanjo A; Jeong, Seok T; Goto-Yamamoto, Nami; Esaka, Muneharu

    2007-09-01

    Acerola (Malpighia glabra L.) is one of the richest natural sources of L-ascorbic acid (AsA; vitamin C). GDP-D-mannose pyrophosphorylase (GMP; EC 2.7.7.13) was found to play a major role in the proposed AsA biosynthetic pathway in plants, considering that Arabidopsis vtc1-1 mutant with point mutation in this gene has a highly reduced AsA content. GMP cDNA was isolated from acerola fruits, designated MgGMP, using rapid amplification of cDNA ends (RACE), and its expression was monitored during fruit ripening. The full-length cDNA was found to have an ORF of 1083bp encoding a polypeptide of 361 amino acids. In silico analysis of the predicted amino acid sequence showed a pI of 6.45 and molecular mass of 39.7kD. MgGMP showed over 80% amino acid sequence identity with other plant GMP homologues. The phylogenetic tree shows the close relation of MgGMP to the GMP of other plants as against those from parasite, yeasts and mammals. Southern analysis indicated that M. glabra contains not less than two copies of GMP genes. Northern blot analysis showed the transcript abundance of MgGMP in all the organs of acerola examined, with the fruit having the highest expression. The relative transcript abundance of MgGMP mRNA levels in the fruits changes as the ripening process progresses, with the unripe green fruits having the highest relative mRNA level, and the lowest was found in the fruits at advanced ripening stage. A strong correlation was also observed between the relative MgGMP mRNA levels and the AsA contents of acerola during fruit ripening. PMID:17764967

  1. Rice GDP-mannose pyrophosphorylase OsVTC1-1 and OsVTC1-3 play different roles in ascorbic acid synthesis.

    PubMed

    Qin, Hua; Deng, Zaian; Zhang, Chuanyu; Wang, Yayun; Wang, Juan; Liu, Hai; Zhang, Zhili; Huang, Rongfeng; Zhang, Zhijin

    2016-02-01

    GDP-D-mannose pyrophosphorylase (GMPase) catalyzes the synthesis of GDP-D-mannose, which is a precursor for ascorbic acid (AsA) synthesis in plants. The rice genome encodes three GMPase homologs OsVTC1-1, OsVTC1-3 and OsVTC1-8, but their roles in AsA synthesis are unclear. The overexpression of OsVTC1-1 or OsVTC1-3 restored the AsA synthesis of vtc1-1 in Arabidopsis, while that of OsVTC1-8 did not, indicating that only OsVTC1-1 and OsVTC1-3 are involved in AsA synthesis in rice. Similar to Arabidopsis VTC1, the expression of OsVTC1-1 was high in leaves, induced by light, and inhibited by dark. Unlike OsVTC1-1, the expression level of OsVTC1-3 was high in roots and quickly induced by the dark, while the transcription level of OsVTC1-8 did not show obvious changes under constant light or dark treatments. In OsVTC1-1 RNAi plants, the AsA content of rice leaves decreased, and the AsA production induced by light was limited. In contrast, OsVTC1-3 RNAi lines altered AsA synthesis levels in rice roots, but not in the leaves or under the light/dark treatment. The enzyme activity showed that OsVTC1-1 and OsVTC1-3 had higher GMPase activities than OsVTC1-8 in vitro. Our data showed that, unlike in Arabidopsis, the rice GPMase homologous proteins illustrated a new model in AsA synthesis: OsVTC1-1 may be involved in the AsA synthesis, which takes place in leaves, while OsVTC1-3 may be responsible for AsA synthesis in roots. The different roles of rice GMPase homologous proteins in AsA synthesis may be due to their differences in transcript levels and enzyme activities. PMID:26715595

  2. Carbohydrate and Enzymic Characterization of a High Sucrose Sugary Inbred Line of Sweet Corn 1

    PubMed Central

    Gonzales, Jorge W.; Rhodes, Ashby M.; Dickinson, David B.

    1976-01-01

    Reserve carbohydrates were determined on developing endosperm of a new line of sugary maize (Zea mays L.). Other entries, included for comparative purposes, were Midway (sugary), Funks G4646 (starchy), and Illini X-tra Sweet (shrunken-2). Sucrose in the new line, Illinois 677a, was more than twice that of Midway at most stages of development, and reached a maximum of 40% of dry weight at 18 days after pollination. Appreciable phytoglycogen accumulated in Illinois 677a, reaching 30% or more of dry weight as endosperm tissue matured. Thus, Illinois 677a is a typical sugary maize concerning phytoglycogen content, but it resembles shrunken-2 concerning the extent of sucrose accumulation. Enhanced sucrose accumulation by Illinois 677a was not accounted for by altered in vitro activities of invertase, sucrose synthase, UDP-glucose pyrophosphorylase, or ADP-glucose pyrophosphorylase. Its normal level of ADP-glucose pyrophosphorylase set Illinois 677a apart from shrunken-2 in which the enzyme was drastically reduced. PMID:16659614

  3. UDP-glucose pyrophosphorylase influences polysaccharide synthesis, cell wall components, and hyphal branching in Ganoderma lucidum via regulation of the balance between glucose-1-phosphate and UDP-glucose.

    PubMed

    Li, Mengjiao; Chen, Tianxi; Gao, Tan; Miao, Zhigang; Jiang, Ailiang; Shi, Liang; Ren, Ang; Zhao, Mingwen

    2015-09-01

    UDP-glucose pyrophosphorylase (UGP) is a key enzyme involved in carbohydrate metabolism, but there are few studies on the functions of this enzyme in fungi. The ugp gene of Ganoderma lucidum was cloned, and enzyme kinetic parameters of the UGP recombinant protein were determined in vitro, revealing that this protein was functional and catalyzed the reversible conversion between Glc-1-P and UDP-Glc. ugp silencing by RNA interference resulted in changes in the levels of the intermediate metabolites Glc-1-P and UDP-Glc. The compounds and structure of the cell wall in the silenced strains were also altered compared with those in the wild-type strains. Moreover, the number of hyphal branches was also changed in the silenced strains. To verify the role of UGP in hyphal branching, a ugp-overexpressing strain was constructed. The results showed that the number of hyphal branches was influenced by UGP. The mechanism underlying hyphal branching was further investigated by adding exogenous Glc-1-P. Our results showed that hyphal branching was regulated by a change in the cytosolic Ca(2+) concentration, which was affected by the level of the intermediate metabolite Glc-1-P, in G. lucidum. Our findings indicate the existence of an interaction between carbon metabolism and Ca(2+) signaling in this fungus. PMID:26235043

  4. Sucrose-to-Starch Metabolism in Tomato Fruit Undergoing Transient Starch Accumulation.

    PubMed Central

    Schaffer, A. A.; Petreikov, M.

    1997-01-01

    Immature green tomato (Lycopersicon esculentum) fruits undergo a period of transient starch accumulation characterized by developmental changes in the activities of key enzymes in the sucrose (Suc)-to-starch metabolic pathway. Activities of Suc synthase, fructokinase, ADP-glucose (Glc) pyrophosphorylase, and soluble and insoluble starch synthases decline dramatically in parallel to the decrease in starch levels in the developing fruit. Comparison of "maximal" in vitro activities of the enzymes in the Suc-to-starch pathway suggests that these same enzymes are limiting to the rate of starch accumulation. In contrast, activities of invertase, UDP-Glc pyrophosphorylase, nucleoside diphosphate kinase, phosphoglucoisomerase, and phosphoglucomutase do not exhibit dramatic decreases in activity and appear to be in excess of starch accumulation rates. Starch accumulation is spatially localized in the inner and radial pericarp and columella, whereas the outer pericarp and seed locule contain little starch. The seed locule is characterized by lower activities of Suc synthase, UDP-Glc pyrophosphorylase, phosphoglucomutase, ADP-Glc pyrophosphorylase, and soluble and insoluble starch synthases. The outer pericarp exhibits comparatively lower activities of ADP-Glc pyrophosphorylase and insoluble starch synthase only. These data are discussed in terms of the developmental and tissue-specific coordinated control of Suc-to-starch metabolism. PMID:12223639

  5. Aeromonas Surface Glucan Attached through the O-Antigen Ligase Represents a New Way to Obtain UDP-Glucose

    PubMed Central

    Merino, Susana; Bouamama, Lamiaa; Knirel, Yuriy A.; Senchenkova, Sofya N.; Regué, Miguel; Tomás, Juan M.

    2012-01-01

    We previously reported that A. hydrophila GalU mutants were still able to produce UDP-glucose introduced as a glucose residue in their lipopolysaccharide core. In this study, we found the unique origin of this UDP-glucose from a branched α-glucan surface polysaccharide. This glucan, surface attached through the O-antigen ligase (WaaL), is common to the mesophilic Aeromonas strains tested. The Aeromonas glucan is produced by the action of the glycogen synthase (GlgA) and the UDP-Glc pyrophosphorylase (GlgC), the latter wrongly indicated as an ADP-Glc pyrophosphorylase in the Aeromonas genomes available. The Aeromonas glycogen synthase is able to react with UDP or ADP-glucose, which is not the case of E. coli glycogen synthase only reacting with ADP-glucose. The Aeromonas surface glucan has a role enhancing biofilm formation. Finally, for the first time to our knowledge, a clear preference on behalf of bacterial survival and pathogenesis is observed when choosing to produce one or other surface saccharide molecules to produce (lipopolysaccharide core or glucan). PMID:22563467

  6. Nitrate Acts as a Signal to Induce Organic Acid Metabolism and Repress Starch Metabolism in Tobacco.

    PubMed Central

    Scheible, W. R.; Gonzalez-Fontes, A.; Lauerer, M.; Muller-Rober, B.; Caboche, M.; Stitt, M.

    1997-01-01

    Nia30(145) transformants with very low nitrate reductase activity provide an in vivo screen to identify processes that are regulated by nitrate. Nia30(145) resembles nitrate-limited wild-type plants with respect to growth rate and protein and amino acid content but accumulates large amounts of nitrate when it is grown on high nitrate. The transcripts for nitrate reductase (NR), nitrite reductase, cytosolic glutamine synthetase, and glutamate synthase increased; NR and nitrite reductase activity increased in leaves and roots; and glutamine synthetase activity increased in roots. The transcripts for phosphoenolpyruvate carboxylase, cytosolic pyruvate kinase, citrate synthase, and NADP-isocitrate dehydrogenase increased; phosphoenolpyruvate carboxylase activity increased; and malate, citrate, isocitrate, and [alpha]-oxoglutarate accumulated in leaves and roots. There was a decrease of the ADP-glucose pyrophosphorylase transcript and activity, and starch decreased in the leaves and roots. After adding 12 mM nitrate to nitrate-limited Nia30(145), the transcripts for NR and phosphoenolpyruvate carboxylase increased, and the transcripts for ADP-glucose pyrophosphorylase decreased within 2 and 4 hr, respectively. Starch was remobilized at almost the same rate as in wild-type plants, even though growth was not stimulated in Nia30(145). It is proposed that nitrate acts as a signal to initiate coordinated changes in carbon and nitrogen metabolism. PMID:12237366

  7. A sensitive method for confocal fluorescence microscopic visualization of starch granules in iodine stained samples

    PubMed Central

    Ovecka, Miroslav; Bahaji, Abdellatif; Muñoz, Francisco José; Almagro, Goizeder; Ezquer, Ignacio; Baroja-Fernández, Edurne; Li, Jun; Pozueta-Romero, Javier

    2012-01-01

    Synthesized by glycogen synthase and starch synthases (SS) using ADP-glucose as the sugar donor molecule, glycogen and starch accumulate as predominant storage carbohydrates in most bacteria and plants, respectively. We have recently shown that the so-called “starch-less” Arabidopsis thaliana adg1–1 and aps1 mutants impaired in ADP-glucose pyrophosphorylase do indeed accumulate low starch content in normal growth conditions, and relatively high starch content when plants were cultured in the presence of microbial volatiles. Our results were strongly supported by data obtained using a highly sensitive method for confocal fluorescence microscopic visualization of iodine stained starch granules. Using Arabidopsis leaves from WT plants, aps1 plants, ss3/ss4 plants lacking both class III and class IV SS, gbss plants lacking the granule-bound SS, and sus1/sus2/sus3/sus4 plants lacking four genes that code for proteins with sucrose synthase activity, in this work we precisely describe the method for preparation of plant samples for starch microscopic examination. Furthermore, we show that this method can be used to visualize glycogen in bacteria, and pure starch granules, amylose and amylopectin. PMID:22899048

  8. Expression of alternansucrase in potato plants

    PubMed Central

    Kok-Jacon, Géraldine A.; Vincken, Jean-Paul; Suurs, Luc C. J. M.; Wang, Denong; Liu, Shaoyi

    2007-01-01

    Alternan, which consists of alternating α-(1→3)/α-(1→6)-linked glucosyl residues, was produced in potato tubers by expressing a mature alternansucrase (Asr) gene from Leuconostoc mesenteroides NRRL B-1355 in potato. Detection of alternan was performed by enzyme-linked immunosorbent assay in tuber juices, revealing a concentration between 0.3 and 1.2 mg g-1 fresh wt. The Asr transcript levels correlated well with alternan accumulation in tuber juices. It appeared that the expression of sucrose-regulated starch-synthesizing genes (ADP-glucose pyrophosphorylase subunit S and granule-bound starch synthase I) was down-regulated. Despite this, the physico-chemical properties of the transgenic starches were unaltered. These results are compared to those obtained with other transgenic potato plants producing mutan [α-(1→3)-linked glucosyl residues] and dextran [α-(1→6)-linked glucosyl residues]. PMID:17380272

  9. Luxury uptake of phosphorus changes the accumulation of starch and lipid in Chlorella sp. under nitrogen depletion.

    PubMed

    Zhu, Shunni; Wang, Yajie; Xu, Jin; Shang, Changhua; Wang, Zhongming; Xu, Jingliang; Yuan, Zhenhong

    2015-12-01

    The aim of this research was to study the effect of phosphorus supply on starch and lipid production under nitrogen starvation using Chlorella sp. as a model. High phosphate level had marginal effect on cell density but increased biomass growth. Massive phosphorus was assimilated quickly and mainly stored in the form of polyphosphate. The algal cells ceased phosphorus uptake when intracellular phosphorus reached a certain level. 5mM phosphate in the culture rendered a 16.7% decrease of starch synthesis and a 22.4% increase of lipid synthesis relative to low phosphate (0.17 mM). It is plausible that phosphate can regulate carbon partitioning between starch and lipid synthesis pathway by influencing ADP-glucose pyrophosphorylase activity. Moreover, high phosphate concentration enhanced the abundance of oleic acid, improving oil quality for biodiesel production. It is a promising cultivation strategy by integration of phosphorus removal from wastewater with biodiesel production for this alga. PMID:26386419

  10. The influence of altered gravity on carbohydrate metabolism in excised wheat leaves

    NASA Technical Reports Server (NTRS)

    Obenland, D. M.; Brown, C. S.

    1994-01-01

    We developed a system to study the influence of altered gravity on carbohydrate metabolism in excised wheat leaves by means of clinorotation. The use of excised leaves in our clinostat studies offered a number of advantages over the use of whole plants, most important of which were minimization of exogenous mechanical stress and a greater amount of carbohydrate accumulation during the time of treatment. We found that horizontal clinorotation of excised wheat leaves resulted in significant reductions in the accumulation of fructose, sucrose, starch and fructan relative to control, vertically clinorotated leaves. Photosynthesis, dark respiration and the extractable activities of ADP glucose pyrophosphorylase (EC 2.7.7.27), sucrose phosphate synthase (EC 2.4.4.14), sucrose sucrose fructosyltransferase (EC 2.4.1.99), and fructan hydrolase (EC 3.2.1.80) were unchanged due to altered gravity treatment.

  11. Molecular signatures in protein sequences that are characteristic of cyanobacteria and plastid homologues.

    PubMed

    Gupta, Radhey S; Pereira, Mark; Chandrasekera, Charu; Johari, Vanessa

    2003-11-01

    Fourteen conserved indels (i.e. inserts or deletions) have been identified in 10 widely distributed proteins that appear to be characteristic of cyanobacterial species and are not found in any other group of bacteria. These signatures include three inserts of 6, 7 and 28 aa in the DNA helicase II (UvrD) protein, an 18-21 aa insert in DNA polymerase I, a 14 aa insert in the enzyme ADP-glucose pyrophosphorylase, a 3 aa insert in the FtsH protein, an 11-13 aa insert in phytoene synthase, a 5 aa insert in elongation factor-Tu, two deletions of 2 and 7 aa in ribosomal S1 protein, a 2 aa insert in the SecA protein, a 1 aa deletion and a 6 aa insert in the enzyme inosine-5'-monophosphate dehydrogenase and a 1 aa deletion in the major sigma factor. These signatures, which are flanked by conserved regions, provide molecular markers for distinguishing cyanobacterial taxa from all other bacteria and they should prove helpful in the identification of cyanobacterial species, simply on the basis of the presence or absence of these markers in the corresponding proteins. The signatures in six of these proteins (SecA, elongation factor-Tu, ADP-glucose pyrophosphorylase, phytoene synthase, FtsH and ribosomal S1 protein) are also commonly present in plastid homologues from plants and algae (chlorophytes, chromophytes and rhodophytes), indicating their specific relationship to cyanobacteria and supporting their endosymbiotic origin from these bacteria. In phylogenetic trees based on a number of these proteins (SecA, UvrD, DNA polymerase I, elongation factor-Tu) that were investigated, the available cyanobacterial homologues grouped together with high affinity (>95 % bootstrap value), supporting the view that the cyanobacterial phylum is monophyletic and that the identified signatures were introduced in a common ancestor of this group. PMID:14657112

  12. Subcellular analysis of starch metabolism in developing barley seeds using a non-aqueous fractionation method

    PubMed Central

    Tiessen, Axel; Nerlich, Annika; Faix, Benjamin; Hümmer, Christine; Fox, Simon; Trafford, Kay; Weber, Hans; Weschke, Winfriede; Geigenberger, Peter

    2012-01-01

    Compartmentation of metabolism in developing seeds is poorly understood due to the lack of data on metabolite distributions at the subcellular level. In this report, a non-aqueous fractionation method is described that allows subcellular concentrations of metabolites in developing barley endosperm to be calculated. (i) Analysis of subcellular volumes in developing endosperm using micrographs shows that plastids and cytosol occupy 50.5% and 49.9% of the total cell volume, respectively, while vacuoles and mitochondria can be neglected. (ii) By using non-aqueous fractionation, subcellular distribution between the cytosol and plastid of the levels of metabolites involved in sucrose degradation, starch synthesis, and respiration were determined. With the exception of ADP and AMP which were mainly located in the plastid, most other metabolites of carbon and energy metabolism were mainly located outside the plastid in the cytosolic compartment. (iii) In developing barley endosperm, the ultimate precursor of starch, ADPglucose (ADPGlc), was mainly located in the cytosol (80–90%), which was opposite to the situation in growing potato tubers where ADPGlc was almost exclusively located in the plastid (98%). This reflects the different subcellular distribution of ADPGlc pyrophosphorylase (AGPase) in these tissues. (iv) Cytosolic concentrations of ADPGlc were found to be close to the published Km values of AGPase and the ADPGlc/ADP transporter at the plastid envelope. Also the concentrations of the reaction partners glucose-1-phosphate, ATP, and inorganic pyrophosphate were close to the respective Km values of AGPase. (v) Knock-out of cytosolic AGPase in Riso16 mutants led to a strong decrease in ADPGlc level, in both the cytosol and plastid, whereas knock-down of the ADPGlc/ADP transporter led to a large shift in the intracellular distribution of ADPGlc. (v) The thermodynamic structure of the pathway of sucrose to starch was determined by calculating the mass–action ratios

  13. Subcellular analysis of starch metabolism in developing barley seeds using a non-aqueous fractionation method.

    PubMed

    Tiessen, Axel; Nerlich, Annika; Faix, Benjamin; Hümmer, Christine; Fox, Simon; Trafford, Kay; Weber, Hans; Weschke, Winfriede; Geigenberger, Peter

    2012-03-01

    Compartmentation of metabolism in developing seeds is poorly understood due to the lack of data on metabolite distributions at the subcellular level. In this report, a non-aqueous fractionation method is described that allows subcellular concentrations of metabolites in developing barley endosperm to be calculated. (i) Analysis of subcellular volumes in developing endosperm using micrographs shows that plastids and cytosol occupy 50.5% and 49.9% of the total cell volume, respectively, while vacuoles and mitochondria can be neglected. (ii) By using non-aqueous fractionation, subcellular distribution between the cytosol and plastid of the levels of metabolites involved in sucrose degradation, starch synthesis, and respiration were determined. With the exception of ADP and AMP which were mainly located in the plastid, most other metabolites of carbon and energy metabolism were mainly located outside the plastid in the cytosolic compartment. (iii) In developing barley endosperm, the ultimate precursor of starch, ADPglucose (ADPGlc), was mainly located in the cytosol (80-90%), which was opposite to the situation in growing potato tubers where ADPGlc was almost exclusively located in the plastid (98%). This reflects the different subcellular distribution of ADPGlc pyrophosphorylase (AGPase) in these tissues. (iv) Cytosolic concentrations of ADPGlc were found to be close to the published K(m) values of AGPase and the ADPGlc/ADP transporter at the plastid envelope. Also the concentrations of the reaction partners glucose-1-phosphate, ATP, and inorganic pyrophosphate were close to the respective K(m) values of AGPase. (v) Knock-out of cytosolic AGPase in Riso16 mutants led to a strong decrease in ADPGlc level, in both the cytosol and plastid, whereas knock-down of the ADPGlc/ADP transporter led to a large shift in the intracellular distribution of ADPGlc. (v) The thermodynamic structure of the pathway of sucrose to starch was determined by calculating the mass-action ratios

  14. Metabolic and photosynthetic consequences of blocking starch biosynthesis in the green alga Chlamydomonas reinhardtii sta6 mutant.

    PubMed

    Krishnan, Anagha; Kumaraswamy, G Kenchappa; Vinyard, David J; Gu, Huiya; Ananyev, Gennady; Posewitz, Matthew C; Dismukes, G Charles

    2015-03-01

    Upon nutrient deprivation, microalgae partition photosynthate into starch and lipids at the expense of protein synthesis and growth. We investigated the role of starch biosynthesis with respect to photosynthetic growth and carbon partitioning in the Chlamydomonas reinhardtii starchless mutant, sta6, which lacks ADP-glucose pyrophosphorylase. This mutant is unable to convert glucose-1-phosphate to ADP-glucose, the precursor of starch biosynthesis. During nutrient-replete culturing, sta6 does not re-direct metabolism to make more proteins or lipids, and accumulates 20% less biomass. The underlying molecular basis for the decreased biomass phenotype was identified using LC-MS metabolomics studies and flux methods. Above a threshold light intensity, photosynthetic electron transport rates (water → CO2) decrease in sta6 due to attenuated rates of NADPH re-oxidation, without affecting photosystems I or II (no change in isolated photosynthetic electron transport). We observed large accumulations of carbon metabolites that are precursors for the biosynthesis of lipids, amino acids and sugars/starch, indicating system-wide consequences of slower NADPH re-oxidation. Attenuated carbon fixation resulted in imbalances in both redox and adenylate energy. The pool sizes of both pyridine and adenylate nucleotides in sta6 increased substantially to compensate for the slower rate of turnover. Mitochondrial respiration partially relieved the reductant stress; however, prolonged high-light exposure caused accelerated photoinhibition. Thus, starch biosynthesis in Chlamydomonas plays a critical role as a principal carbon sink influencing cellular energy balance however, disrupting starch biosynthesis does not redirect resources to other bioproducts (lipids or proteins) during nutrient-replete culturing, resulting in cells that are susceptible to photochemical damage caused by redox stress. PMID:25645872

  15. Metabolic analysis of kiwifruit (Actinidia deliciosa) berries from extreme genotypes reveals hallmarks for fruit starch metabolism

    PubMed Central

    Nardozza, Simona; MacRae, Elspeth A.; Sulpice, Ronan; Clearwater, Michael J.

    2013-01-01

    Tomato, melon, grape, peach, and strawberry primarily accumulate soluble sugars during fruit development. In contrast, kiwifruit (Actinidia Lindl. spp.) and banana store a large amount of starch that is released as soluble sugars only after the fruit has reached maturity. By integrating metabolites measured by gas chromatography–mass spectrometry, enzyme activities measured by a robot-based platform, and transcript data sets during fruit development of Actinidia deliciosa genotypes contrasting in starch concentration and size, this study identified the metabolic changes occurring during kiwifruit development, including the metabolic hallmarks of starch accumulation and turnover. At cell division, a rise in glucose (Glc) concentration was associated with neutral invertase (NI) activity, and the decline of both Glc and NI activity defined the transition to the cell expansion and starch accumulation phase. The high transcript levels of β-amylase 9 (BAM9) during cell division, prior to net starch accumulation, and the correlation between sucrose phosphate synthase (SPS) activity and sucrose suggest the occurrence of sucrose cycling and starch turnover. ADP-Glc pyrophosphorylase (AGPase) is identified as a key enzyme for starch accumulation in kiwifruit berries, as high-starch genotypes had 2- to 5-fold higher AGPase activity, which was maintained over a longer period of time and was also associated with enhanced and extended transcription of the AGPase large subunit 4 (APL4). The data also revealed that SPS and galactinol might affect kiwifruit starch accumulation, and suggest that phloem unloading into kiwifruit is symplastic. These results are relevant to the genetic improvement of quality traits such as sweetness and sugar/acid balance in a range of fruit species. PMID:24058160

  16. Structure, function and regulation of the enzymes in the starch biosynthetic pathway.

    SciTech Connect

    Geiger, Jim

    2013-11-30

    structure of ADP- Glucose pyrophosphorylase from potato in its inhibited conformation, and bound to both ATP and ADP-glucose. In addition, we have determined the first structure of glycogen synthase in its "closed", catalytically active conformation bound to ADP-glucose. We also determined the structure of glycogen synthase bound to malto-oligosaccharides, showing for the first time that an enzyme in the starch biosynthetic pathway recognizes glucans not just in its active site but on binding sites on the surface of the enzyme ten’s of Angstroms from the active site. In addition our structure of a glycogen branching enzyme bound to malto-oligosaccharides identified seven distinct binding sites distributed about the surface of the enzyme. We will now determine the function of these sites to get a molecular-level picture of exactly how these enzymes interact with their polymeric substrates and confer specificity leading to the complex structure of the starch granule. We will extend our studies to other isoforms of the enzymes, to understand how their structures give rise to their distinct function. Our goal is to understand what accounts for the various functional differences between SS and SBE isoforms at a molecular level.

  17. Comparative Proteomic Analysis of Two Barley Cultivars (Hordeum vulgare L.) with Contrasting Grain Protein Content

    PubMed Central

    Guo, Baojian; Luan, Haiye; Lin, Shen; Lv, Chao; Zhang, Xinzhong; Xu, Rugen

    2016-01-01

    Grain protein contents (GPCs) of barley seeds are significantly different between feed and malting barley cultivars. However, there is still no insight into the proteomic analysis of seed proteins between feed and malting barley cultivars. Also, the genetic control of barley GPC is still unclear. GPCs were measured between mature grains of Yangsimai 3 and Naso Nijo. A proteome profiling of differentially expressed protein was established by using a combination of 2-DE and tandem mass spectrometry. In total, 502 reproducible protein spots in barley seed proteome were detected with a pH range of 4–7 and 6–11, among these 41 protein spots (8.17%) were detected differentially expressed between Yangsimai 3 and Naso Nijo. Thirty-four protein spots corresponding to 23 different proteins were identified, which were grouped into eight categories, including stress, protein degradation and post-translational modification, development, cell, signaling, glycolysis, starch metabolism, and other functions. Among the identified proteins, enolase (spot 274) and small subunit of ADP-glucose pyrophosphorylase (spot 271) are exclusively expressed in barley Yangsimai 3, which may be involved in regulating seed protein expression. In addition, malting quality is characterized by an accumulation of serpin protein, Alpha-amylase/trypsin inhibitor CMb and Alpha-amylase inhibitor BDAI-1. Most noticeably, globulin, an important storage protein in barley seed, undergoes post-translational processing in both cultivars, and also displays different expression patterns. PMID:27200019

  18. Changes in photosynthesis and pigmentation in an agp deletion mutant of the cyanobacterium Synechocystis sp.

    PubMed

    Miao, Xiaoling; Wu, Qingyu; Wu, Guifang; Zhao, Nanming

    2003-03-01

    The agp gene encoding ADP-glucose pyrophosphorylase is involved in cyanobacterial glycogen synthesis. By in vitro DNA recombination technology, agp deletion mutant (agp-) of cyanobacterium Synechocystis sp. PCC 6803 was constructed. This mutation led to a complete absence of glycogen biosynthesis. As compared with WT (wild type), a 60% decrease in ratio of the c-phycocyanine/chlorophyll a and no significant change in the carotenoid/chlorophyll a were observed in agp- cells. The agp- mutant had 38% less photosynthetic capacity when grown in light over 600 micromol m(-2) s(-1). Under lower light intensity, the final biomass of the mutant strain was only 1.1 times of that of the WT strain under mixotrophic condition after 6 d culture. Under higher light intensity, however, the final biomass of the WT strain under mixotrophic conditions was 3 times that of the mutant strain after 6 d culture and 1.5 times under photoautotrophic conditions. The results indicate that there is a minimum requirement for glycogen synthesis for normal growth and development in cyanobacteria. PMID:12882559

  19. Experiment 9: ASTROCULTURE: Growth and Starch Accumulation of Potato Tuber

    NASA Technical Reports Server (NTRS)

    Tibbitts, Theodore W.; Brown, Christopher S.; Croxdale, Judith G.; Wheeler, Raymond M.

    1998-01-01

    Potato explants (leaf, small stem section, and axillary bud) flown on STS-73 developed tubers of 1.5 cm diameter and 1.7 g mass during the 16-day period of space flight. The experiment was undertaken in the ASTROCULTURE(TM) experiment package under controlled temperature, humidity, lighting, and carbon dioxide concentrations. The tubers that formed in the explant system under microgravity had the same gross morphology, the same anatomical configuration of cells and tissues, and the same sizes, shapes, and surface character of starch granules as tubers formed in a 1 g environment. The total accumulation of starch and other energy containing compounds was similar in space flight and ground control tubers. Enzyme activity of starch synthase, starch phosphorylase, and total hydrolase was similar in space flight and ground controls, but activity of ADP-glucose pyrophosphorylase was reduced in the space flight tuber tissue. This experiment documented that potatoes will metabolize and accumulate starch as effectively in space flight as on the ground. Thus, this data provides the potential for effective utilization of potatoes in life support systems of space bases.

  20. Malate Plays a Crucial Role in Starch Metabolism, Ripening, and Soluble Solid Content of Tomato Fruit and Affects Postharvest Softening[W][OA

    PubMed Central

    Centeno, Danilo C.; Osorio, Sonia; Nunes-Nesi, Adriano; Bertolo, Ana L.F.; Carneiro, Raphael T.; Araújo, Wagner L.; Steinhauser, Marie-Caroline; Michalska, Justyna; Rohrmann, Johannes; Geigenberger, Peter; Oliver, Sandra N.; Stitt, Mark; Carrari, Fernando; Rose, Jocelyn K.C.; Fernie, Alisdair R.

    2011-01-01

    Despite the fact that the organic acid content of a fruit is regarded as one of its most commercially important quality traits when assessed by the consumer, relatively little is known concerning the physiological importance of organic acid metabolism for the fruit itself. Here, we evaluate the effect of modifying malate metabolism in a fruit-specific manner, by reduction of the activities of either mitochondrial malate dehydrogenase or fumarase, via targeted antisense approaches in tomato (Solanum lycopersicum). While these genetic perturbations had relatively little effect on the total fruit yield, they had dramatic consequences for fruit metabolism, as well as unanticipated changes in postharvest shelf life and susceptibility to bacterial infection. Detailed characterization suggested that the rate of ripening was essentially unaltered but that lines containing higher malate were characterized by lower levels of transitory starch and a lower soluble sugars content at harvest, whereas those with lower malate contained higher levels of these carbohydrates. Analysis of the activation state of ADP-glucose pyrophosphorylase revealed that it correlated with the accumulation of transitory starch. Taken together with the altered activation state of the plastidial malate dehydrogenase and the modified pigment biosynthesis of the transgenic lines, these results suggest that the phenotypes are due to an altered cellular redox status. The combined data reveal the importance of malate metabolism in tomato fruit metabolism and development and confirm the importance of transitory starch in the determination of agronomic yield in this species. PMID:21239646

  1. Space Experiment on Tuber Development and Starch Accumulation for CELSS

    NASA Technical Reports Server (NTRS)

    Tibbitts,Theodore W.; Croxdale, Judith C.; Brown, Christopher S.

