Deregulation of feedback inhibition of phosphoenolpyruvate carboxylase for improved lysine production in Corynebacterium glutamicum.

PubMed

Chen, Zhen; Bommareddy, Rajesh Reddy; Frank, Doinita; Rappert, Sugima; Zeng, An-Ping

2014-02-01

Allosteric regulation of phosphoenolpyruvate carboxylase (PEPC) controls the metabolic flux distribution of anaplerotic pathways. In this study, the feedback inhibition of Corynebacterium glutamicum PEPC was rationally deregulated, and its effect on metabolic flux redistribution was evaluated. Based on rational protein design, six PEPC mutants were designed, and all of them showed significantly reduced sensitivity toward aspartate and malate inhibition. Introducing one of the point mutations (N917G) into the ppc gene, encoding PEPC of the lysine-producing strain C. glutamicum LC298, resulted in ∼37% improved lysine production. In vitro enzyme assays and (13)C-based metabolic flux analysis showed ca. 20 and 30% increases in the PEPC activity and corresponding flux, respectively, in the mutant strain. Higher demand for NADPH in the mutant strain increased the flux toward pentose phosphate pathway, which increased the supply of NADPH for enhanced lysine production. The present study highlights the importance of allosteric regulation on the flux control of central metabolism. The strategy described here can also be implemented to improve other oxaloacetate-derived products.

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

PubMed

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

1989-04-01

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

New insights into the post-translational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination.

PubMed

Ruiz-Ballesta, Isabel; Baena, Guillermo; Gandullo, Jacinto; Wang, Liqun; She, Yi-Min; Plaxton, William Charles; Echevarría, Cristina

2016-05-01

Phosphoenolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) was characterized in developing and germinating sorghum seeds, focusing on the transcript and polypeptide abundance of multiple plant-type phosphoenolpyruvate carboxylase (PTPC) genes, and the post-translational modification of each isoenzyme by phosphorylation versus monoubiquitination during germination. We observed high levels of SbPPC4 (Sb07g014960) transcripts during early development (stage I), and extensive transcript abundance of SbPPC2 (Sb02g021090) and SbPPC3 (Sb04g008720) throughout the entire life cycle of the seed. Although tandem mass spectrometry (MS) analysis of immunopurified PTPC indicated that four different PTPC isoenzymes were expressed in the developing and germinating seeds, SbPPC3 was the most abundant isozyme of the developing seed, and of the embryo and the aleurone layer of germinating seeds. In vivo phosphorylation of the different PTPC isoenzymes at their conserved N-terminal seryl phosphorylation site during germination was also established by MS/MS analysis. Furthermore, three of the four isoenzymes were partially monoubiquitinated, with MS/MS pinpointing SbPPC2 and SbPPC3 monoubiquitination at the conserved Lys-630 and Lys-624 residues, respectively. Our results demonstrate that monoubiquitination and phosphorylation simultaneously occur in vivo with different PTPC isozymes during seed germination. In addition, we show that PTPC monoubiquitination in germinating sorghum seeds always increases at stage II (emergence of the radicle), is maintained during the aerobic period of rapid cell division and reserve mobilization, and remains relatively constant until stage IV-V when coleoptiles initiate the formation of the photosynthetic tissues. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Phosphoenolpyruvate metabolism in Jerusalem artichoke mitochondria.

PubMed

de Bari, Lidia; Valenti, Daniela; Pizzuto, Roberto; Atlante, Anna; Passarella, Salvatore

2007-04-01

We report here initial studies on phosphoenolpyruvate metabolism in coupled mitochondria isolated from Jerusalem artichoke tubers. It was found that: (1) phosphoenolpyruvate can be metabolized by Jerusalem artichoke mitochondria by virtue of the presence of the mitochondrial pyruvate kinase, shown both immunologically and functionally, located in the inner mitochondrial compartments and distinct from the cytosolic pyruvate kinase as shown by the different pH and inhibition profiles. (2) Jerusalem artichoke mitochondria can take up externally added phosphoenolpyruvate in a proton compensated manner, in a carrier-mediated process which was investigated by measuring fluorimetrically the oxidation of intramitochondrial pyridine nucleotide which occurs as a result of phosphoenolpyruvate uptake and alternative oxidase activation. (3) The addition of phosphoenolpyruvate causes pyruvate and ATP production, as monitored via HPLC, with their efflux into the extramitochondrial phase investigated fluorimetrically. Such an efflux occurs via the putative phosphoenolpyruvate/pyruvate and phosphoenolpyruvate/ATP antiporters, which differ from each other and from the pyruvate and the adenine nucleotide carriers, in the light of the different sensitivity to non-penetrant compounds. These carriers were shown to regulate the rate of efflux of both pyruvate and ATP. The appearance of citrate and oxaloacetate outside mitochondria was also found as a result of phosphoenolpyruvate addition.

Influence of NaCl on Growth, Proline, and Phosphoenolpyruvate Carboxylase Levels in Mesembryanthemum crystallinum Suspension Cultures 1

PubMed Central

Thomas, John C.; De Armond, Richard L.; Bohnert, Hans J.

1992-01-01

The facultative halophyte Mesembryanthemum crystallinum responds to salt stress by increasing the levels of phosphoenolpyruvate carboxylase (PEPCase) and other enzymes associated with Crassulacean acid metabolism. A more common response to salt stress in sensitive and tolerant species, including M. crystallinum, is the accumulation of proline. We have established M. crystallinum suspension cultures to investigate whether both these salt-induced responses occur at the cellular level. Leaf-and root-derived cultures maintain 5% of the total soluble amino acids as proline. Cell culture growth slows upon addition of 400 millimolar NaCl, and proline levels increase to 40% of the total soluble amino acids. These results suggest a functional salt-stress and response program in Mesembryanthemum cells. Suspension cultures grown with or without 400 millimolar NaCl have PEPCase levels that compare with those from roots and unstressed leaves. The predominant protein cross-reacting with an anti-PEPCase antibody corresponds to 105 kilodaltons (apparent molecular mass), whereas a second species of approximately 110 kilodaltons is present at low levels. In salt-stressed leaves, the 110 kilodalton protein is more prevalent. Levels of mRNA for both ppc1 (salt stress induced in leaves) and ppc2 (constitutive) genes in salt-treated suspensions cultures are equal to unstressed leaves, and only twice the levels found in untreated suspension cultures. Whereas cells accumulate proline in response to NaCl, PEPCase protein amounts remain similar in salt-treated and untreated cultures. The induction upon salt stress of the 110 kilodalton PEPCase protein and other Crassulacean acid metabolism enzymes in organized tissues is not observed in cell culture and may depend on tissue-dependent or photoautotrophy-dependent programs. ImagesFigure 4Figure 5 PMID:16668687

High activity and stability of codon-optimized phosphoenolpyruvate carboxylase from Photobacterium profundum SS9 at low temperatures and its application for in vitro production of oxaloacetate.

PubMed

Park, Soohyun; Hong, Soohye; Pack, Seung Pil; Lee, Jinwon

2014-02-01

Phosphoenolpyruvate carboxylase (PEPC) of Photobacterium profundum SS9 can be expressed and purified using the Escherichia coli expression system. In this study, a codon-optimized PEPC gene (OPPP) was used to increase expression levels. We confirmed OPPP expression and purified it from extracts of recombinant E. coli SGJS117 harboring the OPPP gene. The purified OPPP showed a specific activity value of 80.3 U/mg protein. The OPPP was stable under low temperature (5-30 °C) and weakly basic conditions (pH 8.5-10). The enzymatic ability of OPPP was investigated for in vitro production of oxaloacetate using phosphoenolpyruvate (PEP) and bicarbonate. Only samples containing the OPPP, PEP, and bicarbonate resulted in oxaloacetate production. OPPP production system using E. coli could be a platform technology to produce high yields of heterogeneous gene and provide the PEPC enzyme, which has high enzyme activity.

Relationship between NH+4 Assimilation Rate and in Vivo Phosphoenolpyruvate Carboxylase Activity 1

PubMed Central

Vanlerberghe, Greg C.; Schuller, Kathryn A.; Smith, Ronald G.; Feil, Regina; Plaxton, William C.; Turpin, David H.

1990-01-01

The rate of NH4+ assimilation by N-limited Selenastrum minutum (Naeg.) Collins cells in the dark was set as an independent variable and the relationship between NH4+ assimilation rate and in vivo activity of phosphoenolpyruvate carboxylase (PEPC) was determined. In vivo activity of PEPC was measured by following the incorporation of H14CO−3 into acid stable products. A linear relationship of 0.3 moles C fixed via PEPC per mole N assimilated was observed. This value agrees extremely well with the PEPC requirement for the synthesis of the amino acids found in total cellular protein. Determinations of metabolite levels in vivo at different rates of N assimilation indicated that the known metabolite effectors of S. minutum PEPC in vitro (KA Schuller, WC Plaxton, DH Turpin, [1990] Plant Physiol 93: 1303-1311) are important regulators of this enzyme during N assimilation. As PEPC activity increased in response to increasing rates of N assimilation, there was a corresponding decline in the level of PEPC inhibitors (2-oxoglutarate, malate), an increase in the level of PEPC activators (glutamine, dihydroxyacetone phosphate), and an increase in the Gln/Glu ratio. Treatment of N-limited cells with azaserine caused an increase in the Gln/Glu ratio resulting in increased PEPC activity in the absence of N assimilation. We suggest glutamate and glutamine play a key role in regulating the anaplerotic function of PEPC in this C3 organism. PMID:16667699

Towards efficient photosynthesis: overexpression of Zea mays phosphoenolpyruvate carboxylase in Arabidopsis thaliana.

PubMed

Kandoi, Deepika; Mohanty, Sasmita; Govindjee; Tripathy, Baishnab C

2016-12-01

Plants with C4 photosynthesis are efficient in carbon assimilation and have an advantage over C3 photosynthesis. In C4 photosynthesis, the primary CO 2 fixation is catalyzed by phosphoenolpyruvate carboxylase (PEPC). Here, we show that overexpression of Zea mays PEPC cDNA, under the control of 35 S promoter, in Arabidopsis thaliana resulted in ~7-10 fold higher protein abundance and ~7-10 fold increase in PEPC activity in the transgenic lines than that in the vector control. We suggest that overexpression of PEPC played an anaplerotic role to increase the supply of 4-carbon carboxylic acids, which provided carbon skeletons for increased amino acid and protein synthesis. Higher protein content must have been responsible for increased metabolic processes including chlorophyll biosynthesis, photosynthesis, and respiration. Consequently, the PEPC-overexpressed transgenic plants had higher chlorophyll content, enhanced electron transport rate (ETR), lower non-photochemical quenching (NPQ) of chlorophyll a fluorescence, and a higher performance index (PI) than the vector control. Consistent with these observations, the rate of CO 2 assimilation, the starch content, and the dry weight of PEPC-overexpressed plants increased by 14-18 %, 10-18 %, and 6.5-16 %, respectively. Significantly, transgenics were tolerant to salt stress as they had increased ability to synthesize amino acids, including the osmolyte proline. NaCl (150 mM)-treated transgenic plants had higher variable to maximum Chl a fluorescence (F v /F m ) ratio, higher PI, higher ETR, and lower NPQ than the salt-treated vector controls. These results suggest that expression of C4 photosynthesis enzyme(s) in a C3 plant can improve its photosynthetic capacity with enhanced tolerance to salinity stress.

Induction of mRNA for Phosphoenolpyruvate Carboxylase Is Correlated with a Decrease in Shoot Water Content in Well-Irrigated Mesembryanthemum crystallinum 1

PubMed Central

Schmitt, Jürgen M.; Piepenbrock, Mechtild

1992-01-01

The abundance of mRNA specific for phosphoenolpyruvate carboxylase (PEPCase) was measured in leaves from well-watered plants of Mesembryanthemum crystallinum. Plants grown side by side in pots of four different volumes (0.16, 0.74, 2.6, 6.5 liters) were compared. The time of increase in the steady-state level of PEPCase mRNA in well-watered plants was dependent on soil volume. The larger the pot, the later PEPCase transcripts were increased. PEPCase mRNA induction started when shoot water content decreased to well below 4000% of dry weight. No positive correlation with the developmental status of the plants could be found. The data indicate that PEPCase mRNA induction in well-watered plants up to 10 weeks of age is controlled environmentally rather than developmentally. ImagesFigure 2 PMID:16668951

Effects of an inhibitor of phosphoenolpyruvate carboxylase on photosynthesis of the terrestrial forms of amphibious Eleocharis species.

PubMed

Ueno, Osamu; Ishimaru, Ken

2002-01-01

The leafless amphibious sedge Eleocharis vivipara develops culms with C(4) traits and Kranz anatomy under terrestrial conditions, but develops culms with C(3) traits and non-Kranz anatomy under submerged conditions. The culms of the terrestrial form have high C(4) enzyme activities, while those of the submerged form have decreased C(4) enzyme activities. The culms accumulate ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the mesophyll cells (MC) and the bundle sheath cells. The Rubisco in the MC may be responsible for the operation of the C(3) pathway in the submerged form. To verify the presence of the C(3) cycle in the MC, we examined the effects of 3,3-dichloro-2-(dihydroxyphosphinoylmethyl) -propenoate (DCDP), an inhibitor of phosphoenolpyruvate carboxylase (PEPC), on photosynthesis in culms of the terrestrial forms of E. vivipara and related amphibious species, E. baldwinii and E. retroflexa ssp. chaetaria. When 1 mM DCDP was fed via the transpiration stream to excised leaves, photosynthesis was inhibited completely in Fimbristylis dichotoma (C(4) control), but by only 20% in potato (C(3) control). In the terrestrial Eleocharis plants, the degree of inhibition of photosynthesis by DCDP was intermediate between those of the C(4) and C(3) plants, at 58-81%. These results suggest that photosynthesis under DCDP treatment in the terrestrial Eleocharis plants is due mainly to fixation of atmospheric CO(2) by Rubisco and probably the C(3) cycle in the MC. These features are reminiscent of those in C(4)-like plants. Differential effects of DCDP on photosynthesis of the 3 Eleocharis species are discussed in relation to differences in the degree of Rubisco accumulation and C(3) activity in the MC.

Changes in PEP carboxylase, rubisco and rubisco activase mRNA levels from maize (Zea mays) exposed to a chronic ozone stress.

PubMed

Leitao, Louis; Maoret, Jean-José; Biolley, Jean-Philippe

2007-01-01

We quantified the ozone impact on levels of Zea mays L. cv. Chambord mRNAs encoding C4-phosphoenolpyruvate carboxylase (C4-PEPc), ribulose-l,5-bisphosphate carboxylase/oxygenase small and large subunits (Rubisco-SSU and Rubisco-LSU, respectively) and Rubisco activase (RCA) using real-time RT-PCR. Foliar pigment content, PEPc and Rubisco protein amounts were simultaneously determined. Two experiments were performed to study the ozone response of the 5th and the 10th leaf. For each experiment, three ozone concentrations were tested in open-top chambers: non-filtered air (NF, control) and non-filtered air containing 40 (+40) and 80 nL L-1 (+80) ozone. Regarding the 5th leaf, +40 atmosphere induced a loss in pigmentation, PEPc and Rubisco activase mRNAs. However, it was unable to notably depress carboxylase protein amounts and mRNAs encoding Rubisco. Except for Rubisco mRNAs, all other measured parameters from 5th leaf were depressed by +80 atmosphere. Regarding the 10th leaf, +40 atmosphere increased photosynthetic pigments and transcripts encoding Rubisco and Rubisco activase. Rubisco and PEPc protein amounts were not drastically changed, even if they tended to be increased. Level of C4-PEPc mRNA remained almost stable. In response to +80 atmosphere, pigments and transcripts encoding PEPc were notably decreased. Rubisco and PEPc protein amounts also declined to a lesser extent. Conversely, the level of transcripts encoding both Rubisco subunits and Rubisco activase that were not consistently disturbed tended to be slightly augmented. So, the present study suggests that maize leaves can respond differentially to a similar ozone stress.

Induction of Crassulacean Acid Metabolism in Mesembryanthemum crystallinum by High Salinity: Mass Increase and de Novo Synthesis of PEP-Carboxylase 1

PubMed Central

Höfner, Roswitha; Vazquez-Moreno, Luz; Winter, Klaus; Bohnert, Hans J.; Schmitt, Jürgen M.

1987-01-01

Intact plants of the halophilic species Mesembryanthemum crystallinum were induced to exhibit Crassulacean acid metabolism by irrigation with nutrient solution containing 500 millimolar NaCl. During the induction period, the extractable activity of phosphoenolpyruvate carboxylase (PEPcase) increased approximately 40-fold. This increase was linearly correlated with a mass increase of PEPcase protein as measured by single radial immunodiffusion. De novo synthesis of PEPcase protein was shown by immunoprecipitation of the newly synthesized, radioactively labeled protein in leaf discs from salt-treated plants. Nontreated plants were characterized by a low level of the enzyme and low rates of PEPcase synthesis. Synthesis of this enzyme in leaf discs was correlated with the concentration of NaCl in the nutrient solution during growth. Images Fig. 1 Fig. 2 Fig. 3 PMID:16665363

Relationship between NH sub 4 sup + assimilation rate and in vivo phosphoenolpyruvate carboxylase activity

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

Vanlerberghe, G.C.; Schuller, K.A.; Smith, R.G.

The rate of NH{sub 4}{sup +} assimilation by N-limited Selenastrum minutum (Naeg.) Collins cells in the dark was set as an independent variable and the relationship between NH{sub 4}{sup +} assimilation rate and in vivo activity of phosphoenolpyruvate carboxylase (PEPC) was determined. In vivo activity of PEPC was measured by following the incorporation of H{sup 14}CO{sub 3}{sup {minus}} into acid stable products. A linear relationship of 0.3 moles C fixed via PEPC per mole N assimilated was observed. This value agrees extremely well with the PEPC requirement for the synthesis of the amino acids found in total cellular protein. Determinationsmore » of metabolite levels in vivo at different rates of N assimilation indicated that the known metabolite effectors of S. minutum PEPC in vitro (KA Schuller, WC Plaxton, DH Turpin, (1990) Plant Physiol 93: 1303-1311) are important regulators of this enzyme during N assimilation. As PEPC activity increased in response to increasing rates of N assimilation, there was a corresponding decline in the level of PEPC inhibitors (2-oxoglutarate, malate), an increase in the level of PEPC activators (glutamine, dihydroxyacetone phosphate), and an increase in the Gln/Glu ratio. Treatment of N-limited cells with azaserine caused an increase in the Gln/Glu ratio resulting in increased PEPC activity in the absence of N assimilation. We suggest glutamate and glutamine play a key role in regulating the anaplerotic function of PEPC in this C{sub 3} organism.« less

Genome-wide Analysis of Phosphoenolpyruvate Carboxylase Gene Family and Their Response to Abiotic Stresses in Soybean

PubMed Central

Wang, Ning; Zhong, Xiujuan; Cong, Yahui; Wang, Tingting; Yang, Songnan; Li, Yan; Gai, Junyi

2016-01-01

Phosphoenolpyruvate carboxylase (PEPC) plays an important role in assimilating atmospheric CO2 during C4 and crassulacean acid metabolism photosynthesis, and also participates in various non-photosynthetic processes, including fruit ripening, stomatal opening, supporting carbon–nitrogen interactions, seed formation and germination, and regulation of plant tolerance to stresses. However, a comprehensive analysis of PEPC family in Glycine max has not been reported. Here, a total of ten PEPC genes were identified in soybean and denominated as GmPEPC1-GmPEPC10. Based on the phylogenetic analysis of the PEPC proteins from 13 higher plant species including soybean, PEPC family could be classified into two subfamilies, which was further supported by analyses of their conserved motifs and gene structures. Nineteen cis-regulatory elements related to phytohormones, abiotic and biotic stresses were identified in the promoter regions of GmPEPC genes, indicating their roles in soybean development and stress responses. GmPEPC genes were expressed in various soybean tissues and most of them responded to the exogenously applied phytohormones. GmPEPC6, GmPEPC8 and GmPEPC9 were significantly induced by aluminum toxicity, cold, osmotic and salt stresses. In addition, the enzyme activities of soybean PEPCs were also up-regulated by these treatments, suggesting their potential roles in soybean response to abiotic stresses. PMID:27924923

Multiple inter-kingdom horizontal gene transfers in the evolution of the phosphoenolpyruvate carboxylase gene family.

PubMed

Peng, Yingmei; Cai, Jing; Wang, Wen; Su, Bing

2012-01-01

Pepcase is a gene encoding phosphoenolpyruvate carboxylase that exists in bacteria, archaea and plants,playing an important role in plant metabolism and development. Most plants have two or more pepcase genes belonging to two gene sub-families, while only one gene exists in other organisms. Previous research categorized one plant pepcase gene as plant-type pepcase (PTPC) while the other as bacteria-type pepcase (BTPC) because of its similarity with the pepcase gene found in bacteria. Phylogenetic reconstruction showed that PTPC is the ancestral lineage of plant pepcase, and that all bacteria, protistpepcase and BTPC in plants are derived from a lineage of pepcase closely related with PTPC in algae. However, their phylogeny contradicts the species tree and traditional chronology of organism evolution. Because the diversification of bacteria occurred much earlier than the origin of plants, presumably all bacterialpepcase derived from the ancestral PTPC of algal plants after divergingfrom the ancestor of vascular plant PTPC. To solve this contradiction, we reconstructed the phylogeny of pepcase gene family. Our result showed that both PTPC and BTPC are derived from an ancestral lineage of gamma-proteobacteriapepcases, possibly via an ancient inter-kingdom horizontal gene transfer (HGT) from bacteria to the eukaryotic common ancestor of plants, protists and cellular slime mold. Our phylogenetic analysis also found 48other pepcase genes originated from inter-kingdom HGTs. These results imply that inter-kingdom HGTs played important roles in the evolution of the pepcase gene family and furthermore that HGTsare a more frequent evolutionary event than previouslythought.

In vivo monoubiquitination of anaplerotic phosphoenolpyruvate carboxylase occurs at Lys624 in germinating sorghum seeds.

PubMed

Ruiz-Ballesta, Isabel; Feria, Ana-Belén; Ni, Hong; She, Yi-Min; Plaxton, William Charles; Echevarría, Cristina

2014-02-01

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is an important cytosolic regulatory enzyme that plays a pivotal role in numerous physiological processes in plants, including seed development and germination. Previous studies demonstrated the occurrence of immunoreactive PEPC polypeptides of ~110 kDa and 107 kDa (p110 and p107, respectively) on immunoblots of clarified extracts of germinating sorghum (Sorghum bicolor) seeds. In order to establish the biochemical basis for this observation, a 460 kDa PEPC heterotetramer composed of an equivalent ratio of p110 and p107 subunits was purified to near homogeneity from the germinated seeds. Mass spectrometry established that p110 and p107 are both encoded by the same plant-type PEPC gene (CP21), but that p107 was in vivo monoubiquitinated at Lys624 to form p110. This residue is absolutely conserved in vascular plant PEPCs and is proximal to a PEP-binding/catalytic domain. Anti-ubiquitin IgG immunodetected p110 but not p107, whereas incubation with a deubiquitinating enzyme (USP-2 core) efficiently converted p110 into p107, while relieving the enzyme's feedback inhibition by L-malate. Partial PEPC monoubiquitination was also detected during sorghum seed development. It is apparent that monoubiquitination at Lys624 is opposed to phosphorylation at Ser7 in terms of regulating the catalytic activity of sorghum seed PEPC. PEPC monoubiquitination is hypothesized to fine-tune anaplerotic carbon flux according to the cell's immediate physiological requirements for tricarboxylic acid cycle intermediates needed in support of biosynthesis and carbon-nitrogen interactions.

In vivo monoubiquitination of anaplerotic phosphoenolpyruvate carboxylase occurs at Lys624 in germinating sorghum seeds

PubMed Central

Echevarría, Cristina

2014-01-01

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is an important cytosolic regulatory enzyme that plays a pivotal role in numerous physiological processes in plants, including seed development and germination. Previous studies demonstrated the occurrence of immunoreactive PEPC polypeptides of ~110kDa and 107kDa (p110 and p107, respectively) on immunoblots of clarified extracts of germinating sorghum (Sorghum bicolor) seeds. In order to establish the biochemical basis for this observation, a 460kDa PEPC heterotetramer composed of an equivalent ratio of p110 and p107 subunits was purified to near homogeneity from the germinated seeds. Mass spectrometry established that p110 and p107 are both encoded by the same plant-type PEPC gene (CP21), but that p107 was in vivo monoubiquitinated at Lys624 to form p110. This residue is absolutely conserved in vascular plant PEPCs and is proximal to a PEP-binding/catalytic domain. Anti-ubiquitin IgG immunodetected p110 but not p107, whereas incubation with a deubiquitinating enzyme (USP-2 core) efficiently converted p110 into p107, while relieving the enzyme’s feedback inhibition by l-malate. Partial PEPC monoubiquitination was also detected during sorghum seed development. It is apparent that monoubiquitination at Lys624 is opposed to phosphorylation at Ser7 in terms of regulating the catalytic activity of sorghum seed PEPC. PEPC monoubiquitination is hypothesized to fine-tune anaplerotic carbon flux according to the cell’s immediate physiological requirements for tricarboxylic acid cycle intermediates needed in support of biosynthesis and carbon–nitrogen interactions. PMID:24288181

Drought tolerance and proteomics studies of transgenic wheat containing the maize C4 phosphoenolpyruvate carboxylase (PEPC) gene.

PubMed

Qin, Na; Xu, Weigang; Hu, Lin; Li, Yan; Wang, Huiwei; Qi, Xueli; Fang, Yuhui; Hua, Xia

2016-11-01

Enhancing drought tolerance of crops has been a great challenge in crop improvement. Here, we report the maize phosphoenolpyruvate carboxylase (PEPC) gene was able to confer drought tolerance and increase grain yield in transgenic wheat (Triticum aestivum L.) plants. The improved of drought tolerance was associated with higher levels of proline, soluble sugar, soluble protein, and higher water use efficiency. The transgenic wheat plants had also a more extensive root system as well as increased photosynthetic capacity during stress treatments. The increased grain yield of the transgenic wheat was contributed by improved biomass, larger spike and grain numbers, and heavier 1000-grain weight under drought-stress conditions. Under non-stressed conditions, there were no significant increases in these of the measured traits except for photosynthetic rate when compared with parental wheat. Proteomic research showed that the expression levels of some proteins, including chlorophyll A-B binding protein and pyruvate, phosphate dikinase, which are related to photosynthesis, PAP fibrillin, which is involved in cytoskeleton synthesis, S-adenosylmethionine synthetase, which catalyzes methionine synthesis, were induced in the transgenic wheat under drought stress. Additionally, the expression of glutamine synthetase, which is involved in ammonia assimilation, was induced by drought stress in the wheat. Our study shows that PEPC can improve both stress tolerance and grain yield in wheat, demonstrating the efficacy of PEPC in crop improvement.

Expression of codon-optmized phosphoenolpyruvate carboxylase gene from Glaciecola sp. HTCC2999 in Escherichia coli and its application for C4 chemical production.

PubMed

Park, Soohyun; Pack, Seung Pil; Lee, Jinwon

2012-08-01

We examined the expression of the phosphoenolpyruvate carboxylase (PEPC) gene from marine bacteria in Escherichia coli using codon optimization. The codon-optimized PEPC gene was expressed in the E. coli K-12 strain W3110. SDS-PAGE analysis revealed that the codon-optimized PEPC gene was only expressed in E. coli, and measurement of enzyme activity indicated the highest PEPC activity in the E. coli SGJS112 strain that contained the codon-optimized PEPC gene. In fermentation assays, the E. coli SGJS112 produced the highest yield of oxaloacetate using glucose as the source and produced a 20-times increase in the yield of malate compared to the control. We concluded that the codon optimization enabled E. coli to express the PEPC gene derived from the Glaciecola sp. HTCC2999. Also, the expressed protein exhibited an enzymatic activity similar to that of E. coli PEPC and increased the yield of oxaloacetate and malate in an E. coli system.

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

PubMed

Potter, J W; Black, C C

1982-08-01

The distribution and molecular weights of cellular proteins in soluble and membrane-associated locations were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining of leaf (Digitaria sanguinalis L. Scop.) extracts and isolated cell extracts. Leaf polypeptides also were pulse-labeled, followed by isolation of the labeled leaf cell types and analysis of the newly synthesized polypeptides in each cell type by electrophoresis and fluorography.Comparison of the electrophoretic patterns of crabgrass whole leaf polypeptides with isolated cell-type polypeptides indicated a difference in protein distribution patterns for the two cell types. The mesophyll cells exhibited a greater allocation of total cellular protein into membrane-associated proteins relative to soluble proteins. In contrast, the bundle sheath cells exhibited a higher percentage of total cellular protein in soluble proteins. Phosphoenolpyruvate carboxylase was the major soluble protein in the mesophyll cell and ribulose bisphosphate carboxylase was the major soluble protein in the bundle sheath cell. The majority of in vivo(35)S-pulse-labeled proteins synthesized by the two crabgrass cell types corresponded in molecular weight to the proteins present in the cell types which were detected by conventional staining techniques. The bundle sheath cell and mesophyll cell fluorograph profiles each had 15 major (35)S-labeled proteins. The major incorporation of (35)S by bundle sheath cells was into products which co-electrophoresed with the large and small subunits of ribulose bisphosphate carboxylase. In contrast, a major (35)S-labeled product in mesophyll cell extracts co-electrophoresed with the subunit of phosphoenolpyruvate carboxylase. Both cell types exhibited equivalent in vivo labeling of a polypeptide with one- and two-dimensional electrophoretic behavior similar to the major apoprotein of the light-harvesting chlorophyll a/b protein. Results from the use of

Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032.

PubMed

Wada, Masaru; Sawada, Kazunori; Ogura, Kotaro; Shimono, Yuta; Hagiwara, Takuya; Sugimoto, Masakazu; Onuki, Akiko; Yokota, Atsushi

2016-02-01

Phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum ATCC13032, a glutamic-acid producing actinobacterium, is subject to feedback inhibition by metabolic intermediates such as aspartic acid and 2-oxoglutaric acid, which implies the importance of PEPC in replenishing oxaloacetic acid into the TCA cycle. Here, we investigated the effects of feedback-insensitive PEPC on glutamic acid production. A single amino-acid substitution in PEPC, D299N, was found to relieve the feedback control by aspartic acid, but not by 2-oxoglutaric acid. A simple mutant, strain R1, having the D299N substitution in PEPC was constructed from ATCC 13032 using the double-crossover chromosome replacement technique. Strain R1 produced glutamic acid at a concentration of 31.0 g/L from 100 g/L glucose in a jar fermentor culture under biotin-limited conditions, which was significantly higher than that of the parent, 26.0 g/L (1.19-fold), indicative of the positive effect of desensitized PEPC on glutamic acid production. Another mutant, strain DR1, having both desensitized PEPC and PYK-gene deleted mutations, was constructed in a similar manner using strain D1 with a PYK-gene deleted mutation as the parent. This mutation had been shown to enhance glutamic acid production in our previous study. Although marginal, strain D1 produced higher glutamic acid, 28.8 g/L, than ATCC13032 (1.11-fold). In contrast, glutamic acid production by strain DR-1 was elevated up to 36.9 g/L, which was 1.42-fold higher than ATCC13032 and significantly higher than the other three strains. The results showed a synergistic effect of these two mutations on glutamic acid production in C. glutamicum. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Discrimination in the dark. Resolving the interplay between metabolic and physical constraints to phosphoenolpyruvate carboxylase activity during the crassulacean acid metabolism cycle.

PubMed

Griffiths, Howard; Cousins, Asaph B; Badger, Murray R; von Caemmerer, Susanne

2007-02-01

A model defining carbon isotope discrimination (delta13C) for crassulacean acid metabolism (CAM) plants was experimentally validated using Kalanchoe daigremontiana. Simultaneous measurements of gas exchange and instantaneous CO2 discrimination (for 13C and 18O) were made from late photoperiod (phase IV of CAM), throughout the dark period (phase I), and into the light (phase II). Measurements of CO2 response curves throughout the dark period revealed changing phosphoenolpyruvate carboxylase (PEPC) capacity. These systematic changes in PEPC capacity were tracked by net CO2 uptake, stomatal conductance, and online delta13C signal; all declined at the start of the dark period, then increased to a maximum 2 h before dawn. Measurements of delta13C were higher than predicted from the ratio of intercellular to external CO2 (p(i)/p(a)) and fractionation associated with CO2 hydration and PEPC carboxylations alone, such that the dark period mesophyll conductance, g(i), was 0.044 mol m(-2) s(-1) bar(-1). A higher estimate of g(i) (0.085 mol m(-2) s(-1) bar(-1)) was needed to account for the modeled and measured delta18O discrimination throughout the dark period. The differences in estimates of g(i) from the two isotope measurements, and an offset of -5.5 per thousand between the 18O content of source and transpired water, suggest spatial variations in either CO2 diffusion path length and/or carbonic anhydrase activity, either within individual cells or across a succulent leaf. Our measurements support the model predictions to show that internal CO2 diffusion limitations within CAM leaves increase delta13C discrimination during nighttime CO2 fixation while reducing delta13C during phase IV. When evaluating the phylogenetic distribution of CAM, carbon isotope composition will reflect these diffusive limitations as well as relative contributions from C3 and C4 biochemistry.

Phosphoenolpyruvate carboxylase: a new era of structural biology.

PubMed

Izui, Katsura; Matsumura, Hiroyoshi; Furumoto, Tsuyoshi; Kai, Yasushi

2004-01-01

There have been remarkable advances in our knowledge of this important enzyme in the last decade. This review focuses on three recent topics: the three-dimensional structure of the protein, molecular mechanisms of catalytic and regulatory functions, and the molecular cloning and characterization of PEPC kinases, which are Ser/Thr kinases involved specifically in regulatory phosphorylation of vascular plant PEPC. Analysis by X-ray crystallography and site-directed mutagenesis for E. coli and maize PEPC identified the catalytic site and allosteric effector binding sites, and revealed the functional importance of mobile loops. We present the reaction mechanism of PEPC in which we assign the roles of individual amino acid residues. We discuss the unique molecular property of PEPC kinase and its possible regulation at the post-translational level.

Variation in levels of some leaf enzymes.

PubMed

Downton, J; Slatyer, R O

1971-03-01

Several procedures were compared for efficiency in the extraction of certain leaf enzymes (phosphoenolpyruvate carboxylase, ribulose 1,5-diphosphate carboxylase and malate dehydrogenase) in Atriplex hastata (a "C3" species exhibiting conventional photosynthetic metabolism), and in A. spongiosa (a "C4" species in which the initial photosynthetic products are C4 dicarboxylic acids). Glycolate oxidase was also assayed in some cases, and Atriplex nummularia and Sorghum bicolor were also used as test material. A simple procedure, involving a mortar and pestle grind with carborundum added to the grinding mixture, was found to be as effective as glass bead grind procedures. In addition, it was more rapid and showed less variability with different operations.Using the carborundum grind procedure, sources of variability in enzyme activity in apparently uniform leaves were compared, as were effects of time of day, leaf age and storage procedure. In general, if apparently uniform leaves could be selected, variability in levels of enzyme activity appeared to be relatively small, not exceeding about 12%. Time of day also appeared to be relatively unimportant for the enzymes examined. However, the ontogentic status of the plant was found to be an important source of variability. Leaf age was also a major source of variability where the activity was expressed on a fresh weight basis, but specific activity (i.e. activity expressed on a protein basis) was relatively constant, at least with the range of species and leaf ages examined here.Storage of fresh samples in liquid nitrogen for 24 h, prior to extraction and assay, led to only a small reduction in activity, but substantial changes occurred if storage was in dry ice or in ice and also where extracts were stored in a deep freeze.

Recombinant thermoactive phosphoenolpyruvate carboxylase (PEPC) from Thermosynechococcus elongatus and its coupling with mesophilic/thermophilic bacterial carbonic anhydrases (CAs) for the conversion of CO2 to oxaloacetate.

PubMed

Del Prete, Sonia; De Luca, Viviana; Capasso, Clemente; Supuran, Claudiu T; Carginale, Vincenzo

2016-01-15

With the continuous increase of atmospheric CO2 in the last decades, efficient methods for carbon capture, sequestration, and utilization are urgently required. The possibility of converting CO2 into useful chemicals could be a good strategy to both decreasing the CO2 concentration and for achieving an efficient exploitation of this cheap carbon source. Recently, several single- and multi-enzyme systems for the catalytic conversion of CO2 mainly to bicarbonate have been implemented. In order to design and construct a catalytic system for the conversion of CO2 to organic molecules, we implemented an in vitro multienzyme system using mesophilic and thermophilic enzymes. The system, in fact, was constituted by a recombinant phosphoenolpyruvate carboxylase (PEPC) from the thermophilic cyanobacterium Thermosynechococcus elongatus, in combination with mesophilic/thermophilic bacterial carbonic anhydrases (CAs), for converting CO2 into oxaloacetate, a compound of potential utility in industrial processes. The catalytic procedure is in two steps: the conversion of CO2 into bicarbonate by CA, followed by the carboxylation of phosphoenolpyruvate with bicarbonate, catalyzed by PEPC, with formation of oxaloacetate (OAA). All tested CAs, belonging to α-, β-, and γ-CA classes, were able to increase OAA production compared to procedures when only PEPC was used. Interestingly, the efficiency of the CAs tested in OAA production was in good agreement with the kinetic parameters for the CO2 hydration reaction of these enzymes. This PEPC also revealed to be thermoactive and thermostable, and when coupled with the extremely thermostable CA from Sulphurhydrogenibium azorense (SazCA) the production of OAA was achieved even if the two enzymes were exposed to temperatures up to 60 °C, suggesting a possible role of the two coupled enzymes in biotechnological processes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Relationship between NH(4) Assimilation Rate and in Vivo Phosphoenolpyruvate Carboxylase Activity : Regulation of Anaplerotic Carbon Flow in the Green Alga Selenastrum minutum.

PubMed

Vanlerberghe, G C; Schuller, K A; Smith, R G; Feil, R; Plaxton, W C; Turpin, D H

1990-09-01

The rate of NH(4) (+) assimilation by N-limited Selenastrum minutum (Naeg.) Collins cells in the dark was set as an independent variable and the relationship between NH(4) (+) assimilation rate and in vivo activity of phosphoenolpyruvate carboxylase (PEPC) was determined. In vivo activity of PEPC was measured by following the incorporation of H(14)CO(-) (3) into acid stable products. A linear relationship of 0.3 moles C fixed via PEPC per mole N assimilated was observed. This value agrees extremely well with the PEPC requirement for the synthesis of the amino acids found in total cellular protein. Determinations of metabolite levels in vivo at different rates of N assimilation indicated that the known metabolite effectors of S. minutum PEPC in vitro (KA Schuller, WC Plaxton, DH Turpin, [1990] Plant Physiol 93: 1303-1311) are important regulators of this enzyme during N assimilation. As PEPC activity increased in response to increasing rates of N assimilation, there was a corresponding decline in the level of PEPC inhibitors (2-oxoglutarate, malate), an increase in the level of PEPC activators (glutamine, dihydroxyacetone phosphate), and an increase in the Gln/Glu ratio. Treatment of N-limited cells with azaserine caused an increase in the Gln/Glu ratio resulting in increased PEPC activity in the absence of N assimilation. We suggest glutamate and glutamine play a key role in regulating the anaplerotic function of PEPC in this C(3) organism.

Synthesis of citrate from phosphoenolpyruvate and acetylcarnitine by mitochondria from rabbit, pigeon and rat liver: implications for lipogenesis.

PubMed

Wiese, T J; Wuensch, S A; Ray, P D

1996-08-01

Rabbit, pigeon and rat liver mitochondria convert exogenous phosphoenolpyruvate and acetylcarnitine to citrate at rates of 14, 74 and 8 nmol/15 min/mg protein. Citrate formation is dependent on exogenous HCO3-, is increased consistently by exogenous nucleotides (GDP, IDP, GTP, ADP, ATP) and inhibited strongly by 3-mercaptopicolinate and 1,2,3-benzenetricarboxylate. Citrate is not made from pyruvate alone or combined with acetylcarnitine. Pigeon and rat liver mitochondria make large amounts of citrate from exogenous succinate, suggesting the presence of an endogenous source of acetyl units or means of converting oxalacetate to acetyl units. Citrate synthesis from succinate by pigeon and rabbit mitochondria is increased significantly by exogenous acetylcarnitine. Pigeon and rat liver contain 80 and 15 times, respectively, more ATP:citrate lyase activity than does rabbit liver. Data suggest that mitochondrial phosphoenolpyruvate carboxykinase in vivo could convert glycolysis-derived phosphoenolpyruvate to oxalacetate that, with acetyl CoA, could form citrate for export to support cytosolic lipogenesis as an activator of acetyl CoA carboxylase, a carbon source via ATP:citrate lyase and NADPH via NADP:malate dehydrogenase or NADP:isocitrate dehydrogenase.

Improved short-term drought response of transgenic rice over-expressing maize C4 phosphoenolpyruvate carboxylase via calcium signal cascade.

PubMed

Liu, Xiaolong; Li, Xia; Dai, Chuanchao; Zhou, Jiayu; Yan, Ting; Zhang, Jinfei

2017-11-01

To understand the link between long-term drought tolerance and short-term drought responses in plants, transgenic rice (Oryza sativa L.) plants over-expressing the maize C 4- pepc gene encoding phosphoenolpyruvate carboxylase (PC) and wild-type (WT) rice plants were subjected to PEG 6000 treatments to simulate drought stress. Compared with WT, PC had the higher survival rate and net photosynthetic rate after 16days of drought treatment, and had higher relative water content in leaves after 2h of drought treatment as well, conferring drought tolerance. WT accumulated higher amounts of malondialdehyde, superoxide radicals, and H 2 O 2 than PC under the 2-h PEG 6000 treatment, indicating greater damages in WT. Results from pretreatments with a Ca 2+ chelator and/or antagonist showed that the regulation of the early drought response in PC was Ca 2+ -dependent. The NO and H 2 O 2 levels in PC lines were also up-regulated via Ca 2+ signals, indicating that Ca 2+ in PC lines also reacted upstream of NO and H 2 O 2 . 2-h drought treatment increased the transcripts of CPK9 and CPK4 in PC via positive up-regulation of Ca 2+ . The transcripts of NAC6 [NACs (NAM, ATAF1, ATAF2, and CUC2)] and bZIP60 (basic leucine zipper, bZIP) were up-regulated, but those of DREB2B (dehydration-responsive element-binding protein, DREB) were down-regulated, both via Ca 2+ signals in PC. PEPC activity, expressions of C 4 -pepc, and the antioxidant enzyme activities in PC lines were up-regulated via Ca 2+ . These results indicated that Ca 2+ signals in PC lines can up-regulate the NAC6 and bZIP60 and the downstream targets for early drought responses, conferring drought tolerance for the long term. Copyright © 2017 Elsevier GmbH. All rights reserved.

Pyramiding expression of maize genes encoding phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) synergistically improve the photosynthetic characteristics of transgenic wheat.

PubMed

Zhang, HuiFang; Xu, WeiGang; Wang, HuiWei; Hu, Lin; Li, Yan; Qi, XueLi; Zhang, Lei; Li, ChunXin; Hua, Xia

2014-09-01

Using particle bombardment transformation, we introduced maize pepc cDNA encoding phosphoenolpyruvate carboxylase (PEPC) and ppdk cDNA encoding pyruvate orthophosphate dikinase (PPDK) into the C3 crop wheat to generate transgenic wheat lines carrying cDNA of pepc (PC lines), ppdk (PK lines) or both (PKC lines). The integration, transcription, and expression of the foreign genes were confirmed by Southern blot, Real-time quantitative reverse transcription PCR (Q-RT-PCR), and Western blot analysis. Q-RT-PCR results indicated that the average relative expression levels of pepc and ppdk in the PKC lines reached 10 and 4.6, respectively, compared to their expressions in untransformed plants (set to 1). The enzyme activities of PEPC and PPDK in the PKC lines were 4.3- and 2.1-fold higher, respectively, than in the untransformed control. The maximum daily net photosynthetic rates of the PKC, PC, and PK lines were enhanced by 26.4, 13.3, and 4.5%, respectively, whereas the diurnal accumulations of photosynthesis were 21.3, 13.9, and 6.9%, respectively, higher than in the control. The Fv/Fm of the transgenic plants decreased less than in the control under high temperature and high light conditions (2 weeks after anthesis), suggesting that the transgenic wheat transports more absorbed light energy into a photochemical reaction. The exogenous maize C4-specific pepc gene was more effective than ppdk at improving the photosynthetic performance and yield characteristics of transgenic wheat, while the two genes showed a synergistic effect when they were transformed into the same genetic background, because the PKC lines exhibited improved photosynthetic and physiological traits.

Ectopic expression of phosphoenolpyruvate carboxylase in Vicia narbonensis seeds: effects of improved nutrient status on seed maturation and transcriptional regulatory networks.

PubMed

Radchuk, Ruslana; Radchuk, Volodymyr; Götz, Klaus-Peter; Weichert, Heiko; Richter, Andreas; Emery, R J Neil; Weschke, Winfriede; Weber, Hans

2007-09-01

Seed maturation responds to endogenous and exogenous signals like nutrient status, energy and hormones. We recently showed that phosphoenolpyruvate carboxylase (PEPC) overexpression in Vicia narbonensis seeds alters seed metabolism and channels carbon into organic acids, resulting in greater seed storage capacity and increased protein content. Thus, these lines represent models with altered sink strength and improved nutrient status. Here we analyse seed developmental and metabolic parameters, and C/N partitioning in these seeds. Transgenic embryos take up more carbon and nitrogen. Changes in dry to FW ratio, seed fill duration and major seed components indicate altered seed development. Array-based gene expression analysis of embryos reveals upregulation of seed metabolism, especially during the transition phase and at late maturation, in terms of protein storage and processing, amino acid metabolism, primary metabolism and transport, energy and mitochondrial activity, transcriptional and translational activity, stress tolerance, photosynthesis, cell proliferation and elongation, signalling and hormone action and regulated protein degradation. Stimulated cell elongation is in accordance with upregulated signalling pathways related to gibberellic acid/brassinosteroids. We discuss that activated organic and amino acid production leads to a wide-range activation of nitrogen metabolism, including the machinery of storage protein synthesis, amino acid synthesis, protein processing and deposition, translational activity and the methylation cycle. We suggest that alpha-ketoglutarate (alpha-KG) and/or oxalacetate provide signals for coordinate upregulation of amino acid biosynthesis. Activation of stress tolerance genes indicates partial overlap between nutrient, stress and abscisic acid (ABA) signals, indicating a common interacting or regulatory mechanism between nutrients, stress and ABA. In conclusion, analysis of PEPC overexpressing seeds identified pathways

Physiological investigation of C4-phosphoenolpyruvate-carboxylase-introduced rice line shows that sucrose metabolism is involved in the improved drought tolerance.

PubMed

Zhang, Chen; Li, Xia; He, Yafei; Zhang, Jinfei; Yan, Ting; Liu, Xiaolong

2017-06-01

We compared the drought tolerance of wild-type (WT) and transgenic rice plants (PC) over-expressing the maize C 4 PEPC gene, which encodes phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) gene, and evaluated the roles of saccharide and sugar-related enzymes in the drought response. Pot-grown seedlings were subjected to real drought conditions outdoors, and the yield components were compared between PC and untransformed wild-type (WT) plants. The stable yield from PC plants was associated with higher net photosynthetic rate under the real drought treatment. The physiological characters of WT and PC seedlings under a simulated drought treatment (25% (w/v) polyethylene glycol-6000 for 3 h; PEG 6000 treatment) were analyzed in detail for the early response of drought. The relative water content was higher in PC than in WT, and PEPC activity and the C 4 -PEPC transcript level in PC were elevated under the simulated drought conditions. The endogenous saccharide responses also differed between PC and WT under simulated drought stress. The higher sugar decomposition rate in PC than in WT under drought analog stress was related to the increased activities of sucrose phosphate synthase, sucrose synthase, acid invertase, and neutral invertase, increased transcript levels of VIN1, CIN1, NIN1, SUT2, SUT4, and SUT5, and increased activities of superoxide dismutase and peroxidase in the leaves. The greater antioxidant defense capacity of PC and its relationship with saccharide metabolism was one of the reasons for the improved drought tolerance. In conclusion, PEPC effectively alleviated oxidative damage and enhanced the drought tolerance in rice plants, which were more related to the increase of the endogenous saccharide decomposition. These findings show that components of C 4 photosynthesis can be used to increase the yield of rice under drought conditions. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Phosphorylation of bacterial-type phosphoenolpyruvate carboxylase at Ser425 provides a further tier of enzyme control in developing castor oil seeds.

PubMed

O'Leary, Brendan; Rao, Srinath K; Plaxton, William C

2011-01-01

PEPC [PEP (phosphoenolpyruvate) carboxylase] is a tightly controlled anaplerotic enzyme situated at a pivotal branch point of plant carbohydrate metabolism. Two distinct oligomeric PEPC classes were discovered in developing COS (castor oil seeds). Class-1 PEPC is a typical homotetramer of 107 kDa PTPC (plant-type PEPC) subunits, whereas the novel 910-kDa Class-2 PEPC hetero-octamer arises from a tight interaction between Class-1 PEPC and 118 kDa BTPC (bacterial-type PEPC) subunits. Mass spectrometric analysis of immunopurified COS BTPC indicated that it is subject to in vivo proline-directed phosphorylation at Ser425. We show that immunoblots probed with phosphorylation site-specific antibodies demonstrated that Ser425 phosphorylation is promoted during COS development, becoming maximal at stage IX (maturation phase) or in response to depodding. Kinetic analyses of a recombinant, chimaeric Class-2 PEPC containing phosphomimetic BTPC mutant subunits (S425D) indicated that Ser425 phosphorylation results in significant BTPC inhibition by: (i) increasing its Km(PEP) 3-fold, (ii) reducing its I50 (L-malate and L-aspartate) values by 4.5- and 2.5-fold respectively, while (iii) decreasing its activity within the physiological pH range. The developmental pattern and kinetic influence of Ser425 BTPC phosphorylation is very distinct from the in vivo phosphorylation/activation of COS Class-1 PEPC's PTPC subunits at Ser11. Collectively, the results establish that BTPC's phospho-Ser425 content depends upon COS developmental and physiological status and that Ser425 phosphorylation attenuates the catalytic activity of BTPC subunits within a Class-2 PEPC complex. To the best of our knowledge, this study provides the first evidence for protein phosphorylation as a mechanism for the in vivo control of vascular plant BTPC activity.

Metabolic networks to generate pyruvate, PEP and ATP from glycerol in Pseudomonas fluorescens.

PubMed

Alhasawi, Azhar; Thomas, Sean C; Appanna, Vasu D

2016-04-01

Glycerol is a major by-product of the biodiesel industry. In this study we report on the metabolic networks involved in its transformation into pyruvate, phosphoenolpyruvate (PEP) and ATP. When the nutritionally-versatile Pseudomonas fluorescens was exposed to hydrogen peroxide (H2O2) in a mineral medium with glycerol as the sole carbon source, the microbe reconfigured its metabolism to generate adenosine triphosphate (ATP) primarily via substrate-level phosphorylation (SLP). This alternative ATP-producing stratagem resulted in the synthesis of copious amounts of PEP and pyruvate. The production of these metabolites was mediated via the enhanced activities of such enzymes as pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC). The high energy PEP was subsequently converted into ATP with the aid of pyruvate phosphate dikinase (PPDK), phosphoenolpyruvate synthase (PEPS) and pyruvate kinase (PK) with the concomitant formation of pyruvate. The participation of the phospho-transfer enzymes like adenylate kinase (AK) and acetate kinase (ACK) ensured the efficiency of this O2-independent energy-generating machinery. The increased activity of glycerol dehydrogenase (GDH) in the stressed bacteria provided the necessary precursors to fuel this process. This H2O2-induced anaerobic life-style fortuitously evokes metabolic networks to an effective pathway that can be harnessed into the synthesis of ATP, PEP and pyruvate. The bioconversion of glycerol to pyruvate will offer interesting economic benefit. Copyright © 2016 Elsevier Inc. All rights reserved.

Stimulation of dihydroxyacetone and glycerol kinase activity in Streptococcus faecalis by phosphoenolpyruvate-dependent phosphorylation catalyzed by enzyme I and HPr of the phosphotransferase systems

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

Deutscher, J.; Sauerwald, H.

1986-06-01

Recently a report was given of the phosphoenolpyruvate (PEP)-dependent phosphorylation of a 55-kilodalton protein of Streptococus faecalis catalyzed by enzyme I and histidine-containing protein (HPr) of the phosphotransferase system. The purified 55-kilodalton protein was found to exhibit dihydroxyacetone kinase activity. Glycerol was six times more slowly phosphorylated than dihydroxyacetone. The K/sub m/s were found to 0.7 mM for ATP, 0.45 mM for dihydroxyacetone, and 0.9 MM for glycerol. PEP-dependent phosphorylation of dihydroxyacetone kinase stimulated phosphorylation of both substrates about 10-fold. Fructose 1,6-diphosphate at concentrations higher than 2 mM inhibited the activity of phosphorylated and unphosphorylated dihydroxyacetone kinase in a noncompetitivemore » manner. The rate of PEP-dependent phosphorylation of dihydroxyacetone kinase was about 200-fold slower than the phosphorylation rate of III proteins (also called enzyme III or factor III), which so far have been considered the only phosphoryl acceptors of histidyl-phosphorylated HPr. P-Dihydroxyacetone kinase was found to be able to transfer its phosphoryl group in a backward reaction to HPr. Following (/sup 32/P)PEP-dependent phosphorylation and tryptic digestion of dihydroxyacetone kinase, the authors isolated a labeled peptide composed of 37 amino acids, as determined by amino acid analysis. The single histidyl residue of this peptide most likely carries the phosphoryl group in phosphorylated dihydroxyacetone kinase.« less

The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs.

PubMed

O'Leary, Brendan; Park, Joonho; Plaxton, William C

2011-05-15

PEPC [PEP (phosphoenolpyruvate) carboxylase] is a tightly controlled enzyme located at the core of plant C-metabolism that catalyses the irreversible β-carboxylation of PEP to form oxaloacetate and Pi. The critical role of PEPC in assimilating atmospheric CO(2) during C(4) and Crassulacean acid metabolism photosynthesis has been studied extensively. PEPC also fulfils a broad spectrum of non-photosynthetic functions, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates consumed during biosynthesis and nitrogen assimilation. An impressive array of strategies has evolved to co-ordinate in vivo PEPC activity with cellular demands for C(4)-C(6) carboxylic acids. To achieve its diverse roles and complex regulation, PEPC belongs to a small multigene family encoding several closely related PTPCs (plant-type PEPCs), along with a distantly related BTPC (bacterial-type PEPC). PTPC genes encode ~110-kDa polypeptides containing conserved serine-phosphorylation and lysine-mono-ubiquitination sites, and typically exist as homotetrameric Class-1 PEPCs. In contrast, BTPC genes encode larger ~117-kDa polypeptides owing to a unique intrinsically disordered domain that mediates BTPC's tight interaction with co-expressed PTPC subunits. This association results in the formation of unusual ~900-kDa Class-2 PEPC hetero-octameric complexes that are desensitized to allosteric effectors. BTPC is a catalytic and regulatory subunit of Class-2 PEPC that is subject to multi-site regulatory phosphorylation in vivo. The interaction between divergent PEPC polypeptides within Class-2 PEPCs adds another layer of complexity to the evolution, physiological functions and metabolic control of this essential CO(2)-fixing plant enzyme. The present review summarizes exciting developments concerning the functions, post-translational controls and subcellular location of plant PTPC and BTPC isoenzymes.

Peach leaf curl disease shifts sugar metabolism in severely infected leaves from source to sink.

PubMed

Moscatello, Stefano; Proietti, Simona; Buonaurio, Roberto; Famiani, Franco; Raggi, Vittorio; Walker, Robert P; Battistelli, Alberto

2017-03-01

Peach leaf curl is a disease that affects the leaves of peach trees, and in severe cases all of the leaf can be similarly affected. This study investigated some effects of this disease on the metabolism of peach leaves in which all parts of the leaf were infected. These diseased leaves contained very little chlorophyll and performed little or no photosynthesis. Compared to uninfected leaves, diseased leaves possessed higher contents of fructose and especially glucose, but lowered contents of sucrose, sorbitol and especially starch. The activities of soluble acid invertase, neutral invertase, sorbitol dehydrogenase and sucrose synthase were all higher in diseased leaves, whereas, those of aldose-6-phosphate reductase and sucrose phosphate synthase were lower. The activities of hexokinase and fructokinase were little changed. In addition, immunblots showed that the contents of Rubisco and ADP-glucose phosphorylase were reduced in diseased leaves, whereas, the content of phosphoenolpyruvate carboxylase was increased. The results show that certain aspects of the metabolism of diseased leaves are similar to immature sink leaves. That is photosynthetic function is reduced, the leaf imports rather than exports sugars, and the contents of non-structural carbohydrates and enzymes involved in their metabolism are similar to sink leaves. Further, the effects of peach leaf curl on the metabolism of peach leaves are comparable to the effects of some other diseases on the metabolism of photosynthetic organs of other plant species. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Light-dependent activation of phosphoenolpyruvate carboxylase by reversible phosphorylation in cluster roots of white lupin plants: diurnal control in response to photosynthate supply

PubMed Central

Feil, Regina; Lunn, John E.; Plaxton, William C.

2016-01-01

Background and Aims Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated enzyme that controls carbohydrate partitioning to organic acid anions (malate, citrate) excreted in copious amounts by cluster roots of inorganic phosphate (Pi)-deprived white lupin plants. Excreted malate and citrate solubilize otherwise inaccessible sources of mineralized soil Pi for plant uptake. The aim of this study was to test the hypotheses that (1) PEPC is post-translationally activated by reversible phosphorylation in cluster roots of illuminated white lupin plants, and (2) light-dependent phosphorylation of cluster root PEPC is associated with elevated intracellular levels of sucrose and its signalling metabolite, trehalose-6-phosphate. Methods White lupin plants were cultivated hydroponically at low Pi levels (≤1 µm) and subjected to various light/dark pretreatments. Cluster root PEPC activity and in vivo phosphorylation status were analysed to assess the enzyme’s diurnal, post-translational control in response to light and dark. Levels of various metabolites, including sucrose and trehalose-6-phosphate, were also quantified in cluster root extracts using enzymatic and spectrometric methods. Key Results During the daytime the cluster root PEPC was activated by phosphorylation at its conserved N-terminal seryl residue. Darkness triggered a progressive reduction in PEPC phosphorylation to undetectable levels, and this was correlated with 75–80 % decreases in concentrations of sucrose and trehalose-6- phosphate. Conclusions Reversible, light-dependent regulatory PEPC phosphorylation occurs in cluster roots of Pi-deprived white lupin plants. This likely facilitates the well-documented light- and sucrose-dependent exudation of Pi-solubilizing organic acid anions by the cluster roots. PEPC’s in vivo phosphorylation status appears to be modulated by sucrose translocated from CO2-fixing leaves into the non-photosynthetic cluster roots. PMID:27063365

Increasing leaf temperature reduces the suppression of isoprene emission by elevated CO₂ concentration.

PubMed

Potosnak, Mark J; Lestourgeon, Lauren; Nunez, Othon

2014-05-15

Including algorithms to account for the suppression of isoprene emission by elevated CO2 concentration affects estimates of global isoprene emission for future climate change scenarios. In this study, leaf-level measurements of isoprene emission were made to determine the short-term interactive effect of leaf temperature and CO2 concentration. For both greenhouse plants and plants grown under field conditions, the suppression of isoprene emission was reduced by increasing leaf temperature. For each of the four different tree species investigated, aspen (Populus tremuloides Michx.), cottonwood (Populus deltoides W. Bartram ex Marshall), red oak (Quercus rubra L.), and tundra dwarf willow (Salix pulchra Cham.), the suppression of isoprene by elevated CO2 was eliminated at increased temperature, and the maximum temperature where suppression was observed ranged from 25 to 35°C. Hypotheses proposed to explain the short-term suppression of isoprene emission by increased CO2 concentration were tested against this observation. Hypotheses related to cofactors in the methylerythritol phosphate (MEP) pathway were consistent with reduced suppression at elevated leaf temperature. Also, reduced solubility of CO2 with increased temperature can explain the reduced suppression for the phosphoenolpyruvate (PEP) carboxylase competition hypothesis. Some global models of isoprene emission include the short-term suppression effect, and should be modified to include the observed interaction. If these results are consistent at longer timescales, there are implications for predicting future global isoprene emission budgets and the reduced suppression at increased temperature could explain some of the variable responses observed in long-term CO2 exposure experiments. Copyright © 2014 Elsevier B.V. All rights reserved.