    1997-01-01

    Potato explants (leaf, small stem section, and axillary bud), flown on STS-73, developed tubers of 1.5 cm diameter and 1.7 g mass during the 16 day period of spaceflight. The experiment was undertaken in the ASTROCULTURE(Trademark) experiment package under controlled temperature, humidity, lighting, and carbon dioxide concentrations. The tubers formed in the explant system under microgravity had the same gross morphology, the same anatomical configuration of cells and tissues, and the same sizes, shapes, and surface character of starch granules as tubers formed in a 1 g environment. The total accumulation of starch and other energy containing compounds was singular in space flight and ground control tubers. Enzyme activity of starch synthase, starch phosphorylase, and total hydrolase was similar in spaceflight and ground controls but activity of ADP-glucose pyrophosphorylase was reduced in the spaceflight tuber tissue. This experiment documented that potatoes will metabolize and accumulate starch as effectively in spaceflight as on the ground and thus this data provides the potential for effective utilization of potatoes in life support systems of space bases.

  2. The failure to express a protein disulphide isomerase-like protein results in a floury endosperm and an endoplasmic reticulum stress response in rice

    PubMed Central

    Han, Xiaohua; Wang, Yihua; Liu, Xi; Jiang, Ling; Ren, Yulong; Liu, Feng; Peng, Cheng; Li, Jingjing; Jin, Ximing; Wu, Fuqing; Wang, Jiulin; Guo, Xiuping; Zhang, Xin; Cheng, Zhijun; Wan, Jianmin

    2012-01-01

    The rice somaclonal mutant T3612 produces small grains with a floury endosperm, caused by the loose packing of starch granules. The positional cloning of the mutation revealed a deletion in a gene encoding a protein disulphide isomerase-like enzyme (PDIL1-1). In the wild type, PDIL1-1 was expressed throughout the plant, but most intensely in the developing grain. In T3612, its expression was abolished, resulting in a decrease in the activity of plastidial phosphorylase and pullulanase, and an increase in that of soluble starch synthase I and ADP-glucose pyrophosphorylase. The amylopectin in the T3612 endosperm showed an increase in chains with a degree of polymerization 8–13 compared with the wild type. The expression in the mutant's endosperm of certain endoplasmic reticulum stress-responsive genes was noticeably elevated. PDIL1-1 appears to play an important role in starch synthesis. Its absence is associated with endoplasmic reticulum stress in the endosperm, which is likely to underlie the formation of the floury endosperm in the T3612 mutant. PMID:21984651

  3. Link between Phosphate Starvation and Glycogen Metabolism in Corynebacterium glutamicum, Revealed by Metabolomics▿ †

    PubMed Central

    Woo, Han Min; Noack, Stephan; Seibold, Gerd M.; Willbold, Sabine; Eikmanns, Bernhard J.; Bott, Michael

    2010-01-01

    In this study, we analyzed the influence of phosphate (Pi) limitation on the metabolism of Corynebacterium glutamicum. Metabolite analysis by gas chromatography-time-of-flight (GC-TOF) mass spectrometry of cells cultivated in glucose minimal medium revealed a greatly increased maltose level under Pi limitation. As maltose formation could be linked to glycogen metabolism, the cellular glycogen content was determined. Unlike in cells grown under Pi excess, the glycogen level in Pi-limited cells remained high in the stationary phase. Surprisingly, even acetate-grown cells, which do not form glycogen under Pi excess, did so under Pi limitation and also retained it in stationary phase. Expression of pgm and glgC, encoding the first two enzymes of glycogen synthesis, phosphoglucomutase and ADP-glucose pyrophosphorylase, was found to be increased 6- and 3-fold under Pi limitation, respectively. Increased glycogen synthesis together with a decreased glycogen degradation might be responsible for the altered glycogen metabolism. Independent from these experimental results, flux balance analysis suggested that an increased carbon flux to glycogen is a solution for C. glutamicum to adapt carbon metabolism to limited Pi concentrations. PMID:20802079

  4. Wheat Drought-Responsive Grain Proteome Analysis by Linear and Nonlinear 2-DE and MALDI-TOF Mass Spectrometry

    PubMed Central

    Jiang, Shan-Shan; Liang, Xiao-Na; Li, Xin; Wang, Shun-Li; Lv, Dong-Wen; Ma, Chao-Ying; Li, Xiao-Hui; Ma, Wu-Jun; Yan, Yue-Ming

    2012-01-01

    A comparative proteomic analysis of drought-responsive proteins during grain development of two wheat varieties Kauz (strong resistance to drought stress) and Janz (sensitive to drought stress) was performed by using linear and nonlinear 2-DE and MALDI-TOF mass spectrometry technologies. Results revealed that the nonlinear 2-DE had much higher resolution than the linear 2-DE. A total of 153 differentially expressed protein spots were detected by both 2-DE maps, of which 122 protein spots were identified by MALDI-TOF and MALDI-TOF/TOF mass spectrometry. The identified differential proteins were mainly involved in carbohydrate metabolism (26%), detoxification and defense (23%), and storage proteins (17%). Some key proteins demonstrated significantly different expression patterns between the two varieties. In particular, catalase isozyme 1, WD40 repeat protein, LEA and alpha-amylase inhibitors displayed an upregulated expression pattern in Kauz, whereas they were downregulated or unchanged in Janz. Small and large subunit ADP glucose pyrophosphorylase, ascorbate peroxidase and G beta-like protein were all downregulated under drought stress in Janz, but had no expression changes in Kauz. Sucrose synthase and triticin precursor showed an upregulated expression pattern under water deficits in both varieties, but their upregulation levels were much higher in Kauz than in Janz. These differentially expressed proteins could be related to the biochemical pathways for stronger drought resistance of Kauz. PMID:23443111

  5. SALT-RESPONSIVE ERF1 is a negative regulator of grain filling and gibberellin-mediated seedling establishment in rice.

    PubMed

    Schmidt, Romy; Schippers, Jos H M; Mieulet, Delphine; Watanabe, Mutsumi; Hoefgen, Rainer; Guiderdoni, Emmanuel; Mueller-Roeber, Bernd

    2014-02-01

    Grain quality is an important agricultural trait that is mainly determined by grain size and composition. Here, we characterize the role of the rice transcription factor (TF) SALT-RESPONSIVE ERF1 (SERF1) during grain development. Through genome-wide expression profiling and chromatin immunoprecipitation, we found that SERF1 directly regulates RICE PROLAMIN-BOX BINDING FACTOR (RPBF), a TF that functions as a positive regulator of grain filling. Loss of SERF1 enhances RPBF expression resulting in larger grains with increased starch content, while SERF1 overexpression represses RPBF resulting in smaller grains. Consistently, during grain filling, starch biosynthesis genes such as GRANULE-BOUND STARCH SYNTHASEI (GBSSI), STARCH SYNTHASEI (SSI), SSIIIa, and ADP-GLUCOSE PYROPHOSPHORYLASE LARGE SUBUNIT2 (AGPL2) are up-regulated in SERF1 knockout grains. Moreover, SERF1 is a direct upstream regulator of GBSSI. In addition, SERF1 negatively regulates germination by controlling RPBF expression, which mediates the gibberellic acid (GA)-induced expression of RICE AMYLASE1A (RAmy1A). Loss of SERF1 results in more rapid seedling establishment, while SERF1 overexpression has the opposite effect. Our study reveals that SERF1 represents a negative regulator of grain filling and seedling establishment by timing the expression of RPBF. PMID:24046061

  6. Thioredoxin f1 and NADPH-Dependent Thioredoxin Reductase C Have Overlapping Functions in Regulating Photosynthetic Metabolism and Plant Growth in Response to Varying Light Conditions1[OPEN

    PubMed Central

    Thormählen, Ina; Meitzel, Tobias; Groysman, Julia; Öchsner, Alexandra Bianca; von Roepenack-Lahaye, Edda; Naranjo, Belén; Cejudo, Francisco J.; Geigenberger, Peter

    2015-01-01

    Two different thiol redox systems exist in plant chloroplasts, the ferredoxin-thioredoxin (Trx) system, which depends on ferredoxin reduced by the photosynthetic electron transport chain and, thus, on light, and the NADPH-dependent Trx reductase C (NTRC) system, which relies on NADPH and thus may be linked to sugar metabolism in the dark. Previous studies suggested, therefore, that the two different systems may have different functions in plants. We now report that there is a previously unrecognized functional redundancy of Trx f1 and NTRC in regulating photosynthetic metabolism and growth. In Arabidopsis (Arabidopsis thaliana) mutants, combined, but not single, deficiencies of Trx f1 and NTRC led to severe growth inhibition and perturbed light acclimation, accompanied by strong impairments of Calvin-Benson cycle activity and starch accumulation. Light activation of key enzymes of these pathways, fructose-1,6-bisphosphatase and ADP-glucose pyrophosphorylase, was almost completely abolished. The subsequent increase in NADPH-NADP+ and ATP-ADP ratios led to increased nitrogen assimilation, NADP-malate dehydrogenase activation, and light vulnerability of photosystem I core proteins. In an additional approach, reporter studies show that Trx f1 and NTRC proteins are both colocalized in the same chloroplast substructure. Results provide genetic evidence that light- and NADPH-dependent thiol redox systems interact at the level of Trx f1 and NTRC to coordinately participate in the regulation of the Calvin-Benson cycle, starch metabolism, and growth in response to varying light conditions. PMID:26338951

  7. Thioredoxin f1 and NADPH-Dependent Thioredoxin Reductase C Have Overlapping Functions in Regulating Photosynthetic Metabolism and Plant Growth in Response to Varying Light Conditions.

    PubMed

    Thormählen, Ina; Meitzel, Tobias; Groysman, Julia; Öchsner, Alexandra Bianca; von Roepenack-Lahaye, Edda; Naranjo, Belén; Cejudo, Francisco J; Geigenberger, Peter

    2015-11-01

    Two different thiol redox systems exist in plant chloroplasts, the ferredoxin-thioredoxin (Trx) system, which depends on ferredoxin reduced by the photosynthetic electron transport chain and, thus, on light, and the NADPH-dependent Trx reductase C (NTRC) system, which relies on NADPH and thus may be linked to sugar metabolism in the dark. Previous studies suggested, therefore, that the two different systems may have different functions in plants. We now report that there is a previously unrecognized functional redundancy of Trx f1 and NTRC in regulating photosynthetic metabolism and growth. In Arabidopsis (Arabidopsis thaliana) mutants, combined, but not single, deficiencies of Trx f1 and NTRC led to severe growth inhibition and perturbed light acclimation, accompanied by strong impairments of Calvin-Benson cycle activity and starch accumulation. Light activation of key enzymes of these pathways, fructose-1,6-bisphosphatase and ADP-glucose pyrophosphorylase, was almost completely abolished. The subsequent increase in NADPH-NADP(+) and ATP-ADP ratios led to increased nitrogen assimilation, NADP-malate dehydrogenase activation, and light vulnerability of photosystem I core proteins. In an additional approach, reporter studies show that Trx f1 and NTRC proteins are both colocalized in the same chloroplast substructure. Results provide genetic evidence that light- and NADPH-dependent thiol redox systems interact at the level of Trx f1 and NTRC to coordinately participate in the regulation of the Calvin-Benson cycle, starch metabolism, and growth in response to varying light conditions. PMID:26338951

  8. Metabolic Consequences of Infection of Grapevine (Vitis vinifera L.) cv. “Modra frankinja” with Flavescence Dorée Phytoplasma

    PubMed Central

    Prezelj, Nina; Covington, Elizabeth; Roitsch, Thomas; Gruden, Kristina; Fragner, Lena; Weckwerth, Wolfram; Chersicola, Marko; Vodopivec, Maja; Dermastia, Marina

    2016-01-01

    Flavescence dorée, caused by the quarantine phytoplasma FDp, represents the most devastating of the grapevine yellows diseases in Europe. In an integrated study we have explored the FDp–grapevine interaction in infected grapevines of cv. “Modra frankinja” under natural conditions in the vineyard. In FDp-infected leaf vein-enriched tissues, the seasonal transcriptional profiles of 14 genes selected from various metabolic pathways showed an FDp-specific plant response compared to other grapevine yellows and uncovered a new association of the SWEET17a vacuolar transporter of fructose with pathogens. Non-targeted metabolome analysis from leaf vein-enriched tissues identified 22 significantly changed compounds with increased levels during infection. Several metabolites corroborated the gene expression study. Detailed investigation of the dynamics of carbohydrate metabolism revealed significant accumulation of sucrose and starch in the mesophyll of FDp-infected leaves, as well as significant up-regulation of genes involved in their biosynthesis. In addition, infected leaves had high activities of ADP-glucose pyrophosphorylase and, more significantly, sucrose synthase. The data support the conclusion that FDp infection inhibits phloem transport, resulting in accumulation of carbohydrates and secondary metabolites that provoke a source-sink transition and defense response status. PMID:27242887

  9. Effects of microgravity and clinorotation on stress ethylene production in two starchless mutants of Arabidopsis thaliana

    NASA Technical Reports Server (NTRS)

    Gallegos, Gregory L.; Hilaire, Emmanuel M.; Peterson, Barbara V.; Brown, Christopher S.; Guikema, James A.

    1995-01-01

    Starch filled plastids termed amyloplasts, contained within columella cells of the root caps of higher plant roots, are believed to play a statolith-like role in the gravitropic response of roots. Plants having amyloplasts containing less starch exhibit a corresponding reduction in gravitropic response. We have observed enhanced ethylene production by sweet clover (Melilotus alba L.) seedlings grown in the altered gravity condition of a slow rotating clinostat, and have suggested that this is a stress response resulting from continuous gravistimulation rather than as a result of the simulation of a microgravity condition. If so, we expect that plants deficient in starch accumulation in amyloplasts may produce less stress ethylene when grown on a clinostat. Therefore, we have grown Arabidopsis thaliana in the small, closed environment of the Fluid Processing Apparatus (FPA). In this preliminary report we compare stationary plants with clinorotated and those grown in microgravity aboard Discovery during the STS-63 flight in February 1995. In addition to wildtype, two mutants deficient in starch biosynthesis, mutants TC7 and TL25, which are, respectively, deficient in the activity of amyloplast phosphoglucomutase and ADP-glucose pyrophosphorylase, were grown for three days before being fixed within the FPA. Gas samples were aspirated from the growth chambers and carbon dioxide and ethylene concentations were measured using a gas chromatograph. The fixed tissue is currently undergoing further morphologic and microscopic characterization.

  10. Use of an osmotically sensitive mutant of Propionibacterium freudenreichii subspp. shermanii for the simultaneous productions of organic acids and trehalose from biodiesel waste based crude glycerol.

    PubMed

    Ruhal, Rohit; Choudhury, Bijan

    2012-04-01

    Recently suitability of crude glycerol for trehalose and propionic acid productions was reported using Propionibacterium freudenreichii subspp. shermanii and it was concluded that presence of KCl in crude glycerol was the probable reason for higher trehalose accumulation with crude glycerol medium. To further improve trehalose production, an osmotic sensitive mutant of this strain (non-viable in medium with 3% NaCl) with higher trehalose yield was isolated. In mutant, trehalose yields achieved with respect to biomass and substrate consumed (391 mg/g of biomass, 90 mg/g of substrate consumed) were three and four times higher, respectively as compared to parent strain when crude glycerol was used as a carbon source. Other major fermentation products obtained were propionic acid (0.42 g/g of substrate consumed) and lactic acid (0.3g/g of substrate consumed). It was also observed that in mutant higher activity of ADP-glucose pyrophosphorylase was probably responsible for higher trehalose accumulation. PMID:22306074

  11. Metabolic Consequences of Infection of Grapevine (Vitis vinifera L.) cv. "Modra frankinja" with Flavescence Dorée Phytoplasma.

    PubMed

    Prezelj, Nina; Covington, Elizabeth; Roitsch, Thomas; Gruden, Kristina; Fragner, Lena; Weckwerth, Wolfram; Chersicola, Marko; Vodopivec, Maja; Dermastia, Marina

    2016-01-01

    Flavescence dorée, caused by the quarantine phytoplasma FDp, represents the most devastating of the grapevine yellows diseases in Europe. In an integrated study we have explored the FDp-grapevine interaction in infected grapevines of cv. "Modra frankinja" under natural conditions in the vineyard. In FDp-infected leaf vein-enriched tissues, the seasonal transcriptional profiles of 14 genes selected from various metabolic pathways showed an FDp-specific plant response compared to other grapevine yellows and uncovered a new association of the SWEET17a vacuolar transporter of fructose with pathogens. Non-targeted metabolome analysis from leaf vein-enriched tissues identified 22 significantly changed compounds with increased levels during infection. Several metabolites corroborated the gene expression study. Detailed investigation of the dynamics of carbohydrate metabolism revealed significant accumulation of sucrose and starch in the mesophyll of FDp-infected leaves, as well as significant up-regulation of genes involved in their biosynthesis. In addition, infected leaves had high activities of ADP-glucose pyrophosphorylase and, more significantly, sucrose synthase. The data support the conclusion that FDp infection inhibits phloem transport, resulting in accumulation of carbohydrates and secondary metabolites that provoke a source-sink transition and defense response status. PMID:27242887

  12. Oligomerization, membrane association and in vivo phosphorylation of sugarcane UDP-glucose pyrophosphorylase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sugarcane is a C4 plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms involved in sucrose accumulation in sugarcane are not well understood and little is known with regard to factors that control the extent of sucrose storage in stalks. UDP-glucose pyroph...

  13. Starch metabolism in leaves.

    PubMed

    Orzechowski, Sławomir

    2008-01-01

    Starch is the most abundant storage carbohydrate produced in plants. The initiation of transitory starch synthesis and degradation in plastids depends mainly on diurnal cycle, post-translational regulation of enzyme activity and starch phosphorylation. For the proper structure of starch granule the activities of all starch synthase isoenzymes, branching enzymes and debranching enzymes are needed. The intensity of starch biosynthesis depends mainly on the activity of AGPase (adenosine 5'-diphosphate glucose pyrophosphorylase). The key enzymes in starch degradation are beta-amylase, isoamylase 3 and disproportionating enzyme. However, it should be underlined that there are some crucial differences in starch metabolism between heterotrophic and autotrophic tissues, e.g. is the ability to build multiprotein complexes responsible for biosynthesis and degradation of starch granules in chloroplasts. The observed huge progress in understanding of starch metabolism was possible mainly due to analyses of the complete Arabidopsis and rice genomes and of numerous mutants with altered starch metabolism in leaves. The aim of this paper is to review current knowledge on transient starch metabolism in higher plants. PMID:18787712

  14. Increasing Phosphatidylinositol (4,5)-Bisphosphate Biosynthesis Affects Basal Signaling and Chloroplast Metabolism in Arabidopsis thaliana

    PubMed Central

    Im, Yang Ju; Smith, Caroline M.; Phillippy, Brian Q.; Strand, Deserah; Kramer, David M.; Grunden, Amy M.; Boss, Wendy F.

    2014-01-01

    One challenge in studying the second messenger inositol(1,4,5)-trisphosphate (InsP3) is that it is present in very low amounts and increases only transiently in response to stimuli. To identify events downstream of InsP3, we generated transgenic plants constitutively expressing the high specific activity, human phosphatidylinositol 4-phosphate 5-kinase Iα (HsPIPKIα). PIP5K is the enzyme that synthesizes phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2); this reaction is flux limiting in InsP3 biosynthesis in plants. Plasma membranes from transgenic Arabidopsis expressing HsPIPKIα had 2–3 fold higher PIP5K specific activity, and basal InsP3 levels in seedlings and leaves were >2-fold higher than wild type. Although there was no significant difference in photosynthetic electron transport, HsPIPKIα plants had significantly higher starch (2–4 fold) and 20% higher anthocyanin compared to controls. Starch content was higher both during the day and at the end of dark period. In addition, transcripts of genes involved in starch metabolism such as SEX1 (glucan water dikinase) and SEX4 (phosphoglucan phosphatase), DBE (debranching enzyme), MEX1 (maltose transporter), APL3 (ADP-glucose pyrophosphorylase) and glucose-6-phosphate transporter (Glc6PT) were up-regulated in the HsPIPKIα plants. Our results reveal that increasing the phosphoinositide (PI) pathway affects chloroplast carbon metabolism and suggest that InsP3 is one component of an inter-organelle signaling network regulating chloroplast metabolism. PMID:27135490

  15. Comparative transcriptome analysis to investigate the high starch accumulation of duckweed (Landoltia punctata) under nutrient starvation

    PubMed Central

    2013-01-01

    Background Duckweed can thrive on anthropogenic wastewater and produce tremendous biomass production. Due to its relatively high starch and low lignin percentage, duckweed is a good candidate for bioethanol fermentation. Previous studies have observed that water devoid of nutrients is good for starch accumulation, but its molecular mechanism remains unrevealed. Results This study globally analyzed the response to nutrient starvation in order to investigate the starch accumulation in duckweed (Landoltia punctata). L. punctata was transferred from nutrient-rich solution to distilled water and sampled at different time points. Physiological measurements demonstrated that the activity of ADP-glucose pyrophosphorylase, the key enzyme of starch synthesis, as well as the starch percentage in duckweed, increased continuously under nutrient starvation. Samples collected at 0 h, 2 h and 24 h time points respectively were used for comparative gene expression analysis using RNA-Seq. A comprehensive transcriptome, comprising of 74,797 contigs, was constructed by a de novo assembly of the RNA-Seq reads. Gene expression profiling results showed that the expression of some transcripts encoding key enzymes involved in starch biosynthesis was up-regulated, while the expression of transcripts encoding enzymes involved in starch consumption were down-regulated, the expression of some photosynthesis-related transcripts were down-regulated during the first 24 h, and the expression of some transporter transcripts were up-regulated within the first 2 h. Very interestingly, most transcripts encoding key enzymes involved in flavonoid biosynthesis were highly expressed regardless of starvation, while transcripts encoding laccase, the last rate-limiting enzyme of lignifications, exhibited very low expression abundance in all three samples. Conclusion Our study provides a comprehensive expression profiling of L. punctata under nutrient starvation, which indicates that nutrient starvation down

  16. Comparative Genomic and Phylogenetic Analyses of Gammaproteobacterial glg Genes Traced the Origin of the Escherichia coli Glycogen glgBXCAP Operon to the Last Common Ancestor of the Sister Orders Enterobacteriales and Pasteurellales

    PubMed Central

    Almagro, Goizeder; Viale, Alejandro M.; Montero, Manuel; Rahimpour, Mehdi; Muñoz, Francisco José; Baroja-Fernández, Edurne; Bahaji, Abdellatif; Zúñiga, Manuel; González-Candelas, Fernando; Pozueta-Romero, Javier

    2015-01-01

    Production of branched α-glucan, glycogen-like polymers is widely spread in the Bacteria domain. The glycogen pathway of synthesis and degradation has been fairly well characterized in the model enterobacterial species Escherichia coli (order Enterobacteriales, class Gammaproteobacteria), in which the cognate genes (branching enzyme glgB, debranching enzyme glgX, ADP-glucose pyrophosphorylase glgC, glycogen synthase glgA, and glycogen phosphorylase glgP) are clustered in a glgBXCAP operon arrangement. However, the evolutionary origin of this particular arrangement and of its constituent genes is unknown. Here, by using 265 complete gammaproteobacterial genomes we have carried out a comparative analysis of the presence, copy number and arrangement of glg genes in all lineages of the Gammaproteobacteria. These analyses revealed large variations in glg gene presence, copy number and arrangements among different gammaproteobacterial lineages. However, the glgBXCAP arrangement was remarkably conserved in all glg-possessing species of the orders Enterobacteriales and Pasteurellales (the E/P group). Subsequent phylogenetic analyses of glg genes present in the Gammaproteobacteria and in other main bacterial groups indicated that glg genes have undergone a complex evolutionary history in which horizontal gene transfer may have played an important role. These analyses also revealed that the E/P glgBXCAP genes (a) share a common evolutionary origin, (b) were vertically transmitted within the E/P group, and (c) are closely related to glg genes of some phylogenetically distant betaproteobacterial species. The overall data allowed tracing the origin of the E. coli glgBXCAP operon to the last common ancestor of the E/P group, and also to uncover a likely glgBXCAP transfer event from the E/P group to particular lineages of the Betaproteobacteria. PMID:25607991

  17. Starch biosynthetic genes and enzymes are expressed and active in the absence of starch accumulation in sugar beet tap-root

    PubMed Central

    2014-01-01

    Background Starch is the predominant storage compound in underground plant tissues like roots and tubers. An exception is sugar beet tap-root (Beta vulgaris ssp altissima) which exclusively stores sucrose. The underlying mechanism behind this divergent storage accumulation in sugar beet is currently not fully known. From the general presence of starch in roots and tubers it could be speculated that the lack in sugar beet tap-roots would originate from deficiency in pathways leading to starch. Therefore with emphasis on starch accumulation, we studied tap-roots of sugar beet using parsnip (Pastinaca sativa) as a comparator. Results Metabolic and structural analyses of sugar beet tap-root confirmed sucrose as the exclusive storage component. No starch granules could be detected in tap-roots of sugar beet or the wild ancestor sea beet (Beta vulgaris ssp. maritima). Analyses of parsnip showed that the main storage component was starch but tap-root tissue was also found to contain significant levels of sugars. Surprisingly, activities of four main starch biosynthetic enzymes, phosphoglucomutase, ADP-glucose pyrophosphorylase, starch synthase and starch branching enzyme, were similar in sugar beet and parsnip tap-roots. Transcriptional analysis confirmed expression of corresponding genes. Additionally, expression of genes involved in starch accumulation such as for plastidial hexose transportation and starch tuning functions could be determined in tap-roots of both plant species. Conclusion Considering underground storage organs, sugar beet tap-root upholds a unique property in exclusively storing sucrose. Lack of starch also in the ancestor sea beet indicates an evolved trait of biological importance. Our findings in this study show that gene expression and enzymatic activity of main starch biosynthetic functions are present in sugar beet tap-root during storage accumulation. In view of this, the complete lack of starch in sugar beet tap-roots is enigmatic. PMID

  18. Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation1[OPEN

    PubMed Central

    Okumura, Masaki; Inoue, Shin-ichiro; Kuwata, Keiko

    2016-01-01

    Plant plasma membrane H+-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H+-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha. However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H+-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H+-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H+-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H+-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H+-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H+-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H+-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism. PMID:27016447

  19. Factors Altering Pyruvate Excretion in a Glycogen Storage Mutant of the Cyanobacterium, Synechococcus PCC7942.

    PubMed

    Benson, Phoebe J; Purcell-Meyerink, Diane; Hocart, Charles H; Truong, Thy T; James, Gabriel O; Rourke, Loraine; Djordjevic, Michael A; Blackburn, Susan I; Price, G D

    2016-01-01

    Interest in the production of carbon commodities from photosynthetically fixed CO2 has focused attention on cyanobacteria as a target for metabolic engineering and pathway investigation. We investigated the redirection of carbon flux in the model cyanobacterial species, Synechococcus elongatus PCC 7942, under nitrogen deprivation, for optimized production of the industrially desirable compound, pyruvate. Under nitrogen limited conditions, excess carbon is naturally stored as the multi-branched polysaccharide, glycogen, but a block in glycogen synthesis, via knockout mutation in the gene encoding ADP-glucose pyrophosphorylase (glgC), results in the accumulation of the organic acids, pyruvate and 2-oxoglutarate, as overflow excretions into the extracellular media. The ΔglgC strain, under 48 h of N-deprivation was shown to excrete pyruvate for the first time in this strain. Additionally, by increasing culture pH, to pH 10, it was possible to substantially elevate excretion of pyruvate, suggesting the involvement of an unknown substrate/proton symporter for export. The ΔglgC mutant was also engineered to express foreign transporters for glucose and sucrose, and then grown photomixotrophically with exogenous organic carbon supply, as added 5 mM glucose or sucrose during N- deprivation. Under these conditions we observed a fourfold increase in extracellular pyruvate excretion when glucose was added, and a smaller increase with added sucrose. Although the magnitude of pyruvate excretion did not correlate with the capacity of the ΔglgC strain for bicarbonate-dependent photosynthetic O2 evolution, or with light intensity, there was, however, a positive correlation observed between the density of the starter culture prior to N-deprivation and the final extracellular pyruvate concentration. The factors that contribute to enhancement of pyruvate excretion are discussed, as well as consideration of whether the source of carbon for pyruvate excretion might be derived from

  20. Feedback Inhibition of Starch Degradation in Arabidopsis Leaves Mediated by Trehalose 6-Phosphate1[W][OPEN

    PubMed Central

    Martins, Marina Camara Mattos; Hejazi, Mahdi; Fettke, Joerg; Steup, Martin; Feil, Regina; Krause, Ursula; Arrivault, Stéphanie; Vosloh, Daniel; Figueroa, Carlos María; Ivakov, Alexander; Yadav, Umesh Prasad; Piques, Maria; Metzner, Daniela; Stitt, Mark; Lunn, John Edward

    2013-01-01

    Many plants accumulate substantial starch reserves in their leaves during the day and remobilize them at night to provide carbon and energy for maintenance and growth. In this paper, we explore the role of a sugar-signaling metabolite, trehalose-6-phosphate (Tre6P), in regulating the accumulation and turnover of transitory starch in Arabidopsis (Arabidopsis thaliana) leaves. Ethanol-induced overexpression of trehalose-phosphate synthase during the day increased Tre6P levels up to 11-fold. There was a transient increase in the rate of starch accumulation in the middle of the day, but this was not linked to reductive activation of ADP-glucose pyrophosphorylase. A 2- to 3-fold increase in Tre6P during the night led to significant inhibition of starch degradation. Maltose and maltotriose did not accumulate, suggesting that Tre6P affects an early step in the pathway of starch degradation in the chloroplasts. Starch granules isolated from induced plants had a higher orthophosphate content than granules from noninduced control plants, consistent either with disruption of the phosphorylation-dephosphorylation cycle that is essential for efficient starch breakdown or with inhibition of starch hydrolysis by β-amylase. Nonaqueous fractionation of leaves showed that Tre6P is predominantly located in the cytosol, with estimated in vivo Tre6P concentrations of 4 to 7 µm in the cytosol, 0.2 to 0.5 µm in the chloroplasts, and 0.05 µm in the vacuole. It is proposed that Tre6P is a component in a signaling pathway that mediates the feedback regulation of starch breakdown by sucrose, potentially linking starch turnover to demand for sucrose by growing sink organs at night. PMID:24043444

  1. Quantitative Multilevel Analysis of Central Metabolism in Developing Oilseeds of Oilseed Rape during in Vitro Culture1[OPEN

    PubMed Central

    Schwender, Jörg; Hebbelmann, Inga; Heinzel, Nicolas; Hildebrandt, Tatjana; Rogers, Alistair; Klapperstück, Matthias; Schreiber, Falk; Borisjuk, Ljudmilla; Rolletschek, Hardy

    2015-01-01

    Seeds provide the basis for many food, feed, and fuel products. Continued increases in seed yield, composition, and quality require an improved understanding of how the developing seed converts carbon and nitrogen supplies into storage. Current knowledge of this process is often based on the premise that transcriptional regulation directly translates via enzyme concentration into flux. In an attempt to highlight metabolic control, we explore genotypic differences in carbon partitioning for in vitro cultured developing embryos of oilseed rape (Brassica napus). We determined biomass composition as well as 79 net fluxes, the levels of 77 metabolites, and 26 enzyme activities with specific focus on central metabolism in nine selected germplasm accessions. Overall, we observed a tradeoff between the biomass component fractions of lipid and starch. With increasing lipid content over the spectrum of genotypes, plastidic fatty acid synthesis and glycolytic flux increased concomitantly, while glycolytic intermediates decreased. The lipid/starch tradeoff was not reflected at the proteome level, pointing to the significance of (posttranslational) metabolic control. Enzyme activity/flux and metabolite/flux correlations suggest that plastidic pyruvate kinase exerts flux control and that the lipid/starch tradeoff is most likely mediated by allosteric feedback regulation of phosphofructokinase and ADP-glucose pyrophosphorylase. Quantitative data were also used to calculate in vivo mass action ratios, reaction equilibria, and metabolite turnover times. Compounds like cyclic 3′,5′-AMP and sucrose-6-phosphate were identified to potentially be involved in so far unknown mechanisms of metabolic control. This study provides a rich source of quantitative data for those studying central metabolism. PMID:25944824

  2. Increasing the energy density of vegetative tissues by diverting carbon from starch to oil biosynthesis in transgenic Arabidopsis.

    PubMed

    Sanjaya; Durrett, Timothy P; Weise, Sean E; Benning, Christoph

    2011-10-01

    Increasing the energy density of biomass by engineering the accumulation of triacylglycerols (TAGs) in vegetative tissues is synergistic with efforts to produce biofuels by conversion of lignocellulosic biomass. Typically, TAG accumulates in developing seeds, and little is known about the regulatory mechanisms and control factors preventing oil biosynthesis in vegetative tissues in most plants. Here, we engineered Arabidopsis thaliana to ectopically overproduce the transcription factor WRINKLED1 (WRI1) involved in the regulation of seed oil biosynthesis. Furthermore, we reduced the expression of APS1 encoding a major catalytic isoform of the small subunit of ADP-glucose pyrophosphorylase involved in starch biosynthesis using an RNAi approach. The resulting AGPRNAi-WRI1 lines accumulated less starch and more hexoses. In addition, these lines produced 5.8-fold more oil in vegetative tissues than plants with WRI1 or AGPRNAi alone. Abundant oil droplets were visible in vegetative tissues. TAG molecular species contained long-chain fatty acids, similar to those found in seed oils. In AGPRNAi-WRI1 lines, the relative expression level of sucrose synthase 2 was considerably elevated and correlated with the level of sugars. The relative expression of the genes encoding plastidic proteins involved in de novo fatty acid synthesis, biotin carboxyl carrier protein isoform 2 and acyl carrier protein 1, was also elevated. The relative contribution of TAG compared to starch to the overall energy density increased 9.5-fold in one AGPRNAi-WRI1 transgenic line consistent with altered carbon partitioning from starch to oil. PMID:22003502

  3. Engineering precursor metabolite pools for increasing production of antitumor mithramycins in Streptomyces argillaceus.

    PubMed

    Zabala, Daniel; Braña, Alfredo F; Flórez, Ana B; Salas, José A; Méndez, Carmen

    2013-11-01

    Mithramycin (MTM) is a polyketide antitumor compound produced by Streptomyces argillaceus constituted by a tricyclic aglycone with two aliphatic side chains, a trisaccharide and a disaccharide chain. The biosynthesis of the polyketide aglycone is initiated by the condensation of ten malonyl-CoA units to render a carbon chain that is modified to a tetracyclic intermediate and sequentially glycosylated by five deoxysugars originated from glucose-1-phosphate. Further oxidation and reduction render the final compound. We aimed to increase the precursor supply of malonyl-CoA and/or glucose-1-phosphate in S. argillaceus to enhance MTM production. We have shown that by overexpressing either the S. coelicolor phosphoglucomutase gene pgm or the acetyl-CoA carboxylase ovmGIH genes from the oviedomycin biosynthesis gene cluster in S. argillaceus, we were able to increase the intracellular pool of glucose-1-phosphate and malonyl-CoA, respectively. Moreover, we have cloned the S. argillaceus ADP-glucose pyrophosphorylase gene glgCa and the acyl-CoA:diacylglycerol acyltransferase gene aftAa, and we showed that by inactivating them, an increase of the intracellular concentration of glucose-1-phosphate/glucose-6-phosphate and malonyl-CoA/acetyl-CoA was observed, respectively. Each individual modification resulted in an enhancement of MTM production but the highest production level was obtained by combining all strategies together. In addition, some of these strategies were successfully applied to increase production of four MTM derivatives with improved pharmacological properties: demycarosyl-mithramycin, demycarosyl-3D-β-D-digitoxosyl-mithramycin, mithramycin SK and mithramycin SDK. PMID:24148183

  4. The intrinsically disordered protein LEA7 from Arabidopsis thaliana protects the isolated enzyme lactate dehydrogenase and enzymes in a soluble leaf proteome during freezing and drying.

    PubMed

    Popova, Antoaneta V; Rausch, Saskia; Hundertmark, Michaela; Gibon, Yves; Hincha, Dirk K

    2015-10-01

    The accumulation of Late Embryogenesis Abundant (LEA) proteins in plants is associated with tolerance against stresses such as freezing and desiccation. Two main functions have been attributed to LEA proteins: membrane stabilization and enzyme protection. We have hypothesized previously that LEA7 from Arabidopsis thaliana may stabilize membranes because it interacts with liposomes in the dry state. Here we show that LEA7, contrary to this expectation, did not stabilize liposomes during drying and rehydration. Instead, it partially preserved the activity of the enzyme lactate dehydrogenase (LDH) during drying and freezing. Fourier-transform infrared (FTIR) spectroscopy showed no evidence of aggregation of LDH in the dry or rehydrated state under conditions that lead to complete loss of activity. To approximate the complex influence of intracellular conditions on the protective effects of a LEA protein in a convenient in-vitro assay, we measured the activity of two Arabidopsis enzymes (glucose-6-P dehydrogenase and ADP-glucose pyrophosphorylase) in total soluble leaf protein extract (Arabidopsis soluble proteome, ASP) after drying and rehydration or freezing and thawing. LEA7 partially preserved the activity of both enzymes under these conditions, suggesting its role as an enzyme protectant in vivo. Further FTIR analyses indicated the partial reversibility of protein aggregation in the dry ASP during rehydration. Similarly, aggregation in the dry ASP was strongly reduced by LEA7. In addition, mixtures of LEA7 with sucrose or verbascose reduced aggregation more than the single additives, presumably through the effects of the protein on the H-bonding network of the sugar glasses. PMID:25988244

  5. Factors Altering Pyruvate Excretion in a Glycogen Storage Mutant of the Cyanobacterium, Synechococcus PCC7942

    PubMed Central

    Benson, Phoebe J.; Purcell-Meyerink, Diane; Hocart, Charles H.; Truong, Thy T.; James, Gabriel O.; Rourke, Loraine; Djordjevic, Michael A.; Blackburn, Susan I.; Price, G. D.

    2016-01-01

    Interest in the production of carbon commodities from photosynthetically fixed CO2 has focused attention on cyanobacteria as a target for metabolic engineering and pathway investigation. We investigated the redirection of carbon flux in the model cyanobacterial species, Synechococcus elongatus PCC 7942, under nitrogen deprivation, for optimized production of the industrially desirable compound, pyruvate. Under nitrogen limited conditions, excess carbon is naturally stored as the multi-branched polysaccharide, glycogen, but a block in glycogen synthesis, via knockout mutation in the gene encoding ADP-glucose pyrophosphorylase (glgC), results in the accumulation of the organic acids, pyruvate and 2-oxoglutarate, as overflow excretions into the extracellular media. The ΔglgC strain, under 48 h of N-deprivation was shown to excrete pyruvate for the first time in this strain. Additionally, by increasing culture pH, to pH 10, it was possible to substantially elevate excretion of pyruvate, suggesting the involvement of an unknown substrate/proton symporter for export. The ΔglgC mutant was also engineered to express foreign transporters for glucose and sucrose, and then grown photomixotrophically with exogenous organic carbon supply, as added 5 mM glucose or sucrose during N- deprivation. Under these conditions we observed a fourfold increase in extracellular pyruvate excretion when glucose was added, and a smaller increase with added sucrose. Although the magnitude of pyruvate excretion did not correlate with the capacity of the ΔglgC strain for bicarbonate-dependent photosynthetic O2 evolution, or with light intensity, there was, however, a positive correlation observed between the density of the starter culture prior to N-deprivation and the final extracellular pyruvate concentration. The factors that contribute to enhancement of pyruvate excretion are discussed, as well as consideration of whether the source of carbon for pyruvate excretion might be derived from

  6. Enhanced Symbiotic Performance by Rhizobium tropici Glycogen Synthase Mutants

    PubMed Central

    Marroquí, Silvia; Zorreguieta, Angeles; Santamaría, Carmen; Temprano, Francisco; Soberón, Mario; Megías, Manuel; Downie, J. Allan

    2001-01-01

    We isolated a Tn5-induced Rhizobium tropici mutant that has enhanced capacity to oxidize N,N-dimethyl-p-phenylendiamine (DMPD) and therefore has enhanced respiration via cytochrome oxidase. The mutant had increased levels of the cytochromes c1 and CycM and a small increase in the amount of cytochrome aa3. In plant tests, the mutant increased the dry weight of Phaseolus vulgaris plants by 20 to 38% compared with the control strain, thus showing significantly enhanced symbiotic performance. The predicted product of the mutated gene is homologous to glycogen synthases from several bacteria, and the mutant lacked glycogen. The DNA sequence of the adjacent gene region revealed six genes predicted to encode products homologous to the following gene products from Escherichia coli: glycogen phosphorylase (glgP), glycogen branching enzyme (glgB), ADP glucose pyrophosphorylase (glgC), glycogen synthase (glgA), phosphoglucomutase (pgm), and glycogen debranching enzyme (glgX). All six genes are transcribed in the same direction, and analysis with lacZ gene fusions suggests that the first five genes are organized in one operon, although pgm appears to have an additional promoter; glgX is transcribed independently. Surprisingly, the glgA mutant had decreased levels of high-molecular-weight exopolysaccharide after growth on glucose, but levels were normal after growth on galactose. A deletion mutant was constructed in order to generate a nonpolar mutation in glgA. This mutant had a phenotype similar to that of the Tn5 mutant, indicating that the enhanced respiration and symbiotic nitrogen fixation and decreased exopolysaccharide were due to mutation of glgA and not to a polar effect on a downstream gene. PMID:11208782

  7. Quantitative Multilevel Analysis of Central Metabolism in Developing Oilseeds of Oilseed Rape During In Vitro Culture

    SciTech Connect

    Schwender, Jorg; Hebbelmann, Inga; Heinzel, Nicholas; Hildebrandt, Tatjana; Rogers, Alistair; Naik, Dhiraj; Klapperstuck, Matthias; Braun, Hans -Peter; Schreiber, Falk; Denolf, Peter; Borisjuk, Ljudmilla; Rolletschek, Hardy

    2015-07-01

    Seeds provide the basis for many food, feed, and fuel products. Continued increases in seed yield, composition, and quality require an improved understanding of how the developing seed converts carbon and nitrogen supplies into storage. Current knowledge of this process is often based on the premise that transcriptional regulation directly translates via enzyme concentration into flux. In an attempt to highlight metabolic control, we explore genotypic differences in carbon partitioning for in vitro cultured developing embryos of oilseed rape (Brassica napus). We determined biomass composition as well as 79 net fluxes, the levels of 77 metabolites, and 26 enzyme activities with specific focus on central metabolism in nine selected germplasm accessions. We observed a tradeoff between the biomass component fractions of lipid and starch. With increasing lipid content over the spectrum of genotypes, plastidic fatty acid synthesis and glycolytic flux increased concomitantly, while glycolytic intermediates decreased. The lipid/starch tradeoff was not reflected at the proteome level, pointing to the significance of (posttranslational) metabolic control. Enzyme activity/flux and metabolite/flux correlations suggest that plastidic pyruvate kinase exerts flux control and that the lipid/starch tradeoff is most likely mediated by allosteric feedback regulation of phosphofructokinase and ADP-glucose pyrophosphorylase. Also, quantitative data were used to calculate in vivo mass action ratios, reaction equilibria, and metabolite turnover times. Compounds like cyclic 3',5'-AMP and sucrose-6-phosphate were identified to potentially be involved in so far unknown mechanisms of metabolic control. This study provides a rich source of quantitative data for those studying central metabolism..

  8. Regulation of starch accumulation by granule-associated plant 14-3-3 proteins.

    PubMed

    Sehnke, P C; Chung, H J; Wu, K; Ferl, R J

    2001-01-16

    In higher plants the production of starch is orchestrated by chloroplast-localized biosynthetic enzymes, namely starch synthases, ADP-glucose pyrophosphorylase, and starch branching and debranching enzymes. Diurnal regulation of these enzymes, as well as starch-degrading enzymes, influences both the levels and composition of starch, and is dependent in some instances upon phosphorylation-linked regulation. The phosphoserine/threonine-binding 14-3-3 proteins participate in environmentally responsive phosphorylation-related regulatory functions in plants, and as such are potentially involved in starch regulation. We report here that reduction of the epsilon subgroup of Arabidopsis 14-3-3 proteins by antisense technology resulted in a 2- to 4-fold increase in leaf starch accumulation. Dark-governed starch breakdown was unaffected in these "antisense plants," indicating an unaltered starch-degradation pathway and suggesting a role for 14-3-3 proteins in regulation of starch synthesis. Absorption spectra and gelatinization properties indicate that the starch from the antisense plants has an altered branched glucan composition. Biochemical characterization of protease-treated starch granules from both Arabidopsis leaves and maize endosperm showed that 14-3-3 proteins are internal intrinsic granule proteins. These data suggest a direct role for 14-3-3 proteins in starch accumulation. The starch synthase III family is a possible target for 14-3-3 protein regulation because, uniquely among plastid-localized starch metabolic enzymes, all members of the family contain the conserved 14-3-3 protein phosphoserine/threonine-binding consensus motif. This possibility is strengthened by immunocapture using antibodies to DU1, a maize starch synthase III family member, and direct interaction with biotinylated 14-3-3 protein, both of which demonstrated an association between 14-3-3 proteins and DU1 or DU1-like proteins. PMID:11149942

  9. Metabolic and enzymatic changes associated with carbon mobilization, utilization and replenishment triggered in grain amaranth (Amaranthus cruentus) in response to partial defoliation by mechanical injury or insect herbivory

    PubMed Central

    2012-01-01

    Background Amaranthus cruentus and A. hypochondriacus are crop plants grown for grain production in subtropical countries. Recently, the generation of large-scale transcriptomic data opened the possibility to study representative genes of primary metabolism to gain a better understanding of the biochemical mechanisms underlying tolerance to defoliation in these species. A multi-level approach was followed involving gene expression analysis, enzyme activity and metabolite measurements. Results Defoliation by insect herbivory (HD) or mechanical damage (MD) led to a rapid and transient reduction of non-structural carbohydrates (NSC) in all tissues examined. This correlated with a short-term induction of foliar sucrolytic activity, differential gene expression of a vacuolar invertase and its inhibitor, and induction of a sucrose transporter gene. Leaf starch in defoliated plants correlated negatively with amylolytic activity and expression of a β-amylase-1 gene and positively with a soluble starch synthase gene. Fatty-acid accumulation in roots coincided with a high expression of a phosphoenolpyruvate/phosphate transporter gene. In all tissues there was a long-term replenishment of most metabolite pools, which allowed damaged plants to maintain unaltered growth and grain yield. Promoter analysis of ADP-glucose pyrophosphorylase and vacuolar invertase genes indicated the presence of cis-regulatory elements that supported their responsiveness to defoliation. HD and MD had differential effects on transcripts, enzyme activities and metabolites. However, the correlation between transcript abundance and enzymatic activities was very limited. A better correlation was found between enzymes, metabolite levels and growth and reproductive parameters. Conclusions It is concluded that a rapid reduction of NSC reserves in leaves, stems and roots followed by their long-term recovery underlies tolerance to defoliation in grain amaranth. This requires the coordinate action of genes

  10. Photosynthesis down-regulation precedes carbohydrate accumulation under sink limitation in Citrus.

    PubMed

    Nebauer, Sergio G; Renau-Morata, Begoña; Guardiola, José Luis; Molina, Rosa-Victoria

    2011-02-01

    Photosynthesis down-regulation due to an imbalance between sources and sinks in Citrus leaves could be mediated by excessive accumulation of carbohydrates. However, there is limited understanding of the physiological role of soluble and insoluble carbohydrates in photosynthesis regulation and the elements triggering the down-regulation process. In this work, the role of non-structural carbohydrates in the regulation of photosynthesis under a broad spectrum of source-sink relationships has been investigated in the Salustiana sweet orange. Soluble sugar and starch accumulation in leaves, induced by girdling experiments, did not induce down-regulation of the photosynthetic rate in the presence of sinks (fruits). The leaf-to-fruit ratio did not modulate photosynthesis but allocation of photoassimilates to the fruits. The lack of strong sink activity led to a decrease in the photosynthetic rate and starch accumulation in leaves. However, photosynthesis down-regulation due to an excess of total soluble sugars or starch was discarded because photosynthesis and stomatal conductance reduction occurred prior to any significant accumulation of these carbohydrates. Gas exchange and fluorescence parameters suggested biochemical limitations to photosynthesis. In addition, the expression of carbon metabolism-related genes was altered within 24 h when strong sinks were removed. Sucrose synthesis and export genes were inhibited, whereas the expression of ADP-glucose pyrophosphorylase was increased to cope with the excess of assimilates. In conclusion, changes in starch and soluble sugar turnover, but not sugar content per se, could provide the signal for photosynthesis regulation. In these conditions, non-stomatal limitations strongly inhibited the photosynthetic rate prior to any significant increase in carbohydrate levels. PMID:21367744

  11. Quantitative Multilevel Analysis of Central Metabolism in Developing Oilseeds of Oilseed Rape during in Vitro Culture.

    PubMed

    Schwender, Jörg; Hebbelmann, Inga; Heinzel, Nicolas; Hildebrandt, Tatjana; Rogers, Alistair; Naik, Dhiraj; Klapperstück, Matthias; Braun, Hans-Peter; Schreiber, Falk; Denolf, Peter; Borisjuk, Ljudmilla; Rolletschek, Hardy

    2015-07-01

    Seeds provide the basis for many food, feed, and fuel products. Continued increases in seed yield, composition, and quality require an improved understanding of how the developing seed converts carbon and nitrogen supplies into storage. Current knowledge of this process is often based on the premise that transcriptional regulation directly translates via enzyme concentration into flux. In an attempt to highlight metabolic control, we explore genotypic differences in carbon partitioning for in vitro cultured developing embryos of oilseed rape (Brassica napus). We determined biomass composition as well as 79 net fluxes, the levels of 77 metabolites, and 26 enzyme activities with specific focus on central metabolism in nine selected germplasm accessions. Overall, we observed a tradeoff between the biomass component fractions of lipid and starch. With increasing lipid content over the spectrum of genotypes, plastidic fatty acid synthesis and glycolytic flux increased concomitantly, while glycolytic intermediates decreased. The lipid/starch tradeoff was not reflected at the proteome level, pointing to the significance of (posttranslational) metabolic control. Enzyme activity/flux and metabolite/flux correlations suggest that plastidic pyruvate kinase exerts flux control and that the lipid/starch tradeoff is most likely mediated by allosteric feedback regulation of phosphofructokinase and ADP-glucose pyrophosphorylase. Quantitative data were also used to calculate in vivo mass action ratios, reaction equilibria, and metabolite turnover times. Compounds like cyclic 3',5'-AMP and sucrose-6-phosphate were identified to potentially be involved in so far unknown mechanisms of metabolic control. This study provides a rich source of quantitative data for those studying central metabolism. PMID:25944824

  12. A Suite of Lotus japonicus Starch Mutants Reveals Both Conserved and Novel Features of Starch Metabolism1[W][OA

    PubMed Central

    Vriet, Cécile; Welham, Tracey; Brachmann, Andreas; Pike, Marilyn; Pike, Jodie; Perry, Jillian; Parniske, Martin; Sato, Shusei; Tabata, Satoshi; Smith, Alison M.; Wang, Trevor L.

    2010-01-01

    The metabolism of starch is of central importance for many aspects of plant growth and development. Information on leaf starch metabolism other than in Arabidopsis (Arabidopsis thaliana) is scarce. Furthermore, its importance in several agronomically important traits exemplified by legumes remains to be investigated. To address this issue, we have provided detailed information on the genes involved in starch metabolism in Lotus japonicus and have characterized a comprehensive collection of forward and TILLING (for Targeting Induced Local Lesions IN Genomes) reverse genetics mutants affecting five enzymes of starch synthesis and two enzymes of starch degradation. The mutants provide new insights into the structure-function relationships of ADP-glucose pyrophosphorylase and glucan, water dikinase1 in particular. Analyses of the mutant phenotypes indicate that the pathways of leaf starch metabolism in L. japonicus and Arabidopsis are largely conserved. However, the importance of these pathways for plant growth and development differs substantially between the two species. Whereas essentially starchless Arabidopsis plants lacking plastidial phosphoglucomutase grow slowly relative to wild-type plants, the equivalent mutant of L. japonicus grows normally even in a 12-h photoperiod. In contrast, the loss of GLUCAN, WATER DIKINASE1, required for starch degradation, has a far greater effect on plant growth and fertility in L. japonicus than in Arabidopsis. Moreover, we have also identified several mutants likely to be affected in new components or regulators of the pathways of starch metabolism. This suite of mutants provides a substantial new resource for further investigations of the partitioning of carbon and its importance for symbiotic nitrogen fixation, legume seed development, and perenniality and vegetative regrowth. PMID:20699404

  13. Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation.

    PubMed

    Okumura, Masaki; Inoue, Shin-Ichiro; Kuwata, Keiko; Kinoshita, Toshinori

    2016-05-01

    Plant plasma membrane H(+)-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H(+)-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H(+)-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H(+)-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H(+)-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H(+)-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H(+)-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H(+)-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H(+)-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism. PMID:27016447

  14. [Enhancement of photoassimilate utilization by manipulation of the ADPglucose pyrophosphorylase gene]. Progress report, April 1993--January 1994

    SciTech Connect

    Okita, T.W.

    1994-04-01

    Part I of this research concerns patterns of gene expression of ADPG-PP in native and transgenic potato plants. The expression of both potato ADPG-PP subunits were analyzed on the transcript and antigen levels. The small and large subunits were coordinately expressed during tuber development suggesting a role for the temporal regulation of ADPG-PP expression as well as providing further support for earlier work in the heterotetrameric subunit structure of the tuber enzyme. Part II involves studies on the structure-function relationships of ADPG-PP, more specifically the mutagenesis of the large and small subunit DNAs of ADPG-PP.

  15. Changes in gravity affect gene expression, protein modulation and metabolite pools of arabidopsis

    NASA Astrophysics Data System (ADS)

    Hampp, R.; Martzivanou, M.; Maier, R. M.; Magel, E.

    Callus cultures of Arabidopsis thaliana (cv. Columbia) in Petri dishes / suspension cultures were exposed to altered g-forces by centrifugation (1 to 10 g), klinorotation, and μ g (sounding rocket flights). Using semi-quantitative RT-PCR, transcripts of genes coding for metabolic key enzymes (ADP-glucose pyrophosphorylase, ADPG-PP; ß-amylase, fructose-1,6-bisphosphatase, FBPase; glyceraldehyde-P dehydrogenase, GAPDH; hydroxymethylglutaryl-CoA reductase, HMG; phenylalanine-ammonium-lyase, PAL; PEP carboxylase, PEPC) were used to monitor threshold conditions for g-number (all) and time of exposure (ß-amylase) which led to altered amounts of the gene product. Exposure to approx. 5 g and higher for 1h resulted in altered transcript levels: transcripts of ß-amylase, PAL, and PEPC were increased, those of ADPG-PP decreased, while those of FBPase, GAPDH, and HMG were not affected. This probably indicates a shift from starch synthesis to starch degradation and increased rates of anaplerosis (PEPC: supply of ketoacids for amino acid synthesis). In order to get more information about g-related effects on gene expression, we used a 1h-exposure to 7 g for a microarray analysis. Transcripts of more than 200 genes were significantly increased in amount (ratio 7g / 1g control; 21.6 and larger). They fall into several categories. Transcripts coding for enzymes of major pathways form the largest group (25%), followed by gene products involved in cellular organisation and cell wall formation / rearrangement (17%), signalling, phosphorylation/dephosphorylation (12%), proteolysis and transport (10% each), hormone synthesis plus related events (8%), defense (4%), stress-response (2%), and gravisensing (2%). Many of the alterations are part of a general stress response, but some changes related to the synthesis / rearrangement of cell wall components could be more hyper-g-specific. Using macroarrays with selected genes according to our hypergravity study (metabolism / signalling

  16. Defects in leaf carbohydrate metabolism compromise acclimation to high light and lead to a high chlorophyll fluorescence phenotype in Arabidopsis thaliana

    PubMed Central

    2012-01-01

    Background We have studied the impact of carbohydrate-starvation on the acclimation response to high light using Arabidopsis thaliana double mutants strongly impaired in the day- and night path of photoassimilate export from the chloroplast. A complete knock-out mutant of the triose phosphate/phosphate translocator (TPT; tpt-2 mutant) was crossed to mutants defective in (i) starch biosynthesis (adg1-1, pgm1 and pgi1-1; knock-outs of ADP-glucose pyrophosphorylase, plastidial phosphoglucomutase and phosphoglucose isomerase) or (ii) starch mobilization (sex1-3, knock-out of glucan water dikinase) as well as in (iii) maltose export from the chloroplast (mex1-2). Results All double mutants were viable and indistinguishable from the wild type when grown under low light conditions, but - except for sex1-3/tpt-2 - developed a high chlorophyll fluorescence (HCF) phenotype and growth retardation when grown in high light. Immunoblots of thylakoid proteins, Blue-Native gel electrophoresis and chlorophyll fluorescence emission analyses at 77 Kelvin with the adg1-1/tpt-2 double mutant revealed that HCF was linked to a specific decrease in plastome-encoded core proteins of both photosystems (with the exception of the PSII component cytochrome b559), whereas nuclear-encoded antennae (LHCs) accumulated normally, but were predominantly not attached to their photosystems. Uncoupled antennae are the major cause for HCF of dark-adapted plants. Feeding of sucrose or glucose to high light-grown adg1-1/tpt-2 plants rescued the HCF- and growth phenotypes. Elevated sugar levels induce the expression of the glucose-6-phosphate/phosphate translocator2 (GPT2), which in principle could compensate for the deficiency in the TPT. A triple mutant with an additional defect in GPT2 (adg1-1/tpt-2/gpt2-1) exhibited an identical rescue of the HCF- and growth phenotype in response to sugar feeding as the adg1-1/tpt-2 double mutant, indicating that this rescue is independent from the sugar

  17. TreT, a novel trehalose glycosyltransferring synthase of the hyperthermophilic archaeon Thermococcus litoralis.

    PubMed

    Qu, Qiuhao; Lee, Sung-Jae; Boos, Winfried

    2004-11-12

    The gene cluster in Thermococcus litoralis encoding a multicomponent and binding protein-dependent ABC transporter for trehalose and maltose contains an open reading frame of unknown function. We cloned this gene (now called treT), expressed it in Escherichia coli, purified the encoded protein, and identified it as an enzyme forming trehalose and ADP from ADP-glucose and glucose. The enzyme can also use UDP- and GDP-glucose but with less efficiency. The reaction is reversible, and ADP-glucose plus glucose can also be formed from trehalose and ADP. The rate of reaction and the equilibrium favor the formation of trehalose. At 90 degrees C, the optimal temperature for the enzymatic reaction, the half-maximal concentration of ADP-glucose at saturating glucose concentrations is 1.14 mm and the V(max) is 160 units/mg protein. In the reverse reaction, the half-maximal concentration of trehalose at saturating ADP concentrations is 11.5 mm and the V(max) was estimated to be 17 units/mg protein. Under non-denaturating in vitro conditions the enzyme behaves as a dimer of identical subunits of 48 kDa. As the transporter encoded in the same gene cluster, TreT is induced by trehalose and maltose in the growth medium. PMID:15364950

  18. Cloning and expression of murine enzymes involved in the salvage pathway of GDP-L-fucose.

    PubMed

    Niittymäki, Jaana; Mattila, Pirkko; Roos, Christophe; Huopaniemi, Laura; Sjöblom, Solveig; Renkonen, Risto

    2004-01-01

    In the salvage pathway of GDP-L-fucose, free cytosolic fucose is phosphorylated by L-fucokinase to form L-fucose-L-phosphate, which is then further converted to GDP-L-fucose in the reaction catalyzed by GDP-L-fucose pyrophosphorylase. We report here the cloning and expression of murine L-fucokinase and GDP-L-fucose pyrophosphorylase. Murine L-fucokinase is expressed as two transcripts of 3057 and 3270 base pairs, encoding proteins of 1019 and 1090 amino acids with predicted molecular masses of 111 kDa and 120 kDa respectively. Only the longer splice variant of L-fucokinase was enzymatically active when expressed in COS-7 cells. Murine GDP-L-fucose pyrophosphorylase has an open reading frame of 1773 base pairs encoding a protein of 591 amino acids with a predicted molecular mass of 65.5 kDa. GDP-L-fucose, the reaction product of GDP-L-pyrophosphorylase, was identified by HPLC and MALDI-TOF MS analysis. The tissue distribution of murine L-fucokinase and GDP-L-fucose pyrophosphorylase was investigated by quantitative real time PCR, which revealed high expression of L-fucokinase and GDP-L-fucose pyrophosphorylase in various tissues. The wide expression of both enzymes can also be observed from the large amount of data collected from a number of expressed sequence tag libraries, which indicate that not only the de novo pathway alone, but also the salvage pathway, could have a significant role in the synthesis of GDP-L-fucose in the cytosol. PMID:14686921

  19. Nucleotide Pyrophosphatase/Phosphodiesterase 1 Exerts a Negative Effect on Starch Accumulation and Growth in Rice Seedlings under High Temperature and CO2 Concentration Conditions

    PubMed Central

    Kaneko, Kentaro; Inomata, Takuya; Masui, Takahiro; Koshu, Tsutomu; Umezawa, Yukiho; Itoh, Kimiko; Pozueta-Romero, Javier; Mitsui, Toshiaki

    2014-01-01

    Nucleotide pyrophosphatase/phosphodiesterase (NPP) is a widely distributed enzymatic activity occurring in both plants and mammals that catalyzes the hydrolytic breakdown of the pyrophosphate and phosphodiester bonds of a number of nucleotides. Unlike mammalian NPPs, the physiological function of plant NPPs remains largely unknown. Using a complete rice NPP1-encoding cDNA as a probe, in this work we have screened a rice shoot cDNA library and obtained complete cDNAs corresponding to six NPP genes (NPP1–NPP6). As a first step to clarify the role of NPPs, recombinant NPP1, NPP2 and NPP6 were purified from transgenic rice cells constitutively expressing NPP1, NPP2 and NPP6, respectively, and their enzymatic properties were characterized. NPP1 and NPP6 exhibited hydrolytic activities toward ATP, UDP-glucose and the starch precursor molecule, ADP-glucose, whereas NPP2 did not recognize nucleotide sugars as substrates, but hydrolyzed UDP, ADP and adenosine 5′-phosphosulfate. To gain insight into the physiological function of rice NPP1, an npp1 knockout mutant was characterized. The ADP-glucose hydrolytic activities in shoots of npp1 rice seedlings were 8% of those of the wild type (WT), thus indicating that NPP1 is a major determinant of ADP-glucose hydrolytic activity in rice shoots. Importantly, when seedlings were cultured at 160 Pa CO2 under a 28°C/23°C (12 h light/12 h dark) regime, npp1 shoots and roots were larger than those of wild-type (WT) seedlings. Furthermore, the starch content in the npp1 shoots was higher than that of WT shoots. Growth and starch accumulation were also enhanced under an atmospheric CO2 concentration (40 Pa) when plants were cultured under a 33°C/28°C regime. The overall data strongly indicate that NPP1 exerts a negative effect on plant growth and starch accumulation in shoots, especially under high CO2 concentration and high temperature conditions. PMID:24092883

  20. Screening of advanced potato breeding clones for resistance to cold-induced-sweetening (CIS)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The advantages of processing potatoes from low temperature storage are well known. It had previously been reported that clones with A-II isozymes of UDP-glucose pyrophosphorylase (UGPase) and low vacuolar acid invertase (VAcInv) activity demonstrate increased resistance to CIS. This study reports o...

  1. Free Sugars in Relation to Starch Accumulation in Developing Rice Grain

    PubMed Central

    Singh, Rangil; Juliano, Bienvenido O.

    1977-01-01

    The changes in sugars (water-soluble carbohydrates) were studied in the developing grain of rice (Oryza sativa L., variety IR28 and IR29) in relation to the role of these sugars as precursors of ADP glucose in starch accumulation. The levels of total sugars, total reducing sugars and free glucose, sucrose and other nonreducing sugars, maltooligosaccharides, and total and nonsucrosyl fructose followed closely the changes in the rate of starch accumulation, in both IR28 and 29; the peak value occurred 9 days after flowering. The level of soluble carbohydrates remained high in the caryopsis and also in milled rice after starch accumulation, suggesting that the supply of sugar precursors does not limit starch accumulation in the rice grain. Because of a higher level of reducing sugars, the level of free sugars in the grain of waxy rice IR29 was higher than that of nonwaxy IR28. PMID:16659864

  2. Metabolic Network for the Biosynthesis of Intra- and Extracellular α-Glucans Required for Virulence of Mycobacterium tuberculosis

    PubMed Central

    van de Weerd, Robert; Chandra, Govind; Appelmelk, Ben; Alber, Marina; Ioerger, Thomas R.; Jacobs, William R.; Geurtsen, Jeroen; Bornemann, Stephen

    2016-01-01

    Mycobacterium tuberculosis synthesizes intra- and extracellular α-glucans that were believed to originate from separate pathways. The extracellular glucose polymer is the main constituent of the mycobacterial capsule that is thought to be involved in immune evasion and virulence. However, the role of the α-glucan capsule in pathogenesis has remained enigmatic due to an incomplete understanding of α-glucan biosynthetic pathways preventing the generation of capsule-deficient mutants. Three separate and potentially redundant pathways had been implicated in α-glucan biosynthesis in mycobacteria: the GlgC-GlgA, the Rv3032 and the TreS-Pep2-GlgE pathways. We now show that α-glucan in mycobacteria is exclusively assembled intracellularly utilizing the building block α-maltose-1-phosphate as the substrate for the maltosyltransferase GlgE, with subsequent branching of the polymer by the branching enzyme GlgB. Some α-glucan is exported to form the α-glucan capsule. There is an unexpected convergence of the TreS-Pep2 and GlgC-GlgA pathways that both generate α-maltose-1-phosphate. While the TreS-Pep2 route from trehalose was already known, we have now established that GlgA forms this phosphosugar from ADP-glucose and glucose 1-phosphate 1000-fold more efficiently than its hitherto described glycogen synthase activity. The two routes are connected by the common precursor ADP-glucose, allowing compensatory flux from one route to the other. Having elucidated this unexpected configuration of the metabolic pathways underlying α-glucan biosynthesis in mycobacteria, an M. tuberculosis double mutant devoid of α-glucan could be constructed, showing a direct link between the GlgE pathway, α-glucan biosynthesis and virulence in a mouse infection model. PMID:27513637

  3. Metabolic Network for the Biosynthesis of Intra- and Extracellular α-Glucans Required for Virulence of Mycobacterium tuberculosis.