Reciprocal Control of Anaplerotic Phosphoenolpyruvate Carboxylase by in Vivo Monoubiquitination and Phosphorylation in Developing Proteoid Roots of Phosphate-Deficient Harsh Hakea1[W][OA

PubMed Central

Shane, Michael W.; Fedosejevs, Eric T.; Plaxton, William C.

2013-01-01

Accumulating evidence indicates important functions for phosphoenolpyruvate (PEP) carboxylase (PEPC) in inorganic phosphate (Pi)-starved plants. This includes controlling the production of organic acid anions (malate, citrate) that are excreted in copious amounts by proteoid roots of nonmycorrhizal species such as harsh hakea (Hakea prostrata). This, in turn, enhances the bioavailability of mineral-bound Pi by solubilizing Al3+, Fe3+, and Ca2+ phosphates in the rhizosphere. Harsh hakea thrives in the nutrient-impoverished, ancient soils of southwestern Australia. Proteoid roots from Pi-starved harsh hakea were analyzed over 20 d of development to correlate changes in malate and citrate exudation with PEPC activity, posttranslational modifications (inhibitory monoubiquitination versus activatory phosphorylation), and kinetic/allosteric properties. Immature proteoid roots contained an equivalent ratio of monoubiquitinated 110-kD and phosphorylated 107-kD PEPC polypeptides (p110 and p107, respectively). PEPC purification, immunoblotting, and mass spectrometry indicated that p110 and p107 are subunits of a 430-kD heterotetramer and that they both originate from the same plant-type PEPC gene. Incubation with a deubiquitinating enzyme converted the p110:p107 PEPC heterotetramer of immature proteoid roots into a p107 homotetramer while significantly increasing the enzyme’s activity under suboptimal but physiologically relevant assay conditions. Proteoid root maturation was paralleled by PEPC activation (e.g. reduced Km [PEP] coupled with elevated I50 [malate and Asp] values) via in vivo deubiquitination of p110 to p107, and subsequent phosphorylation of the deubiquitinated subunits. This novel mechanism of posttranslational control is hypothesized to contribute to the massive synthesis and excretion of organic acid anions that dominates the carbon metabolism of the mature proteoid roots. PMID:23407057

Gluconeogenesis in Leishmania mexicana: contribution of glycerol kinase, phosphoenolpyruvate carboxykinase, and pyruvate phosphate dikinase.

PubMed

Rodriguez-Contreras, Dayana; Hamilton, Nicklas

2014-11-21

Gluconeogenesis is an active pathway in Leishmania amastigotes and is essential for their survival within the mammalian cells. However, our knowledge about this pathway in trypanosomatids is very limited. We investigated the role of glycerol kinase (GK), phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate phosphate dikinase (PPDK) in gluconeogenesis by generating the respective Leishmania mexicana Δgk, Δpepck, and Δppdk null mutants. Our results demonstrated that indeed GK, PEPCK, and PPDK are key players in the gluconeogenesis pathway in Leishmania, although stage-specific differences in their contribution to this pathway were found. GK participates in the entry of glycerol in promastigotes and amastigotes; PEPCK participates in the entry of aspartate in promastigotes, and PPDK is involved in the entry of alanine in amastigotes. Furthermore, the majority of alanine enters into the pathway via decarboxylation of pyruvate in promastigotes, whereas pathway redundancy is suggested for the entry of aspartate in amastigotes. Interestingly, we also found that l-lactate, an abundant glucogenic precursor in mammals, was used by Leishmania amastigotes to synthesize mannogen, entering the pathway through PPDK. On the basis of these new results, we propose a revision in the current model of gluconeogenesis in Leishmania, emphasizing the differences between amastigotes and promastigotes. This work underlines the importance of studying the trypanosomatid intracellular life cycle stages to gain a better understanding of the pathologies caused in humans. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed Central

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

2016-01-01

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

Plant, cell, and molecular mechanisms of abscisic-acid regulation of stomatal apertures. In vivo phosphorylation of phosphoenolpyruvate carboxylase in guard cells of Vicia faba L. is enhanced by fusicoccin and suppressed by abscisic acid

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

Du, Z.; Aghoram, K.; Outlaw, W.H. Jr.

Plants regulate water loss and CO{sub 2} gain by modulating the aperture sizes of stomata that penetrate the epidermis. Aperture size itself is increased by osmolyte accumulation and consequent turgor increase in the pair of guard cells that flank each stoma. Guard-cell phosphoenolpyruvate carboxylase, which catalyzes the regulated step leading to malate synthesis, is crucial for charge and pH maintenance during osmolyte accumulation. Regulation of this cytosolic enzyme by effectors is well documented, but additional regulation by posttranslational modification is predicted by the alteration of PEPC kinetics during stomatal opening. In this study, the authors have investigated whether this alterationmore » is associated with the phosphorylation status of this enzyme. Using sonicated epidermal peels (isolated guard cells) pre-loaded with {sub 32}PO{sub 4}, the authors induced stomatal opening and guard-cell malate accumulation by incubation with 5 {micro}M fusicoccin (FC). In corroboratory experiments, guard cells were incubated with 5 {micro}M fusicoccin (FC). In corroboratory experiments, guard cells were incubated with the FC antagonist, 10 {micro}M abscisic acid (ABA). The phosphorylation status of PEPC was assessed by immunoprecipitation, electrophoresis, immunoblotting, and autoradiography. PEPC was phosphorylated when stomata were stimulated to open, and phosphorylation was lessened by incubation with ABA.« less

Carbon Metabolism in Two Species of Pereskia (Cactaceae) 1

PubMed Central

Rayder, Lisa; Ting, Irwin P.

1981-01-01

The Pereskia are morphologically primitive, leafed members of the Cactaceae. Gas exchange characteristics using a dual isotope porometer to monitor 14CO2 and tritiated water uptake, diurnal malic acid fluctuations, phosphoenolpyruvate carboxylase, and malate dehydrogenase activities were examined in two species of the genus Pereskia, Pereskia grandifolia and Pereskia aculeata. Investigations were done on well watered (control) and water-stressed plants. Nonstressed plants showed a CO2 uptake pattern indicating C3 carbon metabolism. However, diurnal fluctuations in titratable acidity were observed similar to Crassulacean acid metabolism. Plants exposed to 10 days of water stress exhibited stomatal opening only during an early morning period. Titratable acidity, phosphoenolpyruvate carboxylase activity, and malate dehydrogenase activity fluctuations were magnified in the stressed plants, but showed the same diurnal pattern as controls. Water stress causes these cacti to shift to an internal CO2 recycling (“idling”) that has all attributes of Crassulacean acid metabolism except nocturnal stomata opening and CO2 uptake. The consequences of this shift, which has been observed in other succulents, are unknown, and some possibilities are suggested. PMID:16661857

Photosynthetic carbon assimilation in the coccolithophorid Emiliania huxleyi (Haptophyta): Evidence for the predominant operation of the c3 cycle and the contribution of {beta}-carboxylases to the active anaplerotic reaction.

PubMed

Tsuji, Yoshinori; Suzuki, Iwane; Shiraiwa, Yoshihiro

2009-02-01

The coccolithophorid Emiliania huxleyi (Haptophyta) is a representative and unique marine phytoplankton species that fixes inorganic carbon by photosynthesis and calci-fication. We examined the initial process of photosynthetic carbon assimilation by analyses of metabolites, enzymes and genes. When the cells were incubated with a radioactive substrate (2.3 mM NaH(14)CO(3)) for 10 s under illumination, 70% of the (14)C was incorporated into the 80% methanol-soluble fraction. Eighty-five and 15% of (14)C in the soluble fraction was incorporated into phosphate esters (P-esters), including the C(3) cycle intermediates and a C(4) compound, aspartate, respectively. A pulse-chase experiment showed that (14)C in P-esters was mainly transferred into lipids, while [(14)C]aspartate, [(14)C]alanine and [(14)C]glutamate levels remained almost constant. These results indicate that the C(3) cycle functions as the initial pathway of carbon assimilation and that beta-carboxylation contributes to the production of amino acids in subsequent metabolism. Transcriptional analysis of beta-carboxylases such as pyruvate carboxylase (PYC), phosphoenolpyruvate carboxylase (PEPC) and phosphoenolpyruvate carboxykinase (PEPCK) revealed that PYC and PEPC transcripts were greatly increased under illumination, whereas the PEPCK transcript decreased remarkably. PEPC activity was higher in light-grown cells than in dark-adapted cells. PYC activity was detected in isolated chloroplasts of light-grown cells. According to analysis of their deduced N-terminal sequence, PYC and PEPC are predicted to be located in the chloroplasts and mitochondria, respectively. These results suggest that E. huxleyi possesses unique carbon assimila-tion mechanisms in which beta-carboxylation by both PYC and PEPC plays important roles in different organelles.

LCE: leaf carbon exchange data set for tropical, temperate, and boreal species of North and Central America.

PubMed

Smith, Nicholas G; Dukes, Jeffrey S

2017-11-01

Leaf canopy carbon exchange processes, such as photosynthesis and respiration, are substantial components of the global carbon cycle. Climate models base their simulations of photosynthesis and respiration on an empirical understanding of the underlying biochemical processes, and the responses of those processes to environmental drivers. As such, data spanning large spatial scales are needed to evaluate and parameterize these models. Here, we present data on four important biochemical parameters defining leaf carbon exchange processes from 626 individuals of 98 species at 12 North and Central American sites spanning ~53° of latitude. The four parameters are the maximum rate of Rubisco carboxylation (V cmax ), the maximum rate of electron transport for the regeneration of Ribulose-1,5,-bisphosphate (J max ), the maximum rate of phosphoenolpyruvate carboxylase carboxylation (V pmax ), and leaf dark respiration (R d ). The raw net photosynthesis by intercellular CO 2 (A/C i ) data used to calculate V cmax , J max , and V pmax rates are also presented. Data were gathered on the same leaf of each individual (one leaf per individual), allowing for the examination of each parameter relative to others. Additionally, the data set contains a number of covariates for the plants measured. Covariate data include (1) leaf-level traits (leaf mass, leaf area, leaf nitrogen and carbon content, predawn leaf water potential), (2) plant-level traits (plant height for herbaceous individuals and diameter at breast height for trees), (3) soil moisture at the time of measurement, (4) air temperature from nearby weather stations for the day of measurement and each of the 90 d prior to measurement, and (5) climate data (growing season mean temperature, precipitation, photosynthetically active radiation, vapor pressure deficit, and aridity index). We hope that the data will be useful for obtaining greater understanding of the abiotic and biotic determinants of these important biochemical

Distribution of Metabolites between Chloroplast and Cytoplasm during the Induction Phase of Photosynthesis in Leaf Protoplasts 1

PubMed Central

Robinson, Simon P.; Walker, David A.

1980-01-01

A method for rapid separation of the chloroplast and cytoplasmic fractions from isolated leaf protoplasts of wheat and spinach has been used to determine the distribution of 14C-labeled products during photosynthesis. In the dark, CO2 fixation was only 1 to 2% of that in the light and the products were mainly in the cytoplasmic fraction suggesting fixation by phosphoenolpyruvate carboxylase. Label appeared rapidly in the chloroplast fraction following illumination but the amount leveled off after 4 to 5 minutes reflecting the buildup of intermediates to steady state levels. There was only a slight lag before label appeared in the cytoplasmic fraction and it continued to increase at a constant rate reflecting synthesis of neutral products. In the light, the percentage of label in the chloroplast fraction decreased rapidly in the first minute of illumination and was only 10 to 20% in the steady-state. It is suggested that the chloroplast phosphate transporter promotes a rapid transfer of sugar phosphates from the chloroplast to the cytoplasm, even during the induction phase of photosynthesis. PMID:16661305

Integration of Lupinus angustifolius L. (narrow-leafed lupin) genome maps and comparative mapping within legumes.

PubMed

Wyrwa, Katarzyna; Książkiewicz, Michał; Szczepaniak, Anna; Susek, Karolina; Podkowiński, Jan; Naganowska, Barbara

2016-09-01

Narrow-leafed lupin (Lupinus angustifolius L.) has recently been considered a reference genome for the Lupinus genus. In the present work, genetic and cytogenetic maps of L. angustifolius were supplemented with 30 new molecular markers representing lupin genome regions, harboring genes involved in nitrogen fixation during the symbiotic interaction of legumes and soil bacteria (Rhizobiaceae). Our studies resulted in the precise localization of bacterial artificial chromosomes (BACs) carrying sequence variants for early nodulin 40, nodulin 26, nodulin 45, aspartate aminotransferase P2, asparagine synthetase, cytosolic glutamine synthetase, and phosphoenolpyruvate carboxylase. Together with previously mapped chromosomes, the integrated L. angustifolius map encompasses 73 chromosome markers, including 5S ribosomal DNA (rDNA) and 45S rDNA, and anchors 20 L. angustifolius linkage groups to corresponding chromosomes. Chromosomal identification using BAC fluorescence in situ hybridization identified two BAC clones as narrow-leafed lupin centromere-specific markers, which served as templates for preliminary studies of centromere composition within the genus. Bioinformatic analysis of these two BACs revealed that centromeric/pericentromeric regions of narrow-leafed lupin chromosomes consisted of simple sequence repeats ordered into tandem repeats containing the trinucleotide and pentanucleotide simple sequence repeats AGG and GATAC, structured into long arrays. Moreover, cross-genus microsynteny analysis revealed syntenic patterns of 31 single-locus BAC clones among several legume species. The gene and chromosome level findings provide evidence of ancient duplication events that must have occurred very early in the divergence of papilionoid lineages. This work provides a strong foundation for future comparative mapping among legumes and may facilitate understanding of mechanisms involved in shaping legume chromosomes.

Rapid activation by 3,5,3'-L-triiodothyronine of adenosine 5'-monophosphate-activated protein kinase/acetyl-coenzyme a carboxylase and akt/protein kinase B signaling pathways: relation to changes in fuel metabolism and myosin heavy-chain protein content in rat gastrocnemius muscle in vivo.

PubMed

de Lange, Pieter; Senese, Rosalba; Cioffi, Federica; Moreno, Maria; Lombardi, Assunta; Silvestri, Elena; Goglia, Fernando; Lanni, Antonia

2008-12-01

T3 stimulates metabolic rate in many tissues and induces changes in fuel use. The pathways by which T3 induces metabolic/structural changes related to altered fuel use in skeletal muscle have not been fully clarified. Gastrocnemius muscle (isolated at different time points after a single injection of T3 into hypothyroid rats), displayed rapid inductions of AMP-activated protein kinase (AMPK) phosphorylation (threonine 172; within 6 h) and acetyl-coenzyme A carboxylase phosphorylation (serine 79; within 12 h). As a consequence, increases occurred in mitochondrial fatty acid oxidation and carnitine palmitoyl transferase activity. Concomitantly, T3 stimulated signaling toward increased glycolysis through a rapid increase in Akt/protein kinase B (serine 473) phosphorylation (within 6 h) and a directly related increase in the activity of phosphofructokinase. The kinase specificity of the above effects was verified by treatment with inhibitors of AMPK and Akt activity (compound C and wortmannin, respectively). In contrast, glucose transporter 4 translocation to the membrane (activated by T3 within 6 h) was maintained when either AMPK or Akt activity was inhibited. The metabolic changes were accompanied by a decline in myosin heavy-chain Ib protein [causing a shift toward the fast-twitch (glycolytic) phenotype]. The increases in AMPK and acetyl-coenzyme A carboxylase phosphorylation were transient events, both levels declining from 12 h after the T3 injection, but Akt phosphorylation remained elevated until at least 48h after the injection. These data show that in skeletal muscle, T3 stimulates both fatty acid and glucose metabolism through rapid activations of the associated signaling pathways involving AMPK and Akt/protein kinase B.

C(3)-C(4) Intermediate Species in Alternanthera (Amaranthaceae) : Leaf Anatomy, CO(2) Compensation Point, Net CO(2) Exchange and Activities of Photosynthetic Enzymes.

PubMed

Rajendrudu, G; Prasad, J S; Das, V S

1986-02-01

Two naturally occurring species of the genus Alternanthera, namely A. ficoides and A. tenella, were identified as C(3)-C(4) intermediates based on leaf anatomy, photosynthetic CO(2) compensation point (Gamma), O(2) response of small ghe, Cyrillic, light intensity response of small ghe, Cyrillic, and the activities of key enzymes of photosynthesis. A. ficoides and A. tenella exhibited a less distinct Kranz-like leaf anatomy with substantial accumulation of starch both in mesophyll and bundle sheath cells. Photosynthetic CO(2) compensation points of these two intermediate species at 29 degrees C were much lower than in C(3) plants and ranged from 18 to 22 microliters per liter. Although A. ficoides and A. tenella exhibited similar intermediacy in small ghe, Cyrillic, the apparent photorespiratory component of O(2) inhibition in A. ficoides is lower than in A. tenella. The small ghe, Cyrillic progressively decreases from 35 microliters per liter at lowest light intensity to 18 microliters per liter at highest light intensity in A. tenella. It was, however, constant in A. ficoides at 20 to 25 microliters per liter between light intensities measured. The rates of net photosynthesis at 21% O(2) and 29 degrees C by A. ficoides and A. tenella were 25 to 28 milligrams CO(2) per square decimeter per hour which are intermediate between values obtained for Tridax procumbens and A. pungens, C(3) and C(4) species, respectively. The activities of key enzymes of C(4) photosynthesis, phosphoenolpyruvate carboxylase, pyruvate Pi dikinase, NAD malic enzyme, NADP malic enzyme and phosphoenolpyruvate carboxykinase in the two intermediates, A. ficoides and A. tenella are very low or insignificant. Results indicated that the relatively low apparent photorespiratory component in these two species is presumably the basis for the C(3)-C(4) intermediate photosynthesis.

Functional Analysis of Corn Husk Photosynthesis[W][OA

PubMed Central

Pengelly, Jasper J.L.; Kwasny, Scott; Bala, Soumi; Evans, John R.; Voznesenskaya, Elena V.; Koteyeva, Nuria K.; Edwards, Gerald E.; Furbank, Robert T.; von Caemmerer, Susanne

2011-01-01

The husk surrounding the ear of corn/maize (Zea mays) has widely spaced veins with a number of interveinal mesophyll (M) cells and has been described as operating a partial C3 photosynthetic pathway, in contrast to its leaves, which use the C4 photosynthetic pathway. Here, we characterized photosynthesis in maize husk and leaf by measuring combined gas exchange and carbon isotope discrimination, the oxygen dependence of the CO2 compensation point, and photosynthetic enzyme activity and localization together with anatomy. The CO2 assimilation rate in the husk was less than that in the leaves and did not saturate at high CO2, indicating CO2 diffusion limitations. However, maximal photosynthetic rates were similar between the leaf and husk when expressed on a chlorophyll basis. The CO2 compensation points of the husk were high compared with the leaf but did not vary with oxygen concentration. This and the low carbon isotope discrimination measured concurrently with gas exchange in the husk and leaf suggested C4-like photosynthesis in the husk. However, both Rubisco activity and the ratio of phosphoenolpyruvate carboxylase to Rubisco activity were reduced in the husk. Immunolocalization studies showed that phosphoenolpyruvate carboxylase is specifically localized in the layer of M cells surrounding the bundle sheath cells, while Rubisco and glycine decarboxylase were enriched in bundle sheath cells but also present in M cells. We conclude that maize husk operates C4 photosynthesis dispersed around the widely spaced veins (analogous to leaves) in a diffusion-limited manner due to low M surface area exposed to intercellular air space, with the functional role of Rubisco and glycine decarboxylase in distant M yet to be explained. PMID:21511990

The phosphoenolpyruvate/phosphate translocator is required for phenolic metabolism, palisade cell development, and plastid-dependent nuclear gene expression.

PubMed

Streatfield, S J; Weber, A; Kinsman, E A; Häusler, R E; Li, J; Post-Beittenmiller, D; Kaiser, W M; Pyke, K A; Flügge, U I; Chory, J

1999-09-01

The Arabidopsis chlorophyll a/b binding protein (CAB) gene underexpressed 1 (cue1) mutant underexpresses light-regulated nuclear genes encoding chloroplast-localized proteins. cue1 also exhibits mesophyll-specific chloroplast and cellular defects, resulting in reticulate leaves. Both the gene underexpression and the leaf cell morphology phenotypes are dependent on light intensity. In this study, we determine that CUE1 encodes the plastid inner envelope phosphoenolpyruvate/phosphate translocator (PPT) and define amino acid residues that are critical for translocator function. The biosynthesis of aromatics is compromised in cue1, and the reticulate phenotype can be rescued by feeding aromatic amino acids. Determining that CUE1 encodes PPT indicates the in vivo role of the translocator in metabolic partitioning and reveals a mesophyll cell-specific requirement for the translocator in Arabidopsis leaves. The nuclear gene expression defects in cue1 suggest that a light intensity-dependent interorganellar signal is modulated through metabolites dependent on a plastid supply of phosphoenolpyruvate.

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

PubMed Central

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

1997-01-01

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

Contraction-induced changes in acetyl-CoA carboxylase and 5'-AMP-activated kinase in skeletal muscle.

PubMed

Vavvas, D; Apazidis, A; Saha, A K; Gamble, J; Patel, A; Kemp, B E; Witters, L A; Ruderman, N B

1997-05-16

The concentration of malonyl-CoA, a negative regulator of fatty acid oxidation, diminishes acutely in contracting skeletal muscle. To determine how this occurs, the activity and properties of acetyl-CoA carboxylase beta (ACC-beta), the skeletal muscle isozyme that catalyzes malonyl-CoA formation, were examined in rat gastrocnemius-soleus muscles at rest and during contractions induced by electrical stimulation of the sciatic nerve. To avoid the problem of contamination of the muscle extract by mitochondrial carboxylases, an assay was developed in which ACC-beta was first purified by immunoprecipitation with a monoclonal antibody. ACC-beta was quantitatively recovered in the immunopellet and exhibited a high sensitivity to citrate (12-fold activation) and a Km for acetyl-CoA (120 microM) similar to that reported for ACC-beta purified by other means. After 5 min of contraction, ACC-beta activity was decreased by 90% despite an apparent increase in the cytosolic concentration of citrate, a positive regulator of ACC. SDS-polyacrylamide gel electrophoresis of both homogenates and immunopellets from these muscles showed a decrease in the electrophoretic mobility of ACC, suggesting that phosphorylation could account for the decrease in ACC activity. In keeping with this notion, citrate activation of ACC purified from contracting muscle was markedly depressed. In addition, homogenization of the muscles in a buffer free of phosphatase inhibitors and containing the phosphatase activators glutamate and MgCl2 or treatment of immunoprecipitated ACC-beta with purified protein phosphatase 2A abolished the decreases in both ACC-beta activity and electrophoretic mobility caused by contraction. The rapid decrease in ACC-beta activity after the onset of contractions (50% by 20 s) and its slow restoration to initial values during recovery (60-90 min) were paralleled temporally by reciprocal changes in the activity of the alpha2 but not the alpha1 isoform of 5'-AMP-activated protein

Interaction Between the Biotin Carboxyl Carrier Domain and the Biotin Carboxylase Domain in Pyruvate Carboxylase from Rhizobium etli†

PubMed Central

Lietzan, Adam D.; Menefee, Ann L.; Zeczycki, Tonya N.; Kumar, Sudhanshu; Attwood, Paul V.; Wallace, John C.; Cleland, W. Wallace; Maurice, Martin St.

2011-01-01

Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To effect catalysis, the tethered biotin of PC must gain access to active sites in both the biotin carboxylase domain and the carboxyl transferase domain. Previous studies have demonstrated that a mutation of threonine 882 to alanine in PC from Rhizobium etli renders the carboxyl transferase domain inactive and favors the positioning of biotin in the biotin carboxylase domain. We report the 2.4 Å resolution X-ray crystal structure of the Rhizobium etli PC T882A mutant which reveals the first high-resolution description of the domain interaction between the biotin carboxyl carrier protein domain and the biotin carboxylase domain. The overall quaternary arrangement of Rhizobium etli PC remains highly asymmetrical and is independent of the presence of allosteric activator. While biotin is observed in the biotin carboxylase domain, its access to the active site is precluded by the interaction between Arg353 and Glu248, revealing a mechanism for regulating carboxybiotin access to the BC domain active site. The binding location for the biotin carboxyl carrier protein domain demonstrates that tethered biotin cannot bind in the biotin carboxylase domain active site in the same orientation as free biotin, helping to explain the difference in catalysis observed between tethered biotin and free biotin substrates in biotin carboxylase enzymes. Electron density located in the biotin carboxylase domain active site is assigned to phosphonoacetate, offering a probable location for the putative carboxyphosphate intermediate formed during biotin carboxylation. The insights gained from the T882A Rhizobium etli PC crystal structure provide a new series of catalytic snapshots in PC and offer a revised perspective on catalysis in the biotin-dependent enzyme family. PMID:21958016

Structural analysis, plastid localization, and expression of the biotin carboxylase subunit of acetyl-coenzyme A carboxylase from tobacco.

PubMed

Shorrosh, B S; Roesler, K R; Shintani, D; van de Loo, F J; Ohlrogge, J B

1995-06-01

Acetyl-coenzyme A carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-coenzyme A, which is utilized in the plastid for de novo fatty acid synthesis and outside the plastid for a variety of reactions, including the synthesis of very long chain fatty acids and flavonoids. Recent evidence for both multifunctional and multisubunit ACCase isozymes in dicot plants has been obtained. We describe here the isolation of a tobacco (Nicotiana tabacum L. cv bright yellow 2 [NT1]) cDNA clone (E3) that encodes a 58.4-kD protein that shares 80% sequence similarity and 65% identity with the Anabaena biotin carboxylase subunit of ACCase. Similar to other biotin carboxylase subunits of acetyl-CoA carboxylase, the E3-encoded protein contains a putative ATP-binding motif but lacks a biotin-binding site (methionine-lysine-methionine or methionine-lysine-leucine). The deduced protein sequence contains a putative transit peptide whose function was confirmed by its ability to direct in vitro chloroplast uptake. The subcellular localization of this biotin carboxylase has also been confirmed to be plastidial by western blot analysis of pea (Pisum sativum), alfalfa (Medicago sativa L.), and castor (Ricinus communis L.) plastid preparations. Northern blot analysis indicates that the plastid biotin carboxylase transcripts are expressed at severalfold higher levels in castor seeds than in leaves.

Structural analysis, plastid localization, and expression of the biotin carboxylase subunit of acetyl-coenzyme A carboxylase from tobacco.

PubMed Central

Shorrosh, B S; Roesler, K R; Shintani, D; van de Loo, F J; Ohlrogge, J B

1995-01-01

Acetyl-coenzyme A carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-coenzyme A, which is utilized in the plastid for de novo fatty acid synthesis and outside the plastid for a variety of reactions, including the synthesis of very long chain fatty acids and flavonoids. Recent evidence for both multifunctional and multisubunit ACCase isozymes in dicot plants has been obtained. We describe here the isolation of a tobacco (Nicotiana tabacum L. cv bright yellow 2 [NT1]) cDNA clone (E3) that encodes a 58.4-kD protein that shares 80% sequence similarity and 65% identity with the Anabaena biotin carboxylase subunit of ACCase. Similar to other biotin carboxylase subunits of acetyl-CoA carboxylase, the E3-encoded protein contains a putative ATP-binding motif but lacks a biotin-binding site (methionine-lysine-methionine or methionine-lysine-leucine). The deduced protein sequence contains a putative transit peptide whose function was confirmed by its ability to direct in vitro chloroplast uptake. The subcellular localization of this biotin carboxylase has also been confirmed to be plastidial by western blot analysis of pea (Pisum sativum), alfalfa (Medicago sativa L.), and castor (Ricinus communis L.) plastid preparations. Northern blot analysis indicates that the plastid biotin carboxylase transcripts are expressed at severalfold higher levels in castor seeds than in leaves. PMID:7610168

Effect of biotin on activity and gene expression of biotin-dependent carboxylases in the liver of dairy cows.

PubMed

Ferreira, G; Weiss, W P

2007-03-01

Biotin is a cofactor of the gluconeogenic enzymes pyruvate carboxylase (PC) and propionyl-coenzyme A carboxylase (PCC). We hypothesized that biotin supplementation increases the activity and gene expression of PC and PCC and the gene expression of phosphoenol-pyruvate carboxykinase (PEPCK) in the liver of lactating dairy cows. Eight multiparous Holstein cows (40 +/- 2 kg/d of milk yield and 162 +/- 35 d in milk) were randomly assigned to 1 of 2 diet sequences in a crossover design with two 22-d periods. Treatments consisted of a basal diet (60% concentrate) containing 0 or 0.96 mg/kg of supplemental biotin. On d 21 of each period, liver tissue was collected by percutaneous liver biopsy. Activities of PC and PCC were determined by measuring the fixation of [14C]O2 in liver homogenates. Abundance of mRNA for PCC, PC, and PEPCK was determined by quantitative reverse-transcription PCR. Biotin supplementation did not affect milk production or composition. Biotin supplementation increased the activity of PC but had no effect on PCC activity. Biotin supplementation did not affect the gene expression of PC, PCC, and PEPCK. The increased activity of PC without changes in mRNA abundance may have been caused by increased activation of the apoenzymes by holocarboxylase synthetase. In conclusion, biotin supplementation affected the activity of PC in the liver of lactating dairy cows, but whether biotin supplementation increases glucose production in the liver remains to be determined.

Molecular cloning and characterization of two genes for the biotin carboxylase and carboxyltransferase subunits of acetyl coenzyme A carboxylase in Myxococcus xanthus.

PubMed

Kimura, Y; Miyake, R; Tokumasu, Y; Sato, M

2000-10-01

We have cloned a DNA fragment from a genomic library of Myxococcus xanthus using an oligonucleotide probe representing conserved regions of biotin carboxylase subunits of acetyl coenzyme A (acetyl-CoA) carboxylases. The fragment contained two open reading frames (ORF1 and ORF2), designated the accB and accA genes, capable of encoding a 538-amino-acid protein of 58.1 kDa and a 573-amino-acid protein of 61.5 kDa, respectively. The protein (AccA) encoded by the accA gene was strikingly similar to biotin carboxylase subunits of acetyl-CoA and propionyl-CoA carboxylases and of pyruvate carboxylase. The putative motifs for ATP binding, CO(2) fixation, and biotin binding were found in AccA. The accB gene was located upstream of the accA gene, and they formed a two-gene operon. The protein (AccB) encoded by the accB gene showed high degrees of sequence similarity with carboxyltransferase subunits of acetyl-CoA and propionyl-CoA carboxylases and of methylmalonyl-CoA decarboxylase. Carboxybiotin-binding and acyl-CoA-binding domains, which are conserved in several carboxyltransferase subunits of acyl-CoA carboxylases, were found in AccB. An accA disruption mutant showed a reduced growth rate and reduced acetyl-CoA carboxylase activity compared with the wild-type strain. Western blot analysis indicated that the product of the accA gene was a biotinylated protein that was expressed during the exponential growth phase. Based on these results, we propose that this M. xanthus acetyl-CoA carboxylase consists of two subunits, which are encoded by the accB and accA genes, and occupies a position between prokaryotic and eukaryotic acetyl-CoA carboxylases in terms of evolution.

Direct and selective small-molecule inhibition of photosynthetic PEP carboxylase: New approach to combat C4 weeds in arable crops.

PubMed

Paulus, Judith Katharina; Förster, Kerstin; Groth, Georg

2014-06-05

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of C4 photosynthesis. Besides, non-photosynthetic isoforms of PEPC are found in bacteria and all types of plants, although not in animals or fungi. A single residue in the allosteric feedback inhibitor site of PEPC was shown to adjust the affinity of the photosynthetic and non-photosynthetic isoforms for feedback inhibition by metabolites of the C4 pathway. Here, we applied computational screening and biochemical analyses to identify molecules that selectively inhibit C4 PEPC, but have no effect on the activity of non-photosynthetic PEPCs. We found two types of selective inhibitors, catechins and quinoxalines. Binding constants in the lower μM range and a strong preference for C4 PEPC qualify the quinoxaline compounds as potential selective herbicides to combat C4 weeds. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Expression of PEP carboxylase from Escherichia coli complements the phenotypic effects of pyruvate carboxylase mutations in Saccharomyces cerevisiae.

PubMed

Flores, C L; Gancedo, C

1997-08-04

We investigated the effects of the expression of the Escherichia coli ppc gene encoding PEP carboxylase in Saccharomyces cerevisiae mutants devoid of pyruvate carboxylase. Functional expression of the ppc gene restored the ability of the yeast mutants to grow in glucose-ammonium medium. Growth yield in this medium was the same in the transformed yeast than in the wild type although the growth rate of the transformed yeast was slower. Growth in pyruvate was slowed down in the transformed strain, likely due to a futile cycle produced by the simultaneous action of PEP carboxykinase and PEP carboxylase.

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

PubMed Central

Salvucci, Michael E.; Bowes, George

1981-01-01

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

Path of carbon flow during NO/sub 3//sup -/-induced photosynthetic suppression in N-limited Selenastrum minutum

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

Elrifi, I.R.; Turpin, D.H.

Nitrate addition to nitrate-limited cultures of Selenastrum minutum Naeg. Collins (Chlorophyta) resulted in a 70% suppression of photosynthetic carbon fixation. In /sup 14/CO/sub 2/ pulse/chase experiments nitrate resupply increased radiolabel incorporation into amino and organic acids and decreased radiolabel incorporation into insoluble material. Nitrate resupply increased the concentration of phosphoenolpyruvate and increased the radiolabeling of phosphoenolpyruvate, pyruvate and tricarboxylic acid cycle intermediates, notably citrate, fumarate, and malate. Furthermore, nitrate also increased the pool sizes and radiolabeling of most amino acids, with alanine, aspartate, glutamate, and glutamine showing the largest changes. Nitrate resupply increased the proportion of radiolabel in the C-4more » position of malate and increased the ratios of radiolabel in aspartate to phosphoenolpyruvate and in pyruvate to phosphoenolpyruvate, indicative of increased phosphoenolpyruvate carboxylase and pyruvate kinase activities. Analysis of these data showed that the rate of carbon flow through glutamate (10.6 ..mu..moles glutamate per milligram chlorophyll per hour) and the rate of net glutamate production (7.9 ..mu..moles glutamate per milligram chlorophyll per hour) were both greater than the maximum rate of carbon export from the Calvin cycle which could be maintained during steady state photosynthesis. These results are consistent with the hypothesis that nitrogen resupply to nitrogen-limited microalgae results in a transient suppression of photosynthetic carbon fixation due, in part, to the severity of competition for carbon skeletons between the Calvin cycle and nitrogen assimilation.« less

The Path of Carbon Flow during NO3−-Induced Photosynthetic Suppression in N-Limited Selenastrum minutum1

PubMed Central

Elrifi, Ivor R.; Turpin, David H.

1987-01-01

Nitrate addition to nitrate-limited cultures of Selenastrum minutum Naeg. Collins (Chlorophyta) resulted in a 70% suppression of photosynthetic carbon fixation. In 14CO2 pulse/chase experiments nitrate resupply increased radiolabel incorporation into amino and organic acids and decreased radiolabel incorporation into insoluble material. Nitrate resupply increased the concentration of phosphoenolpyruvate and increased the radiolabeling of phosphoenolpyruvate, pyruvate and tricarboxylic acid cycle intermediates, notably citrate, fumarate, and malate. Furthermore, nitrate also increased the pool sizes and radiolabeling of most amino acids, with alanine, aspartate, glutamate, and glutamine showing the largest changes. Nitrate resupply increased the proportion of radiolabel in the C-4 position of malate and increased the ratios of radiolabel in aspartate to phosphoenolpyruvate and in pyruvate to phosphoenolpyruvate, indicative of increased phosphoenolpyruvate carboxylase and pyruvate kinase activities. Analysis of these data showed that the rate of carbon flow through glutamate (10.6 μmoles glutamate per milligram chlorophyll per hour) and the rate of net glutamate production (7.9 μmoles glutamate per milligram chlorophyll per hour) were both greater than the maximum rate of carbon export from the Calvin cycle which could be maintained during steady state photosynthesis. These results are consistent with the hypothesis that nitrogen resupply to nitrogen-limited microalgae results in a transient suppression of photosynthetic carbon fixation due, in part, to the severity of competition for carbon skeletons between the Calvin cycle and nitrogen assimilation (IR Elrifi, DH Turpin 1986 Plant Physiol 81: 273-279). PMID:16665223

The Path of Carbon Flow during NO(3)-Induced Photosynthetic Suppression in N-Limited Selenastrum minutum.

PubMed

Elrifi, I R; Turpin, D H

1987-01-01

Nitrate addition to nitrate-limited cultures of Selenastrum minutum Naeg. Collins (Chlorophyta) resulted in a 70% suppression of photosynthetic carbon fixation. In (14)CO(2) pulse/chase experiments nitrate resupply increased radiolabel incorporation into amino and organic acids and decreased radiolabel incorporation into insoluble material. Nitrate resupply increased the concentration of phosphoenolpyruvate and increased the radiolabeling of phosphoenolpyruvate, pyruvate and tricarboxylic acid cycle intermediates, notably citrate, fumarate, and malate. Furthermore, nitrate also increased the pool sizes and radiolabeling of most amino acids, with alanine, aspartate, glutamate, and glutamine showing the largest changes. Nitrate resupply increased the proportion of radiolabel in the C-4 position of malate and increased the ratios of radiolabel in aspartate to phosphoenolpyruvate and in pyruvate to phosphoenolpyruvate, indicative of increased phosphoenolpyruvate carboxylase and pyruvate kinase activities. Analysis of these data showed that the rate of carbon flow through glutamate (10.6 mumoles glutamate per milligram chlorophyll per hour) and the rate of net glutamate production (7.9 mumoles glutamate per milligram chlorophyll per hour) were both greater than the maximum rate of carbon export from the Calvin cycle which could be maintained during steady state photosynthesis. These results are consistent with the hypothesis that nitrogen resupply to nitrogen-limited microalgae results in a transient suppression of photosynthetic carbon fixation due, in part, to the severity of competition for carbon skeletons between the Calvin cycle and nitrogen assimilation (IR Elrifi, DH Turpin 1986 Plant Physiol 81: 273-279).

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

PubMed

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

2013-03-01

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

Aspartate Biosynthesis Is Essential for the Growth of Streptococcus thermophilus in Milk, and Aspartate Availability Modulates the Level of Urease Activity▿

PubMed Central

Arioli, Stefania; Monnet, Christophe; Guglielmetti, Simone; Parini, Carlo; De Noni, Ivano; Hogenboom, Johannes; Halami, Prakash M.; Mora, Diego

2007-01-01

We investigated the carbon dioxide metabolism of Streptococcus thermophilus, evaluating the phenotype of a phosphoenolpyruvate carboxylase-negative mutant obtained by replacement of a functional ppc gene with a deleted and inactive version, Δppc. The growth of the mutant was compared to that of the parent strain in a chemically defined medium and in milk, supplemented or not with l-aspartic acid, the final product of the metabolic pathway governed by phosphoenolpyruvate carboxylase. It was concluded that aspartate present in milk is not sufficient for the growth of S. thermophilus. As a consequence, phosphoenolpyruvate carboxylase activity was considered fundamental for the biosynthesis of l-aspartic acid in S. thermophilus metabolism. This enzymatic activity is therefore essential for growth of S. thermophilus in milk even if S. thermophilus was cultured in association with proteinase-positive Lactobacillus delbrueckii subsp. bulgaricus. It was furthermore observed that the supplementation of milk with aspartate significantly affected the level of urease activity. Further experiments, carried out with a pureI-gusA recombinant strain, revealed that expression of the urease operon was sensitive to the aspartate concentration in milk and to the cell availability of glutamate, glutamine, and ammonium ions. PMID:17660309

The unique structural and biochemical development of single cell C4 photosynthesis along longitudinal leaf gradients in Bienertia sinuspersici and Suaeda aralocaspica (Chenopodiaceae)

PubMed Central

Koteyeva, Nuria K.; Voznesenskaya, Elena V.; Berry, James O.; Cousins, Asaph B.; Edwards, Gerald E.

2016-01-01

Temporal and spatial patterns of photosynthetic enzyme expression and structural maturation of chlorenchyma cells along longitudinal developmental gradients were characterized in young leaves of two single cell C4 species, Bienertia sinuspersici and Suaeda aralocaspica. Both species partition photosynthetic functions between distinct intracellular domains. In the C4-C domain, C4 acids are formed in the C4 cycle during capture of atmospheric CO2 by phosphoenolpyruvate carboxylase. In the C4-D domain, CO2 released in the C4 cycle via mitochondrial NAD-malic enzyme is refixed by Rubisco. Despite striking differences in origin and intracellular positioning of domains, these species show strong convergence in C4 developmental patterns. Both progress through a gradual developmental transition towards full C4 photosynthesis, with an associated increase in levels of photosynthetic enzymes. Analysis of longitudinal sections showed undeveloped domains at the leaf base, with Rubisco rbcL mRNA and protein contained within all chloroplasts. The two domains were first distinguishable in chlorenchyma cells at the leaf mid-regions, but still contained structurally similar chloroplasts with equivalent amounts of rbcL mRNA and protein; while mitochondria had become confined to just one domain (proto-C4-D). The C4 state was fully formed towards the leaf tips, Rubisco transcripts and protein were compartmentalized specifically to structurally distinct chloroplasts in the C4-D domains indicating selective regulation of Rubisco expression may occur by control of transcription or stability of rbcL mRNA. Determination of CO2 compensation points showed young leaves were not functionally C4, consistent with cytological observations of the developmental progression from C3 default to intermediate to C4 photosynthesis. PMID:26957565

D-Xylose as a sugar complement regulates blood glucose levels by suppressing phosphoenolpyruvate carboxylase (PEPCK) in streptozotocin-nicotinamide-induced diabetic rats and by enhancing glucose uptake in vitro

PubMed Central

Kim, Eunju; Kim, Yoo-Sun; Kim, Kyung-Mi; Jung, Sangwon; Yoo, Sang-Ho

2016-01-01

BACKGROUND/OBJECTIVES Type 2 diabetes (T2D) is more frequently diagnosed and is characterized by hyperglycemia and insulin resistance. D-Xylose, a sucrase inhibitor, may be useful as a functional sugar complement to inhibit increases in blood glucose levels. The objective of this study was to investigate the anti-diabetic effects of D-xylose both in vitro and stretpozotocin (STZ)-nicotinamide (NA)-induced models in vivo. MATERIALS/METHODS Wistar rats were divided into the following groups: (i) normal control; (ii) diabetic control; (iii) diabetic rats supplemented with a diet where 5% of the total sucrose content in the diet was replaced with D-xylose; and (iv) diabetic rats supplemented with a diet where 10% of the total sucrose content in the diet was replaced with D-xylose. These groups were maintained for two weeks. The effects of D-xylose on blood glucose levels were examined using oral glucose tolerance test, insulin secretion assays, histology of liver and pancreas tissues, and analysis of phosphoenolpyruvate carboxylase (PEPCK) expression in liver tissues of a STZ-NA-induced experimental rat model. Levels of glucose uptake and insulin secretion by differentiated C2C12 muscle cells and INS-1 pancreatic β-cells were analyzed. RESULTS In vivo, D-xylose supplementation significantly reduced fasting serum glucose levels (P < 0.05), it slightly reduced the area under the glucose curve, and increased insulin levels compared to the diabetic controls. D-Xylose supplementation enhanced the regeneration of pancreas tissue and improved the arrangement of hepatocytes compared to the diabetic controls. Lower levels of PEPCK were detected in the liver tissues of D-xylose-supplemented rats (P < 0.05). In vitro, both 2-NBDG uptake by C2C12 cells and insulin secretion by INS-1 cells were increased with D-xylose supplementation in a dose-dependent manner compared to treatment with glucose alone. CONCLUSIONS In this study, D-xylose exerted anti-diabetic effects in vivo by

Regulation of phosphoenolpyruvate carboxykinase and pyruvate kinase in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources and the role of mitochondrial function on gluconeogenesis.

PubMed

Wilson, A J; Bhattacharjee, J K

1986-12-01

Phosphoenolpyruvate carboxykinase (PEPCKase) and pyruvate kinase (PKase) were measured in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources. The PEPCKase activity was highest in ethanol-grown cells. However, high PEPCKase activity was also observed in cells grown in 1% glucose, especially as compared with the activity of sucrose-, maltose-, or galactose-grown cells. Activity was first detected after 12 h when glucose was exhausted from the growth medium. The PKase activity was very high in glucose-grown cells; considerable activity was also present in ethanol- and pyruvate-grown cells. The absolute requirement of respiration for gluconeogenesis was demonstrated by the absence or significantly low levels of PEPCKase and fructose-1,6-bisphosphatase activities observed in respiratory deficient mutants, as well as in wild-type S. cerevisiae cells grown in the presence of glucose and antimycin A or chloramphenicol. Obligate glycolytic and gluconeogenic enzymes were present simultaneously only in stationary phase cells, but not in exponential phase cells; hence futile cycling could not occur in log phase cells regardless of the presence of carbon source in the growth medium.

Regulatory Tasks of the Phosphoenolpyruvate-Phosphotransferase System of Pseudomonas putida in Central Carbon Metabolism

PubMed Central

Chavarría, Max; Kleijn, Roelco J.; Sauer, Uwe; Pflüger-Grau, Katharina; de Lorenzo, Víctor

2012-01-01

ABSTRACT Two branches of the phosphoenolpyruvate-phosphotransferase system (PTS) operate in the soil bacterium Pseudomonas putida KT2440. One branch encompasses a complete set of enzymes for fructose intake (PTSFru), while the other (N-related PTS, or PTSNtr) controls various cellular functions unrelated to the transport of carbohydrates. The potential of these two systems for regulating central carbon catabolism has been investigated by measuring the metabolic fluxes of isogenic strains bearing nonpolar mutations in PTSFru or PTSNtr genes and grown on either fructose (a PTS substrate) or glucose, the transport of which is not governed by the PTS in this bacterium. The flow of carbon from each sugar was distinctly split between the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways in a ratio that was maintained in each of the PTS mutants examined. However, strains lacking PtsN (EIIANtr) displayed significantly higher fluxes in the reactions of the pyruvate shunt, which bypasses malate dehydrogenase in the TCA cycle. This was consistent with the increased activity of the malic enzyme and the pyruvate carboxylase found in the corresponding PTS mutants. Genetic evidence suggested that such a metabolic effect of PtsN required the transfer of high-energy phosphate through the system. The EIIANtr protein of the PTSNtr thus helps adjust central metabolic fluxes to satisfy the anabolic and energetic demands of the overall cell physiology. PMID:22434849

Ribulose 1,5-bisphosphate carboxylase and phosphoribulokinase in Prochloron

NASA Technical Reports Server (NTRS)

Berhow, M. A.; Mcfadden, B. A.

1983-01-01

Ribulose 1,5-bisphosphate (RuBP) carboxylase and phosphoribulokinase, enzymes in the reductive pentose-phosphate cycle, were measured in cell-free extracts of Prochloran didemni. The partial purification and characterization of RuBP carboxylase were described. Prochloron RuBP carboxylase, when purified by isopycnic centrifugation in reoriented linear 0.2 to 0.8 M sucrose gradients, sedimented to a position which corresponded to that of the 520,000-dalton spinach enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the Prochloron enzyme was composed of large and small subunits (MW = 57,500 and 18,800). Though results established that the enzymes RuBP carboxylase and phosphoribulokinase were present in levels comparable to other CO2-fixing microorganisms, it was suggested that other enzymes in the Calvin cycle limit growth or that additional enzymic insufficiencies exist.

The contribution of stored malate and citrate to the substrate requirements of metabolism of ripening peach (Prunus persica L. Batsch) flesh is negligible. Implications for the occurrence of phosphoenolpyruvate carboxykinase and gluconeogenesis.

PubMed

Famiani, Franco; Farinelli, Daniela; Moscatello, Stefano; Battistelli, Alberto; Leegood, Richard C; Walker, Robert P

2016-04-01

The first aim of this study was to determine the contribution of stored malate and citrate to the substrate requirements of metabolism in the ripening flesh of the peach (Prunus persica L. Batsch) cultivar Adriatica. In the flesh, stored malate accumulated before ripening could contribute little or nothing to the net substrate requirements of metabolism. This was because there was synthesis and not dissimilation of malate throughout ripening. Stored citrate could potentially contribute a very small amount (about 5.8%) of the substrate required by metabolism when the whole ripening period was considered, and a maximum of about 7.5% over the latter part of ripening. The second aim of this study was to investigate why phosphoenolpyruvate carboxykinase (PEPCK) an enzyme utilised in gluconeogenesis from malate and citrate is present in peach flesh. The occurrence and localisation of enzymes utilised in the metabolism of malate, citrate and amino acids were determined in peach flesh throughout its development. Phosphoenolpyruvate carboxylase (essential for the synthesis of malate and citrate) was present in the same cells and at the same time as PEPCK and NADP-malic enzyme (both utilised in the dissimilation of malate and citrate). A hypothesis is presented to explain the presence of these enzymes and to account for the likely occurrence of gluconeogenesis. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Alteration in Auxin Homeostasis and Signaling by Overexpression Of PINOID Kinase Causes Leaf Growth Defects in Arabidopsis thaliana

PubMed Central

Saini, Kumud; Markakis, Marios N.; Zdanio, Malgorzata; Balcerowicz, Daria M.; Beeckman, Tom; De Veylder, Lieven; Prinsen, Els; Beemster, Gerrit T. S.; Vissenberg, Kris

2017-01-01

In plants many developmental processes are regulated by auxin and its directional transport. PINOID (PID) kinase helps to regulate this transport by influencing polar recruitment of PIN efflux proteins on the cellular membranes. We investigated how altered auxin levels affect leaf growth in Arabidopsis thaliana. Arabidopsis mutants and transgenic plants with altered PID expression levels were used to study the effect on auxin distribution and leaf development. Single knockouts showed small pleiotropic growth defects. Contrastingly, several leaf phenotypes related to changes in auxin concentrations and transcriptional activity were observed in PID overexpression (PIDOE) lines. Unlike in the knockout lines, the leaves of PIDOE lines showed an elevation in total indole-3-acetic acid (IAA). Accordingly, enhanced DR5-visualized auxin responses were detected, especially along the leaf margins. Kinematic analysis revealed that ectopic expression of PID negatively affects cell proliferation and expansion rates, yielding reduced cell numbers and small-sized cells in the PIDOE leaves. We used PIDOE lines as a tool to study auxin dose effects on leaf development and demonstrate that auxin, above a certain threshold, has a negative affect on leaf growth. RNA sequencing further showed how subtle PIDOE-related changes in auxin levels lead to transcriptional reprogramming of cellular processes. PMID:28659952

Isolation and characterization of an Arabidopsis biotin carboxylase gene and its promoter.

PubMed

Bao, X; Shorrosh, B S; Ohlrogge, J B

1997-11-01

In the plastids of most plants, acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) is a multisubunit complex consisting of biotin carboxylase (BC), biotin-carboxyl carrier protien (BCCP), and carboxytransferase (alpha-CT, beta-CT) subunits. To better understand the regulation of this enzyme, we have isolated and sequenced a BC genomic clone from Arabidopsis and partially characterized its promoter. Fifteen introns were identified. The deduced amino acid sequence of the mature BC protein is highly conserved between Arabidopsis and tobacco (92.6% identity). BC expression was evaluated using northern blots and BC/GUS fusion constructs in transgenic Arabidopsis. GUS activity in the BC/GUS transgenics as well as transcript level of the native gene were both found to be higher in silique and flower than in root and leaf. Analysis of tobacco suspension cells transformed with truncated BC promoter/GUS gene fusions indicated the region from -140 to +147 contained necessary promoter elements which supported basal gene expression. A positive regulatory region was found to be located between -2100 and -140, whereas a negative element was possibly located in the first intron. In addition, several conserved regulatory elements were identified in the BC promoter. Surprisingly, although BC is a low-abundance protein, the expression of BC/GUS fusion constructs was similar to 35S/GUS constructs.

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

PubMed

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

2009-02-01

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

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

PubMed

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

2017-08-01

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

Phosphoenolpyruvate-dependent sucrose phosphotransferase activity in five serotypes of Streptococcus mutans.

PubMed

Slee, A M; Tanzer, J M

1979-11-01

An inducible phosphoenolpyruvate-dependent sucrose phosphotransferase system has been demonstrated in decryptified cell suspensions of the various common serotypes of the cariogenic microorganism Streptococcus mutans.

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

PubMed Central

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

1985-01-01

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

Dimerization of the Bacterial Biotin Carboxylase Subunit Is Required for Acetyl Coenzyme A Carboxylase Activity In Vivo

PubMed Central

Smith, Alexander C.

2012-01-01

Acetyl coenzyme A (acteyl-CoA) carboxylase (ACC) is the first committed enzyme of the fatty acid synthesis pathway. Escherichia coli ACC is composed of four different proteins. The first enzymatic activity of the ACC complex, biotin carboxylase (BC), catalyzes the carboxylation of the protein-bound biotin moiety of another subunit with bicarbonate in an ATP-dependent reaction. Although BC is found as a dimer in cell extracts and the carboxylase activities of the two subunits of the dimer are interdependent, mutant BC proteins deficient in dimerization are reported to retain appreciable activity in vitro (Y. Shen, C. Y. Chou, G. G. Chang, and L. Tong, Mol. Cell 22:807–818, 2006). However, in vivo BC must interact with the other proteins of the complex, and thus studies of the isolated BC may not reflect the intracellular function of the enzyme. We have tested the abilities of three BC mutant proteins deficient in dimerization to support growth and report that the two BC proteins most deficient in dimerization fail to support growth unless expressed at high levels. In contrast, the wild-type protein supports growth at low expression levels. We conclude that BC must be dimeric to fulfill its physiological function. PMID:22037404

Photosynthetic carbon fixation characteristics of fruiting structures of Brassica campestris L

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

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

1987-04-01

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

Short-term acclimation to warmer temperatures accelerates leaf carbon exchange processes across plant types.

PubMed

Smith, Nicholas G; Dukes, Jeffrey S

2017-11-01

While temperature responses of photosynthesis and plant respiration are known to acclimate over time in many species, few studies have been designed to directly compare process-level differences in acclimation capacity among plant types. We assessed short-term (7 day) temperature acclimation of the maximum rate of Rubisco carboxylation (V cmax ), the maximum rate of electron transport (J max ), the maximum rate of phosphoenolpyruvate carboxylase carboxylation (V pmax ), and foliar dark respiration (R d ) in 22 plant species that varied in lifespan (annual and perennial), photosynthetic pathway (C 3 and C 4 ), and climate of origin (tropical and nontropical) grown under fertilized, well-watered conditions. In general, acclimation to warmer temperatures increased the rate of each process. The relative increase in different photosynthetic processes varied by plant type, with C 3 species tending to preferentially accelerate CO 2 -limited photosynthetic processes and respiration and C 4 species tending to preferentially accelerate light-limited photosynthetic processes under warmer conditions. R d acclimation to warmer temperatures caused a reduction in temperature sensitivity that resulted in slower rates at high leaf temperatures. R d acclimation was similar across plant types. These results suggest that temperature acclimation of the biochemical processes that underlie plant carbon exchange is common across different plant types, but that acclimation to warmer temperatures tends to have a relatively greater positive effect on the processes most limiting to carbon assimilation, which differ by plant type. The acclimation responses observed here suggest that warmer conditions should lead to increased rates of carbon assimilation when water and nutrients are not limiting. © 2017 John Wiley & Sons Ltd.

Carrier detection of pyruvate carboxylase deficiency in fibroblasts and lymphocytes.

PubMed

Atkin, B M

1979-10-01

Pyruvate carboxylase (E.C. 6.4.1.1) activity was determined in the circulating peripheral lymphocytes and cultured skin fibroblasts from the family of a patient with hepatic, cerebral, renal cortical, leukocyte, and fibroblast pyruvate carboxylase deficiency (PC Portland deficiency). Lymphocyte activities were: mother, 33--39%; father, 11--29%; brother, 82--103%; and sister, 38--48% of the lowest normal. Fibroblasts from the patient's mother and father had 42 and 34%, respectively, of the activity of the lowest normal. These data demonstrate that the disease is inherited in an autosomal recessive manner and that lymphocytes and fibroblasts can be used to detect carriers. Neither pyruvate carboxylase nor mitochondrial PEPCK activity in lymphocytes was increased by a 21-hr fast.

Genetics Home Reference: pyruvate carboxylase deficiency

MedlinePlus

... infants have severe lactic acidosis, a buildup of ammonia in the blood (hyperammonemia), and liver failure. They ... carboxylase allows compounds such as lactic acid and ammonia to build up and damage organs and tissues. ...

Appearance and accumulation of C/sub 4/ carbon pathway enzymes in developing maize leaves and differentiating maize A188 callus

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

Aoyagi, K.; Bassham, J.A.

1986-02-01

Regenerating maize A188 tissue cultures were examined for the presence of enzymes involved in C/sub 4/ photosynthesis, for cell morphology, and for /sup 14/C labeling kinetics to study the implementation of this pathway during plant development. For comparison, sections of maize seedling leaves were examined. Protein blot analysis using antibodies to leaf enzymes showed a different profile of these enzymes during the early stages of shoot regeneration from callus from the closely-coordinated profile observed in seedling leaves. Pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1) and phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.31) were found in nonchlorophyllous callus while ribulose 1,5-bisphosphate carboxylase (RuBPC, ECmore » 4.1.1.39) and malic enzyme, NADP-specific (ME-NADP) (EC 1.3.1.37) were not detectable until later. Enzyme activity assays showed the presence of ME-NADP as well as PEPC and PPDK in nonchlorophyllous callus. However, the activities of ME-NADP and PEPC had properties similar to those of the enzymes from C/sub 3/ leaves and from etiolated C/sub 4/ leaf tissues, but differing from the corresponding enzymes in the mature leaf. Immunoprecipitation of in vitro translation products of poly(A)RNA extracted from embryoid-forming callus showed both the 110 kilodalton precursor to chloroplast PPDK and the 94 kilodalton polypeptide. Therefore, the chloroplast tye of PPDK mRNA is present prior to the appearance of leaf morphology. Analysis of the labeled products of /sup 14/CO/sub 2/ fixation by nonchlorophyllous calli indicated ..beta..-carboxylation to give acids of the tricarboxylic acid cycle, but no incorporation into phosphoglycerate. With greening of the callus, some incorporation into phosphoglycerate and sugar phosphates occurred, and this increased in shoots as they developed, although with older shoots the increase in ..beta..-carboxylation products was even greater.« less

High-Throughput Phenotyping of Maize Leaf Physiological and Biochemical Traits Using Hyperspectral Reflectance1[OPEN

PubMed Central

Yendrek, Craig R.; Tomaz, Tiago; Montes, Christopher M.; Cao, Youyuan; Morse, Alison M.; Brown, Patrick J.; McIntyre, Lauren M.; Leakey, Andrew D.B.

2017-01-01

High-throughput, noninvasive field phenotyping has revealed genetic variation in crop morphological, developmental, and agronomic traits, but rapid measurements of the underlying physiological and biochemical traits are needed to fully understand genetic variation in plant-environment interactions. This study tested the application of leaf hyperspectral reflectance (λ = 500–2,400 nm) as a high-throughput phenotyping approach for rapid and accurate assessment of leaf photosynthetic and biochemical traits in maize (Zea mays). Leaf traits were measured with standard wet-laboratory and gas-exchange approaches alongside measurements of leaf reflectance. Partial least-squares regression was used to develop a measure of leaf chlorophyll content, nitrogen content, sucrose content, specific leaf area, maximum rate of phosphoenolpyruvate carboxylation, [CO2]-saturated rate of photosynthesis, and leaf oxygen radical absorbance capacity from leaf reflectance spectra. Partial least-squares regression models accurately predicted five out of seven traits and were more accurate than previously used simple spectral indices for leaf chlorophyll, nitrogen content, and specific leaf area. Correlations among leaf traits and statistical inferences about differences among genotypes and treatments were similar for measured and modeled data. The hyperspectral reflectance approach to phenotyping was dramatically faster than traditional measurements, enabling over 1,000 rows to be phenotyped during midday hours over just 2 to 4 d, and offers a nondestructive method to accurately assess physiological and biochemical trait responses to environmental stress. PMID:28049858

Metabolic adaptation and oxaloacetate homeostasis in P. fluorescens exposed to aluminum toxicity.