    PubMed

    Koliwer-Brandl, Hendrik; Syson, Karl; van de Weerd, Robert; Chandra, Govind; Appelmelk, Ben; Alber, Marina; Ioerger, Thomas R; Jacobs, William R; Geurtsen, Jeroen; Bornemann, Stephen; Kalscheuer, Rainer

    2016-08-01

    Mycobacterium tuberculosis synthesizes intra- and extracellular α-glucans that were believed to originate from separate pathways. The extracellular glucose polymer is the main constituent of the mycobacterial capsule that is thought to be involved in immune evasion and virulence. However, the role of the α-glucan capsule in pathogenesis has remained enigmatic due to an incomplete understanding of α-glucan biosynthetic pathways preventing the generation of capsule-deficient mutants. Three separate and potentially redundant pathways had been implicated in α-glucan biosynthesis in mycobacteria: the GlgC-GlgA, the Rv3032 and the TreS-Pep2-GlgE pathways. We now show that α-glucan in mycobacteria is exclusively assembled intracellularly utilizing the building block α-maltose-1-phosphate as the substrate for the maltosyltransferase GlgE, with subsequent branching of the polymer by the branching enzyme GlgB. Some α-glucan is exported to form the α-glucan capsule. There is an unexpected convergence of the TreS-Pep2 and GlgC-GlgA pathways that both generate α-maltose-1-phosphate. While the TreS-Pep2 route from trehalose was already known, we have now established that GlgA forms this phosphosugar from ADP-glucose and glucose 1-phosphate 1000-fold more efficiently than its hitherto described glycogen synthase activity. The two routes are connected by the common precursor ADP-glucose, allowing compensatory flux from one route to the other. Having elucidated this unexpected configuration of the metabolic pathways underlying α-glucan biosynthesis in mycobacteria, an M. tuberculosis double mutant devoid of α-glucan could be constructed, showing a direct link between the GlgE pathway, α-glucan biosynthesis and virulence in a mouse infection model. PMID:27513637

  4. Mutan produced in potato amyloplasts adheres to starch granules.

    PubMed

    Kok-Jacon, Géraldine A; Vincken, Jean-Paul; Suurs, Luc C J M; Visser, Richard G F

    2005-05-01

    Production of water-insoluble mutan polymers in Kardal potato tubers was investigated after expression of a full-length (GtfI) and a truncated mutansucrase gene referred to as GtfICAT (GtfI without glucan-binding domain) from Streptococcus downei. Subsequent effects on starch biosynthesis at the molecular and biochemical levels were studied. Expression of the GtfICAT gene resulted in the adhesion of mutan material on starch granules, which stained red with erythrosine, and which was hydrolysed by exo-mutanase. In addition, GtfICAT-expressing plants exhibited a severely altered tuber phenotype and starch granule morphology in comparison to those expressing the full-length GtfI gene. In spite of that, no structural changes at the starch level were observed. Expression levels of the sucrose-regulated, AGPase and GBSSI genes were down-regulated in only the GTFICAT transformants, showing that GtfICAT expression interfered with the starch biosynthetic pathway. In accordance with the down-regulated AGPase gene, a lower starch content was observed in the GTFICAT transformants. Finally, the rheological properties of the GTFICAT starches were modified; they showed a higher retrogradation during cooling of the starch paste. PMID:17129316

  5. Arabidopsis AMY1 expressions and early flowering mutant phenotype.

    PubMed

    Jie, Wang; Dashi, Yu; XinHong, Guo; Xuanming, Liu

    2009-02-28

    The homozygous T-DNA mutant of the AMY1 gene in Arabidopsis was identified and importantly, shown to cause an early flowering phenotype. We found that the disruption of AMY1 enhanced expression of CO and FT. The expression analyses of genes related to starch metabolism revealed that expression of the AGPase small subunit APS1 in the wild type was higher than in the amy1 mutant. However, there were no significant differences in expression levels of the AGPase large subunit genes ApL1, AMY2, or AMY3 between wild type and the amy1 mutant. Expression profiling showed that AMY1 was highly expressed in leaves, stems, and flowers, and expressed less in leafstalks and roots. Furthermore, the level of AMY1 mRNA was highly elevated with age and in senescing leaves. RT-PCR analyses showed that the expression of AMY1 was induced by heat shock, GA, and ABA, while salt stress had no apparent effect on its expression. PMID:19250611

  6. Physicochemical properties of starches and expression and activity of starch biosynthesis-related genes in sweet potatoes.

    PubMed

    Lai, Yung C; Wang, Shu Y; Gao, Huan Y; Nguyen, Khiem M; Nguyen, Chinh H; Shih, Ming C; Lin, Kuan H

    2016-05-15

    The functional properties of starches from six sweet potato varieties containing various starch components and structures were studied in an attempt to identify starch sources for industrial uses. Tainan 18 (TNN18) with high-amylose (AM) starch exhibited high setback and breakdown viscosities, high water solubility at 85°C but low swelling volume at 65°C, and high hardness and adhesiveness; in contrast, the low-AM starch of Tainung 31 (TNG31) had opposite characteristics. Seven genes related to starch biosynthesis were tested, and GBSS, SS, SBEII, ISA, and AGPase were highly expressed in TNN18 and TNG31; however, transcript levels in DBE and SBE were extremely low. GBSS and SS activity reflected the abundance of GBSS and SS mRNA in TNG31 and TNN18, and expression of AGPase, GBSS, SS, and SBE in TNN18 substantially increased content of AM. The expression and activity of DBE had a significant effect on TNG31 with increased AP content. PMID:26776008

  7. Influence of Water Deficit on Maize Endosperm Development 1

    PubMed Central

    Ober, Eric S.; Setter, Tim L.; Madison, James T.; Thompson, John F.; Shapiro, Paul S.

    1991-01-01

    In maize (Zea mays L.), drought during the post-pollination stage decreases kernel growth and often leads to grain yield losses. Kernels in the apical region of the ear are more severely affected than basally positioned kernels. We hypothesized that water deficit during early endosperm development might inhibit kernel growth by decreasing endosperm cell division, and that this response might be mediated by changes in endosperm abscisic acid (ABA) levels. Greenhouse-grown maize, cultivar Pioneer 3925, was subjected to water limitation from 1 to 15 days after pollination (DAP), spanning the period of endosperm cell division and induction of storage product accumulation. Water deficit decreased the number of endosperm nuclei during the treatment period; the most substantial effect was in the apical region of ears. Correspondingly, endosperm fresh weight, starch accumulation and dry mass at maturity were decreased by water limitation. Abscisic acid concentrations in endosperm were quantified by enzyme-linked immunosorbent assay. Water deficit increased ABA concentration in apical-region endosperm by four-fold compared to controls. ABA concentrations were also increased in middle and basal regions of the ear, but to a lesser extent. Two key enzymes in the starch synthesis pathway, sucrose synthase and granule-bound ADP-glucose starch synthase, and zein, the major storage protein in maize endosperm, were studied as markers of storage product synthesis. Water deficit did not affect sucrose synthase enzyme activity or RNA transcript abundance relative to total RNA. However, ADP-glucose starch synthase activity and RNA transcript abundance decreased slightly in apical-region endosperm of water-limited plants by 15 DAP, compared with well-watered controls. In contrast to starch, there was no treatment effect on the accumulation of zein, evaluated at either the polypeptide or RNA level. We conclude that under the conditions tested, the establishment of starch and zein

  8. Trehalose-phosphate synthase of Mycobacterium tuberculosis. Cloning, expression and properties of the recombinant enzyme.

    PubMed

    Pan, Y T; Carroll, J D; Elbein, A D

    2002-12-01

    The trehalose-phosphate synthase (TPS) of Mycobacterium smegmatis was previously purified to apparent homogeneity and several peptides from the 58 kDa protein were sequenced. Based on that sequence information, the gene for TPS was identified in the Mycobacterium tuberculosis genome, and the gene was cloned and expressed in Escherichia coli with a (His)6 tag at the amino terminus. The TPS was expressed in good yield and as active enzyme, and was purified on a metal ion column to give a single band of approximately 58 kDa on SDS/PAGE. Approximately 1.3 mg of purified TPS were obtained from a 1-L culture of E. coli ( approximately 2.3 g cell paste). The purified recombinant enzyme showed a single band of approximately 58 kDa on SDS/PAGE, but a molecular mass of approximately 220 kDa by gel filtration, indicating that the active TPS is probably a tetrameric protein. Like the enzyme originally purified from M. smegmatis, the recombinant enzyme is an unusual glycosyltransferase as it can utilize any of the nucleoside diphosphate glucose derivatives as glucosyl donors, i.e. ADP-glucose, CDP-glucose, GDP-glucose, TDP-glucose and UDP-glucose, with ADP-glucose, GDP-glucose and UDP-glucose being the preferred substrates. These studies prove conclusively that the mycobacterial TPS is indeed responsible for catalyzing the synthesis of trehalose-P from any of the nucleoside diphosphate glucose derivatives. Although the original enzyme from M. smegmatis was greatly stimulated in its utilization of UDP-glucose by polyanions such as heparin, the recombinant enzyme was stimulated only modestly by heparin. The Km for UDP-glucose as the glucosyl donor was approximately 18 mm, and that for GDP-glucose was approximately 16 mm. The enzyme was specific for glucose-6-P as the glucosyl acceptor, and the Km for this substrate was approximately 7 mm when UDP-glucose was the glucosyl donor and approximately 4 mm with GDP-glucose. TPS did not show an absolute requirement for divalent cations

  9. CO2-Responsive CONSTANS, CONSTANS-Like, and Time of Chlorophyll a/b Binding Protein Expression1 Protein Is a Positive Regulator of Starch Synthesis in Vegetative Organs of Rice1[OPEN

    PubMed Central

    Sugino, Miho; Hatanaka, Tomoko; Misoo, Shuji

    2015-01-01

    A unique CO2-Responsive CONSTANS, CONSTANS-like, and Time of Chlorophyll a/b Binding Protein1 (CCT) Protein (CRCT) containing a CCT domain but not a zinc finger motif is described, which is up-regulated under elevated CO2 in rice (Oryza sativa). The expression of CRCT showed diurnal oscillation peaked at the end of the light period and was also increased by sugars such as glucose and sucrose. Promoter β-glucuronidase analysis showed that CRCT was highly expressed in the phloem of various tissues such as leaf blade and leaf sheath. Overexpression or RNA interference knockdown of CRCT had no appreciable effect on plant growth and photosynthesis except that tiller angle was significantly increased by the overexpression. More importantly, starch content in leaf sheath, which serves as a temporary storage organ for photoassimilates, was markedly increased in overexpression lines and decreased in knockdown lines. The expressions of several genes related to starch synthesis, such as ADP-glucose pyrophospholylase and α-glucan phospholylase, were significantly changed in transgenic lines and positively correlated with the expression levels of CRCT. Given these observations, we suggest that CRCT is a positive regulator of starch accumulation in vegetative tissues, regulating coordinated expression of starch synthesis genes in response to the levels of photoassimilates. PMID:25717036

  10. Metabolic symbiosis and the birth of the plant kingdom.

    PubMed

    Deschamps, Philippe; Colleoni, Christophe; Nakamura, Yasunori; Suzuki, Eiji; Putaux, Jean-Luc; Buléon, Alain; Haebel, Sophie; Ritte, Gerhard; Steup, Martin; Falcón, Luisa I; Moreira, David; Löffelhardt, Wolfgang; Raj, Jenifer Nirmal; Plancke, Charlotte; d'Hulst, Christophe; Dauvillée, David; Ball, Steven

    2008-03-01

    Eukaryotic cells are composed of a variety of membrane-bound organelles that are thought to derive from symbiotic associations involving bacteria, archaea, or other eukaryotes. In addition to acquiring the plastid, all Archaeplastida and some of their endosymbiotic derivatives can be distinguished from other organisms by the fact that they accumulate starch, a semicrystalline-storage polysaccharide distantly related to glycogen and never found elsewhere. We now provide the first evidence for the existence of starch in a particular species of single-cell diazotrophic cyanobacterium. We provide evidence for the existence in the eukaryotic host cell at the time of primary endosymbiosis of an uridine diphosphoglucose (UDP-glucose)-based pathway similar to that characterized in amoebas. Because of the monophyletic origin of plants, we can define the genetic makeup of the Archaeplastida ancestor with respect to storage polysaccharide metabolism. The most likely enzyme-partitioning scenario between the plastid's ancestor and its eukaryotic host immediately suggests the precise nature of the ancient metabolic symbiotic relationship. The latter consisted in the export of adenosine diphosphoglucose (ADP-glucose) from the cyanobiont in exchange for the import of reduced nitrogen from the host. We further speculate that the monophyletic origin of plastids may lie in an organism with close relatedness to present-day group V cyanobacteria. PMID:18093994

  11. Plastid uridine salvage activity is required for photoassimilate allocation and partitioning in Arabidopsis.

    PubMed

    Chen, Mingjie; Thelen, Jay J

    2011-08-01

    Nucleotides are synthesized from de novo and salvage pathways. To characterize the uridine salvage pathway, two genes, UKL1 and UKL2, that tentatively encode uridine kinase (UK) and uracil phosphoribosyltransferase (UPRT) bifunctional enzymes were studied in Arabidopsis thaliana. T-DNA insertions in UKL1 and UKL2 reduced transcript expression and increased plant tolerance to toxic analogs 5-fluorouridine and 5-fluorouracil. Enzyme activity assays using purified recombinant proteins indicated that UKL1 and UKL2 have UK but not UPRT activity. Subcellular localization using a C-terminal enhanced yellow fluorescent protein fusion indicated that UKL1 and UKL2 localize to plastids. The ukl2 mutant shows reduced transient leaf starch during the day. External application of orotate rescued this phenotype in ukl2, indicating pyrimidine pools are limiting for starch synthesis in ukl2. Intermediates for lignin synthesis were upregulated, and there was increased lignin and reduced cellulose content in the ukl2 mutant. Levels of ATP, ADP, ADP-glucose, UTP, UDP, and UDP-glucose were altered in a light-dependent manner. Seed composition of the ukl1 and ukl2 mutants included lower oil and higher protein compared with the wild type. Unlike single gene mutants, the ukl1 ukl2 double mutant has severe developmental defects and reduced biomass accumulation, indicating these enzymes catalyze redundant reactions. These findings point to crucial roles played by uridine salvage for photoassimilate allocation and partitioning. PMID:21828290

  12. Identification and characterization of a novel plastidic adenine nucleotide uniporter from Solanum tuberosum.

    PubMed

    Leroch, Michaela; Kirchberger, Simon; Haferkamp, Ilka; Wahl, Markus; Neuhaus, H Ekkehard; Tjaden, Joachim

    2005-05-01

    Homologs of BT1 (the Brittle1 protein) are found to be phylogenetically related to the mitochondrial carrier family and appear to occur in both mono- and dicotyledonous plants. Whereas BT1 from cereals is probably involved in the transport of ADP-glucose, which is essential for starch metabolism in endosperm plastids, BT1 from a noncereal plant, Solanum tuberosum (StBT1), catalyzes an adenine nucleotide uniport when functionally integrated into the bacterial cytoplasmic membrane. Import studies into intact Escherichia coli cells harboring StBT1 revealed a narrow substrate spectrum with similar affinities for AMP, ADP, and ATP of about 300-400 mum. Transiently expressed StBT1-green fluorescent protein fusion protein in tobacco leaf protoplasts showed a plastidic localization of the StBT1. In vitro synthesized radioactively labeled StBT1 was targeted to the envelope membranes of isolated spinach chloroplasts. Furthermore, we showed by real time reverse transcription-PCR a ubiquitous expression pattern of the StBT1 in autotrophic and heterotrophic potato tissues. We therefore propose that StBT1 is a plastidic adenine nucleotide uniporter used to provide the cytosol and other compartments with adenine nucleotides exclusively synthesized inside plastids. PMID:15737999

  13. Nicotinamide nucleotide synthesis in regenerating rat liver

    PubMed Central

    Ferris, G. M.; Clark, J. B.

    1971-01-01

    1. The concentrations and total content of the nicotinamide nucleotides were measured in the livers of rats at various times after partial hepatectomy and laparotomy (sham hepatectomy) and correlated with other events in the regeneration process. 2. The NAD content and concentration in rat liver were relatively unaffected by laparotomy, but fell to a minimum, 25 and 33% below control values respectively, 24h after partial hepatectomy. NADP content and concentration were affected similarly by both laparotomy and partial hepatectomy, falling rapidly and remaining depressed for up to 48h. 3. The effect of injecting various doses of nicotinamide on the liver DNA and NAD 18h after partial hepatectomy was studied and revealed an inverse correlation between NAD content and DNA content. 4. Injections of nicotinamide at various times after partial hepatectomy revealed that the ability to synthesize NAD from nicotinamide was impaired during the first 12h, rose to a peak at 26h and fell again by 48h after partial hepatectomy. 5. The total liver activity of NAD pyrophosphorylase (EC 2.7.7.1) remained at or slightly above the initial value for 12h after partial hepatectomy and then rose continuously until 48h after operation. The activity of NMN pyrophosphorylase (EC 2.4.2.12) showed a similar pattern of change after partial hepatectomy, but was at no time greater than 5% of the activity of NAD pyrophosphorylase. 6. The results are discussed with reference to the control of NAD synthesis in rapidly dividing tissue. It is suggested that the availability of cofactors and substrates for NAD synthesis is more important as a controlling factor than the maximum enzyme activities. It is concluded that the low concentrations of nicotinamide nucleotides in rapidly dividing tissues are the result of competition between NAD synthesis and nucleic acid synthesis for common precursor and cofactors. PMID:4398891

  14. [Synthesis of nicotinamide adenine dinucleotide in the nuclei of pigeon erythrocytes].

    PubMed

    Nemchinskaia, V L; Makarova, T G; Mozhenok, T P; Braun, A D

    1975-08-01

    The nuclei of pigeon erythrocytes are capable of synthesizing NAD from nicotinamid-mononucleotides and ATP. Some data on the kinetics of NAD-pyrophosphorylase have been obtained: the optimal concentration of nuclei and the effect of various incubation time. The pretreatment of nuclei by Triton X-100, or by ultrasonics enhances NAD synthesis. The results suggest that cyclic 3',5'-AMP (Fluka) may have no effect on NAD synthesis. The control of the cell metabolism by NAD formation is considered. PMID:181878

  15. A Potential Pathway for Galactose Metabolism in Cucumis sativus L., A Stachyose Transporting Species 1

    PubMed Central

    Gross, Kenneth C.; Pharr, David M.

    1982-01-01

    Conversion of [14C]galactose (Gal) 1-P, UDP-[14C]Gal, or UDP-[14C]glucose to [14C]sucrose was observed when cell-free homogenates of cucumber (Cucumis sativus L.) fruit peduncles were incubated with individual 14C-labeled substrates, appropriate cofactors, and fructose. The sucrose product was labeled only in the glucose moiety. Conversion of [14C]Gal-1-P to [14C]sucrose was catalyzed by extracts of peduncles from all other stachyose transporting species tested, as well as green bean (a sucrose transporter) but was not catalyzed by peduncle extracts from three other sucrose transporting species. In cucumber, the ability of extracts to form [14C]sucrose from [14C]Gal-1-P was greater when peduncles were harvested from growing fruit than from unpollinated ovaries. [14C]Sucrose formation from [14C]Gal-1-P was inhibited by Mg · PPi, Mg · UDP, UMP, and sucrose. α-Galactosidase, galactokinase, UDP-gal pyrophosphorylase, UDP-Gal-4′-epimerase, UDP-glucose pyrophosphorylase, and sucrose synthase activities were detected in peduncle extracts. Neither sucrose phosphate synthetase nor hexose-1-P uridyltransferase were detected. Peduncle tissue contained a small pool of free galactose. These results suggest a potential pathway for the metabolism of galactose moieties hydrolyzed from stachyose, the major sugar transported by cucumber plants. PMID:16662141

  16. Depletion of UDP-Glucose and UDP-Galactose Using a Degron System Leads to Growth Cessation of Leishmania major

    PubMed Central

    Bandini, Giulia; Zarnovican, Patricia; Buettner, Falk R.; Lüder, Carsten G. K.; Ferguson, Michael A. J.; Routier, Françoise H.

    2015-01-01

    Interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) by the UDP-Glc 4´-epimerase intimately connects the biosynthesis of these two nucleotide sugars. Their de novo biosynthesis involves transformation of glucose-6-phosphate into glucose-1-phosphate by the phosphoglucomutase and subsequent activation into UDP-Glc by the specific UDP-Glc pyrophosphorylase (UGP). Besides UGP, Leishmania parasites express an uncommon UDP-sugar pyrophosphorylase (USP) able to activate both galactose-1-phosphate and glucose-1-phosphate in vitro. Targeted gene deletion of UGP alone was previously shown to principally affect expression of lipophosphoglycan, resulting in a reduced virulence. Since our attempts to delete both UGP and USP failed, deletion of UGP was combined with conditional destabilisation of USP to control the biosynthesis of UDP-Glc and UDP-Gal. Stabilisation of the enzyme produced by a single USP allele was sufficient to maintain the steady-state pools of these two nucleotide sugars and preserve almost normal glycoinositolphospholipids galactosylation, but at the apparent expense of lipophosphoglycan biosynthesis. However, under destabilising conditions, the absence of both UGP and USP resulted in depletion of UDP-Glc and UDP-Gal and led to growth cessation and cell death, suggesting that either or both of these metabolites is/are essential. PMID:26529232

  17. Regulation of starch synthesis in potato tubers

    SciTech Connect

    Davies, H.; Oparka, K.; Viola, R.; Wright, K.; Ross, H. )

    1990-05-01

    Following tuber excision from the mother plant sucrose synthase activity fell from 3,120 to 960 nmol/g.f. wt./h within 7 days and starch synthesis ({sup 14}C sucrose incorporated into isolated discs) from 23 to 7 nmol/g.f. wt./h. While the maximum catalytic activity of sucrose synthase was more than sufficient to account for the observed rate of starch synthesis a maximum of 27% of sucrose incorporated by discs was converted into starch within 3 h. This compared with 80% conversion of {sup 14}C glucose incorporated. Tuber excision also reduced the rate of starch biosynthesis with glucose as a substrate (from 206 to 64 nmol/g.f. wt./h). The activities of UDPG-pyrophosphorylase, PPi-PFK, ATP-PFK, starch synthase and hexokinase (glucose or fructose substrates) were unaffected by tuber removal. ADPG pyrophosphorylase activity was reduced from 8,000 to 4,500 nmol/g.f. wt./h. Preliminary experiments indicate that the decline in sucrose synthease activity is prevented by maintaining sucrose flux into tubers through the cut stolon.

  18. Heterologous Production of Hyaluronic Acid in an ε-Poly-l-Lysine Producer, Streptomyces albulus

    PubMed Central

    Yoshimura, Tomohiro; Shibata, Nobuyuki; Hamano, Yoshimitsu

    2015-01-01

    Hyaluronic acid (HA) is used in a wide range of medical applications, where its performance and therapeutic efficacy are highly dependent on its molecular weight. In the microbial production of HA, it has been suggested that a high level of intracellular ATP enhances the productivity and molecular weight of HA. Here, we report on heterologous HA production in an ε-poly-l-lysine producer, Streptomyces albulus, which has the potential to generate ATP at high level. The hasA gene from Streptococcus zooepidemicus, which encodes HA synthase, was refactored and expressed under the control of a late-log growth phase-operating promoter. The expression of the refactored hasA gene, along with genes coding for UDP-glucose dehydrogenase, UDP-N-acetylglucosamine pyrophosphorylase, and UDP-glucose pyrophosphorylase, which are involved in HA precursor sugar biosynthesis, resulted in efficient production of HA in the 2.0 MDa range, which is greater than typical bacterial HA, demonstrating that a sufficient amount of ATP was provided to support the biosynthesis of the precursor sugars, which in turn promoted HA production. In addition, unlike in the case of streptococcal HA, S. albulus-derived HA was not cell associated. Based on these findings, our heterologous production system appears to have several advantages for practical HA production. We propose that the present system could be applicable to the heterologous production of a wide variety of molecules other than HA in the case their biosynthesis pathways require ATP in vivo. PMID:25795665

  19. Unidirectional steady state rates of central metabolism enzymes measured simultaneously in a living plant tissue.

    PubMed

    Roscher, A; Emsley, L; Raymond, P; Roby, C

    1998-09-25

    The unidirectional steady state reaction rates of several enzymes and metabolic fluxes of distinct processes were measured simultaneously in hypoxic maize root tips using two-dimensional phosphorus NMR exchange spectroscopy. A single spectrum monitors ATP synthesis and hydrolysis as well as the activities of four enzymes involved in key pathways of central metabolism: UDP-glucose pyrophosphorylase, phosphoglucomutase, hexose-phosphate isomerase, and enolase. The corresponding unidirectional reaction rates and net metabolic fluxes were calculated from spectral intensities. This method provides a unique picture, at enzyme resolution, of how metabolism reacts in a concerted fashion to changes in external parameters such as temperature and oxygen concentration. By increasing hypoxia via an increase in temperature, we measured the expected increase in glycolysis through enolase activity while total ATP synthesis settled. At the same time, we observed a net flux through phosphoglucomutase and UDP-glucose pyrophosphorylase toward carbohydrate synthesis. This result is discussed in relation to the current hypothesis on the turnover of cell walls and sucrose. This reaction also produces a net flux of pyrophosphate, which is needed by pyrophosphate:fructose-6-phosphate 1-phosphotransferase to work as a glycolytic enzyme. PMID:9737962

  20. The Crystal Structure of Nitrosomonas europaea Sucrose Synthase Reveals Critical Conformational Changes and Insights into Sucrose Metabolism in Prokaryotes

    PubMed Central

    Wu, Rui; Asención Diez, Matías D.; Figueroa, Carlos M.; Machtey, Matías; Iglesias, Alberto A.; Ballicora, Miguel A.

    2015-01-01

    ABSTRACT In this paper we report the first crystal structure of a prokaryotic sucrose synthase from the nonphotosynthetic bacterium Nitrosomonas europaea. The obtained structure was in an open form, whereas the only other available structure, from the plant Arabidopsis thaliana, was in a closed conformation. Comparative structural analysis revealed a “hinge-latch” combination, which is critical to transition between the open and closed forms of the enzyme. The N. europaea sucrose synthase shares the same fold as the GT-B family of the retaining glycosyltransferases. In addition, a triad of conserved homologous catalytic residues in the family was shown to be functionally critical in the N. europaea sucrose synthase (Arg567, Lys572, and Glu663). This implies that sucrose synthase shares not only a common origin with the GT-B family but also a similar catalytic mechanism. The enzyme preferred transferring glucose from ADP-glucose rather than UDP-glucose like the eukaryotic counterparts. This predicts that these prokaryotic organisms have a different sucrose metabolic scenario from plants. Nucleotide preference determines where the glucose moiety is targeted after sucrose is degraded. IMPORTANCE We obtained biochemical and structural evidence of sucrose metabolism in nonphotosynthetic bacteria. Until now, only sucrose synthases from photosynthetic organisms have been characterized. Here, we provide the crystal structure of the sucrose synthase from the chemolithoautotroph N. europaea. The structure supported that the enzyme functions with an open/close induced fit mechanism. The enzyme prefers as the substrate adenine-based nucleotides rather than uridine-based like the eukaryotic counterparts, implying a strong connection between sucrose and glycogen metabolism in these bacteria. Mutagenesis data showed that the catalytic mechanism must be conserved not only in sucrose synthases but also in all other retaining GT-B glycosyltransferases. PMID:26013491

  1. Growth, /sup 14/C-sucrose uptake, and metabolites of starch synthesis in apical and basal kernels of corn (Zea mays L. )

    SciTech Connect

    Greenberg, J.M.

    1985-01-01

    Developing field-grown kernels of corn (Zea mays L. cv. Cornell 175) from the base and apex of the ear were sampled from seven to 70 days after pollination (DAP) an compared with respect to dry weight, ability to take up /sup 14/C-sucrose from solution in vitro, and content of sucrose, glucose, starch, glucose-1-P (G1P), glucose-6-P (G6P), fructose-6-P (F6P), ADP-glucose (ADPG), and UDP-glucose (UDPG). ADPG and UDPG were analyzed by HPLC. All other metabolites were analyzed enzymatically. Simultaneous hand-pollination of all ovaries in an ear did not reduce the difference between apical and basal kernels in dry weight, indicating that the latter fertilization of apical kernels was not responsible for their lesser mature dry weight. Detached kernels took up /sup 14/C-sucrose (0.3-400 mM) and glucose (5-100 mM) at rates linearly proportional to the sugar concentration. Glucose, fructose, and sorbitol did not inhibit uptake of /sup 14/C-sucrose. Uptake was not stimulated by 5 mM CaCl/sup 2/ or the addition of buffers (pH 4.5-6.7) to the medium. Sulfhydryl reagents (PCMBS, NEM) and metabolic inhibitors (TNBS, DNP, NaF) did not reduce uptake. These observations suggest that sucrose is taken up by a non-saturable, non-energy-requiring mechanism. Sucrose uptake increased throughout development, especially at the stage when basal kernels began to accumulate more dry weight than apical kernels (10-20 DAP in freely pollinated ears; 25 DAP in synchronously pollinated ears). Hydrolysis of incorporated sucrose increased from 87% at 14 DAP to 99% by 57 DAP.

  2. Two Alternative Pathways for the Synthesis of the Rare Compatible Solute Mannosylglucosylglycerate in Petrotoga mobilis▿

    PubMed Central

    Fernandes, Chantal; Mendes, Vitor; Costa, Joana; Empadinhas, Nuno; Jorge, Carla; Lamosa, Pedro; Santos, Helena; da Costa, Milton S.