PubMed

Lemire, Joseph; Kumar, Puja; Mailloux, Ryan; Cossar, Kathyrn; Appanna, Vasu D

2008-08-01

Microbial systems are known to elaborate intricate metabolic strategies in an effort to fend the toxic impact of numerous metals. In this study, we show that the exposure of Pseudomonas fluorescens to aluminum (Al) resulted in a metabolic shift aimed at diverting oxaloacetate towards the biogenesis of an aluminophore. This metabolic alteration was characterized by uncoupling of two gluconeogenic enzymes, namely pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK). While PC displayed a sharp increase in activity and expression, PEPCK was severely diminished. Malic enzyme (ME) and NAD kinase (NADK), two enzymes involved in maintaining a reductive environment, were markedly increased in the Al-stressed cells. Hence, Al-exposed Pseudomonas fluorescens evoked a metabolic response aimed at generating oxaloacetate and promoting an intracellular reductive environment. (c) 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Differences between magnesium-activated and manganese-activated pyruvate kinase from the muscle of Concholepas concholepas.

PubMed

González, R; Carvajal, N; Morán, A

1984-01-01

In contrast to the Mg2+-activated enzyme, in the presence of Mn2+ pyruvate kinase exhibits hyperbolic kinetics with respect to the substrate phosphoenolpyruvate and is insensitive to fructose 1,6-biphosphate, phenylalanine and alanine. However, with both metal activated species inhibition by excess ADP is observed. In contrast with Mg2+, which affords significant protection against inactivation caused by 5,5'-dithiobis (2-nitrobenzoic acid), the rate of inactivation by this reagent is increased in the presence of Mn2+. Differences in conformational changes induced by combination of pyruvate kinase with Mg2+ or Mn2+ were indicated by u.v. difference spectra.

Gene encoding acetyl-coenzyme A carboxylase

DOEpatents

Roessler, Paul G.; Ohlrogge, John B.

1996-01-01

A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives thereof which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides.

Phosphoenolpyruvate:glucose phosphotransferase system modification increases the conversion rate during L-tryptophan production in Escherichia coli.

PubMed

Liu, Lina; Chen, Sheng; Wu, Jing

2017-10-01

Escherichia coli FB-04(pta1), a recombinant L-tryptophan production strain, was constructed in our laboratory. However, the conversion rate (L-tryptophan yield per glucose) of this strain is somewhat low. In this study, additional genes have been deleted in an effort to increase the conversion rate of E. coli FB-04(pta1). Initially, the pykF gene, which encodes pyruvate kinase I (PYKI), was inactivated to increase the accumulation of phosphoenolpyruvate, a key L-tryptophan precursor. The resulting strain, E. coli FB-04(pta1)ΔpykF, showed a slightly higher L-tryptophan yield and a higher conversion rate in fermentation processes. To further improve the conversion rate, the phosphoenolpyruvate:glucose phosphotransferase system (PTS) was disrupted by deleting the ptsH gene, which encodes the phosphocarrier protein (HPr). The levels of biomass, L-tryptophan yield, and conversion rate of this strain, E. coli FB-04(pta1)ΔpykF/ptsH, were especially low during fed-batch fermentation process, even though it achieved a significant increase in conversion rate during shake-flask fermentation. To resolve this issue, four HPr mutations (N12S, N12A, S46A, and S46N) were introduced into the genomic background of E. coli FB-04(pta1)ΔpykF/ptsH, respectively. Among them, the strain harboring the N12S mutation (E. coli FB-04(pta1)ΔpykF-ptsHN12S) showed a prominently increased conversion rate of 0.178 g g -1 during fed-batch fermentation; an increase of 38.0% compared with parent strain E. coli FB-04(pta1). Thus, mutation of the genomic of ptsH gene provided an alternative method to weaken the PTS and improve the efficiency of carbon source utilization.

A Tale of Two Sugars: Trehalose 6-Phosphate and Sucrose1[OPEN

PubMed Central

2016-01-01

Trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is an essential signal metabolite in plants, linking growth and development to carbon status. The Suc-Tre6P nexus model postulates that Tre6P is both a signal and negative feedback regulator of Suc levels, forming part of a mechanism to maintain Suc levels within an optimal range and functionally comparable to the insulin-glucagon system for regulating blood Glc levels in animals. The target range and sensitivity of the Tre6P-Suc feedback control circuit can be adjusted according to the cell type, developmental stage, and environmental conditions. In source leaves, Tre6P modulates Suc levels by affecting Suc synthesis, whereas in sink organs it regulates Suc consumption. In illuminated leaves, Tre6P influences the partitioning of photoassimilates between Suc, organic acids, and amino acids via posttranslational regulation of phosphoenolpyruvate carboxylase and nitrate reductase. At night, Tre6P regulates the remobilization of leaf starch reserves to Suc, potentially linking starch turnover in source leaves to carbon demand from developing sink organs. Use of Suc for growth in developing tissues is strongly influenced by the antagonistic activities of two protein kinases: SUC-NON-FERMENTING-1-RELATED KINASE1 (SnRK1) and TARGET OF RAPAMYCIN (TOR). The relationship between Tre6P and SnRK1 in developing tissues is complex and not yet fully resolved, involving both direct and indirect mechanisms, and positive and negative effects. No direct connection between Tre6P and TOR has yet been described. The roles of Tre6P in abiotic stress tolerance and stomatal regulation are also discussed. PMID:27482078

Gene encoding acetyl-coenzyme A carboxylase

DOEpatents

Roessler, P.G.; Ohlrogge, J.B.

1996-09-24

A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives are disclosed which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides. 5 figs.

Transgene silencing of sucrose synthase in alfalfa (Medicago sativa L.) stem vascular tissue suggests a role for invertase in cell wall cellulose synthesis

USDA-ARS?s Scientific Manuscript database

Alfalfa (Medicago sativa L.) plants were transformed with two constructs: (1) a truncated phosphoenolpyruvate carboxylase promoter isolated from alfalfa nodules (PEPC-4) fused to GUS; and (2) PEPC-4 fused with sucrose synthase (SUS) isolated from alfalfa nodules. Histochemical staining for GUS in st...

Structural Analysis of Substrate, Reaction Intermediate, and Product Binding in Haemophilus influenzae Biotin Carboxylase

PubMed Central

Broussard, Tyler C.; Pakhomova, Svetlana; Neau, David B.; Bonnot, Ross; Waldrop, Grover L.

2015-01-01

Acetyl-CoA carboxylase catalyzes the first and regulated step in fatty acid synthesis. In most Gram-negative and Gram-positive bacteria, the enzyme is composed of three proteins: biotin carboxylase, a biotin carboxyl carrier protein (BCCP), and carboxyltransferase. The reaction mechanism involves two half-reactions with biotin carboxylase catalyzing the ATP-dependent carboxylation of biotin-BCCP in the first reaction. In the second reaction, carboxyltransferase catalyzes the transfer of the carboxyl group from biotin-BCCP to acetyl-CoA to form malonyl-CoA. In this report, high-resolution crystal structures of biotin carboxylase from Haemophilus influenzae were determined with bicarbonate, the ATP analogue AMPPCP; the carboxyphosphate intermediate analogues, phosphonoacetamide and phosphonoformate; the products ADP and phosphate; and the carboxybiotin analogue N1′-methoxycarbonyl biotin methyl ester. The structures have a common theme in that bicarbonate, phosphate, and the methyl ester of the carboxyl group of N1′-methoxycarbonyl biotin methyl ester all bound in the same pocket in the active site of biotin carboxylase and as such utilize the same set of amino acids for binding. This finding suggests a catalytic mechanism for biotin carboxylase in which the binding pocket that binds tetrahedral phosphate also accommodates and stabilizes a tetrahedral dianionic transition state resulting from direct transfer of CO2 from the carboxyphosphate intermediate to biotin. PMID:26020841

Chemical and Physical Characterization of the Activation of Ribulosebiphosphate Carboxylase/Oxygenase

DOE R&D Accomplishments Database

Donnelly, M. I.; Ramakrishnan, V.; Hartman, F. C.

1983-08-01

Molecular structure of ribulosebiphosphate carboxylase/oxygenase isolated from Rhodospirillium was compared with the enzyme isolated from Alcaligens eutrophus. Peptides derived from the active center of the bacterial enzyme were highly homologous with those isolated from spinach. Molecular shapes of the carboxylases were estimated using neutron scattering data. These studies suggested that the enzyme as isolated from R. rubrum is a solid prolate ellipsoid or cylinder, while the spinach enzyme resembles a hollow sphere.

Interaction of renin-angiotensin system and adenosine monophosphate-activated protein kinase signaling pathway in renal carcinogenesis of uninephrectomized rats.

PubMed

Yang, Ke-Ke; Sui, Yi; Zhou, Hui-Rong; Zhao, Hai-Lu

2017-05-01

Renin-angiotensin system and adenosine monophosphate-activated protein kinase signaling pathway both play important roles in carcinogenesis, but the interplay of renin-angiotensin system and adenosine monophosphate-activated protein kinase in carcinogenesis is not clear. In this study, we researched the interaction of renin-angiotensin system and adenosine monophosphate-activated protein kinase in renal carcinogenesis of uninephrectomized rats. A total of 96 rats were stratified into four groups: sham, uninephrectomized, and uninephrectomized treated with angiotensin-converting enzyme inhibitor or angiotensin receptor blocker. Renal adenosine monophosphate-activated protein kinase and its downstream molecule acetyl coenzyme A carboxylase were detected by immunohistochemistry and western blot at 10 months after uninephrectomy. Meanwhile, we examined renal carcinogenesis by histological transformation and expressions of Ki67 and mutant p53. During the study, fasting lipid profiles were detected dynamically at 3, 6, 8, and 10 months. The results indicated that adenosine monophosphate-activated protein kinase expression in uninephrectomized rats showed 36.8% reduction by immunohistochemistry and 89.73% reduction by western blot. Inversely, acetyl coenzyme A carboxylase expression increased 83.3% and 19.07% in parallel to hyperlipidemia at 6, 8, and 10 months. The histopathology of carcinogenesis in remnant kidneys was manifested by atypical proliferation and carcinoma in situ, as well as increased expressions of Ki67 and mutant p53. Intervention with angiotensin-converting enzyme inhibitor or angiotensin receptor blocker significantly prevented the inhibition of adenosine monophosphate-activated protein kinase signaling pathway and renal carcinogenesis in uninephrectomized rats. In conclusion, the novel findings suggest that uninephrectomy-induced disturbance in adenosine monophosphate-activated protein kinase signaling pathway resulted in hyperlipidemia and

Mutations in CsPID encoding a Ser/Thr protein kinase are responsible for round leaf shape in cucumber (Cucumis sativus L.).

PubMed

Zhang, Chaowen; Chen, Feifan; Zhao, Ziyao; Hu, Liangliang; Liu, Hanqiang; Cheng, Zhihui; Weng, Yiqun; Chen, Peng; Li, Yuhong

2018-06-01

Two round-leaf mutants, rl-1 and rl-2, were identified from EMS-induced mutagenesis. High throughput sequencing and map-based cloning suggested CsPID encoding a Ser/Thr protein kinase as the most possible candidate for rl-1. Rl-2 was allelic to Rl-1. Leaf shape is an important plant architecture trait that is affected by plant hormones, especially auxin. In Arabidopsis, PINOID (PID), a regulator for the auxin polar transporter PIN (PIN-FORMED) affects leaf shape formation, but this function of PID in crop plants has not been well studied. From an EMS mutagenesis population, we identified two round-leaf (rl) mutants, C356 and C949. Segregation analysis suggested that both mutations were controlled by single recessive genes, rl-1 and rl-2, respectively. With map-based cloning, we show that CsPID as the candidate gene of rl-1; a non-synonymous SNP in the second exon of CsPID resulted in an amino acid substitution and the round leaf phenotype. As compared in the wild type plant, CsPID had significantly lower expression in the root, leaf and female flowers in C356, which may result in the less developed roots, round leaves and abnormal female flowers, respectively in the rl-1 mutant. Among the three copies of PID genes, CsPID, CsPID2 and CSPID2L (CsPID2-like) in the cucumber genome, CsPID was the only one with significantly differential expression in adult leaves between WT and C356 suggesting CsPID plays a main role in leaf shape formation. The rl-2 mutation in C949 was also cloned, which was due to another SNP in a nearby location of rl-1 in the same CsPID gene. The two round leaf mutants and the work presented herein provide a good foundation for understanding the molecular mechanisms of CsPID in cucumber leaf development.

Immunomodulatory Efficacy of Standardized Annona muricata (Graviola) Leaf Extract via Activation of Mitogen-Activated Protein Kinase Pathways in RAW 264.7 Macrophages

PubMed Central

2016-01-01

Annona muricata, commonly known as Graviola, has been utilized as a traditional medicine to treat various human diseases. The aim of this study was to examine the immune-enhancing activity of Graviola leaf extracts in RAW 264.7 macrophage cells. Active ingredients in Graviola leaf extracts (GE) were identified as kaempferol-3-O-rutinoside and quercetin-3-O-rutinoside by LC-MS/MS. When treated with steam or 50% ethanol GE, cell morphology was altered due to initiation of cell differentiation. While the cell viability was not altered by the steam GE, it was reduced by the ethanol GE. Both steam and ethanol GE induced the transcriptional expression of cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-1β, but only the steam extract upregulated inducible nitric oxide synthase (iNOS). In consistence with mRNA expression, the production of TNF-α and nitrite was elevated by both steam and ethanol extracts of Graviola leaves. This is mainly due to activation of mitogen-activated protein (MAP) kinase signaling pathways. These results suggest that Graviola leaves enhance immunity by activation of the MAP kinase pathways. These bioactive properties of Graviola indicate its potential as a health-promoting ingredient to boost the immune system. PMID:28096884

Structural properties of pyruvate carboxylases from chicken liver and other sources

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

Barden, R.E.; Taylor, B.L.; Isohashi, F.

1975-11-01

Varieties of pyruvate carboxylase (pyruvate: CO/sub 2/ ligase (ADP-forming), EC 6.4.1.1) obtained from the livers of several species of vertebrates, including humans, all show the same basic structure. They are composed of large polypeptide chains of molecular weights ranging from 1.2 to 1.3 x 10/sup 5/ for the different varieties of the enzyme. The native form of the enzyme appears to be a tetramer with a molecular weight of about 5 x 10/sup 5/. In the case of pyruvate carboxylase from chicken liver each polypeptide chain contains a biotin moiety, thus supporting the thesis that the tetramer contains four identicalmore » polypeptide chains. Pyruvate carboxylase from yeast appears to be basically similar to those from the vertebrate species and has a tetrameric structure. Each protomer contains a single polypeptide chain with a molecular weight of 1.25 x 10/sup 5/. In contrast, pyruvate carboxylase from two bacterial species, Pseudomonas citronellolis and Azotobacter vinelandii, appears to be a dimer with a molecular weight (2.5 x 10/sup 5/) about half that of the animal and yeast species. As a further difference, each of the protomers of the bacterial enzymes contain two polypeptides of 6.5 and 5.4 x 10/sup 5/ molecular weight in the case of the Pseudomonas enzyme. The larger of the two polypeptides contains the biotin moiety. The functional units of the bacterial enzyme thus appear to contain two polypeptides while that of the liver and yeast enzymes is made up of a single chain. Neither of these arrangements corresponds with those of other biotin enzymes whose structure has been extensively studied (acetyl-CoA carboxylases from liver or Escherichia coli, and transcarboxylase from Propionibacterium). (auth)« less

Light versus Dark Carbon Metabolism in Cherry Tomato Fruits

PubMed Central

Laval-Martin, Danielle; Farineau, Jack; Diamond, Jeffrey

1977-01-01

The photosynthetic properties of the internal and peripheral tissues of the cherry tomato fruit (Lycopersicum esculentum var. cerasiforme Dun A. Gray) were investigated. Whole fruit and their isolated tissues evolve large amounts of CO2 in darkness. In the light, this evolution decreases but nevertheless remains a net evolution; 3-(3,4-dichlorophenyl)-1,1-dimethylurea abolishes the effects of light. Incorporation of 14CO2 by leaves and fruit tissues demonstrates that the outer region of the fruit has the highest photosynthetic efficiency on a chlorophyll basis; the internal fruit tissue, richer in chlorophyll, has a much lower efficiency. The identification of intermediates following short term incubations with 14CO2 shows that in darkness the fruit accumulates the majority of label in malate. In the light, leaf tissue exhibits a pattern of incorporation characteristic of C-3 metabolism, whereas fruit tissue exhibits a decreased labeling of malate with a concomitant appearance of label in Calvin cycle intermediates. This is in agreement with the levels and types of carboxylating activities demonstrated in vitro; especially noteworthy is the very low ribulose diphosphate carboxylase activity in the internal fruit tissue. The photosynthetic potential, phosphoenolpyruvate carboxylase activity, and quantities of malate accumulated by fruit tissues are parallel to their chlorophyll content during growth and maturation. PMID:16660204

The complex character of photosynthesis in cucumber fruit

PubMed Central

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

2017-01-01

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

Photosynthesis-related characteristics of the midrib and the interveinal lamina in leaves of the C3–CAM intermediate plant Mesembryanthemum crystallinum

PubMed Central

Kuźniak, Elżbieta; Kornas, Andrzej; Kaźmierczak, Andrzej; Rozpądek, Piotr; Nosek, Michał; Kocurek, Maciej; Zellnig, Günther; Müller, Maria; Miszalski, Zbigniew

2016-01-01

Background and Aims Leaf veins are usually encircled by specialized bundle sheath cells. In C4 plants, they play an important role in CO2 assimilation, and the photosynthetic activity is compartmentalized between the mesophyll and the bundle sheath. In C3 and CAM (Crassulacean acid metabolism) plants, the photosynthetic activity is generally attributed to the leaf mesophyll cells, and the vascular parenchymal cells are rarely considered for their role in photosynthesis. Recent studies demonstrate that enzymes required for C4 photosynthesis are also active in the veins of C3 plants, and their vascular system contains photosynthetically competent parenchyma cells. However, our understanding of photosynthesis in veins of C3 and CAM plants still remains insufficient. Here spatial analysis of photosynthesis-related properties were applied to the midrib and the interveinal lamina cells in leaves of Mesembryanthemum crystallinum, a C3–CAM intermediate plant. Methods The midrib anatomy as well as chloroplast structure and chlorophyll fluorescence, diurnal gas exchange profiles, the immunoblot patterns of PEPC (phosphoenolpyruvate carboxylase) and RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase), H2O2 localization and antioxidant enzyme activities were compared in the midrib and in the interveinal mesophyll cells in leaves of C3 and CAM plants. Key Results Leaf midribs were structurally competent to perform photosynthesis in C3 and CAM plants. The midrib chloroplasts resembled those in the bundle sheath cells of C4 plants and were characterized by limited photosynthetic activity. Conclusions The metabolic roles of midrib chloroplasts differ in C3 and CAM plants. It is suggested that in leaves of C3 plants the midrib chloroplasts could be involved in the supply of CO2 for carboxylation, and in CAM plants they could provide malate to different metabolic processes and mediate H2O2 signalling. PMID:27091507

Conserved and divergent rhythms of crassulacean acid metabolism-related and core clock gene expression in the cactus Opuntia ficus-indica.

PubMed

Mallona, Izaskun; Egea-Cortines, Marcos; Weiss, Julia

2011-08-01

The cactus Opuntia ficus-indica is a constitutive Crassulacean acid metabolism (CAM) species. Current knowledge of CAM metabolism suggests that the enzyme phosphoenolpyruvate carboxylase kinase (PPCK) is circadian regulated at the transcriptional level, whereas phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), NADP-malic enzyme (NADP-ME), and pyruvate phosphate dikinase (PPDK) are posttranslationally controlled. As little transcriptomic data are available from obligate CAM plants, we created an expressed sequence tag database derived from different organs and developmental stages. Sequences were assembled, compared with sequences in the National Center for Biotechnology Information nonredundant database for identification of putative orthologs, and mapped using Kyoto Encyclopedia of Genes and Genomes Orthology and Gene Ontology. We identified genes involved in circadian regulation and CAM metabolism for transcriptomic analysis in plants grown in long days. We identified stable reference genes for quantitative polymerase chain reaction and found that OfiSAND, like its counterpart in Arabidopsis (Arabidopsis thaliana), and OfiTUB are generally appropriate standards for use in the quantification of gene expression in O. ficus-indica. Three kinds of expression profiles were found: transcripts of OfiPPCK oscillated with a 24-h periodicity; transcripts of the light-active OfiNADP-ME and OfiPPDK genes adapted to 12-h cycles, while transcript accumulation patterns of OfiPEPC and OfiMDH were arrhythmic. Expression of the circadian clock gene OfiTOC1, similar to Arabidopsis, oscillated with a 24-h periodicity, peaking at night. Expression of OfiCCA1 and OfiPRR9, unlike in Arabidopsis, adapted best to a 12-h rhythm, suggesting that circadian clock gene interactions differ from those of Arabidopsis. Our results indicate that the evolution of CAM metabolism could be the result of modified circadian regulation at both the transcriptional and posttranscriptional

Conserved and Divergent Rhythms of Crassulacean Acid Metabolism-Related and Core Clock Gene Expression in the Cactus Opuntia ficus-indica1[C][W

PubMed Central

Mallona, Izaskun; Egea-Cortines, Marcos; Weiss, Julia

2011-01-01

The cactus Opuntia ficus-indica is a constitutive Crassulacean acid metabolism (CAM) species. Current knowledge of CAM metabolism suggests that the enzyme phosphoenolpyruvate carboxylase kinase (PPCK) is circadian regulated at the transcriptional level, whereas phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), NADP-malic enzyme (NADP-ME), and pyruvate phosphate dikinase (PPDK) are posttranslationally controlled. As little transcriptomic data are available from obligate CAM plants, we created an expressed sequence tag database derived from different organs and developmental stages. Sequences were assembled, compared with sequences in the National Center for Biotechnology Information nonredundant database for identification of putative orthologs, and mapped using Kyoto Encyclopedia of Genes and Genomes Orthology and Gene Ontology. We identified genes involved in circadian regulation and CAM metabolism for transcriptomic analysis in plants grown in long days. We identified stable reference genes for quantitative polymerase chain reaction and found that OfiSAND, like its counterpart in Arabidopsis (Arabidopsis thaliana), and OfiTUB are generally appropriate standards for use in the quantification of gene expression in O. ficus-indica. Three kinds of expression profiles were found: transcripts of OfiPPCK oscillated with a 24-h periodicity; transcripts of the light-active OfiNADP-ME and OfiPPDK genes adapted to 12-h cycles, while transcript accumulation patterns of OfiPEPC and OfiMDH were arrhythmic. Expression of the circadian clock gene OfiTOC1, similar to Arabidopsis, oscillated with a 24-h periodicity, peaking at night. Expression of OfiCCA1 and OfiPRR9, unlike in Arabidopsis, adapted best to a 12-h rhythm, suggesting that circadian clock gene interactions differ from those of Arabidopsis. Our results indicate that the evolution of CAM metabolism could be the result of modified circadian regulation at both the transcriptional and posttranscriptional

Mechanism of α-Glycerophosphate Regulation of Acetyl-Coenzyme A Carboxylase of Saccharomyces cerevisiae

PubMed Central

Rasmussen, Roberta K.; Klein, Harold P.

1968-01-01

The mechanism proposed for the activation of animal acetyl-coenzyme A (CoA) carboxylase by α-glycerophosphate, namely, the removal of inhibitory palmityl-CoA via glyceride synthesis, is not the only possible one in the yeast system because extracts exhibiting marked stimulation of acetyl-CoA carboxylase activity by α-glyerophosphate show a lack of acyl-CoA compounds. PMID:5643049

Brassinosteroid insensitive 1-associated kinase 1 (OsI-BAK1) is associated with grain filling and leaf development in rice.

PubMed

Khew, Choy-Yuen; Teo, Chin-Jit; Chan, Wai-Sun; Wong, Hann-Ling; Namasivayam, Parameswari; Ho, Chai-Ling

2015-06-15

Brassinosteroid Insensitive 1 (BRI1)-Associated Kinase I (BAK1) has been reported to interact with BRI1 for brassinosteroid (BR) perception and signal transduction that regulate plant growth and development. The aim of this study is to investigate the functions of a rice OsBAK1 homologue, designated as OsI-BAK1, which is highly expressed after heading. Silencing of OsI-BAK1 in rice plants produced a high number of undeveloped green and unfilled grains compared to the untransformed plants. Histological analyses demonstrated that embryos were either absent or retarded in their development in these unfilled rice grains of OsI-BAK1 RNAi plants. Down regulation of OsI-BAK1 caused a reduction in cell number and enlargement in leaf bulliform cells. Furthermore, transgenic rice plants overexpressing OsI-BAK1 were demonstrated to have corrugated and twisted leaves probably due to increased cell number that caused abnormal bulliform cell structure which were enlarged and plugged deep into leaf epidermis. The current findings suggest that OsI-BAK1 may play an important role in the developmental processes of rice grain filling and leaf cell including the bulliform cells. Copyright © 2015 Elsevier GmbH. All rights reserved.

Decreased expression of γ-carboxylase in diabetes-associated arterial stiffness: impact on matrix Gla protein.

PubMed

Doyon, Marielle; Mathieu, Patrick; Moreau, Pierre

2013-02-01

Arterial stiffness is accelerated in type 1 diabetic patients. Medial artery calcification (MAC) contributes to the development of arterial stiffness. Vitamin K oxidoreductase (VKOR) reduces the vitamin K required by γ-carboxylase to activate matrix γ-carboxyglutamic acid (Gla) protein (MGP), an inhibitor of vascular calcification. This study aimed to evaluate the hypothesis that diabetes reduces the γ-carboxylation of MGP in the aortic wall, leading to increased vascular calcification, and the role of γ-carboxylase and VKOR in this γ-carboxylation deficit. Type 1 diabetes was induced in male Wistar rats with a single ip injection of streptozotocin. Augmentation of arterial stiffness in diabetic rats was shown by a 44% increase in aortic pulse wave velocity. Aortic and femoral calcification were increased by 26 and 56%, respectively. γ-Carboxylated MGP (cMGP, active) was reduced by 36% and the aortic expression of γ-carboxylase was reduced by 58%. Expression of γ-carboxylase correlated with cMGP (r= 0.59) and aortic calcification (r = -0.57). VKOR aortic expression and activity were not modified by diabetes. Vitamin K plasma concentrations were increased by 191% in diabetic rats. In ex vivo experiments with aortic rings, vitamin K supplementation prevented the glucose-induced decrease in γ-carboxylase expression. Our results suggest that reduced cMGP, through an impaired expression of γ-carboxylase, is involved in the early development of MAC in diabetes, and therefore, in the acceleration of arterial stiffness. A defect in vitamin K uptake by target cells could also be involved.

Antidiabetic effects of pterosin A, a small-molecular-weight natural product, on diabetic mouse models.

PubMed

Hsu, Feng-Lin; Huang, Chun-Fa; Chen, Ya-Wen; Yen, Yuan-Peng; Wu, Cheng-Tien; Uang, Biing-Jiun; Yang, Rong-Sen; Liu, Shing-Hwa

2013-02-01

The therapeutic effect of pterosin A, a small-molecular-weight natural product, on diabetes was investigated. Pterosin A, administered orally for 4 weeks, effectively improved hyperglycemia and glucose intolerance in streptozotocin, high-fat diet-fed, and db/db diabetic mice. There were no adverse effects in normal or diabetic mice treated with pterosin A for 4 weeks. Pterosin A significantly reversed the increased serum insulin and insulin resistance (IR) in dexamethasone-IR mice and in db/db mice. Pterosin A significantly reversed the reduced muscle GLUT-4 translocation and the increased liver phosphoenolpyruvate carboxyl kinase (PEPCK) expression in diabetic mice. Pterosin A also significantly reversed the decreased phosphorylations of AMP-activated protein kinase (AMPK) and Akt in muscles of diabetic mice. The decreased AMPK phosphorylation and increased p38 phosphorylation in livers of db/db mice were effectively reversed by pterosin A. Pterosin A enhanced glucose uptake and AMPK phosphorylation in cultured human muscle cells. In cultured liver cells, pterosin A inhibited inducer-enhanced PEPCK expression, triggered the phosphorylations of AMPK, acetyl CoA carboxylase, and glycogen synthase kinase-3, decreased glycogen synthase phosphorylation, and increased the intracellular glycogen level. These findings indicate that pterosin A may be a potential therapeutic option for diabetes.

Sensitivity of Photosynthesis in a C4 Plant, Maize, to Heat Stress

PubMed Central

Crafts-Brandner, Steven J.; Salvucci, Michael E.

2002-01-01

Our objective was to determine the sensitivity of components of the photosynthetic apparatus of maize (Zea mays), a C4 plant, to high temperature stress. Net photosynthesis (Pn) was inhibited at leaf temperatures above 38°C, and the inhibition was much more severe when the temperature was increased rapidly rather than gradually. Transpiration rate increased progressively with leaf temperature, indicating that inhibition was not associated with stomatal closure. Nonphotochemical fluorescence quenching (qN) increased at leaf temperatures above 30°C, indicating increased thylakoid energization even at temperatures that did not inhibit Pn. Compared with CO2 assimilation, the maximum quantum yield of photosystem II (Fv/Fm) was relatively insensitive to leaf temperatures up to 45°C. The activation state of phosphoenolpyruvate carboxylase decreased marginally at leaf temperatures above 40°C, and the activity of pyruvate phosphate dikinase was insensitive to temperature up to 45°C. The activation state of Rubisco decreased at temperatures exceeding 32.5°C, with nearly complete inactivation at 45°C. Levels of 3-phosphoglyceric acid and ribulose-1,5-bisphosphate decreased and increased, respectively, as leaf temperature increased, consistent with the decrease in Rubisco activation. When leaf temperature was increased gradually, Rubisco activation acclimated in a similar manner as Pn, and acclimation was associated with the expression of a new activase polypeptide. Rates of Pn calculated solely from the kinetics of Rubisco were remarkably similar to measured rates if the calculation included adjustment for temperature effects on Rubisco activation. We conclude that inactivation of Rubisco was the primary constraint on the rate of Pn of maize leaves as leaf temperature increased above 30°C. PMID:12177490

Genetics Home Reference: 3-methylcrotonyl-CoA carboxylase deficiency

MedlinePlus

... Targets Orphanet: 3-methylcrotonyl-CoA carboxylase deficiency Screening, Technology, and Research in Genetics Virginia Department of Health (PDF) Patient Support and Advocacy Resources (3 links) Children Living with Inherited Metabolic Diseases (CLIMB) (UK) Organic ...

Genes encoding biotin carboxylase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution

USDA-ARS?s Scientific Manuscript database

Comparative genomics is a useful tool to investigate gene and genome evolution. Biotin carboxylase (BC), an important subunit of heteromeric ACCase that is a rate-limiting enzyme in fatty acid biosynthesis in dicots, catalyzes ATP, biotin-carboxyl-carrier protein and CO2 to form carboxybiotin-carbo...

Stem juice production of the C4 sugarcane (Saccharum officinarum) is enhanced by growth at double-ambient CO2 and high temperature.

PubMed

Vu, Joseph C V; Allen, Leon H

2009-07-15

Two cultivars of sugarcane (Saccharum officinarum cv. CP73-1547 and CP88-1508) were grown for 3 months in paired-companion, temperature-gradient, sunlit greenhouses under daytime [CO2] of 360 (ambient) and 720 (double ambient) micromol mol(-1) and at temperatures of 1.5 degrees C (near ambient) and 6.0 degrees C higher than outside ambient temperature. Leaf area and biomass, stem biomass and juice and CO2 exchange rate (CER) and activities of ribulose bisphosphate carboxylase-oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC) of fully developed leaves were measured at harvest. On a main stem basis, leaf area, leaf dry weight, stem dry weight and stem juice volume were increased by growth at doubled [CO2] or high temperature. Such increases were even greater under combination of doubled [CO2]/high temperature. Plants grown at doubled [CO2]/high temperature combination averaged 50%, 26%, 84% and 124% greater in leaf area, leaf dry weight, stem dry weight and stem juice volume, respectively, compared with plants grown at ambient [CO2]/near-ambient temperature combination. In addition, plants grown at doubled [CO2]/high temperature combination were 2-3-fold higher in stem soluble solids than those at ambient [CO2]/near-ambient temperature combination. Although midday CER of fully developed leaves was not affected by doubled [CO2] or high temperature, plants grown at doubled [CO2] were 41-43% less in leaf stomatal conductance and 69-79% greater in leaf water-use efficiency, compared with plants grown at ambient [CO2]. Activity of PEPC was down-regulated 23-32% at doubled [CO2], while high temperature did not have a significant impact on this enzyme. Activity of Rubisco was not affected by growth at doubled [CO2], but was reduced 15-28% at high temperature. The increases in stem juice production and stem juice soluble solids concentration for sugarcane grown at doubled [CO2] or high temperature, or at doubled [CO2]/high temperature combination, were partially

Activation of acetyl-coenzyme A carboxylase is involved in Taxol-induced ovarian cancer cell death

PubMed Central

WU, JIANG; JI, FANG; DI, WEN; CHEN, HONGDUO; WAN, YINSHENG

2011-01-01

Acetyl-coenzyme A carboxylase (ACC) is an attractive target for research into the treatment of a variety of human diseases, including diabetes, obesity and cancer. Mounting evidence suggests that the inhibition of ACC induced of cancer cell apoptosis. However, whether the inhibition of ACC regulates apoptosis in CaOV3 cancer cells has yet to be addressed. This study investigated the cytotoxic mechanism of action of ACC inhibition. Results showed that 5-(tetradecyloxy)-2-furoic acid (TOFA), an ACC inhibitor, enhanced Taxol-induced CaOV3 human ovarian cancer cell apoptosis. Notably, when TOFA was administered as a monotherapy, it induced CaOV3 cell apoptosis. Pre-treatment with the EGFR inhibitor PD153035 was found to markedly enhance ACC phosphorylation, whereas AMP-activated protein kinase (AMPK) activator AICAR was found to marginally enhance ACC phosphorylation. Taken together, the data showed ACC is a potential novel molecular target of Taxol. Additionally, ACC inhibition partially contributed to the cytotoxic effect of Taxol in ovarian cancer cells. PMID:22866118

Activation of acetyl-coenzyme A carboxylase is involved in Taxol-induced ovarian cancer cell death.

PubMed

Wu, Jiang; Ji, Fang; DI, Wen; Chen, Hongduo; Wan, Yinsheng

2011-05-01

Acetyl-coenzyme A carboxylase (ACC) is an attractive target for research into the treatment of a variety of human diseases, including diabetes, obesity and cancer. Mounting evidence suggests that the inhibition of ACC induced of cancer cell apoptosis. However, whether the inhibition of ACC regulates apoptosis in CaOV3 cancer cells has yet to be addressed. This study investigated the cytotoxic mechanism of action of ACC inhibition. Results showed that 5-(tetradecyloxy)-2-furoic acid (TOFA), an ACC inhibitor, enhanced Taxol-induced CaOV3 human ovarian cancer cell apoptosis. Notably, when TOFA was administered as a monotherapy, it induced CaOV3 cell apoptosis. Pre-treatment with the EGFR inhibitor PD153035 was found to markedly enhance ACC phosphorylation, whereas AMP-activated protein kinase (AMPK) activator AICAR was found to marginally enhance ACC phosphorylation. Taken together, the data showed ACC is a potential novel molecular target of Taxol. Additionally, ACC inhibition partially contributed to the cytotoxic effect of Taxol in ovarian cancer cells.

Emodin Regulates Glucose Utilization by Activating AMP-activated Protein Kinase*

PubMed Central

Song, Parkyong; Kim, Jong Hyun; Ghim, Jaewang; Yoon, Jong Hyuk; Lee, Areum; Kwon, Yonghoon; Hyun, Hyunjung; Moon, Hyo-Youl; Choi, Hueng-Sik; Berggren, Per-Olof; Suh, Pann-Ghill; Ryu, Sung Ho

2013-01-01

AMP-activated protein kinase has been described as a key signaling protein that can regulate energy homeostasis. Here, we aimed to characterize novel AMP-activated kinase (AMPK)-activating compounds that have a much lower effective concentration than metformin. As a result, emodin, a natural anthraquinone derivative, was shown to stimulate AMPK activity in skeletal muscle and liver cells. Emodin enhanced GLUT4 translocation and [14C]glucose uptake into the myotube in an AMPK-dependent manner. Also, emodin inhibited glucose production by suppressing the expression of key gluconeogenic genes, such as phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, in hepatocytes. Furthermore, we found that emodin can activate AMPK by inhibiting mitochondrial respiratory complex I activity, leading to increased reactive oxygen species and Ca2+/calmodulin-dependent protein kinase kinase activity. Finally, we confirmed that a single dose administration of emodin significantly decreased the fasting plasma glucose levels and improved glucose tolerance in C57Bl/6J mice. Increased insulin sensitivity was also confirmed after daily injection of emodin for 8 days using an insulin tolerance test and insulin-stimulated PI3K phosphorylation in wild type and high fat diet-induced diabetic mouse models. Our study suggests that emodin regulates glucose homeostasis in vivo by AMPK activation and that this may represent a novel therapeutic principle in the treatment of type 2 diabetic models. PMID:23303186

Pig liver pyruvate carboxylase. The reaction pathway for the decarboxylation of oxaloacetate

PubMed Central

Warren, Graham B.; Tipton, Keith F.

1974-01-01

1. The reaction pathway for the decarboxylation of oxaloacetate, catalysed by pig liver pyruvate carboxylase, was studied in the presence of saturating concentrations of K+ and acetyl-CoA. 2. Free Mg2+ binds to the enzyme in an equilibrium fashion and remains bound during all further catalytic cycles. MgADP− and Pi bind randomly, at equilibrium, followed by the binding of oxaloacetate. Pyruvate is released before the ordered steay-state release of HCO3− and MgATP2−. 3. These results are entirely consistent with studies on the carboxylation of pyruvate presented in the preceding paper (Warren & Tipton, 1974b) and together they allow a quantitative description of the reaction mechanism of pig liver pyruvate carboxylase. 4. In the absence of other substrates of the back reaction pig liver pyruvate carboxylase will decarboxylate oxaloacetate in a manner that is not inhibited by avidin. 5. Reciprocal plots involving oxaloacetate are non-linear curves, which suggest a negatively co-operative interaction between this substrate and the enzyme. PMID:4447613

How to pattern a leaf.

PubMed

Bolduc, N; O'Connor, D; Moon, J; Lewis, M; Hake, S

2012-01-01

Leaf development presents a tremendous resource for tackling the question of patterning in biology. Leaves can be simple or highly dissected. They may have elaborated parts such as the tendrils of a pea leaf or the rolled blade of a carnivorous pitcher plant. Despite the variation in size, shape, and function, all leaves initiate in the same manner: from the flanks of a meristem. The maize leaf is useful for analysis of patterning due to the wealth of mutants and the distinct tissues along the proximal distal axis. The blade is distal, the sheath is proximal, and the ligule forms at the blade/sheath boundary. Establishment of this boundary involves the transcription factors LIGULELESS1 and LIGULELESS2 and the kinase LIGULELESS NARROW. The meristem-specific protein KNOTTED1 (KN1) binds and modulates the lg2 gene. Given the localization of KN1 at the proximal end of the leaf from the time of inception, we hypothesize that KN1 has a role in establishing the very proximal end of the leaf, whereas an auxin maximum guides the growing distal tip.

Increased leaf angle1, a Raf-like MAPKKK that interacts with a nuclear protein family, regulates mechanical tissue formation in the Lamina joint of rice.

PubMed

Ning, Jing; Zhang, Baocai; Wang, Nili; Zhou, Yihua; Xiong, Lizhong

2011-12-01

Mitogen-activated protein kinase kinase kinases (MAPKKKs), which function at the top level of mitogen-activated protein kinase cascades, are clustered into three groups. However, no Group C Raf-like MAPKKKs have yet been functionally identified. We report here the characterization of a rice (Oryza sativa) mutant, increased leaf angle1 (ila1), resulting from a T-DNA insertion in a Group C MAPKKK gene. The increased leaf angle in ila1 is caused by abnormal vascular bundle formation and cell wall composition in the leaf lamina joint, as distinct from the mechanism observed in brassinosteroid-related mutants. Phosphorylation assays revealed that ILA1 is a functional kinase with Ser/Thr kinase activity. ILA1 is predominantly resident in the nucleus and expressed in the vascular bundles of leaf lamina joints. Yeast two-hybrid screening identified six closely related ILA1 interacting proteins (IIPs) of unknown function. Using representative IIPs, the interaction of ILA1 and IIPs was confirmed in vivo. IIPs were localized in the nucleus and showed transactivation activity. Furthermore, ILA1 could phosphorylate IIP4, indicating that IIPs may be the downstream substrates of ILA1. Microarray analyses of leaf lamina joints provided additional evidence for alterations in mechanical strength in ila1. ILA1 is thus a key factor regulating mechanical tissue formation at the leaf lamina joint.

Computational redesign of bacterial biotin carboxylase inhibitors using structure-based virtual screening of combinatorial libraries.

PubMed

Brylinski, Michal; Waldrop, Grover L

2014-04-02

As the spread of antibiotic resistant bacteria steadily increases, there is an urgent need for new antibacterial agents. Because fatty acid synthesis is only used for membrane biogenesis in bacteria, the enzymes in this pathway are attractive targets for antibacterial agent development. Acetyl-CoA carboxylase catalyzes the committed and regulated step in fatty acid synthesis. In bacteria, the enzyme is composed of three distinct protein components: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase. Fragment-based screening revealed that amino-oxazole inhibits biotin carboxylase activity and also exhibits antibacterial activity against Gram-negative organisms. In this report, we redesigned previously identified lead inhibitors to expand the spectrum of bacteria sensitive to the amino-oxazole derivatives by including Gram-positive species. Using 9,411 small organic building blocks, we constructed a diverse combinatorial library of 1.2×10⁸ amino-oxazole derivatives. A subset of 9×10⁶ of these compounds were subjected to structure-based virtual screening against seven biotin carboxylase isoforms using similarity-based docking by eSimDock. Potentially broad-spectrum antibiotic candidates were selected based on the consensus ranking by several scoring functions including non-linear statistical models implemented in eSimDock and traditional molecular mechanics force fields. The analysis of binding poses of the top-ranked compounds docked to biotin carboxylase isoforms suggests that: (1) binding of the amino-oxazole anchor is stabilized by a network of hydrogen bonds to residues 201, 202 and 204; (2) halogenated aromatic moieties attached to the amino-oxazole scaffold enhance interactions with a hydrophobic pocket formed by residues 157, 169, 171 and 203; and (3) larger substituents reach deeper into the binding pocket to form additional hydrogen bonds with the side chains of residues 209 and 233. These structural insights into drug

The dynamic organization of fungal acetyl-CoA carboxylase

NASA Astrophysics Data System (ADS)

Hunkeler, Moritz; Stuttfeld, Edward; Hagmann, Anna; Imseng, Stefan; Maier, Timm

2016-04-01

Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control.

Expression and activity of the 5'-adenosine monophosphate-activated protein kinase pathway in selected tissues during chicken embryonic development.

PubMed

Proszkowiec-Weglarz, M; Richards, M P

2009-01-01

The 5'-adenosine monophosphate-activated protein kinase (AMPK) is a highly conserved serine-threonine protein kinase and a key part of a kinase-signaling cascade that senses cellular energy status (adenosine monophosphate:adenosine triphosphate ratio) and acts to maintain energy homeostasis by coordinately regulating energy-consuming and energy-generating metabolic pathways. The objective of this study was to investigate aspects of the AMPK pathway in the liver, brain, breast muscle, and heart from d 12 of incubation through hatch in chickens. We first determined mRNA and protein expression profiles for a major upstream AMPK kinase, LKB1, which is known to activate (phosphorylate) AMPK in response to increases in the adenosine monophosphate:adenosine triphosphate ratio. Expression of LKB1 protein was greatest in the brain, which demonstrated tissue-specific patterns for phosphorylation. Next, AMPK subunit mRNA and protein expression profiles were determined. Significant changes in AMPK subunit mRNA expression occurred in all tissues from d 12 of incubation to hatch. Differences in the levels of active (phosphorylated) AMPK as well as alpha and beta subunit proteins were observed in all 4 tissues during embryonic development. Finally, we determined the protein level and phosphorylation status of an important downstream target for AMPK, acetyl-coenzyme A carboxylase. The expression of acetyl-co-enzyme A carboxylase and phosphorylated acetyl-coenzyme A was greater in the brain than the liver, but was undetectable by Western blotting in the breast muscle and heart throughout the period of study. Together, our results are the first to demonstrate the expression and activity of the AMPK pathway in key tissues during the transition from embryonic to posthatch development in chickens.

C3-C4 Intermediate Species in Alternanthera (Amaranthaceae) 1

PubMed Central

Rajendrudu, Gedupudi; Prasad, Jasty S. R.; Das, V. S. Rama

1986-01-01

Two naturally occurring species of the genus Alternanthera, namely A. ficoides and A. tenella, were identified as C3-C4 intermediates based on leaf anatomy, photosynthetic CO2 compensation point (Γ), O2 response of г, light intensity response of г, and the activities of key enzymes of photosynthesis. A. ficoides and A. tenella exhibited a less distinct Kranz-like leaf anatomy with substantial accumulation of starch both in mesophyll and bundle sheath cells. Photosynthetic CO2 compensation points of these two intermediate species at 29°C were much lower than in C3 plants and ranged from 18 to 22 microliters per liter. Although A. ficoides and A. tenella exhibited similar intermediacy in г, the apparent photorespiratory component of O2 inhibition in A. ficoides is lower than in A. tenella. The г progressively decreases from 35 microliters per liter at lowest light intensity to 18 microliters per liter at highest light intensity in A. tenella. It was, however, constant in A. ficoides at 20 to 25 microliters per liter between light intensities measured. The rates of net photosynthesis at 21% O2 and 29°C by A. ficoides and A. tenella were 25 to 28 milligrams CO2 per square decimeter per hour which are intermediate between values obtained for Tridax procumbens and A. pungens, C3 and C4 species, respectively. The activities of key enzymes of C4 photosynthesis, phosphoenolpyruvate carboxylase, pyruvate Pi dikinase, NAD malic enzyme, NADP malic enzyme and phosphoenolpyruvate carboxykinase in the two intermediates, A. ficoides and A. tenella are very low or insignificant. Results indicated that the relatively low apparent photorespiratory component in these two species is presumably the basis for the C3-C4 intermediate photosynthesis. Images Fig. 2 PMID:16664634

Phosphoenolpyruvate carboxykinase and gluconeogenesis in grape pericarp.

PubMed

Walker, Robert P; Battistelli, Alberto; Moscatello, Stefano; Técsi, László; Leegood, Richard C; Famiani, Franco

2015-12-01

Glycolysis from sugars is necessary at all stages of development of grape pericarp, and this raises the question as to why gluconeogenesis from malate occurs. Phosphoenolpyruvate carboxykinase (PEPCK) is required for gluconeogenesis in grape pericarp. In this study we determined the abundance of PEPCK protein and activity in different parts of grape pericarp during its development. Both PEPCK protein and activity were present throughout development, however, in both the skin and the flesh their abundance increased greatly at the start of ripening. This coincided with the onset of the decrease in the malate content of the berry. The location of PEPCK in the pericarp at different stages of development was determined using both immunohistochemistry and dissection. We provide a possible explanation for the occurrence of gluconeogenesis in grape pericarp. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Central Role of Pyruvate Kinase in Carbon Co-catabolism of Mycobacterium tuberculosis*

PubMed Central

Noy, Tahel; Vergnolle, Olivia; Hartman, Travis E.; Rhee, Kyu Y.; Jacobs, William R.; Berney, Michael; Blanchard, John S.

2016-01-01

Mycobacterium tuberculosis (Mtb) displays a high degree of metabolic plasticity to adapt to challenging host environments. Genetic evidence suggests that Mtb relies mainly on fatty acid catabolism in the host. However, Mtb also maintains a functional glycolytic pathway and its role in the cellular metabolism of Mtb has yet to be understood. Pyruvate kinase catalyzes the last and rate-limiting step in glycolysis and the Mtb genome harbors one putative pyruvate kinase (pykA, Rv1617). Here we show that pykA encodes an active pyruvate kinase that is allosterically activated by glucose 6-phosphate (Glc-6-P) and adenosine monophosphate (AMP). Deletion of pykA prevents Mtb growth in the presence of fermentable carbon sources and has a cidal effect in the presence of glucose that correlates with elevated levels of the toxic catabolite methylglyoxal. Growth attenuation was also observed in media containing a combination of short chain fatty acids and glucose and surprisingly, in media containing odd and even chain fatty acids alone. Untargeted high sensitivity metabolomics revealed that inactivation of pyruvate kinase leads to accumulation of phosphoenolpyruvate (P-enolpyruvate), citrate, and aconitate, which was consistent with allosteric inhibition of isocitrate dehydrogenase by P-enolpyruvate. This metabolic block could be relieved by addition of the α-ketoglutarate precursor glutamate. Taken together, our study identifies an essential role of pyruvate kinase in preventing metabolic block during carbon co-catabolism in Mtb. PMID:26858255

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

PubMed

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

2014-11-01

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

Photosynthesis-related characteristics of the midrib and the interveinal lamina in leaves of the C3-CAM intermediate plant Mesembryanthemum crystallinum.

PubMed

Kuźniak, Elżbieta; Kornas, Andrzej; Kaźmierczak, Andrzej; Rozpądek, Piotr; Nosek, Michał; Kocurek, Maciej; Zellnig, Günther; Müller, Maria; Miszalski, Zbigniew

2016-06-01

Leaf veins are usually encircled by specialized bundle sheath cells. In C4 plants, they play an important role in CO2 assimilation, and the photosynthetic activity is compartmentalized between the mesophyll and the bundle sheath. In C3 and CAM (Crassulacean acid metabolism) plants, the photosynthetic activity is generally attributed to the leaf mesophyll cells, and the vascular parenchymal cells are rarely considered for their role in photosynthesis. Recent studies demonstrate that enzymes required for C4 photosynthesis are also active in the veins of C3 plants, and their vascular system contains photosynthetically competent parenchyma cells. However, our understanding of photosynthesis in veins of C3 and CAM plants still remains insufficient. Here spatial analysis of photosynthesis-related properties were applied to the midrib and the interveinal lamina cells in leaves of Mesembryanthemum crystallinum, a C3-CAM intermediate plant. The midrib anatomy as well as chloroplast structure and chlorophyll fluorescence, diurnal gas exchange profiles, the immunoblot patterns of PEPC (phosphoenolpyruvate carboxylase) and RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase), H2O2 localization and antioxidant enzyme activities were compared in the midrib and in the interveinal mesophyll cells in leaves of C3 and CAM plants. Leaf midribs were structurally competent to perform photosynthesis in C3 and CAM plants. The midrib chloroplasts resembled those in the bundle sheath cells of C4 plants and were characterized by limited photosynthetic activity. The metabolic roles of midrib chloroplasts differ in C3 and CAM plants. It is suggested that in leaves of C3 plants the midrib chloroplasts could be involved in the supply of CO2 for carboxylation, and in CAM plants they could provide malate to different metabolic processes and mediate H2O2 signalling. © The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For

Unique response of lung acetyl-CoA carboxylase to inhibitors

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

Patterson, C.E.; Davis, K.S.; Rhoades, R.A.

1986-05-01

Fatty acid synthesis (FAS) in lung is not inhibited by c-AMP analogs or aminophylline although these agents inhibit FAS in other lipogenic tissues. To further characterize FAS in lung, the authors examined the response of cultured fetal lung explants to known inhibitors of FAS in liver: t-butyl benzoic acid (tBB-which binds CoA and inhibits acetyl-CoA carboxylase) and palmitate (an allosteric effector of acetyl-CoA carboxylase). Explants derived from d18 fetuses (term=22d) were cultured 2d in F12k media containing 10mM lactate, 2mM glucose, and 10mM Hepes. At 48h, FAS was determined by incubation with /sup 3/H/sub 2/O (control = 3892 +/- 755more » nmoles C2 units/g/h) and surfactant lipid production estimated by incorporation of /sup 14/C-choline into DSPC (control = 35.8 +/- 9.0 nmoles/g/h). Addition of tBB (50uM) did not significantly alter FAS or choline incorporation. Addition of palmitate (0.15mM) in either ethanol (1% final conc.) or albumin (3% final conc.) did not result in diminished FAS. Palmitate did increase DSPC labeling 20%, indicating that in these cultures the rate of surfactant synthesis is partially dependent upon palmitate availability. These data show that lung is unique in its unresponsiveness to various inhibitors of FAS which act at the level acetyl-CoA carboxylase and suggest that FAS is maintained in order to insure a de novo palmitate supply for surfactant lipid synthesis.« less

Biochemical signatures mimicking multiple carboxylase deficiency in children with mutations in MT-ATP6.

PubMed

Larson, Austin A; Balasubramaniam, Shanti; Christodoulou, John; Burrage, Lindsay C; Marom, Ronit; Graham, Brett H; Diaz, George A; Glamuzina, Emma; Hauser, Natalie; Heese, Bryce; Horvath, Gabriella; Mattman, Andre; van Karnebeek, Clara; Lane Rutledge, S; Williamson, Amy; Estrella, Lissette; Van Hove, Johan K L; Weisfeld-Adams, James D

2018-01-04

Elevations of specific acylcarnitines in blood reflect carboxylase deficiencies, and have utility in newborn screening for life-threatening organic acidemias and other inherited metabolic diseases. In this report, we describe a newly-identified association of biochemical features of multiple carboxylase deficiency in individuals harboring mitochondrial DNA (mtDNA) mutations in MT-ATP6 and in whom organic acidemias and multiple carboxylase deficiencies were excluded. Using retrospective chart review, we identified eleven individuals with abnormally elevated propionylcarnitine (C3) or hydroxyisovalerylcarnitine (C5OH) with mutations in MT-ATP6, most commonly m.8993T>G in high heteroplasmy or homoplasmy. Most patients were ascertained on newborn screening; most had normal enzymatic or molecular genetic testing to exclude biotinidase and holocarboxylase synthetase deficiencies. MT-ATP6 is associated with some cases of Leigh disease; clinical outcomes in our cohort ranged from death from neurodegenerative disease in early childhood to clinically and developmentally normal after several years of follow-up. These cases expand the biochemical phenotype associated with MT-ATP6 mutations, especially m.8993T>G, to include acylcarnitine abnormalities mimicking carboxylase deficiency states. Clinicians should be aware of this association and its implications for newborn screening, and consider mtDNA sequencing in patients exhibiting similar acylcarnitine abnormalities that are biotin-unresponsive and in whom other enzymatic deficiencies have been excluded. Copyright © 2018 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

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

NASA Technical Reports Server (NTRS)

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

1968-01-01

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

Trends in leaf photosynthesis in historical rice varieties developed in the Philippines since 1966.

PubMed

Hubbart, S; Peng, S; Horton, P; Chen, Y; Murchie, E H

2007-01-01

Crop improvement in terms of yield is rarely linked to leaf photosynthesis. However, in certain crop plants such as rice, it is predicted that an increase in photosynthetic rate will be required to support future grain yield potential. In order to understand the relationships between yield improvement and leaf photosynthesis, controlled environment conditions were used to grow 10 varieties which were released from the International Rice Research Institute (IRRI) between 1966 and 1995 and one newly developed line. Two growth light intensities were used: high light (1500 micromol m(-2) s(-1)) and low light (300 micromol m(-2) s(-1)). Gas exchange, leaf protein, chlorophyll, and leaf morphology were measured in the ninth leaf on the main stem. A high level of variation was observed among high light-grown plants for light-saturated photosynthetic rate per unit leaf area (P(max)), stomatal conductance (g), content of ribulose bisphosphate carboxylase-oxygenase (Rubisco), and total leaf protein content. Notably, between 1966 and 1980 there was a decline in P(max), g, leaf protein, chlorophyll, and Rubisco content. Values recovered in those varieties released after 1980. This striking trend coincides with a previous published observation that grain yield in IRRI varieties released prior to 1980 correlated with harvest index whereas that for those released after 1980 correlated with biomass. P(max) showed significant correlations with both g and Rubisco content. Large differences were observed between high light- and low light-grown plants (photoacclimation). The photoacclimation 'range' for P(max) correlated with P(max) in high light-grown plants. It is concluded that (i) leaf photosynthesis may be systematically affected by breeding strategy; (ii) P(max) is a useful target for yield improvements where yield is limited by biomass production rather than partitioning; and (iii) the capacity for photoacclimation is related to high P(max) values.

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

USDA-ARS?s Scientific Manuscript database

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

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

PubMed Central

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

1992-01-01

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

Evidence that Ribulose 1,5-Bisphosphate (RuBP) Binds to Inactive Sites of RuBP Carboxylase in Vivo and an Estimate of the Rate Constant for Dissociation 1

PubMed Central

Cardon, Zoe G.; Mott, Keith A.

1989-01-01

The binding of ribulose 1,5-bisphosphate (RuBP) to inactive (noncarbamylated) sites of the enzyme RuBP carboxylase in vivo was investigated in Spinacia oleracea and Helianthus annuus. The concentrations of RuBP and inactive sites were determined in leaf tissue as a function of time after a change to darkness. RuBP concentrations fell rapidly after the change to darkness and were approximately equal to the concentration of inactive sites after 60 s. Variations in the concentration of inactive sites, which were induced by differences in the light intensity before the light-dark transition, correlated with the concentration of RuBP between 60 and 120 s after the change to darkness. These data are discussed as evidence that RuBP binds to inactive sites of RuBP carboxylase in vivo. After the concentration of RuBP fell below that of inactive sites (at times longer than 60 s of darkness), the decline in RuBP was logarithmic with time. This would be expected if the dissociation of RuBP from inactive sites controlled the decline in RuBP concentration. These data were used to estimate the rate constant for dissociation of RuBP from inactive sites in vivo. PMID:16666692

Properties of ribulose diphosphate carboxylase immobilized on porous glass

NASA Technical Reports Server (NTRS)

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

1974-01-01

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

Arctigenin protects against steatosis in WRL68 hepatocytes through activation of phosphoinositide 3-kinase/protein kinase B and AMP-activated protein kinase pathways.

PubMed

Chen, Kung-Yen; Lin, Jui-An; Yao, Han-Yun; Hsu, An-Chih; Tai, Yu-Ting; Chen, Jui-Tai; Hsieh, Mao-Chih; Shen, Tang-Long; Hsu, Ren-Yi; Wu, Hong-Tan; Wang, Guey Horng; Ho, Bing-Ying; Chen, Yu-Pei

2018-04-01

Arctigenin (ATG), a lignin extracted from Arctium lappa (L.), exerts antioxidant and anti-inflammatory effects. We hypothesized that ATG exerts a protective effect on hepatocytes by preventing nonalcoholic fatty liver disease (NAFLD) progression associated with lipid oxidation-associated lipotoxicity and inflammation. We established an in vitro NAFLD cell model by using normal WRL68 hepatocytes to investigate oleic acid (OA) accumulation and the potential bioactive role of ATG. The results revealed that ATG inhibited OA-induced lipid accumulation, lipid peroxidation, and inflammation in WRL68 hepatocytes, as determined using Oil Red O staining, thiobarbituric acid reactive substance assay, and inflammation antibody array assays. Quantitative RT-PCR analysis demonstrated that ATG significantly mitigated the expression of acetylcoenzyme A carboxylase 1 and sterol regulatory element-binding protein-1 and significantly increased the expression of carnitine palmitoyltransferase 1 and peroxisome proliferator-activated receptor alpha. The 40 targets of the Human Inflammation Antibody Array indicated that ATG significantly inhibited the elevation of the U937 lymphocyte chemoattractant, ICAM-1, IL-1β, IL-6, IL-6sR, IL-7, and IL-8. ATG could activate the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and AMP-activated protein kinase (AMPK) pathways and could increase the phosphorylation levels of Akt and AMPK to mediate cell survival, lipid metabolism, oxidation stress, and inflammation. Thus, we demonstrated that ATG could inhibit NAFLD progression associated with lipid oxidation-associated lipotoxicity and inflammation, and we provided insights into the underlying mechanisms and revealed potential targets to enable a thorough understanding of NAFLD progression. Copyright © 2018 Elsevier Inc. All rights reserved.

Differences in properties and proteomes of the midribs contribute to the size of the leaf angle in two near-isogenic maize lines.

PubMed

Wang, Ning; Cao, Di; Gong, Fangping; Ku, Lixia; Chen, Yanhui; Wang, Wei

2015-10-14

The midrib of maize leaves provides the primary support for the blade and is largely associated with leaf angle size. To elucidate the role of the midrib in leaf angle formation, the maize line Shen137 (larger leaf angle) and a near isogenic line (NIL, smaller leaf angle) were used in the present study. The results of the analysis showed that both the puncture forces and proximal collenchyma number of the midribs of the first and second leaves above the ear were higher in NIL than in Shen137. Comparative proteomic analysis was performed to reveal protein profile differences in the midribs of the 5th, 10th and 19th newly expanded leaves between Shen137 and NIL. Quantitative analysis of 24 identified midrib proteins indicated that the maximum changes in abundance of 22 proteins between Shen137 and NIL appeared at the 10th leaf stage, of which phosphoglycerate kinase, adenosine kinase, fructose-bisphosphate aldolase and adenylate kinase were implicated in glycometabolism. Thus, glycometabolism might be associated with leaf angle formation and the physical and mechanical properties of the midribs. These results provide insight into the mechanism underlying maize leaf angle formation. Copyright © 2015 Elsevier B.V. All rights reserved.

Pyruvate kinase deletion as an effective phenotype to enhance lysine production in Corynebacterium glutamicum ATCC13032: Redirecting the carbon flow to a precursor metabolite.

PubMed

Yanase, Masaki; Aikoh, Tohru; Sawada, Kazunori; Ogura, Kotaro; Hagiwara, Takuya; Imai, Keita; Wada, Masaru; Yokota, Atsushi

2016-08-01

Various attempts have been made to enhance lysine production in Corynebacterium glutamicum. Pyruvate kinase (PYK) defect is one of the strategies used to enhance the supply of oxaloacetic acid (OAA), a precursor metabolite for lysine biosynthesis. However, inconsistent effects of this mutation have been reported: positive effects of PYK defect in mutants having phosphoenolpyruvate carboxylase (PEPC) desensitized to feedback inhibition by aspartic acid, while negative effects in simple PYK gene (pyk) knockout mutants. To address these discrepancies, the effects of pyk deletion on lysine yield were investigated with or without the D299N mutation in ppc rendering PEPC desensitization. C. glutamicum ATCC13032 mutant strain P with a feedback inhibition-desensitized aspartokinase was used as the parent strain, producing 9.36 g/L lysine from 100 g/L glucose in a jar fermentor culture. Under these conditions, while the simple mutant D2 with pyk deletion or R2 with the PEPC-desensitization mutation showed marginally increased lysine yield (∼1.1-fold, not significant), the mutant DR2 strain having both mutations showed synergistically increased lysine productivity (1.38-fold, 12.9 g/L). Therefore, the pyk deletion is effective under a PEPC-desensitized background, which ensures enhanced supply of OAA, thus clarifying the discrepancies. A citrate synthase defective mutation (S252C in gltA) further increased the lysine yield in strain DR2 (1.68-fold, 15.7 g/L). Thus, these three mutations coordinately enhanced the lysine yield. Both the malate:quinone oxidoreductase activity and respiration rate were significantly reduced in strains D2 and DR2. Overall, these results provide valuable knowledge for engineering the anaplerotic reaction to increase lysine yield in C. glutamicum. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Insect acetyl-CoA carboxylase: activity during the larval, pupal and adult stages of insect development.

PubMed

Goldring, J P; Read, J S

1993-12-01

1. The activity of the lipogenic enzyme, acetyl-CoA carboxylase, was investigated in four insect species; Bombyx mori (Lepidoptera), Tenebrio molitor (Coleoptera), Glossina morsitans and Sarcophaga nodosa (Diptera). 2. Acetyl-CoA carboxylase activity in larval, pupal and adult forms was compared with the saponifiable lipid mass at each stage of the life-cycle, and found to follow similar patterns except for Tenebrio molitor. 3. The results are examined in relation to known metabolic requirements for each insect.

The structural and photosynthetic characteristics of the exposed peduncle of wheat (Triticum aestivum L.): an important photosynthate source for grain-filling.

PubMed

Kong, Lingan; Wang, Fahong; Feng, Bo; Li, Shengdong; Si, Jisheng; Zhang, Bin

2010-07-11

In wheat (Triticum aestivum L), the flag leaf has been thought of as the main source of assimilates for grain growth, whereas the peduncle has commonly been thought of as a transporting organ. The photosynthetic characteristics of the exposed peduncle have therefore been neglected. In this study, we investigated the anatomical traits of the exposed peduncle during wheat grain ontogenesis, and we compared the exposed peduncle to the flag leaf with respect to chloroplast ultrastructure, photosystem II (PSII) quantum yield, and phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) activity. Transmission electron microscope observations showed well-developed chloroplasts with numerous granum stacks at grain-filling stages 1, 2 and 3 in both the flag leaf and the exposed peduncle. In the exposed peduncle, the membranes constituting the thylakoids were very distinct and plentiful, but in the flag leaf, there was a sharp breakdown at stage 4 and complete disintegration of the thylakoid membranes at stage 5. PSII quantum yield assays revealed that the photosynthetic efficiency remained constant at stages 1, 2 and 3 and then declined in both organs. However, the decline occurred more dramatically in the flag leaf than in the exposed peduncle. An enzyme assay showed that at stages 1 and 2 the PEPCase activity was lower in the exposed peduncle than in the flag leaf; but at stages 3, 4 and 5 the value was higher in the exposed peduncle, with a particularly significant difference observed at stage 5. Subjecting the exposed part of the peduncle to darkness following anthesis reduced the rate of grain growth. Our results suggest that the exposed peduncle is a photosynthetically active organ that produces photosynthates and thereby makes a crucial contribution to grain growth, particularly during the late stages of grain-filling.

Arabidopsis Type II Phosphatidylinositol 4-Kinase PI4Kγ5 Regulates Auxin Biosynthesis and Leaf Margin Development through Interacting with Membrane-Bound Transcription Factor ANAC078

PubMed Central

Tan, Shu-Tang; Xue, Hong-Wei

2016-01-01

Normal leaf margin development is important for leaf morphogenesis and contributes to diverse leaf shapes in higher plants. We here show the crucial roles of an atypical type II phosphatidylinositol 4-kinase, PI4Kγ5, in Arabidopsis leaf margin development. PI4Kγ5 presents a dynamics expression pattern along with leaf development and a T-DNA mutant lacking PI4Kγ5, pi4kγ5–1, presents serrated leaves, which is resulted from the accelerated cell division and increased auxin concentration at serration tips. Studies revealed that PI4Kγ5 interacts with and phosphorylates a membrane-bound NAC transcription factor, ANAC078. Previous studies demonstrated that membrane-bound transcription factors regulate gene transcription by undergoing proteolytic process to translocate into nucleus, and ANAC078 undergoes proteolysis by cleaving off the transmembrane region and carboxyl terminal. Western blot analysis indeed showed that ANAC078 deleting of carboxyl terminal is significantly reduced in pi4kγ5–1, indicating that PI4Kγ5 is important for the cleavage of ANAC078. This is consistent with the subcellular localization observation showing that fluorescence by GFP-ANAC078 is detected at plasma membrane but not nucleus in pi4kγ5–1 mutant and that expression of ANAC078 deleting of carboxyl terminal, driven by PI4Kγ5 promoter, could rescue the leaf serration defects of pi4kγ5–1. Further analysis showed that ANAC078 suppresses the auxin synthesis by directly binding and regulating the expression of auxin synthesis-related genes. These results indicate that PI4Kγ5 interacts with ANAC078 to negatively regulate auxin synthesis and hence influences cell proliferation and leaf development, providing informative clues for the regulation of in situ auxin synthesis and cell division, as well as the cleavage and functional mechanism of membrane-bound transcription factors. PMID:27529511

Acetone and Butanone Metabolism of the Denitrifying Bacterium “Aromatoleum aromaticum” Demonstrates Novel Biochemical Properties of an ATP-Dependent Aliphatic Ketone Carboxylase

PubMed Central

Schühle, Karola

2012-01-01

The anaerobic and aerobic metabolism of acetone and butanone in the betaproteobacterium “Aromatoleum aromaticum” is initiated by their ATP-dependent carboxylation to acetoacetate and 3-oxopentanoic acid, respectively. Both reactions are catalyzed by the same enzyme, acetone carboxylase, which was purified and characterized. Acetone carboxylase is highly induced under growth on acetone or butanone and accounts for at least 5.5% of total cell protein. The enzyme consists of three subunits of 85, 75, and 20 kDa, respectively, in a (αβγ)2 composition and contains 1 Zn and 2 Fe per heterohexamer but no organic cofactors. Chromatographic analysis of the ATP hydrolysis products indicated that ATP was exclusively cleaved to AMP and 2 Pi. The stoichiometry was determined to be 2 ATP consumed per acetone carboxylated. Purified acetone carboxylase from A. aromaticum catalyzes the carboxylation of acetone and butanone as the only substrates. However, the enzyme shows induced (uncoupled) ATPase activity with many other substrates that were not carboxylated. Acetone carboxylase is a member of a protein family that also contains acetone carboxylases of various other organisms, acetophenone carboxylase of A. aromaticum, and ATP-dependent hydantoinases/oxoprolinases. While the members of this family share several characteristic features, they differ with respect to the products of ATP hydrolysis, subunit composition, and metal content. PMID:22020645

A plant/fungal-type phosphoenolpyruvate carboxykinase located in the parasite mitochondrion ensures glucose-independent survival of Toxoplasma gondii.

PubMed

Nitzsche, Richard; Günay-Esiyok, Özlem; Tischer, Maximilian; Zagoriy, Vyacheslav; Gupta, Nishith

2017-09-15

Toxoplasma gondii is considered to be one of the most successful intracellular pathogens, because it can reproduce in varied nutritional milieus, encountered in diverse host cell types of essentially any warm-blooded organism. Our earlier work demonstrated that the acute (tachyzoite) stage of T. gondii depends on cooperativity of glucose and glutamine catabolism to meet biosynthetic demands. Either of these two nutrients can sustain the parasite survival; however, what determines the metabolic plasticity has not yet been resolved. Here, we reveal two discrete phosphoenolpyruvate carboxykinase (PEPCK) enzymes in the parasite, one of which resides in the m i t ochondrion ( Tg PEPCK mt ), whereas the other protein is n ot e xpressed in t achyzoites ( Tg PEPCK net ). Parasites with an intact glycolysis can tolerate genetic deletions of Tg PEPCK mt as well as of Tg PEPCK net , indicating their nonessential roles for tachyzoite survival. Tg PEPCK net can also be ablated in a glycolysis-deficient mutant, while Tg PEPCK mt is refractory to deletion. Consistent with this, the lytic cycle of a conditional mutant of Tg PEPCK mt in the glycolysis-impaired strain was aborted upon induced repression of the mitochondrial isoform, demonstrating its essential role for the glucose-independent survival of parasites. Isotope-resolved metabolomics of the conditional mutant revealed defective flux of glutamine-derived carbon into RNA-bound ribose sugar as well as metabolites associated with gluconeogenesis, entailing a critical nodal role of PEPCK mt in linking catabolism of glucose and glutamine with anabolic pathways. Our data also suggest a homeostatic function of Tg PEPCK mt in cohesive operation of glycolysis and the tricarboxylic acid cycle in a normal glucose-replete milieu. Conversely, we found that the otherwise integrative enzyme pyruvate carboxylase ( Tg PyC) is dispensable not only in glycolysis-competent but also in glycolysis-deficient tachyzoites despite a mitochondrial

Overexpression of ubiquitous 6-phosphofructo-2-kinase in the liver of transgenic mice results in weight gain

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

Duran, Joan; Navarro-Sabate, Aurea; Pujol, Anna

2008-01-11

Fructose 2,6-bisphosphate (Fru-2,6-P{sub 2}) is an important metabolite that controls glycolytic and gluconeogenic pathways in several cell types. Its synthesis and degradation are catalyzed by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2). Four genes, designated Pfkfb1-4, codify the different PFK-2 isozymes. The Pfkfb3 gene product, ubiquitous PFK-2 (uPFK-2), has the highest kinase/bisphosphatase activity ratio and is associated with proliferation and tumor metabolism. A transgenic mouse model that overexpresses uPFK-2 under the control of the phosphoenolpyruvate carboxykinase promoter was designed to promote sustained and elevated Fru-2,6-P{sub 2} levels in the liver. Our results demonstrate that in diet-induced obesity, high Fru-2,6-P{sub 2} levelsmore » in transgenic livers caused changes in hepatic gene expression profiles for key gluconeogenic and lipogenic enzymes, as well as an accumulation of lipids in periportal cells, and weight gain.« less

Inhibition of AMP Kinase by the Protein Phosphatase 2A Heterotrimer, PP2APpp2r2d*

PubMed Central

Joseph, Biny K.; Liu, Hsing-Yin; Francisco, Jamie; Pandya, Devanshi; Donigan, Melissa; Gallo-Ebert, Christina; Giordano, Caroline; Bata, Adam; Nickels, Joseph T.

2015-01-01

AMP kinase is a heterotrimeric serine/threonine protein kinase that regulates a number of metabolic processes, including lipid biosynthesis and metabolism. AMP kinase activity is regulated by phosphorylation, and the kinases involved have been uncovered. The particular phosphatases counteracting these kinases remain elusive. Here we discovered that the protein phosphatase 2A heterotrimer, PP2APpp2r2d, regulates the phosphorylation state of AMP kinase by dephosphorylating Thr-172, a residue that activates kinase activity when phosphorylated. Co-immunoprecipitation and co-localization studies indicated that PP2APpp2r2d directly interacted with AMP kinase. PP2APpp2r2d dephosphorylated Thr-172 in rat aortic and human vascular smooth muscle cells. A positive correlation existed between decreased phosphorylation, decreased acetyl-CoA carboxylase Acc1 phosphorylation, and sterol response element-binding protein 1c-dependent gene expression. PP2APpp2r2d protein expression was up-regulated in the aortas of mice fed a high fat diet, and the increased expression correlated with increased blood lipid levels. Finally, we found that the aortas of mice fed a high fat diet had decreased AMP kinase Thr-172 phosphorylation, and contained an Ampk-PP2APpp2r2d complex. Thus, PP2APpp2r2d may antagonize the aortic AMP kinase activity necessary for maintaining normal aortic lipid metabolism. Inhibiting PP2APpp2r2d or activating AMP kinase represents a potential pharmacological treatment for many lipid-related diseases. PMID:25694423

Targeted manipulation of leaf form via local growth repression.

PubMed

Malinowski, Robert; Kasprzewska, Ania; Fleming, Andrew J

2011-06-01

A classical view is that leaf shape is the result of local promotion of growth linked to cell proliferation. However, an alternative hypothesis is that leaf form is the result of local repression of growth in an otherwise growing system. Here we show that leaf form can indeed be manipulated in a directed fashion by local repression of growth. We show that targeting expression of an inhibitor of a cyclin-dependent kinase (KRP1) to the sinus area of developing leaves of Arabidopsis leads to local growth repression and the formation of organs with extreme lobing, including generation of leaflet-like organs. Directing KRP1 expression to other regions of the leaf using an miRNA target sequence tagging approach also leads to predictable novel leaf forms, and repression of growth in the leaf margin blocks the outgrowth of lobes, leading to a smoother perimeter. In addition, we show that decreased growth around the perimeter and across the leaf abaxial surface leads to a change in 3D form, as predicted by mechanical models of leaf growth. Our analysis provides experimental evidence that local repression of growth influences leaf shape, suggesting that it could be part of the mechanism of morphogenesis in plants in the context of an otherwise growing system. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

The AMP-activated protein kinase is involved in the regulation of ketone body production by astrocytes.

PubMed

Blázquez, C; Woods, A; de Ceballos, M L; Carling, D; Guzmán, M

1999-10-01

The possible role of the AMP-activated protein kinase (AMPK), a highly conserved stress-activated kinase, in the regulation of ketone body production by astrocytes was studied. AMPK activity in rat cortical astrocytes was three times higher than in rat cortical neurons. AMPK in astrocytes was shown to be functionally active. Thus, incubation of astrocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a cell-permeable activator of AMPK, stimulated both ketogenesis from palmitate and carnitine palmitoyltransferase I. This was concomitant to a decrease of intracellular malonyl-CoA levels and an inhibition of acetyl-CoA carboxylase/fatty acid synthesis and 3-hydroxy-3-methylglutaryl-CoA reductase/cholesterol synthesis. Moreover, in microdialysis experiments AICAR was shown to stimulate brain ketogenesis markedly. The effect of chemical hypoxia on AMPK and the ketogenic pathway was studied subsequently. Incubation of astrocytes with azide led to a remarkable drop of fatty acid beta-oxidation. However, activation of AMPK during hypoxia compensated the depression of beta-oxidation, thereby sustaining ketone body production. This effect seemed to rely on the cascade hypoxia --> increase of the AMP/ATP ratio --> AMPK stimulation --> acetyl-CoA carboxylase inhibition --> decrease of malonyl-CoA concentration --> carnitine palmitoyltransferase I deinhibition --> enhanced ketogenesis. Furthermore, incubation of neurons with azide blunted lactate oxidation, but not 3-hydroxybutyrate oxidation. Results show that (a) AMPK plays an active role in the regulation of ketone body production by astrocytes, and (b) ketone bodies produced by astrocytes during hypoxia might be a substrate for neuronal oxidative metabolism.

Biochemical, molecular, and phylogenetic analysis of pyruvate carboxylase in the yellow fever mosquito, Aedes aegypti.

PubMed

Tu, Z; Hagedorn, H H

1997-02-01

Pyruvate carboxylase (PC, pyruvate: carbon dioxide ligase [ADP-forming], EC 6.4.1.1) was purified from the yellow fever mosquito, Aedes aegypti. The purified PC showed two polypeptides of similar M(r) (133 and 128 k). The N-terminal sequences of both polypeptides were shown to be very similar, if not identical. A polyclonal antiserum against the 133 kDa polypeptide cross-reacted strongly with the 128 kDa polypeptide. PC was found in all tissues examined. Using a semi-quantitative Western blot assay, PC was shown to be concentrated in the indirect flight muscles and fat body preparations. The ratios of the 133 to 128 kDa polypeptides were shown to differ in various tissues and an Aedes albopictus cell line. The indirect flight muscle was the only tissue in which the 128 kDa polypeptide was more abundant, while both the midgut and the cell line showed almost exclusively the 133 kDa polypeptide. Both peptides were present in varying amounts in brain, malpighian tubule, ovary and fat body preparation. The two isoforms of PC could play different roles in the flight muscle and other tissues. Clones covering a complete cDNA of PC of A. aegypti were obtained using a directional approach. The 3952 bp nucleotide sequence, including a 3585 bp coding region, was determined from these cDNA clones. The deduced 1195 amino acid sequence has a calculated M(r) of 132,200. A putative mitochondrial targeting sequence was determined by comparing the deduced amino acid sequence to the N-terminal sequences of the mature protein. The presence of a mitochondrial targeting sequence indicates that the mosquito PC encoded by the cloned cDNA may be localized in the mitochondria. After the targeting sequence, three functional domains were identified in the following order; biotin carboxylase (BC), carboxyltransferase (CT) and biotin carboxyl carrier protein (BCCP). The mosquito PC showed very high similarity to PCs from other sources (55.1-75.2% identity). Genomic Southern analysis indicated

Glycolysis without pyruvate kinase in Clostridium thermocellum

DOE PAGES

Olson, Daniel G.; Horl, Manuel; Fuhrer, Tobias; ...

2016-12-01

The metabolism of Clostridium thermocellum is notable in that it assimilates sugar via the EMP pathway but does not possess a pyruvate kinase enzyme. In the wild type organism, there are three proposed pathways for conversion of phosphoenolpyruvate (PEP) to pyruvate, which differ in their cofactor usage. One path uses pyruvate phosphate dikinase (PPDK), another pathway uses the combined activities of PEP carboxykinase (PEPCK) and oxaloacetate decarboxylase (ODC). Yet another pathway, the malate shunt, uses the combined activities of PEPCK, malate dehydrogenase and malic enzyme. First we showed that there is no flux through the ODC pathway by enzyme assay.more » Flux through the remaining two pathways (PPDK and malate shunt) was determined by dynamic 13C labeling. In the wild-type strain, the malate shunt accounts for about 33 ± 2% of the flux to pyruvate, with the remainder via the PPDK pathway. Deletion of the ppdk gene resulted in a redirection of all pyruvate flux through the malate shunt. Lastly, this provides the first direct evidence of the in-vivo function of the malate shunt.« less

Leptin activates hypothalamic acetyl-CoA carboxylase to inhibit food intake

PubMed Central

Gao, Su; Kinzig, Kimberly P.; Aja, Susan; Scott, Karen A.; Keung, Wendy; Kelly, Sandra; Strynadka, Ken; Chohnan, Shigeru; Smith, Wanli W.; Tamashiro, Kellie L. K.; Ladenheim, Ellen E.; Ronnett, Gabriele V.; Tu, Yajun; Birnbaum, Morris J.; Lopaschuk, Gary D.; Moran, Timothy H.

2007-01-01

Hypothalamic fatty acid metabolism has recently been implicated in the controls of food intake and energy homeostasis. We report that intracerebroventricular (ICV) injection of leptin, concomitant with inhibiting AMP-activated kinase (AMPK), activates acetyl-CoA carboxylase (ACC), the key regulatory enzyme in fatty acid biosynthesis, in the arcuate nucleus (Arc) and paraventricular nucleus (PVN) in the hypothalamus. Arc overexpression of constitutively active AMPK prevents the Arc ACC activation in response to ICV leptin, supporting the hypothesis that AMPK lies upstream of ACC in leptin's Arc intracellular signaling pathway. Inhibiting hypothalamic ACC with 5-tetradecyloxy-2-furoic acid, a specific ACC inhibitor, blocks leptin-mediated decreases in food intake, body weight, and mRNA level of the orexigenic neuropeptide NPY. These results show that hypothalamic ACC activation makes an important contribution to leptin's anorectic effects. Furthermore, we find that ICV leptin up-regulates the level of malonyl-CoA (the intermediate of fatty acid biosynthesis) specifically in the Arc and increases the level of palmitoyl-CoA (a major product of fatty acid biosynthesis) specifically in the PVN. The rises of both levels are blocked by 5-tetradecyloxy-2-furoic acid along with the blockade of leptin-mediated hypophagia. These data suggest malonyl-CoA as a downstream mediator of ACC in leptin's signaling pathway in the Arc and imply that palmitoyl-CoA, instead of malonyl-CoA, could be an effector in relaying ACC signaling in the PVN. Together, these findings highlight site-specific impacts of hypothalamic ACC activation in leptin's anorectic signaling cascade. PMID:17956983

Leaf nitrogen assimilation and partitioning differ among subtropical forest plants in response to canopy addition of nitrogen treatments.

PubMed

Liu, Nan; Wu, Shuhua; Guo, Qinfeng; Wang, Jiaxin; Cao, Ce; Wang, Jun

2018-05-12

Global increases in nitrogen deposition may alter forest structure and function by interfering with plant nitrogen metabolism (e.g., assimilation and partitioning) and subsequent carbon assimilation, but it is unclear how these responses to nitrogen deposition differ among species. In this study, we conducted a 2-year experiment to investigate the effects of canopy addition of nitrogen (CAN) on leaf nitrogen assimilation and partitioning in three subtropical forest plants (Castanea henryi, Ardisia quinquegona, and Blastus cochinchinensis). We hypothesized that responses of leaf nitrogen assimilation and partitioning to CAN differ among subtropical forest plants. CAN increased leaf nitrate reductase (NR) activity, and leaf nitrogen and chlorophyll contents but reduced leaf maximum photosynthetic rate (A max ), photosynthetic nitrogen use efficiency (PNUE), ribulose-1,5-bisphosphate carboxylase (Rubisco) activity, and metabolic protein content of an overstory tree species C. henryi. In an understory tree A. quinquegona, CAN increased NR activity and glutamine synthetase activity and therefore increased metabolic protein synthesis (e.g., Rubisco) in leaves. In the shrub B. cochinchinensis, CAN increased A max , PNUE, Rubisco content, metabolic protein content, and Rubisco activity in leaves. Leaf nitrogen assimilation and partitioning results indicated that A. quinquegona and B. cochinchinensis may better acclimate to CAN than C. henryi and that the acclimation mechanism differs among the species. Results from this study suggest that long-term elevated atmospheric nitrogen deposition has contributed to the ongoing transformation of subtropical forests into communities dominated by small trees and shrubs. Copyright © 2018 Elsevier B.V. All rights reserved.

Regulation of Ribulose-1,5-Bisphosphate Carboxylase Activity by the Activase System in Lysed Spinach Chloroplasts

PubMed Central

Parry, Martin A. J.; Keys, Alfred J.; Foyer, Christine H.; Furbank, Robert T.; Walker, David A.

1988-01-01

Ribulose-1,5-bisphosphate (RuBP) carboxylase in lysed spinach (Spinacia oleracea L. cv virtuosa) chloroplasts that had been partly inactivated at low CO2 and Mg2+ by incubating in darkness with 4 millimolar partially purified RuBP was reactivated by light. If purified RuBP was used to inhibit dark activation of the enzyme, reactivation by light was not observed unless fructose-1,6-bisphosphate, ATP, or ADP plus inorganic phosphate were also added. Presumably, ADP plus inorganic phosphate acted as an ATP-generating system with a requirement for the generation of ΔpH across the thylakoid membrane. When the RuBP obtained from Sigma Chemical Co. was used, light did not reactivate the enzyme. There was no direct correlation between ΔpH and activation. Therefore, thylakoids are required in the ribulose-1,5-bisphosphate carboxylase activase system largely to synthesize ATP. Inactivation of RuBP carboxylase in isolated chloroplasts or in the lysed chloroplast system was not promoted simply by a transition from light to dark conditions but was caused by low CO2 and Mg2+. PMID:16666184

Evidence for an Inducible Nucleotide-Dependent Acetone Carboxylase in Rhodococcus rhodochrous B276

PubMed Central

Clark, Daniel D.; Ensign, Scott A.

1999-01-01

The metabolism of acetone was investigated in the actinomycete Rhodococcus rhodochrous (formerly Nocardia corallina) B276. Suspensions of acetone- and isopropanol-grown R. rhodochrous readily metabolized acetone. In contrast, R. rhodochrous cells cultured with glucose as the carbon source lacked the ability to metabolize acetone at the onset of the assay but gained the ability to do so in a time-dependent fashion. Chloramphenicol and rifampin prevented the time-dependent increase in this activity. Acetone metabolism by R. rhodochrous was CO2 dependent, and 14CO2 fixation occurred concomitant with this process. A nucleotide-dependent acetone carboxylase was partially purified from cell extracts of acetone-grown R. rhodochrous by DEAE-Sepharose chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that the acetone carboxylase was composed of three subunits with apparent molecular masses of 85, 74, and 16 kDa. Acetone metabolism by the partially purified enzyme was dependent on the presence of a divalent metal and a nucleoside triphosphate. GTP and ITP supported the highest rates of acetone carboxylation, while CTP, UTP, and XTP supported carboxylation at 10 to 50% of these rates. ATP did not support acetone carboxylation. Acetoacetate was determined to be the stoichiometric product of acetone carboxylation. The longer-chain ketones butanone, 2-pentanone, 3-pentanone, and 2-hexanone were substrates. This work has identified an acetone carboxylase with a novel nucleotide usage and broader substrate specificity compared to other such enzymes studied to date. These results strengthen the proposal that carboxylation is a common strategy used for acetone catabolism in aerobic acetone-oxidizing bacteria. PMID:10217764

Screening Phosphorylation Site Mutations in Yeast Acetyl-CoA Carboxylase Using Malonyl-CoA Sensor to Improve Malonyl-CoA-Derived Product.

PubMed

Chen, Xiaoxu; Yang, Xiaoyu; Shen, Yu; Hou, Jin; Bao, Xiaoming

2018-01-01

Malonyl-coenzyme A (malonyl-CoA) is a critical precursor for the biosynthesis of a variety of biochemicals. It is synthesized by the catalysis of acetyl-CoA carboxylase (Acc1p), which was demonstrated to be deactivated by the phosphorylation of Snf1 protein kinase in yeast. In this study, we designed a synthetic malonyl-CoA biosensor and used it to screen phosphorylation site mutations of Acc1p in Saccharomyces cerevisiae . Thirteen phosphorylation sites were mutated, and a combination of three site mutations in Acc1p, S686A, S659A, and S1157A, was found to increase malonyl-CoA availability. ACC1 S686AS659AS1157A expression also improved the production of 3-hydroxypropionic acid, a malonyl-CoA-derived chemical, compared to both wild type and the previously reported ACC1 S659AS1157A mutation. This mutation will also be beneficial for other malonyl-CoA-derived products.

The HPr(Ser) Kinase of Streptococcus salivarius: Purification, Properties, and Cloning of the hprK Gene

PubMed Central

Brochu, Denis; Vadeboncoeur, Christian

1999-01-01

In gram-positive bacteria, HPr, a protein of the phosphoenolpyruvate:sugar phosphotransferase system, is phosphorylated on a serine residue at position 46 by an ATP-dependent protein kinase. The HPr(Ser) kinase of Streptococcus salivarius ATCC 25975 was purified, and the encoding gene (hprK) was cloned by using a nucleotide probe designed from the N-terminal amino acid sequence. The predicted amino acid sequence of the S. salivarius enzyme showed 45% identity with the Bacillus subtilis enzyme, the conserved residues being located mainly in the C-terminal half of the protein. The predicted hprK gene product has a molecular mass of 34,440 Da and a pI of 5.6. These values agree well with those found experimentally by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, molecular sieve chromatography in the presence of guanidine hydrochloride, and chromatofocusing using the purified protein. The native protein migrates on a Superdex 200 HR column as a 330,000-Da protein, suggesting that the HPr(Ser) kinase is a decamer. The enzyme requires Mg2+ for activity and functions optimally at pH 7.5. Unlike the enzyme from other gram-positive bacteria, the HPr(Ser) kinase from S. salivarius is not stimulated by FDP or other glycolytic intermediates. The enzyme is inhibited by inorganic phosphate, and its Kms for HPr and ATP are 31 μM and 1 mM, respectively. PMID:9922231

Major diffusion leaks of clamp-on leaf cuvettes still unaccounted: how erroneous are the estimates of Farquhar et al. model parameters?

PubMed

Rodeghiero, Mirco; Niinemets, Ulo; Cescatti, Alessandro

2007-08-01

Estimates of leaf gas-exchange characteristics using standard clamp-on leaf chambers are prone to errors because of diffusion leaks. While some consideration has been given to CO(2) diffusion leaks, potential water vapour diffusion leaks through chamber gaskets have been neglected. We estimated diffusion leaks of two clamp-on Li-Cor LI-6400 (Li-Cor, Inc., Lincoln, NE, USA) leaf chambers with polymer foam gaskets and enclosing either 2 or 6 cm(2) leaf area, and conducted a sensitivity analysis of the diffusion leak effects on Farquhar et al. photosynthesis model parameters - the maximum carboxylase activity of ribulose 1 x 5-bisphosphate carboxylase/oxygenase (Rubisco) (V(cmax)), capacity for photosynthetic electron transport (J(max)) and non-photorespiratory respiration rate in light (R(d)). In addition, net assimilation rate (A(n)) versus intercellular CO(2) (C(i)) responses were measured in leaves of Mediterranean evergreen species Quercus ilex L. enclosing the whole leaf chamber in a polyvinyl fluoride bag flushed with the exhaust air of leaf chamber, thereby effectively reducing the CO(2) and water vapour gradients between ambient air and leaf chamber. For the empty chambers, average diffusion leak for CO(2), K(CO2), (molar flow rate corresponding to unit CO(2) mole fraction difference) was ca. 0.40 micromol s(-1). K(CO2) increased ca. 50% if a dead leaf was clamped between the leaf chamber. Average diffusion leak for H(2)O was ca. 5- to 10-fold larger than the diffusion leak for CO(2). Sensitivity analyses demonstrated that the consequence of a CO(2) diffusion leak was apparent enhancement of A(n) at high CO(2) mole fraction and reduction at lower CO(2) mole fraction, and overall compression of C(i) range. As the result of these modifications, Farquhar et al. model parameters were overestimated. The degree of overestimation increased in the order of V(cmax) < J(max) < R(d), and was larger for smaller chambers and for leaves with lower photosynthetic capacity

Genistein promotes insulin action through adenosine monophosphate-activated protein kinase activation and p70 ribosomal protein S6 kinase 1 inhibition in the skeletal muscle of mice fed a high energy diet.

PubMed

Arunkumar, Elumalai; Anuradha, Carani Venkatraman

2012-08-01

Genistein (GEN), a soy isoflavone, exerts insulin-sensitizing actions in animals; however, the underlying mechanisms have not been determined. Because GEN is a known activator of adenosine monophosphate-activated protein kinase (AMPK), we hypothesize that GEN activates insulin signaling through AMPK activation. To test this hypothesis, a high fat-high fructose diet (HFFD)-fed mice model of insulin resistance was administered GEN, and the insulin signaling pathway proteins in the skeletal muscle were examined. Hyperglycemia and hyperinsulinemia observed in HFFD-fed mice were significantly lowered by GEN. GEN increased insulin-stimulated tyrosine phosphorylation of insulin receptor-β and insulin receptor substrate (IRS) 1 but down-regulated IRS-1 serine phosphorylation in the skeletal muscle of HFFD-fed mice. Furthermore, GEN treatment improved muscle IRS-1-associated phospatidylinositol-3 kinase expression, phosphorylation of Akt at Ser(473), and translocation of glucose transporter subtype 4. Phosphorylation of AMPK at Thr(172) and acetyl coenzyme A carboxylase (ACC) at Ser(79) was augmented, whereas phosphorylation of p70 ribosomal protein S6 kinase 1 at Thr(389) was significantly decreased after GEN treatment in the skeletal muscle of HFFD-fed mice. These results suggest that GEN might improve insulin action in the skeletal muscle by targeting AMPK. Copyright © 2012 Elsevier Inc. All rights reserved.

Nitrate-Dependent Degradation of Acetone by Alicycliphilus and Paracoccus Strains and Comparison of Acetone Carboxylase Enzymes ▿

PubMed Central

Dullius, Carlos Henrique; Chen, Ching-Yuan; Schink, Bernhard

2011-01-01

A novel acetone-degrading, nitrate-reducing bacterium, strain KN Bun08, was isolated from an enrichment culture with butanone and nitrate as the sole sources of carbon and energy. The cells were motile short rods, 0.5 to 1 by 1 to 2 μm in size, which gave Gram-positive staining results in the exponential growth phase and Gram-negative staining results in the stationary-growth phase. Based on 16S rRNA gene sequence analysis, the isolate was assigned to the genus Alicycliphilus. Besides butanone and acetone, the strain used numerous fatty acids as substrates. An ATP-dependent acetone-carboxylating enzyme was enriched from cell extracts of this bacterium and of Alicycliphilus denitrificans K601T by two subsequent DEAE Sepharose column procedures. For comparison, acetone carboxylases were enriched from two additional nitrate-reducing bacterial species, Paracoccus denitrificans and P. pantotrophus. The products of the carboxylase reaction were acetoacetate and AMP rather than ADP. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of cell extracts and of the various enzyme preparations revealed bands corresponding to molecular masses of 85, 78, and 20 kDa, suggesting similarities to the acetone carboxylase enzymes described in detail for the aerobic bacterium Xanthobacter autotrophicus strain Py2 (85.3, 78.3, and 19.6 kDa) and the phototrophic bacterium Rhodobacter capsulatus. Protein bands were excised and compared by mass spectrometry with those of acetone carboxylases of aerobic bacteria. The results document the finding that the nitrate-reducing bacteria studied here use acetone-carboxylating enzymes similar to those of aerobic and phototrophic bacteria. PMID:21841031

Glucose-Specific Enzyme IIA of the Phosphoenolpyruvate:Carbohydrate Phosphotransferase System Modulates Chitin Signaling Pathways in Vibrio cholerae.

PubMed

Yamamoto, Shouji; Ohnishi, Makoto

2017-09-15

In Vibrio cholerae , the genes required for chitin utilization and natural competence are governed by the chitin-responsive two-component system (TCS) sensor kinase ChiS. In the classical TCS paradigm, a sensor kinase specifically phosphorylates a cognate response regulator to activate gene expression. However, our previous genetic study suggested that ChiS stimulates the non-TCS transcriptional regulator TfoS by using mechanisms distinct from classical phosphorylation reactions (S. Yamamoto, J. Mitobe, T. Ishikawa, S. N. Wai, M. Ohnishi, H. Watanabe, and H. Izumiya, Mol Microbiol 91:326-347, 2014, https://doi.org/10.1111/mmi.12462). TfoS specifically activates the transcription of tfoR , encoding a small regulatory RNA essential for competence gene expression. Whether ChiS and TfoS interact directly remains unknown. To determine if other factors mediate the communication between ChiS and TfoS, we isolated transposon mutants that turned off tfoR :: lacZ expression but possessed intact chiS and tfoS genes. We demonstrated an unexpected association of chitin-induced signaling pathways with the glucose-specific enzyme IIA (EIIA glc ) of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) for carbohydrate uptake and catabolite control of gene expression. Genetic and physiological analyses revealed that dephosphorylated EIIA glc inactivated natural competence and tfoR transcription. Chitin-induced expression of the chb operon, which is required for chitin transport and catabolism, was also repressed by dephosphorylated EIIA glc Furthermore, the regulation of tfoR and chb expression by EIIA glc was dependent on ChiS and intracellular levels of ChiS were not affected by disruption of the gene encoding EIIA glc These results define a previously unknown connection between the PTS and chitin signaling pathways in V. cholerae and suggest a strategy whereby this bacterium can physiologically adapt to the existing nutrient status. IMPORTANCE The EIIA glc

A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore

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

Miller, J. Richard; Dunham, Steve; Mochalkin, Igor

2009-06-25

As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidinesmore » target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious Gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity.« less

FGF21 does not require adipocyte AMP-activated protein kinase (AMPK) or the phosphorylation of acetyl-CoA carboxylase (ACC) to mediate improvements in whole-body glucose homeostasis.

PubMed

Mottillo, Emilio P; Desjardins, Eric M; Fritzen, Andreas M; Zou, Vito Z; Crane, Justin D; Yabut, Julian M; Kiens, Bente; Erion, Derek M; Lanba, Adhiraj; Granneman, James G; Talukdar, Saswata; Steinberg, Gregory R

2017-06-01

Fibroblast growth factor 21 (FGF21) shows great potential for the treatment of obesity and type 2 diabetes, as its long-acting analogue reduces body weight and improves lipid profiles of participants in clinical studies; however, the intracellular mechanisms mediating these effects are poorly understood. AMP-activated protein kinase (AMPK) is an important energy sensor of the cell and a molecular target for anti-diabetic medications. This work examined the role of AMPK in mediating the glucose and lipid-lowering effects of FGF21. Inducible adipocyte AMPK β1β2 knockout mice (iβ1β2AKO) and littermate controls were fed a high fat diet (HFD) and treated with native FGF21 or saline for two weeks. Additionally, HFD-fed mice with knock-in mutations on the AMPK phosphorylation sites of acetyl-CoA carboxylase (ACC)1 and ACC2 (DKI mice) along with wild-type (WT) controls received long-acting FGF21 for two weeks. Consistent with previous studies, FGF21 treatment significantly reduced body weight, adiposity, and liver lipids in HFD fed mice. To add, FGF21 improved circulating lipids, glycemic control, and insulin sensitivity. These effects were independent of adipocyte AMPK and were not associated with changes in browning of white (WAT) and brown adipose tissue (BAT). Lastly, we assessed whether FGF21 exerted its effects through the AMPK/ACC axis, which is critical in the therapeutic benefits of the anti-diabetic medication metformin. ACC DKI mice had improved glucose and insulin tolerance and a reduction in body weight, body fat and hepatic steatosis similar to WT mice in response to FGF21 administration. These data illustrate that the metabolic improvements upon FGF21 administration are independent of adipocyte AMPK, and do not require the inhibitory action of AMPK on ACC. This is in contrast to the anti-diabetic medication metformin and suggests that the treatment of obesity and diabetes with the combination of FGF21 and AMPK activators merits consideration.

Coordination between leaf CO2 diffusion and Rubisco properties allows maximizing photosynthetic efficiency in Limonium species.

PubMed

Galmés, Jeroni; Molins, Arántzazu; Flexas, Jaume; Conesa, Miquel À

2017-10-01

High photosynthetic efficiency intrinsically demands tight coordination between traits related to CO 2 diffusion capacity and leaf biochemistry. Although this coordination constitutes the basis of existing mathematical models of leaf photosynthesis, it has been barely explored among closely related species, which could reveal rapid adaptation clues in the recent past. With this aim, we characterized the photosynthetic capacity of 12 species of Limonium, possessing contrasting Rubisco catalytic properties, grown under optimal (WW) and extreme drought conditions (WD). The availability of CO 2 at the site of carboxylation (C c ) determined the photosynthetic capacity of Limonium under WD, while both diffusional and biochemical components governed the photosynthetic performance under WW. The variation in the in vivo caboxylation efficiency correlated with both the concentration of active Rubisco sites and the in vitro-based properties of Rubisco, such as the maximum carboxylase turnover rate (k cat c ) and the Michaelis-Menten constant for CO 2 (K c ). Notably, the results confirmed the hypothesis of coordination between the CO 2 offer and demand functions of photosynthesis: those Limonium species with high total leaf conductance to CO 2 have evolved towards increased velocity (i.e. higher k cat c ), at the penalty of lower affinity for CO 2 (i.e. lower specificity factor, S c/o ). © 2017 John Wiley & Sons Ltd.

Streptococcal phosphoenolpyruvate-sugar phosphotransferase system: amino acid sequence and site of ATP-dependent phosphorylation of HPr

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

Deutscher, J.; Pevec, B.; Beyreuther, K.

1986-10-21

The amino acid sequence of histidine-containing protein (HPr) from Streptococcus faecalis has been determined by direct Edman degradation of intact HPr and by amino acid sequence analysis of tryptic peptides, V8 proteolyptic peptides, thermolytic peptides, and cyanogen bromide cleavage products. HPr from S. faecalis was found to contain 89 amino acid residues, corresponding to a molecular weight of 9438. The amino acid sequence of HPr from S. faecalis shows extended homology to the primary structure of HPr proteins from other bacteria. Besides the phosphoenolpyruvate-dependent phosphorylation of a histidyl residue in HPr, catalyzed by enzyme I of the bacterial phosphotransferase system,more » HPr was also found to be phosphorylated at a seryl residue in an ATP-dependent protein kinase catalyzed reaction. The site of ATP-dependent phosphorylation in HPr of S faecalis has now been determined. (/sup 32/P)P-Ser-HPr was digested with three different proteases, and in each case, a single labeled peptide was isolated. Following digestion with subtilisin, they obtained a peptide with the sequence -(P)Ser-Ile-Met-. Using chymotrypsin, they isolated a peptide with the sequence -Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-Gly-Val-Met-. The longest labeled peptide was obtained with V8 staphylococcal protease. According to amino acid analysis, this peptide contained 36 out of the 89 amino acid residues of HPr. The following sequence of 12 amino acid residues of the V8 peptide was determined: -Tyr-Lys-Gly-Lys-Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-. Thus, the site of ATP-dependent phosphorylation was determined to be Ser-46 within the primary structure of HPr.« less

Carboxylate metabolism in sugar beet plants grown with excess Zn.

PubMed

Sagardoy, R; Morales, F; Rellán-Álvarez, R; Abadía, A; Abadía, J; López-Millán, A F

2011-05-01

The effects of Zn excess on carboxylate metabolism were investigated in sugar beet (Beta vulgaris L.) plants grown hydroponically in a growth chamber. Root extracts of plants grown with 50 or 100μM Zn in the nutrient solution showed increases in several enzymatic activities related to organic acid metabolism, including citrate synthase and phosphoenolpyruvate carboxylase, when compared to activities in control root extracts. Root citric and malic acid concentrations increased in plants grown with 100μM Zn, but not in plants grown with 50μM Zn. In the xylem sap, plants grown with 50 and 100μM Zn showed increases in the concentrations of citrate and malate compared to the controls. Leaves of plants grown with 50 or 100μM Zn showed increases in the concentrations of citric and malic acid and in the activities of citrate synthase and fumarase. Leaf isocitrate dehydrogenase increased only in plants grown with 50μM Zn when compared to the controls. In plants grown with 300μM Zn, the only enzyme showing activity increases in root extracts was citrate synthase, whereas the activities of other enzymes decreased compared to the controls, and root citrate concentrations increased. In the 300μM Zn-grown plants, the xylem concentrations of citric and malic acids were higher than those of controls, whereas in leaf extracts the activity of fumarase increased markedly, and the leaf citric acid concentration was higher than in the controls. Based on our data, a metabolic model of the carboxylate metabolism in sugar beet plants grown under Zn excess is proposed. Copyright © 2010 Elsevier GmbH. All rights reserved.

Expression, purification, and characterization of human acetyl-CoA carboxylase 2.

PubMed

Kim, Ki Won; Yamane, Harvey; Zondlo, James; Busby, James; Wang, Minghan

2007-05-01

The full-length human acetyl-CoA carboxylase 1 (ACC1) was expressed and purified to homogeneity by two separate groups (Y.G. Gu, M. Weitzberg, R.F. Clark, X. Xu, Q. Li, T. Zhang, T.M. Hansen, G. Liu, Z. Xin, X. Wang, T. McNally, H. Camp, B.A. Beutel, H.I. Sham, Synthesis and structure-activity relationships of N-{3-[2-(4-alkoxyphenoxy)thiazol-5-yl]-1-methylprop-2-ynyl}carboxy derivatives as selective acetyl-CoA carboxylase 2 inhibitors, J. Med. Chem. 49 (2006) 3770-3773; D. Cheng, C.H. Chu, L. Chen, J.N. Feder, G.A. Mintier, Y. Wu, J.W. Cook, M.R. Harpel, G.A. Locke, Y. An, J.K. Tamura, Expression, purification, and characterization of human and rat acetyl coenzyme A carboxylase (ACC) isozymes, Protein Expr. Purif., in press). However, neither group was successful in expressing the full-length ACC2 due to issues of solubility and expression levels. The two versions of recombinant human ACC2 in these reports are either truncated (lacking 1-148 aa) or have the N-terminal 275 aa replaced with the corresponding ACC1 region (1-133 aa). Despite the fact that ACC activity was observed in both cases, these constructs are not ideal because the N-terminal region of ACC2 could be important for the correct folding of the catalytic domains. Here, we report the high level expression and purification of full-length human ACC2 that lacks only the N-terminal membrane attachment sequence (1-20 and 1-26 aa, respectively) in Trichoplusia ni cells. In addition, we developed a sensitive HPLC assay to analyze the kinetic parameters of the recombinant enzyme. The recombinant enzyme is a soluble protein and has a K(m) value of 2 microM for acetyl-CoA, almost 30-fold lower than that reported for the truncated human ACC2. Our recombinant enzyme also has a lower K(m) value for ATP (K(m)=52 microM). Although this difference could be ascribed to different assay conditions, our data suggest that the longer human ACC2 produced in our system may have higher affinities for the substrates and could

5-Aminoimidazole-4-carboxamide ribonucleoside-mediated adenosine monophosphate-activated protein kinase activation induces protective innate responses in bacterial endophthalmitis.

PubMed

Kumar, Ajay; Giri, Shailendra; Kumar, Ashok

2016-12-01

The retina is considered to be the most metabolically active tissue in the body. However, the link between energy metabolism and retinal inflammation, as incited by microbial infection such as endophthalmitis, remains unexplored. In this study, using a mouse model of Staphylococcus aureus (SA) endophthalmitis, we demonstrate that the activity (phosphorylation) of 5' adenosine monophosphate-activated protein kinase alpha (AMPKα), a cellular energy sensor and its endogenous substrate; acetyl-CoA carboxylase is down-regulated in the SA-infected retina. Intravitreal administration of an AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), restored AMPKα and acetyl-CoA carboxylase phosphorylation. AICAR treatment reduced both the bacterial burden and intraocular inflammation in SA-infected eyes by inhibiting NF-kB and MAP kinases (p38 and JNK) signalling. The anti-inflammatory effects of AICAR were diminished in eyes pretreated with AMPK inhibitor, Compound C. The bioenergetics (Seahorse) analysis of SA-infected microglia and bone marrow-derived macrophages revealed an increase in glycolysis, which was reinstated by AICAR treatment. AICAR also reduced the expression of SA-induced glycolytic genes, including hexokinase 2 and glucose transporter 1 in microglia, bone marrow-derived macrophages and the mouse retina. Interestingly, AICAR treatment enhanced the bacterial phagocytic and intracellular killing activities of cultured microglia, macrophages and neutrophils. Furthermore, AMPKα1 global knockout mice exhibited increased susceptibility towards SA endophthalmitis, as evidenced by increased inflammatory mediators and bacterial burden and reduced retinal function. Together, these findings provide the first evidence that AMPK activation promotes retinal innate defence in endophthalmitis by modulating energy metabolism and that it can be targeted therapeutically to treat ocular infections. © 2016 John Wiley & Sons Ltd.

C4 Photosynthesis in the Rice Paddy: Insights from the Noxious Weed Echinochloa glabrescens1[OPEN

PubMed Central

Covshoff, Sarah; Szecowka, Marek; Hughes, Thomas E.; Kelly, Steven; Bailey, Karen J.; Sage, Tammy L.; Pachebat, Justin A.; Leegood, Richard

2016-01-01

The C4 pathway is a highly complex trait that increases photosynthetic efficiency in more than 60 plant lineages. Although the majority of C4 plants occupy disturbed, arid, and nutrient-poor habitats, some grow in high-nutrient, waterlogged conditions. One such example is Echinochloa glabrescens, which is an aggressive weed of rice paddies. We generated comprehensive transcriptome datasets for C4 E. glabrescens and C3 rice to identify genes associated with adaption to waterlogged, nutrient-replete conditions, but also used the data to better understand how C4 photosynthesis operates in these conditions. Leaves of E. glabrescens exhibited classical Kranz anatomy with lightly lobed mesophyll cells having low chloroplast coverage. As with rice and other hygrophytic C3 species, leaves of E. glabrescens accumulated a chloroplastic phosphoenolpyruvate carboxylase protein, albeit at reduced amounts relative to rice. The arid-grown species Setaria italica (C4) and Brachypodium distachyon (C3) were also found to accumulate chloroplastic phosphoenolpyruvate carboxylase. We identified a molecular signature associated with C4 photosynthesis in nutrient-replete, waterlogged conditions that is highly similar to those previously reported from C4 plants that grow in more arid conditions. We also identified a cohort of genes that have been subjected to a selective sweep associated with growth in paddy conditions. Overall, this approach highlights the value of using wild species such as weeds to identify adaptions to specific conditions associated with high-yielding crops in agriculture. PMID:26527656

Herbivory Rapidly Activates MAPK Signaling in Attacked and Unattacked Leaf Regions but Not between Leaves of Nicotiana attenuata[W

PubMed Central

Wu, Jianqiang; Hettenhausen, Christian; Meldau, Stefan; Baldwin, Ian T.

2007-01-01

Mitogen-activated protein kinase (MAPK) signaling plays a central role in transducing extracellular stimuli into intracellular responses, but its role in mediating plant responses to herbivore attack remains largely unexplored. When Manduca sexta larvae attack their host plant, Nicotiana attenuata, the plant's wound response is reconfigured at transcriptional, phytohormonal, and defensive levels due to the introduction of oral secretions (OS) into wounds during feeding. We show that OS dramatically amplify wound-induced MAPK activity and that fatty acid–amino acid conjugates in M. sexta OS are the elicitors. Virus-induced gene silencing of salicylic acid–induced protein kinase (SIPK) and wound-induced protein kinase revealed their importance in mediating wound and OS-elicited hormonal responses and transcriptional regulation of defense-related genes. We found that after applying OS to wounds created in one portion of a leaf, SIPK is activated in both wounded and specific unwounded regions of the leaf but not in phylotactically connected adjacent leaves. We propose that M. sexta attack elicits a mobile signal that travels to nonwounded regions of the attacked leaf where it activates MAPK signaling and, thus, downstream responses; subsequently, a different signal is transported by the vascular system to systemic leaves to initiate defense responses without activating MAPKs in systemic leaves. PMID:17400894

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

PubMed

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

2013-01-01

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

Enhancing the supply of oxaloacetate for L-glutamate production by pyc overexpression in different Corynebacterium glutamicum.

PubMed

Guo, Xuan; Wang, Jing; Xie, Xixian; Xu, Qingyang; Zhang, Chenglin; Chen, Ning

2013-06-01

During L-glutamate production, phosphoenolpyruvate carboxylase and pyruvate carboxylase (PCx) play important roles in supplying oxaloacetate to the tricarboxylic acid cycle. To explore the significance of PCx for L-glutamate overproduction, the pyc gene encoding PCx was amplified in Corynebacterium glutamicum GDK-9 triggered by biotin limitation and CN1021 triggered by a temperature shock, respectively. In the fed-batch cultures, GDK-9pXMJ19pyc exhibited 7.4 % lower L-alanine excretion and no improved L-glutamate production. In contrast, CN1021pXMJ19pyc finally exhibited 13 % lower L-alanine excretion and identical L-glutamate production, however, 8.5 % higher L-glutamate production was detected during a short period of the fermentation. It was indicated that pyc overexpression in L-glutamate producer strains, especially CN1021, increased the supply of oxaloacetate for L-glutamate synthesis and decreased byproduct excretion at the pyruvate node.

Adenosine monophosphate-activated protein kinase activation and suppression of inflammatory response by cell stretching in rabbit synovial fibroblasts.

PubMed

Kunanusornchai, Wanlop; Muanprasat, Chatchai; Chatsudthipong, Varanuj

2016-12-01

Joint mobilization is known to be beneficial in osteoarthritis (OA) patients. This study aimed to investigate the effect of stretching on adenosine monophosphate-activated protein kinase (AMPK) activity and its role in modulating inflammation in rabbit synovial fibroblasts. Uniaxial stretching of isolated rabbit synovial fibroblasts for ten min was performed. Stretching-induced AMPK activation, its underlying mechanism, and its anti-inflammatory effect were investigated using Western blot. Static stretching at 20 % of initial length resulted in AMPK activation characterized by expression of phosphorylated AMPK and phosphorylated acetyl-Co A carboxylase. AMP-activated protein kinase phosphorylation peaked 1 h after stretching and declined toward resting activity. Using cell viability assays, static stretching did not appear to cause cellular damage. Activation of AMPK involves Ca 2+ influx via a mechanosensitive L-type Ca 2+ channel, which subsequently raises intracellular Ca 2+ and activates AMPK via Ca 2+ /calmodulin-dependent protein kinase kinase β (CaMKKβ). Interestingly, stretching suppressed TNFα-induced expression of COX-2, iNOS, and phosphorylated NF-κB. These effects were prevented by pretreatment with compound C, an AMPK inhibitor. These results suggest that mechanical stretching suppressed inflammatory responses in synovial fibroblasts via a L-type Ca 2+ -channel-CaMKKβ-AMPK-dependent pathway which may underlie joint mobilization's ability to alleviate OA symptoms.

Silibinin activates AMP-activated protein kinase to protect neuronal cells from oxygen and glucose deprivation-re-oxygenation.

PubMed

Xie, Zhi; Ding, Sheng-quan; Shen, Ya-fang

2014-11-14

In this study, we explored the cytoprotective potential of silibinin against oxygen-glucose deprivation (OGD)-induced neuronal cell damages, and studied underling mechanisms. In vitro model of ischemic stroke was created by keeping neuronal cells (SH-SY5Y cells and primary mouse cortical neurons) in an OGD condition followed by re-oxygenation. Pre-treatment of silibinin significantly inhibited OGD/re-oxygenation-induced necrosis and apoptosis of neuronal cells. OGD/re-oxygenation-induced reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) reduction were also inhibited by silibinin. At the molecular level, silibinin treatment in SH-SY5Y cells and primary cortical neurons led to significant AMP-activated protein kinase (AMPK) signaling activation, detected by phosphorylations of AMPKα1, its upstream kinase liver kinase B1 (LKB1) and the downstream target acetyl-CoA Carboxylase (ACC). Pharmacological inhibition or genetic depletion of AMPK alleviated the neuroprotective ability of silibinin against OGD/re-oxygenation. Further, ROS scavenging ability by silibinin was abolished with AMPK inhibition or silencing. While A-769662, the AMPK activator, mimicked silibinin actions and suppressed ROS production and neuronal cell death following OGD/re-oxygenation. Together, these results show that silibinin-mediated neuroprotection requires activation of AMPK signaling. Copyright © 2014 Elsevier Inc. All rights reserved.

Conversion of carbon dioxide to oxaloacetate using integrated carbonic anhydrase and phosphoenolpyruvate carboxylase.

PubMed

Chang, Kwang Suk; Jeon, Hancheol; Gu, Man Bock; Pack, Seung Pil; Jin, EonSeon

2013-12-01

The development and implementation of strategies for CO2 mitigation are necessary to counteract the greenhouse gas effect of carbon dioxide emissions. To demonstrate the possibility of simultaneously capturing CO2 and utilizing four-carbon compounds, an integrated system using CA and PEPCase was developed, which mimics an in vivo carbon dioxide concentration mechanism. We first cloned the PEPCase 1 gene of the marine diatom Phaeodactylum tricornutum and produced a recombinant PtPEPCase 1. The affinity column purified PtPEPCase 1 exhibited specific enzymatic activity (5.89 U/mg). When the simultaneous and coordinated reactions of CA from Dunaliella sp. and the PtPEPCase 1 occurred, more OAA was produced than when only PEPCase was present. Therefore, this integrated CA-PEPCase system can be used not only to capture CO2 but also for a new technology to produce value-added four-carbon platform chemicals.

Resistance to herbicides caused by single amino acid mutations in acetyl-CoA carboxylase in resistant populations of grassy weeds.

PubMed

Jang, SoRi; Marjanovic, Jasmina; Gornicki, Piotr

2013-03-01

Eleven spontaneous mutations of acetyl-CoA carboxylase have been identified in many herbicide-resistant populations of 42 species of grassy weeds, hampering application of aryloxyphenoxypropionate, cyclohexadione and phenylpyrazoline herbicides in agriculture. IC(50) shifts (resistance indices) caused by herbicide-resistant mutations were determined using a recombinant yeast system that allows comparison of the effects of single amino acid mutations in the same biochemical background, avoiding the complexity inherent in the in planta experiments. The effect of six mutations on the sensitivity of acetyl-CoA carboxylase to nine herbicides representing the three chemical classes was studied. A combination of partially overlapping binding sites of the three classes of herbicides and the structure of their variable parts explains cross-resistance among and between the three classes of inhibitors, as well as differences in their specificity. Some degree of resistance was detected for 51 of 54 herbicide/mutation combinations. Introduction of new herbicides targeting acetyl-CoA carboxylase will depend on their ability to overcome the high degree of cross-resistance already existing in weed populations. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

A guanine insert in OsBBS1 leads to early leaf senescence and salt stress sensitivity in rice (Oryza sativa L.).

PubMed

Zeng, Dong-Dong; Yang, Cheng-Cong; Qin, Ran; Alamin, Md; Yue, Er-Kui; Jin, Xiao-Li; Shi, Chun-Hai

2018-06-01

A rice receptor-like kinase gene OSBBS1/OsRLCK109 was identified; this gene played vital roles in leaf senescence and the salt stress response. Early leaf senescence can cause negative effects on rice yield, but the underlying molecular regulation is not fully understood. bilateral blade senescence 1 (bbs1), an early leaf senescence mutant with a premature senescence phenotype that occurs mainly performing at the leaf margins, was isolated from a rice mutant population generated by ethylmethane sulfonate (EMS) treatment. The mutant showed premature leaf senescence beginning at the tillering stage and exhibited severe symptoms at the late grain-filling stage. bbs1 showed accelerated dark-induced leaf senescence. The OsBBS1 gene was cloned by a map-based cloning strategy, and a guanine (G) insertion was found in the first exon of LOC_Os03g24930. This gene encodes a receptor-like cytoplasmic kinase and was named OsRLCK109 in a previous study. Transgenic LOC_Os03g24930 knockout plants generated by a CRISPR/Cas9 strategy exhibited similar early leaf senescence phenotypes as did the bbs1 mutant, which confirmed that LOC_Os03g24930 was the OsBBS1 gene. OsBBS1/OsRLCK109 was expressed in all detected tissues and was predominantly expressed in the main vein region of mature leaves. The expression of OsBBS1 could be greatly induced by salt stress, and the bbs1 mutant exhibited hypersensitivity to salt stress. In conclusion, this is the first identification of OsRLCKs participating in leaf senescence and playing critical roles in the salt stress response in rice (Oryza sativa L.).

Photosynthetic activity during olive (Olea europaea) leaf development correlates with plastid biogenesis and Rubisco levels.