    2010-01-01

    The compatible solute mannosylglucosylglycerate (MGG), recently identified in Petrotoga miotherma, also accumulates in Petrotoga mobilis in response to hyperosmotic conditions and supraoptimal growth temperatures. Two functionally connected genes encoding a glucosyl-3-phosphoglycerate synthase (GpgS) and an unknown glycosyltransferase (gene Pmob_1143), which we functionally characterized as a mannosylglucosyl-3-phosphoglycerate synthase and designated MggA, were identified in the genome of Ptg. mobilis. This enzyme used the product of GpgS, glucosyl-3-phosphoglycerate (GPG), as well as GDP-mannose to produce mannosylglucosyl-3-phosphoglycerate (MGPG), the phosphorylated precursor of MGG. The MGPG dephosphorylation was determined in cell extracts, and the native enzyme was partially purified and characterized. Surprisingly, a gene encoding a putative glucosylglycerate synthase (Ggs) was also identified in the genome of Ptg. mobilis, and an active Ggs capable of producing glucosylglycerate (GG) from ADP-glucose and d-glycerate was detected in cell extracts and the recombinant enzyme was characterized, as well. Since GG has never been identified in this organism nor was it a substrate for the MggA, we anticipated the existence of a nonphosphorylating pathway for MGG synthesis. We putatively identified the corresponding gene, whose product had some sequence homology with MggA, but it was not possible to recombinantly express a functional enzyme from Ptg. mobilis, which we named mannosylglucosylglycerate synthase (MggS). In turn, a homologous gene from Thermotoga maritima was successfully expressed, and the synthesis of MGG was confirmed from GDP-mannose and GG. Based on the measurements of the relevant enzyme activities in cell extracts and on the functional characterization of the key enzymes, we propose two alternative pathways for the synthesis of the rare compatible solute MGG in Ptg. mobilis. PMID:20061481

  3. Alanine aminotransferase 1 (OsAlaAT1) plays an essential role in the regulation of starch storage in rice endosperm.

    PubMed

    Yang, Jungil; Kim, Sung-Ryul; Lee, Sang-Kyu; Choi, Heebak; Jeon, Jong-Seong; An, Gynheung

    2015-11-01

    Alteration of storage substances, in particular the major storage form starch, leads to floury endosperm. Because floury mutants have physical attributes for milling processes, identification and characterization of those mutants are valuable. In this study we identified a floury endosperm mutant caused by a T-DNA insertion in Oryza sativa alanine-aminotransferase1 (OsAlaAT1). OsAlaAT1 is localized in the cytosol and has aminotransferase enzyme activity. The osalaat1 mutant has less amylose and its amylopectin is structurally altered. OsAlaAT1 is predominantly expressed in developing seeds during active starch synthesis. AlaAT catalyzes the interconversion of pyruvate to alanine, and this pathway is activated under low-oxygen conditions. Consistently, OsAlaAT1 is induced by such conditions. Expression of the starch synthesis genes AGPases, OsSSI, OsSSIIa, and OsPPDKB is decreased in the mutant. Thus, our observations suggest that OsAlaAT1 plays an essential role in starch synthesis in developing seeds that are exposed to low concentrations of oxygen. PMID:26475189

  4. Common reed accumulates starch in its stem by metabolic adaptation under Cd stress conditions

    PubMed Central

    Higuchi, Kyoko; Kanai, Masatake; Tsuchiya, Masahisa; Ishii, Haruka; Shibuya, Naofumi; Fujita, Naoko; Nakamura, Yasunori; Suzui, Nobuo; Fujimaki, Shu; Miwa, Eitaro

    2015-01-01

    In a previous study, we reported that the common reed accumulates water-soluble Cd complexed with an α-glucan-like molecule, and that the synthesis of this molecule is induced in the stem of the common reed under Cd stress. We studied the metabolic background to ensure α-glucan accumulation under the Cd stress conditions that generally inhibit photosynthesis. We found that the common reed maintained an adequate CO2 assimilation rate, tended to allocate more assimilated 11C to the stem, and accumulated starch granules in its stem under Cd stress conditions. AGPase activity, which is the rate-limiting enzyme for starch synthesis, increased in the stem of common reed grown in the presence of Cd. Starch accumulation in the stem of common reed was not obvious under other excess metal conditions. Common reed may preferentially allocate assimilated carbon as the carbon source for the formation of Cd and α-glucan complexes in its stem followed by prevention of Cd transfer to leaves acting as the photosynthetic organ. These responses may allow the common reed to grow even under severe Cd stress conditions. PMID:25806040

  5. Cold-night responses in grapevine inflorescences.

    PubMed

    Sawicki, Mélodie; Ait Barka, Essaid; Clément, Christophe; Gilard, Françoise; Tcherkez, Guillaume; Baillieul, Fabienne; Vaillant-Gaveau, Nathalie; Jacquard, Cédric

    2015-10-01

    Cold nights impact grapevine flower development and fruit set. Regulation at the female meiosis stepmay be of considerable importance for further understanding on how flower reacts to cold stress. In this study, the impact of chilling temperature (0 °C overnight) on carbon metabolism was investigated in the inflorescencesof two cultivars, Pinot noir (Pinot) and Gewurztraminer (Gewurtz.). Fluctuations in photosynthetic activity and carbohydrate metabolism were monitored by analyzing gas exchanges, simultaneous photosystem I and II activities, andcarbohydrate content. Further, the expression of PEPc, PC, FNR, ISO, OXO, AGPase, amylases and invertase genes, activities of various enzymes, as well as metabolomic analysis were attained. Results showed that the chilling night has different impacts depending on cultivars. Thus, in Gewurtz., net photosynthesis (Pn) was transiently increased whereas, in Pinot, the Pn increase was persistent and concomitant with an inhibition of respiration. However, during the days following the cold night, photosynthetic activity was decreased, and the cyclic electron flow was inhibited in Gewurtz., suggesting lower efficient energy dissipation. Likewise, metabolomic analysis revealed that several metabolites contents (namely alanine, GABA, lysine and succinate)were distinctly modulated in the two cultivars. Taking together, these results reflect a photosynthetic metabolism alteration or internal CO2 conductance in Gewurtz. explaining partly why Pinot is less susceptible to cold stress. PMID:26398796

  6. Proteomic analysis of heat treated bitter gourd (Momordica charantia L. var. Hong Kong Green) using 2D-DIGE.

    PubMed

    Ng, Zhi Xiang; Chua, Kek Heng; Kuppusamy, Umah Rani

    2014-04-01

    This study aimed to investigate the changes in the proteome of bitter gourd prior to and after subjecting to boiling and microwaving. A comparative analysis of the proteome profiles of raw and thermally treated bitter gourds was performed using 2D-DIGE. The protein content and number of protein spots in raw sample was higher when compared to the cooked samples. Qualitative analysis revealed that 103 (boiled sample) and 110 (microwaved sample) protein spots were up regulated whereas 120 (boiled sample) and 107 (microwaved sample) protein spots were down regulated. Ten protein spots with the highest significant fold change in the cooked samples were involved in carbohydrate/energy metabolisms and stress responses. Small heat shock proteins, superoxide dismutase, quinone oxidoreductase, UDP-glucose pyrophosphorylase and phosphoglycerate kinase play a role in heat-stress-mediated protection of bitter gourd. This study suggests that appropriate heat treatment (cooking methods) can lead to induction of selected proteins in bitter gourd. PMID:24262540

  7. Comparative Intermediary Metabolism of Vegetative Cells and Microcysts of Myxococcus xanthus1

    PubMed Central

    Watson, Benjamin F.; Dworkin, Martin

    1968-01-01

    Crude extracts of both vegetative cells and glycerol-induced microcysts of Myxococcus xanthus contained the following enzyme activities: phosphofructokinase, phosphoglucoisomerase, fructose-1,6-diphosphatase, fructosediphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphopyruvate carboxylase, citrate synthase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, and uridine diphosphate glucose pyrophosphorylase. With the exception of isocitrate dehydrogenase, which was present at a fivefold higher concentration in microcysts, all activities in extracts from both types of cells were essentially equal. Hexokinase and pyruvate kinase could not be detected in extracts from either type of cell. Microcysts metabolized acetate at a lower rate than did vegetative cells. Most of this decrease was reflected in a substantial decrease in ability of microcysts to oxidize acetate to CO2. In addition, microcysts and vegetative cells showed a different distribution of 14C-label from incorporated acetate. PMID:4302296

  8. Improved one-pot multienzyme (OPME) systems for synthesizing UDP-uronic acids and glucuronides†

    PubMed Central

    Muthana, Musleh M.; Qu, Jingyao; Xue, Mengyang; Klyuchnik, Timofey; Siu, Alex; Li, Yanhong; Zhang, Lei; Yu, Hai; Li, Lei; Wang, Peng G.

    2015-01-01

    Arabidopsis thaliana glucuronokinase (AtGlcAK) was cloned and shown to be able to use various uronic acids as substrates to produce the corresponding uronic acid-1-phosphates. AtGlcAK or Bifidobacterium infantis galactokinase (BiGalK) was used with a UDP-sugar pyrophosphorylase, an inorganic pyrophosphatase, with or without a glycosyltransferase for highly efficient synthesis of UDP-uronic acids and glucuronides. These improved cost-effective one-pot multienzyme (OPME) systems avoid the use of nicotinamide adenine dinucleotide (NAD+)-cofactor in dehydrogenase-dependent UDP-glucuronic acid production processes and can be broadly applied for synthesizing various glucuronic acid-containing molecules. PMID:25686901

  9. Comparative intermediary metabolism of vegetative cells and microcysts of Myxococcus xanthus.

    PubMed

    Watson, B F; Dworkin, M

    1968-11-01

    Crude extracts of both vegetative cells and glycerol-induced microcysts of Myxococcus xanthus contained the following enzyme activities: phosphofructokinase, phosphoglucoisomerase, fructose-1,6-diphosphatase, fructosediphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphopyruvate carboxylase, citrate synthase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, and uridine diphosphate glucose pyrophosphorylase. With the exception of isocitrate dehydrogenase, which was present at a fivefold higher concentration in microcysts, all activities in extracts from both types of cells were essentially equal. Hexokinase and pyruvate kinase could not be detected in extracts from either type of cell. Microcysts metabolized acetate at a lower rate than did vegetative cells. Most of this decrease was reflected in a substantial decrease in ability of microcysts to oxidize acetate to CO(2). In addition, microcysts and vegetative cells showed a different distribution of (14)C-label from incorporated acetate. PMID:4302296

  10. Carbon Metabolism Enzymes of Rhizobium tropici Cultures and Bacteroids.

    PubMed

    Romanov, V I; Hernández-Lucas, I; Martínez-Romero, E

    1994-07-01

    We determined the activities of selected enzymes involved in carbon metabolism in free-living cells of Rhizobium tropici CFN299 grown in minimal medium with different carbon sources and in bacteroids of the same strain. The set of enzymatic activities in sucrose-grown cells suggests that the pentose phosphate pathway, with the participation of the Entner-Doudoroff pathway, is probably the primary route for sugar catabolism. In glutamate- and malate-grown cells, high activities of the gluconeogenic enzymes (phosphoenolpyruvate carboxykinase, fructose-6-phosphate aldolase, and fructose bisphosphatase) were detected. In bacteroids, isolated in Percoll gradients, the levels of activity for many of the enzymes measured were similar to those of malate-grown cells, except that higher activities of glucokinase, glucose-6-phosphate dehydrogenase, and NAD-dependent phosphogluconate dehydrogenase were detected. Phosphoglucomutase and UDP glucose pyrophosphorylase showed high and constant levels under all growth conditions and in bacteroids. PMID:16349319

  11. Measurement of the inorganic pyrophosphate in tissues of Pisum sativum L.

    PubMed

    Edwards, J; Rees, T A; Wilson, P M; Morrell, S

    1984-09-01

    Purified pyrophosphate: fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90) was used to measure the inorganic pyrophosphate in unfractionated extracts of tissues of Pisum sativum L. The fructose 1,6-bisphosphate produced by the above enzyme was measured by coupling to NADH oxidation via aldolase (EC 4.1.2.13), triosephosphate isomerase (EC 5.3.1.1) and glycerol-3-phosphate dehydrogenase (EC 1.1.1.8). Amounts of pyrophosphate as low as 1 nmol could be measured. The contents of pyrophosphate in the developing embryo of pea, and in the apical 2 cm of the roots, were appreciable; 9.4 and 8.9 nmol g(-1) fresh weight, respectively. The possibility that pyrophosphate acts in vivo as an energy source for pyrophosphate: fructose 6-phosphate 1-phosphotransferase and for UDPglucose pyrophosphorylase (EC 2.7.7.9) is considered. PMID:24254055

  12. Purification and biochemical characterisation of GlmU from Yersinia pestis.

    PubMed

    Patin, Delphine; Bayliss, Marc; Mengin-Lecreulx, Dominique; Oyston, Petra; Blanot, Didier

    2015-04-01

    Antibiotic resistance has emerged as a real threat to mankind, rendering many compounds ineffective in the fight against bacterial infection, including for significant diseases such as plague caused by Yersinia pestis. Essential genes have been identified as promising targets for inhibiting with new classes of compounds. Previously, the gene encoding the bifunctional UDP-N-acetylglucosamine pyrophosphorylase/glucosamine-1-phosphate N-acetyltransferase enzyme GlmU was confirmed as an essential gene in Yersinia. As a step towards exploiting this target for antimicrobial screening, we undertook a biochemical characterisation of the Yersinia GlmU. Effects of pH and magnesium concentration on the acetyltransferase and uridyltransferase activities were analysed, and kinetic parameters were determined. The acetyltransferase activity, which is strongly increased in the presence of reducing agent, was shown to be susceptible to oxidation and thiol-specific reagents. PMID:25417006

  13. Metabolic engineering of Pichia pastoris for production of hyaluronic acid with high molecular weight.

    PubMed

    Jeong, Euijoon; Shim, Woo Yong; Kim, Jung Hoe

    2014-09-20

    The high molecular weight (>1 MDa) of hyaluronic acid (HA) is important for its biological functions. The reported limiting factors for the production of HA with high molecular weight (MW) by microbial fermentation are the insufficient HA precursor pool and cell growth inhibition. To overcome these issues, the Xenopus laevis xhasA2 and xhasB genes encoding hyaluronan synthase 2 (xhasA2) and UDP-glucose dehydrogenase (xhasB), were expressed in Pichia pastoris widely used for production of heterologous proteins. In this study, expression vectors containing various combination cassettes of HA pathway genes including xhasA2 and xhasB from X. laevis as well as UDP-glucose pyrophosphorylase (hasC), UDP-N-acetylglucosamine pyrophosphorylase (hasD) and phosphoglucose isomerase (hasE) from P. pastoris were constructed and tested. First, HA pathway genes were overexpressed using pAO815 and pGAPZB vectors, resulting in the production of 1.2 MDa HA polymers. Second, in order to decrease hyaluronan synthase expression a strong AOX1 promoter in the xhasA2 gene was replaced by a weak AOX2 promoter which increased the mean MW of HA to 2.1 MDa. Finally, the MW of HA polymer was further increased to 2.5 MDa by low-temperature cultivation (26 °C) which reduced cell growth inhibition. The yield of HA production by the P. pastoris recombinant strains in 1L of fermentation culture was 0.8-1.7 g/L. PMID:24892811

  14. Identification of Intersubunit Domain Interactions within Eukaryotic Initiation Factor (eIF) 2B, the Nucleotide Exchange Factor for Translation Initiation*

    PubMed Central

    Reid, Peter J.; Mohammad-Qureshi, Sarah S.; Pavitt, Graham D.

    2012-01-01

    In eukaryotic translation initiation, eIF2B is the guanine nucleotide exchange factor (GEF) required for reactivation of the G protein eIF2 between rounds of protein synthesis initiation. eIF2B is unusually complex with five subunits (α–ϵ) necessary for GEF activity and its control by phosphorylation of eIF2α. In addition, inherited mutations in eIF2B cause a fatal leukoencephalopathy. Here we describe experiments examining domains of eIF2Bγ and ϵ that both share sequence and predicted tertiary structure similarity with a family of phospho-hexose sugar nucleotide pyrophosphorylases. Firstly, using a genetic approach, we find no evidence to support a significant role for a potential nucleotide-binding region within the pyrophosphorylase-like domain (PLD) of eIF2Bϵ for nucleotide exchange. These findings are at odds with one mechanism for nucleotide exchange proposed previously. By using a series of constructs and a co-expression and precipitation strategy, we find that the eIF2Bϵ and -γ PLDs and a shared second domain predicted to form a left-handed β helix are all critical for interprotein interactions between eIF2B subunits necessary for eIF2B complex formation. We have identified extensive interactions between the PLDs and left-handed β helix domains that form the eIF2Bγϵ subcomplex and propose a model for domain interactions between eIF2B subunits. PMID:22238343

  15. The transfer of mannose from guanosine diphosphate mannose to dolichol phosphate and protein by pig liver endoplasmic reticulum

    PubMed Central

    Richards, J. B.; Hemming, F. W.

    1972-01-01

    incubation was transferred to protein. A scheme is proposed to explain the variations with time in the production of radioactive mannolipid, mannoprotein, mannose 1-phosphate and mannose from GDP-[14C]mannose that takes account of the above observations. ATP, ADP, UTP, GDP, ADP-glucose and UDP-glucose markedly inhibited the transfer of mannose to the mannolipid. PMID:4655455

  16. Functional Characterization of a Nudix Hydrolase AtNUDX8 upon Pathogen Attack Indicates a Positive Role in Plant Immune Responses

    PubMed Central

    Fonseca, Jose Pedro; Dong, Xinnian

    2014-01-01

    Nudix hydrolases comprise a large gene family of twenty nine members in Arabidopsis, each containing a conserved motif capable of hydrolyzing specific substrates like ADP-glucose and NADH. Until now only two members of this family, AtNUDX6 and AtNUDX7, have been shown to be involved in plant immunity. RPP4 is a resistance gene from a multigene family that confers resistance to downy mildew. A time course expression profiling after Hyaloperonospora arabidopsidis inoculation in both wild-type (WT) and the rpp4 mutant was carried out to identify differentially expressed genes in RPP4-mediated resistance. AtNUDX8 was one of several differentially expressed, downregulated genes identified. A T-DNA knockout mutant (KO-nudx8) was obtained from a Salk T-DNA insertion collection, which exhibited abolished AtNUDX8 expression. The KO-nudx8 mutant was infected separately from the oomycete pathogen Hpa and the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. The mutant displayed a significantly enhanced disease susceptibility to both pathogens when compared with the WT control. We observed a small, stunted phenotype for KO-nudx8 mutant plants when grown over a 12/12 hour photoperiod but not over a 16/8 hour photoperiod. AtNUDX8 expression peaked at 8 hours after the lights were turned on and this expression was significantly repressed four-fold by salicylic acid (SA). The expression of three pathogen-responsive thioredoxins (TRX-h2, TRX-h3 and TRX-h5) were downregulated at specific time points in the KO-nudx8 mutant when compared with the WT. Furthermore, KO-nudx8 plants like the npr1 mutant, displayed SA hypersensitivity. Expression of a key SA biosynthetic gene ICS1 was repressed at specific time points in the KO-nudx8 mutant suggesting that AtNUDX8 is involved in SA signaling in plants. Similarly, NPR1 and PR1 transcript levels were also downregulated at specific time points in the KO-nudx8 mutant. This study shows that AtNUDX8 is involved in plant immunity as

  17. The Role of Cysteine Residues in Redox Regulation and Protein Stability of Arabidopsis thaliana Starch Synthase 1

    PubMed Central

    Skryhan, Katsiaryna; Cuesta-Seijo, Jose A.; Nielsen, Morten M.; Marri, Lucia; Mellor, Silas B.; Glaring, Mikkel A.; Jensen, Poul E.; Palcic, Monica M.; Blennow, Andreas

    2015-01-01

    Starch biosynthesis in Arabidopsis thaliana is strictly regulated. In leaf extracts, starch synthase 1 (AtSS1) responds to the redox potential within a physiologically relevant range. This study presents data testing two main hypotheses: 1) that specific thiol-disulfide exchange in AtSS1 influences its catalytic function 2) that each conserved Cys residue has an impact on AtSS1 catalysis. Recombinant AtSS1 versions carrying combinations of cysteine-to-serine substitutions were generated and characterized in vitro. The results demonstrate that AtSS1 is activated and deactivated by the physiological redox transmitters thioredoxin f1 (Trxf1), thioredoxin m4 (Trxm4) and the bifunctional NADPH-dependent thioredoxin reductase C (NTRC). AtSS1 displayed an activity change within the physiologically relevant redox range, with a midpoint potential equal to -306 mV, suggesting that AtSS1 is in the reduced and active form during the day with active photosynthesis. Cys164 and Cys545 were the key cysteine residues involved in regulatory disulfide formation upon oxidation. A C164S_C545S double mutant had considerably decreased redox sensitivity as compared to wild type AtSS1 (30% vs 77%). Michaelis-Menten kinetics and molecular modeling suggest that both cysteines play important roles in enzyme catalysis, namely, Cys545 is involved in ADP-glucose binding and Cys164 is involved in acceptor binding. All the other single mutants had essentially complete redox sensitivity (98–99%). In addition of being part of a redox directed activity “light switch”, reactivation tests and low heterologous expression levels indicate that specific cysteine residues might play additional roles. Specifically, Cys265 in combination with Cys164 can be involved in proper protein folding or/and stabilization of translated protein prior to its transport into the plastid. Cys442 can play an important role in enzyme stability upon oxidation. The physiological and phylogenetic relevance of these findings

  18. Changes in the activities of starch metabolism enzymes in rice grains in response to elevated CO2 concentration

    NASA Astrophysics Data System (ADS)

    Xie, Li-Yong; Lin, Er-Da; Zhao, Hong-Liang; Feng, Yong-Xiang

    2016-05-01

    The global atmospheric CO2 concentration is currently (2012) 393.1 μmol mol-1, an increase of approximately 42 % over pre-industrial levels. In order to understand the responses of metabolic enzymes to elevated CO2 concentrations, an experiment was conducted using the Free Air CO2 Enrichment (FACE )system. Two conventional japonica rice varieties ( Oryza sativa L. ssp. japonica) grown in North China, Songjing 9 and Daohuaxiang 2, were used in this study. The activities of ADPG pyrophosphorylase, soluble and granule-bound starch synthases, and soluble and granule-bound starch branching enzymes were measured in rice grains, and the effects of elevated CO2 on the amylose and protein contents of the grains were analyzed. The results showed that elevated CO2 levels significantly increased the activity of ADPG pyrophosphorylase at day 8, 24, and 40 after flower, with maximum increases of 56.67 % for Songjing 9 and 21.31 % for Daohuaxiang 2. Similarly, the activities of starch synthesis enzymes increased significantly from the day 24 after flower to the day 40 after flower, with maximum increases of 36.81 % for Songjing 9 and 66.67 % for Daohuaxiang 2 in soluble starch synthase (SSS), and 25.00 % for Songjing 9 and 36.44 % for Daohuaxiang 2 in granule-bound starch synthase (GBSS), respectively. The elevated CO2 concentration significantly increased the activity of soluble starch branching enzyme (SSBE) at day 16, 32, and 40 after flower, and also significantly increased the activity of granule-bound starch branching enzyme (GBSBE) at day 8, 32, and 40 after flower. The elevated CO2 concentration increased the peak values of enzyme activity, and the timing of the activity peaks for SSS and GBSBE were earlier in Songjing 9 than in Daohuaxiang 2. There were obvious differences in developmental stages between the two varieties of rice, which indicated that the elevated CO2 concentration increased enzyme activity expression and starch synthesis, affecting the final contents

  19. Changes in the activities of starch metabolism enzymes in rice grains in response to elevated CO2 concentration.

    PubMed

    Xie, Li-Yong; Lin, Er-Da; Zhao, Hong-Liang; Feng, Yong-Xiang

    2016-05-01

    The global atmospheric CO(2) concentration is currently (2012) 393.1 μmol mol(-1), an increase of approximately 42 % over pre-industrial levels. In order to understand the responses of metabolic enzymes to elevated CO(2) concentrations, an experiment was conducted using the Free Air CO(2) Enrichment (FACE )system. Two conventional japonica rice varieties (Oryza sativa L. ssp. japonica) grown in North China, Songjing 9 and Daohuaxiang 2, were used in this study. The activities of ADPG pyrophosphorylase, soluble and granule-bound starch synthases, and soluble and granule-bound starch branching enzymes were measured in rice grains, and the effects of elevated CO(2) on the amylose and protein contents of the grains were analyzed. The results showed that elevated CO(2) levels significantly increased the activity of ADPG pyrophosphorylase at day 8, 24, and 40 after flower, with maximum increases of 56.67 % for Songjing 9 and 21.31 % for Daohuaxiang 2. Similarly, the activities of starch synthesis enzymes increased significantly from the day 24 after flower to the day 40 after flower, with maximum increases of 36.81 % for Songjing 9 and 66.67 % for Daohuaxiang 2 in soluble starch synthase (SSS), and 25.00 % for Songjing 9 and 36.44 % for Daohuaxiang 2 in granule-bound starch synthase (GBSS), respectively. The elevated CO(2) concentration significantly increased the activity of soluble starch branching enzyme (SSBE) at day 16, 32, and 40 after flower, and also significantly increased the activity of granule-bound starch branching enzyme (GBSBE) at day 8, 32, and 40 after flower. The elevated CO(2) concentration increased the peak values of enzyme activity, and the timing of the activity peaks for SSS and GBSBE were earlier in Songjing 9 than in Daohuaxiang 2. There were obvious differences in developmental stages between the two varieties of rice, which indicated that the elevated CO(2) concentration increased enzyme activity expression and starch synthesis, affecting the

  20. Genes involved in the synthesis of the exopolysaccharide methanolan by the obligate methylotroph Methylobacillus sp strain 12S.

    PubMed

    Yoshida, Takako; Ayabe, Yuko; Yasunaga, Masaaki; Usami, Yusuke; Habe, Hiroshi; Nojiri, Hideaki; Omori, Toshio

    2003-02-01

    Methylobacillus sp. strain 12S produces an exopolysaccharide (EPS), methanolan, composed of glucose, mannose and galactose. Twenty-four ORFs flanking a Tn5 insertion site in an EPS-deficient mutant were identified, and 21 genes (epsCBAKLDEFGHIJMNOPQRSTU) were predicted to participate in methanolan synthesis on the basis of the features of the primary sequence. Gene disruption analyses revealed that epsABCEFGIJNOP and epsR are required for methanolan synthesis, whereas epsKD and epsH are not essential. EpsFG and EpsE showed homology with Wzc (chain length regulator) and Wza (export protein) of group 1 capsule-producing Escherichia coli, suggesting that methanolan was synthesized via a Wzy-like biosynthesis system. This possibility was supported by the fact that the putative hydropathy profiles of EpsH and EpsM were similar to those of Wzx and Wzy, which are also involved in the flipping of the repeating unit in the cytoplasmic membrane and the polymerization of the capsule in the Wzy-dependent system. EpsBJNOP and EpsR are probably glycosyltransferases involved in the synthesis of the repeating unit onto the lipid carrier. In particular, EpsB appeared to catalyse the initial transfer of the glucose moiety. On the basis of their predicted location in the cells, it is proposed that EpsI and EpsL are involved in methanolan export to the cell surface. E. coli strains expressing EpsQ, EpsS and EpsT showed enhanced activities of GDP-mannose pyrophosphorylase, UDP-galactose 4-epimerase and UDP-glucose pyrophosphorylase, respectively, revealing that they were responsible for the production of the activated compositional sugars of methanolan. EpsU contains a conserved a lytic transglycosylase motif, indicating that it could participate in the degradation of polysaccharides. EpsA and EpsK, which have conserved DNA-binding and cAMP-binding motifs, respectively, were deduced to be transcriptional regulators. In particular, EpsA seems to positively regulate the transcription of

  1. The functional O-mannose glycan on α-dystroglycan contains a phospho-ribitol primed for matriglycan addition.

    PubMed

    Praissman, Jeremy L; Willer, Tobias; Sheikh, M Osman; Toi, Ants; Chitayat, David; Lin, Yung-Yao; Lee, Hane; Stalnaker, Stephanie H; Wang, Shuo; Prabhakar, Pradeep Kumar; Nelson, Stanley F; Stemple, Derek L; Moore, Steven A; Moremen, Kelley W; Campbell, Kevin P; Wells, Lance

    2016-01-01

    Multiple glycosyltransferases are essential for the proper modification of alpha-dystroglycan, as mutations in the encoding genes cause congenital/limb-girdle muscular dystrophies. Here we elucidate further the structure of an O-mannose-initiated glycan on alpha-dystroglycan that is required to generate its extracellular matrix-binding polysaccharide. This functional glycan contains a novel ribitol structure that links a phosphotrisaccharide to xylose. ISPD is a CDP-ribitol (ribose) pyrophosphorylase that generates the reduced sugar nucleotide for the insertion of ribitol in a phosphodiester linkage to the glycoprotein. TMEM5 is a UDP-xylosyl transferase that elaborates the structure. We demonstrate in a zebrafish model as well as in a human patient that defects in TMEM5 result in muscular dystrophy in combination with abnormal brain development. Thus, we propose a novel structure-a ribitol in a phosphodiester linkage-for the moiety on which TMEM5, B4GAT1, and LARGE act to generate the functional receptor for ECM proteins having LG domains. PMID:27130732

  2. A new strategy to enhance gellan production by two-stage culture in Sphingomonas paucimobilis.

    PubMed

    Zhu, Guilan; Sheng, Long; Tong, Qunyi

    2013-10-15

    The effects of different initial sucrose concentrations and temperatures on gellan biosynthesis by Sphingomonas paucimobilis ATCC 31461 were investigated. Lower sucrose concentrations and higher temperatures were favorable for cell growth. Higher sucrose concentrations and lower temperatures promoted gellan production but retarded cell growth. Based on these results, a two-stage culture strategy was developed to improve gellan production. During the first 24 h, S. paucimobilis was cultured in a pulse fed-batch mode with an initial sucrose concentration 10 g/L. Ten grams per liter of sucrose were added at 12 h and 24 h, and the temperature was controlled at 33 °C. Batch culture was performed, and the temperature was reduced to 28 °C to achieve a high gellan accumulation. The two-stage culture strategy achieved the highest gellan production (22.61 g/L) at 60 h that was 35.71% higher than the result of the best conventional batch operation (16.66 g/L). Meanwhile, high gellan yield was related to high UDPG-pyrophosphorylase activity and glucosyltransferase activity. PMID:23987418

  3. Starch Biosynthesis in Guard Cells But Not in Mesophyll Cells Is Involved in CO2-Induced Stomatal Closing.

    PubMed

    Azoulay-Shemer, Tamar; Bagheri, Andisheh; Wang, Cun; Palomares, Axxell; Stephan, Aaron B; Kunz, Hans-Henning; Schroeder, Julian I

    2016-06-01

    Starch metabolism is involved in stomatal movement regulation. However, it remains unknown whether starch-deficient mutants affect CO2-induced stomatal closing and whether starch biosynthesis in guard cells and/or mesophyll cells is rate limiting for high CO2-induced stomatal closing. Stomatal responses to [CO2] shifts and CO2 assimilation rates were compared in Arabidopsis (Arabidopsis thaliana) mutants that were either starch deficient in all plant tissues (ADP-Glc-pyrophosphorylase [ADGase]) or retain starch accumulation in guard cells but are starch deficient in mesophyll cells (plastidial phosphoglucose isomerase [pPGI]). ADGase mutants exhibited impaired CO2-induced stomatal closure, but pPGI mutants did not, showing that starch biosynthesis in guard cells but not mesophyll functions in CO2-induced stomatal closing. Nevertheless, starch-deficient ADGase mutant alleles exhibited partial CO2 responses, pointing toward a starch biosynthesis-independent component of the response that is likely mediated by anion channels. Furthermore, whole-leaf CO2 assimilation rates of both ADGase and pPGI mutants were lower upon shifts to high [CO2], but only ADGase mutants caused impairments in CO2-induced stomatal closing. These genetic analyses determine the roles of starch biosynthesis for high CO2-induced stomatal closing. PMID:27208296

  4. Influence of fermentation conditions on polysaccharide production and the activities of enzymes involved in the polysaccharide synthesis of Cordyceps militaris.

    PubMed

    Zhu, Zhen-Yuan; Liu, Xiao-Cui; Dong, Feng-Ying; Guo, Ming-Zhu; Wang, Xiao-Ting; Wang, Zheng; Zhang, Yong-Min

    2016-05-01

    The influence of different fermentation conditions on intracellular polysaccharide (IPS) production and activities of the phosphoglucomutase (PGM), UDPG-pyrophosphorylase (UGP), phosphoglucose isomerase (PGI), UDPG-dehydrogenase (UGD), and glucokinase (GK) implicated in metabolite synthesis in Cordyceps militaris was evaluated. The highest IPS production (327.57 ± 6.27 mg/100 mL) was obtained when the strain was grown in the optimal medium containing glucose (40 g · L(-1)), beef extract (10 g · L(-1)), and CaCO3 (0.5 g · L(-1)), and the initial pH and temperature were 7 and 25 °C, respectively. The activities of PGM, UGP, and PGI were proved to be influenced by the fermentation conditions. A strong correlation between the activities of these enzymes and the production of IPS was found. The transcription level of the pgm gene (encoding PGM) was 1.049 times and 1.467 times compared to the ugp gene and pgi gene (encoding UGP and PGI), respectively, in the optimal culture medium. This result indicated that PGM might be the highly key enzyme to regulate the biosynthesis of IPS of C. militaris in a liquid-submerged culture. Our study might be helpful for further research on the pathway of polysaccharide biosynthesis aimed to improve the IPS production of C. militaris. PMID:26685672

  5. Review: Mechanisms of ammonium toxicity and the quest for tolerance.

    PubMed

    Esteban, Raquel; Ariz, Idoia; Cruz, Cristina; Moran, Jose Fernando

    2016-07-01

    Ammonium sensitivity of plants is a worldwide problem, constraining crop production. Prolonged application of ammonium as the sole nitrogen source may result in physiological and morphological disorders that lead to decreased plant growth and toxicity. The main causes of ammonium toxicity/tolerance described until now include high ammonium assimilation by plants and/or low sensitivity to external pH acidification. The various ammonium transport-related components, especially the non-electrogenic influx of NH3 (related to the depletion of (15)N) and the electrogenic influx of NH4(+), may contribute to ammonium accumulation, and therefore to NH3 toxicity. However, this accumulation may be influenced by increasing K(+) concentration in the root medium. Recently, new insights have been provided by "omics" studies, leading to a suggested involvement of GDP mannose-pyrophosphorylase in the response pathways of NH4(+) stress. In this review, we highlight the cross-talk signaling between nitrate, auxins and NO, and the importance of the connection of the plants' urea cycle to metabolism of polyamines. Overall, the tolerance and amelioration of ammonium toxicity are outlined to improve the yield of ammonium-grown plants. This review identifies future directions of research, focusing on the putative importance of aquaporins in ammonium influx, and on genes involved in ammonium sensitivity and tolerance. PMID:27181951

  6. The functional O-mannose glycan on α-dystroglycan contains a phospho-ribitol primed for matriglycan addition

    PubMed Central

    Praissman, Jeremy L; Willer, Tobias; Sheikh, M Osman; Toi, Ants; Chitayat, David; Lin, Yung-Yao; Lee, Hane; Stalnaker, Stephanie H; Wang, Shuo; Prabhakar, Pradeep Kumar; Nelson, Stanley F; Stemple, Derek L; Moore, Steven A; Moremen, Kelley W; Campbell, Kevin P; Wells, Lance

    2016-01-01

    Multiple glycosyltransferases are essential for the proper modification of alpha-dystroglycan, as mutations in the encoding genes cause congenital/limb-girdle muscular dystrophies. Here we elucidate further the structure of an O-mannose-initiated glycan on alpha-dystroglycan that is required to generate its extracellular matrix-binding polysaccharide. This functional glycan contains a novel ribitol structure that links a phosphotrisaccharide to xylose. ISPD is a CDP-ribitol (ribose) pyrophosphorylase that generates the reduced sugar nucleotide for the insertion of ribitol in a phosphodiester linkage to the glycoprotein. TMEM5 is a UDP-xylosyl transferase that elaborates the structure. We demonstrate in a zebrafish model as well as in a human patient that defects in TMEM5 result in muscular dystrophy in combination with abnormal brain development. Thus, we propose a novel structure—a ribitol in a phosphodiester linkage—for the moiety on which TMEM5, B4GAT1, and LARGE act to generate the functional receptor for ECM proteins having LG domains. DOI: http://dx.doi.org/10.7554/eLife.14473.001 PMID:27130732

  7. Ethylene Response Factor TERF1, Regulated by ETHYLENE-INSENSITIVE3-like Factors, Functions in Reactive Oxygen Species (ROS) Scavenging in Tobacco (Nicotiana tabacum L.).