PubMed

Maayan, Inbar; Shaya, Felix; Ratner, Kira; Mani, Yair; Lavee, Shimon; Avidan, Benjamin; Shahak, Yosepha; Ostersetzer-Biran, Oren

2008-11-01

Olive leaves are known to mature slowly, reaching their maximum photosynthetic activity only after full leaf expansion. Poor assimilation rates, typical to young olive leaves, were previously associated with low stomata conductance. Yet, very little is known about chloroplast biogenesis throughout olive leaf development. Here, the photosynthetic activity and plastids development throughout leaf maturation is characterized by biochemical and ultrastructural analyses. Although demonstrated only low photosynthetic activity, the plastids found in young leaves accumulated both photosynthetic pigments and proteins required for photophosphorylation and carbon fixation. However, Rubisco (ribulose-1,5-bisphosphate carboxylase-oxygenase), which catalyzes the first major step of carbon fixation and one of the most abundant proteins in plants, could not be detected in the young leaves and only slowly accumulated throughout development. In fact, Rubisco levels seemed tightly correlated with the observed photosynthetic activities. Unlike Rubisco, numerous proteins accumulated in the young olive leaves. These included the early light induced proteins, which may be required to reduce the risk of photodamage, because of light absorption by photosynthetic pigments. Also, high levels of ribosomal L11 subunit, transcription factor elF-5A, Histones H2B and H4 were observed in the apical leaves, and in particular a plastidic-like aldolase, which accounted for approximately 30% of the total proteins. These proteins may upregulate in their levels to accommodate the high demand for metabolic energy in the young developing plant tissue, further demonstrating the complex sink-to-source relationship between young and photosynthetically active mature leaves.

Crystal Structures of Human and Staphylococcus aureus Pyruvate Carboxylase and Molecular Insights into the Carboxyltransfer Reaction

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

Xiang,S.; Tong, L.

2008-01-01

Pyruvate carboxylase (PC) catalyzes the biotin-dependent production of oxaloacetate and has important roles in gluconeogenesis, lipogenesis, insulin secretion and other cellular processes. PC contains the biotin carboxylase (BC), carboxyltransferase (CT) and biotin-carboxyl carrier protein (BCCP) domains. We report here the crystal structures at 2.8-Angstroms resolution of full-length PC from Staphylococcus aureus and the C-terminal region (missing only the BC domain) of human PC. A conserved tetrameric association is observed for both enzymes, and our structural and mutagenesis studies reveal a previously uncharacterized domain, the PC tetramerization (PT) domain, which is important for oligomerization. A BCCP domain is located in themore » active site of the CT domain, providing the first molecular insights into how biotin participates in the carboxyltransfer reaction. There are dramatic differences in domain positions in the monomer and the organization of the tetramer between these enzymes and the PC from Rhizobium etli.« less

Pharmacological AMP-kinase activators have compartment-specific effects on cell physiology.

PubMed

Kodiha, Mohamed; Ho-Wo-Cheong, Dennis; Stochaj, Ursula

2011-12-01

5'-AMP-activated kinase (AMPK) regulates numerous biological events and is an essential target for the treatment of type 2 diabetes. The objectives of the present study were first to determine the compartment-specific effects of three established AMPK activators on Thr172 phosphorylation of the α-subunit, an indicator of AMPK activation. Second, we examined how cytoplasmic and nuclear processes are modulated by pharmacological AMPK activators. Specifically, the impact of phenformin, resveratrol, and 5-aminoimidazole-4-carboxamide riboside (AICAR) on Thr172 phosphorylation in the cytoplasm and nucleus was quantified by different methods. To analyze how these activators change cell physiology, we measured the inactivation of acetyl-CoA-carboxylase 1, a predominantly cytoplasmic enzyme that is crucial for lipid metabolism. As a criterion for activities associated with the nucleus, de novo RNA synthesis in nucleoli was quantified. Our studies demonstrate that pharmacological activators of AMPK can alter the balance between nuclear and cytoplasmic AMPK pools. Thus, phenformin and resveratrol caused a strong activation of AMPK in the cytoplasm, whereas the effect was less pronounced in nuclei. By contrast, AICAR elicited a comparable rise in Thr172 phosphorylation in both compartments. Notably, these activators differed drastically in their effects on physiological processes that are located in distinct subcellular compartments. All compounds led to a substantial inactivation of acetyl-CoA-carboxylase 1 in the cytoplasm, with only minor changes to the nuclear enzyme. In the nucleolus, transcription was strongly inhibited by resveratrol, while a moderate inhibition was observed with phenformin and AICAR. Taken together, the compartment-specific phosphorylation of AMPK and downstream events are determined by the activator.

Hydrogen sulphide improves adaptation of Zea mays seedlings to iron deficiency

PubMed Central

Chen, Juan; Wu, Fei-Hua; Shang, Yu-Ting; Wang, Wen-Hua; Hu, Wen-Jun; Simon, Martin; Liu, Xiang; Shangguan, Zhou-Ping; Zheng, Hai-Lei

2015-01-01

Hydrogen sulphide (H2S) is emerging as a potential molecule involved in physiological regulation in plants. However, whether H2S regulates iron-shortage responses in plants is largely unknown. Here, the role of H2S in modulating iron availability in maize (Zea mays L. cv Canner) seedlings grown in iron-deficient culture solution is reported. The main results are as follows: Firstly, NaHS, a donor of H2S, completely prevented leaf interveinal chlorosis in maize seedlings grown in iron-deficient culture solution. Secondly, electron micrographs of mesophyll cells from iron-deficient maize seedlings revealed plastids with few photosynthetic lamellae and rudimentary grana. On the contrary, mesophyll chloroplasts appeared completely developed in H2S-treated maize seedlings. Thirdly, H2S treatment increased iron accumulation in maize seedlings by changing the expression levels of iron homeostasis- and sulphur metabolism-related genes. Fourthly, phytosiderophore (PS) accumulation and secretion were enhanced by H2S treatment in seedlings grown in iron-deficient solution. Indeed, the gene expression of ferric-phytosiderophore transporter (ZmYS1) was specifically induced by iron deficiency in maize leaves and roots, whereas their abundance was decreased by NaHS treatment. Lastly, H2S significantly enhanced photosynthesis through promoting the protein expression of ribulose-1,5-bisphosphate carboxylase large subunit (RuBISCO LSU) and phosphoenolpyruvate carboxylase (PEPC) and the expression of genes encoding RuBISCO large subunit (RBCL), small subunit (RBCS), D1 protein (psbA), and PEPC in maize seedlings grown in iron-deficient solution. These results indicate that H2S is closely related to iron uptake, transport, and accumulation, and consequently increases chlorophyll biosynthesis, chloroplast development, and photosynthesis in plants. PMID:26208645

Hydrogen sulphide improves adaptation of Zea mays seedlings to iron deficiency.

PubMed

Chen, Juan; Wu, Fei-Hua; Shang, Yu-Ting; Wang, Wen-Hua; Hu, Wen-Jun; Simon, Martin; Liu, Xiang; Shangguan, Zhou-Ping; Zheng, Hai-Lei

2015-11-01

Hydrogen sulphide (H2S) is emerging as a potential molecule involved in physiological regulation in plants. However, whether H2S regulates iron-shortage responses in plants is largely unknown. Here, the role of H2S in modulating iron availability in maize (Zea mays L. cv Canner) seedlings grown in iron-deficient culture solution is reported. The main results are as follows: Firstly, NaHS, a donor of H2S, completely prevented leaf interveinal chlorosis in maize seedlings grown in iron-deficient culture solution. Secondly, electron micrographs of mesophyll cells from iron-deficient maize seedlings revealed plastids with few photosynthetic lamellae and rudimentary grana. On the contrary, mesophyll chloroplasts appeared completely developed in H2S-treated maize seedlings. Thirdly, H2S treatment increased iron accumulation in maize seedlings by changing the expression levels of iron homeostasis- and sulphur metabolism-related genes. Fourthly, phytosiderophore (PS) accumulation and secretion were enhanced by H2S treatment in seedlings grown in iron-deficient solution. Indeed, the gene expression of ferric-phytosiderophore transporter (ZmYS1) was specifically induced by iron deficiency in maize leaves and roots, whereas their abundance was decreased by NaHS treatment. Lastly, H2S significantly enhanced photosynthesis through promoting the protein expression of ribulose-1,5-bisphosphate carboxylase large subunit (RuBISCO LSU) and phosphoenolpyruvate carboxylase (PEPC) and the expression of genes encoding RuBISCO large subunit (RBCL), small subunit (RBCS), D1 protein (psbA), and PEPC in maize seedlings grown in iron-deficient solution. These results indicate that H2S is closely related to iron uptake, transport, and accumulation, and consequently increases chlorophyll biosynthesis, chloroplast development, and photosynthesis in plants. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

The role of biotin and oxamate in the carboxyl transferase reaction of pyruvate carboxylase

PubMed Central

Lietzan, Adam D.; Lin, Yi; St. Maurice, Martin

2014-01-01

Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. During catalysis, carboxybiotin is translocated to the carboxyltransferase domain where the carboxyl group is transferred to the acceptor substrate, pyruvate. Many studies on the carboxyltransferase domain of PC have demonstrated an enhanced oxaloacetate decarboxylation activity in the presence of oxamate and it has been shown that oxamate accepts a carboxyl group from carboxybiotin during oxaloacetate decarboxylation. The X-ray crystal structure of the carboxyltransferase domain from Rhizobium etli PC reveals that oxamate is positioned in the active site in an identical manner to the substrate, pyruvate, and kinetic data are consistent with the oxamate-stimulated decarboxylation of oxaloacetate proceeding through a simple ping-pong bi bi mechanism in the absence of the biotin carboxylase domain. Additionally, analysis of truncated PC enzymes indicates that the BCCP domain devoid of biotin does not contribute directly to the enzymatic reaction and conclusively demonstrates a biotin-independent oxaloacetate decarboxylation activity in PC. These findings advance the description of catalysis in PC and can be extended to the study of related biotin-dependent enzymes. PMID:25157442

Mulberry leaf polyphenol extract induced apoptosis involving regulation of adenosine monophosphate-activated protein kinase/fatty acid synthase in a p53-negative hepatocellular carcinoma cell.

PubMed

Yang, Tzi-Peng; Lee, Huei-Jane; Ou, Ting-Tsz; Chang, Ya-Ju; Wang, Chau-Jong

2012-07-11

The polyphenols in mulberry leaf possess the ability to inhibit cell proliferation, invasion, and metastasis of tumors. It was reported that the p53 status plays an important role in switching apoptosis and the cell cycle following adenosine monophosphate-activated protein kinase (AMPK) activation. In this study, we aimed to detect the effect of the mulberry leaf polyphenol extract (MLPE) on inducing cell death in p53-negative (Hep3B) and p53-positive (Hep3B with transfected p53) hepatocellular carcinoma cells and also to clarify the role of p53 in MLPE-treated cells. After treatment of the Hep3B cells with MLPE, apoptosis was induced via the AMPK/PI3K/Akt and Bcl-2 family pathways. Transient transfection of p53 into Hep3B cells led to switching autophagy instead of apoptosis by MLPE treatment. We demonstrated that acridine orange staining and protein expressions of LC-3 and beclin-1 were increased in p53-transfected cells. These results implied induction of apoptosis or autophagy in MLPE-treated hepatocellular carcinoma cells can be due to the p53 status. We also found MLPE can not only activate AMPK but also diminish fatty acid synthase, a molecular target for cancer inhibition. At present, our results indicate MLPE can play an active role in mediating the cell death of hepatocellular carcinoma cells and the p53 might play an important role in regulating the death mechanisms.

Acetyl-CoA carboxylase rewires cancer metabolism to allow cancer cells to survive inhibition of the Warburg effect by cetuximab

PubMed Central

Luo, Jingtao; Hong, Yun; Lu, Yang; Qiu, Songbo; Chaganty, Bharat K. R.; Zhang, Lun; Wang, Xudong; Li, Qiang; Fan, Zhen

2016-01-01

Cetuximab inhibits HIF-1-regulated glycolysis in cancer cells, thereby reversing the Warburg effect and leading to inhibition of cancer cell metabolism. AMP-activated protein kinase (AMPK) is activated after cetuximab treatment, and a sustained AMPK activity is a mechanism contributing to cetuximab resistance. Here, we investigated how acetyl-CoA carboxylase (ACC), a downstream target of AMPK, rewires cancer metabolism in response to cetuximab treatment. We found that introduction of experimental ACC mutants lacking the AMPK phosphorylation sites (ACC1_S79A and ACC2_S212A) into head and neck squamous cell carcinoma (HNSCC) cells protected HNSCC cells from cetuximab-induced growth inhibition. HNSCC cells with acquired cetuximab resistance contained not only high levels of T172-phosphorylated AMPK and S79-phosphorylated ACC1 but also an increased level of total ACC. These findings were corroborated in tumor specimens of HNSCC patients treated with cetuximab. Cetuximab plus TOFA (an allosteric inhibitor of ACC) achieved remarkable growth inhibition of cetuximab-resistant HNSCC xenografts. Our data suggest a novel paradigm in which cetuximab-mediated activation of AMPK and subsequent phosphorylation and inhibition of ACC is followed by a compensatory increase in total ACC, which rewires cancer metabolism from glycolysis-dependent to lipogenesis-dependent. PMID:27693630

Acetyl-CoA carboxylase rewires cancer metabolism to allow cancer cells to survive inhibition of the Warburg effect by cetuximab.

PubMed

Luo, Jingtao; Hong, Yun; Lu, Yang; Qiu, Songbo; Chaganty, Bharat K R; Zhang, Lun; Wang, Xudong; Li, Qiang; Fan, Zhen

2017-01-01

Cetuximab inhibits HIF-1-regulated glycolysis in cancer cells, thereby reversing the Warburg effect and leading to inhibition of cancer cell metabolism. AMP-activated protein kinase (AMPK) is activated after cetuximab treatment, and a sustained AMPK activity is a mechanism contributing to cetuximab resistance. Here, we investigated how acetyl-CoA carboxylase (ACC), a downstream target of AMPK, rewires cancer metabolism in response to cetuximab treatment. We found that introduction of experimental ACC mutants lacking the AMPK phosphorylation sites (ACC1_S79A and ACC2_S212A) into head and neck squamous cell carcinoma (HNSCC) cells protected HNSCC cells from cetuximab-induced growth inhibition. HNSCC cells with acquired cetuximab resistance contained not only high levels of T172-phosphorylated AMPK and S79-phosphorylated ACC1 but also an increased level of total ACC. These findings were corroborated in tumor specimens of HNSCC patients treated with cetuximab. Cetuximab plus TOFA (an allosteric inhibitor of ACC) achieved remarkable growth inhibition of cetuximab-resistant HNSCC xenografts. Our data suggest a novel paradigm in which cetuximab-mediated activation of AMPK and subsequent phosphorylation and inhibition of ACC is followed by a compensatory increase in total ACC, which rewires cancer metabolism from glycolysis-dependent to lipogenesis-dependent. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Effector analogues detect varied allosteric roles for conserved protein-effector interactions in pyruvate kinase isozymes†

PubMed Central

Alontaga, Aileen Y.; Fenton, Aron W.

2011-01-01

The binding site for allosteric inhibitor (amino acid) is highly conserved between human liver pyruvate kinase (hL-PYK) and the rabbit muscle isozyme (rM1-PYK). To detail similarities/differences in the allosteric function of these two homologs, we quantified the binding of 45 amino acid analogues to hL-PYK and their allosteric impact on affinity for the substrate, phosphoenolpyruvate (PEP). This complements a similar study previously completed for rM1-PYK. In hL-PYK, the minimum chemical requirements for effector binding are the same as those identified for rM1-PYK (i.e. the L-2-aminopropanaldehyde substructure of the effector is primarily responsible for binding). However different regions of the effector determine the magnitude of the allosteric response in hL-PYK vs. rM1-PYK. This finding is inconsistent with the idea that allosteric pathways are conserved between homologs of a protein family. PMID:21261284

Isolation of nucleotide binding site-leucine rich repeat and kinase resistance gene analogues from sugarcane (Saccharum spp.).

PubMed

Glynn, Neil C; Comstock, Jack C; Sood, Sushma G; Dang, Phat M; Chaparro, Jose X

2008-01-01

Resistance gene analogues (RGAs) have been isolated from many crops and offer potential in breeding for disease resistance through marker-assisted selection, either as closely linked or as perfect markers. Many R-gene sequences contain kinase domains, and indeed kinase genes have been reported as being proximal to R-genes, making kinase analogues an additionally promising target. The first step towards utilizing RGAs as markers for disease resistance is isolation and characterization of the sequences. Sugarcane clone US01-1158 was identified as resistant to yellow leaf caused by the sugarcane yellow leaf virus (SCYLV) and moderately resistant to rust caused by Puccinia melanocephala Sydow & Sydow. Degenerate primers that had previously proved useful for isolating RGAs and kinase analogues in wheat and soybean were used to amplify DNA from sugarcane (Saccharum spp.) clone US-01-1158. Sequences generated from 1512 positive clones were assembled into 134 contigs of between two and 105 sequences. Comparison of the contig consensuses with the NCBI sequence database using BLASTx showed that 20 had sequence homology to nuclear binding site and leucine rich repeat (NBS-LRR) RGAs, and eight to kinase genes. Alignment of the deduced amino acid sequences with similar sequences from the NCBI database allowed the identification of several conserved domains. The alignment and resulting phenetic tree showed that many of the sequences had greater similarity to sequences from other species than to one another. The use of degenerate primers is a useful method for isolating novel sugarcane RGA and kinase gene analogues. Further studies are needed to evaluate the role of these genes in disease resistance.

Purification and Characterization of the Acetone Carboxylase of Cupriavidus metallidurans Strain CH34

PubMed Central

Rosier, Caroline; Leys, Natalie; Henoumont, Céline; Mergeay, Max

2012-01-01

Acetone carboxylase (Acx) is a key enzyme involved in the biodegradation of acetone by bacteria. Except for the Helicobacteraceae family, genome analyses revealed that bacteria that possess an Acx, such as Cupriavidus metallidurans strain CH34, are associated with soil. The Acx of CH34 forms the heterohexameric complex α2β2γ2 and can carboxylate only acetone and 2-butanone in an ATP-dependent reaction to acetoacetate and 3-keto-2-methylbutyrate, respectively. PMID:22492439

[Role of hypothalamic AMP-activated protein kinase in the control of food intake].

PubMed

Fijałkowski, Franciszek; Jarzyna, Robert

2010-05-21

AMP-activated kinase is an evolutionarily conserved enzyme found in every eukaryotic organism examined for its presence. It plays a critical role in the shift between catabolic and anabolic metabolism. Its activity is under the control of many factors, but basically it integrates the level of intracellular AMP with signals transduced by upstream kinases. It acts through the control of the activities of other enzymes, mitochondrial biogenesis, vesicular transport, and gene expression. From a physiological point of view its effects are pleiotropic and tissue dependent. In 2004, the control of food intake in hypothalamic neurons was added to the long list of its varied functions. Since then, its crucial role in transmitting signals from all important factors that inform the brain about the body's energy level, including leptin, insulin, glucose, ghrelin, and adiponectin, has been well established. Much attention was also paid to the molecular basis of this regulation. It seems that the main targets of hypothalamic AMPK are acetyl-CoA carboxylase and mTOR and the main candidate for upstream kinase is CaMKKbeta. These discoveries seem interesting not only due to their cognitive value, but because they may also carry significant practical aspects, both in the context of AMPK activators, such as the use of metformin in diabetes mellitus therapy, and in the recent trend to look for new ways to deal with the increase in obesity in well-developed countries. A better understanding of the role of AMPK in the control of food intake may create the possibility for new therapeutic approaches in this disease.

Hepatic transcriptional changes in critical genes for gluconeogenesis following castration of bulls

PubMed Central

Fassah, Dilla Mareistia; Jeong, Jin Young

2018-01-01

Objective This study was performed to understand transcriptional changes in the genes involved in gluconeogenesis and glycolysis pathways following castration of bulls. Methods Twenty Korean bulls were weaned at average 3 months of age, and castrated at 6 months. Liver tissues were collected from bulls (n = 10) and steers (n = 10) of Korean cattle, and hepatic gene expression levels were measured using quantitative real-time polymerase chain reaction. We examined hepatic transcription levels of genes encoding enzymes for irreversible reactions in both gluconeogenesis and glycolysis as well as genes encoding enzymes for the utilization of several glucogenic substrates. Correlations between hepatic gene expression and carcass characteristics were performed to understand their associations. Results Castration increased the mRNA (3.6 fold; p<0.01) and protein levels (1.4 fold; p< 0.05) of pyruvate carboxylase and mitochondrial phosphoenolpyruvate carboxykinase genes (1.7 fold; p<0.05). Hepatic mRNA levels of genes encoding the glycolysis enzymes were not changed by castration. Castration increased mRNA levels of both lactate dehydrogenase A (1.5 fold; p<0.05) and lactate dehydrogenase B (2.2 fold; p<0.01) genes for lactate utilization. Castration increased mRNA levels of glycerol kinase (2.7 fold; p<0.05) and glycerol-3-phosphate dehydrogenase 1 (1.5 fold; p<0.05) genes for glycerol utilization. Castration also increased mRNA levels of propionyl-CoA carboxylase beta (mitochondrial) (3.5 fold; p<0.01) and acyl-CoA synthetase short chain family member 3 (1.3 fold; p = 0.06) genes for propionate incorporation. Conclusion Castration increases transcription levels of critical genes coding for enzymes involved in irreversible gluconeogenesis reactions from pyruvate to glucose and enzymes responsible for incorporation of glucogenic substrates including lactate, glycerol, and propionate. Hepatic gluconeogenic gene expression levels were associated with intramuscular fat

Hepatic transcriptional changes in critical genes for gluconeogenesis following castration of bulls.

PubMed

Fassah, Dilla Mareistia; Jeong, Jin Young; Baik, Myunggi

2018-04-01

This study was performed to understand transcriptional changes in the genes involved in gluconeogenesis and glycolysis pathways following castration of bulls. Twenty Korean bulls were weaned at average 3 months of age, and castrated at 6 months. Liver tissues were collected from bulls (n = 10) and steers (n = 10) of Korean cattle, and hepatic gene expression levels were measured using quantitative real-time polymerase chain reaction. We examined hepatic transcription levels of genes encoding enzymes for irreversible reactions in both gluconeogenesis and glycolysis as well as genes encoding enzymes for the utilization of several glucogenic substrates. Correlations between hepatic gene expression and carcass characteristics were performed to understand their associations. Castration increased the mRNA (3.6 fold; p<0.01) and protein levels (1.4 fold; p< 0.05) of pyruvate carboxylase and mitochondrial phosphoenolpyruvate carboxykinase genes (1.7 fold; p<0.05). Hepatic mRNA levels of genes encoding the glycolysis enzymes were not changed by castration. Castration increased mRNA levels of both lactate dehydrogenase A (1.5 fold; p<0.05) and lactate dehydrogenase B (2.2 fold; p<0.01) genes for lactate utilization. Castration increased mRNA levels of glycerol kinase (2.7 fold; p<0.05) and glycerol-3-phosphate dehydrogenase 1 (1.5 fold; p<0.05) genes for glycerol utilization. Castration also increased mRNA levels of propionyl-CoA carboxylase beta (mitochondrial) (3.5 fold; p<0.01) and acyl-CoA synthetase short chain family member 3 (1.3 fold; p = 0.06) genes for propionate incorporation. Castration increases transcription levels of critical genes coding for enzymes involved in irreversible gluconeogenesis reactions from pyruvate to glucose and enzymes responsible for incorporation of glucogenic substrates including lactate, glycerol, and propionate. Hepatic gluconeogenic gene expression levels were associated with intramuscular fat deposition.

Deer predation on leaf miners via leaf abscission

NASA Astrophysics Data System (ADS)

Yamazaki, Kazuo; Sugiura, Shinji

2008-03-01

The evergreen oak Quercus gilva Blume sheds leaves containing mines of the leaf miner Stigmella sp. (Lepidoptera: Nepticulidae) earlier than leaves with no mines in early spring in Nara, central Japan. The eclosion rates of the leaf miner in abscised and retained leaves were compared in the laboratory to clarify the effects of leaf abscission on leaf miner survival in the absence of deer. The leaf miner eclosed successfully from both fallen leaves and leaves retained on trees. However, sika deer ( Cervus nippon centralis Kishida) feed on the fallen mined leaves. Field observations showed that deer consume many fallen leaves under Q. gilva trees, suggesting considerable mortality of leaf miners due to deer predation via leaf abscission. This is a previously unreported relationship between a leaf miner and a mammalian herbivore via leaf abscission.

Cyanidin-3-O-β-glucoside regulates fatty acid metabolism via an AMP-activated protein kinase-dependent signaling pathway in human HepG2 cells

PubMed Central

2012-01-01

Background Hepatic metabolic derangements are key components in the development of fatty liver disease. AMP-activated protein kinase (AMPK) plays a central role in controlling hepatic lipid metabolism through modulating the downstream acetyl CoA carboxylase (ACC) and carnitine palmitoyl transferase 1 (CPT-1) pathway. In this study, cyanidin-3-O-β-glucoside (Cy-3-g), a typical anthocyanin pigment was used to examine its effects on AMPK activation and fatty acid metabolism in human HepG2 hepatocytes. Results Anthocyanin Cy-3-g increased cellular AMPK activity in a calmodulin kinase kinase dependent manner. Furthermore, Cy-3-g substantially induced AMPK downstream target ACC phosphorylation and inactivation, and then decreased malonyl CoA contents, leading to stimulation of CPT-1 expression and significant increase of fatty acid oxidation in HepG2 cells. These effects of Cy-3-g are largely abolished by pharmacological and genetic inhibition of AMPK. Conclusion This study demonstrates that Cy-3-g regulates hepatic lipid homeostasis via an AMPK-dependent signaling pathway. Targeting AMPK activation by anthocyanin may represent a promising approach for the prevention and treatment of obesity-related nonalcoholic fatty liver disease. PMID:22243683

A novel ATP-generating machinery to counter nitrosative stress is mediated by substrate-level phosphorylation.

PubMed

Auger, Christopher; Appanna, Vasu D

2015-01-01

It is well-known that elevated amounts of nitric oxide and other reactive nitrogen species (RNS) impact negatively on the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. These perturbations severely compromise O2-dependent energy production. While bacteria are known to adapt to RNS, a key tool employed by macrophages to combat infections, the exact mechanisms are unknown. The bacterium was cultured in a defined mineral medium and cell-free extracts obtained at the same growth phase were utilized for various biochemical studies Blue native polyacrylamide gel electrophoresis followed by in-gel activity assays, high performance liquid chromatography and co-immunoprecipitaton are applied to investigate the effects of RNS on the model microbe Pseudomonas fluorescens. Citrate is channeled away from the tricarboxylic acid cycle using a novel metabolon consisting of citrate lyase (CL), phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase (PPDK). This metabolic engine comprising three disparate enzymes appears to transiently assemble as a supercomplex aimed at ATP synthesis. The up-regulation in the activities of adenylate kinase (AK) and nucleoside diphosphate kinase (NDPK) ensured the efficacy of this ATP-making machine. Microbes may escape the effects of nitrosative stress by re-engineering metabolic networks in order to generate and store ATP anaerobically when the electron transport chain is defective. The molecular configuration described herein provides further understanding of how metabolism plays a key role in the adaptation to nitrosative stress and reveals novel targets that will inform the development of antimicrobial agents to counter RNS-resistant pathogens. Copyright © 2014 Elsevier B.V. All rights reserved.

Gene expression of regulatory enzymes of glycolysis/gluconeogenesis in regenerating rat liver.

PubMed Central

Rosa, J L; Bartrons, R; Tauler, A

1992-01-01

Levels of mRNA for glucokinase, L-pyruvate kinase, fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase were analysed during liver regeneration. Levels of mRNA for glycolytic enzymes (glucokinase and L-pyruvate kinase) decreased rapidly after partial hepatectomy. Glucokinase mRNA increased at 16-24 h, returning to normal values after this time. L-pyruvate kinase mRNA recovered control levels at 168 h. In contrast, phosphoenolpyruvate carboxykinase mRNA increased rapidly after liver resection and remained high during the regenerative process. However, the levels of fructose-1,6-bisphosphatase mRNA were not modified significantly. These results correlate with the reported increased rate of gluconeogenesis and changes in enzyme levels after partial hepatectomy. The effect of stress on the mRNA levels was also studied. All enzymes showed variations in their mRNA levels after the surgical stress. In general, the differences were more pronounced in regenerating liver than in sham-operated animals, being practically normalized at 24 h. Images Fig. 2. Fig. 3. PMID:1329724

Phosphatidylinositol 3-Kinase Promotes V-ATPase Activation and Vacuolar Acidification and Delays Methyl Jasmonate-Induced Leaf Senescence1

PubMed Central

Liu, Jian; Ji, Yingbin; Zhou, Jun; Xing, Da

2016-01-01

PI3K and its product PI3P are both involved in plant development and stress responses. In this study, the down-regulation of PI3K activity accelerated leaf senescence induced by methyl jasmonate (MeJA) and suppressed the activation of vacuolar H+-ATPase (V-ATPase). Yeast two-hybrid analyses indicated that PI3K bound to the V-ATPase B subunit (VHA-B). Analysis of bimolecular fluorescence complementation in tobacco guard cells showed that PI3K interacted with VHA-B2 in the tonoplasts. Through the use of pharmacological and genetic tools, we found that PI3K and V-ATPase promoted vacuolar acidification and stomatal closure during leaf senescence. Vacuolar acidification was suppressed by the PIKfyve inhibitor in 35S:AtVPS34-YFP Arabidopsis during MeJA-induced leaf senescence, but the decrease was lower than that in YFP-labeled Arabidopsis. These results suggest that PI3K promotes V-ATPase activation and consequently induces vacuolar acidification and stomatal closure, thereby delaying MeJA-induced leaf senescence. PMID:26739232

Anaplerotic roles of pyruvate carboxylase in mammalian tissues.

PubMed

Jitrapakdee, S; Vidal-Puig, A; Wallace, J C

2006-04-01

Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate. PC serves an anaplerotic role for the tricarboxylic acid cycle, when intermediates are removed for different biosynthetic purposes. In liver and kidney, PC provides oxaloacetate for gluconeogenesis. In adipocytes PC is involved in de novo fatty acid synthesis and glyceroneogenesis, and is regulated by the peroxisome proliferator-activated receptor-gamma, suggesting that PC is involved in the metabolic switch controlling fuel partitioning toward lipogenesis. In islets, PC is necessary for glucose-induced insulin secretion by providing oxaloacetate to form malate that participates in the 'pyruvate/malate cycle' to shuttle 3C or 4C between mitochondria and cytoplasm. Hyperglycemia and hyperlipidemia impair this cycle and affect glucose-stimulated insulin release. In astrocytes, PC is important for de novo synthesis of glutamate, an important excitatory neurotransmitter supplied to neurons. Transcriptional studies of the PC gene pinpoint some transcription factors that determine tissue-specific expression.

C75, a fatty acid synthase inhibitor, modulates AMP-activated protein kinase to alter neuronal energy metabolism.

PubMed

Landree, Leslie E; Hanlon, Andrea L; Strong, David W; Rumbaugh, Gavin; Miller, Ian M; Thupari, Jagan N; Connolly, Erin C; Huganir, Richard L; Richardson, Christine; Witters, Lee A; Kuhajda, Francis P; Ronnett, Gabriele V

2004-01-30

C75, a synthetic inhibitor of fatty acid synthase (FAS), is hypothesized to alter the metabolism of neurons in the hypothalamus that regulate feeding behavior to contribute to the decreased food intake and profound weight loss seen with C75 treatment. In the present study, we characterize the suitability of primary cultures of cortical neurons for studies designed to investigate the consequences of C75 treatment and the alteration of fatty acid metabolism in neurons. We demonstrate that in primary cortical neurons, C75 inhibits FAS activity and stimulates carnitine palmitoyltransferase-1 (CPT-1), consistent with its effects in peripheral tissues. C75 alters neuronal ATP levels and AMP-activated protein kinase (AMPK) activity. Neuronal ATP levels are affected in a biphasic manner with C75 treatment, decreasing initially, followed by a prolonged increase above control levels. Cerulenin, a FAS inhibitor, causes a similar biphasic change in ATP levels, although levels do not exceed control. C75 and cerulenin modulate AMPK phosphorylation and activity. TOFA, an inhibitor of acetyl-CoA carboxylase, increases ATP levels, but does not affect AMPK activity. Several downstream pathways are affected by C75 treatment, including glucose metabolism and acetyl-CoA carboxylase (ACC) phosphorylation. These data demonstrate that C75 modulates the levels of energy intermediates, thus, affecting the energy sensor AMPK. Similar effects in hypothalamic neurons could form the basis for the effects of C75 on feeding behavior.

Stevioside improves pancreatic beta-cell function during glucotoxicity via regulation of acetyl-CoA carboxylase.

PubMed

Chen, Jianguo; Jeppesen, Per Bendix; Nordentoft, Iver; Hermansen, Kjeld

2007-06-01

Chronic hyperglycemia is detrimental to pancreatic beta-cells, causing impaired insulin secretion and beta-cell turnover. The characteristic secretory defects are increased basal insulin secretion (BIS) and a selective loss of glucose-stimulated insulin secretion (GSIS). Several recent studies support the view that the acetyl-CoA carboxylase (ACC) plays a pivotal role for GSIS. We have shown that stevioside (SVS) enhances insulin secretion and ACC gene expression. Whether glucotoxicity influences ACC and whether this action can be counteracted by SVS are not known. To investigate this, we exposed isolated mouse islets as well as clonal INS-1E beta-cells for 48 h to 27 or 16.7 mM glucose, respectively. We found that 48-h exposure to high glucose impairs GSIS from mouse islets and INS-1E cells, an effect that is partly counteracted by SVS. The ACC dephosphorylation inhibitor okadaic acid (OKA, 10(-8) M), and 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR, 10(-4) M), an activator of 5'-AMP protein kinase that phosphorylates ACC, eliminated the beneficial effect of SVS. 5-Tetrade-cyloxy-2-furancarboxylic acid (TOFA), the specific ACC inhibitor, blocked the effect of SVS as well. During glucotoxity, ACC gene expression, ACC protein, and phosphorylated ACC protein were increased in INS-1E beta-cells. SVS pretreatment further increased ACC gene expression with strikingly elevated ACC activity and increased glucose uptake accompanied by enhanced GSIS. Our studies show that glucose is a potent stimulator of ACC and that SVS to some extent counteracts glucotoxicity via increased ACC activity. SVS possesses the potential to alleviate negative effects of glucotoxicity in beta-cells via a unique mechanism of action.

AMPK activation represses the human gene promoter of the cardiac isoform of acetyl-CoA carboxylase: Role of nuclear respiratory factor-1

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

Adam, Tasneem; Opie, Lionel H.; Essop, M. Faadiel, E-mail: mfessop@sun.ac.za

Research highlights: {yields} AMPK inhibits acetyl-CoA carboxylase beta gene promoter activity. {yields} Nuclear respiratory factor-1 inhibits acetyl-CoA carboxylase beta promoter activity. {yields} AMPK regulates acetyl-CoA carboxylase beta at transcriptional level. -- Abstract: The cardiac-enriched isoform of acetyl-CoA carboxylase (ACC{beta}) produces malonyl-CoA, a potent inhibitor of carnitine palmitoyltransferase-1. AMPK inhibits ACC{beta} activity, lowering malonyl-CoA levels and promoting mitochondrial fatty acid {beta}-oxidation. Previously, AMPK increased promoter binding of nuclear respiratory factor-1 (NRF-1), a pivotal transcriptional modulator controlling gene expression of mitochondrial proteins. We therefore hypothesized that NRF-1 inhibits myocardial ACC{beta} promoter activity via AMPK activation. A human ACC{beta} promoter-luciferase construct was transientlymore » transfected into neonatal cardiomyocytes {+-} a NRF-1 expression construct. NRF-1 overexpression decreased ACC{beta} gene promoter activity by 71 {+-} 4.6% (p < 0.001 vs. control). Transfections with 5'-end serial promoter deletions revealed that NRF-1-mediated repression of ACC{beta} was abolished with a pPII{beta}-18/+65-Luc deletion construct. AMPK activation dose-dependently reduced ACC{beta} promoter activity, while NRF-1 addition did not further decrease it. We also investigated NRF-1 inhibition in the presence of upstream stimulatory factor 1 (USF1), a known transactivator of the human ACC{beta} gene promoter. Here NRF-1 blunted USF1-dependent induction of ACC{beta} promoter activity by 58 {+-} 7.5% (p < 0.001 vs. control), reversed with a dominant negative NRF-1 construct. NRF-1 also suppressed endogenous USF1 transcriptional activity by 55 {+-} 6.2% (p < 0.001 vs. control). This study demonstrates that NRF-1 is a novel transcriptional inhibitor of the human ACC{beta} gene promoter in the mammalian heart. Our data extends AMPK regulation of ACC{beta} to the transcriptional

Assessing soybean leaf area and leaf biomass by spectral measurements

NASA Technical Reports Server (NTRS)

Holben, B. N.; Tucker, C. J.; Fan, C. J.

1979-01-01

Red and photographic infrared spectral radiances were correlated with soybean total leaf area index, green leaf area index, chlorotic leaf area index, green leaf biomass, chlorotic leaf biomass, and total biomass. The most significant correlations were found to exist between the IR/red radiance ratio data and green leaf area index and/or green leaf biomass (r squared equals 0.85 and 0.86, respectively). These findings demonstrate that remote sensing data can supply information basic to soybean canopy growth, development, and status by nondestructive determination of the green leaf area or green leaf biomass.

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

DOEpatents

Houtz, Robert L.

1999-01-01

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

Relationships of leaf dark respiration to leaf nitrogen, specific leaf area and leaf life-span: a test across biomes and functional groups.

PubMed

Reich, Peter B; Walters, Michael B; Ellsworth, David S; Vose, James M; Volin, John C; Gresham, Charles; Bowman, William D

1998-05-01

Based on prior evidence of coordinated multiple leaf trait scaling, we hypothesized that variation among species in leaf dark respiration rate (R d ) should scale with variation in traits such as leaf nitrogen (N), leaf life-span, specific leaf area (SLA), and net photosynthetic capacity (A max ). However, it is not known whether such scaling, if it exists, is similar among disparate biomes and plant functional types. We tested this idea by examining the interspecific relationships between R d measured at a standard temperature and leaf life-span, N, SLA and A max for 69 species from four functional groups (forbs, broad-leafed trees and shrubs, and needle-leafed conifers) in six biomes traversing the Americas: alpine tundra/subalpine forest, Colorado; cold temperate forest/grassland, Wisconsin; cool temperate forest, North Carolina; desert/shrubland, New Mexico; subtropical forest, South Carolina; and tropical rain forest, Amazonas, Venezuela. Area-based R d was positively related to area-based leaf N within functional groups and for all species pooled, but not when comparing among species within any site. At all sites, mass-based R d (R d-mass ) decreased sharply with increasing leaf life-span and was positively related to SLA and mass-based A max and leaf N (leaf N mass ). These intra-biome relationships were similar in shape and slope among sites, where in each case we compared species belonging to different plant functional groups. Significant R d-mass -N mass relationships were observed in all functional groups (pooled across sites), but the relationships differed, with higher R d at any given leaf N in functional groups (such as forbs) with higher SLA and shorter leaf life-span. Regardless of biome or functional group, R d-mass was well predicted by all combinations of leaf life-span, N mass and/or SLA (r 2 ≥ 0.79, P < 0.0001). At any given SLA, R d-mass rises with increasing N mass and/or decreasing leaf life-span; and at any level of N mass , R d

Relationships of leaf dark respiration to leaf nitrogen, specific leaf area and leaf life-span: a test across biomes and functional groups

Treesearch

Peter B. Reich; Michael B. Walters; David S. Ellsworth; [and others; [Editor’s note: James M.. Vose is the SRS co-author for this publication.

1998-01-01

Based on prior evidence of coordinated multiple leaf trait scaling, the authors hypothesized that variation among species in leaf dark respiration rate (Rd) should scale with variation in traits such as leaf nitrogen (N), leaf life-span, specific leaf area (SLA), and net photosynthetic capacity (Amax). However, it is not known whether such scaling, if it exists, is...

Characterization of a Dopaminergic Stimulatory Factor Derived From Monoclonal Cell Lines of Striatal Origin

DTIC Science & Technology

2005-12-01

purification scheme that appears most advantageous. The purest product obtained will be subjected to NMR analysis by Josh Kurutz and mass spectroscopy by...Phosphoenolpyruvate carboxykinase 2 Phosphomevalonate kinase Protein phosphatase 1A, magnesium dependent, alpha isoform Uridine- cytidine kinase 1-like 1...more purified preparation should permit structural analysis of the molecules responsible for the activity using mass spectroscopy and nuclear

Leaf-IT: An Android application for measuring leaf area.

PubMed

Schrader, Julian; Pillar, Giso; Kreft, Holger

2017-11-01

The use of plant functional traits has become increasingly popular in ecological studies because plant functional traits help to understand key ecological processes in plant species and communities. This also includes changes in diversity, inter- and intraspecific interactions, and relationships of species at different spatiotemporal scales. Leaf traits are among the most important traits as they describe key dimensions of a plant's life history strategy. Further, leaf area is a key parameter with relevance for other traits such as specific leaf area, which in turn correlates with leaf chemical composition, photosynthetic rate, leaf longevity, and carbon investment. Measuring leaf area usually involves the use of scanners and commercial software and can be difficult under field conditions. We present Leaf-IT, a new smartphone application for measuring leaf area and other trait-related areas. Leaf-IT is free, designed for scientific purposes, and runs on Android 4 or higher. We tested the precision and accuracy using objects with standardized area and compared the area measurements of real leaves with the well-established, commercial software WinFOLIA using the Altman-Bland method. Area measurements of standardized objects show that Leaf-IT measures area with high accuracy and precision. Area measurements with Leaf-IT of real leaves are comparable to those of WinFOLIA. Leaf-IT is an easy-to-use application running on a wide range of smartphones. That increases the portability and use of Leaf-IT and makes it possible to measure leaf area under field conditions typical for remote locations. Its high accuracy and precision are similar to WinFOLIA. Currently, its main limitation is margin detection of damaged leaves or complex leaf morphologies.

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

PubMed Central

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

2015-01-01

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

Contrasting cDNA-AFLP profiles between crown and leaf tissues of cold-acclimated wheat plants indicate differing regulatory circuitries for low temperature tolerance.

PubMed

Ganeshan, Seedhabadee; Sharma, Pallavi; Young, Lester; Kumar, Ashwani; Fowler, D Brian; Chibbar, Ravindra N

2011-03-01

Low-temperature (LT) tolerance in winter wheat (Triticum aestivum L.) is an economically important but complex trait. Four selected wheat genotypes, a winter hardy cultivar, Norstar, a tender spring cultivar, Manitou and two near-isogenic lines with Vrn-A1 (spring Norstar) and vrn-A1 (winter Manitou) alleles of Manitou and Norstar were cold-acclimated at 6°C and crown and leaf tissues were collected at 0, 2, 14, 21, 35, 42, 56 and 70 days of cold acclimation. cDNA-AFLP profiling was used to determine temporal expression profiles of transcripts during cold-acclimation in crown and leaf tissues, separately to determine if LT regulatory circuitries in crown and leaf tissues could be delineated using this approach. Screening 64 primer combinations identified 4,074 and 2,757 differentially expressed transcript-derived fragments (TDFs) out of which 38 and 16% were up-regulated as compared to 3 and 6% that were down-regulated in crown and leaf tissues, respectively. DNA sequencing of TDFs revealed sequences common to both tissues including genes coding for DEAD-box RNA helicase, choline-phosphate cytidylyltransferase and delta-1-pyrroline carboxylate synthetase. TDF specific to crown tissues included genes coding for phospahtidylinositol kinase, auxin response factor protein and brassinosteroid insensitive 1-associated receptor kinase. In leaf, genes such as methylene tetrahydrofolate reductase, NADH-cytochrome b5 reductase and malate dehydrogenase were identified. However, 30 and 14% of the DNA sequences from the crown and leaf tissues, respectively, were hypothetical or unknown proteins. Cluster analysis of up-, down-regulated and unique TDFs, DNA sequence and real-time PCR validation, infer that mechanisms operating in crown and leaf tissue in response to LT are differently regulated and warrant further studies.

Interactive response of photosynthetic characteristics in Haloxylon ammodendron and Hedysarum scoparium exposed to soil water and air vapor pressure deficits.

PubMed

Gong, Chunmei; Wang, Jiajia; Hu, Congxia; Wang, Junhui; Ning, Pengbo; Bai, Juan

2015-08-01

C4 plants possess better drought tolerance than C3 plants. However, Hedysarum scoparium, a C3 species, is dominant and widely distributed in the desert areas of northwestern China due to its strong drought tolerance. This study compared it with Haloxylon ammodendron, a C4 species, regarding the interactive effects of drought stress and different leaf-air vapor pressure deficits. Variables of interest included gas exchange, the activity levels of key C4 photosynthetic enzymes, and cellular anatomy. In both species, gas exchange parameters were more sensitive to high vapor pressure deficit than to strong water stress, and the net CO2 assimilation rate (An) was enhanced as vapor pressure deficits increased. A close relationship between An and stomatal conductance (gs) suggested that the species shared a similar response mechanism. In H. ammodendron, the activity levels of key C4 enzymes were higher, including those of phosphoenolpyruvate carboxylase (PEPC) and nicotinamide adenine dinucleotide phosphate-malate enzyme (NADP-ME), whereas in H. scoparium, the activity level of nicotinamide adenine dinucleotide-malate enzyme (NAD-ME) was higher. Meanwhile, H. scoparium utilized adaptive structural features, including a larger relative vessel area and a shorter distance from vein to stomata, which facilitated the movement of water. These findings implied that some C4 biochemical pathways were present in H. scoparium to respond to environmental challenges. Copyright © 2015. Published by Elsevier B.V.

CaMKKβ Is Involved in AMP-Activated Protein Kinase Activation by Baicalin in LKB1 Deficient Cell Lines

PubMed Central

Wang, Ying; Du, Zhiyan; Liu, Daihua; Guo, Hongxia; Shen, Jingkang; Peng, Hongli

2012-01-01

AMP-activated protein kinase (AMPK) plays an important role in mediating energy metabolism and is controlled mainly by two upstream kinases, LKB1 or Ca2+/calmodulin-dependent protein kinase kinase-β (CaMKKβ). Previously, we found that baicalin, one of the major flavonoids in a traditional Chinese herb medicine, Scutellaria baicalensis, protects against the development of hepatic steatosis in rats feeding with a high-fat diet by the activation of AMPK, but, the underlying mechanism for AMPK activation is unknown. Here we show that in two LKB1-deficient cells, HeLa and A549 cells, baicalin activates AMPK by α Thr-172 phosphorylation and subsequent phosphorylation of its downstream target, acetyl CoA carboxylase, at Ser-79, to a similar degree as does in HepG2 cells (that express LKB1). Pharmacologic inhibition of CaMKKβ by its selective inhibitor STO-609 markedly inhibits baicalin-induced AMPK activation in both HeLa and HepG2 cells, indicating that CaMKKβ is the responsible AMPK kinase. We also show that treatment of baicalin causes a larger increase in intracellular Ca2+ concentration ([Ca2+]i), although the maximal level of [Ca2+]i is lower in HepG2 cells compared to HeLa cells. Chelation of intracellular free Ca2+ by EDTA and EGTA, or depletion of intracellular Ca2+ stores by the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin abrogates baicalin-induced activation of AMPK in HeLa cells. Neither cellular ATP nor the production of reactive oxygen species is altered by baicalin. Finally, in HeLa cells, baicalin treatment no longer decreases intracellular lipid accumulation caused by oleic acid after inhibition of CaMKKβ by STO-609. These results demonstrate that a potential Ca2+/CaMKKβ dependent pathway is involved in the activation of AMPK by baicalin and suggest that CaMKKβ likely acts as an upstream kinase of AMPK in response to baicalin. PMID:23110126

Final report on the safety assessment of AloeAndongensis Extract, Aloe Andongensis Leaf Juice,aloe Arborescens Leaf Extract, Aloe Arborescens Leaf Juice, Aloe Arborescens Leaf Protoplasts, Aloe Barbadensis Flower Extract, Aloe Barbadensis Leaf, Aloe Barbadensis Leaf Extract, Aloe Barbadensis Leaf Juice,aloe Barbadensis Leaf Polysaccharides, Aloe Barbadensis Leaf Water, Aloe Ferox Leaf Extract, Aloe Ferox Leaf Juice, and Aloe Ferox Leaf Juice Extract.

PubMed

2007-01-01

Plant materials derived from the Aloe plant are used as cosmetic ingredients, including Aloe Andongensis Extract, Aloe Andongensis Leaf Juice, Aloe Arborescens Leaf Extract, Aloe Arborescens Leaf Juice, Aloe Arborescens Leaf Protoplasts, Aloe Barbadensis Flower Extract, Aloe Barbadensis Leaf, Aloe Barbadensis Leaf Extract, Aloe Barbadensis Leaf Juice, Aloe Barbadensis Leaf Polysaccharides, Aloe Barbadensis Leaf Water, Aloe Ferox Leaf Extract, Aloe Ferox Leaf Juice, and Aloe Ferox Leaf Juice Extract. These ingredients function primarily as skin-conditioning agents and are included in cosmetics only at low concentrations. The Aloe leaf consists of the pericyclic cells, found just below the plant's skin, and the inner central area of the leaf, i.e., the gel, which is used for cosmetic products. The pericyclic cells produce a bitter, yellow latex containing a number of anthraquinones, phototoxic compounds that are also gastrointestinal irritants responsible for cathartic effects. The gel contains polysaccharides, which can be acetylated, partially acetylated, or not acetylated. An industry established limit for anthraquinones in aloe-derived material for nonmedicinal use is 50 ppm or lower. Aloe-derived ingredients are used in a wide variety of cosmetic product types at concentrations of raw material that are 0.1% or less, although can be as high as 20%. The concentration of Aloe in the raw material also may vary from 100% to a low of 0.0005%. Oral administration of various anthraquinone components results in a rise in their blood concentrations, wide systemic distribution, accumulation in the liver and kidneys, and excretion in urine and feces; polysaccharide components are distributed systemically and metabolized into smaller molecules. aloe-derived material has fungicidal, antimicrobial, and antiviral activities, and has been effective in wound healing and infection treatment in animals. Aloe barbadensis (also known as Aloe vera)-derived ingredients were not toxic

Biotin augments acetyl CoA carboxylase 2 gene expression in the hypothalamus, leading to the suppression of food intake in mice.

PubMed

Sone, Hideyuki; Kamiyama, Shin; Higuchi, Mutsumi; Fujino, Kaho; Kubo, Shizuka; Miyazawa, Masami; Shirato, Saya; Hiroi, Yuka; Shiozawa, Kota

2016-07-29

It is known that biotin prevents the development of diabetes by increasing the functions of pancreatic beta-cells and improving insulin sensitivity in the periphery. However, its anti-obesity effects such as anorectic effects remain to be clarified. Acetyl CoA carboxylase (ACC), a biotin-dependent enzyme, has two isoforms (ACC1 and ACC2) and serves to catalyze the reaction of acetyl CoA to malonyl CoA. In the hypothalamus, ACC2 increases the production of malonyl CoA, which acts as a satiety signal. In this study, we investigated whether biotin increases the gene expression of ACC2 in the hypothalamus and suppresses food intake in mice administered excessive biotin. Food intake was significantly decreased by biotin, but plasma regulators of appetite, including glucose, ghrelin, and leptin, were not affected. On the other hand, biotin notably accumulated in the hypothalamus and enhanced ACC2 gene expression there, but it did not change the gene expression of ACC1, malonyl CoA decarboxylase (a malonyl CoA-degrading enzyme), and AMP-activated protein kinase α-2 (an ACC-inhibitory enzyme). These findings strongly suggest that biotin potentiates the suppression of appetite by upregulating ACC2 gene expression in the hypothalamus. This effect of biotin may contribute to the prevention of diabetes by biotin treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

The phosphoenolpyruvate:sugar phosphotransferase system is involved in sensitivity to the glucosylated bacteriocin sublancin.

PubMed

Garcia De Gonzalo, C V; Denham, E L; Mars, R A T; Stülke, J; van der Donk, W A; van Dijl, J M

2015-11-01

The mode of action of a group of glycosylated antimicrobial peptides known as glycocins remains to be elucidated. In the current study of one glycocin, sublancin, we identified the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of Bacillus species as a key player in bacterial sensitivity. Sublancin kills several Gram-positive bacteria, such as Bacillus species and Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA). Unlike other classes of bacteriocins for which the PTS is involved in their mechanism of action, we show that the addition of PTS-requiring sugars leads to increased resistance rather than increased sensitivity, suggesting that sublancin has a distinct mechanism of action. Collectively, our present mutagenesis and genomic studies demonstrate that the histidine-containing phosphocarrier protein (HPr) and domain A of enzyme II (PtsG) in particular are critical determinants for bacterial sensitivity to sublancin. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Nutrient Dependence of RNase E Essentiality in Escherichia coli

PubMed Central

Tamura, Masaru; Moore, Christopher J.

2013-01-01

Escherichia coli cells normally require RNase E activity to form colonies (colony-forming ability [CFA]). The CFA-defective phenotype of cells lacking RNase E is partly reversed by overexpression of the related endoribonuclease RNase G or by mutation of the gene encoding the RNA helicase DeaD. We found that the carbon source utilization by rne deaD doubly mutant bacteria differs from that of rne+ cells and from that of cells mutated in deaD alone and that the loss of rne function in these bacteria limits conversion of the glycolytic pathway product phosphoenolpyruvate to the tricarboxylic acid (TCA) cycle intermediate oxaloacetic acid. We show that the mechanism underlying this effect is reduced production of the enzyme phosphoenolpyruvate carboxylase (PPC) and that adventitious overexpression of PPC, which facilitates phosphoenolpyruvate utilization and connects the glycolytic pathway with the TCA cycle, restored CFA to rne deaD mutant bacteria cultured on carbon sources that otherwise were unable to sustain growth. We further show that bacteria producing full-length RNase E, which allows formation of degradosomes, have nutritional requirements different from those of cells supplied with only the N-terminal catalytic region of RNase E and that mitigation of RNase E deficiency by overexpression of a related RNase, RNase G, is also affected by carbon source. Our results reveal previously unsuspected effects of RNase E deficiency and degradosome formation on nutrient utilization by E. coli cells. PMID:23275245

Brown Pine Leaf Extract and Its Active Component Trans-Communic Acid Inhibit UVB-Induced MMP-1 Expression by Targeting PI3K

PubMed Central

Park, Gaeun; Lim, Tae-gyu; Kwon, Jung Yeon; Song, Da Som; Jeong, Eun Hee; Lee, Charles C.; Son, Joe Eun; Seo, Sang Gwon; Lee, Eunjung; Kim, Jong Rhan; Lee, Chang Yong; Park, Jun Seong; Lee, Ki Won

2015-01-01

Japanese red pine (Pinus densiflora) is widely present in China, Japan, and Korea. Its green pine leaves have traditionally been used as a food as well as a coloring agent. After being shed, pine leaves change their color from green to brown within two years, and although the brown pine leaves are abundantly available, their value has not been closely assessed. In this study, we investigated the potential anti-photoaging properties of brown pine leaves for skin. Brown pine leaf extract (BPLE) inhibited UVB-induced matrix metalloproteinase-1 (MMP-1) expression to a greater extent than pine leaf extract (PLE) in human keratinocytes and a human skin equivalent model. HPLC analysis revealed that the quantity of trans-communic acid (TCA) and dehydroabietic acid (DAA) significantly increases when the pine leaf color changes from green to brown. BPLE and TCA elicited reductions in UVB-induced MMP-1 mRNA expression and activator protein-1 (AP-1) transactivation by reducing DNA binding activity of phospho-c-Jun, c-fos and Fra-1. BPLE and TCA also inhibited UVB-induced Akt phosphorylation, but not mitogen activated protein kinase (MAPK), known regulators of AP-1 transactivation. We additionally found that BPLE and TCA inhibited phosphoinositide 3-kinase (PI3K), the upstream kinase of Akt, in vitro. In summary, both BPLE and its active component TCA exhibit protective effects against UVB-induced skin aging. Taken together, these findings underline the potential for BPLE and TCA to be utilized as anti-wrinkling agents and cosmetic ingredients, as they suppress UVB-induced MMP-1 expression. PMID:26066652

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

PubMed

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

2011-11-01

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

Maize YABBY genes drooping leaf1 and drooping leaf2 affect agronomic traits by regulating leaf architecture

USDA-ARS?s Scientific Manuscript database

Leaf architectural traits, such as length, width and angle, directly influence canopy structure and light penetration, photosynthate production and overall yield. We discovered and characterized a maize (Zea mays) mutant with aberrant leaf architecture we named drooping leaf1 (drl1), as leaf blades ...

Cerebral Gluconeogenesis and Diseases

PubMed Central

Yip, James; Geng, Xiaokun; Shen, Jiamei; Ding, Yuchuan

2017-01-01

The gluconeogenesis pathway, which has been known to normally present in the liver, kidney, intestine, or muscle, has four irreversible steps catalyzed by the enzymes: pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase, and glucose 6-phosphatase. Studies have also demonstrated evidence that gluconeogenesis exists in brain astrocytes but no convincing data have yet been found in neurons. Astrocytes exhibit significant 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 activity, a key mechanism for regulating glycolysis and gluconeogenesis. Astrocytes are unique in that they use glycolysis to produce lactate, which is then shuttled into neurons and used as gluconeogenic precursors for reduction. This gluconeogenesis pathway found in astrocytes is becoming more recognized as an important alternative glucose source for neurons, specifically in ischemic stroke and brain tumor. Further studies are needed to discover how the gluconeogenesis pathway is controlled in the brain, which may lead to the development of therapeutic targets to control energy levels and cellular survival in ischemic stroke patients, or inhibit gluconeogenesis in brain tumors to promote malignant cell death and tumor regression. While there are extensive studies on the mechanisms of cerebral glycolysis in ischemic stroke and brain tumors, studies on cerebral gluconeogenesis are limited. Here, we review studies done to date regarding gluconeogenesis to evaluate whether this metabolic pathway is beneficial or detrimental to the brain under these pathological conditions. PMID:28101056

Cerebral Gluconeogenesis and Diseases.

PubMed

Yip, James; Geng, Xiaokun; Shen, Jiamei; Ding, Yuchuan

2016-01-01

The gluconeogenesis pathway, which has been known to normally present in the liver, kidney, intestine, or muscle, has four irreversible steps catalyzed by the enzymes: pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase, and glucose 6-phosphatase. Studies have also demonstrated evidence that gluconeogenesis exists in brain astrocytes but no convincing data have yet been found in neurons. Astrocytes exhibit significant 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 activity, a key mechanism for regulating glycolysis and gluconeogenesis. Astrocytes are unique in that they use glycolysis to produce lactate, which is then shuttled into neurons and used as gluconeogenic precursors for reduction. This gluconeogenesis pathway found in astrocytes is becoming more recognized as an important alternative glucose source for neurons, specifically in ischemic stroke and brain tumor. Further studies are needed to discover how the gluconeogenesis pathway is controlled in the brain, which may lead to the development of therapeutic targets to control energy levels and cellular survival in ischemic stroke patients, or inhibit gluconeogenesis in brain tumors to promote malignant cell death and tumor regression. While there are extensive studies on the mechanisms of cerebral glycolysis in ischemic stroke and brain tumors, studies on cerebral gluconeogenesis are limited. Here, we review studies done to date regarding gluconeogenesis to evaluate whether this metabolic pathway is beneficial or detrimental to the brain under these pathological conditions.

Difference in C3-C4 metabolism underlies tradeoff between growth rate and biomass yield in Methylobacterium extorquens AM1.

PubMed

Fu, Yanfen; Beck, David A C; Lidstrom, Mary E

2016-07-19

Two variants of Methylobacterium extorquens AM1 demonstrated a trade-off between growth rate and biomass yield. In addition, growth rate and biomass yield were also affected by supplementation of growth medium with different amounts of cobalt. The metabolism changes relating to these growth phenomena as well as the trade-off were investigated in this study. (13)C metabolic flux analysis was used to generate a detailed central carbon metabolic flux map with both absolute and normalized flux values. The major differences between the two variants occurred at the formate node as well as within C3-C4 inter-conversion pathways. Higher relative fluxes through formyltetrahydrofolate ligase, phosphoenolpyruvate carboxylase, and malic enzyme led to higher biomass yield, while higher relative fluxes through pyruvate kinase and pyruvate dehydrogenase led to higher growth rate. These results were then tested by phenotypic studies on three mutants (null pyk, null pck mutant and null dme mutant) in both variants, which agreed with the model prediction. In this study, (13)C metabolic flux analysis for two strain variants of M. extorquens AM1 successfully identified metabolic pathways contributing to the trade-off between cell growth and biomass yield. Phenotypic analysis of mutants deficient in corresponding genes supported the conclusion that C3-C4 inter-conversion strategies were the major response to the trade-off.

The relationship of leaf photosynthetic traits V cmax and Jmax - to leaf nitrogen, leaf phosphorus, and specific leaf area: A meta-analysis and modeling study

DOE PAGES

Walker, Anthony P.; Beckerman, Andrew P.; Gu, Lianhong; ...

2014-07-25

Great uncertainty exists in the global exchange of carbon between the atmosphere and the terrestrial biosphere. An important source of this uncertainty lies in the dependency of photosynthesis on the maximum rate of carboxylation (Vcmax) and the maximum rate of electron transport (Jmax). Understanding and making accurate prediction of C fluxes thus requires accurate characterization of these rates and their relationship with plant nutrient status over large geographic scales. Plant nutrient status is indicated by the traits: leaf nitrogen (N), leaf phosphorus (P), and specific leaf area (SLA). Correlations between Vcmax and Jmax and leaf nitrogen (N) are typically derivedmore » from local to global scales, while correlations with leaf phosphorus (P) and specific leaf area (SLA) have typically been derived at a local scale. Thus, there is no global-scale relationship between Vcmax and Jmax and P or SLA limiting the ability of global-scale carbon flux models do not account for P or SLA. We gathered published data from 24 studies to reveal global relationships of Vcmax and Jmax with leaf N, P, and SLA. Vcmax was strongly related to leaf N, and increasing leaf P substantially increased the sensitivity of Vcmax to leaf N. Jmax was strongly related to Vcmax, and neither leaf N, P, or SLA had a substantial impact on the relationship. Although more data are needed to expand the applicability of the relationship, we show leaf P is a globally important determinant of photosynthetic rates. In a model of photosynthesis, we showed that at high leaf N (3 gm 2), increasing leaf P from 0.05 to 0.22 gm 2 nearly doubled assimilation rates. Lastly, we show that plants may employ a conservative strategy of Jmax to Vcmax coordination that restricts photoinhibition when carboxylation is limiting at the expense of maximizing photosynthetic rates when light is limiting.« less

The relationship of leaf photosynthetic traits V cmax and Jmax - to leaf nitrogen, leaf phosphorus, and specific leaf area: A meta-analysis and modeling study

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

Walker, Anthony P.; Beckerman, Andrew P.; Gu, Lianhong

Great uncertainty exists in the global exchange of carbon between the atmosphere and the terrestrial biosphere. An important source of this uncertainty lies in the dependency of photosynthesis on the maximum rate of carboxylation (Vcmax) and the maximum rate of electron transport (Jmax). Understanding and making accurate prediction of C fluxes thus requires accurate characterization of these rates and their relationship with plant nutrient status over large geographic scales. Plant nutrient status is indicated by the traits: leaf nitrogen (N), leaf phosphorus (P), and specific leaf area (SLA). Correlations between Vcmax and Jmax and leaf nitrogen (N) are typically derivedmore » from local to global scales, while correlations with leaf phosphorus (P) and specific leaf area (SLA) have typically been derived at a local scale. Thus, there is no global-scale relationship between Vcmax and Jmax and P or SLA limiting the ability of global-scale carbon flux models do not account for P or SLA. We gathered published data from 24 studies to reveal global relationships of Vcmax and Jmax with leaf N, P, and SLA. Vcmax was strongly related to leaf N, and increasing leaf P substantially increased the sensitivity of Vcmax to leaf N. Jmax was strongly related to Vcmax, and neither leaf N, P, or SLA had a substantial impact on the relationship. Although more data are needed to expand the applicability of the relationship, we show leaf P is a globally important determinant of photosynthetic rates. In a model of photosynthesis, we showed that at high leaf N (3 gm 2), increasing leaf P from 0.05 to 0.22 gm 2 nearly doubled assimilation rates. Lastly, we show that plants may employ a conservative strategy of Jmax to Vcmax coordination that restricts photoinhibition when carboxylation is limiting at the expense of maximizing photosynthetic rates when light is limiting.« less

Activation of AMP-activated Protein Kinase by Metformin Induces Protein Acetylation in Prostate and Ovarian Cancer Cells*

PubMed Central

Galdieri, Luciano; Gatla, Himavanth; Vancurova, Ivana; Vancura, Ales

2016-01-01

AMP-activated protein kinase (AMPK) is an energy sensor and master regulator of metabolism. AMPK functions as a fuel gauge monitoring systemic and cellular energy status. Activation of AMPK occurs when the intracellular AMP/ATP ratio increases and leads to a metabolic switch from anabolism to catabolism. AMPK phosphorylates and inhibits acetyl-CoA carboxylase (ACC), which catalyzes carboxylation of acetyl-CoA to malonyl-CoA, the first and rate-limiting reaction in de novo synthesis of fatty acids. AMPK thus regulates homeostasis of acetyl-CoA, a key metabolite at the crossroads of metabolism, signaling, chromatin structure, and transcription. Nucleocytosolic concentration of acetyl-CoA affects histone acetylation and links metabolism and chromatin structure. Here we show that activation of AMPK with the widely used antidiabetic drug metformin or with the AMP mimetic 5-aminoimidazole-4-carboxamide ribonucleotide increases the inhibitory phosphorylation of ACC and decreases the conversion of acetyl-CoA to malonyl-CoA, leading to increased protein acetylation and altered gene expression in prostate and ovarian cancer cells. Direct inhibition of ACC with allosteric inhibitor 5-(tetradecyloxy)-2-furoic acid also increases acetylation of histones and non-histone proteins. Because AMPK activation requires liver kinase B1, metformin does not induce protein acetylation in liver kinase B1-deficient cells. Together, our data indicate that AMPK regulates the availability of nucleocytosolic acetyl-CoA for protein acetylation and that AMPK activators, such as metformin, have the capacity to increase protein acetylation and alter patterns of gene expression, further expanding the plethora of metformin's physiological effects. PMID:27733682

Increased Phosphoenolpyruvate Carboxykinase (PEPCK) Gene Expression and Steatosis During Hepatitis C Virus (HCV) Subgenome Replication: Role of Nonstructural Component-5A (NS5A) and CCAAT/Enhancer Binding Protein ß (C/EBPß)

USDA-ARS?s Scientific Manuscript database

Chronic hepatitis C virus (HCV) infection greatly increases the risk for type 2 diabetes and nonalcoholic steatohepatitis; however, the pathogenic mechanisms remain incompletely understood. Here we report gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) transcription and associated tra...

Maternal obesity reduces milk lipid production in lactating mice by inhibiting acetyl-CoA carboxylase and impairing fatty acid synthesis.

PubMed

Saben, Jessica L; Bales, Elise S; Jackman, Matthew R; Orlicky, David; MacLean, Paul S; McManaman, James L

2014-01-01

Maternal metabolic and nutrient trafficking adaptations to lactation differ among lean and obese mice fed a high fat (HF) diet. Obesity is thought to impair milk lipid production, in part, by decreasing trafficking of dietary and de novo synthesized lipids to the mammary gland. Here, we report that de novo lipogenesis regulatory mechanisms are disrupted in mammary glands of lactating HF-fed obese (HF-Ob) mice. HF feeding decreased the total levels of acetyl-CoA carboxylase-1 (ACC), and this effect was exacerbated in obese mice. The relative levels of phosphorylated (inactive) ACC, were elevated in the epithelium, and decreased in the adipose stroma, of mammary tissue from HF-Ob mice compared to those of HF-fed lean (HF-Ln) mice. Mammary gland levels of AMP-activated protein kinase (AMPK), which catalyzes formation of inactive ACC, were also selectively elevated in mammary glands of HF-Ob relative to HF-Ln dams or to low fat fed dams. These responses correlated with evidence of increased lipid retention in mammary adipose, and decreased lipid levels in mammary epithelial cells, of HF-Ob dams. Collectively, our data suggests that maternal obesity impairs milk lipid production, in part, by disrupting the balance of de novo lipid synthesis in the epithelial and adipose stromal compartments of mammary tissue through processes that appear to be related to increased mammary gland AMPK activity, ACC inhibition, and decreased fatty acid synthesis.

Maternal Obesity Reduces Milk Lipid Production in Lactating Mice by Inhibiting Acetyl-CoA Carboxylase and Impairing Fatty Acid Synthesis

PubMed Central

Saben, Jessica L.; Bales, Elise S.; Jackman, Matthew R.; Orlicky, David; MacLean, Paul S.; McManaman, James L.

2014-01-01

Maternal metabolic and nutrient trafficking adaptations to lactation differ among lean and obese mice fed a high fat (HF) diet. Obesity is thought to impair milk lipid production, in part, by decreasing trafficking of dietary and de novo synthesized lipids to the mammary gland. Here, we report that de novo lipogenesis regulatory mechanisms are disrupted in mammary glands of lactating HF-fed obese (HF-Ob) mice. HF feeding decreased the total levels of acetyl-CoA carboxylase-1 (ACC), and this effect was exacerbated in obese mice. The relative levels of phosphorylated (inactive) ACC, were elevated in the epithelium, and decreased in the adipose stroma, of mammary tissue from HF-Ob mice compared to those of HF-fed lean (HF-Ln) mice. Mammary gland levels of AMP-activated protein kinase (AMPK), which catalyzes formation of inactive ACC, were also selectively elevated in mammary glands of HF-Ob relative to HF-Ln dams or to low fat fed dams. These responses correlated with evidence of increased lipid retention in mammary adipose, and decreased lipid levels in mammary epithelial cells, of HF-Ob dams. Collectively, our data suggests that maternal obesity impairs milk lipid production, in part, by disrupting the balance of de novo lipid synthesis in the epithelial and adipose stromal compartments of mammary tissue through processes that appear to be related to increased mammary gland AMPK activity, ACC inhibition, and decreased fatty acid synthesis. PMID:24849657

Potassium deficiency affects the carbon-nitrogen balance in cotton leaves.

PubMed

Hu, Wei; Coomer, Taylor D; Loka, Dimitra A; Oosterhuis, Derrick M; Zhou, Zhiguo

2017-06-01

Potassium (K) plays important roles in the metabolism of carbon (C) and nitrogen (N), but studies of K deficiency affecting C-N balance are lacking. This study explored the influence of K deficiency on C-N interaction in cotton leaves by conducting a field experiment with cotton cultivar DP0912 under two K rates (K0: 0 kg K 2 O ha -1 and K67: 67 kg K 2 O ha -1 ) and a controlled environment experiment with K-deficient solution (K1: 0 mM K + ) and K-sufficient solution (K2: 6 mM K + ). The results showed that leaf K content, leaf number, leaf area, boll number, reproductive dry weight and total dry weight were significant lower under K deficiency (K0 or K1). Lower total chlorophyll content and Chl a/b ratio, and decreased Pn along with lower Gs and higher Ci were measured under K deficiency, suggesting that the decrease in Pn was resulted from non-stomatal limitation. Leaf glucose, fructose, sucrose and starch contents were higher under K deficiency, because lower sucrose export was detected in phloem. Although leaf nitrate and ammonium contents significantly decreased, free amino acid content was increased by 40-63% under K deficiency, since lower amino acid export was also measured in phloem. K deficiency also induced lower soluble protein content in leaves. Leaf ATP level was significantly increased under K deficiency, indicating ATP utilization was lower, so that less energy was supplied to C and N metabolism. The ratio of soluble sugar to free amino acid and the C/N ratio markedly increased under K deficiency, and one reason was that the phloem export reduced more prominent for sucrose (54.6-78.0%) than amino acid (36.7-85.4%) under K deficiency. In addition, lower phosphoenolpyruvate carboxylase activity limited malate and citrate biosynthesis under K deficiency, causing a decrease of C flux into the amino acids, which was not beneficial for maintaining C-N balance. Sucrose phosphate synthase and nitrate reductase activities were lower under K deficiency

Metformin and phenformin activate AMP-activated protein kinase in the heart by increasing cytosolic AMP concentration.

PubMed

Zhang, Li; He, Huamei; Balschi, James A

2007-07-01

AMP-activated protein kinase (AMPK) acts as a cellular energy sensor: it responds to an increase in AMP concentration ([AMP]) or the AMP-to-ATP ratio (AMP/ATP). Metformin and phenformin, which are biguanides, have been reported to increase AMPK activity without increasing AMP/ATP. This study tests the hypothesis that these biguanides increase AMPK activity in the heart by increasing cytosolic [AMP]. Groups of isolated rat hearts (n = 5-7 each) were perfused with Krebs-Henseleit buffer with or without 0.2 mM phenformin or 10 mM metformin, and (31)P-NMR-measured phosphocreatine, ATP, and intracellular pH were used to calculate cytosolic [AMP]. At various times, hearts were freeze-clamped and assayed for AMPK activity, phosphorylation of Thr(172) on AMPK-alpha, and phosphorylation of Ser(79) on acetyl-CoA carboxylase, an AMPK target. In hearts treated with phenformin for 18 min and then perfused for 20 min with Krebs-Henseleit buffer, [AMP] began to increase at 26 min and AMPK activity was elevated at 36 min. In hearts treated with metformin, [AMP] was increased at 50 min and AMPK activity, phosphorylated AMPK, and phosphorylated acetyl-CoA carboxylase were elevated at 61 min. In metformin-treated hearts, HPLC-measured total AMP content and total AMP/ATP did not increase. In summary, phenformin and metformin increase AMPK activity and phosphorylation in the isolated heart. The increase in AMPK activity was always preceded by and correlated with increased cytosolic [AMP]. Total AMP content and total AMP/ATP did not change. Cytosolic [AMP] reported metabolically active AMP, which triggered increased AMPK activity, but measures of total AMP did not.

Transport of Phosphoenolpyruvate by Chloroplasts from Mesembryanthemum crystallinum L. Exhibiting Crassulacean Acid Metabolism 1

PubMed Central

Neuhaus, H. Ekkehard; Holtum, Joseph A. M.; Latzko, Erwin

1988-01-01

Chloroplasts from CAM-Mesembryanthemum crystallinum can transport phosphoenolpyruvate (PEP) across the envelope. The initial velocities of PEP uptake in the dark at 4°C exhibited saturation kinetics with increasing external PEP concentration. PEP uptake had a Vmax of 6.46 (±0.05) micromoles per milligram chlorophyll per hour and an apparent Kmpep of 0.148 (±0.004) millimolar. The uptake was competitively inhibited by Pi (apparent Ki = 0.19 millimolar), by glycerate 3-phosphate (apparent Ki = 0.13 millimolar), and by dihydroxyacetone phosphate, but malate and pyruvate were without effect. The chloroplasts were able to synthesize PEP when presented with pyruvate. PEP synthesis was light dependent. The prolonged synthesis and export of PEP from the chloroplasts required the presence of Pi or glycerate 3-phosphate in the external medium. It is suggested that the transport of pyruvate and PEP across the chloroplasts envelope is required during the gluconeogenic conversion of carbon from malate to storage carbohydrate in the light. PMID:16666128

A crotonyl-CoA reductase-carboxylase independent pathway for assembly of unusual alkylmalonyl-CoA polyketide synthase extender units

NASA Astrophysics Data System (ADS)

Ray, Lauren; Valentic, Timothy R.; Miyazawa, Takeshi; Withall, David M.; Song, Lijiang; Milligan, Jacob C.; Osada, Hiroyuki; Takahashi, Shunji; Tsai, Shiou-Chuan; Challis, Gregory L.

2016-12-01

Type I modular polyketide synthases assemble diverse bioactive natural products. Such multienzymes typically use malonyl and methylmalonyl-CoA building blocks for polyketide chain assembly. However, in several cases more exotic alkylmalonyl-CoA extender units are also known to be incorporated. In all examples studied to date, such unusual extender units are biosynthesized via reductive carboxylation of α, β-unsaturated thioesters catalysed by crotonyl-CoA reductase/carboxylase (CCRC) homologues. Here we show using a chemically-synthesized deuterium-labelled mechanistic probe, and heterologous gene expression experiments that the unusual alkylmalonyl-CoA extender units incorporated into the stambomycin family of polyketide antibiotics are assembled by direct carboxylation of medium chain acyl-CoA thioesters. X-ray crystal structures of the unusual β-subunit of the acyl-CoA carboxylase (YCC) responsible for this reaction, alone and in complex with hexanoyl-CoA, reveal the molecular basis for substrate recognition, inspiring the development of methodology for polyketide bio-orthogonal tagging via incorporation of 6-azidohexanoic acid and 8-nonynoic acid into novel stambomycin analogues.

A rapid screening method to detect specific inhibitors of pyruvate orthophosphate dikinase as leads for C4 plant-selective herbicides.

PubMed

Doyle, Jason R; Burnell, James N; Haines, Dianne S; Llewellyn, Lyndon E; Motti, Cherie A; Tapiolas, Dianne M

2005-02-01

Plants using the C(4) photosynthetic pathway are highly represented among the world's worst weeds, with only 4 C(4) species being agriculturally productive (maize, sorghum, millet, and sugar cane). With the C(4) acid cycle operating as a biochemical appendage of C(3) photosynthesis, the additional enzymes involved in C(4) photosynthesis represent an attractive target for the development of weed-specific herbicides. The rate-limiting enzyme of this metabolic pathway is pyruvate orthophosphate dikinase (PPDK). PPDK, coupled with phosphoenolpyruvate carboxylase and nicotinamide adenine dinucleotide-malate dehydrogenase, was used to develop a microplate-based assay to detect inhibitors of enzymes of the C(4) acid cycle. The resulting assay had a Z' factor of 0.61, making it a high-quality assay able to reliably identify active test samples. Organic extracts of 6679 marine macroscopic organisms were tested within the assay, and 343 were identified that inhibited the 3 enzyme-coupled reaction. A high confirmation rate was achieved, with 95% of these hit extracts proving active again upon retesting. Sequential addition of phosphoenolpyruvate and oxaloacetate to the assay facilitated identification of 83 extracts that specifically inhibited PPDK.

Woodward's reagent K reacts with histidine and cysteine residues in Escherichia coli and Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases.

PubMed

Bustos, P; Gajardo, M I; Gómez, C; Goldie, H; Cardemil, E; Jabalquinto, A M

1996-07-01

The reaction of Woordward's reagent K (WRK) with model amino acids and proteins has been analyzed. Our results indicate that WRK forms 340-nm-absorbing adducts with sulfhydryl- and imidazol-containing compounds, but not with carboxylic acid derivatives, in agreement with Liamas et al. [(1986), J. Am. Chem. Soc. 108, 5543-5548], but not with Sinha and Brewer [(1985), Anal. Biochem. 151, 327-333]. The chemical modification of Escherichia coli and Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases with WRK leads to an increase in the absorption at 340 nm, and we have demonstrated its reaction with His and Cys residues in these proteins. These results caution against claims of glutamic or aspartic acid modification by WRK based on the absorption at 340 nm of protein- WRK adducts.

Metabolic Regulation of Invadopodia and Invasion by Acetyl-CoA Carboxylase 1 and De novo Lipogenesis

PubMed Central

Scott, Kristen E. N.; Wheeler, Frances B.; Davis, Amanda L.; Thomas, Michael J.; Ntambi, James M.; Seals, Darren F.; Kridel, Steven J.

2012-01-01

Invadopodia are membrane protrusions that facilitate matrix degradation and cellular invasion. Although lipids have been implicated in several aspects of invadopodia formation, the contributions of de novo fatty acid synthesis and lipogenesis have not been defined. Inhibition of acetyl-CoA carboxylase 1 (ACC1), the committed step of fatty acid synthesis, reduced invadopodia formation in Src-transformed 3T3 (3T3-Src) cells, and also decreased the ability to degrade gelatin. Inhibition of fatty acid synthesis through AMP-activated kinase (AMPK) activation and ACC phosphorylation also decreased invadopodia incidence. The addition of exogenous 16∶0 and 18∶1 fatty acid, products of de novo fatty acid synthesis, restored invadopodia and gelatin degradation to cells with decreased ACC1 activity. Pharmacological inhibition of ACC also altered the phospholipid profile of 3T3-Src cells, with the majority of changes occurring in the phosphatidylcholine (PC) species. Exogenous supplementation with the most abundant PC species, 34∶1 PC, restored invadopodia incidence, the ability to degrade gelatin and the ability to invade through matrigel to cells deficient in ACC1 activity. On the other hand, 30∶0 PC did not restore invadopodia and 36∶2 PC only restored invadopodia incidence and gelatin degradation, but not cellular invasion through matrigel. Pharmacological inhibition of ACC also reduced the ability of MDA-MB-231 breast, Snb19 glioblastoma, and PC-3 prostate cancer cells to invade through matrigel. Invasion of PC-3 cells through matrigel was also restored by 34∶1 PC supplementation. Collectively, the data elucidate the novel metabolic regulation of invadopodia and the invasive process by de novo fatty acid synthesis and lipogenesis. PMID:22238651

Dendrobium mixture regulates hepatic gluconeogenesis in diabetic rats via the phosphoinositide-3-kinase/protein kinase B signaling pathway.

PubMed

Lin, Xinjun; Shi, Hong; Cui, Yi; Wang, Xiaoning; Zhang, Jieping; Yu, Wenzhen; Wei, Min

2018-07-01

The present study aimed to evaluate the impact of dendrobium mixture (DMix) on the gene and protein expression of insulin signaling pathway-associated factors in the livers of diabetic rats. The molecular mechanisms by which DMix inhibits gluconeogenesis were also investigated. A total of 47 female Wistar rats were used in the present study. Of these, 11 rats were randomly selected as healthy controls and diabetes was induced in the remaining 36 rats by administering a high-fat and high-sugar diet for 6 weeks, followed by two intraperitoneal injections of streptozotocin. The 36 rats were screened for diabetes and then randomly divided into three groups: Model, metformin and DMix groups. Following 12 weeks of treatment, the fasting blood glucose (FBG), glycosylated serum protein (GSP), serum insulin, blood lipids [total cholesterol (Tch) and triglycerides (TG)], alanine transaminase (ALT) and aspartate transaminase (AST) were assessed. In addition, hematoxylin and eosin staining was used for histomorphological examination of the liver tissues. The mRNA expression of insulin receptor (InsR), forkhead box protein O1 (FoxO1), phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase) in the liver was measured with reverse transcription-quantitative polymerase chain reaction and the protein expression of InsR, phosphoinositide-3-kinase (PI3K), phosphorylated (p)-PI3K, protein kinase B (Akt), p-Akt, FoxO1, PEPCK and G6Pase in the liver was measured by western blot analysis. The FBG, GSP, InsR, Tch, TG, ALT and AST levels were significantly lower in the DMix-treated group compared with the model group (P<0.05). In addition, DMix treatment notably improved liver histopathology and significantly increased the gene and protein expression of InsR, PI3K and Akt (P<0.05). DMix treatment also significantly reduced the gene and protein expression of FoxO1, PEPCK and G6Pase (P<0.05). DMix effectively reduced FBG and blood lipids and significantly improved liver

Characterization of PsMPK2, the first C1 subgroup MAP kinase from pea (Pisum sativum L.).

PubMed

Ortiz-Masia, Dolores; Perez-Amador, Miguel A; Carbonell, Pablo; Aniento, Fernando; Carbonell, Juan; Marcote, Maria J

2008-05-01

Mitogen-activated protein kinase (MAPK) cascades play a key role in plant growth and development as well as in biotic and abiotic stress responses. They are classified according to their sequence homology into four major groups (A-D). A large amount of information about MAPKs in groups A and B is available but few data of the C group have been reported. In this study, a C1 subgroup MAP kinase cDNA, PsMPK2, was isolated from Pisum sativum. PsMPK2 is expressed in vegetative (root and leaf) and reproductive (stamen, pistil and fruit) organs. Expression of PsMPK2 in Arabidopsis thaliana shows that mechanical injury and other stress signals as abscisic acid, jasmonic acid and hydrogen peroxide increase its kinase activity, extending previous results indicating that C1 subgroup MAPKs may be involved in the response to stress.

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

PubMed

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

2014-03-01

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

Mutations in CsPID encoding a Ser/Thr protein kinase are responsible for round leaf shape in cucumber (Cucumis sativus L.)

USDA-ARS?s Scientific Manuscript database

Leaf shape is an important plant architecture trait that is affected by plant hormones, especially auxin. In Arabidopsis, PINOID (PID), a regulator for the auxin polar transporter PIN (PIN-FORMED) affects leaf shape formation, but this function of PID in crop plants has not been well studied. From a...

Effect of plant age, larval age, and fertilizer treatment on resistance of a cry1Ab-transformed aromatic rice to lepidopterous stem borers and foliage feeders.

PubMed

Alinia, F; Ghareyazie, B; Rubia, L; Bennett, J; Cohen, M B

2000-04-01

The resistance of vegetative, booting, and flowering stage plants of a variety of an aromatic rice, Oryza sativa L., transformed with a Bacillus thuringiensis Berliner cry1Ab gene under control of the maize phosphoenolpyruvate carboxylase (PEPC) promoter was evaluated against four lepidopterous rice pests--the stem borers Chilo suppressalis (Walker) (Lepidoptera: Crambidae) and Scirpophaga incertulas (Walker) (Lepidoptera: Pyralidae), and the foliage feeders Cnaphalocrocis medinalis Guenée (Lepidoptera: Pyralidae) and Naranga aenescens Moore (Lepidoptera: Noctuidae). Plants of the cry1Ab-transformed line (no. 827) were more resistant to young larvae of S. incertulas, C. suppressalis, and C. medinalis than control plants at the vegetative stage but not at the flowering stage. Survival of 10-d-old stem borer larvae did not differ on cry1Ab plants and control plants at either the vegetative or flowering stage, but the development of 10-d-old C. suppressalis larvae was retarded on the vegetative stage cry1Ab plants. Immunological analysis also showed an apparent decline in Cry1Ab titer in leaf blades and leaf sheaths at the reproductive stage. In experiments comparing three fertilizer treatments (NPK, PK, and none), there was a significant interaction between fertilizer treatment and variety on larval survival only in whole-plant assays at booting stage with C. suppressalis. On cry1Ab plants, larval survival did not differ significantly among the three fertilizer levels, whereas on control plants survival was highest with the NPK treatment. cry1Ab plants tested at the sixth and seventh generations after transformation were more resistant than control plants to N. aenescens and C. suppressalis, respectively, suggesting that gene silencing will not occur in line 827. The results of the experiments are discussed in terms of resistance management for B. thuringiensis toxins in rice.

Timing Is Everything: Highly Specific and Transient Expression of a MAP Kinase Determines Auxin-Induced Leaf Venation Patterns in Arabidopsis

PubMed Central

Stanko, Vera; Giuliani, Concetta; Retzer, Katarzyna; Djamei, Armin; Wahl, Vanessa; Wurzinger, Bernhard; Wilson, Cathal; Heberle-Bors, Erwin; Teige, Markus; Kragler, Friedrich

2014-01-01

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules present in all eukaryotes. In plants, MAPK cascades were shown to regulate cell division, developmental processes, stress responses, and hormone pathways. The subgroup A of Arabidopsis MAPKs consists of AtMPK3, AtMPK6, and AtMPK10. AtMPK3 and AtMPK6 are activated by their upstream MAP kinase kinases (MKKs) AtMKK4 and AtMKK5 in response to biotic and abiotic stress. In addition, they were identified as key regulators of stomatal development and patterning. AtMPK10 has long been considered as a pseudo-gene, derived from a gene duplication of AtMPK6. Here we show that AtMPK10 is expressed highly but very transiently in seedlings and at sites of local auxin maxima leaves. MPK10 encodes a functional kinase and interacts with the upstream MAP kinase kinase (MAPKK) AtMKK2. mpk10 mutants are delayed in flowering in long-day conditions and in continuous light. Moreover, cotyledons of mpk10 and mkk2 mutants have reduced vein complexity, which can be reversed by inhibiting polar auxin transport (PAT). Auxin does not affect AtMPK10 expression while treatment with the PAT inhibitor HFCA extends the expression in leaves and reverses the mpk10 mutant phenotype. These results suggest that the AtMKK2–AtMPK10 MAPK module regulates venation complexity by altering PAT efficiency. PMID:25064848

Response of Leaf Water Potential, Stomatal Resistance, and Leaf Rolling to Water Stress

PubMed Central

O'Toole, John C.; Cruz, Rolando T.

1980-01-01

Numerous studies have associated increased stomatal resistance with response to water deficit in cereals. However, consideration of change in leaf form seems to have been neglected. The response of adaxial and abaxial stomatal resistance and leaf rolling in rice to decreasing leaf water potential was investigated. Two rice cultivars were subjected to control and water stress treatments in a deep (1-meter) aerobic soil. Concurrent measurements of leaf water potential, stomatal resistance, and degree of leaf rolling were made through a 29-day period after cessation of irrigation. Kinandang Patong, an upland adapted cultivar, maintained higher dawn and midday leaf water potential than IR28, a hybrid selected in irrigated conditions. This was not explained by differences in leaf diffusive resistance or leaf rolling, and is assumed to result from a difference in root system extent. Stomatal resistance increased more on the abaxial than the adaxial leaf surface in both cultivars. This was associated with a change in leaf form or rolling inward of the upper leaf surface. Both responses, increased stomatal resistance and leaf rolling, were initiated in a similar leaf water potential range (−8 to −12 bars). Leaves of IR28 became fully rolled at leaf water potential of about −22 bars; however, total leaf diffusive resistance was only about 4 to 5 seconds per centimeter (conductance 0.25 to 0.2 centimeter per second) at that stage. Leaf diffusive resistance and degree of leaf rolling were linearly related to leaf water potential. Thus, leaf rolling in rice may be used as an estimate of the other two less obvious effects of water deficit. PMID:16661206

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

PubMed

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

2004-01-01

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

"Breath figures" on leaf surfaces-formation and effects of microscopic leaf wetness.

PubMed

Burkhardt, Juergen; Hunsche, Mauricio

2013-01-01

"Microscopic leaf wetness" means minute amounts of persistent liquid water on leaf surfaces which are invisible to the naked eye. The water is mainly maintained by transpired water vapor condensing onto the leaf surface and to attached leaf surface particles. With an estimated average thickness of less than 1 μm, microscopic leaf wetness is about two orders of magnitude thinner than morning dewfall. The most important physical processes which reduce the saturation vapor pressure and promote condensation are cuticular absorption and the deliquescence of hygroscopic leaf surface particles. Deliquescent salts form highly concentrated solutions. Depending on the type and concentration of the dissolved ions, the physicochemical properties of microscopic leaf wetness can be considerably different from those of pure water. Microscopic leaf wetness can form continuous thin layers on hydrophobic leaf surfaces and in specific cases can act similar to surfactants, enabling a strong potential influence on the foliar exchange of ions. Microscopic leaf wetness can also enhance the dissolution, the emission, and the reaction of specific atmospheric trace gases e.g., ammonia, SO2, or ozone, leading to a strong potential role for microscopic leaf wetness in plant/atmosphere interaction. Due to its difficult detection, there is little knowledge about the occurrence and the properties of microscopic leaf wetness. However, based on the existing evidence and on physicochemical reasoning it can be hypothesized that microscopic leaf wetness occurs on almost any plant worldwide and often permanently, and that it significantly influences the exchange processes of the leaf surface with its neighboring compartments, i.e., the plant interior and the atmosphere. The omission of microscopic water in general leaf wetness concepts has caused far-reaching, misleading conclusions in the past.

Protoplast isolation, transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid gene expression analysis.

PubMed

Nanjareddy, Kalpana; Arthikala, Manoj-Kumar; Blanco, Lourdes; Arellano, Elizabeth S; Lara, Miguel

2016-06-24

Phaseolus vulgaris is one of the most extensively studied model legumes in the world. The P. vulgaris genome sequence is available; therefore, the need for an efficient and rapid transformation system is more imperative than ever. The functional characterization of P. vulgaris genes is impeded chiefly due to the non-amenable nature of Phaseolus sp. to stable genetic transformation. Transient transformation systems are convenient and versatile alternatives for rapid gene functional characterization studies. Hence, the present work focuses on standardizing methodologies for protoplast isolation from multiple tissues and transient transformation protocols for rapid gene expression analysis in the recalcitrant grain legume P. vulgaris. Herein, we provide methodologies for the high-throughput isolation of leaf mesophyll-, flower petal-, hypocotyl-, root- and nodule-derived protoplasts from P. vulgaris. The highly efficient polyethylene glycol-mannitol magnesium (PEG-MMG)-mediated transformation of leaf mesophyll protoplasts was optimized using a GUS reporter gene. We used the P. vulgaris SNF1-related protein kinase 1 (PvSnRK1) gene as proof of concept to demonstrate rapid gene functional analysis. An RT-qPCR analysis of protoplasts that had been transformed with PvSnRK1-RNAi and PvSnRK1-OE vectors showed the significant downregulation and ectopic constitutive expression (overexpression), respectively, of the PvSnRK1 transcript. We also demonstrated an improved transient transformation approach, sonication-assisted Agrobacterium-mediated transformation (SAAT), for the leaf disc infiltration of P. vulgaris. Interestingly, this method resulted in a 90 % transformation efficiency and transformed 60-85 % of the cells in a given area of the leaf surface. The constitutive expression of YFP further confirmed the amenability of the system to gene functional characterization studies. We present simple and efficient methodologies for protoplast isolation from multiple P

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

PubMed Central

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

1980-01-01

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

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

PubMed Central

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

1993-01-01

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

Green tissue-specific co-expression of chitinase and oxalate oxidase 4 genes in rice for enhanced resistance against sheath blight.

PubMed

Karmakar, Subhasis; Molla, Kutubuddin Ali; Chanda, Palas K; Sarkar, Sailendra Nath; Datta, Swapan K; Datta, Karabi

2016-01-01

Green tissue-specific simultaneous overexpression of two defense-related genes ( OsCHI11 & OsOXO4 ) in rice leads to significant resistance against sheath blight pathogen ( R. solani ) without distressing any agronomically important traits. Overexpressing two defense-related genes (OsOXO4 and OsCHI11) cloned from rice is effective at enhancing resistance against sheath blight caused by Rhizoctonia solani. These genes were expressed under the control of two different green tissue-specific promoters, viz. maize phosphoenolpyruvate carboxylase gene promoter, PEPC, and rice cis-acting 544-bp DNA element, immediately upstream of the D54O translational start site, P D54O-544 . Putative T0 transgenic rice plants were screened by PCR and integration of genes was confirmed by Southern hybridization of progeny (T1) rice plants. Successful expression of OsOXO4 and OsCHI11 in all tested plants was confirmed. Expression of PR genes increased significantly following pathogen infection in overexpressing transgenic plants. Following infection, transgenic plants exhibited elevated hydrogen peroxide levels, significant changes in activity of ROS scavenging enzymes and reduced membrane damage when compared to their wild-type counterpart. In a Rhizoctonia solani toxin assay, a detached leaf inoculation test and an in vivo plant bioassay, transgenic plants showed a significant reduction in disease symptoms in comparison to non-transgenic control plants. This is the first report of overexpression of two different PR genes driven by two green tissue-specific promoters providing enhanced sheath blight resistance in transgenic rice.

Heterologous expression of key C and N metabolic enzymes improves re-assimilation of photorespired CO2 and NH3, and growth.

PubMed

Kaachra, Anish; Vats, Surender Kumar; Kumar, Sanjay

2018-06-11

We investigated the effect of heterologous expression of phosphoenolpyruvate carboxylase (ZmPepcase), aspartate aminotransferase (GmAspAT), and glutamine synthetase (NtGS) on carbon (C) and nitrogen (N) metabolism in Arabidopsis (Arabidopsis thaliana). These transgenes were expressed either separately or in different combinations. The highest gains in shoot dry weight were observed in transgenic lines co-expressing all three genes. Tracer experiments using NaH14CO3 suggested that the co-expression of ZmPepcase, GmAspAT,and NtGS resulted in a higher flux of assimilated CO2 towards sugars and amino acids. Upon feeding the leaf discs with glycine-1-14C, transgenic lines evolved significantly lower 14CO2 levels than the WT, suggesting a higher re-assimilation of CO2 evolved during photorespiration. Leaves of transgenic plants accumulated significantly lower ammonium without any significant difference in the levels of photorespiratory ammonium relative to the WT, suggesting higher re-assimilation of photorespired NH3. Transgenic lines also showed improved photosynthetic rates, higher shoot biomass accumulation, and improved seed yield in comparison to WT plants under both optimum and limiting N conditions. The present work demonstrates that the heterologous co-expression of ZmPepcase, GmAspAT, and NtGS reduced the photorespiratory loss of C and N with concomitant enhancements in shoot biomass and seed yield. {copyright, serif} 2018 American Society of Plant Biologists. All rights reserved.

Ammonium intensifies CAM photosynthesis and counteracts drought effects by increasing malate transport and antioxidant capacity in Guzmania monostachia.

PubMed

Pereira, Paula Natália; Gaspar, Marília; Smith, J Andrew C; Mercier, Helenice

2018-04-09

Guzmania monostachia (Bromeliaceae) is a tropical epiphyte capable of up-regulating crassulacean acid metabolism (CAM) in its photosynthetic tissues in response to changing nutrient and water availability. Previous studies have shown that under drought there is a gradient of increasing CAM expression from the basal (youngest) to the apical (oldest) portion of the leaves, and additionally that nitrogen deficiency can further increase CAM intensity in the leaf apex of this bromeliad. The present study investigated the inter-relationships between nitrogen source (nitrate and/or ammonium) and water deficit in regulating CAM expression in G. monostachia leaves. The highest CAM activity was observed under ammonium nutrition in combination with water deficit. This was associated with enhanced activity of the key enzyme phosphoenolpyruvate carboxylase, elevated rates of ATP- and PPi-dependent proton transport at the vacuolar membrane in the presence of malate, and increased transcript levels of the vacuolar malate channel-encoding gene, ALMT. Water deficit was consistently associated with higher levels of total soluble sugars, which were maximal under ammonium nutrition, as were the activities of several antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase). Thus, ammonium nutrition, whilst associated with the highest degree of CAM induction in G. monostachia, also mitigates the effects of water deficit by osmotic adjustment and can limit oxidative damage in the leaves of this bromeliad under conditions that may be typical of its epiphytic habitat.

Mesophyll conductance in Zea mays responds transiently to CO2 availability: implications for transpiration efficiency in C4 crops.

PubMed

Kolbe, Allison R; Cousins, Asaph B

2018-03-01

Mesophyll conductance (g m ) describes the movement of CO 2 from the intercellular air spaces below the stomata to the site of initial carboxylation in the mesophyll. In contrast with C 3 -g m , little is currently known about the intraspecific variation in C 4 -g m or its responsiveness to environmental stimuli. To address these questions, g m was measured on five maize (Zea mays) lines in response to CO 2 , employing three different estimates of g m . Each of the methods indicated a significant response of g m to CO 2 . Estimates of g m were similar between methods at ambient and higher CO 2 , but diverged significantly at low partial pressures of CO 2 . These differences are probably driven by incomplete chemical and isotopic equilibrium between CO 2 and bicarbonate under these conditions. Carbonic anhydrase and phosphoenolpyruvate carboxylase in vitro activity varied significantly despite similar values of g m and leaf anatomical traits. These results provide strong support for a CO 2 response of g m in Z. mays, and indicate that g m in maize is probably driven by anatomical constraints rather than by biochemical limitations. The CO 2 response of g m indicates a potential role for facilitated diffusion in C 4 -g m . These results also suggest that water-use efficiency could be enhanced in C 4 species by targeting g m . © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

PubMed Central

Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

2005-01-01

The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

Crystal Structure of Biotin Carboxylase in Complex with Substrates and Implications for Its Catalytic Mechanism

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

Chou, C.; Yu, L; Tong, L

2009-01-01

Biotin-dependent carboxylases are widely distributed in nature and have important functions in many cellular processes. These enzymes share a conserved biotin carboxylase (BC) component, which catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as the donor. Despite the availability of a large amount of biochemical and structural information on BC, the molecular basis for its catalysis is currently still poorly understood. We report here the crystal structure at 2.0 {angstrom} resolution of wild-type Escherichia coli BC in complex with its substrates biotin, bicarbonate, and Mg-ADP. The structure suggests that Glu{sup 296} is the general base that extracts the proton frommore » bicarbonate, and Arg{sup 338} is the residue that stabilizes the enolate biotin intermediate in the carboxylation reaction. The B domain of BC is positioned closer to the active site, leading to a 2-{angstrom} shift in the bound position of the adenine nucleotide and bringing it near the bicarbonate for catalysis. One of the oxygen atoms of bicarbonate is located in the correct position to initiate the nucleophilic attack on ATP to form the carboxyphosphate intermediate. This oxygen is also located close to the N1' atom of biotin, providing strong evidence that the phosphate group, derived from decomposition of carboxyphosphate, is the general base that extracts the proton on this N1' atom. The structural observations are supported by mutagenesis and kinetic studies. Overall, this first structure of BC in complex with substrates offers unprecedented insights into the molecular mechanism for the catalysis by this family of enzymes.« less

Evidence against translational repression by the carboxyltransferase component of Escherichia coli acetyl coenzyme A carboxylase.

PubMed

Smith, Alexander C; Cronan, John E

2014-11-01

In Escherichia coli, synthesis of the malonyl coenzyme A (malonyl-CoA) required for membrane lipid synthesis is catalyzed by acetyl-CoA carboxylase, a large complex composed of four subunits. The subunit proteins are needed in a defined stoichiometry, and it remains unclear how such production is achieved since the proteins are encoded at three different loci. Meades and coworkers (G. Meades, Jr., B. K. Benson, A. Grove, and G. L. Waldrop, Nucleic Acids Res. 38:1217-1227, 2010, doi:http://dx.doi.org/10.1093/nar/gkp1079) reported that coordinated production of the AccA and AccD subunits is due to a translational repression mechanism exerted by the proteins themselves. The AccA and AccD subunits form the carboxyltransferase (CT) heterotetramer that catalyzes the second partial reaction of acetyl-CoA carboxylase. Meades et al. reported that CT tetramers bind the central portions of the accA and accD mRNAs and block their translation in vitro. However, long mRNA molecules (500 to 600 bases) were required for CT binding, but such long mRNA molecules devoid of ribosomes seemed unlikely to exist in vivo. This, plus problematical aspects of the data reported by Meades and coworkers, led us to perform in vivo experiments to test CT tetramer-mediated translational repression of the accA and accD mRNAs. We report that increased levels of CT tetramer have no detectable effect on translation of the CT subunit mRNAs. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Evidence against Translational Repression by the Carboxyltransferase Component of Escherichia coli Acetyl Coenzyme A Carboxylase

PubMed Central

Smith, Alexander C.

2014-01-01

In Escherichia coli, synthesis of the malonyl coenzyme A (malonyl-CoA) required for membrane lipid synthesis is catalyzed by acetyl-CoA carboxylase, a large complex composed of four subunits. The subunit proteins are needed in a defined stoichiometry, and it remains unclear how such production is achieved since the proteins are encoded at three different loci. Meades and coworkers (G. Meades, Jr., B. K. Benson, A. Grove, and G. L. Waldrop, Nucleic Acids Res. 38:1217–1227, 2010, doi:http://dx.doi.org/10.1093/nar/gkp1079) reported that coordinated production of the AccA and AccD subunits is due to a translational repression mechanism exerted by the proteins themselves. The AccA and AccD subunits form the carboxyltransferase (CT) heterotetramer that catalyzes the second partial reaction of acetyl-CoA carboxylase. Meades et al. reported that CT tetramers bind the central portions of the accA and accD mRNAs and block their translation in vitro. However, long mRNA molecules (500 to 600 bases) were required for CT binding, but such long mRNA molecules devoid of ribosomes seemed unlikely to exist in vivo. This, plus problematical aspects of the data reported by Meades and coworkers, led us to perform in vivo experiments to test CT tetramer-mediated translational repression of the accA and accD mRNAs. We report that increased levels of CT tetramer have no detectable effect on translation of the CT subunit mRNAs. PMID:25157077

Ixodes scapularis Tick Cells Control Anaplasma phagocytophilum Infection by Increasing the Synthesis of Phosphoenolpyruvate from Tyrosine.

PubMed

Cabezas-Cruz, Alejandro; Espinosa, Pedro J; Obregón, Dasiel A; Alberdi, Pilar; de la Fuente, José

2017-01-01

The obligate intracellular pathogen, Anaplasma phagocytophilum , is the causative agent of life-threatening diseases in humans and animals. A. phagocytophilum is an emerging tick-borne pathogen in the United States, Europe, Africa and Asia, with increasing numbers of infected people and animals every year. It is increasingly recognized that intracellular pathogens modify host cell metabolic pathways to increase infection and transmission in both vertebrate and invertebrate hosts. Recent reports have shown that amino acids are central to the host-pathogen metabolic interaction. In this study, a genome-wide search for components of amino acid metabolic pathways was performed in Ixodes scapularis , the main tick vector of A. phagocytophilum in the United States, for which the genome was recently published. The enzymes involved in the synthesis and degradation pathways of the twenty amino acids were identified. Then, the available transcriptomics and proteomics data was used to characterize the mRNA and protein levels of I. scapularis amino acid metabolic pathway components in response to A. phagocytophilum infection of tick tissues and ISE6 tick cells. Our analysis was focused on the interplay between carbohydrate and amino acid metabolism during A. phagocytophilum infection in ISE6 cells. The results showed that tick cells increase the synthesis of phosphoenolpyruvate (PEP) from tyrosine to control A. phagocytophilum infection. Metabolic pathway analysis suggested that this is achieved by (i) increasing the transcript and protein levels of mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M), (ii) shunting tyrosine into the tricarboxylic acid (TCA) cycle to increase fumarate and oxaloacetate which will be converted into PEP by PEPCK-M, and (iii) blocking all the pathways that use PEP downstream gluconeogenesis (i.e., de novo serine synthesis pathway (SSP), glyceroneogenesis and gluconeogenesis). While sequestering host PEP may be critical for this bacterium

Activation of Rubisco by activase in leaf extracts

USDA-ARS?s Scientific Manuscript database

The activation of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) is catalyzed by a chaperone called Rubisco activase. Rubisco activation is an early target of moderate heat stress, due largely to the acute sensitivity of Rubisco activase to thermal inactivation. In the past, assay...

Larger temperature response of autumn leaf senescence than spring leaf-out phenology.

PubMed

Fu, Yongshuo H; Piao, Shilong; Delpierre, Nicolas; Hao, Fanghua; Hänninen, Heikki; Liu, Yongjie; Sun, Wenchao; Janssens, Ivan A; Campioli, Matteo

2018-05-01

Climate warming is substantially shifting the leaf phenological events of plants, and thereby impacting on their individual fitness and also on the structure and functioning of ecosystems. Previous studies have largely focused on the climate impact on spring phenology, and to date the processes underlying leaf senescence and their associated environmental drivers remain poorly understood. In this study, experiments with temperature gradients imposed during the summer and autumn were conducted on saplings of European beech to explore the temperature responses of leaf senescence. An additional warming experiment during winter enabled us to assess the differences in temperature responses of spring leaf-out and autumn leaf senescence. We found that warming significantly delayed the dates of leaf senescence both during summer and autumn warming, with similar temperature sensitivities (6-8 days delay per °C warming), suggesting that, in the absence of water and nutrient limitation, temperature may be a dominant factor controlling the leaf senescence in European beech. Interestingly, we found a significantly larger temperature response of autumn leaf senescence than of spring leaf-out. This suggests a possible larger contribution of delays in autumn senescence, than of the advancement in spring leaf-out, to extending the growing season under future warmer conditions. © 2017 John Wiley & Sons Ltd.

The mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK-M) and glucose homeostasis: has it been overlooked?

PubMed Central

Stark, Romana; Kibbey, Richard G.

2013-01-01

Background Plasma glucose levels are tightly regulated within a narrow physiologic range. Insulin-mediated glucose uptake by tissues must be balanced by the appearance of glucose from nutritional sources, glycogen stores, or gluconeogenesis. In this regard, a common pathway regulating both glucose clearance and appearance has not been described. The metabolism of glucose to produce ATP is generally considered to be the primary stimulus for insulin release from beta-cells. Similarly, gluconeogenesis from phosphoenolpyruvate (PEP) is believed to be the primarily pathway via the cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK-C). These models cannot adequately explain the regulation of insulin secretion or gluconeogenesis. Scope of review A metabolic sensing pathway involving mitochondrial GTP (mtGTP) and PEP synthesis by the mitochondrial isoform of PEPCK (PEPCK-M) is associated with glucose-stimulated insulin secretion from pancreatic beta-cells. Here we examine whether there is evidence for a similar mtGTP-dependent pathway involved in gluconeogenesis. In both islets and the liver, mtGTP is produced at the substrate level by the enzyme succinyl CoA synthetase (SCS-GTP) with a rate proportional to the TCA cycle. In the beta-cell PEPCK-M then hydrolyzes mtGTP in the production of PEP that, unlike mtGTP, can escape the mitochondria to generate a signal for insulin release. Similarly, PEPCK-M and mtGTP might also provide a significant source of PEP in gluconeogenic tissues for the production of glucose. This review will focus on the possibility that PEPCK-M, as a sensor for TCA cycle flux, is a key mechanism to regulate both insulin secretion and gluconeogenesis suggesting conservation of this biochemical mechanism in regulating multiple aspects of glucose homeostasis. Moreover, we propose that this mechanism may be more important for regulating insulin secretion and gluconeogenesis compared to canonical nutrient sensing pathways. Major conclusions PEPCK

Alterations in leaf nitrogen metabolism indicated the structural changes of subtropical forest by canopy addition of nitrogen.

PubMed

Liu, Nan; Wang, Jiaxin; Guo, Qinfeng; Wu, Shuhua; Rao, Xingquan; Cai, Xi'an; Lin, Zhifang

2018-09-30

Globally, nitrogen deposition increment has caused forest structural changes due to imbalanced plant nitrogen metabolism and subsequent carbon assimilation. Here, a 2 consecutive-year experiment was conducted to reveal the effects of canopy addition of nitrogen (CAN) on nitrogen absorption, assimilation, and allocation in leaves of three subtropical forest woody species (Castanea henryi, Ardisia quinquegona, and Blastus cochinchinensis). We hypothesized that CAN altered leaf nitrogen absorption, assimilation and partitioning of different plants in different ways in subtropical forest. It shows that CAN increased maximum photosynthetic rate (A max ), photosynthetic nitrogen use efficiency (PNUE), and metabolic protein content of the two understory species A. quinquegona and B. cochinchinensis. By contrary, for the overstory species, C. henryi, A max , PNUE, and metabolic protein content were significantly reduced in response to CAN. We found that changes in leaf nitrogen metabolism were mainly due to the differences in enzyme (e.g. Ribulose-1,5-bisphosphate carboxylase, nitrate reductase, nitrite reductase and glutamine synthetase) activities under CAN treatment. Our results indicated that C. henryi may be more susceptible to CAN treatment, and both A. quinquegona and B. cochinchinensis could better adapt to CAN treatment but in different ways. Our findings may partially explain the ongoing degradation of subtropical forest into a community dominated by small trees and shrubs in recent decades. It is possible that persistent high levels of atmospheric nitrogen deposition will lead to the steady replacement of dominant woody species in this subtropical forest. Copyright © 2018 Elsevier Inc. All rights reserved.

Sequence analyses and evolutionary relationships among the energy-coupling proteins Enzyme I and HPr of the bacterial phosphoenolpyruvate: sugar phosphotransferase system.

PubMed Central

Reizer, J.; Hoischen, C.; Reizer, A.; Pham, T. N.; Saier, M. H.

1993-01-01

We have previously reported the overexpression, purification, and biochemical properties of the Bacillus subtilis Enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) (Reizer, J., et al., 1992, J. Biol. Chem. 267, 9158-9169). We now report the sequencing of the ptsI gene of B. subtilis encoding Enzyme I (570 amino acids and 63,076 Da). Putative transcriptional regulatory signals are identified, and the pts operon is shown to be subject to carbon source-dependent regulation. Multiple alignments of the B. subtilis Enzyme I with (1) six other sequenced Enzymes I of the PTS from various bacterial species, (2) phosphoenolpyruvate synthase of Escherichia coli, and (3) bacterial and plant pyruvate: phosphate dikinases (PPDKs) revealed regions of sequence similarity as well as divergence. Statistical analyses revealed that these three types of proteins comprise a homologous family, and the phylogenetic tree of the 11 sequenced protein members of this family was constructed. This tree was compared with that of the 12 sequence HPr proteins or protein domains. Antibodies raised against the B. subtilis and E. coli Enzymes I exhibited immunological cross-reactivity with each other as well as with PPDK of Bacteroides symbiosus, providing support for the evolutionary relationships of these proteins suggested from the sequence comparisons. Putative flexible linkers tethering the N-terminal and the C-terminal domains of protein members of the Enzyme I family were identified, and their potential significance with regard to Enzyme I function is discussed. The codon choice pattern of the B. subtilis and E. coli ptsI and ptsH genes was found to exhibit a bias toward optimal codons in these organisms.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7686067

Unraveling the evolutionary history of the phosphoryl-transfer chain of the phosphoenolpyruvate:phosphotransferase system through phylogenetic analyses and genome context

PubMed Central

2008-01-01

Background The phosphoenolpyruvate phosphotransferase system (PTS) plays a major role in sugar transport and in the regulation of essential physiological processes in many bacteria. The PTS couples solute transport to its phosphorylation at the expense of phosphoenolpyruvate (PEP) and it consists of general cytoplasmic phosphoryl transfer proteins and specific enzyme II complexes which catalyze the uptake and phosphorylation of solutes. Previous studies have suggested that the evolution of the constituents of the enzyme II complexes has been driven largely by horizontal gene transfer whereas vertical inheritance has been prevalent in the general phosphoryl transfer proteins in some bacterial groups. The aim of this work is to test this hypothesis by studying the evolution of the phosphoryl transfer proteins of the PTS. Results We have analyzed the evolutionary history of the PTS phosphoryl transfer chain (PTS-ptc) components in 222 complete genomes by combining phylogenetic methods and analysis of genomic context. Phylogenetic analyses alone were not conclusive for the deepest nodes but when complemented with analyses of genomic context and functional information, the main evolutionary trends of this system could be depicted. Conclusion The PTS-ptc evolved in bacteria after the divergence of early lineages such as Aquificales, Thermotogales and Thermus/Deinococcus. The subsequent evolutionary history of the PTS-ptc varied in different bacterial lineages: vertical inheritance and lineage-specific gene losses mainly explain the current situation in Actinobacteria and Firmicutes whereas horizontal gene transfer (HGT) also played a major role in Proteobacteria. Most remarkably, we have identified a HGT event from Firmicutes or Fusobacteria to the last common ancestor of the Enterobacteriaceae, Pasteurellaceae, Shewanellaceae and Vibrionaceae. This transfer led to extensive changes in the metabolic and regulatory networks of these bacteria including the development of a

Vaccinium angustifolium (lowbush blueberry) leaf extract increases extravillous trophoblast cell migration and invasion in vitro.

PubMed

Ly, Christina; Ferrier, Jonathan; Gaudet, Jeremiah; Yockell-Lelièvre, Julien; Arnason, John Thor; Gruslin, Andrée; Bainbridge, Shannon

2018-04-01

Perturbations to extravillous trophoblast (EVT) cell migration and invasion are associated with the development of placenta-mediated diseases. Phytochemicals found in the lowbush blueberry plant (Vaccinium angustifolium) have been shown to influence cell migration and invasion in models of tumorigenesis and noncancerous, healthy cells, however never in EVT cells. We hypothesized that the phenolic compounds present in V. angustifolium leaf extract promote trophoblast migration and invasion. Using the HTR-8/SVneo human EVT cell line and Boyden chamber assays, the influence of V. angustifolium leaf extract (0 to 2 × 10 4  ng/ml) on trophoblast cell migration (n = 4) and invasion (n = 4) was determined. Cellular proliferation and viability were assessed using immunoreactivity to Ki67 (n = 3) and trypan blue exclusion assays (n = 3), respectively. At 20 ng/ml, V. angustifolium leaf extract increased HTR-8/SVneo cell migration and invasion (p < .01) and did not affect cell proliferation or viability. Chlorogenic acid was identified as a major phenolic compound of the leaf extract and the most active compound. Evidence from Western blot analysis (n = 3) suggests that the effects of the leaf extract and chlorogenic acid on trophoblast migration and invasion are mediated through an adenosine monophosphate-activated protein (AMP) kinase-dependent mechanism. Further investigations examining the potential therapeutic applications of this natural health product extract and its major chemical compounds in the context of placenta-mediated diseases are warranted. Copyright © 2018 John Wiley & Sons, Ltd.

Increasing leaf hydraulic conductance with transpiration rate minimizes the water potential drawdown from stem to leaf

PubMed Central

Simonin, Kevin A.; Burns, Emily; Choat, Brendan; Barbour, Margaret M.; Dawson, Todd E.; Franks, Peter J.

2015-01-01

Leaf hydraulic conductance (k leaf) is a central element in the regulation of leaf water balance but the properties of k leaf remain uncertain. Here, the evidence for the following two models for k leaf in well-hydrated plants is evaluated: (i) k leaf is constant or (ii) k leaf increases as transpiration rate (E) increases. The difference between stem and leaf water potential (ΔΨstem–leaf), stomatal conductance (g s), k leaf, and E over a diurnal cycle for three angiosperm and gymnosperm tree species growing in a common garden, and for Helianthus annuus plants grown under sub-ambient, ambient, and elevated atmospheric CO2 concentration were evaluated. Results show that for well-watered plants k leaf is positively dependent on E. Here, this property is termed the dynamic conductance, k leaf(E), which incorporates the inherent k leaf at zero E, which is distinguished as the static conductance, k leaf(0). Growth under different CO2 concentrations maintained the same relationship between k leaf and E, resulting in similar k leaf(0), while operating along different regions of the curve owing to the influence of CO2 on g s. The positive relationship between k leaf and E minimized variation in ΔΨstem–leaf. This enables leaves to minimize variation in Ψleaf and maximize g s and CO2 assimilation rate over the diurnal course of evaporative demand. PMID:25547915

The impact of ions on allosteric functions in human liver pyruvate kinase

PubMed Central

Alontaga, Aileen Y.

2010-01-01

Experimental designs used to monitor the magnitude of an allosteric response can greatly influence observed values. We report here the impact of buffer, monovalent cation, divalent cation and anion on the magnitude of the allosteric regulation of the affinity of human liver pyruvate kinase (hL-PYK) for substrate, phosphoenolpyruvate (PEP). The magnitudes of the allosteric activation by fructose-1,6-bisphosphate (Fru-1,6-BP) and the allosteric inhibition by alanine are independent of most, but not all buffers tested. However, these magnitudes are dependent on whether Mg2+ or Mn2+ is included as the divalent cation. In the presence of Mn2+, any change in Kapp-PEP caused by Fru-1,6-BP is minimal. hL-PYK activity does not appear to require monovalent cation. Monovalent cation binding in the active site impacts PEP affinity with minimum influence on the magnitude of allosteric coupling. However, Na+ and Li+ reduce the magnitude of the allosteric response to Fru-1,6-BP, likely due to mechanisms outside of the active site. Which anion is used to maintain a constant monovalent cation concentration also influences the magnitude of the allosteric response. The value of determining the impact of ions on allosteric function can be appreciated by considering that representative structures used in comparative studies have often been determined using protein crystals grown in diverse buffer and salt conditions. PMID:21609859

Difference in C3–C4 metabolism underlies tradeoff between growth rate and biomass yield in Methylobacterium extorquens AM1

DOE PAGES

Fu, Yanfen; Beck, David A. C.; Lidstrom, Mary E.

2016-07-19

In this study, two variants of Methylobacterium extorquens AM1 demonstrated a trade-off between growth rate and biomass yield. In addition, growth rate and biomass yield were also affected by supplementation of growth medium with different amounts of cobalt. The metabolism changes relating to these growth phenomena as well as the trade-off were investigated in this study. 13C metabolic flux analysis was used to generate a detailed central carbon metabolic flux map with both absolute and normalized flux values. As a result, the major differences between the two variants occurred at the formate node as well as within C3-C4 inter-conversion pathways.more » Higher relative fluxes through formyltetrahydrofolate ligase, phosphoenolpyruvate carboxylase, and malic enzyme led to higher biomass yield, while higher relative fluxes through pyruvate kinase and pyruvate dehydrogenase led to higher growth rate. These results were then tested by phenotypic studies on three mutants (null pyk, null pck mutant and null dme mutant) in both variants, which agreed with the model prediction. In this study, 13C metabolic flux analysis for two strain variants of M. extorquens AM1 successfully identified metabolic pathways contributing to the trade-off between cell growth and biomass yield. Phenotypic analysis of mutants deficient in corresponding genes supported the conclusion that C3-C4 inter-conversion strategies were the major response to the trade-off.« less

Difference in C3–C4 metabolism underlies tradeoff between growth rate and biomass yield in Methylobacterium extorquens AM1

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

Fu, Yanfen; Beck, David A. C.; Lidstrom, Mary E.