    PubMed

    Zhang, Hongbo; Li, Ang; Zhang, Zhijin; Huang, Zejun; Lu, Pingli; Zhang, Dingyu; Liu, Xinmin; Zhang, Zhong-Feng; Huang, Rongfeng

    2016-01-01

    The phytohormone ethylene plays a crucial role in the production and accumulation of reactive oxygen species (ROS) in plants under stress conditions. Ethylene response factors (ERFs) are important ethylene-signaling regulators functioning in plant defense responses against biotic and abiotic stresses. However, the roles of ERFs during plant adapting to ROS stress have not yet been well documented. Our studies previously reported that a tomato ERF transcription factor TERF1 functions in the regulation of plant ethylene responses and stress tolerance. Here, we report our findings regarding the roles of TERF1 in ROS scavenging. In this study, we revealed that the transcription of TERF1 is regulated by upstream EIN3-like (EIN3, ethylene-insensitive 3) regulators LeEIL3 and LeEIL4 in tomato (Solanum lycopersicum), and is also inducible by exogenous applied ROS-generating reagents. Ectopic expression of TERF1 in tobacco promoted the expression of genes involved in oxidative stress responses, including carbonic anhydrase functioning in hypersensitive defense, catalase and glutathione peroxidase catalyzing oxidative reactions, and GDP-D-mannose pyrophosphorylase functioning in ascorbic acid biosynthesis, reduced the ROS content induced by ethylene treatment, and enhanced stress tolerance of tobacco seedlings to hydrogen peroxide (H2O2). Cumulatively, these findings suggest that TERF1 is an ethylene inducible factor regulating ROS scavenging during stress responses. PMID:27435661

  8. Use of differential fluorescence induction and optical trapping to isolate environmentally induced genes.

    PubMed

    Allaway, D; Schofield, N A; Leonard, M E; Gilardoni, L; Finan, T M; Poole, P S

    2001-06-01

    The techniques of differential fluorescence induction (DFI) and optical trapping (OT) have been combined to allow the identification of environmentally induced genes in single bacterial cells. Designated DFI-OT, this technique allows the in situ isolation of genes driving the expression of green fluorescent protein (Gfp) using temporal and spatial criteria. A series of plasmid-based promoter probe vectors (pOT) was developed for the construction of random genomic libraries that are linked to gfpUV or egfp. Bacteria that do not express Gfp on laboratory medium (i.e. non-fluorescent) were inoculated into the environment, and induced genes were detected with a combined fluorescence/optical trapping microscope. Using this selection strategy, rhizosphere-induced genes with homology to thiamine pyrophosphorylase (thiE) and cyclic glucan synthase (ndvB) were isolated. Other genes were expressed late in the stationary phase or as a consequence of surface-dependent growth, including fixND and metX, and a putative ABC transporter of putrescine. This strategy provides a unique ability to combine spatial, temporal and physical information to identify environmental regulation of bacterial gene expression. PMID:11472504

  9. Improved polysaccharide production in a submerged culture of Ganoderma lucidum by the heterologous expression of Vitreoscilla hemoglobin gene.

    PubMed

    Li, Huan-Jun; Zhang, De-Huai; Yue, Tong-Hui; Jiang, Lu-Xi; Yu, Xuya; Zhao, Peng; Li, Tao; Xu, Jun-Wei

    2016-01-10

    Expression of Vitreoscilla hemoglobin (VHb) gene was used to improve polysaccharide production in Ganoderma lucidum. The VHb gene, vgb, under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase gene promoter was introduced into G. lucidum. The activity of expressed VHb was confirmed by the observation of VHb specific CO-difference spectrum with a maximal absorption at 419 nm for the transformant. The effects of VHb expression on intracellular polysaccharide (IPS) content, extracellular polysaccharide (EPS) production and transcription levels of three genes encoding the enzymes involved in polysaccharide biosynthesis, including phosphoglucomutase (PGM), uridine diphosphate glucose pyrophosphorylase (UGP), and β-1,3-glucan synthase (GLS), were investigated. The maximum IPS content and EPS production in the vgb-bearing G. lucidum were 26.4 mg/100mg dry weight and 0.83 g/L, respectively, which were higher by 30.5% and 88.2% than those of the wild-type strain. The transcription levels of PGM, UGP and GLS were up-regulated by 1.51-, 1.55- and 3.83-fold, respectively, in the vgb-bearing G. lucidum. This work highlights the potential of VHb to enhance G. lucidum polysaccharide production by large scale fermentation. PMID:26603122

  10. One-pot multienzyme synthesis of Lewis x and sialyl Lewis x antigens

    PubMed Central

    Yu, Hai; Lau, Kam; Li, Yanhong; Sugiarto, Go; Chen, Xi

    2012-01-01

    L-Fucose has been found abundantly in human milk oligosaccharides, bacterial lipopolysaccharides, glycolipids, and many N- and O-linked glycans produced by mammalian cells. Fucose-containing carbohydrates have important biological functions. Alterations in the expression of fucosylated oligosaccharides have been observed in several pathological processes such as cancer and atherosclerosis. Chemical formation of fucosidic bonds is challenging due to its acid lability. Enzymatic construction of fucosidic bonds by fucosyltransferases is highly efficient and selective but requires the expensive sugar nucleotide donor guanosine 5′- diphosphate-L-fucose (GDP-Fuc). Here, we describe a protocol for applying a one-pot three-enzyme system in synthesizing structurally defined fucose-containing oligosaccharides from free L-fucose. In this system, GDP-Fuc is generated from L-fucose, adenosine 5′-triphosphate (ATP), and guanosine 5′-triphosphate (GTP) by a bifunctional L-fucokinase/GDP-fucose pyrophosphorylase (FKP). An inorganic pyrophosphatase (PpA) is used to degrade the by-product pyrophosphate (PPi) to drive the reaction towards the formation of GDP-Fuc. In situ generated GDP-Fuc is then used by a suitable fucosyltransferase for the formation of fucosides. The three-enzyme reactions are carried out in one pot without the need for high cost sugar nucleotide or isolation of intermediates. The time for the synthesis is 4–24 hours. Purification and characterization of products can be completed in 2–3 days. PMID:25000293

  11. Inactivation of the UGPase1 gene causes genic male sterility and endosperm chalkiness in rice (Oryza sativa L.)

    PubMed Central

    Woo, Mi-Ok; Ham, Tae-Ho; Ji, Hyeon-So; Choi, Min-Seon; Jiang, Wenzhu; Chu, Sang-Ho; Piao, Rihua; Chin, Joong-Hyoun; Kim, Jung-A; Park, Bong Soo; Seo, Hak Soo; Jwa, Nam-Soo; McCouch, Susan; Koh, Hee-Jong

    2008-01-01

    A rice genic male-sterility gene ms-h is recessive and has a pleiotropic effect on the chalky endosperm. After fine mapping, nucleotide sequencing analysis of the ms-h gene revealed a single nucleotide substitution at the 3′-splice junction of the 14th intron of the UDP-glucose pyrophosphorylase 1 (UGPase1; EC2.7.7.9) gene, which causes the expression of two mature transcripts with abnormal sizes caused by the aberrant splicing. An in vitro functional assay showed that both proteins encoded by the two abnormal transcripts have no UGPase activity. The suppression of UGPase by the introduction of a UGPase1-RNAi construct in wild-type plants nearly eliminated seed set because of the male defect, with developmental retardation similar to the ms-h mutant phenotype, whereas overexpression of UGPase1 in ms-h mutant plants restored male fertility and the transformants produced T1 seeds that segregated into normal and chalky endosperms. In addition, both phenotypes were co-segregated with the UGPase1 transgene in segregating T1 plants, which demonstrates that UGPase1 has functional roles in both male sterility and the development of a chalky endosperm. Our results suggest that UGPase1 plays a key role in pollen development as well as seed carbohydrate metabolism. PMID:18182026

  12. Donor Substrate Regeneration for Efficient Synthesis of Globotetraose and Isoglobotetraose

    PubMed Central

    Shao, Jun; Zhang, Jianbo; Kowal, Przemyslaw; Wang, Peng George

    2002-01-01

    Here we describe the efficient synthesis of two oligosaccharide moieties of human glycosphingolipids, globotetraose (GalNAcβ1→3Galα1→4Galβ1→4Glc) and isoglobotetraose (GalNAcβ1→3Galα1→3Galβ1→4Glc), with in situ enzymatic regeneration of UDP-N-acetylgalactosamine (UDP-GalNAc). We demonstrate that the recombinant β-1,3-N-acetylgalactosaminyltransferase from Haemophilus influenzae strain Rd can transfer N-acetylgalactosamine to a wide range of acceptor substrates with a terminal galactose residue. The donor substrate UDP-GalNAc can be regenerated by a six-enzyme reaction cycle consisting of phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, phosphate acetyltransferase, pyruvate kinase, and inorganic pyrophosphatase from Escherichia coli, as well as UDP-N-acetylglucosamine C4 epimerase from Plesiomonas shigelloides. All these enzymes were overexpressed in E. coli with six-histidine tags and were purified by one-step nickel-nitrilotriacetic acid affinity chromatography. Multiple-enzyme synthesis of globotetraose or isoglobotetraose with the purified enzymes was achieved with relatively high yields. PMID:12406759

  13. Enzyme Activities of Starch and Sucrose Pathways and Growth of Apical and Basal Maize Kernels 1

    PubMed Central

    Ou-Lee, Tsai-Mei; Setter, Tim Lloyd

    1985-01-01

    Apical kernels of maize (Zea mays L.) ears have smaller size and lower growth rates than basal kernels. To improve our understanding of this difference, the developmental patterns of starch-synthesis-pathway enzyme activities and accumulation of sugars and starch was determined in apical- and basal-kernel endosperm of greenhouse-grown maize (cultivar Cornell 175) plants. Plants were synchronously pollinated, kernels were sampled from apical and basal ear positions throughout kernel development, and enzyme activities were measured in crude preparations. Several factors were correlated with the higher dry matter accumulation rate and larger mature kernel size of basal-kernel endosperm. During the period of cell expansion (7 to 19 days after pollination), the activity of insoluble (acid) invertase and sucose concentration in endosperm of basal kernels exceeded that in apical kernels. Soluble (alkaline) invertase was also high during this stage but was the same in endosperm of basal and apical kernels, while glucose concentration was higher in apical-kernel endosperm. During the period of maximal starch synthesis, the activities of sucrose synthase, ADP-Glc-pyrophosphorylase, and insoluble (granule-bound) ADP-Glc-starch synthase were higher in endosperm of basal than apical kernels. Soluble ADP-Glc-starch synthase, which was maximal during the early stage before starch accumulated, was the same in endosperm from apical and basal kernels. It appeared that differences in metabolic potential between apical and basal kernels were established at an early stage in kernel development. PMID:16664503

  14. Identification and subcellular localization of starch-metabolizing enzymes in the green alga Dunaliella marina.

    PubMed

    Kombrink, E; Wöber, G

    1980-07-01

    Enzymes of starch synthesis and degradation were identified in crude extracts of the unicellular green alga Dunaliella marina (Volvocales). By polyacrylamide gel electrophoresis and specific staining for enzyme activities, 4 multiple forms of starch synthase, 2 amylases, and at least 2 forms of α-glucan phosphorylase were visible. Using specific α-glucans incorporated into the gel before electrophoresis we have tentatively correlated α-amylase and β-amylase with both hydrolytic activities. The activities of α-glucan phosphorylase and amylase(s) were measured quantitatively in crude extracts, and the concomitant action of α-glucan phosphorylase and amylase(s) was found to account for the fastest rate of starch mobilization observed in vivo. Isolated chloroplasts retained both typical plastid marker enzymes and ADPglucose pyrophosphorylase, starch synthase, amylase(s), and α-glucan phosphorylase to a similar percentage. Gel electrophoretic analysis followed by staining for enzyme activity of a stromal fraction resulted in a pattern of multiple forms of starch-metabolizing enzymes analogous to that found in a crude extract. We interpret the combined data as indicating the exclusive location in vivo of starch-metabolizing enzymes in chloroplasts of D. marina. PMID:24306243

  15. Biochemical and genetic analysis of the effects of amylose-extender mutation in rice endosperm.

    PubMed

    Nishi, A; Nakamura, Y; Tanaka, N; Satoh, H

    2001-10-01

    Biochemical analysis of amylose-extender (ae) mutant of rice (Oryza sativa) revealed that the mutation in the gene for starch-branching enzyme IIb (BEIIb) specifically altered the structure of amylopectin in the endosperm by reducing short chains with degree of polymerization of 17 or less, with the greatest decrease in chains with degree of polymerization of 8 to 12. The extent of such change was correlated with the gelatinization properties of the starch granules, as determined in terms of solubility in urea solution. The ae mutation caused a dramatic reduction in the activity of BEIIb. The activity of soluble starch synthase I (SSI) in the ae mutant was significantly lower than in the wild type, suggesting that the mutation had a pleiotropic effect on the SSI activity. In contrast, the activities of BEI, BEIIa, ADP-Glc pyrophosphorylase, isoamylase, isoamylase, pullulanase, and Suc synthase were not affected by the mutation. Therefore, it is stressed that the function of BEIIb cannot be complemented by BEIIa and BEI. These results strongly suggest that BEIIb plays a specific role in the transfer of short chains, which might then be extended by SS to form the A and B(1) chains of amylopectin cluster in rice endosperm. PMID:11598221

  16. Towards a better understanding of the metabolic system for amylopectin biosynthesis in plants: rice endosperm as a model tissue.

    PubMed

    Nakamura, Yasunori

    2002-07-01

    Starch is made up of amylose (linear alpha-1,4-polyglucans) and amylopectin (alpha-1,6-branched polyglucans). Amylopectin has a distinct fine structure called multiple cluster structure and is synthesized by multiple subunits or isoforms of four classes of enzymes: ADPglucose pyrophosphorylase, soluble starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE). In the present paper, based on analyses of mutants and transgenic lines of rice in which each enzyme activity is affected, the contribution of the individual isoform to the fine structure of amylopectin in rice endosperm is evaluated, and a new model referred to as the "two-step branching and improper branch clearing model" is proposed to explain how amylopectin is synthesized. The model emphasizes that two sets of reactions, alpha-1,6-branch formation and the subsequent alpha-1,4-chain elongation, are catalyzed by distinct BE and SS isoforms, respectively, are fundamental to the construction of the cluster structure. The model also assesses the role of DBE, namely isoamylase or in addition pullulanase, to remove unnecessary alpha-1,6-glucosidic linkages that are occasionally formed at improper positions apart from two densely branched regions of the cluster. PMID:12154134

  17. Sucrose metabolism gene families and their biological functions

    PubMed Central

    Jiang, Shu-Ye; Chi, Yun-Hua; Wang, Ji-Zhou; Zhou, Jun-Xia; Cheng, Yan-Song; Zhang, Bao-Lan; Ma, Ali; Vanitha, Jeevanandam; Ramachandran, Srinivasan

    2015-01-01

    Sucrose, as the main product of photosynthesis, plays crucial roles in plant development. Although studies on general metabolism pathway were well documented, less information is available on the genome-wide identification of these genes, their expansion and evolutionary history as well as their biological functions. We focused on four sucrose metabolism related gene families including sucrose synthase, sucrose phosphate synthase, sucrose phosphate phosphatase and UDP-glucose pyrophosphorylase. These gene families exhibited different expansion and evolutionary history as their host genomes experienced differentiated rates of the whole genome duplication, tandem and segmental duplication, or mobile element mediated gene gain and loss. They were evolutionarily conserved under purifying selection among species and expression divergence played important roles for gene survival after expansion. However, we have detected recent positive selection during intra-species divergence. Overexpression of 15 sorghum genes in Arabidopsis revealed their roles in biomass accumulation, flowering time control, seed germination and response to high salinity and sugar stresses. Our studies uncovered the molecular mechanisms of gene expansion and evolution and also provided new insight into the role of positive selection in intra-species divergence. Overexpression data revealed novel biological functions of these genes in flowering time control and seed germination under normal and stress conditions. PMID:26616172

  18. GMPPB-Associated Dystroglycanopathy: Emerging Common Variants with Phenotype Correlation.

    PubMed

    Jensen, Braden S; Willer, Tobias; Saade, Dimah N; Cox, Mary O; Mozaffar, Tahseen; Scavina, Mena; Stefans, Vikki A; Winder, Thomas L; Campbell, Kevin P; Moore, Steven A; Mathews, Katherine D

    2015-12-01

    Mutations in GDP-mannose pyrophosphorylase B (GMPPB), a catalyst for the formation of the sugar donor GDP-mannose, were recently identified as a cause of muscular dystrophy resulting from abnormal glycosylation of α-dystroglycan. In this series, we report nine unrelated individuals with GMPPB-associated dystroglycanopathy. The most mildly affected subject has normal strength at 25 years, whereas three severely affected children presented in infancy with intellectual disability and epilepsy. Muscle biopsies of all subjects are dystrophic with abnormal immunostaining for glycosylated α-dystroglycan. This cohort, together with previously published cases, allows preliminary genotype-phenotype correlations to be made for the emerging GMPPB common variants c.79G>C (p.D27H) and c.860G>A (p.R287Q). We observe that c.79G>C (p.D27H) is associated with a mild limb-girdle muscular dystrophy phenotype, whereas c.860G>A (p.R287Q) is associated with a relatively severe congenital muscular dystrophy typically involving brain development. Sixty-six percent of GMPPB families to date have one of these common variants. PMID:26310427

  19. Evidences for Chlorogenic Acid — A Major Endogenous Polyphenol Involved in Regulation of Ripening and Senescence of Apple Fruit

    PubMed Central

    Xi, Yu; Cheng, Dai; Zeng, Xiangquan; Cao, Jiankang; Jiang, Weibo

    2016-01-01

    To learn how the endogenous polyphenols may play a role in fruit ripening and senescence, apple pulp discs were used as a model to study the influences of chlorogenic acid (CHA, a major polyphenol in apple pulp) on fruit ripening and senescence. Apple (‘Golden Delicious’) pulp discs prepared from pre-climacteric fruit were treated with 50 mg L-1 CHA and incubated in flasks with 10 mM MES buffer (pH 6.0, 11% sorbitol). Compared to the control samples, treatment with CHA significantly reduced ethylene production and respiration rate, and enhanced levels of firmness and soluble solids content of the pulp discs during incubation at 25°C. These results suggested that CHA could retard senescence of the apple pulp discs. Proteomics analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry (MALDI-TOF/TOF) revealed that the expressions of several key proteins correlated to fruit ripening and senescence were affected by the treatment with CHA. Further study showed that treating the pulp discs with CHA remarkably reduced levels of lipoxygenase, β-galactosidase, NADP-malic enzyme, and enzymatic activities of lipoxygenase and UDP-glucose pyrophosphorylase, all of which are known as promoters of fruit ripening and senescence. These results could provide new insights into the functions of endogenous phenolic compounds in fruit ripening and senescence. PMID:26756813

  20. Examination of the mechanism of sucrose synthetase by positional isotope exchange

    SciTech Connect

    Singh, A.N.; Hester, L.S.; Raushel, F.M.

    1987-02-25

    The mechanism of the sucrose synthetase reaction has been probed by the technique of positional isotope exchange. (beta-/sup 18/O/sub 2/, alpha beta-/sup 18/O)UDP-Glc has been synthesized starting from oxygen-18-labeled phosphate and the combined activities of carbamate kinase, hexokinase, phosphoglucomutase, and uridine diphosphoglucose pyrophosphorylase. The oxygen-18 at the alpha beta-bridge position of the labeled UDP-Glc has been shown to cause a 0.014 ppm upfield chemical shift in the 31P NMR spectrum of both the alpha- and beta-phosphorus atoms in UDP-Glc relative to the unlabeled compound. The chemical shift induced by each of the beta-nonbridge oxygen-18 atoms was 0.030 ppm. Incubation of (beta-/sup 18/O/sub 2/, alpha beta-/sup 18/O)UDP-Glc with sucrose synthetase in the presence and absence of 2,5-anhydromannitol did not result in any significant exchange of an oxygen-18 from the beta-nonbridge position to the anomeric oxygen of the glucose moiety. It can thus be concluded that either sucrose synthetase does not catalyze the cleavage of the scissile carbon-oxygen bond of UDP-Glc in the absence of fructose or, alternatively, the beta-phosphoryl group of the newly formed UDP is rotationally immobilized.

  1. Starch Biosynthesis in Guard Cells But Not in Mesophyll Cells Is Involved in CO2-Induced Stomatal Closing1[OPEN

    PubMed Central

    Stephan, Aaron B.; Schroeder, Julian I.

    2016-01-01

    Starch metabolism is involved in stomatal movement regulation. However, it remains unknown whether starch-deficient mutants affect CO2-induced stomatal closing and whether starch biosynthesis in guard cells and/or mesophyll cells is rate limiting for high CO2-induced stomatal closing. Stomatal responses to [CO2] shifts and CO2 assimilation rates were compared in Arabidopsis (Arabidopsis thaliana) mutants that were either starch deficient in all plant tissues (ADP-Glc-pyrophosphorylase [ADGase]) or retain starch accumulation in guard cells but are starch deficient in mesophyll cells (plastidial phosphoglucose isomerase [pPGI]). ADGase mutants exhibited impaired CO2-induced stomatal closure, but pPGI mutants did not, showing that starch biosynthesis in guard cells but not mesophyll functions in CO2-induced stomatal closing. Nevertheless, starch-deficient ADGase mutant alleles exhibited partial CO2 responses, pointing toward a starch biosynthesis-independent component of the response that is likely mediated by anion channels. Furthermore, whole-leaf CO2 assimilation rates of both ADGase and pPGI mutants were lower upon shifts to high [CO2], but only ADGase mutants caused impairments in CO2-induced stomatal closing. These genetic analyses determine the roles of starch biosynthesis for high CO2-induced stomatal closing. PMID:27208296

  2. Effects of elevated ammonium on glycosylation gene expression in CHO cells.

    PubMed

    Chen, Peifeng; Harcum, Sarah W

    2006-03-01

    The negative effects of ammonium on recombinant protein productivity and glycosylation have been well documented, but the interaction of ammonium on glycosylation genes has not been completely elucidated. In this study, the effects of elevated ammonium on 12 glycosylation related genes in Chinese hamster ovary cells were evaluated by quantitative real time reverse transcriptase polymerase chain reaction. Numerous cytosol and endoplasmic reticulum (ER) localized genes associated with early glycosylation steps were insensitive to the ammonium condition. The initial expression of uridine diphosphate (UDP)-galactose transporter was higher for the ammonium-treated culture, while the initial expressions of cytosine monophosphate (CMP)-sialic acid transporter, beta(1,4)-galactosyltransferase, and UDP-glucose pyrophosphorylase were higher for the control culture. alpha(2,3)-sialyltransferase was observed to have lower expression level under the elevated ammonium condition compared to the control culture. This study indicates that galactosylation and sialylation inhibition is mainly due to decreased gene expression of galactosyltransferase, sialyltransferase, and CMP-sialic acid transporter and not due to sialidase. These unbalanced initial glycosylation and branching steps can explain the higher molecular heterogeneity under ammonium stress. Moreover, this study indicates that elevated ammonium has limited effects on the glycosylation genes associated with the ER and cytosol compared to the genes associated with the Golgi. PMID:16380282

  3. Molecular Typing of IberoAmerican Cryptococcus neoformans Isolates

    PubMed Central

    Castañeda, Alexandra; Jackson, Stuart; Huynh, Matthew; Castañeda, Elizabeth

    2003-01-01

    A network was established to acquire basic knowledge of Cryptococcus neoformans in IberoAmerican countries. To this effect, 340 clinical, veterinary, and environmental isolates from Argentina, Brazil, Chile, Colombia, Mexico, Peru, Venezuela, Guatemala, and Spain were typed by using M13 polymerase chain reaction-fingerprinting and orotidine monophosphate pyrophosphorylase (URA5) gene restriction fragment length polymorphsm analysis with HhaI and Sau96I in a double digest. Both techniques grouped all isolates into eight previously established molecular types. The majority of the isolates, 68.2% (n=232), were VNI (var. grubii, serotype A), which accords with the fact that this variety causes most human cryptococcal infections worldwide. A smaller proportion, 5.6% (n=19), were VNII (var. grubii, serotype A); 4.1% (n=14), VNIII (AD hybrid), with 9 isolates having a polymorphism in the URA5 gene; 1.8% (n=6), VNIV (var. neoformans, serotype D); 3.5% (n=12), VGI; 6.2% (n=21), VGII; 9.1% (n=31), VGIII, and 1.5% (n=5) VGIV, with all four VG types containing var. gattii serotypes B and C isolates. PMID:12603989

  4. Starch Synthesis in Arabidopsis Is Achieved by Spatial Cotranscription of Core Starch Metabolism Genes1[W][OA

    PubMed Central

    Tsai, Huang-Lung; Lue, Wei-Ling; Lu, Kuan-Jen; Hsieh, Ming-Hsiun; Wang, Shue-Mei; Chen, Jychian

    2009-01-01

    Starch synthesis and degradation require the participation of many enzymes, occur in both photosynthetic and nonphotosynthetic tissues, and are subject to environmental and developmental regulation. We examine the distribution of starch in vegetative tissues of Arabidopsis (Arabidopsis thaliana) and the expression of genes encoding core enzymes for starch synthesis. Starch is accumulated in plastids of epidermal, mesophyll, vascular, and root cap cells but not in root proper cells. We also identify cells that can synthesize starch heterotrophically in albino mutants. Starch synthesis in leaves is regulated by developmental stage and light. Expression of gene promoter-β-glucuronidase fusion constructs in transgenic seedlings shows that starch synthesis genes are transcriptionally active in cells with starch synthesis and are inactive in root proper cells except the plastidial phosphoglucose isomerase. In addition, ADG2 (for ADPG PYROPHOSPHORYLASE2) is not required for starch synthesis in root cap cells. Expression profile analysis reveals that starch metabolism genes can be clustered into two sets based on their tissue-specific expression patterns. Starch distribution and expression pattern of core starch synthesis genes are common in Arabidopsis and rice (Oryza sativa), suggesting that the regulatory mechanism for starch metabolism genes may be conserved evolutionarily. We conclude that starch synthesis in Arabidopsis is achieved by spatial coexpression of core starch metabolism genes regulated by their promoter activities and is fine-tuned by cell-specific endogenous and environmental controls. PMID:19759345

  5. Evidences for Chlorogenic Acid--A Major Endogenous Polyphenol Involved in Regulation of Ripening and Senescence of Apple Fruit.

    PubMed

    Xi, Yu; Cheng, Dai; Zeng, Xiangquan; Cao, Jiankang; Jiang, Weibo

    2016-01-01

    To learn how the endogenous polyphenols may play a role in fruit ripening and senescence, apple pulp discs were used as a model to study the influences of chlorogenic acid (CHA, a major polyphenol in apple pulp) on fruit ripening and senescence. Apple ('Golden Delicious') pulp discs prepared from pre-climacteric fruit were treated with 50 mg L(-1) CHA and incubated in flasks with 10 mM MES buffer (pH 6.0, 11% sorbitol). Compared to the control samples, treatment with CHA significantly reduced ethylene production and respiration rate, and enhanced levels of firmness and soluble solids content of the pulp discs during incubation at 25°C. These results suggested that CHA could retard senescence of the apple pulp discs. Proteomics analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry (MALDI-TOF/TOF) revealed that the expressions of several key proteins correlated to fruit ripening and senescence were affected by the treatment with CHA. Further study showed that treating the pulp discs with CHA remarkably reduced levels of lipoxygenase, β-galactosidase, NADP-malic enzyme, and enzymatic activities of lipoxygenase and UDP-glucose pyrophosphorylase, all of which are known as promoters of fruit ripening and senescence. These results could provide new insights into the functions of endogenous phenolic compounds in fruit ripening and senescence. PMID:26756813

  6. Efficient use of energy in anoxia-tolerant plants with focus on germinating rice seedlings.

    PubMed

    Atwell, Brian J; Greenway, Hank; Colmer, Timothy D

    2015-04-01

    Anoxia tolerance in plants is distinguished by direction of the sparse supply of energy to processes crucial to cell maintenance and sometimes to growth, as in rice seedlings. In anoxic rice coleoptiles energy is used to synthesise proteins, take up K(+) , synthesise cell walls and lipids, and in cell maintenance. Maintenance of electrochemical H(+) gradients across the tonoplast and plasma membrane is crucial for solute compartmentation and thus survival. These gradients sustain some H(+) -solute cotransport and regulate cytoplasmic pH. Pyrophosphate (PPi ), the alternative energy donor to ATP, allows direction of energy to the vacuolar H(+) -PPi ase, sustaining H(+) gradients across the tonoplast. When energy production is critically low, operation of a biochemical pHstat allows H(+) -solute cotransport across plasma membranes to continue for at least for 18 h. In active (e.g. growing) cells, PPi produced during substantial polymer synthesis allows conversion of PPi to ATP by PPi -phosphofructokinase (PFK). In quiescent cells with little polymer synthesis and associated PPi formation, the PPi required by the vacuolar H(+) -PPi ase and UDPG pyrophosphorylase involved in sucrose mobilisation via sucrose synthase might be produced by conversion of ATP to PPi through reversible glycolytic enzymes, presumably pyruvate orthophosphate dikinase. These hypotheses need testing with species characterised by contrasting anoxia tolerance. PMID:25472708

  7. Ethylene Response Factor TERF1, Regulated by ETHYLENE-INSENSITIVE3-like Factors, Functions in Reactive Oxygen Species (ROS) Scavenging in Tobacco (Nicotiana tabacum L.)

    PubMed Central

    Zhang, Hongbo; Li, Ang; Zhang, Zhijin; Huang, Zejun; Lu, Pingli; Zhang, Dingyu; Liu, Xinmin; Zhang, Zhong-Feng; Huang, Rongfeng

    2016-01-01

    The phytohormone ethylene plays a crucial role in the production and accumulation of reactive oxygen species (ROS) in plants under stress conditions. Ethylene response factors (ERFs) are important ethylene-signaling regulators functioning in plant defense responses against biotic and abiotic stresses. However, the roles of ERFs during plant adapting to ROS stress have not yet been well documented. Our studies previously reported that a tomato ERF transcription factor TERF1 functions in the regulation of plant ethylene responses and stress tolerance. Here, we report our findings regarding the roles of TERF1 in ROS scavenging. In this study, we revealed that the transcription of TERF1 is regulated by upstream EIN3-like (EIN3, ethylene-insensitive 3) regulators LeEIL3 and LeEIL4 in tomato (Solanum lycopersicum), and is also inducible by exogenous applied ROS-generating reagents. Ectopic expression of TERF1 in tobacco promoted the expression of genes involved in oxidative stress responses, including carbonic anhydrase functioning in hypersensitive defense, catalase and glutathione peroxidase catalyzing oxidative reactions, and GDP-D-mannose pyrophosphorylase functioning in ascorbic acid biosynthesis, reduced the ROS content induced by ethylene treatment, and enhanced stress tolerance of tobacco seedlings to hydrogen peroxide (H2O2). Cumulatively, these findings suggest that TERF1 is an ethylene inducible factor regulating ROS scavenging during stress responses. PMID:27435661

  8. Molecular insights into how a deficiency of amylose affects carbon allocation – carbohydrate and oil analyses and gene expression profiling in the seeds of a rice waxy mutant

    PubMed Central

    2012-01-01

    rice waxy mutant GM077 revealed several candidate genes implicated in the carbon reallocation response to an amylose deficiency, including genes encoding AGPase and SUSIBA2-like. We believe that AGP and SUSIBA2 are two promising targets for classical breeding and/or transgenic plant improvement to control the carbon flux between starch and other components in cereal seeds. PMID:23217057

  9. Photosynthate Metabolism in the Source Leaves of N2-Fixing Soybean Plants

    PubMed Central

    de Veau, Edward J. I.; Robinson, J. Michael; Warmbrodt, Robert D.; Kremer, Diane F.