In this study, two variants of Methylobacterium extorquens AM1 demonstrated a trade-off between growth rate and biomass yield. In addition, growth rate and biomass yield were also affected by supplementation of growth medium with different amounts of cobalt. The metabolism changes relating to these growth phenomena as well as the trade-off were investigated in this study. 13C metabolic flux analysis was used to generate a detailed central carbon metabolic flux map with both absolute and normalized flux values. As a result, the major differences between the two variants occurred at the formate node as well as within C3-C4 inter-conversion pathways.more » Higher relative fluxes through formyltetrahydrofolate ligase, phosphoenolpyruvate carboxylase, and malic enzyme led to higher biomass yield, while higher relative fluxes through pyruvate kinase and pyruvate dehydrogenase led to higher growth rate. These results were then tested by phenotypic studies on three mutants (null pyk, null pck mutant and null dme mutant) in both variants, which agreed with the model prediction. In this study, 13C metabolic flux analysis for two strain variants of M. extorquens AM1 successfully identified metabolic pathways contributing to the trade-off between cell growth and biomass yield. Phenotypic analysis of mutants deficient in corresponding genes supported the conclusion that C3-C4 inter-conversion strategies were the major response to the trade-off.« less

Long term leaf phenology and leaf exchange strategies of a cerrado savanna community

NASA Astrophysics Data System (ADS)

de Camargo, Maria Gabriela G.; Costa Alberton, Bruna; de Carvalho, Gustavo H.; Magalhães, Paula A. N. R.; Morellato, Leonor Patrícia C.

2017-04-01

Leaf development and senescence cycles are linked to a range of ecosystem processes, affecting seasonal patterns of atmosphere-ecosystem carbon and energy exchanges, resource availability and nutrient cycling. The degree of deciduousness of tropical trees and communities depend on ecosystems characteristics such as amount of biomass, species diversity and the strength and length of the dry season. Besides defining the growing season, deciduousness can also be an indicator of species response to climate changes in the tropics, mainly because severity of dry season can intensify leaf loss. Based on seven-years of phenological observations (2005 to 2011) we describe the long-term patterns of leafing phenology of a Brazilian cerrado savanna, aiming to (i) identify leaf exchange strategies of species, quantifying the degree of deciduousness, and verify whether these strategies vary among years depending on the length and strength of the dry seasons; (ii) define the growing seasons along the years and the main drivers of leaf flushing in the cerrado. We analyzed leafing patterns of 107 species and classified 69 species as deciduous (11 species), semi-deciduous (29) and evergreen (29). Leaf exchange was markedly seasonal, as expected for seasonal tropical savannas. Leaf fall predominated in the dry season, peaking in July, and leaf flushing in the transition between dry to wet seasons, peaking in September. Leafing patterns were similar among years with the growing season starting at the end of dry season, in September, for most species. However, leaf exchange strategies varied among years for most species (65%), except for evergreen strategy, mainly constant over years. Leafing patterns of cerrado species were strongly constrained by rainfall. The length of the dry season and rainfall intensity were likely affecting the individuals' leaf exchange strategies and suggesting a differential resilience of species to changes of rainfall regime, predicted on future global

Evaluation of Methane from Sisal Leaf Residue and Palash Leaf Litter

NASA Astrophysics Data System (ADS)

Arisutha, S.; Baredar, P.; Deshpande, D. M.; Suresh, S.

2014-12-01

The aim of this study is to evaluate methane production from sisal leaf residue and palash leaf litter mixed with different bulky materials such as vegetable market waste, hostel kitchen waste and digested biogas slurry in a laboratory scale anaerobic reactor. The mixture was prepared with 1:1 proportion. Maximum methane content of 320 ml/day was observed in the case of sisal leaf residue mixed with vegetable market waste as the feed. Methane content was minimum (47 ml/day), when palash leaf litter was used as feed. This was due to the increased content of lignin and polyphenol in the feedstock which were of complex structure and did not get degraded directly by microorganisms. Sisal leaf residue mixtures also showed highest content of volatile fatty acids (VFAs) as compared to palash leaf litter mixtures. It was observed that VFA concentration in the digester first increased, reached maximum (when pH was minimum) and then decreased.

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

PubMed Central

Tomimatsu, Yoshio; Donovan, John W.

1981-01-01

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

The pH dependence of the allosteric response of human liver pyruvate kinase to fructose-1,6-bisphosphate, ATP, and alanine

PubMed Central

Fenton, Aron W.; Hutchinson, Myra

2009-01-01

The allosteric regulation of human liver pyruvate kinase (hL-PYK) by fructose-1,6-bisphosphate (Fru-1,6-BP; activator), ATP (inhibitor) and alanine (Ala; inhibitor) was monitored over a pH range from 6.5 to 8.0 at 37°C. As a function of increasing pH, hL-PYK's affinity for the substrate phosphoenolpyruvate (PEP), and for Fru-1,6-BP decreases, while affinities for ATP and Ala slightly increases. At pH 6.5, Fru-1,6-BP and ATP elicit only small allosteric impacts on PEP affinity. As pH increases, Fru-1,6-BP and ATP elicit greater allosteric responses, but the response to Ala is relatively constant. Since the magnitudes of the allosteric coupling for ATP and for Ala inhibition are different and the pH dependences of these magnitudes are not similar, these inhibitors likely elicit their responses using different molecular mechanisms. In addition, our results fail to support a general correlation between pH dependent changes in effector affinity and pH dependent changes in the corresponding allosteric response. PMID:19467627

Drought effects on leaf abscission and leaf production in Populus clones

Treesearch

Stephen G. Pallardy; Julie L. Rhoads

1997-01-01

Leaf abscission and foliation responses to water stress were studied in potted plants of five Populus clones grown in a greenhouse. As predawn leaf water potential (Ψ1) fell to -3 MPa, drought-induced leaf abscission increased progressively to 30% for data pooled across clones. As predawn Ψ1...

7 CFR 29.3033 - Leaf.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 2 2013-01-01 2013-01-01 false Leaf. 29.3033 Section 29.3033 Agriculture Regulations... Leaf. Whole, unstemmed leaf. Leaf, when applied to tobacco in strip form, shall describe the divided unit of a whole leaf. [49 FR 16758, Apr. 20, 1984] ...

7 CFR 29.3033 - Leaf.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 2 2011-01-01 2011-01-01 false Leaf. 29.3033 Section 29.3033 Agriculture Regulations... Leaf. Whole, unstemmed leaf. Leaf, when applied to tobacco in strip form, shall describe the divided unit of a whole leaf. [49 FR 16758, Apr. 20, 1984] ...

7 CFR 29.3033 - Leaf.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 2 2012-01-01 2012-01-01 false Leaf. 29.3033 Section 29.3033 Agriculture Regulations... Leaf. Whole, unstemmed leaf. Leaf, when applied to tobacco in strip form, shall describe the divided unit of a whole leaf. [49 FR 16758, Apr. 20, 1984] ...

7 CFR 29.3033 - Leaf.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 2 2014-01-01 2014-01-01 false Leaf. 29.3033 Section 29.3033 Agriculture Regulations... Leaf. Whole, unstemmed leaf. Leaf, when applied to tobacco in strip form, shall describe the divided unit of a whole leaf. [49 FR 16758, Apr. 20, 1984] ...

7 CFR 29.3033 - Leaf.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false Leaf. 29.3033 Section 29.3033 Agriculture Regulations... Leaf. Whole, unstemmed leaf. Leaf, when applied to tobacco in strip form, shall describe the divided unit of a whole leaf. [49 FR 16758, Apr. 20, 1984] ...

Seasonal Changes in Leaf Area of Amazon Forests from Leaf Flushing and Abscission

NASA Astrophysics Data System (ADS)

Samanta, A.; Knyazikhin, Y.; Xu, L.; Dickinson, R.; Fu, R.; Costa, M. H.; Ganguly, S.; Saatchi, S. S.; Nemani, R. R.; Myneni, R.

2011-12-01

A large increase in near-infrared (NIR) reflectance of Amazon forests during the light-rich dry season and a corresponding decrease during the light-poor wet season has been observed in satellite measurements. This has been variously interpreted as seasonal changes in leaf area resulting from net leaf flushing in the dry season and net leaf abscission in the wet season, enhanced photosynthetic activity during the dry season from flushing new leaves and as change in leaf scattering and absorption properties between younger and older leaves covered with epiphylls. Reconciling these divergent views using theory and observations is the goal of this article. The observed changes in NIR reflectance of Amazon forests could be due to similar, but small, changes in NIR leaf albedo (reflectance plus transmittance) only, from exchanging older leaves with newer ones, with total leaf area unchanged. However, this argument ignores accumulating evidence from ground-based studies of higher leaf area in the dry season relative to the wet season, seasonal changes in litterfall and does not satisfactorily explain why NIR reflectance of these forests decreases in the wet season. A more convincing explanation for the observed increase in NIR reflectance during the dry season and decrease during the wet season is one that invokes changes in both leaf area and leaf optical properties. Such an argument is consistent with known phonological behavior of tropical forests, ground-based reports of seasonal changes in leaf area, litterfall, leaf optical properties and fluxes of evapotranspiration, and thus, reconciles the various seemingly divergent views.

Seasonal changes in leaf area of Amazon forests from leaf flushing and abscission

NASA Astrophysics Data System (ADS)

Samanta, Arindam; Knyazikhin, Yuri; Xu, Liang; Dickinson, Robert E.; Fu, Rong; Costa, Marcos H.; Saatchi, Sassan S.; Nemani, Ramakrishna R.; Myneni, Ranga B.

2012-03-01

A large increase in near-infrared (NIR) reflectance of Amazon forests during the light-rich dry season and a corresponding decrease during the light-poor wet season has been observed in satellite measurements. This increase has been variously interpreted as seasonal change in leaf area resulting from net leaf flushing in the dry season or net leaf abscission in the wet season, enhanced photosynthetic activity during the dry season from flushing new leaves and as change in leaf scattering and absorption properties between younger and older leaves covered with epiphylls. Reconciling these divergent views using theory and observations is the goal of this article. The observed changes in NIR reflectance of Amazon forests could be due to similar, but small, changes in NIR leaf albedo (reflectance plus transmittance) resulting from the exchange of older leaves for newer ones, but with the total leaf area unchanged. However, this argument ignores accumulating evidence from ground-based reports of higher leaf area in the dry season than the wet season, seasonal changes in litterfall and does not satisfactorily explain why NIR reflectance of these forests decreases in the wet season. More plausibly, the increase in NIR reflectance during the dry season and the decrease during the wet season would result from changes in both leaf area and leaf optical properties. Such change would be consistent with known phenological behavior of tropical forests, ground-based reports of seasonal changes in leaf area, litterfall, leaf optical properties and fluxes of evapotranspiration, and thus, would reconcile the various seemingly divergent views.

Leaf Relative Water Content Estimated from Leaf Reflectance and Transmittance

NASA Technical Reports Server (NTRS)

Vanderbilt, Vern; Daughtry, Craig; Dahlgren, Robert

2016-01-01

Remotely sensing the water status of plants and the water content of canopies remain long term goals of remote sensing research. In the research we report here, we used optical polarization techniques to monitor the light reflected from the leaf interior, R, as well as the leaf transmittance, T, as the relative water content (RWC) of corn (Zea mays) leaves decreased. Our results show that R and T both change nonlinearly. The result show that the nonlinearities cancel in the ratio R/T, which appears linearly related to RWC for RWC less than 90%. The results suggest that potentially leaf water status and perhaps even canopy water status could be monitored starting from leaf and canopy optical measurements.

Leaf phenomics: a systematic reverse genetic screen for Arabidopsis leaf mutants.

PubMed

Wilson-Sánchez, David; Rubio-Díaz, Silvia; Muñoz-Viana, Rafael; Pérez-Pérez, José Manuel; Jover-Gil, Sara; Ponce, María Rosa; Micol, José Luis

2014-09-01

The study and eventual manipulation of leaf development in plants requires a thorough understanding of the genetic basis of leaf organogenesis. Forward genetic screens have identified hundreds of Arabidopsis mutants with altered leaf development, but the genome has not yet been saturated. To identify genes required for leaf development we are screening the Arabidopsis Salk Unimutant collection. We have identified 608 lines that exhibit a leaf phenotype with full penetrance and almost constant expressivity and 98 additional lines with segregating mutant phenotypes. To allow indexing and integration with other mutants, the mutant phenotypes were described using a custom leaf phenotype ontology. We found that the indexed mutation is present in the annotated locus for 78% of the 553 mutants genotyped, and that in half of these the annotated T-DNA is responsible for the phenotype. To quickly map non-annotated T-DNA insertions, we developed a reliable, cost-effective and easy method based on whole-genome sequencing. To enable comprehensive access to our data, we implemented a public web application named PhenoLeaf (http://genetics.umh.es/phenoleaf) that allows researchers to query the results of our screen, including text and visual phenotype information. We demonstrated how this new resource can facilitate gene function discovery by identifying and characterizing At1g77600, which we found to be required for proximal-distal cell cycle-driven leaf growth, and At3g62870, which encodes a ribosomal protein needed for cell proliferation and chloroplast function. This collection provides a valuable tool for the study of leaf development, characterization of biomass feedstocks and examination of other traits in this fundamental photosynthetic organ. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Kranz and single-cell forms of C4 plants in the subfamily Suaedoideae show kinetic C4 convergence for PEPC and Rubisco with divergent amino acid substitutions

PubMed Central

Rosnow, Josh J.; Evans, Marc A.; Kapralov, Maxim V.; Cousins, Asaph B.; Edwards, Gerald E.; Roalson, Eric H.

2015-01-01

The two carboxylation reactions performed by phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) are vital in the fixation of inorganic carbon for C4 plants. The abundance of PEPC is substantially elevated in C4 leaves, while the location of Rubisco is restricted to one of two chloroplast types. These differences compared with C3 leaves have been shown to result in convergent enzyme optimization in some C4 species. Investigation into the kinetic properties of PEPC and Rubisco from Kranz C4, single cell C4, and C3 species in Chenopodiaceae s. s. subfamily Suaedoideae showed that these major carboxylases in C4 Suaedoideae species lack the same mutations found in other C4 systems which have been examined; but still have similar convergent kinetic properties. Positive selection analysis on the N-terminus of PEPC identified residues 364 and 368 to be under positive selection with a posterior probability >0.99 using Bayes empirical Bayes. Compared with previous analyses on other C4 species, PEPC from C4 Suaedoideae species have different convergent amino acids that result in a higher K m for PEP and malate tolerance compared with C3 species. Kinetic analysis of Rubisco showed that C4 species have a higher catalytic efficiency of Rubisco (k catc in mol CO2 mol–1 Rubisco active sites s–1), despite lacking convergent substitutions in the rbcL gene. The importance of kinetic changes to the two-carboxylation reactions in C4 leaves related to amino acid selection is discussed. PMID:26417023

7 CFR 29.3525 - Leaf.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 2 2014-01-01 2014-01-01 false Leaf. 29.3525 Section 29.3525 Agriculture Regulations... Type 95) § 29.3525 Leaf. Whole, unstemmed leaf. Leaf, when applied to tobacco in strip form, shall describe the divided unit of a whole leaf. [49 FR 16759, Apr. 20, 1984] ...

7 CFR 29.3525 - Leaf.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 2 2012-01-01 2012-01-01 false Leaf. 29.3525 Section 29.3525 Agriculture Regulations... Type 95) § 29.3525 Leaf. Whole, unstemmed leaf. Leaf, when applied to tobacco in strip form, shall describe the divided unit of a whole leaf. [49 FR 16759, Apr. 20, 1984] ...

7 CFR 29.3525 - Leaf.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 2 2011-01-01 2011-01-01 false Leaf. 29.3525 Section 29.3525 Agriculture Regulations... Type 95) § 29.3525 Leaf. Whole, unstemmed leaf. Leaf, when applied to tobacco in strip form, shall describe the divided unit of a whole leaf. [49 FR 16759, Apr. 20, 1984] ...

7 CFR 29.3525 - Leaf.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false Leaf. 29.3525 Section 29.3525 Agriculture Regulations... Type 95) § 29.3525 Leaf. Whole, unstemmed leaf. Leaf, when applied to tobacco in strip form, shall describe the divided unit of a whole leaf. [49 FR 16759, Apr. 20, 1984] ...

7 CFR 29.3525 - Leaf.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 2 2013-01-01 2013-01-01 false Leaf. 29.3525 Section 29.3525 Agriculture Regulations... Type 95) § 29.3525 Leaf. Whole, unstemmed leaf. Leaf, when applied to tobacco in strip form, shall describe the divided unit of a whole leaf. [49 FR 16759, Apr. 20, 1984] ...

Regulation of Ca(2+)/calmodulin-dependent protein kinase kinase alpha by cAMP-dependent protein kinase: I. Biochemical analysis.

PubMed

Okuno, S; Kitani, T; Fujisawa, H

2001-10-01

Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) I and IV are activated upon phosphorylation of their Thr(177) and Thr(196), respectively, by the upstream Ca(2+)/calmodulin-dependent protein kinases CaM-kinase kinase alpha and beta, and deactivated upon dephosphorylation by protein phosphatases such as CaM-kinase phosphatase. Recent studies demonstrated that the activity of CaM-kinase kinase alpha is decreased upon phosphorylation by cAMP-dependent protein kinase (PKA), and the relationship between the inhibition and phosphorylation of CaM-kinase kinase alpha by PKA has been studied. In the present study, we demonstrate that the activity of CaM-kinase kinase alpha toward PKIV peptide, which contains the sequence surrounding Thr(196) of CaM-kinase IV, is increased by incubation with PKA in the presence of Ca(2+)/calmodulin but decreased in its absence, while the activity toward CaM-kinase IV is decreased by incubation with PKA in both the presence and absence of Ca(2+)/calmodulin. Six phosphorylation sites on CaM-kinase kinase alpha, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA, were identified by amino acid sequence analysis of the phosphopeptides purified from the tryptic digest of the phosphorylated enzymes. The presence of Ca(2+)/calmodulin suppresses phosphorylation on Ser(52), Ser(74), Thr(108), and Ser(458) by PKA, but accelerates phosphorylation on Ser(475). The changes in the activity of the enzyme upon phosphorylation appear to occur as a result of conformational changes induced by phosphorylation on several sites.

Leaf habit and woodiness regulate different leaf economy traits at a given nutrient supply.

PubMed

Ordoñez, Jenny C; van Bodegom, Peter M; Witte, Jan-Philip M; Bartholomeus, Ruud P; van Dobben, Han F; Aerts, Rien

2010-11-01

The large variation in the relationships between environmental factors and plant traits observed in natural communities exemplifies the alternative solutions that plants have developed in response to the same environmental limitations. Qualitative attributes, such as growth form, woodiness, and leaf habit can be used to approximate these alternative solutions. Here, we quantified the extent to which these attributes affect leaf trait values at a given resource supply level, using measured plant traits from 105 different species (254 observations) distributed across 50 sites in mesic to wet plant communities in The Netherlands. For each site, soil total N, soil total P, and water supply estimates were obtained by field measurements and modeling. Effects of growth forms, woodiness, and leaf habit on relations between leaf traits (SLA, specific leaf area; LNC, leaf nitrogen concentration; and LPC, leaf phosphorus concentration) vs. nutrient and water supply were quantified using maximum-likelihood methods and Bonferroni post hoc tests. The qualitative attributes explained 8-23% of the variance within sites in leaf traits vs. soil fertility relationships, and therefore they can potentially be used to make better predictions of global patterns of leaf traits in relation to nutrient supply. However, at a given soil fertility, the strength of the effect of each qualitative attribute was not the same for all leaf traits. These differences may imply a differential regulation of the leaf economy traits at a given nutrient supply, in which SLA and LPC seem to be regulated in accordance to changes in plant size and architecture while LNC seems to be primarily regulated at the leaf level by factors related to leaf longevity.

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

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

Vera, P.; Conejero, V.

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

Autoregulation of kinase dephosphorylation by ATP binding in AGC protein kinases.

PubMed

Chan, Tung O; Pascal, John M; Armen, Roger S; Rodeck, Ulrich

2012-02-01

AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non- ATP-competitive kinase inhibitors that discriminate within and between protein kinase families.

7 CFR 29.2528 - Leaf.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false Leaf. 29.2528 Section 29.2528 Agriculture Regulations...-Cured Tobacco (u.s. Types 22, 23, and Foreign Type 96) § 29.2528 Leaf. Whole, unstemmed leaf. Leaf, when applied to tobacco in strip form, shall describe the divided unit of a whole leaf. [49 FR 16757, Apr. 20...

7 CFR 29.1028 - Leaf.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false Leaf. 29.1028 Section 29.1028 Agriculture Regulations... Type 92) § 29.1028 Leaf. Whole, unstemmed leaf. Leaf, when applied to tobacco in strip form, shall describe the divided unit of a whole leaf. [49 FR 16755, Apr. 20, 1984. Redesignated at 51 FR 25027, July...

7 CFR 29.2528 - Leaf.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 2 2011-01-01 2011-01-01 false Leaf. 29.2528 Section 29.2528 Agriculture Regulations...-Cured Tobacco (u.s. Types 22, 23, and Foreign Type 96) § 29.2528 Leaf. Whole, unstemmed leaf. Leaf, when applied to tobacco in strip form, shall describe the divided unit of a whole leaf. [49 FR 16757, Apr. 20...

7 CFR 29.2528 - Leaf.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 2 2012-01-01 2012-01-01 false Leaf. 29.2528 Section 29.2528 Agriculture Regulations...-Cured Tobacco (u.s. Types 22, 23, and Foreign Type 96) § 29.2528 Leaf. Whole, unstemmed leaf. Leaf, when applied to tobacco in strip form, shall describe the divided unit of a whole leaf. [49 FR 16757, Apr. 20...

7 CFR 29.1028 - Leaf.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 2 2013-01-01 2013-01-01 false Leaf. 29.1028 Section 29.1028 Agriculture Regulations... Type 92) § 29.1028 Leaf. Whole, unstemmed leaf. Leaf, when applied to tobacco in strip form, shall describe the divided unit of a whole leaf. [49 FR 16755, Apr. 20, 1984. Redesignated at 51 FR 25027, July...

7 CFR 29.1028 - Leaf.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 2 2012-01-01 2012-01-01 false Leaf. 29.1028 Section 29.1028 Agriculture Regulations... Type 92) § 29.1028 Leaf. Whole, unstemmed leaf. Leaf, when applied to tobacco in strip form, shall describe the divided unit of a whole leaf. [49 FR 16755, Apr. 20, 1984. Redesignated at 51 FR 25027, July...

7 CFR 29.1028 - Leaf.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 2 2011-01-01 2011-01-01 false Leaf. 29.1028 Section 29.1028 Agriculture Regulations... Type 92) § 29.1028 Leaf. Whole, unstemmed leaf. Leaf, when applied to tobacco in strip form, shall describe the divided unit of a whole leaf. [49 FR 16755, Apr. 20, 1984. Redesignated at 51 FR 25027, July...

7 CFR 29.1028 - Leaf.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 2 2014-01-01 2014-01-01 false Leaf. 29.1028 Section 29.1028 Agriculture Regulations... Type 92) § 29.1028 Leaf. Whole, unstemmed leaf. Leaf, when applied to tobacco in strip form, shall describe the divided unit of a whole leaf. [49 FR 16755, Apr. 20, 1984. Redesignated at 51 FR 25027, July...

7 CFR 29.2528 - Leaf.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 2 2013-01-01 2013-01-01 false Leaf. 29.2528 Section 29.2528 Agriculture Regulations...-Cured Tobacco (u.s. Types 22, 23, and Foreign Type 96) § 29.2528 Leaf. Whole, unstemmed leaf. Leaf, when applied to tobacco in strip form, shall describe the divided unit of a whole leaf. [49 FR 16757, Apr. 20...

7 CFR 29.2528 - Leaf.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 2 2014-01-01 2014-01-01 false Leaf. 29.2528 Section 29.2528 Agriculture Regulations...-Cured Tobacco (u.s. Types 22, 23, and Foreign Type 96) § 29.2528 Leaf. Whole, unstemmed leaf. Leaf, when applied to tobacco in strip form, shall describe the divided unit of a whole leaf. [49 FR 16757, Apr. 20...

Metformin attenuates ovarian cancer cell growth in an AMP-kinase dispensable manner

PubMed Central

Rattan, R; Giri, S; Hartmann, LC; Shridhar, V

2011-01-01

Abstract Metformin, the most widely used drug for type 2 diabetes activates 59 adenosine monophosphate (AMP)-activated protein kinase (AMPK), which regulates cellular energy metabolism. Here, we report that ovarian cell lines VOSE, A2780, CP70, C200, OV202, OVCAR3, SKOV3ip, PE01 and PE04 predominantly express -α1, -β1, -γ1 and -γ2 isoforms of AMPK subunits. Our studies show that metformin treatment (1) significantly inhibited proliferation of diverse chemo-responsive and -resistant ovarian cancer cell lines (A2780, CP70, C200, OV202, OVCAR3, SKVO3ip, PE01 and PE04), (2) caused cell cycle arrest accompanied by decreased cyclin D1 and increased p21 protein expression, (3) activated AMPK in various ovarian cancer cell lines as evident from increased phosphorylation of AMPKα and its downstream substrate; acetyl co-carboxylase (ACC) and enhanced β-oxidation of fatty acid and (4) attenuated mTOR-S6RP phosphorylation, inhibited protein translational and lipid biosynthetic pathways, thus implicating metformin as a growth inhibitor of ovarian cancer cells. We also show that metformin-mediated effect on AMPK is dependent on liver kinase B1 (LKB1) as it failed to activate AMPK-ACC pathway and cell cycle arrest in LKB1 null mouse embryo fibroblasts (mefs). This observation was further supported by using siRNA approach to down-regulate LKB1 in ovarian cancer cells. In contrast, met formin inhibited cell proliferation in both wild-type and AMPKα1/2 null mefs as well as in AMPK silenced ovarian cancer cells. Collectively, these results provide evidence on the role of metformin as an anti-proliferative therapeutic that can act through both AMPK-dependent as well as AMPK-independent pathways. PMID:19874425

Global variability in leaf respiration in relation to climate and leaf traits

NASA Astrophysics Data System (ADS)

Atkin, Owen K.

2015-04-01

Leaf respiration plays a vital role in regulating ecosystem functioning and the Earth's climate. Because of this, it is imperative that that Earth-system, climate and ecosystem-level models be able to accurately predict variations in rates of leaf respiration. In the field of photosynthesis research, the F/vC/B model has enabled modellers to accurately predict variations in photosynthesis through time and space. By contrast, we lack an equivalent biochemical model to predict variations in leaf respiration. Consequently, we need to rely on phenomenological approaches to model variations in respiration across the Earth's surface. Such approaches require that we develop a thorough understanding of how rates of respiration vary among species and whether global environmental gradients play a role in determining variations in leaf respiration. Dealing with these issues requires that data sets be assembled on rates of leaf respiration in biomes across the Earth's surface. In this talk, I will use a newly-assembled global database on leaf respiration and associated traits (including photosynthesis) to highlight variation in leaf respiration (and the balance between respiration and photosynthesis) across global gradients in growth temperature and aridity.

Antidiabetic effect of gomisin N via activation of AMP-activated protein kinase.

PubMed

Jung, Dae Young; Kim, Ji-Hyun; Lee, Hoyoung; Jung, Myeong Ho

2017-12-16

Gomisin N (GN) is a lignan derived from Schisandra chinensis. AMP-activated kinase (AMPK) has gained attention as a therapeutic target for the treatment of metabolic syndrome. Previously, we reported that GN activated the AMPK pathway and ameliorated high-fat diet (HFD)-induced hepatic steatosis. In this study, we investigated the anti-diabetic effects of GN in C2C12 myotubes and HFD obese mice. GN enhanced the phosphorylation of AMPK/acetyl-CoA carboxylase (ACC) and Akt. In addition, GN promoted glucose uptake in C2C12 myotubes, which was accompanied by the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Treatment with compound C, an AMPK inhibitor, suppressed GN-mediated stimulation of glucose uptake. Furthermore, GN increased the expression of mitochondria biogenesis and fatty acid oxidation genes in C2C12 myotubes. In the in vivo study, administration of GN to HFD mice decreased the levels of fasting blood glucose and insulin, and improved glucose tolerance in HFD obese mice. GN administration rescued the decreased phosphorylation of AMPK and Akt and stimulated the expression of mitochondria biogenesis genes in the skeletal muscle of HFD mice. These findings suggested that GN exerted anti-hyperglycemic effects through AMPK activation. Copyright © 2017 Elsevier Inc. All rights reserved.

Autoregulation of kinase dephosphorylation by ATP binding to AGC protein kinases

PubMed Central

Pascal, John M; Armen, Roger S

2012-01-01

AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non-ATP-competitive kinase inhibitors that discriminate within and between protein kinase families. PMID:22262182

Successful synthesis of active human coagulation factor VII by co-expression of mammalian gamma-glutamyl carboxylase and modification of vit.K cycle in Drosophila Schneider S2 cells.

PubMed

Nagahashi, Kotomi; Umemura, Kazuo; Kanayama, Naohiro; Iwaki, Takayuki

2017-04-01

Mammalian gamma-glutamyl carboxylase and reduced vitamin K are indispensable for synthesis of mature mammalian vitamin K dependent proteins including some of blood coagulation factors (factors II, VII, IX, and X). It was well known that Drosophila melanogaster expressed gamma-glutamyl carboxylase and possessed a vit.K cycle although native substrates for them have not been identified yet. Despite the potential capability of gamma carboxylation in D. melanogaster derived cells such as S2 cells, Drosophila gamma-glutamyl carboxylase failed to gamma carboxylate a peptide fused to the human coagulation factor IX propeptide. Thus, it had been believed that the Drosophila system was not adequate to synthesize mammalian vit.K dependent proteins. Indeed, we previously attempted to synthesize biologically active factor VII in S2 cells although we were not able to obtain it. However, recently, a successful transient expression of biologically active human factor IX from S2 cells was reported. In the present study, several expression vectors which enable expressing mammalian GGCX, VKORC1, and/or PDIA2 along with F7 were developed. S2 cells transfected with pMKA85, pMAK86, and pMAK219 successfully synthesized active FVII. Thus, mammalian GGCX was indispensable to synthesize active FVII while mammalian VKORC1 and PDIA2 were not critical but supportive factors for S2 cells.

Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia.

PubMed

Roth Flach, Rachel J; Danai, Laura V; DiStefano, Marina T; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K; Bortell, Rita; Alonso, Laura C; Czech, Michael P

2016-07-29

Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural and Biochemical Studies on the Regulation of Biotin Carboxylase by Substrate Inhibition and Dimerization

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

C Chou; L Tong

2011-12-31

Biotin carboxylase (BC) activity is shared among biotin-dependent carboxylases and catalyzes the Mg-ATP-dependent carboxylation of biotin using bicarbonate as the CO{sub 2} donor. BC has been studied extensively over the years by structural, kinetic, and mutagenesis analyses. Here we report three new crystal structures of Escherichia coli BC at up to 1.9 {angstrom} resolution, complexed with different ligands. Two structures are wild-type BC in complex with two ADP molecules and two Ca{sup 2+} ions or two ADP molecules and one Mg{sup 2+} ion. One ADP molecule is in the position normally taken by the ATP substrate, whereas the other ADPmore » molecule occupies the binding sites of bicarbonate and biotin. One Ca{sup 2+} ion and the Mg{sup 2+} ion are associated with the ADP molecule in the active site, and the other Ca{sup 2+} ion is coordinated by Glu-87, Glu-288, and Asn-290. Our kinetic studies confirm that ATP shows substrate inhibition and that this inhibition is competitive against bicarbonate. The third structure is on the R16E mutant in complex with bicarbonate and Mg-ADP. Arg-16 is located near the dimer interface. The R16E mutant has only a 2-fold loss in catalytic activity compared with the wild-type enzyme. Analytical ultracentrifugation experiments showed that the mutation significantly destabilized the dimer, although the presence of substrates can induce dimer formation. The binding modes of bicarbonate and Mg-ADP are essentially the same as those to the wild-type enzyme. However, the mutation greatly disrupted the dimer interface and caused a large re-organization of the dimer. The structures of these new complexes have implications for the catalysis by BC.« less

Structural and Biochemical Studies on the Regulation of Biotin Carboxylase by Substrate Inhibition and Dimerization

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

Chou, Chi-Yuan; Tong, Liang

2012-06-19

Biotin carboxylase (BC) activity is shared among biotin-dependent carboxylases and catalyzes the Mg-ATP-dependent carboxylation of biotin using bicarbonate as the CO{sub 2} donor. BC has been studied extensively over the years by structural, kinetic, and mutagenesis analyses. Here we report three new crystal structures of Escherichia coli BC at up to 1.9 {angstrom} resolution, complexed with different ligands. Two structures are wild-type BC in complex with two ADP molecules and two Ca{sup 2+} ions or two ADP molecules and one Mg{sup 2+} ion. One ADP molecule is in the position normally taken by the ATP substrate, whereas the other ADPmore » molecule occupies the binding sites of bicarbonate and biotin. One Ca{sup 2+} ion and the Mg{sup 2+} ion are associated with the ADP molecule in the active site, and the other Ca{sup 2+} ion is coordinated by Glu-87, Glu-288, and Asn-290. Our kinetic studies confirm that ATP shows substrate inhibition and that this inhibition is competitive against bicarbonate. The third structure is on the R16E mutant in complex with bicarbonate and Mg-ADP. Arg-16 is located near the dimer interface. The R16E mutant has only a 2-fold loss in catalytic activity compared with the wild-type enzyme. Analytical ultracentrifugation experiments showed that the mutation significantly destabilized the dimer, although the presence of substrates can induce dimer formation. The binding modes of bicarbonate and Mg-ADP are essentially the same as those to the wild-type enzyme. However, the mutation greatly disrupted the dimer interface and caused a large re-organization of the dimer. The structures of these new complexes have implications for the catalysis by BC.« less

Stomatal Density Influences Leaf Water and Leaf Wax D/H Values in Arabidopsis

NASA Astrophysics Data System (ADS)

Lee, H.; Feakins, S. J.; Sternberg, L. O.

2014-12-01

The hydrogen isotopic composition (δD) of plant leaf wax is a powerful tool to study the hydrology of past and present environments. The δD value of leaf waxes is known to primarily reflect the δD value of source water, modified by biological fractionations commonly summarized as the 'net or apparent' fractionation. It remains a challenge, however, to quantitatively relate the isotopic composition of the end product (wax) back to that of the precursor (water) because multiple isotope effects contributing to the net fractionation are not yet well understood. Transgenic variants have heretofore unexplored potential to isolate individual isotope effects. Here we report the first hydrogen isotopic measurements from transgenic Arabidopsis thaliana plants with calculations of leaf water enrichment, net and biosynthetic fractionation values from measured δD of plant waters and leaf wax n-alkanes. We employed transgenic Arabidopsis leaves, engineered to have different stomatal density, by differential expression of the stomatal growth hormone stomagen. Comparison of variants and wild types allow us to isolate the effects of stomatal density on leaf water and the net fractionation expressed by leaf wax biomarkers. Results show that transgenic leaves with denser pores have more enriched leaf water and leaf wax δD values than wild type and even more so than transgenic leaves with sparse stomata (difference of 10 ‰). Our findings that stomatal density controls leaf water and leaf wax δD values adds insights into the cause of variations in net fractionations between species, as well as suggesting that geological variations in stomatal density may modulate the sedimentary leaf wax δD record. In nature, stomatal density varies between species and environments, and all other factors being equal, this will contribute to variations in fractionations observed. Over geological history, lower stomatal densities occur at times of elevated pCO2; our findings predict reduced leaf

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

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

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

Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates

PubMed Central

Xue, Liang; Wang, Wen-Horng; Iliuk, Anton; Hu, Lianghai; Galan, Jacob A.; Yu, Shuai; Hans, Michael; Geahlen, Robert L.; Tao, W. Andy

2012-01-01

Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity. PMID:22451900

Ethanol Extract of Pinus koraiensis Leaf Ameliorates Alcoholic Fatty Liver via the Activation of LKB1-AMPK Signaling In Vitro and In Vivo.

PubMed

Hong, Sang-Hyuk; Lee, Hyemin; Lee, Hyo-Jung; Kim, Bonglee; Nam, Min-Ho; Shim, Bum-Sang; Kim, Sung-Hoon

2017-05-01

Although Pinus koraiensis leaf (PKL) was reported for its anti-diabetes, anti-obesity and anticancer effects as a folk remedy, the inhibitory effect of PKL on alcoholic fatty liver has never been elucidated yet. This study investigated the molecular mechanisms of PKL on alcoholic fatty liver in HepG2 cells, Sprague Dawley (SD) rats and Imprinting Control Region (ICR) mice. Pinus koraiensis leaf increased phosphorylation of liver kinase B1 (LKB1)/AMP-activated protein kinase signaling, low-density lipoprotein receptor and decreased fatty acid biosynthesis-related proteins such as sterol regulatory element-binding protein 1c, fatty acid synthase, 3-hydroxy-3-methylglutaryl-CoA reductase in HepG2 cells. In SD rats with 25% alcohol-induced fatty liver, PKL suppressed the levels of aspartate aminotransferase and triglyceride and also enhanced the activities of antioxidant enzymes including superoxide dismutase, glutathione peroxidase and glutathione s-transferase compared with untreated control. Furthermore, PKL increased serum alcohol dehydrogenase and serum aldehyde dehydrogenase, but decreased serum alcohol concentration in ICR mice after alcohol administration. Consistently, histochemical analysis revealed that PKL attenuated alcohol-induced fatty liver in SD rats. Overall, these findings suggest that PKL ameliorates alcohol-induced fatty liver via activation of LKB1-AMP-activated protein kinase and modulation of proteins related to lipogenesis synthesis, cholesterol synthesis and fatty acid oxidation. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Maize YABBY Genes drooping leaf1 and drooping leaf2 Regulate Plant Architecture[OPEN

PubMed Central

Briggs, Sarah; Bradbury, Peter J.

2017-01-01

Leaf architecture directly influences canopy structure, consequentially affecting yield. We discovered a maize (Zea mays) mutant with aberrant leaf architecture, which we named drooping leaf1 (drl1). Pleiotropic mutations in drl1 affect leaf length and width, leaf angle, and internode length and diameter. These phenotypes are enhanced by natural variation at the drl2 enhancer locus, including reduced expression of the drl2-Mo17 allele in the Mo17 inbred. A second drl2 allele, produced by transposon mutagenesis, interacted synergistically with drl1 mutants and reduced drl2 transcript levels. The drl genes are required for proper leaf patterning, development and cell proliferation of leaf support tissues, and for restricting auricle expansion at the midrib. The paralogous loci encode maize CRABS CLAW co-orthologs in the YABBY family of transcriptional regulators. The drl genes are coexpressed in incipient and emergent leaf primordia at the shoot apex, but not in the vegetative meristem or stem. Genome-wide association studies using maize NAM-RIL (nested association mapping-recombinant inbred line) populations indicated that the drl loci reside within quantitative trait locus regions for leaf angle, leaf width, and internode length and identified rare single nucleotide polymorphisms with large phenotypic effects for the latter two traits. This study demonstrates that drl genes control the development of key agronomic traits in maize. PMID:28698237

Leaf anatomy mediates coordination of leaf hydraulic conductance and mesophyll conductance to CO2 in Oryza.

PubMed

Xiong, Dongliang; Flexas, Jaume; Yu, Tingting; Peng, Shaobing; Huang, Jianliang

2017-01-01

Leaf hydraulic conductance (K leaf ) and mesophyll conductance (g m ) both represent major constraints to photosynthetic rate (A), and previous studies have suggested that K leaf and g m is correlated in leaves. However, there is scarce empirical information about their correlation. In this study, K leaf , leaf hydraulic conductance inside xylem (K x ), leaf hydraulic conductance outside xylem (K ox ), A, stomatal conductance (g s ), g m , and anatomical and structural leaf traits in 11 Oryza genotypes were investigated to elucidate the correlation of H 2 O and CO 2 diffusion inside leaves. All of the leaf functional and anatomical traits varied significantly among genotypes. K leaf was not correlated with the maximum theoretical stomatal conductance calculated from stomatal dimensions (g smax ), and neither g s nor g smax were correlated with K x . Moreover, K ox was linearly correlated with g m and both were closely related to mesophyll structural traits. These results suggest that K leaf and g m are related to leaf anatomical and structural features, which may explain the mechanism for correlation between g m and K leaf . © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Rewiring the reductive tricarboxylic acid pathway and L-malate transport pathway of Aspergillus oryzae for overproduction of L-malate.

PubMed

Liu, Jingjing; Xie, Zhipeng; Shin, Hyun-Dong; Li, Jianghua; Du, Guocheng; Chen, Jian; Liu, Long

2017-07-10

Aspergillus oryzae finds wide application in the food, feed, and wine industries, and is an excellent cell factory platform for production of organic acids. In this work, we achieved the overproduction of L-malate by rewiring the reductive tricarboxylic acid (rTCA) pathway and L-malate transport pathway of A. oryzae NRRL 3488. First, overexpression of native pyruvate carboxylase and malate dehydrogenase in the rTCA pathway improved the L-malate titer from 26.1gL -1 to 42.3gL -1 in shake flask culture. Then, the oxaloacetate anaplerotic reaction was constructed by heterologous expression of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase from Escherichia coli, increasing the L-malate titer to 58.5gL -1 . Next, the export of L-malate from the cytoplasm to the external medium was strengthened by overexpression of a C4-dicarboxylate transporter gene from A. oryzae and an L-malate permease gene from Schizosaccharomyces pombe, improving the L-malate titer from 58.5gL -1 to 89.5gL -1 . Lastly, guided by transcription analysis of the expression profile of key genes related to L-malate synthesis, the 6-phosphofructokinase encoded by the pfk gene was identified as a potential limiting step for L-malate synthesis. Overexpression of pfk with the strong sodM promoter increased the L-malate titer to 93.2gL -1 . The final engineered A. oryzae strain produced 165gL -1 L-malate with a productivity of 1.38gL -1 h -1 in 3-L fed-batch culture. Overall, we constructed an efficient L-malate producer by rewiring the rTCA pathway and L-malate transport pathway of A. oryzae NRRL 3488, and the engineering strategy adopted here may be useful for the construction of A. oryzae cell factories to produce other organic acids. Copyright © 2017 Elsevier B.V. All rights reserved.

A Constrained Maximization Model for inspecting the impact of leaf shape on optimal leaf size and stoma resistance

NASA Astrophysics Data System (ADS)

Ding, J.; Johnson, E. A.; Martin, Y. E.

2017-12-01

Leaf is the basic production unit of plants. Water is the most critical resource of plants. Its availability controls primary productivity of plants by affecting leaf carbon budget. To avoid the damage of cavitation from lowering vein water potential t caused by evapotranspiration, the leaf must increase the stomatal resistance to reduce evapotranspiration rate. This comes at the cost of reduced carbon fixing rate as increasing stoma resistance meanwhile slows carbon intake rate. Studies suggest that stoma will operate at an optimal resistance to maximize the carbon gain with respect to water. Different plant species have different leaf shapes, a genetically determined trait. Further, on the same plant leaf size can vary many times in size that is related to soil moisture, an indicator of water availability. According to metabolic scaling theory, increasing leaf size will increase total xylem resistance of vein, which may also constrain leaf carbon budget. We present a Constrained Maximization Model of leaf (leaf CMM) that incorporates metabolic theory into the coupling of evapotranspiration and carbon fixation to examine how leaf size, stoma resistance and maximum net leaf primary productivity change with petiole xylem water potential. The model connects vein network structure to leaf shape and use the difference between petiole xylem water potential and the critical minor vein cavitation forming water potential as the budget. The CMM shows that both maximum net leaf primary production and optimal leaf size increase with petiole xylem water potential while optimal stoma resistance decreases. Narrow leaf has overall lower optimal leaf size and maximum net leaf carbon gain and higher optimal stoma resistance than those of broad leaf. This is because with small width to length ratio, total xylem resistance increases faster with leaf size. Total xylem resistance of narrow leaf increases faster with leaf size causing higher average and marginal cost of xylem water

On the temporal variation of leaf magnetic parameters: seasonal accumulation of leaf-deposited and leaf-encapsulated particles of a roadside tree crown.

PubMed

Hofman, Jelle; Wuyts, Karen; Van Wittenberghe, Shari; Samson, Roeland

2014-09-15

Understanding the accumulation behaviour of atmospheric particles inside tree leaves is of great importance for the interpretation of biomagnetic monitoring results. In this study, we evaluated the temporal variation of the saturation isothermal remanent magnetisation (SIRM) of leaves of a roadside urban Platanus × acerifolia Willd. tree in Antwerp, Belgium. We hereby examined the seasonal development of the total leaf SIRM signal as well as the leaf-encapsulated fraction of the deposited dust, by washing the leaves before biomagnetic analysis. On average 38% of the leaf SIRM signal was exhibited by the leaf-encapsulated particles. Significant correlations were found between the SIRM and the cumulative daily average atmospheric PM10 and PM2.5 measurements. Moreover, a steady increase of the SIRM throughout the in-leaf season was observed endorsing the applicability of biomagnetic monitoring as a proxy for the time-integrated PM exposure of urban tree leaves. Strongest correlations were obtained for the SIRM of the leaf-encapsulated particles which confirms the dynamic nature of the leaf surface-accumulated particles. Copyright © 2014 Elsevier B.V. All rights reserved.

Seasonal variability of multiple leaf traits captured by leaf spectroscopy at two temperate deciduous forests

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

Yang, Xi; Tang, Jianwu; Mustard, John F.

Understanding the temporal patterns of leaf traits is critical in determining the seasonality and magnitude of terrestrial carbon, water, and energy fluxes. However, we lack robust and efficient ways to monitor the temporal dynamics of leaf traits. Here we assessed the potential of leaf spectroscopy to predict and monitor leaf traits across their entire life cycle at different forest sites and light environments (sunlit vs. shaded) using a weekly sampled dataset across the entire growing season at two temperate deciduous forests. In addition, the dataset includes field measured leaf-level directional-hemispherical reflectance/transmittance together with seven important leaf traits [total chlorophyll (chlorophyllmore » a and b), carotenoids, mass-based nitrogen concentration (N mass), mass-based carbon concentration (C mass), and leaf mass per area (LMA)]. All leaf traits varied significantly throughout the growing season, and displayed trait-specific temporal patterns. We used a Partial Least Square Regression (PLSR) modeling approach to estimate leaf traits from spectra, and found that PLSR was able to capture the variability across time, sites, and light environments of all leaf traits investigated (R 2 = 0.6–0.8 for temporal variability; R 2 = 0.3–0.7 for cross-site variability; R 2 = 0.4–0.8 for variability from light environments). We also tested alternative field sampling designs and found that for most leaf traits, biweekly leaf sampling throughout the growing season enabled accurate characterization of the seasonal patterns. Compared with the estimation of foliar pigments, the performance of N mass, C mass and LMA PLSR models improved more significantly with sampling frequency. Our results demonstrate that leaf spectra-trait relationships vary with time, and thus tracking the seasonality of leaf traits requires statistical models calibrated with data sampled throughout the growing season. In conclusion, our results have broad implications for future

Seasonal variability of multiple leaf traits captured by leaf spectroscopy at two temperate deciduous forests

DOE PAGES

Yang, Xi; Tang, Jianwu; Mustard, John F.; ...

2016-04-02

Understanding the temporal patterns of leaf traits is critical in determining the seasonality and magnitude of terrestrial carbon, water, and energy fluxes. However, we lack robust and efficient ways to monitor the temporal dynamics of leaf traits. Here we assessed the potential of leaf spectroscopy to predict and monitor leaf traits across their entire life cycle at different forest sites and light environments (sunlit vs. shaded) using a weekly sampled dataset across the entire growing season at two temperate deciduous forests. In addition, the dataset includes field measured leaf-level directional-hemispherical reflectance/transmittance together with seven important leaf traits [total chlorophyll (chlorophyllmore » a and b), carotenoids, mass-based nitrogen concentration (N mass), mass-based carbon concentration (C mass), and leaf mass per area (LMA)]. All leaf traits varied significantly throughout the growing season, and displayed trait-specific temporal patterns. We used a Partial Least Square Regression (PLSR) modeling approach to estimate leaf traits from spectra, and found that PLSR was able to capture the variability across time, sites, and light environments of all leaf traits investigated (R 2 = 0.6–0.8 for temporal variability; R 2 = 0.3–0.7 for cross-site variability; R 2 = 0.4–0.8 for variability from light environments). We also tested alternative field sampling designs and found that for most leaf traits, biweekly leaf sampling throughout the growing season enabled accurate characterization of the seasonal patterns. Compared with the estimation of foliar pigments, the performance of N mass, C mass and LMA PLSR models improved more significantly with sampling frequency. Our results demonstrate that leaf spectra-trait relationships vary with time, and thus tracking the seasonality of leaf traits requires statistical models calibrated with data sampled throughout the growing season. In conclusion, our results have broad implications for future

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

PubMed

Ueno, Osamu; Wakayama, Masataka

2004-12-01

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

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

PubMed

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

2017-01-01

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

Target of Rapamycin Regulates Development and Ribosomal RNA Expression through Kinase Domain in Arabidopsis1[W][OA

PubMed Central

Ren, Maozhi; Qiu, Shuqing; Venglat, Prakash; Xiang, Daoquan; Feng, Li; Selvaraj, Gopalan; Datla, Raju

2011-01-01

Target of rapamycin (TOR) is a central regulator of cell growth, cell death, nutrition, starvation, hormone, and stress responses in diverse eukaryotes. However, very little is known about TOR signaling and the associated functional domains in plants. We have taken a genetic approach to dissect TOR functions in Arabidopsis (Arabidopsis thaliana) and report here that the kinase domain is essential for the role of TOR in embryogenesis and 45S rRNA expression. Twelve new T-DNA insertion mutants, spanning 14.2 kb of TOR-encoding genomic region, have been characterized. Nine of these share expression of defective kinase domain and embryo arrest at 16 to 32 cell stage. However, three T-DNA insertion lines affecting FATC domain displayed normal embryo development, indicating that FATC domain was dispensable in Arabidopsis. Genetic complementation showed that the TOR kinase domain alone in tor-10/tor-10 mutant background can rescue early embryo lethality and restore normal development. Overexpression of full-length TOR or kinase domain in Arabidopsis displayed developmental abnormalities in meristem, leaf, root, stem, flowering time, and senescence. We further show that TOR, especially the kinase domain, plays a role in ribosome biogenesis by activating 45S rRNA production. Of the six putative nuclear localization sequences in the kinase domain, nuclear localization sequence 6 was identified to confer TOR nuclear targeting in transient expression assays. Chromatin immunoprecipitation studies revealed that the HEAT repeat domain binds to 45S rRNA promoter and the 5′ external transcribed spacer elements motif. Together, these results show that TOR controls the embryogenesis, postembryonic development, and 45S rRNA production through its kinase domain in Arabidopsis. PMID:21266656

Cell cycle regulation of the BRCA1/acetyl-CoA-carboxylase complex.

PubMed

Ray, H; Suau, F; Vincent, A; Dalla Venezia, N

2009-01-16

Germ-line alterations in BRCA1 are associated with an increased susceptibility to breast and ovarian cancer. The BRCA1 protein has been implicated in multiple cellular functions. We have recently demonstrated that BRCA1 reduces acetyl-CoA-carboxylase alpha (ACCA) activity through its phospho-dependent binding to ACCA, and further established that the phosphorylation of the Ser1263 of ACCA is required for this interaction. Here, to gain more insight into the cellular conditions that trigger the BRCA1/ACCA interaction, we designed an anti-pSer1263 antibody and demonstrated that the Ser1263 of ACCA is phosphorylated in vivo, in a cell cycle-dependent manner. We further showed that the interaction between BRCA1 and ACCA is regulated during cell cycle progression. Taken together, our findings reveal a novel mechanism of regulation of ACCA distinct from the previously described phosphorylation of Ser79, and provide new insights into the control of lipogenesis through the cell cycle.

SU-F-T-350: Continuous Leaf Optimization (CLO) for IMRT Leaf Sequencing

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

Long, T; Chen, M; Jiang, S

Purpose: To study a new step-and-shoot IMRT leaf sequencing model that avoids the two main pitfalls of conventional leaf sequencing: (1) target fluence being stratified into a fixed number of discrete levels and/or (2) aperture leaf positions being restricted to a discrete set of locations. These assumptions induce error into the sequence or reduce the feasible region of potential plans, respectively. Methods: We develop a one-dimensional (single leaf pair) methodology that does not make assumptions (1) or (2) that can be easily extended to a multi-row model. The proposed continuous leaf optimization (CLO) methodology takes in an existing set ofmore » apertures and associated intensities, or solution “seed,” and improves the plan without the restrictiveness of 1or (2). It then uses a first-order descent algorithm to converge onto a locally optimal solution. A seed solution can come from models that assume (1) and (2), thus allowing the CLO model to improve upon existing leaf sequencing methodologies. Results: The CLO model was applied to 208 generated target fluence maps in one dimension. In all cases for all tested sequencing strategies, the CLO model made improvements on the starting seed objective function. The CLO model also was able to keep MUs low. Conclusion: The CLO model can improve upon existing leaf sequencing methods by avoiding the restrictions of (1) and (2). By allowing for more flexible leaf positioning, error can be reduced when matching some target fluence. This study lays the foundation for future models and solution methodologies that can incorporate continuous leaf positions explicitly into the IMRT treatment planning model. Supported by Cancer Prevention & Research Institute of Texas (CPRIT) - ID RP150485.« less

The adenosine triphosphate inhibition of the pyruvate kinase reaction and its dependence on the total magnesium ion concentration

PubMed Central

Holmsen, Holm; Storm, Eva

1969-01-01

1. The effects of ATP, PPi and EDTA on the skeletal-muscle pyruvate kinase reaction at various concentrations of magnesium (where `magnesium' refers to total Mg2+, both free and in the form of complexes) were investigated. The reaction rate was determined as the amount of pyruvate formed in a recorded time of incubation. 2. At 44mm-magnesium the Km values for ADP and phosphoenolpyruvate were unaltered by the presence of ATP up to 6·8mm in systems buffered with either tris–hydrochloric acid or glycylglycine–sodium hydroxide, but the Km values were different in these systems. The Km for one substrate was independent of the concentration of the second substrate. 3. At 10mm-magnesium in the tris–hydrochloric acid system ATP inhibited the reaction competitively with respect to ADP and phosphoenolpyruvate. In the glycylglycine–sodium hydroxide system the inhibition appeared to be non-competitive. At 10mm-magnesium the Km values were lower than at 44mm-magnesium and dependent on the system used. 4. In the tris–hydrochloric acid system the reaction rate rose with increasing magnesium concentration up to a maximum at a concentration 10–20 times that of ADP. Further increase inhibited the reaction and at 44mm-magnesium the rate was 25–50% of its maximum. This inhibition paralleled that produced by increasing trimethylammonium chloride concentrations and was not due to a specific effect of the Mg2+ ion. 5. In the presence of 6·8mm-ATP no reaction occurred below 4–6mm-magnesium, and further increase apparently abolished the inhibition as the reaction rate increased and became equal to those obtained in the absence of ATP at 10–25mm-magnesium. Further increase in magnesium concentration gave reaction rates that were slightly higher in the presence of ATP than in its absence. The maximal rate in the presence of ATP was distinctly lower than in its absence. When 6·8mm-PPi or 6·8mm-EDTA was present the variations in reaction rate with rising magnesium

Are leaf physiological traits related to leaf water isotopic enrichment in restinga woody species?

PubMed

Rosado, Bruno H P; De Mattos, Eduardo A; Sternberg, Leonel Da S L

2013-09-01

During plant-transpiration, water molecules having the lighter stable isotopes of oxygen and hydrogen evaporate and diffuse at a faster rate through the stomata than molecules having the heavier isotopes, which cause isotopic enrichment of leaf water. Although previous models have assumed that leaf water is well-mixed and isotopically uniform, non-uniform stomatal closure, promoting different enrichments between cells, and different pools of water within leaves, due to morpho-physiological traits, might lead to inaccuracies in isotopic models predicting leaf water enrichment. We evaluate the role of leaf morpho-physiological traits on leaf water isotopic enrichment in woody species occurring in a coastal vegetation of Brazil known as restinga. Hydrogen and oxygen stable isotope values of soil, plant stem and leaf water and leaf traits were measured in six species from restinga vegetation during a drought and a wet period. Leaf water isotopic enrichment relative to stem water was more homogeneous among species during the drought in contrast to the wet period suggesting convergent responses to deal to temporal heterogeneity in water availability. Average leaf water isotopic enrichment relative to stem water during the drought period was highly correlated with relative apoplastic water content. We discuss this observation in the context of current models of leaf water isotopic enrichment as a function of the Péclet effect. We suggest that future studies should include relative apoplastic water content in isotopic models.

How do leaf veins influence the worldwide leaf economic spectrum? Review and synthesis.

PubMed

Sack, Lawren; Scoffoni, Christine; John, Grace P; Poorter, Hendrik; Mason, Chase M; Mendez-Alonzo, Rodrigo; Donovan, Lisa A

2013-10-01

Leaf vein traits are implicated in the determination of gas exchange rates and plant performance. These traits are increasingly considered as causal factors affecting the 'leaf economic spectrum' (LES), which includes the light-saturated rate of photosynthesis, dark respiration, foliar nitrogen concentration, leaf dry mass per area (LMA) and leaf longevity. This article reviews the support for two contrasting hypotheses regarding a key vein trait, vein length per unit leaf area (VLA). Recently, Blonder et al. (2011, 2013) proposed that vein traits, including VLA, can be described as the 'origin' of the LES by structurally determining LMA and leaf thickness, and thereby vein traits would predict LES traits according to specific equations. Careful re-examination of leaf anatomy, published datasets, and a newly compiled global database for diverse species did not support the 'vein origin' hypothesis, and moreover showed that the apparent power of those equations to predict LES traits arose from circularity. This review provides a 'flux trait network' hypothesis for the effects of vein traits on the LES and on plant performance, based on a synthesis of the previous literature. According to this hypothesis, VLA, while virtually independent of LMA, strongly influences hydraulic conductance, and thus stomatal conductance and photosynthetic rate. We also review (i) the specific physiological roles of VLA; (ii) the role of leaf major veins in influencing LES traits; and (iii) the role of VLA in determining photosynthetic rate per leaf dry mass and plant relative growth rate. A clear understanding of leaf vein traits provides a new perspective on plant function independently of the LES and can enhance the ability to explain and predict whole plant performance under dynamic conditions, with applications towards breeding improved crop varieties.

Leaf age dependent changes in within-canopy variation in leaf functional traits: a meta-analysis

PubMed Central

Niinemets, Ülo

2018-01-01

Within-canopy variation in leaf structural and photosynthetic characteristics is a major means by which whole canopy photosynthesis is maximized at given total canopy nitrogen. As key acclimatory modifications, leaf nitrogen content (NA) and photosynthetic capacity (AA) per unit area increase with increasing light availability in the canopy and these increases are associated with increases in leaf dry mass per unit area (MA) and/or nitrogen content per dry mass and/or allocation. However, leaf functional characteristics change with increasing leaf age during leaf development and aging, but the importance of these alterations for within-canopy trait gradients is unknown. I conducted a meta-analysis based on 71 canopies that were sampled at different time periods or, in evergreens, included measurements for different-aged leaves to understand how within-canopy variations in leaf traits (trait plasticity) depend on leaf age. The analysis demonstrated that in evergreen woody species, MA and NA plasticity decreased with increasing leaf age, but the change in AA plasticity was less suggesting a certain re-acclimation of AA to altered light. In deciduous woody species, MA and NA gradients in flush-type species increased during leaf development and were almost invariable through the rest of the season, while in continuously leaf-forming species, trait gradients increased constantly with increasing leaf age. In forbs, NA plasticity increased, while in grasses, NA plasticity decreased with increasing leaf age, reflecting life form differences in age-dependent changes in light availability and in nitrogen resorption for growth of generative organs. Although more work is needed to improve the coverage of age-dependent plasticity changes in some plant life forms, I argue that the age-dependent variation in trait plasticity uncovered in this study is large enough to warrant incorporation in simulations of canopy photosynthesis through the growing period. PMID:27033356

Overcoming diabetes-induced hyperglycemia through inhibition of hepatic phosphoenolpyruvate carboxykinase (GTP) with RNAi.