    1992-01-01

    Soybean plants (Glycine max [L.] Merr. cv Williams), which were symbiotic with Bradyrhizobium japonicum, and which grew well upon reduced nitrogen supplied solely through N2 fixation processes, often exhibited excess accumulation of starch and sucrose and diminished soluble protein in their source leaves. Nitrate and ammonia, when supplied to the nodulated roots of N2-fixing plants, mediated a reduction of foliar starch accumulation and a corresponding increase in soluble protein in the source leaves. This provided an opportunity to examine the potential metabolic adjustments by which NO3− and NH4+ (N) sufficiency or deficiency exerted an influence upon soybean leaf starch synthesis. When compared with soybean plants supplied with N, elevated starch accumulation was focused in leaf palisade parenchyma tissue of N2-fixing plants. Foliar activities of starch synthesis pathway enzymes including fructose-1,6-bisphosphate phosphatase, phosphohexoisomerase, phosphoglucomutase (PGM), as well as adenosine diphosphate glucose pyrophosphorylase (in some leaves) exhibited highest activities in leaf extracts of N2-fixing plants when expressed on a leaf protein basis. This was interpreted to mean that there was an adaptation of these enzyme activities in the leaves of N2-fixing plants, and this contributed to an increase in starch accumulation. Another major causal factor associated with increased starch accumulation was the elevation in foliar levels of fructose-6-phosphate, glucose-6-phosphate, and glucose-1-phosphate (G1P), which had risen to chloroplast concentrations considerably in excess of the Km values for their respective target enzymes associated with starch synthesis, e.g. elevated G1P with respect to adenosine diphosphate glucose pyrophosphorylase (ADPG-PPiase) binding sites. The cofactor glucose-1,6-bisphosphate (G1,6BP) was found to be obligate for maximal PGM activity in soybean leaf extracts of N2-fixing as well as N-supplemented plants, and G1,6BP levels in

  10. Identification of Novel Short Ragweed Pollen Allergens Using Combined Transcriptomic and Immunoproteomic Approaches

    PubMed Central

    Bouley, Julien; Groeme, Rachel; Jain, Karine; Baron-Bodo, Véronique; Nony, Emmanuel; Mascarell, Laurent; Moingeon, Philippe

    2015-01-01

    Background Allergy to short ragweed (Ambrosia artemisiifolia) pollen is a serious and expanding health problem in North America and Europe. Whereas only 10 short ragweed pollen allergens are officially recorded, patterns of IgE reactivity observed in ragweed allergic patients suggest that other allergens contribute to allergenicity. The objective of the present study was to identify novel allergens following extensive characterization of the transcriptome and proteome of short ragweed pollen. Methods Following a Proteomics-Informed-by-Transcriptomics approach, a comprehensive transcriptomic data set was built up from RNA-seq analysis of short ragweed pollen. Mass spectrometry-based proteomic analyses and IgE reactivity profiling after high resolution 2D-gel electrophoresis were then combined to identify novel allergens. Results Short ragweed pollen transcripts were assembled after deep RNA sequencing and used to inform proteomic analyses, thus leading to the identification of 573 proteins in the short ragweed pollen. Patterns of IgE reactivity of individual sera from 22 allergic patients were assessed using an aqueous short ragweed pollen extract resolved over 2D-gels. Combined with information derived from the annotated pollen proteome, those analyses revealed the presence of multiple unreported IgE reactive proteins, including new Amb a 1 and Amb a 3 isoallergens as well as 7 novel candidate allergens reacting with IgEs from 20–70% of patients. The latter encompass members of the carbonic anhydrase, enolase, galactose oxidase, GDP dissociation inhibitor, pathogenesis related-17, polygalacturonase and UDP-glucose pyrophosphorylase families. Conclusions We extended the list of allergens identified in short ragweed pollen. These findings have implications for both diagnosis and allergen immunotherapy purposes. PMID:26317427

  11. Inhibitory Role of Greatwall-Like Protein Kinase Rim15p in Alcoholic Fermentation via Upregulating the UDP-Glucose Synthesis Pathway in Saccharomyces cerevisiae.

    PubMed

    Watanabe, Daisuke; Zhou, Yan; Hirata, Aiko; Sugimoto, Yukiko; Takagi, Kenichi; Akao, Takeshi; Ohya, Yoshikazu; Takagi, Hiroshi; Shimoi, Hitoshi

    2016-01-01

    The high fermentation rate of Saccharomyces cerevisiae sake yeast strains is attributable to a loss-of-function mutation in the RIM15 gene, which encodes a Greatwall-family protein kinase that is conserved among eukaryotes. In the present study, we performed intracellular metabolic profiling analysis and revealed that deletion of the RIM15 gene in a laboratory strain impaired glucose-anabolic pathways through the synthesis of UDP-glucose (UDPG). Although Rim15p is required for the synthesis of trehalose and glycogen from UDPG upon entry of cells into the quiescent state, we found that Rim15p is also essential for the accumulation of cell wall β-glucans, which are also anabolic products of UDPG. Furthermore, the impairment of UDPG or 1,3-β-glucan synthesis contributed to an increase in the fermentation rate. Transcriptional induction of PGM2 (phosphoglucomutase) and UGP1 (UDPG pyrophosphorylase) was impaired in Rim15p-deficient cells in the early stage of fermentation. These findings demonstrate that the decreased anabolism of glucose into UDPG and 1,3-β-glucan triggered by a defect in the Rim15p-mediated upregulation of PGM2 and UGP1 redirects the glucose flux into glycolysis. Consistent with this, sake yeast strains with defective Rim15p exhibited impaired expression of PGM2 and UGP1 and decreased levels of β-glucans, trehalose, and glycogen during sake fermentation. We also identified a sake yeast-specific mutation in the glycogen synthesis-associated glycogenin gene GLG2, supporting the conclusion that the glucose-anabolic pathway is impaired in sake yeast. These findings demonstrate that downregulation of the UDPG synthesis pathway is a key mechanism accelerating alcoholic fermentation in industrially utilized S. cerevisiae sake strains. PMID:26497456

  12. A Novel GDP-d-glucose Phosphorylase Involved in Quality Control of the Nucleoside Diphosphate Sugar Pool in Caenorhabditis elegans and Mammals*

    PubMed Central

    Adler, Lital N.; Gomez, Tara A.; Clarke, Steven G.; Linster, Carole L.

    2011-01-01

    The plant VTC2 gene encodes GDP-l-galactose phosphorylase, a rate-limiting enzyme in plant vitamin C biosynthesis. Genes encoding apparent orthologs of VTC2 exist in both mammals, which produce vitamin C by a distinct metabolic pathway, and in the nematode worm Caenorhabditis elegans where vitamin C biosynthesis has not been demonstrated. We have now expressed cDNAs of the human and worm VTC2 homolog genes (C15orf58 and C10F3.4, respectively) and found that the purified proteins also display GDP-hexose phosphorylase activity. However, as opposed to the plant enzyme, the major reaction catalyzed by these enzymes is the phosphorolysis of GDP-d-glucose to GDP and d-glucose 1-phosphate. We detected activities with similar substrate specificity in worm and mouse tissue extracts. The highest expression of GDP-d-glucose phosphorylase was found in the nervous and male reproductive systems. A C. elegans C10F3.4 deletion strain was found to totally lack GDP-d-glucose phosphorylase activity; this activity was also found to be decreased in human HEK293T cells transfected with siRNAs against the human C15orf58 gene. These observations confirm the identification of the worm C10F3.4 and the human C15orf58 gene expression products as the GDP-d-glucose phosphorylases of these organisms. Significantly, we found an accumulation of GDP-d-glucose in the C10F3.4 mutant worms, suggesting that the GDP-d-glucose phosphorylase may function to remove GDP-d-glucose formed by GDP-d-mannose pyrophosphorylase, an enzyme that has previously been shown to lack specificity for its physiological d-mannose 1-phosphate substrate. We propose that such removal may prevent the misincorporation of glucosyl residues for mannosyl residues into the glycoconjugates of worms and mammals. PMID:21507950

  13. 2-Fluoro-L-Fucose Is a Metabolically Incorporated Inhibitor of Plant Cell Wall Polysaccharide Fucosylation.

    PubMed

    Villalobos, Jose A; Yi, Bo R; Wallace, Ian S

    2015-01-01

    The monosaccharide L-fucose (L-Fuc) is a common component of plant cell wall polysaccharides and other plant glycans, including the hemicellulose xyloglucan, pectic rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II), arabinogalactan proteins, and N-linked glycans. Mutations compromising the biosynthesis of many plant cell wall polysaccharides are lethal, and as a result, small molecule inhibitors of plant cell wall polysaccharide biosynthesis have been developed because these molecules can be applied at defined concentrations and developmental stages. In this study, we characterize novel small molecule inhibitors of plant fucosylation. 2-fluoro-L-fucose (2F-Fuc) analogs caused severe growth phenotypes when applied to Arabidopsis seedlings, including reduced root growth and altered root morphology. These phenotypic defects were dependent upon the L-Fuc salvage pathway enzyme L-Fucose Kinase/ GDP-L-Fucose Pyrophosphorylase (FKGP), suggesting that 2F-Fuc is metabolically converted to the sugar nucleotide GDP-2F-Fuc, which serves as the active inhibitory molecule. The L-Fuc content of cell wall matrix polysaccharides was reduced in plants treated with 2F-Fuc, suggesting that this molecule inhibits the incorporation of L-Fuc into these polysaccharides. Additionally, phenotypic defects induced by 2F-Fuc treatment could be partially relieved by the exogenous application of boric acid, suggesting that 2F-Fuc inhibits RG-II biosynthesis. Overall, the results presented here suggest that 2F-Fuc is a metabolically incorporated inhibitor of plant cellular fucosylation events, and potentially suggest that other 2-fluorinated monosaccharides could serve as useful chemical probes for the inhibition of cell wall polysaccharide biosynthesis. PMID:26414071

  14. Identification of differentially accumulating pistil proteins associated with self-incompatibility of non-heading Chinese cabbage.

    PubMed

    Wang, L; Peng, H; Ge, T; Liu, T; Hou, X; Li, Y

    2014-01-01

    Non-heading Chinese cabbage (Brassica campestris L. ssp. chinensis Makino), an important vegetable crop in China, exhibits a typical sporophytic self-incompatibility (SI) system. To better understand the mechanism of SI response and identify potential candidate proteins involved in the SI system of this vegetable crop, the proteomic approach was taken to identify differential accumulating pistil proteins. Pistils were collected at 0 h and 2 h after self-pollination at anthesis in self-incompatible and compatible lines of non-heading Chinese cabbage, and total proteins were extracted and separated by two-dimensional gel electrophoresis (2-DE). A total of 25 protein spots that displayed differential abundance were identified by matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF/TOF MS) and peptide mass fingerprinting (PMF). Among them, 22 protein spots were confidently established. The mRNA levels of the corresponding genes were detected by quantitative RT-PCR. The 22 identified protein spots are involved in energy metabolism (four), protein biosynthesis (three), photosynthesis (six), stress response and defence (five), and protein degradation (four). Among these potential candidate proteins, UDP-sugar pyrophosphorylase could be involved in sucrose degradation to influence pollen germination and growth. Glutathione S-transferases could be involved in pollen maturation, and affect pollen fertility. Senescence-associated cysteine protease, which is related to programmed cell death, could be mainly related to self pollen recognition of non-heading Chinese cabbage. The study will contribute to further investigations of molecular mechanism of sporophytic SI in Brassicaceae. PMID:23581423

  15. A single gene mutation that increases maize seed weight

    SciTech Connect

    Giroux, M.J.; Shaw, J.; Hannah, L.C. |

    1996-06-11

    The maize endosperm-specific gene shrunken2 (Sh2) encodes the large subunit of the heterotetrameric starch synthetic enzyme adenosine diphosphoglucose pyrophosphorylase (AGP; EC 2.7.7.27). Here we exploit an in vivo, site-specific mutagenesis system to create short insertion mutations in a region of the gene known to be involved in the allosteric regulation of AGP. The site-specific mutagen is the transposable element dissociation (Ds). Approximately one-third (8 of 23) of the germinal revertants sequenced restored the wild-type sequence, whereas the remaining revertants contained insertions of 3 or 6 bp. All revertants retained the original reading frame 3 feet to the insertion site and involved the addition of tyrosine and/or serine. Each insertion revertant reduced total AGP activity and the amount of the SH2 protein. The revertant containing additional tyrosine and serine residues increased seed weight 11-18% without increasing or decreasing the percentage of starch. Other insertion revertants lacking an additional serine reduced seed weight. Reduced sensitivity to phosphate, a long-known inhibitor of AGP, was found in the high seed-weight revertant. This alteration is likely universally important since insertion of tyrosine and serine in the potato large subunit of AGP at the comparable position and expression in Escherichia coli also led to a phosphate-insensitive enzyme. These results show that single gene mutations giving rise to increased seed weight, and therefore perhaps yield, are clearly possible in a plant with a long history of intensive and successful breeding efforts. 20 refs., 5 figs., 5 tabs.

  16. Biochemical characterization of an α1,2-colitosyltransferase from Escherichia coli O55:H7.

    PubMed

    Wu, Zhigang; Zhao, Guohui; Li, Tiehai; Qu, Jingyao; Guan, Wanyi; Wang, Jiajia; Ma, Cheng; Li, Xu; Zhao, Wei; Wang, Peng G; Li, Lei

    2016-05-01

    Colitose, also known as 3,6-dideoxy-L-galactose or 3-deoxy-L-fucose, is one of only five naturally occurring 3,6-dideoxyhexoses. Colitose was found in lipopolysaccharide of a number of infectious bacteria, including Escherichia coli O55 & O111 and Vibrio cholera O22 & O139. To date, no colitosyltransferase (ColT) has been characterized, probably due to the inaccessibility of the sugar donor, GDP-colitose. In this study, starting with chemically prepared colitose, 94.6 mg of GDP-colitose was prepared via a facile and efficient one-pot two-enzyme system involving an L-fucokinase/GDP-L-Fuc pyrophosphorylase and an inorganic pyrophosphatase (EcPpA). WbgN, a putative ColT from E. coliO55:H5 was then cloned, overexpressed, purified and biochemically characterized by using GDP-colitose as a sugar donor. Activity assay and structural identification of the synthetic product clearly demonstrated that wbgN encodes an α1,2-ColT. Biophysical study showed that WbgN does not require metal ion, and is highly active at pH 7.5-9.0. In addition, acceptor specificity study indicated that WbgN exclusively recognizes lacto-N-biose (Galβ1,3-GlcNAc). Most interestingly, it was found that WbgN exhibits similar activity toward GDP-l-Fuc (kcat/Km= 9.2 min(-1)mM(-1)) as that toward GDP-colitose (kcat/Km= 12 min(-1)mM(-1)). Finally, taking advantage of this, type 1 H-antigen was successfully synthesized in preparative scale. PMID:26703456

  17. Analysis of expressed sequence tags derived from a compatible Mycosphaerella fijiensis-banana interaction.

    PubMed

    Portal, Orelvis; Izquierdo, Yovanny; De Vleesschauwer, David; Sánchez-Rodríguez, Aminael; Mendoza-Rodríguez, Milady; Acosta-Suárez, Mayra; Ocaña, Bárbara; Jiménez, Elio; Höfte, Monica

    2011-05-01

    Mycosphaerella fijiensis, a hemibiotrophic fungus, is the causal agent of black leaf streak disease, the most serious foliar disease of bananas and plantains. To analyze the compatible interaction of M. fijiensis with Musa spp., a suppression subtractive hybridization (SSH) cDNA library was constructed to identify transcripts induced at late stages of infection in the host and the pathogen. In addition, a full-length cDNA library was created from the same mRNA starting material as the SSH library. The SSH procedure was effective in identifying specific genes predicted to be involved in plant-fungal interactions and new information was obtained mainly about genes and pathways activated in the plant. Several plant genes predicted to be involved in the synthesis of phenylpropanoids and detoxification compounds were identified, as well as pathogenesis-related proteins that could be involved in the plant response against M. fijiensis infection. At late stages of infection, jasmonic acid and ethylene signaling transduction pathways appear to be active, which corresponds with the necrotrophic life style of M. fijiensis. Quantitative PCR experiments revealed that antifungal genes encoding PR proteins and GDSL-like lipase are only transiently induced 30 days post inoculation (dpi), indicating that the fungus is probably actively repressing plant defense. The only fungal gene found was induced 37 dpi and encodes UDP-glucose pyrophosphorylase, an enzyme involved in the biosynthesis of trehalose. Trehalose biosynthesis was probably induced in response to prior activation of plant antifungal genes and may act as an osmoprotectant against membrane damage. PMID:21279642

  18. Transcriptional Profiles of Hybrid Eucalyptus Genotypes with Contrasting Lignin Content Reveal That Monolignol Biosynthesis-related Genes Regulate Wood Composition

    PubMed Central

    Shinya, Tomotaka; Iwata, Eiji; Nakahama, Katsuhiko; Fukuda, Yujiroh; Hayashi, Kazunori; Nanto, Kazuya; Rosa, Antonio C.; Kawaoka, Akiyoshi

    2016-01-01

    Eucalyptus species constitutes the most widely planted hardwood trees in temperate and subtropical regions. In this study, we compared the transcript levels of genes involved in lignocellulose formation such as cellulose, hemicellulose and lignin biosynthesis in two selected 3-year old hybrid Eucalyptus (Eucalyptus urophylla × Eucalyptus grandis) genotypes (AM063 and AM380) that have different lignin content. AM063 and AM380 had 20.2 and 35.5% of Klason lignin content and 59.0 and 48.2%, α-cellulose contents, respectively. We investigated the correlation between wood properties and transcript levels of wood formation-related genes using RNA-seq with total RNAs extracted from developing xylem tissues at a breast height. Transcript levels of cell wall construction genes such as cellulose synthase (CesA) and sucrose synthase (SUSY) were almost the same in both genotypes. However, AM063 exhibited higher transcript levels of UDP-glucose pyrophosphorylase and xyloglucan endotransglucoxylase than those in AM380. Most monolignol biosynthesis-related isozyme genes showed higher transcript levels in AM380. These results indicate monolignol biosynthesis-related genes may regulate wood composition in Eucalyptus. Flavonoids contents were also observed at much higher levels in AM380 as a result of the elevated transcript levels of common phenylpropanoid pathway genes, phenylalanine ammonium lyase, cinnamate-4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL). Secondary plant cell wall formation is regulated by many transcription factors. We analyzed genes encoding NAC, WRKY, AP2/ERF, and KNOX transcription factors and found higher transcript levels of these genes in AM380. We also observed increased transcription of some MYB and LIM domain transcription factors in AM380 compared to AM063. All these results show that genes related to monolignol biosynthesis may regulate the wood composition and help maintain the ratio of cellulose and lignin contents in Eucalyptus plants. PMID

  19. 2-Fluoro-L-Fucose Is a Metabolically Incorporated Inhibitor of Plant Cell Wall Polysaccharide Fucosylation

    PubMed Central

    Wallace, Ian S.

    2015-01-01

    The monosaccharide L-fucose (L-Fuc) is a common component of plant cell wall polysaccharides and other plant glycans, including the hemicellulose xyloglucan, pectic rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II), arabinogalactan proteins, and N-linked glycans. Mutations compromising the biosynthesis of many plant cell wall polysaccharides are lethal, and as a result, small molecule inhibitors of plant cell wall polysaccharide biosynthesis have been developed because these molecules can be applied at defined concentrations and developmental stages. In this study, we characterize novel small molecule inhibitors of plant fucosylation. 2-fluoro-L-fucose (2F-Fuc) analogs caused severe growth phenotypes when applied to Arabidopsis seedlings, including reduced root growth and altered root morphology. These phenotypic defects were dependent upon the L-Fuc salvage pathway enzyme L-Fucose Kinase/ GDP-L-Fucose Pyrophosphorylase (FKGP), suggesting that 2F-Fuc is metabolically converted to the sugar nucleotide GDP-2F-Fuc, which serves as the active inhibitory molecule. The L-Fuc content of cell wall matrix polysaccharides was reduced in plants treated with 2F-Fuc, suggesting that this molecule inhibits the incorporation of L-Fuc into these polysaccharides. Additionally, phenotypic defects induced by 2F-Fuc treatment could be partially relieved by the exogenous application of boric acid, suggesting that 2F-Fuc inhibits RG-II biosynthesis. Overall, the results presented here suggest that 2F-Fuc is a metabolically incorporated inhibitor of plant cellular fucosylation events, and potentially suggest that other 2-fluorinated monosaccharides could serve as useful chemical probes for the inhibition of cell wall polysaccharide biosynthesis. PMID:26414071

  20. Manipulation of the rice L-galactose pathway: evaluation of the effects of transgene overexpression on ascorbate accumulation and abiotic stress tolerance.

    PubMed

    Zhang, Gui-Yun; Liu, Ru-Ru; Zhang, Chang-Quan; Tang, Ke-Xuan; Sun, Ming-Fa; Yan, Guo-Hong; Liu, Qiao-Quan

    2015-01-01

    Ascorbic acid (AsA) is the most abundant water-soluble antioxidant in plants, and it plays a crucial role in plant growth, development and abiotic stress tolerance. In the present study, six key Arabidopsis or rapeseed genes involved in AsA biosynthesis were constitutively overexpressed in an elite Japonica rice cultivar. These genes encoded the GDP-mannose pyrophosphorylase (GMP), GDP-mannose-3',5'-epimerase (GME), GDP-L-galactose phosphorylase (GGP), L-galactose-1-phosphate phosphatase (GPP), L-galactose dehydrogenase (GDH), and L-galactono-1,4-lactone dehydrogenase (GalLDH). The effects of transgene expression on rice leaf AsA accumulation were carefully evaluated. In homozygous transgenic seedlings, AtGGP transgenic lines had the highest AsA contents (2.55-fold greater than the empty vector transgenic control), followed by the AtGME and AtGDH transgenic lines. Moreover, with the exception of the AtGPP lines, the increased AsA content also provoked an increase in the redox state (AsA/DHA ratio). To evaluate salt tolerance, AtGGP and AtGME transgenic seedlings were exposed to salt stress for one week. The relative plant height, root length and fresh weight growth rates were significantly higher for the transgenic lines compared with the control plants. Altogether, our results suggest that GGP may be a key rate-limiting step in rice AsA biosynthesis, and the plants with elevated AsA contents demonstrated enhanced tolerance for salt stress. PMID:25938231

  1. High-throughput screen for inhibitors of transglycosylase and/or transpeptidase activities of Escherichia coli penicillin binding protein 1b.

    PubMed

    Chandrakala, B; Shandil, Radha K; Mehra, Upasana; Ravishankar, Sudha; Kaur, Parvinder; Usha, Veeraraghavan; Joe, Bina; deSousa, Sunita M

    2004-01-01

    Penicillin binding protein (PBP) 1b of Escherichia coli has both transglycosylase and transpeptidase activities, which are attractive targets for the discovery of new antibacterial agents. A high-throughput assay that detects inhibitors of the PBPs was described previously, but it cannot distinguish them from inhibitors of the MraY, MurG, and lipid pyrophosphorylase. We report on a method that distinguishes inhibitors of both activities of the PBPs from those of the other three enzymes. Radioactive peptidoglycan was synthesized by using E. coli membranes. Following termination of the reaction the products were analyzed in three ways. Wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads were added to one set, and the same beads together with a detergent were added to a second set. Type A polyethylenimine-coated WGA-coated SPA beads were added to a third set. By comparison of the results of assays run in parallel under the first two conditions, inhibitors of the transpeptidase and transglycosylase could be distinguished from inhibitors of the other enzymes, as the inhibitors of the other enzymes showed similar inhibitory concentrations (IC(50)s) under both conditions but the inhibitors of the PBPs showed insignificant inhibition in the absence of detergent. Furthermore, comparison of the results of assays run under conditions two and three enabled the distinction of transpeptidase inhibitors. Penicillin and other beta-lactams showed insignificant inhibition with type A beads compared with that shown with WGA-coated SPA beads plus detergent. However, inhibitors of the other four enzymes (tunicamycin, nisin, bacitracin, and moenomycin) showed similar IC(50)s under both conditions. We show that the main PBP being measured under these conditions is PBP 1b. This screen can be used to find novel transglycosylase or transpeptidase inhibitors. PMID:14693515

  2. Inhibitory Role of Greatwall-Like Protein Kinase Rim15p in Alcoholic Fermentation via Upregulating the UDP-Glucose Synthesis Pathway in Saccharomyces cerevisiae

    PubMed Central

    Watanabe, Daisuke; Zhou, Yan; Hirata, Aiko; Sugimoto, Yukiko; Takagi, Kenichi; Akao, Takeshi; Ohya, Yoshikazu; Takagi, Hiroshi

    2015-01-01

    The high fermentation rate of Saccharomyces cerevisiae sake yeast strains is attributable to a loss-of-function mutation in the RIM15 gene, which encodes a Greatwall-family protein kinase that is conserved among eukaryotes. In the present study, we performed intracellular metabolic profiling analysis and revealed that deletion of the RIM15 gene in a laboratory strain impaired glucose-anabolic pathways through the synthesis of UDP-glucose (UDPG). Although Rim15p is required for the synthesis of trehalose and glycogen from UDPG upon entry of cells into the quiescent state, we found that Rim15p is also essential for the accumulation of cell wall β-glucans, which are also anabolic products of UDPG. Furthermore, the impairment of UDPG or 1,3-β-glucan synthesis contributed to an increase in the fermentation rate. Transcriptional induction of PGM2 (phosphoglucomutase) and UGP1 (UDPG pyrophosphorylase) was impaired in Rim15p-deficient cells in the early stage of fermentation. These findings demonstrate that the decreased anabolism of glucose into UDPG and 1,3-β-glucan triggered by a defect in the Rim15p-mediated upregulation of PGM2 and UGP1 redirects the glucose flux into glycolysis. Consistent with this, sake yeast strains with defective Rim15p exhibited impaired expression of PGM2 and UGP1 and decreased levels of β-glucans, trehalose, and glycogen during sake fermentation. We also identified a sake yeast-specific mutation in the glycogen synthesis-associated glycogenin gene GLG2, supporting the conclusion that the glucose-anabolic pathway is impaired in sake yeast. These findings demonstrate that downregulation of the UDPG synthesis pathway is a key mechanism accelerating alcoholic fermentation in industrially utilized S. cerevisiae sake strains. PMID:26497456

  3. An alpha-glucose-1-phosphate phosphodiesterase is present in rat liver cytosol

    SciTech Connect

    Srisomsap, C.; Richardson, K.L.; Jay, J.C.; Marchase, R.B. )

    1989-12-05

    UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of alpha-Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins. The predominant acceptor for this transfer in both mammalian cells and Paramecium is a cytoplasmic glycoprotein of 62-63 kDa. When cytoplasmic proteins from rat liver were fractionated by preparative isoelectric focusing following incubation of a liver homogenate with the 35S-labeled phosphorothioate analogue of UDP-Glc ((beta-35S)UDP-Glc), the acceptor was found to have a pI of about 6.0. This fraction, when not labeled prior to the focusing, became very heavily labeled when mixed with (beta-35S). UDP-Glc and intact liver microsomes, a rich source of the Glc-phosphotransferase. In addition, it was observed that the isoelectric fractions of the cytosol having pI values of 2-3.2 contained a degradative activity, alpha-Glc-1-P phosphodiesterase, that was capable of removing alpha-Glc-1-P, monitored through radioactive labeling both in the sugar and the phosphate, as an intact unit from the 62-kDa acceptor. Identification of the product of this cleavage was substantiated by its partial transformation to UDP-Glc in the presence of UTP and UDP-Glc pyrophosphorylase. The alpha-Glc-1-P phosphodiesterase had a pH optimum of 7.5 and was not effectively inhibited by any of the potential biochemical inhibitors that were tested. Specificity for the Glc-alpha-1-P-6-Man diester was suggested by the diesterase's inability to degrade UDP-Glc or glucosylphosphoryldolichol. This enzyme may be important in the regulation of secretion since the alpha-Glc-1-P present on the 62-kDa phosphoglycoprotein appears to be removed and then rapidly replaced in response to secretagogue.

  4. Comparative Transcriptome Analysis of Cultivated and Wild Watermelon during Fruit Development.

    PubMed

    Guo, Shaogui; Sun, Honghe; Zhang, Haiying; Liu, Jingan; Ren, Yi; Gong, Guoyi; Jiao, Chen; Zheng, Yi; Yang, Wencai; Fei, Zhangjun; Xu, Yong

    2015-01-01

    Watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] is an important vegetable crop world-wide. Watermelon fruit quality is a complex trait determined by various factors such as sugar content, flesh color and flesh texture. Fruit quality and developmental process of cultivated and wild watermelon are highly different. To systematically understand the molecular basis of these differences, we compared transcriptome profiles of fruit tissues of cultivated watermelon 97103 and wild watermelon PI296341-FR. We identified 2,452, 826 and 322 differentially expressed genes in cultivated flesh, cultivated mesocarp and wild flesh, respectively, during fruit development. Gene ontology enrichment analysis of these genes indicated that biological processes and metabolic pathways related to fruit quality such as sweetness and flavor were significantly changed only in the flesh of 97103 during fruit development, while those related to abiotic stress response were changed mainly in the flesh of PI296341-FR. Our comparative transcriptome profiling analysis identified critical genes potentially involved in controlling fruit quality traits including α-galactosidase, invertase, UDP-galactose/glucose pyrophosphorylase and sugar transporter genes involved in the determination of fruit sugar content, phytoene synthase, β-carotene hydroxylase, 9-cis-epoxycarotenoid dioxygenase and carotenoid cleavage dioxygenase genes involved in carotenoid metabolism, and 4-coumarate:coenzyme A ligase, cellulose synthase, pectinesterase, pectinesterase inhibitor, polygalacturonase inhibitor and α-mannosidase genes involved in the regulation of flesh texture. In addition, we found that genes in the ethylene biosynthesis and signaling pathway including ACC oxidase, ethylene receptor and ethylene responsive factor showed highly ripening-associated expression patterns, indicating a possible role of ethylene in fruit development and ripening of watermelon, a non-climacteric fruit. Our analysis provides

  5. The Autophagic Degradation of Chloroplasts via Rubisco-Containing Bodies Is Specifically Linked to Leaf Carbon Status But Not Nitrogen Status in Arabidopsis1[W][OA

    PubMed Central

    Izumi, Masanori; Wada, Shinya; Makino, Amane; Ishida, Hiroyuki

    2010-01-01

    Autophagy is an intracellular process facilitating the vacuolar degradation of cytoplasmic components and is important for nutrient recycling during starvation. We previously demonstrated that chloroplasts can be partially mobilized to the vacuole by autophagy via spherical bodies named Rubisco-containing bodies (RCBs). Although chloroplasts contain approximately 80% of total leaf nitrogen and represent a major carbon and nitrogen source for new growth, the relationship between leaf nutrient status and RCB production remains unclear. We examined the effects of nutrient factors on the appearance of RCBs in leaves of transgenic Arabidopsis (Arabidopsis thaliana) expressing stroma-targeted fluorescent proteins. In excised leaves, the appearance of RCBs was suppressed by the presence of metabolic sugars, which were added externally or were produced during photosynthesis in the light. The light-mediated suppression was relieved by the inhibition of photosynthesis. During a diurnal cycle, RCB production was suppressed in leaves excised at the end of the day with high starch content. Starchless mutants phosphoglucomutase and ADP-Glc pyrophosphorylase1 produced a large number of RCBs, while starch-excess mutants starch-excess1 and maltose-excess1 produced fewer RCBs. In nitrogen-limited plants, as leaf carbohydrates were accumulated, RCB production was suppressed. We propose that there exists a close relationship between the degradation of chloroplast proteins via RCBs and leaf carbon but not nitrogen status in autophagy. We also found that the appearance of non-RCB-type autophagic bodies was not suppressed in the light and somewhat responded to nitrogen in excised leaves, unlike RCBs. These results imply that the degradation of chloroplast proteins via RCBs is specifically controlled in autophagy. PMID:20807997

  6. NAD[S], an NAD analogue with reduced susceptibility to phosphodiesterase. Chemical synthesis and enzymic properties.

    PubMed

    Meyer, T; Wielckens, K; Thiem, J; Hilz, H

    1984-05-01

    The chemical synthesis of adenosine(5') [alpha-thio]diphospho(5')ribofuranosyl-nicotinamide (NAD[S]) is described. The product occurs as a pair of diastereomers with different configuration at the sulfur-bearing phosphorus atom. The diastereomers were separated by high-performance liquid chromatography and their absolute configuration was determined after chemical degradation to the ADP[alpha S] diastereomers and chromatographic comparison with enzymically synthesized ADP[alpha S] diastereomers of known absolute configuration. Additional support for this assignment is based on different rates in the phosphodiesterase-catalyzed hydrolysis. Furthermore the synthesis of [14C]NAD[S] is described. The coenzyme activity of NAD[S] in the reaction with alcohol dehydrogenase from baker's yeast and lactate dehydrogenase from pig heart is very similar to that of beta-NAD. Also, NAD and NAD[S] serve equally well as substrates for NAD glycohydrolase from calf spleen. In contrast, no reaction was detected with NAD pyrophosphorylase, and hydrolysis of the separated NAD[S] diastereomers with snake venom phosphodiesterase showed a 26-fold and a 33-fold slower reaction rate than that of NAD. Nucleotide pyrophosphatase was less sensitive to the S substitution, hydrolyzing NAD[S] 14-times slower than NAD. Poly(ADP-ribose) polymerase from Ehrlich ascites tumor cell nuclei accepted NAD[S] as a substrate but the reaction was significantly slower and approached saturation at much lower values than with NAD. Alkaline hydrolysis of the products insoluble in trichloroacetic acid yielded AMP[S] as the main derivative. It is concluded that with NAD[S] as a substrate the nuclear acceptors were nearly exclusively mono(ADP-ribosyl) ated . PMID:6144544

  7. UDP-glucose is a potential intracellular signal molecule in the control of expression of sigma S and sigma S-dependent genes in Escherichia coli.

    PubMed Central

    Böhringer, J; Fischer, D; Mosler, G; Hengge-Aronis, R

    1995-01-01

    The sigma S subunit of RNA polymerase is the master regulator of a regulatory network that controls stationary-phase induction as well as osmotic regulation of many genes in Escherichia coli. In an attempt to identify additional regulatory components in this network, we have isolated Tn10 insertion mutations that in trans alter the expression of osmY and other sigma S-dependent genes. One of these mutations conferred glucose sensitivity and was localized in pgi (encoding phosphoglucose isomerase). pgi::Tn10 strains exhibit increased basal levels of expression of osmY and otsBA in exponentially growing cells and reduced osmotic inducibility of these genes. A similar phenotype was also observed for pgm and galU mutants, which are deficient in phosphoglucomutase and UDP-glucose pyrophosphorylase, respectively. This indicates that the observed effects on gene expression are related to the lack of UDP-glucose (or a derivative thereof), which is common to all three mutants. Mutants deficient in UDP-galactose epimerase (galE mutants) and trehalose-6-phosphate synthase (otsA mutants) do not exhibit such an effect on gene expression, and an mdoA mutant that is deficient in the first step of the synthesis of membrane-derived oligosaccharides, shows only a partial increase in the expression of osmY. We therefore propose that the cellular content of UDP-glucose serves as an internal signal that controls expression of osmY and other sigma S-dependent genes. In addition, we demonstrate that pgi, pgm, and galU mutants contain increased levels of sigma S during steady-state growth, indicating that UDP-glucose interferes with the expression of sigma S itself. PMID:7814331

  8. Investigation of ascorbate metabolism during inducement of storage disorders in pear.

    PubMed

    Cascia, Giuseppe; Bulley, Sean M; Punter, Matthew; Bowen, Judith; Rassam, Maysoon; Schotsmans, Wendy C; Larrigaudière, Christian; Johnston, Jason W

    2013-02-01

    In pear and apple, depletion of ascorbate has previously been associated with development of stress-related flesh browning. This disorder occurs in intact fruit and differs from browning associated with tissue maceration and processing. We investigated changes in ascorbate content, ascorbate peroxidase (APX) activities and gene expression of l-galactose pathway genes, ascorbate recycling genes and APXs from harvest to 30 days storage for three pear varieties ['Williams Bon Chretien' (WBC), 'Doyenne du Comice' and 'Beurre Bosc']. The pears were stored at 0.5°C in air or controlled atmosphere (CA, 2 kPa O(2) and 5 kPa CO(2)). Storage in CA caused significant amounts of storage disorders in WBC only. Ascorbate content generally declined after harvest, although a transient increase in ascorbate in the form of dehydroascorbate (DHA) between harvest and 3 days was observed in CA stored WBC, possibly due to low at-harvest monodehydroascorbate reductase and CA-decreased dehydroascorbate reductase expression. Quantitative polymerase chain reaction indicated that all cultivars responded to CA storage by increasing transcripts for APXs, and surprisingly the pre-l-galactose pathway gene GDP-mannose pyrophosphorylase, of which the product GDP mannose, is utilized either for cell wall polysaccharides, protein N-glycosylation or ascorbate production. Overall, the small differences in ascorbate we observed suggest how ascorbate is utilized, rather than ascorbate content, determines the potential to develop internal browning. Moreover, a transitory increase in DHA postharvest may indicate that fruits are at risk of developing the disorder. PMID:22568767

  9. Comparative Transcriptome Analysis of Cultivated and Wild Watermelon during Fruit Development

    PubMed Central

    Guo, Shaogui; Sun, Honghe; Zhang, Haiying; Liu, Jingan; Ren, Yi; Gong, Guoyi; Jiao, Chen; Zheng, Yi; Yang, Wencai; Fei, Zhangjun; Xu, Yong

    2015-01-01

    Watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] is an important vegetable crop world-wide. Watermelon fruit quality is a complex trait determined by various factors such as sugar content, flesh color and flesh texture. Fruit quality and developmental process of cultivated and wild watermelon are highly different. To systematically understand the molecular basis of these differences, we compared transcriptome profiles of fruit tissues of cultivated watermelon 97103 and wild watermelon PI296341-FR. We identified 2,452, 826 and 322 differentially expressed genes in cultivated flesh, cultivated mesocarp and wild flesh, respectively, during fruit development. Gene ontology enrichment analysis of these genes indicated that biological processes and metabolic pathways related to fruit quality such as sweetness and flavor were significantly changed only in the flesh of 97103 during fruit development, while those related to abiotic stress response were changed mainly in the flesh of PI296341-FR. Our comparative transcriptome profiling analysis identified critical genes potentially involved in controlling fruit quality traits including α-galactosidase, invertase, UDP-galactose/glucose pyrophosphorylase and sugar transporter genes involved in the determination of fruit sugar content, phytoene synthase, β-carotene hydroxylase, 9-cis-epoxycarotenoid dioxygenase and carotenoid cleavage dioxygenase genes involved in carotenoid metabolism, and 4-coumarate:coenzyme A ligase, cellulose synthase, pectinesterase, pectinesterase inhibitor, polygalacturonase inhibitor and α-mannosidase genes involved in the regulation of flesh texture. In addition, we found that genes in the ethylene biosynthesis and signaling pathway including ACC oxidase, ethylene receptor and ethylene responsive factor showed highly ripening-associated expression patterns, indicating a possible role of ethylene in fruit development and ripening of watermelon, a non-climacteric fruit. Our analysis provides

  10. Differential transcriptional regulation of L-ascorbic acid content in peel and pulp of citrus fruits during development and maturation.

    PubMed

    Alós, Enriqueta; Rodrigo, María J; Zacarías, Lorenzo

    2014-05-01

    Citrus fruits are an important source of ascorbic acid (AsA) for human nutrition, but the main pathways involved in its biosynthesis and their regulation are still not fully characterized. To study the transcriptional regulation of AsA accumulation, expression levels of 13 genes involved in AsA biosynthesis, 5 in recycling and 5 in degradation were analyzed in peel and pulp of fruit of two varieties with different AsA concentration: Navel orange (Citrus sinensis) and Satsuma mandarin (Citrus unshiu). AsA accumulation in peel and pulp correlated with the transcriptional profiling of the L-galactose pathway genes, and the myo-inositol pathway appeared to be also relevant in the peel of immature-green orange. Differences in AsA content between varieties were associated with differential gene expression of GDP-mannose pyrophosphorylase (GMP), GDP-L-galactose phosphorylase (GGP) and L-galactose-1-phosphate phosphatase (GPP), myo-inositol oxygenase in peel, and GGP and GPP in pulp. Relative expressions of monodehydroascorbate reductase 3 (MDHAR3) and dehydroascorbate reductase1 (DHAR1) correlated with AsA accumulation during development and ripening in peel and pulp, respectively, and were more highly expressed in the variety with higher AsA contents. Collectively, results indicated a differential regulation of AsA concentration in peel and pulp of citrus fruits that may change during the different stages of fruit development. The L-galactose pathway appears to be predominant in both tissues, but AsA concentration is regulated by complex mechanisms in which degradation and recycling also play important roles. PMID:24567029

  11. Biochemical consequences of resistance to tiazofurin in human myelogenous leukemic K562 cells.

    PubMed

    Jayaram, H N; Zhen, W; Gharehbaghi, K

    1993-05-15

    Tiazofurin exhibits antitumor activity in murine and human tumor cells. In a recent phase I/II trial in patients with end-stage leukemia, tiazofurin showed good response; however, repeated treatment resulted in clinical resistance to the drug. To elucidate the mechanisms of resistance in human leukemic cells, two variants of human myelogenous leukemia K652 cells resistant to tiazofurin were developed by drug-selection pressure. Compared to a concentration producing 50% cell proliferation reduction that was 9.1 microM in sensitive cells, the resistant variants displayed concentrations producing 50% cell proliferation reductions of 12 and 16 mM. The activity of the target enzyme, IMP dehydrogenase, was not altered in the resistant cells. Studies on tiazofurin metabolism revealed that resistant variants formed < 10% of the active metabolite, thiazole-4-carboxamide adenine dinucleotide. This correlated with the activity of NAD pyrophosphorylase, the enzyme that synthesizes thiazole-4-carboxamide adenine dinucleotide, which was reduced to 10% in the resistant lines. Concurrently, the activity of thiazole-4-carboxamide adenine dinucleotide phosphodiesterase was elevated in the refractory cells. Compared to the sensitive counterpart, the levels of GMP and NAD were lower in the resistant lines. Guanine salvage activity was decreased in the resistant cells. Basal dGTP and dATP concentrations were elevated in the resistant line; nevertheless, tiazofurin incubation decreased dGTP levels in only the sensitive cells. Although there was no difference in the Km of tiazofurin transport or efflux, the Vmax of uptake of the drug was reduced in the resistant lines. Sensitive and resistant cells exhibit similar cytotoxicity to agents which do not share the mechanism of action of tiazofurin, suggesting that refractory cells are still sensitive to other standard antileukemic drugs. PMID:8097964

  12. Glucose-1-Phosphate Transport into Protoplasts and Chloroplasts from Leaves of Arabidopsis1

    PubMed Central

    Fettke, Joerg; Malinova, Irina; Albrecht, Tanja; Hejazi, Mahdi; Steup, Martin

    2011-01-01

    Almost all glucosyl transfer reactions rely on glucose-1-phosphate (Glc-1-P) that either immediately acts as glucosyl donor or as substrate for the synthesis of the more widely used Glc dinucleotides, ADPglucose or UDPglucose. In this communication, we have analyzed two Glc-1-P-related processes: the carbon flux from externally supplied Glc-1-P to starch by either mesophyll protoplasts or intact chloroplasts from Arabidopsis (Arabidopsis thaliana). When intact protoplasts or chloroplasts are incubated with [U-14C]Glc-1-P, starch is rapidly labeled. Incorporation into starch is unaffected by the addition of unlabeled Glc-6-P or Glc, indicating a selective flux from Glc-1-P to starch. However, illuminated protoplasts incorporate less 14C into starch when unlabeled bicarbonate is supplied in addition to the 14C-labeled Glc-1-P. Mesophyll protoplasts incubated with [U-14C]Glc-1-P incorporate 14C into the plastidial pool of adenosine diphosphoglucose. Protoplasts prepared from leaves of mutants of Arabidopsis that lack either the plastidial phosphorylase or the phosphoglucomutase isozyme incorporate 14C derived from external Glc-1-P into starch, but incorporation into starch is insignificant when protoplasts from a mutant possessing a highly reduced ADPglucose pyrophosphorylase activity are studied. Thus, the path of assimilatory starch biosynthesis initiated by extraplastidial Glc-1-P leads to the plastidial pool of adenosine diphosphoglucose, and at this intermediate it is fused with the Calvin cycle-driven route. Mutants lacking the plastidial phosphoglucomutase contain a small yet significant amount of transitory starch. PMID:21115809

  13. Transcriptional Profiles of Hybrid Eucalyptus Genotypes with Contrasting Lignin Content Reveal That Monolignol Biosynthesis-related Genes Regulate Wood Composition.

    PubMed

    Shinya, Tomotaka; Iwata, Eiji; Nakahama, Katsuhiko; Fukuda, Yujiroh; Hayashi, Kazunori; Nanto, Kazuya; Rosa, Antonio C; Kawaoka, Akiyoshi

    2016-01-01

    Eucalyptus species constitutes the most widely planted hardwood trees in temperate and subtropical regions. In this study, we compared the transcript levels of genes involved in lignocellulose formation such as cellulose, hemicellulose and lignin biosynthesis in two selected 3-year old hybrid Eucalyptus (Eucalyptus urophylla × Eucalyptus grandis) genotypes (AM063 and AM380) that have different lignin content. AM063 and AM380 had 20.2 and 35.5% of Klason lignin content and 59.0 and 48.2%, α-cellulose contents, respectively. We investigated the correlation between wood properties and transcript levels of wood formation-related genes using RNA-seq with total RNAs extracted from developing xylem tissues at a breast height. Transcript levels of cell wall construction genes such as cellulose synthase (CesA) and sucrose synthase (SUSY) were almost the same in both genotypes. However, AM063 exhibited higher transcript levels of UDP-glucose pyrophosphorylase and xyloglucan endotransglucoxylase than those in AM380. Most monolignol biosynthesis-related isozyme genes showed higher transcript levels in AM380. These results indicate monolignol biosynthesis-related genes may regulate wood composition in Eucalyptus. Flavonoids contents were also observed at much higher levels in AM380 as a result of the elevated transcript levels of common phenylpropanoid pathway genes, phenylalanine ammonium lyase, cinnamate-4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL). Secondary plant cell wall formation is regulated by many transcription factors. We analyzed genes encoding NAC, WRKY, AP2/ERF, and KNOX transcription factors and found higher transcript levels of these genes in AM380. We also observed increased transcription of some MYB and LIM domain transcription factors in AM380 compared to AM063. All these results show that genes related to monolignol biosynthesis may regulate the wood composition and help maintain the ratio of cellulose and lignin contents in Eucalyptus plants. PMID

  14. The tomato HD-Zip I transcription factor SlHZ24 modulates ascorbate accumulation through positive regulation of the D-mannose/L-galactose pathway.

    PubMed

    Hu, Tixu; Ye, Jie; Tao, Peiwen; Li, Hanxia; Zhang, Junhong; Zhang, Yuyang; Ye, Zhibiao

    2016-01-01

    Ascorbate (AsA) is an antioxidant that can scavenge the reactive oxygen species (ROS) produced when plants encounter stressful conditions. Here, it was revealed by a yeast one-hybrid assay that a tomato (Solanum lycopersicum) HD-Zip I family transcription factor, SlHZ24, binds to the promoter of an AsA biosynthetic gene encoding GDP-D-mannose pyrophosphorylase 3 (SlGMP3). Both the transient expression system and electrophoretic mobility shift assay (EMSA) showed that SlHZ24 binds to a regulatory cis-element in the SlGMP3 promoter, and further overexpression of SlHZ24 in transgenic tomato lines resulted in increased AsA levels. In contrast, suppressing expression of the gene using RNA interference (RNAi) had the opposite effect. These data suggest that SlHZ24 can positively regulate the accumulation of AsA, and in support of this it was shown that SlGMP3 expression increased in the SlHZ24-overexpressing lines and declined in SlHZ24-RNAi lines. SlHZ24 also affected the expression of other genes in the D-mannose/L-galactose pathway, such as genes encoding GDP-mannose-3',5'-epimerase 2 (SlGME2), GDP-L-galactose phosphorylase (SlGGP) and SlGMP4. The EMSA indicated that SlHZ24 bound to the promoters of SlGME2 and SlGGP, suggesting multi-targeted regulation of AsA biosynthesis. Finally, SlHZ24-overexpressing plants showed less sensitivity to oxidative stress; we therefore conclude that SlHZ24 promotes AsA biosynthesis, which in turn enhances oxidative stress tolerance. PMID:26610866

  15. Climate Extreme Effects on the Chemical Composition of Temperate Grassland Species under Ambient and Elevated CO2: A Comparison of Fructan and Non-Fructan Accumulators

    PubMed Central

    Zinta, Gaurav; Van den Ende, Wim; Janssens, Ivan A.; Asard, Han

    2014-01-01

    Elevated CO2 concentrations and extreme climate events, are two increasing components of the ongoing global climatic change factors, may alter plant chemical composition and thereby their economic and ecological characteristics, e.g. nutritional quality and decomposition rates. To investigate the impact of climate extremes on tissue quality, four temperate grassland species: the fructan accumulating grasses Lolium perenne, Poa pratensis, and the nitrogen (N) fixing legumes Medicago lupulina and Lotus corniculatus were subjected to water deficit at elevated temperature (+3°C), under ambient CO2 (392 ppm) and elevated CO2 (620 ppm). As a general observation, the effects of the climate extreme were larger and more ubiquitous in combination with elevated CO2. The imposed climate extreme increased non-structural carbohydrate and phenolics in all species, whereas it increased lignin in legumes and decreased tannins in grasses. However, there was no significant effect of climate extreme on structural carbohydrates, proteins, lipids and mineral contents and stoichiometric ratios. In combination with elevated CO2, climate extreme elicited larger increases in fructan and sucrose content in the grasses without affecting the total carbohydrate content, while it significantly increased total carbohydrates in legumes. The accumulation of carbohydrates in legumes was accompanied by higher activity of sucrose phosphate synthase, sucrose synthase and ADP-Glc pyrophosphorylase. In the legumes, elevated CO2 in combination with climate extreme reduced protein, phosphorus (P) and magnesium (Mg) contents and the total element:N ratio and it increased phenol, lignin, tannin, carbon (C), nitrogen (N) contents and C:N, C:P and N:P ratios. On the other hand, the tissue composition of the fructan accumulating grasses was not affected at this level, in line with recent views that fructans contribute to cellular homeostasis under stress. It is speculated that quality losses will be less

  16. Gene expression of ascorbic acid biosynthesis related enzymes of the Smirnoff-Wheeler pathway in acerola (Malpighia glabra).

    PubMed

    Badejo, Adebanjo A; Fujikawa, Yukichi; Esaka, Muneharu

    2009-04-01

    The Smirnoff-Wheeler (SW) pathway has been proven to be the only significant source of l-ascorbic acid (AsA; vitamin C) in the seedlings of the model plant Arabidopsis thaliana. It is yet uncertain whether the same pathway holds for all other plants and their various organs as AsA may also be synthesized through alternative pathways. In this study, we have cloned some of the genes involved in the SW-pathway from acerola (Malpighia glabra), a plant containing enormous amount of AsA, and examined the expression patterns of these genes in the plant. The AsA contents of acerola leaves were about 8-fold more than that of Arabidopsis with 5-700-fold higher mRNA abundance in AsA-biosynthesizing genes. The unripe fruits have the highest AsA content but the accumulation was substantially repressed as the fruit transitions to maturation. The mRNAs encoding these genes showed correlation in their expression with the AsA contents of the fruits. Although very little AsA was recorded in the seeds the mRNAs encoding all the genes, with the exception of the mitochondrially located L-galactono-1,4-lactone dehydrogenase, were clearly detected in the seeds of the unripe fruits. In young leaves of acerola, the expression of most genes were repressed by the dark and induced by light. However, the expression of GDP-D-mannose pyrophosphorylase similar to that encoded by A. thaliana VTC1 was induced in the dark. The expressions of all the genes surged after 24h following wounding stress on the young leaves. These findings will advance the investigation into the molecular factors regulating the biosynthesis of abundant AsA in acerola. PMID:18952318

  17. Gain of function AMP-activated protein kinase γ3 mutation (AMPKγ3R200Q) in pig muscle increases glycogen storage regardless of AMPK activation.

    PubMed

    Scheffler, Tracy L; Park, Sungkwon; Roach, Peter J; Gerrard, David E

    2016-06-01

    Chronic activation of AMP-activated protein kinase (AMPK) increases glycogen content in skeletal muscle. Previously, we demonstrated that a mutation in the ryanodine receptor (RyR1(R615C)) blunts AMPK phosphorylation in longissimus muscle of pigs with a gain of function mutation in the AMPKγ3 subunit (AMPKγ3(R200Q)); this may decrease the glycogen storage capacity of AMPKγ3(R200Q) + RyR1(R615C) muscle. Therefore, our aim in this study was to utilize our pig model to understand how AMPKγ3(R200Q) and AMPK activation contribute to glycogen storage and metabolism in muscle. We selected and bred pigs in order to generate offspring with naturally occurring AMPKγ3(R200Q), RyR1(R615C), and AMPKγ3(R200Q) + RyR1(R615C) mutations, and also retained wild-type littermates (control). We assessed glycogen content and parameters of glycogen metabolism in longissimus muscle. Regardless of RyR1(R615C), AMPKγ3(R200Q) increased the glycogen content by approximately 70%. Activity of glycogen synthase (GS) without the allosteric activator glucose 6-phosphate (G6P) was decreased in AMPKγ3(R200Q) relative to all other genotypes, whereas both AMPKγ3(R200Q) and AMPKγ3(R200Q) + RyR1(R615C) muscle exhibited increased GS activity with G6P. Increased activity of GS with G6P was not associated with increased abundance of GS or hexokinase 2. However, AMPKγ3(R200Q) enhanced UDP-glucose pyrophosphorylase 2 (UGP2) expression approximately threefold. Although UGP2 is not generally considered a rate-limiting enzyme for glycogen synthesis, our model suggests that UGP2 plays an important role in increasing flux to glycogen synthase. Moreover, we have shown that the capacity for glycogen storage is more closely related to the AMPKγ3(R200Q) mutation than activity. PMID:27302990

  18. HPLC-MS/MS Analyses Show That the Near-Starchless aps1 and pgm Leaves Accumulate Wild Type Levels of ADPglucose: Further Evidence for the Occurrence of Important ADPglucose Biosynthetic Pathway(s) Alternative to the pPGI-pPGM-AGP Pathway

    PubMed Central

    Muñoz, Francisco José; Li, Jun; Almagro, Goizeder; Montero, Manuel; Pujol, Pablo; Galarza, Regina; Kaneko, Kentaro; Oikawa, Kazusato; Wada, Kaede; Mitsui, Toshiaki; Pozueta-Romero, Javier

    2014-01-01

    In leaves, it is widely assumed that starch is the end-product of a metabolic pathway exclusively taking place in the chloroplast that (a) involves plastidic phosphoglucomutase (pPGM), ADPglucose (ADPG) pyrophosphorylase (AGP) and starch synthase (SS), and (b) is linked to the Calvin-Benson cycle by means of the plastidic phosphoglucose isomerase (pPGI). This view also implies that AGP is the sole enzyme producing the starch precursor molecule, ADPG. However, mounting evidence has been compiled pointing to the occurrence of important sources, other than the pPGI-pPGM-AGP pathway, of ADPG. To further explore this possibility, in this work two independent laboratories have carried out HPLC-MS/MS analyses of ADPG content in leaves of the near-starchless pgm and aps1 mutants impaired in pPGM and AGP, respectively, and in leaves of double aps1/pgm mutants grown under two different culture conditions. We also measured the ADPG content in wild type (WT) and aps1 leaves expressing in the plastid two different ADPG cleaving enzymes, and in aps1 leaves expressing in the plastid GlgC, a bacterial AGP. Furthermore, we measured the ADPG content in ss3/ss4/aps1 mutants impaired in starch granule initiation and chloroplastic ADPG synthesis. We found that, irrespective of their starch contents, pgm and aps1 leaves, WT and aps1 leaves expressing in the plastid ADPG cleaving enzymes, and aps1 leaves expressing in the plastid GlgC accumulate WT ADPG content. In clear contrast, ss3/ss4/aps1 leaves accumulated ca. 300 fold-more ADPG than WT leaves. The overall data showed that, in Arabidopsis leaves, (a) there are important ADPG biosynthetic pathways, other than the pPGI-pPGM-AGP pathway, (b) pPGM and AGP are not major determinants of intracellular ADPG content, and (c) the contribution of the chloroplastic ADPG pool to the total ADPG pool is low. PMID:25133777

  19. Proteomic responses of drought-tolerant and drought-sensitive cotton varieties to drought stress.

    PubMed

    Zhang, Haiyan; Ni, Zhiyong; Chen, Quanjia; Guo, Zhongjun; Gao, Wenwei; Su, Xiujuan; Qu, Yanying

    2016-06-01

    Drought, one of the most widespread factors reducing agricultural crop productivity, affects biological processes such as development, architecture, flowering and senescence. Although protein analysis techniques and genome sequencing have made facilitated the proteomic study of cotton, information on genetic differences associated with proteomic changes in response to drought between different cotton genotypes is lacking. To determine the effects of drought stress on cotton seedlings, we used two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry to comparatively analyze proteome of drought-responsive proteins during the seedling stage in two cotton (Gossypium hirsutum L.) cultivars, drought-tolerant KK1543 and drought-sensitive Xinluzao26. A total of 110 protein spots were detected on 2-DE maps, of which 56 were identified by MALDI-TOF and MALDI-TOF/TOF mass spectrometry. The identified proteins were mainly associated with metabolism (46.4 %), antioxidants (14.2 %), and transport and cellular structure (23.2 %). Some key proteins had significantly different expression patterns between the two genotypes. In particular, 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, UDP-D-glucose pyrophosphorylase and ascorbate peroxidase were up-regulated in KK1543 compared with Xinluzao26. Under drought stress conditions, the vacuolar H(+)-ATPase catalytic subunit, a 14-3-3g protein, translation initiation factor 5A and pathogenesis-related protein 10 were up-regulated in KK1543, whereas ribosomal protein S12, actin, cytosolic copper/zinc superoxide dismutase, protein disulfide isomerase, S-adenosylmethionine synthase and cysteine synthase were down-regulated in Xinluzao26. This work represents the first characterization of proteomic changes that occur in response to drought in roots of cotton plants. These differentially expressed proteins may be related to

  20. The Regulation of Sugar Uptake and Accumulation in Bean Pod Tissue 1

    PubMed Central

    Sacher, J. A.

    1966-01-01

    The identity, localization and physiological significance of enzymes involved in sugar uptake and accumulation were determined for endocarp tissue of pods of Kentucky Wonder pole beans (Phaseolus vulgaris). An intracellular, alkaline invertase (pH optimum, 8) was assayed in extracted protein, as well as enzymes involved in sucrose synthesis, namely, uridinediphosphate (UDP-glucose pyrophosphorylase and UDP-glucose-fructose transglucosylase). Indirect evidence indicated the presence also of hexokinase, phosphohexoseisomerase and phosphoglucomutase. The data suggested that sucrose synthesis occurred in the cytoplasm, and that both sugar storage and an alkaline invertase occurred in the vacuole. The latter functions to hydrolyze accumulated sucrose. An outer space invertase (pH optimum, 4.0) was detected, but was variable in occurrence. Although its activity at the cell surface enhanced sucrose uptake, sucrose may be taken up unaltered. Over a wide range of concentrations of exogenous glucose the sucrose/reducing sugar ratio of accumulated sugars remained unchanged at about 20. Synthesis of sucrose appears to be requisite to initial accumulation from glucose or fructose, as free hexoses do not increase at the apparent saturating concentration for uptake. Sucrose accumulation from exogenous hexose represents a steady-state value, in which sucrose is transported across the tonoplast into the vacuole at a rate equivalent to its rate of synthesis. Evidence indicates that this component of the accumulation process involves active transport of sucrose against a concentration gradient. The ratio of sucrose/reducing sugars in the accumulated sugars immediately after a period of uptake was inversely related to the level of inner space invertase. Within 16 hours after a period of accumulation, practically all of the sugar occurs as glucose and fructose. The absence of competition among hexoses and sucrose indicated that a common carrier was not involved in their uptake. From a

  1. Characterization of the GDP-D-mannose biosynthesis pathway in Coxiella burnetii: the initial steps for GDP-β-D-virenose biosynthesis.

    PubMed

    Narasaki, Craig T; Mertens, Katja; Samuel, James E

    2011-01-01

    Coxiella burnetii, the etiologic agent of human Q fever, is a gram-negative and naturally obligate intracellular bacterium. The O-specific polysaccharide chain (O-PS) of the lipopolysaccharide (LPS) of C. burnetii is considered a heteropolymer of the two unusual sugars β-D-virenose and dihydrohydroxystreptose and mannose. We hypothesize that GDP-D-mannose is a metabolic intermediate to GDP-β-D-virenose. GDP-D-mannose is synthesized from fructose-6-phosphate in 3 successive reactions; Isomerization to mannose-6-phosphate catalyzed by a phosphomannose isomerase (PMI), followed by conversion to mannose-1-phosphate mediated by a phosphomannomutase (PMM) and addition of GDP by a GDP-mannose pyrophosphorylase (GMP). GDP-D-mannose is then likely converted to GDP-6-deoxy-D-lyxo-hex-4-ulopyranose (GDP-Sug), a virenose intermediate, by a GDP-mannose-4,6-dehydratase (GMD). To test the validity of this pathway in C. burnetii, three open reading frames (CBU0671, CBU0294 and CBU0689) annotated as bifunctional type II PMI, as PMM or GMD were functionally characterized by complementation of corresponding E. coli mutant strains and in enzymatic assays. CBU0671, failed to complement an Escherichia coli manA (PMM) mutant strain. However, complementation of an E. coli manC (GMP) mutant strain restored capsular polysaccharide biosynthesis. CBU0294 complemented a Pseudomonas aeruginosa algC (GMP) mutant strain and showed phosphoglucomutase activity (PGM) in a pgm E. coli mutant strain. Despite the inability to complement a manA mutant, recombinant C. burnetii PMI protein showed PMM enzymatic activity in biochemical assays. CBU0689 showed dehydratase activity and determined kinetic parameters were consistent with previously reported data from other organisms. These results show the biological function of three C. burnetii LPS biosynthesis enzymes required for the formation of GDP-D-mannose and GDP-Sug. A fundamental understanding of C. burnetii genes that encode PMI, PMM and GMP is

  2. Enzymatic organization of the subcommissural organ.

    PubMed

    Köhl, W

    1975-01-01

    In the subcommissural organ (SCO) of the guinea pig, rat, golden hamster, and mouse the activity and distribution of enzymes related to the energy-supplying metabolism and of some marker enzymes of different cell organelles have been investigated by means of mostly modified histochemical methods. The results were compared with findings in the ciliated ependyma of the ventricular wall and with those in the ependyma of the choroid plexus of the third ventricle. In the ependymal part of the SCO only a moderate activity of hexokinase is observed in its specialized columnar cells whereas a high activity is present both in the ciliated ependyma and the choroid plexus. - The staining pattern of glucose-6-phosphatase is similar to that of hexokinase but this enzyme is found is the SCO only. - Likewise hexokinase, glycogen granules and enzymes related to glycogen metabolism (phosphoglucomutase, uridine-diphosphoglucose pyrophosphorylase, glycogen synthetase and phosphorylase) are regularly found most numerous and active in the nuclear and supra-nuclear area of the ependymal part. These enzymes are less active in both the other ependymal regions. - Uridine-diphosphoglucose dehydrogenase could not be demonstrated in the SCO. The NADP-linked enzymes of the pentose phosphate shunt, glucose-6-phosphate and 6-phosphogluconate dehydrogenase, show a moderate activity which decreases also from the nuclear towards the apical area of the ependymal cells of the SCO. Enzymes of the glycolytic pathway, such as glucosephosphate isomerase, fructose-6-phosphate kinase, fructose-I,6-diphosphate aldolase, glyceraldehyde-3-phosphate and lactate dehydrogenase, are highly active in the SCO and are located mainly in the supranuclear area, too. Fructose-1,6-diphosphatase could not be demonstrated thus indicating that in the SCO the pathway is most probably only glycolytic but not gluconeogenetic. Compared to the ependyma of the ventricular wall and of the choroid plexus, in the SCO the M type

  3. Synthesis of the A-band polysaccharide sugar D-rhamnose requires Rmd and WbpW: identification of multiple AlgA homologues, WbpW and ORF488, in Pseudomonas aeruginosa.

    PubMed

    Rocchetta, H L; Pacan, J C; Lam, J S

    1998-09-01

    Pseudomonas aeruginosa is capable of producing various cell-surface polysaccharides including alginate, A-band and B-band lipopolysaccharides (LPS). The D-mannuronic acid residues of alginate and the D-rhamnose (D-Rha) residues of A-band polysaccharide are both derived from the common sugar nucleotide precursor GDP-D-mannose (D-Man). Three genes, rmd, gmd and wbpW, which encode proteins involved in the synthesis of GDP-D-Rha, have been localized to the 5' end of the A-band gene cluster. In this study, WbpW was found to be homologous to phosphomannose isomerases (PMIs) and GDP-mannose pyrophosphorylases (GMPs) involved in GDP-D-Man biosynthesis. To confirm the enzymatic activity of WbpW, Escherichia coli PMI and GMP mutants deficient in the K30 capsule were complemented with wbpW, and restoration of K30 capsule production was observed. This indicates that WbpW, like AlgA, is a bifunctional enzyme that possesses both PMI and GMP activities for the synthesis of GDP-D-Man. No gene encoding a phosphomannose mutase (PMM) enzyme could be identified within the A-band gene cluster. This suggests that the PMM activity of AlgC may be essential for synthesis of the precursor pool of GDP-D-Man, which is converted to GDP-D-Rha for A-band synthesis. Gmd, a previously reported A-band enzyme, and Rmd are predicted to perform the two-step conversion of GDP-D-Man to GDP-D-Rha. Chromosomal mutants were generated in both rmd and wbpW. The Rmd mutants do not produce A-band LPS, while the WbpW mutants synthesize very low amounts of A band after 18 h of growth. The latter observation was thought to result from the presence of the functional homologue AlgA, which may compensate for the WbpW deficiency in these mutants. Thus, WbpW AlgA double mutants were constructed. These mutants also produced low levels of A-band LPS. A search of the PAO1 genome sequence identified a second AlgA homologue, designated ORF488, which may be responsible for the synthesis of GDP-D-Man in the absence of Wbp