PubMed

Gómez-Valadés, Alicia G; Vidal-Alabró, Anna; Molas, Maria; Boada, Jordi; Bermúdez, Jordi; Bartrons, Ramon; Perales, José C

2006-02-01

Phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) is the rate-controlling enzyme in gluconeogenesis. In diabetic individuals, altered rates of gluconeogenesis are responsible for increased hepatic glucose output and sustained hyperglycemia. Liver-specific inhibition of PEPCK has not been assessed to date as a treatment for diabetes. We have designed a therapeutic, vector-based RNAi approach to induce posttranscriptional gene silencing of hepatic PEPCK using nonviral gene delivery. A transient reduction of PEPCK enzymatic activity (7.6 +/- 0.6 vs 9.7 +/- 1.1 mU/mg, P < 0.05) that correlated with decreased protein content of up to 50% was achieved using this strategy in diabetic mice. PEPCK partial silencing was sufficient to demonstrate lowered blood glucose (218 +/- 26 vs 364 +/- 33 mg/dl, P < 0.001) and improved glucose tolerance together with decreased circulating FFA (0.89 +/- 0.10 vs 1.44 +/- 0.11 mEq/dl, P < 0.001) and TAG (65 +/- 11 vs 102 +/- 16 mg/dl, P < 0.01), in the absence of liver steatosis or lactic acidosis. SREBP1c was down-regulated in PEPCK-silenced animals, suggesting a role for this pathway in the alterations of lipid metabolism. These data reinforce the significance of PEPCK in sustaining diabetes-induced hyperglycemia and validate liver-specific intervention at the level of PEPCK for diabetes gene therapy.

Stereochemical course of the reactions catalyzed by the bacterial phosphoenolpyruvate: Mannitol phosphotransferase system

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

Mueller, E.G.; Knowles, J.R.; Khandekar, S.S.

1990-07-24

The authors have determined the overall stereochemical course of the reactions leading to the phosphorylation of D-mannitol by mannitol-specific enzyme II (EII{sup Mtl}) of the Escherichia coli phosphoenolpyruvate- (PEP) dependent phosphotransferase system (PTS). In the presence of enzyme I and HPr of the PTS, and of membranes containing EII{sup Mtl}, the phospho group from ((R)-{sup 16}O, {sup 17}O, {sup 18}O)PEP was transferred to D-mannitol to form mannitol 1-phosphate with overall inversion of the configuration at phosphorus with respect to that of PEP. Since in the course of these reactions enzyme I and HPr are each covalently phosphorylated at a singlemore » site and inversion of the chiral phospho group from PEP indicates an odd number of transfer steps overall, transfer from phospho-HPr to mannitol via EII{sup Mtl} must also occur in an odd number of steps. Taken together with the fact that catalytically important phospho-EII{sup Mtl} intermediates have been demonstrated biochemically, the results imply that EII{sup Mtl} is sequentially phosphorylated at two different sites during phospho transfer from phospho-HPr to mannitol. This conclusion is consistent with the available evidence on phospho-EII{sup Mtl} intermediates and in particular with the recent report that two different phospho peptides can be isolated from the fully phosphorylated protein.« less

A system dynamics model integrating physiology and biochemical regulation predicts extent of crassulacean acid metabolism (CAM) phases.

PubMed

Owen, Nick A; Griffiths, Howard

2013-12-01

A system dynamics (SD) approach was taken to model crassulacean acid metabolism (CAM) expression from measured biochemical and physiological constants. SD emphasizes state-dependent feedback interaction to describe the emergent properties of a complex system. These mechanisms maintain biological systems with homeostatic limits on a temporal basis. Previous empirical studies on CAM have correlated biological constants (e.g. enzyme kinetic parameters) with expression over the CAM diel cycle. The SD model integrates these constants within the architecture of the CAM 'system'. This allowed quantitative causal connections to be established between biological inputs and the four distinct phases of CAM delineated by gas exchange and malic acid accumulation traits. Regulation at flow junctions (e.g. stomatal and mesophyll conductance, and malic acid transport across the tonoplast) that are subject to feedback control (e.g. stomatal aperture, malic acid inhibition of phosphoenolpyruvate carboxylase, and enzyme kinetics) was simulated. Simulated expression for the leaf-succulent Kalanchoë daigremontiana and more succulent tissues of Agave tequilana showed strong correlation with measured gas exchange and malic acid accumulation (R(2)  = 0.912 and 0.937, respectively, for K. daigremontiana and R(2)  = 0.928 and 0.942, respectively, for A. tequilana). Sensitivity analyses were conducted to quantitatively identify determinants of diel CO2 uptake. The transition in CAM expression from low to high volume/area tissues (elimination of phase II-IV carbon-uptake signatures) was achieved largely by the manipulation three input parameters. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Plasticity of crassulacean acid metabolism at subtropical latitudes: a pineapple case study.

PubMed

Rainha, Nuno; Medeiros, Violante P; Câmara, Mariana; Faustino, Hélder; Leite, João P; Barreto, Maria do Carmo; Cruz, Cristina; Pacheco, Carlos A; Ponte, Duarte; Bernardes da Silva, Anabela

2016-01-01

Plants with the crassulacean acid metabolism (CAM) express high-metabolic plasticity, to adjust to environmental stresses. This article hypothesizes that irradiance and nocturnal temperatures are the major limitations for CAM at higher latitudes such as the Azores (37°45'N). Circadian CAM expression in Ananas comosus L. Merr. (pineapple) was assessed by the diurnal pattern of leaf carbon fixation into l-malate at the solstices and equinoxes, and confirmed by determining maximal phosphoenolpyruvate carboxylase (PEPC) activity in plant material. Metabolic adjustments to environmental conditions were confirmed by gas exchange measurements, and integrated with environmental data to determine CAM's limiting factors: light and temperature. CAM plasticity was observed at the equinoxes, under similar photoperiods, but different environmental conditions. In spring, CAM expression was similar between vegetative and flowering plants, while in autumn, flowering (before anthesis) and fructifying (with fully developed fruit before ripening) plants accumulated more l-malate. Below 100 µmol m(-2) s(-1) , CAM phase I was extended, reducing CAM phase III during the day. Carbon fixation inhibition may occur by two major pathways: nocturnal temperature (<15°C) inhibiting PEPC activity and l-malate accumulation; and low irradiance influencing the interplay between CAM phase I and III, affecting carboxylation and decarboxylation. Both have important consequences for plant development in autumn and winter. Observations were confirmed by flowering time prediction using environmental data, emphasizing that CAM expression had a strong seasonal regulation due to a complex network response to light and temperature, allowing pineapple to survive in environments not suitable for high productivity. © 2015 Scandinavian Plant Physiology Society.

Leaf Activities.

ERIC Educational Resources Information Center

Mingie, Walter

Leaf activities can provide a means of using basic concepts of outdoor education to learn in elementary level subject areas. Equipment needed includes leaves, a clipboard with paper, and a pencil. A bag of leaves may be brought into the classroom if weather conditions or time do not permit going outdoors. Each student should pick a leaf, examine…

Pancreatic β-Cell Dysfunction in Diet-Induced Obese Mice: Roles of AMP-Kinase, Protein Kinase Cε, Mitochondrial and Cholesterol Metabolism, and Alterations in Gene Expression

PubMed Central

Pepin, Émilie; Al-Mass, Anfal; Attané, Camille; Zhang, Kezhuo; Lamontagne, Julien; Lussier, Roxane; Madiraju, S. R. Murthy; Joly, Erik; Ruderman, Neil B.; Sladek, Robert; Prentki, Marc; Peyot, Marie-Line

2016-01-01

Diet induced obese (DIO) mice can be stratified according to their weight gain in response to high fat diet as low responders (LDR) and high responders (HDR). This allows the study of β-cell failure and the transitions to prediabetes (LDR) and early diabetes (HDR). C57BL/6N mice were fed for 8 weeks with a normal chow diet (ND) or a high fat diet and stratified as LDR and HDR. Freshly isolated islets from ND, LDR and HDR mice were studied ex-vivo for mitochondrial metabolism, AMPK activity and signalling, the expression and activity of key enzymes of energy metabolism, cholesterol synthesis, and mRNA profiling. Severely compromised glucose-induced insulin secretion in HDR islets, as compared to ND and LDR islets, was associated with suppressed AMP-kinase activity. HDR islets also showed reduced acetyl-CoA carboxylase activity and enhanced activity of 3-hydroxy-3-methylglutaryl-CoA reductase, which led respectively to elevated fatty acid oxidation and increased cholesterol biosynthesis. HDR islets also displayed mitochondrial membrane hyperpolarization and reduced ATP turnover in the presence of elevated glucose. Expression of protein kinase Cε, which reduces both lipolysis and production of signals for insulin secretion, was elevated in DIO islets. Genes whose expression increased or decreased by more than 1.2-fold were minor between LDR and ND islets (17 differentially expressed), but were prominent between HDR and ND islets (1508 differentially expressed). In HDR islets, particularly affected genes were related to cell cycle and proliferation, AMPK signaling, mitochondrial metabolism and cholesterol metabolism. In conclusion, chronically reduced AMPK activity, mitochondrial dysfunction, elevated cholesterol biosynthesis in islets, and substantial alterations in gene expression accompany β-cell failure in HDR islets. The β-cell compensation process in the prediabetic state (LDR) is largely independent of transcriptional adaptive changes, whereas the transition

Antihypertensive and vasorelaxant effects of water-soluble proanthocyanidins from persimmon leaf tea in spontaneously hypertensive rats.

PubMed

Kawakami, Kayoko; Aketa, Saiko; Sakai, Hiroki; Watanabe, Yusuke; Nishida, Hiroshi; Hirayama, Masao

2011-01-01

The antihypertensive and vasorelaxant effects of water-soluble proanthocyanidins, extracted in persimmon leaf tea, were investigated in spontaneously hypertensive rats, rat aortas, and human umbilical vein endothelial cells. Oral administration of proanthocyanidins significantly decreased the systolic blood pressure of the rats after 4 h, as compared with distilled water controls. A vasorelaxant effect on rat aortas was induced by proanthocyanidins, and it was abolished by removal of the endothelium and inhibition of endothelial nitric oxide synthase and soluble guanylyl cyclase activity. The phosphorylation levels of endothelial nitric oxide synthase (Ser-1177) and the upstream kinase Akt (Ser-473) in umbilical cells also increased in a time-dependent manner after the addition of a proanthocyanidin-rich fraction. These results suggest that the antihypertensive effect of proanthocyanidins in persimmon leaf tea is due to vasorelaxation via an endothelium-dependent nitric oxide/cGMP pathway, and that proanthocyanidins might be useful in dietary lowering of blood pressure.

Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity

PubMed Central

Anastassiadis, Theonie; Deacon, Sean W.; Devarajan, Karthik; Ma, Haiching; Peterson, Jeffrey R.

2011-01-01

Small-molecule protein kinase inhibitors are central tools for elucidating cellular signaling pathways and are promising therapeutic agents. Due to evolutionary conservation of the ATP-binding site, most kinase inhibitors that target this site promiscuously inhibit multiple kinases. Interpretation of experiments utilizing these compounds is confounded by a lack of data on the comprehensive kinase selectivity of most inhibitors. Here we profiled the activity of 178 commercially available kinase inhibitors against a panel of 300 recombinant protein kinases using a functional assay. Quantitative analysis revealed complex and often unexpected kinase-inhibitor interactions, with a wide spectrum of promiscuity. Many off-target interactions occur with seemingly unrelated kinases, revealing how large-scale profiling can be used to identify multi-targeted inhibitors of specific, diverse kinases. The results have significant implications for drug development and provide a resource for selecting compounds to elucidate kinase function and for interpreting the results of experiments that use them. PMID:22037377

Trypanosoma evansi contains two auxiliary enzymes of glycolytic metabolism: Phosphoenolpyruvate carboxykinase and pyruvate phosphate dikinase.

PubMed

Rivero, Luz Amira; Concepción, Juan Luis; Quintero-Troconis, Ender; Quiñones, Wilfredo; Michels, Paul A M; Acosta, Héctor

2016-06-01

Trypanosoma evansi is a monomorphic protist that can infect horses and other animal species of economic importance for man. Like the bloodstream form of the closely related species Trypanosoma brucei, T. evansi depends exclusively on glycolysis for its free-energy generation. In T. evansi as in other kinetoplastid organisms, the enzymes of the major part of the glycolytic pathway are present within organelles called glycosomes, which are authentic but specialized peroxisomes. Since T. evansi does not undergo stage-dependent differentiations, it occurs only as bloodstream forms, it has been assumed that the metabolic pattern of this parasite is identical to that of the bloodstream form of T. brucei. However, we report here the presence of two additional enzymes, phosphoenolpyruvate carboxykinase and PPi-dependent pyruvate phosphate dikinase in T. evansi glycosomes. Their colocalization with glycolytic enzymes within the glycosomes of this parasite has not been reported before. Both enzymes can make use of PEP for contributing to the production of ATP within the organelles. The activity of these enzymes in T. evansi glycosomes drastically changes the model assumed for the oxidation of glucose by this parasite. Copyright © 2016 Elsevier Inc. All rights reserved.

Light-regulated phosphorylation of maize phosphoenolpyruvate carboxykinase plays a vital role in its activity.

PubMed

Chao, Qing; Liu, Xiao-Yu; Mei, Ying-Chang; Gao, Zhi-Fang; Chen, Yi-Bo; Qian, Chun-Rong; Hao, Yu-Bo; Wang, Bai-Chen

2014-05-01

Phosphoenolpyruvate carboxykinase (PEPCK)-the major decarboxylase in PEPCK-type C4 plants-is also present in appreciable amounts in the bundle sheath cells of NADP-malic enzyme-type C4 plants, such as maize (Zea mays), where it plays an apparent crucial role during photosynthesis (Wingler et al., in Plant Physiol 120(2):539-546, 1999; Furumoto et al., in Plant Mol Biol 41(3):301-311, 1999). Herein, we describe the use of mass spectrometry to demonstrate phosphorylation of maize PEPCK residues Ser55, Thr58, Thr59, and Thr120. Western blotting indicated that the extent of Ser55 phosphorylation dramatically increases in the leaves of maize seedlings when the seedlings are transferred from darkness to light, and decreases in the leaves of seedlings transferred from light to darkness. The effect of light on phosphorylation of this residue is opposite that of the effect of light on PEPCK activity, with the decarboxylase activity of PEPCK being less in illuminated leaves than in leaves left in the dark. This inverse relationship between PEPCK activity and the extent of phosphorylation suggests that the suppressive effect of light on PEPCK decarboxylation activity might be mediated by reversible phosphorylation of Ser55.

Exploring metabolic engineering design principles for the photosynthetic production of lactic acid by Synechocystis sp. PCC6803

PubMed Central

2014-01-01

Background Molecular engineering of the intermediary physiology of cyanobacteria has become important for the sustainable production of biofuels and commodity compounds from CO2 and sunlight by “designer microbes.” The chemical commodity product L-lactic acid can be synthesized in one step from a key intermediary metabolite of these organisms, pyruvate, catalyzed by a lactate dehydrogenase. Synthetic biology engineering to make “designer microbes” includes the introduction and overexpression of the product-forming biochemical pathway. For further optimization of product formation, modifications in the surrounding biochemical network of intermediary metabolism have to be made. Results To improve light-driven L-lactic acid production from CO2, we explored several metabolic engineering design principles, using a previously engineered L-lactic acid producing mutant strain of Synechocystis sp. PCC6803 as the benchmark. These strategies included: (i) increasing the expression level of the relevant product-forming enzyme, lactate dehydrogenase (LDH), for example, via expression from a replicative plasmid; (ii) co-expression of a heterologous pyruvate kinase to increase the flux towards pyruvate; and (iii) knockdown of phosphoenolpyruvate carboxylase to decrease the flux through a competing pathway (from phosphoenolpyruvate to oxaloacetate). In addition, we tested selected lactate dehydrogenases, some of which were further optimized through site-directed mutagenesis to improve the enzyme’s affinity for the co-factor nicotinamide adenine dinucleotide phosphate (NADPH). The carbon partitioning between biomass and lactic acid was increased from about 5% to over 50% by strain optimization. Conclusion An efficient photosynthetic microbial cell factory will display a high rate and extent of conversion of substrate (CO2) into product (here: L-lactic acid). In the existing CO2-based cyanobacterial cell factories that have been described in the literature, by far most of

Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase

NASA Technical Reports Server (NTRS)

Bachmann, M.; Shiraishi, N.; Campbell, W. H.; Yoo, B. C.; Harmon, A. C.; Huber, S. C.; Davies, E. (Principal Investigator)

1996-01-01

Spinach leaf NADH:nitrate reductase (NR) responds to light/dark signals and photosynthetic activity in part as a result of rapid regulation by reversible protein phosphorylation. We have identified the major regulatory phosphorylation site as Ser-543, which is located in the hinge 1 region connecting the cytochrome b domain with the molybdenum-pterin cofactor binding domain of NR, using recombinant NR fragments containing or lacking the phosphorylation site sequence. Studies with NR partial reactions indicated that the block in electron flow caused by phosphorylation also could be localized to the hinge 1 region. A synthetic peptide (NR6) based on the phosphorylation site sequence was phosphorylated readily by NR kinase (NRk) in vitro. NR6 kinase activity tracked the ATP-dependent inactivation of NR during several chromatographic steps and completely inhibited inactivation/phosphorylation of native NR in vitro. Two forms of NRk were resolved by using anion exchange chromatography. Studies with synthetic peptide analogs indicated that both forms of NRk had similar specificity determinants, requiring a basic residue at P-3 (i.e., three amino acids N-terminal to the phosphorylated serine) and a hydrophobic residue at P-5. Both forms are strictly calcium dependent but belong to distinct families of protein kinases because they are distinct immunochemically.

Easy Leaf Area: Automated digital image analysis for rapid and accurate measurement of leaf area.

PubMed

Easlon, Hsien Ming; Bloom, Arnold J

2014-07-01

Measurement of leaf areas from digital photographs has traditionally required significant user input unless backgrounds are carefully masked. Easy Leaf Area was developed to batch process hundreds of Arabidopsis rosette images in minutes, removing background artifacts and saving results to a spreadsheet-ready CSV file. • Easy Leaf Area uses the color ratios of each pixel to distinguish leaves and calibration areas from their background and compares leaf pixel counts to a red calibration area to eliminate the need for camera distance calculations or manual ruler scale measurement that other software methods typically require. Leaf areas estimated by this software from images taken with a camera phone were more accurate than ImageJ estimates from flatbed scanner images. • Easy Leaf Area provides an easy-to-use method for rapid measurement of leaf area and nondestructive estimation of canopy area from digital images.

Archaeal Shikimate Kinase, a New Member of the GHMP-Kinase Family

PubMed Central

Daugherty, Matthew; Vonstein, Veronika; Overbeek, Ross; Osterman, Andrei

2001-01-01

Shikimate kinase (EC 2.7.1.71) is a committed enzyme in the seven-step biosynthesis of chorismate, a major precursor of aromatic amino acids and many other aromatic compounds. Genes for all enzymes of the chorismate pathway except shikimate kinase are found in archaeal genomes by sequence homology to their bacterial counterparts. In this study, a conserved archaeal gene (gi|1500322 in Methanococcus jannaschii) was identified as the best candidate for the missing shikimate kinase gene by the analysis of chromosomal clustering of chorismate biosynthetic genes. The encoded hypothetical protein, with no sequence similarity to bacterial and eukaryotic shikimate kinases, is distantly related to homoserine kinases (EC 2.7.1.39) of the GHMP-kinase superfamily. The latter functionality in M. jannaschii is assigned to another gene (gi|1591748), in agreement with sequence similarity and chromosomal clustering analysis. Both archaeal proteins, overexpressed in Escherichia coli and purified to homogeneity, displayed activity of the predicted type, with steady-state kinetic parameters similar to those of the corresponding bacterial kinases: Km,shikimate = 414 ± 33 μM, Km,ATP = 48 ± 4 μM, and kcat = 57 ± 2 s−1 for the predicted shikimate kinase and Km,homoserine = 188 ± 37 μM, Km,ATP = 101 ± 7 μM, and kcat = 28 ± 1 s−1 for the homoserine kinase. No overlapping activity could be detected between shikimate kinase and homoserine kinase, both revealing a >1,000-fold preference for their own specific substrates. The case of archaeal shikimate kinase illustrates the efficacy of techniques based on reconstruction of metabolism from genomic data and analysis of gene clustering on chromosomes in finding missing genes. PMID:11114929

Leaf anatomical traits determine the 18O enrichment of leaf water in coastal halophytes

NASA Astrophysics Data System (ADS)

Liang, J.; Lin, G., Sr.; Sternberg, L. O.

2017-12-01

Foliar anatomical adaptations to high-salinity environment in mangroves may be recorded by leaf water isotopes. Recent studies observed that a few mangrove species have lower 18O enrichment of leaf water (ΔL) relative to source water than the adjacent terrestrial trees, but what factors actually control this phenomenon is still disputable at present. To resolve this issue, we collected 15 species of true mangrove plants, 14 species of adjacent freshwater trees and 4 species of semi-mangrove plants at five study sites on the southeastern coast of China. Leaf stomatal density and pore size, water content, ΔL and other related leaf physiological traits were determined for the selected leaves of these plants. Our results confirmed that ΔL values of mangroves were generally 3 4 ‰ lower than those of the adjacent freshwater or semi-mangrove species. Higher leaf water per area (LWC) and lower leaf stomatal density (LS) of mangroves played co-dominant roles in lowering ΔL through elongating effective leaf mixing length by about 20%. The Péclet model incorporated by LWC and LS performed well in predicting ΔL. The demonstrated general law between leaf anatomy and ΔL in this paper based on a large pool of species bridges the gap between leaf functional traits and metabolic proxies derived ΔL, which will have considerable potential applications in vegetation succession and reconstruction of paleoclimate research.

Coming of leaf age: control of growth by hydraulics and metabolics during leaf ontogeny.

PubMed

Pantin, Florent; Simonneau, Thierry; Muller, Bertrand

2012-10-01

Leaf growth is the central process facilitating energy capture and plant performance. This is also one of the most sensitive processes to a wide range of abiotic stresses. Because hydraulics and metabolics are two major determinants of expansive growth (volumetric increase) and structural growth (dry matter increase), we review the interaction nodes between water and carbon. We detail the crosstalks between water and carbon transports, including the dual role of stomata and aquaporins in regulating water and carbon fluxes, the coupling between phloem and xylem, the interactions between leaf water relations and photosynthetic capacity, the links between Lockhart's hydromechanical model and carbon metabolism, and the central regulatory role of abscisic acid. Then, we argue that during leaf ontogeny, these interactions change dramatically because of uncoupled modifications between several anatomical and physiological features of the leaf. We conclude that the control of leaf growth switches from a metabolic to a hydromechanical limitation during the course of leaf ontogeny. Finally, we illustrate how taking leaf ontogeny into account provides insights into the mechanisms underlying leaf growth responses to abiotic stresses that affect water and carbon relations, such as elevated CO2, low light, high temperature and drought. © 2012 INRA. New Phytologist © 2012 New Phytologist Trust.

Active suppression of a leaf meristem orchestrates determinate leaf growth

PubMed Central

Alvarez, John Paul; Furumizu, Chihiro; Efroni, Idan; Eshed, Yuval; Bowman, John L

2016-01-01

Leaves are flat determinate organs derived from indeterminate shoot apical meristems. The presence of a specific leaf meristem is debated, as anatomical features typical of meristems are not present in leaves. Here we demonstrate that multiple NGATHA (NGA) and CINCINNATA-class-TCP (CIN-TCP) transcription factors act redundantly, shortly after leaf initiation, to gradually restrict the activity of a leaf meristem in Arabidopsis thaliana to marginal and basal domains, and that their absence confers persistent marginal growth to leaves, cotyledons and floral organs. Following primordia initiation, the restriction of the broadly acting leaf meristem to the margins is mediated by the juxtaposition of adaxial and abaxial domains and maintained by WOX homeobox transcription factors, whereas other marginal elaboration genes are dispensable for its maintenance. This genetic framework parallels the morphogenetic program of shoot apical meristems and may represent a relic of an ancestral shoot system from which seed plant leaves evolved. DOI: http://dx.doi.org/10.7554/eLife.15023.001 PMID:27710768

Kinase inhibitor profiling reveals unexpected opportunities to inhibit disease-associated mutant kinases

PubMed Central

Duong-Ly, Krisna C.; Devarajan, Karthik; Liang, Shuguang; Horiuchi, Kurumi Y.; Wang, Yuren; Ma, Haiching; Peterson, Jeffrey R.

2016-01-01

Summary Small-molecule kinase inhibitors have typically been designed to inhibit wild-type kinases rather than the mutant forms that frequently arise in diseases such as cancer. Mutations can have serious clinical implications by increasing kinase catalytic activity or conferring therapeutic resistance. To identify opportunities to repurpose inhibitors against disease-associated mutant kinases, we conducted a large-scale functional screen of 183 known kinase inhibitors against 76 recombinant, mutant kinases. The results revealed lead compounds with activity against clinically important mutant kinases including ALK, LRRK2, RET, and EGFR as well as unexpected opportunities for repurposing FDA-approved kinase inhibitors as leads for additional indications. Furthermore, using T674I PDGFRα as an example, we show how single-dose screening data can provide predictive structure-activity data to guide subsequent inhibitor optimization. This study provides a resource for the development of inhibitors against numerous disease-associated mutant kinases and illustrates the potential of unbiased profiling as an approach to compound-centric inhibitor development. PMID:26776524

Oncogenic Receptor Tyrosine Kinases Directly Phosphorylate Focal Adhesion Kinase (FAK) as a Resistance Mechanism to FAK-kinase Inhibitors

PubMed Central

Marlowe, Timothy A.; Lenzo, Felicia L.; Figel, Sheila A.; Grapes, Abigail T.; Cance, William G.

2016-01-01

Focal adhesion kinase (FAK) is a major drug target in cancer and current inhibitors targeted to the ATP-binding pocket of the kinase domain have entered clinical trials. However, preliminary results have shown limited single-agent efficacy in patients. Despite these unfavorable data, the molecular mechanisms which drive intrinsic and acquired resistance to FAK-kinase inhibitors are largely unknown. We have demonstrated that receptor tyrosine kinases (RTKs) can directly bypass FAK-kinase inhibition in cancer cells through phosphorylation of FAK’s critical tyrosine 397 (Y397). We also showed that HER2 forms a direct protein-protein interaction with the FAK-FERM-F1 lobe, promoting direct phosphorylation of Y397. Additionally, FAK-kinase inhibition induced two forms of compensatory RTK reprogramming: 1) the rapid phosphorylation and activation of RTK signaling pathways in RTKHigh cells and 2) the long-term acquisition of RTKs novel to the parental cell line in RTKLow cells. Finally, HER2+ cancer cells displayed resistance to FAK-kinase inhibition in 3D–growth assays using a HER2 isogenic system and HER2+ cancer cell lines. Our data indicate a novel drug resistance mechanism to FAK-kinase inhibitors whereby HER2 and other RTKs can rescue and maintain FAK activation (pY397) even in the presence of FAK-kinase inhibition. These data may have important ramifications for existing clinical trials of FAK inhibitors and suggest that individual tumor stratification by RTK expression would be important to predict patient response to FAK-kinase inhibitors. PMID:27638858

Molecular interactions between tomato and the leaf mold pathogen Cladosporium fulvum.

PubMed

Rivas, Susana; Thomas, Colwyn M

2005-01-01

The interaction between tomato and the leaf mold pathogen Cladosporium fulvum is controlled in a gene-for-gene manner. This interaction has provided useful insights to the molecular basis of recognition specificity in plant disease resistance (R) proteins, disease resistance (R) gene evolution, R-protein mediated signaling, and cellular responses to pathogen attack. Tomato Cf genes encode type I membrane-associated receptor-like proteins (RLPs) comprised predominantly of extracellular leucine-rich repeats (eLRRs) and which are anchored in the plasma membrane. Cf proteins recognize fungal avirulence (Avr) peptides secreted into the leaf apoplast during infection. A direct interaction of Cf proteins with their cognate Avr proteins has not been demonstrated and the molecular mechanism of Avr protein perception is not known. Following ligand perception Cf proteins trigger a hypersensitive response (HR) and the arrest of pathogen development. Cf proteins lack an obvious signaling domain, suggesting that defense response activation is mediated through interactions with other partners. Avr protein perception results in the rapid accumulation of active oxygen species (AOS), changes in cellular ion fluxes, activation of protein kinase cascades, changes in gene expression and, possibly, targeted protein degradation. Here we review our current understanding of Cf-mediated responses in resistance to C. fulvum.

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

DOEpatents

Houtz, Robert L.

1998-01-01

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

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

DOEpatents

Houtz, R.L.

1998-03-03

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

Association of tomato leaf curl Gujarat virus and tomato leaf curl Bangladesh betasatellite on papaya showing typical leaf curl symptoms in North India.

PubMed

Varun, Priyanka; Saxena, Sangeeta

2018-05-01

Papaya leaf curl is an economically important disease occurring in papaya growing tropical and subtropical areas. Papaya leaf curl virus, a begomovirus, is the main causative agent for the disease, but many other begomoviruses as well as betasatellites have also been reported on papaya leaf curl disease. Rapidly evolving host range of begomoviruses is a major issue for developing successful resistance strategies against begomoviral infection considering their expanding host range and mixed infection. In our study, we have identified the presence of begomovirus and associated satellite molecule on papaya showing severe leaf curl symptoms in Lucknow, India. Analysis of complete DNA-A component of this isolate (MG757245) revealed the highest similarity (91%) with tomato leaf curl Gujarat virus (ToLCuGuV), while sequence data of betasatellite (MG478451) showed maximum (89%) identity with tomato leaf curl Bangladesh betasatellite (ToLCuBB). This is the first report on identification of ToLCuGuV and ToLCuBB coinfecting papaya plants in Lucknow, Uttar Pradesh (India).

Identification of regions of rabbit muscle pyruvate kinase important for allosteric regulation by phenylalanine, detected by H/D exchange mass spectrometry†

PubMed Central

Prasannan, Charulata B.; Villar, Maria T.; Artigues, Antonio; Fenton, Aron W.

2013-01-01

Mass spectrometry has been used to determine the number of exchangeable backbone amide protons and the associated rate constants that are altered when rabbit muscle pyruvate kinase (rM1-PYK) binds either the allosteric inhibitor (phenylalanine) or a non-allosteric analogue of the inhibitor. Alanine is used as the non-allosteric analogue since it binds competitively with phenylalanine, but elicits a negligible allosteric inhibition, i.e. a negligible reduction of the affinity of rM1-PYK for the substrate, phosphoenolpyruvate (PEP). This experimental design is expected to distinguish changes in the protein caused by effector binding (i.e. those changes common upon the addition of alanine vs. phenylalanine) from changes associated with allosteric regulation (i.e. those elicited by the addition of phenylalanine binding, but not alanine binding). High quality peptic fragments covering 98% of the protein were identified. Changes in both the number of exchangeable protons per peptide and in the rate constant associated with exchange highlight regions of the protein with allosteric roles. The set of allosterically relevant peptides identified by this technique include residues previously identified by mutagenesis to have roles in the allosteric regulation by phenylalanine. PMID:23418858

LKB1, an upstream AMPK kinase, regulates glucose and lipid metabolism in cultured liver and muscle cells.

PubMed

Imai, Kenta; Inukai, Kouichi; Ikegami, Yuichi; Awata, Takuya; Katayama, Shigehiro

2006-12-22

LKB1 is a 50 kDa serine/threonine kinase that phosphorylates and activates the catalytic subunit of AMPK at its T-loop residue Thr 172. We prepared adenoviruses expressing the constitutive active (wild-type) form (CA) or dominant negative (kinase inactive, D194A mutant) form (DN) of LKB1 and overexpressed these proteins in cultured myotubes (C2C12 cells) and rat hepatoma cells (FAO cells). When analyzed by immunoblotting with the antibody against Thr172-phosphorylated AMPK, the phosphorylation of AMPK was increased (2.5-fold) and decreased (0.4-fold) in cells expressing CA and DN LKB1, respectively, as compared with Lac-Z expressing control cells. Immunoprecipitation experiments, using isoform-specific antibody, revealed these alterations of AMPK phosphorylation to be attributable to altered phosphorylation of AMPK alpha2, but not alpha1 catalytic subunits, strongly suggesting the alpha2 catalytic subunit to be the major substrate for LKB1 in mammalian cells. In addition, adiponectin or AICAR-stimulated AMPK phosphorylation was inhibited by overexpression of DN LKB1, while phenformin-stimulated phosphorylation was unaffected. These results may explain the difference in AMPK activation mechanisms between AMP and phenformin, and also indicate that AMPK phosphorylation by LKB1 is involved in AMP-stimulated AMPK activation. As a downstream target for AMPK, AICAR-induced glucose uptake and ACCbeta phosphorylation were found to be significantly reduced in DN LKB1 expressing C2C12 cells. The expression of key enzymes for gluconeogenesis, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, was also dependent on LKB1 activities in FAO cells. These results demonstrate that LKB1 is a crucial regulator of AMPK activation in muscle and liver cells and, therefore, that LKB1 activity is potentially of importance to our understanding of glucose and lipid metabolism.

Investigation of the Roles of Allosteric Domain Arginine, Aspartate, and Glutamate Residues of Rhizobium etli Pyruvate Carboxylase in Relation to Its Activation by Acetyl CoA.

PubMed

Sirithanakorn, Chaiyos; Jitrapakdee, Sarawut; Attwood, Paul V

2016-08-02

The mechanism of allosteric activation of pyruvate carboxylase by acetyl CoA is not fully understood. Here we have examined the roles of residues near the acetyl CoA binding site in the allosteric activation of Rhizobium etli pyruvate carboxylase using site-directed mutagenesis. Arg429 was found to be especially important for acetyl CoA binding as substitution with serine resulted in a 100-fold increase in the Ka of acetyl CoA activation and a large decrease in the cooperativity of this activation. Asp420 and Arg424, which do not make direct contact with bound acetyl CoA, were nonetheless found to affect acetyl CoA binding when mutated, probably through changed interactions with another acetyl CoA binding residue, Arg427. Thermodynamic activation parameters for the pyruvate carboxylation reaction were determined from modified Arrhenius plots and showed that acetyl CoA acts to decrease the activation free energy of the reaction by both increasing the activation entropy and decreasing the activation enthalpy. Most importantly, mutations of Asp420, Arg424, and Arg429 enhanced the activity of the enzyme in the absence of acetyl CoA. A main focus of this work was the detailed investigation of how this increase in activity occurred in the R424S mutant. This mutation decreased the activation enthalpy of the pyruvate carboxylation reaction by an amount consistent with removal of a single hydrogen bond. It is postulated that Arg424 forms a hydrogen bonding interaction with another residue that stabilizes the asymmetrical conformation of the R. etli pyruvate carboxylase tetramer, constraining its interconversion to the symmetrical conformer that is required for catalysis.

Genome-wide association study of rice (Oryza sativa L.) leaf traits with a high-throughput leaf scorer.

PubMed

Yang, Wanneng; Guo, Zilong; Huang, Chenglong; Wang, Ke; Jiang, Ni; Feng, Hui; Chen, Guoxing; Liu, Qian; Xiong, Lizhong

2015-09-01

Leaves are the plant's solar panel and food factory, and leaf traits are always key issues to investigate in plant research. Traditional methods for leaf trait measurement are time-consuming. In this work, an engineering prototype has been established for high-throughput leaf scoring (HLS) of a large number of Oryza sativa accessions. The mean absolute per cent of errors in traditional measurements versus HLS were below 5% for leaf number, area, shape, and colour. Moreover, HLS can measure up to 30 leaves per minute. To demonstrate the usefulness of HLS in dissecting the genetic bases of leaf traits, a genome-wide association study (GWAS) was performed for 29 leaf traits related to leaf size, shape, and colour at three growth stages using HLS on a panel of 533 rice accessions. Nine associated loci contained known leaf-related genes, such as Nal1 for controlling the leaf width. In addition, a total of 73, 123, and 177 new loci were detected for traits associated with leaf size, colour, and shape, respectively. In summary, after evaluating the performance with a large number of rice accessions, the combination of GWAS and high-throughput leaf phenotyping (HLS) has proven a valuable strategy to identify the genetic loci controlling rice leaf traits. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Kranz and single-cell forms of C4 plants in the subfamily Suaedoideae show kinetic C4 convergence for PEPC and Rubisco with divergent amino acid substitutions.

PubMed

Rosnow, Josh J; Evans, Marc A; Kapralov, Maxim V; Cousins, Asaph B; Edwards, Gerald E; Roalson, Eric H

2015-12-01

The two carboxylation reactions performed by phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) are vital in the fixation of inorganic carbon for C4 plants. The abundance of PEPC is substantially elevated in C4 leaves, while the location of Rubisco is restricted to one of two chloroplast types. These differences compared with C3 leaves have been shown to result in convergent enzyme optimization in some C4 species. Investigation into the kinetic properties of PEPC and Rubisco from Kranz C4, single cell C4, and C3 species in Chenopodiaceae s. s. subfamily Suaedoideae showed that these major carboxylases in C4 Suaedoideae species lack the same mutations found in other C4 systems which have been examined; but still have similar convergent kinetic properties. Positive selection analysis on the N-terminus of PEPC identified residues 364 and 368 to be under positive selection with a posterior probability >0.99 using Bayes empirical Bayes. Compared with previous analyses on other C4 species, PEPC from C4 Suaedoideae species have different convergent amino acids that result in a higher K m for PEP and malate tolerance compared with C3 species. Kinetic analysis of Rubisco showed that C4 species have a higher catalytic efficiency of Rubisco (k catc in mol CO2 mol(-1) Rubisco active sites s(-1)), despite lacking convergent substitutions in the rbcL gene. The importance of kinetic changes to the two-carboxylation reactions in C4 leaves related to amino acid selection is discussed. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Regulation of Acetate Kinase Isozymes and Its Importance for Mixed-Acid Fermentation in Lactococcus lactis

PubMed Central

Puri, Pranav; Goel, Anisha; Bochynska, Agnieszka

2014-01-01

Acetate kinase (ACK) converts acetyl phosphate to acetate along with the generation of ATP in the pathway for mixed-acid fermentation in Lactococcus lactis. The reverse reaction yields acetyl phosphate for assimilation purposes. Remarkably, L. lactis has two ACK isozymes, and the corresponding genes are present in an operon. We purified both enzymes (AckA1 and AckA2) from L. lactis MG1363 and determined their oligomeric state, specific activities, and allosteric regulation. Both proteins form homodimeric complexes, as shown by size exclusion chromatography and static light-scattering measurements. The turnover number of AckA1 is about an order of magnitude higher than that of AckA2 for the reaction in either direction. The Km values for acetyl phosphate, ATP, and ADP are similar for both enzymes. However, AckA2 has a higher affinity for acetate than does AckA1, suggesting an important role under acetate-limiting conditions despite the lower activity. Fructose-1,6-bisphosphate, glyceraldehyde-3-phosphate, and phospho-enol-pyruvate inhibit the activities of AckA1 and AckA2 to different extents. The allosteric regulation of AckA1 and AckA2 and the pool sizes of the glycolytic intermediates are consistent with a switch from homolactic to mixed-acid fermentation upon slowing of the growth rate. PMID:24464460

Crystal structure of casein kinase-1, a phosphate-directed protein kinase.

PubMed Central

Xu, R M; Carmel, G; Sweet, R M; Kuret, J; Cheng, X

1995-01-01

The structure of a truncated variant of casein kinase-1 from Schizosaccharomyces pombe, has been determined in complex with MgATP at 2.0 A resolution. The model resembles the 'closed', ATP-bound conformations of the cyclin-dependent kinase 2 and the cAMP-dependent protein kinase, with clear differences in the structure of surface loops that impart unique features to casein kinase-1. The structure is of unphosphorylated, active conformation of casein kinase-1 and the peptide-binding site is fully accessible to substrate. Images PMID:7889932

Oncogenic Receptor Tyrosine Kinases Directly Phosphorylate Focal Adhesion Kinase (FAK) as a Resistance Mechanism to FAK-Kinase Inhibitors.

PubMed

Marlowe, Timothy A; Lenzo, Felicia L; Figel, Sheila A; Grapes, Abigail T; Cance, William G

2016-12-01

Focal adhesion kinase (FAK) is a major drug target in cancer and current inhibitors targeted to the ATP-binding pocket of the kinase domain have entered clinical trials. However, preliminary results have shown limited single-agent efficacy in patients. Despite these unfavorable data, the molecular mechanisms that drive intrinsic and acquired resistance to FAK-kinase inhibitors are largely unknown. We have demonstrated that receptor tyrosine kinases (RTK) can directly bypass FAK-kinase inhibition in cancer cells through phosphorylation of FAK's critical tyrosine 397 (Y397). We also showed that HER2 forms a direct protein-protein interaction with the FAK-FERM-F1 lobe, promoting direct phosphorylation of Y397. In addition, FAK-kinase inhibition induced two forms of compensatory RTK reprogramming: (i) the rapid phosphorylation and activation of RTK signaling pathways in RTK High cells and (ii) the long-term acquisition of RTKs novel to the parental cell line in RTK Low cells. Finally, HER2 +: cancer cells displayed resistance to FAK-kinase inhibition in 3D growth assays using a HER2 isogenic system and HER2 + cancer cell lines. Our data indicate a novel drug resistance mechanism to FAK-kinase inhibitors whereby HER2 and other RTKs can rescue and maintain FAK activation (pY397) even in the presence of FAK-kinase inhibition. These data may have important ramifications for existing clinical trials of FAK inhibitors and suggest that individual tumor stratification by RTK expression would be important to predict patient response to FAK-kinase inhibitors. Mol Cancer Ther; 15(12); 3028-39. ©2016 AACR. ©2016 American Association for Cancer Research.

Leaf drop affects herbivory in oaks.

PubMed

Pearse, Ian S; Karban, Richard

2013-11-01

Leaf phenology is important to herbivores, but the timing and extent of leaf drop has not played an important role in our understanding of herbivore interactions with deciduous plants. Using phylogenetic general least squares regression, we compared the phenology of leaves of 55 oak species in a common garden with the abundance of leaf miners on those trees. Mine abundance was highest on trees with an intermediate leaf retention index, i.e. trees that lost most, but not all, of their leaves for 2-3 months. The leaves of more evergreen species were more heavily sclerotized, and sclerotized leaves accumulated fewer mines in the summer. Leaves of more deciduous species also accumulated fewer mines in the summer, and this was consistent with the idea that trees reduce overwintering herbivores by shedding leaves. Trees with a later leaf set and slower leaf maturation accumulated fewer herbivores. We propose that both leaf drop and early leaf phenology strongly affect herbivore abundance and select for differences in plant defense. Leaf drop may allow trees to dispose of their herbivores so that the herbivores must recolonize in spring, but trees with the longest leaf retention also have the greatest direct defenses against herbivores.

Automated Leaf Tracking using Multi-view Image Sequences of Maize Plants for Leaf-growth Monitoring

NASA Astrophysics Data System (ADS)

Das Choudhury, S.; Awada, T.; Samal, A.; Stoerger, V.; Bashyam, S.

2017-12-01

Extraction of phenotypes with botanical importance by analyzing plant image sequences has the desirable advantages of non-destructive temporal phenotypic measurements of a large number of plants with little or no manual intervention in a relatively short period of time. The health of a plant is best interpreted by the emergence timing and temporal growth of individual leaves. For automated leaf growth monitoring, it is essential to track each leaf throughout the life cycle of the plant. Plants are constantly changing organisms with increasing complexity in architecture due to variations in self-occlusions and phyllotaxy, i.e., arrangements of leaves around the stem. The leaf cross-overs pose challenges to accurately track each leaf using single view image sequence. Thus, we introduce a novel automated leaf tracking algorithm using a graph theoretic approach by multi-view image sequence analysis based on the determination of leaf-tips and leaf-junctions in the 3D space. The basis of the leaf tracking algorithm is: the leaves emerge using bottom-up approach in the case of a maize plant, and the direction of leaf emergence strictly alternates in terms of direction. The algorithm involves labeling of the individual parts of a plant, i.e., leaves and stem, following graphical representation of the plant skeleton, i.e., one-pixel wide connected line obtained from the binary image. The length of the leaf is measured by the number of pixels in the leaf skeleton. To evaluate the performance of the algorithm, a benchmark dataset is indispensable. Thus, we publicly release University of Nebraska-Lincoln Component Plant Phenotyping dataset-2 (UNL-CPPD-2) consisting of images of the 20 maize plants captured by visible light camera of the Lemnatec Scanalyzer 3D high throughout plant phenotyping facility once daily for 60 days from 10 different views. The dataset is aimed to facilitate the development and evaluation of leaf tracking algorithms and their uniform comparisons.

Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase). Evidence that auto-kinase belongs to a member of the p21-activated kinase family.

PubMed

Yu, J S; Chen, W J; Ni, M H; Chan, W H; Yang, S D

1998-08-15

Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421-9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or gamma-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both

Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase). Evidence that auto-kinase belongs to a member of the p21-activated kinase family.

PubMed Central

Yu, J S; Chen, W J; Ni, M H; Chan, W H; Yang, S D

1998-01-01

Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421-9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or gamma-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both

Spatial patterns of photosynthesis in thin- and thick-leaved epiphytic orchids: unravelling C3–CAM plasticity in an organ-compartmented way

PubMed Central

Rodrigues, Maria Aurineide; Matiz, Alejandra; Cruz, Aline Bertinatto; Matsumura, Aline Tiemi; Takahashi, Cassia Ayumi; Hamachi, Leonardo; Félix, Lucas Macedo; Pereira, Paula Natália; Latansio-Aidar, Sabrina Ribeiro; Aidar, Marcos Pereira Marinho; Demarco, Diego; Freschi, Luciano; Mercier, Helenice; Kerbauy, Gilberto Barbante

2013-01-01

Background and Aims A positive correlation between tissue thickness and crassulacean acid metabolism (CAM) expression has been frequently suggested. Therefore, this study addressed the question of whether water availability modulates photosynthetic plasticity in different organs of two epiphytic orchids with distinct leaf thickness. Methods Tissue morphology and photosynthetic mode (C3 and/or CAM) were examined in leaves, pseudobulbs and roots of a thick-leaved (Cattleya walkeriana) and a thin-leaved (Oncidium ‘Aloha’) epiphytic orchid. Morphological features were studied comparing the drought-induced physiological responses observed in each organ after 30 d of either drought or well-watered treatments. Key Results Cattleya walkeriana, which is considered a constitutive CAM orchid, displayed a clear drought-induced up-regulation of CAM in its thick leaves but not in its non-leaf organs (pseudobulbs and roots). The set of morphological traits of Cattleya leaves suggested the drought-inducible CAM up-regulation as a possible mechanism of increasing water-use efficiency and carbon economy. Conversely, although belonging to an orchid genus classically considered as performing C3 photosynthesis, Oncidium ‘Aloha’ under drought seemed to express facultative CAM in its roots and pseudobulbs but not in its leaves, indicating that such photosynthetic responses might compensate for the lack of capacity to perform CAM in its thin leaves. Morphological features of Oncidium leaves also indicated lower efficiency in preventing water and CO2 losses, while aerenchyma ducts connecting pseudobulbs and leaves suggested a compartmentalized mechanism of nighttime carboxylation via phosphoenolpyruvate carboxylase (PEPC) (pseudobulbs) and daytime carboxylation via Rubisco (leaves) in drought-exposed Oncidium plants. Conclusions Water availability modulated CAM expression in an organ-compartmented manner in both orchids studied. As distinct regions of the same orchid could perform

Redefinition of rubisco carboxylase reaction reveals origin of water for hydration and new roles for active-site residues.

PubMed

Kannappan, Babu; Gready, Jill E

2008-11-12

Crystallographic, mutagenesis, kinetic, and computational studies on Rubisco over three decades have revealed much about its catalytic mechanism and the role played by several active-site residues. However, key questions remain unanswered. Specific details of the carboxylase and oxygenase mechanisms, required to underpin the rational re-engineering of Rubisco, are still speculative. Here we address critical gaps in knowledge with a definitive comprehensive computational investigation of the mechanism of carboxylase activity at the Rubisco active site. Density functional theory calculations (B3LYP/6-31G(d,p)) were performed on active-site fragment models of a size up to 77 atoms, not previously possible computationally. All amino acid residues suspected to play roles in the acid-base chemistry in the multistep reaction, and interacting directly with the central Mg (2+) atom and the reactive moiety of substrate and intermediates, were included. The results provide a firm basis for us to propose a novel mechanism for the entire sequence of reactions in the carboxylase catalysis and to define precise roles for the active-site residues, singly and in concert. In this mechanism, the carbamylated LYS201 plays a more limited role than previously proposed but is crucial for initiating the reaction by acting as a base in the enolization. We suggest a wider role for HIS294, with involvement in the carboxylation, hydration, and C2-C3 bond-scission steps, consistent with the suggestion of Harpel et al. (1998) but contrary to the consensus view of Cleland et al. (1998). In contrast to the common assumption that the water molecule for the hydration step comes from within the active site, we propose that the Mg-coordinated water is not dissociated at the start of the gas-addition reaction but rather remains coordinated and is used for the hydration of the C3 carbon atom. New roles are also proposed for LYS175, GLU204, and HIS294. The mechanism suggests roles in the gas

Leaf area dynamics of conifer forests

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

Margolis, H.; Oren, R.; Whitehead, D.

1995-07-01

Estimating the surface area of foliage supported by a coniferous forest canopy is critical for modeling its biological properties. Leaf area represents the surface area available for the interception of energy, the absorption of carbon dioxide, and the diffusion of water from the leaf to the atmosphere. The concept of leaf area is pertinent to the physiological and ecological dynamics of conifers at a wide range of spatial scales, from individual leaves to entire biomes. In fact, the leaf area of vegetation at a global level can be thought of as a carbon-absorbing, water-emitting membrane of variable thickness, which canmore » have an important influence on the dynamics and chemistry of the Earth`s atmosphere over both the short and the long term. Unless otherwise specified, references to leaf area herein refer to projected leaf area, i.e., the vertical projection of needles placed on a flat plane. Total leaf surface area is generally from 2.0 to 3.14 times that of projected leaf area for conifers. It has recently been suggested that hemisurface leaf area, i.e., one-half of the total surface area of a leaf, a more useful basis for expressing leaf area than is projected area. This chapter is concerned with the dynamics of coniferous forest leaf area at different spatial and temporal scales. In the first part, we consider various hypotheses related to the control of leaf area development, ranging from simple allometric relations with tree size to more complex mechanistic models that consider the movement of water and nutrients to tree canopies. In the second part, we consider various aspects of leaf area dynamics at varying spatial and temporal scales, including responses to perturbation, seasonal dynamics, genetic variation in crown architecture, the responses to silvicultural treatments, the causes and consequences of senescence, and the direct measurement of coniferous leaf area at large spatial scales using remote sensing.« less

Hypolipidemic activity of Taraxacum mongolicum associated with the activation of AMP-activated protein kinase in human HepG2 cells.

PubMed

Liu, Yan-Jin; Shieh, Po-Chuen; Lee, Jang-Chang; Chen, Fu-An; Lee, Chih-Hung; Kuo, Sheng-Chu; Ho, Chi-Tang; Kuo, Daih-Huang; Huang, Li-Jiau; Way, Tzong-Der

2014-08-01

This study investigated the hypolipidemic effect and potential mechanisms of T. mongolicum extracts. T. mongolicum was extracted by refluxing three times with water (TM-1), 50% ethanol (TM-2) and 95% ethanol (TM-3). TM-2 contained components with the most effective hypolipidemic potentials in HepG2 cells. Extended administration of TM-2 stimulated a significant reduction in body weight and levels of serum triglyceride LDL-C and total cholesterol in rats. To evaluate the bioactive compounds, we successively fractionated TM-2 with n-hexane (TM-4), dichloromethane (TM-5), ethyl acetate (TM-6), and water (TM-7). TM-4 fraction had the most effective hypolipidemic potential in HepG2 cells, and it decreased the expression of fatty acid synthase (FASN) and inhibited the activity of acetyl-coenzyme A carboxylase (ACC) through the phosphorylation of AMP-activated protein kinase (AMPK). Linoleic acid, phytol and tetracosanol are bioactive compounds identified from TM-4. These results suggest that T. mongolicum is expected to be useful for hypolipidemic effects.

CcpN Controls Central Carbon Fluxes in Bacillus subtilis▿ ‡

PubMed Central

Tännler, Simon; Fischer, Eliane; Le Coq, Dominique; Doan, Thierry; Jamet, Emmanuel; Sauer, Uwe; Aymerich, Stéphane

2008-01-01

The transcriptional regulator CcpN of Bacillus subtilis has been recently characterized as a repressor of two gluconeogenic genes, gapB and pckA, and of a small noncoding regulatory RNA, sr1, involved in arginine catabolism. Deletion of ccpN impairs growth on glucose and strongly alters the distribution of intracellular fluxes, rerouting the main glucose catabolism from glycolysis to the pentose phosphate (PP) pathway. Using transcriptome analysis, we show that during growth on glucose, gapB and pckA are the only protein-coding genes directly repressed by CcpN. By quantifying intracellular fluxes in deletion mutants, we demonstrate that derepression of pckA under glycolytic condition causes the growth defect observed in the ccpN mutant due to extensive futile cycling through the pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and pyruvate kinase. Beyond ATP dissipation via this cycle, PckA activity causes a drain on tricarboxylic acid cycle intermediates, which we show to be the main reason for the reduced growth of a ccpN mutant. The high flux through the PP pathway in the ccpN mutant is modulated by the flux through the alternative glyceraldehyde-3-phosphate dehydrogenases, GapA and GapB. Strongly increased concentrations of intermediates in upper glycolysis indicate that GapB overexpression causes a metabolic jamming of this pathway and, consequently, increases the relative flux through the PP pathway. In contrast, derepression of sr1, the third known target of CcpN, plays only a marginal role in ccpN mutant phenotypes. PMID:18586936

Metabolism of organic acids, nitrogen and amino acids in chlorotic leaves of 'Honeycrisp' apple (Malus domestica Borkh) with excessive accumulation of carbohydrates.

PubMed

Wang, Huicong; Ma, Fangfang; Cheng, Lailiang

2010-07-01

Metabolite profiles and activities of key enzymes in the metabolism of organic acids, nitrogen and amino acids were compared between chlorotic leaves and normal leaves of 'Honeycrisp' apple to understand how accumulation of non-structural carbohydrates affects the metabolism of organic acids, nitrogen and amino acids. Excessive accumulation of non-structural carbohydrates and much lower CO(2) assimilation were found in chlorotic leaves than in normal leaves, confirming feedback inhibition of photosynthesis in chlorotic leaves. Dark respiration and activities of several key enzymes in glycolysis and tricarboxylic acid (TCA) cycle, ATP-phosphofructokinase, pyruvate kinase, citrate synthase, aconitase and isocitrate dehydrogenase were significantly higher in chlorotic leaves than in normal leaves. However, concentrations of most organic acids including phosphoenolpyruvate (PEP), pyruvate, oxaloacetate, 2-oxoglutarate, malate and fumarate, and activities of key enzymes involved in the anapleurotic pathway including PEP carboxylase, NAD-malate dehydrogenase and NAD-malic enzyme were significantly lower in chlorotic leaves than in normal leaves. Concentrations of soluble proteins and most free amino acids were significantly lower in chlorotic leaves than in normal leaves. Activities of key enzymes in nitrogen assimilation and amino acid synthesis, including nitrate reductase, glutamine synthetase, ferredoxin and NADH-dependent glutamate synthase, and glutamate pyruvate transaminase were significantly lower in chlorotic leaves than in normal leaves. It was concluded that, in response to excessive accumulation of non-structural carbohydrates, glycolysis and TCA cycle were up-regulated to "consume" the excess carbon available, whereas the anapleurotic pathway, nitrogen assimilation and amino acid synthesis were down-regulated to reduce the overall rate of amino acid and protein synthesis.

7 CFR 29.1162 - Leaf (B Group).

Code of Federal Regulations, 2010 CFR

2010-01-01

... Specifications, and Tolerances B1L—Choice Quality Lemon Leaf Ripe, firm leaf structure, medium body, rich in oil... percent. B2L—Fine Quality Lemon Leaf Ripe, firm leaf structure, medium body, rich in oil, deep color.... B3L—Good Quality Lemon Leaf Ripe, firm leaf structure, medium body, oily, strong color intensity...

7 CFR 29.1162 - Leaf (B Group).

Code of Federal Regulations, 2011 CFR

2011-01-01

... Specifications, and Tolerances B1L—Choice Quality Lemon Leaf Ripe, firm leaf structure, medium body, rich in oil... percent. B2L—Fine Quality Lemon Leaf Ripe, firm leaf structure, medium body, rich in oil, deep color.... B3L—Good Quality Lemon Leaf Ripe, firm leaf structure, medium body, oily, strong color intensity...

Two chalcones, 4-hydroxyderricin and xanthoangelol, stimulate GLUT4-dependent glucose uptake through the LKB1/AMP-activated protein kinase signaling pathway in 3T3-L1 adipocytes.

PubMed

Ohta, Mitsuhiro; Fujinami, Aya; Kobayashi, Norihiro; Amano, Akiko; Ishigami, Akihito; Tokuda, Harukuni; Suzuki, Nobutaka; Ito, Fumitake; Mori, Taisuke; Sawada, Morio; Iwasa, Koichi; Kitawaki, Jo; Ohnishi, Katsunori; Tsujikawa, Muneo; Obayashi, Hiroshi

2015-07-01

4-Hydroxyderricin (4HD) and xanthoangelol (XAG) are major components of n-hexane/ethyl acetate (5:1) extract of the yellow-colored stem juice of Angelica keiskei. 4-Hydroxyderricin and XAG have been reported to increase glucose transporter 4 (GLUT4)-dependent glucose uptake in 3T3-L1 adipocytes, but the detailed mechanism of this phenomenon remains unknown. This present study was aimed at clarifying the detailed mechanism by which 4HD and XAG increase GLUT4-dependent glucose uptake in 3T3-L1 adipocytes. Both 4HD and XAG increased glucose uptake and GLUT4 translocation to the plasma membrane. 4-Hydroxyderricin and XAG also stimulated the phosphorylation of 5' adenosine monophosphate-activated protein kinase (AMPK) and its downstream target acetyl-CoA carboxylase. In addition, phosphorylation of liver kinase B1 (LKB1), which acts upstream of AMPK, was also increased by 4HD and XAG treatment. Small interfering RNA knockdown of LKB1 attenuated 4HD- and XAG-stimulated AMPK phosphorylation and suppressed glucose uptake. These findings demonstrate that 4HD and XAG can increase GLUT4-dependent glucose uptake through the LKB1/AMPK signaling pathway in 3T3-L1 adipocytes. Copyright © 2015 Elsevier Inc. All rights reserved.

7 CFR 29.3036 - Leaf surface.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 2 2014-01-01 2014-01-01 false Leaf surface. 29.3036 Section 29.3036 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Leaf surface. The smoothness or roughness of the web or lamina of a tobacco leaf. Leaf surface is...

7 CFR 29.3036 - Leaf surface.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 2 2012-01-01 2012-01-01 false Leaf surface. 29.3036 Section 29.3036 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Leaf surface. The smoothness or roughness of the web or lamina of a tobacco leaf. Leaf surface is...

7 CFR 29.3036 - Leaf surface.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false Leaf surface. 29.3036 Section 29.3036 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Leaf surface. The smoothness or roughness of the web or lamina of a tobacco leaf. Leaf surface is...

7 CFR 30.2 - Leaf tobacco.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 2 2011-01-01 2011-01-01 false Leaf tobacco. 30.2 Section 30.2 Agriculture... AND STANDARDS Classification of Leaf Tobacco Covering Classes, Types and Groups of Grades § 30.2 Leaf... stemming, sweating or fermenting, and conditioning are not regarded as manufacturing processes. Leaf...

7 CFR 29.3036 - Leaf surface.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 2 2011-01-01 2011-01-01 false Leaf surface. 29.3036 Section 29.3036 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Leaf surface. The smoothness or roughness of the web or lamina of a tobacco leaf. Leaf surface is...

7 CFR 30.2 - Leaf tobacco.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false Leaf tobacco. 30.2 Section 30.2 Agriculture... AND STANDARDS Classification of Leaf Tobacco Covering Classes, Types and Groups of Grades § 30.2 Leaf... stemming, sweating or fermenting, and conditioning are not regarded as manufacturing processes. Leaf...

7 CFR 30.2 - Leaf tobacco.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 2 2012-01-01 2012-01-01 false Leaf tobacco. 30.2 Section 30.2 Agriculture... AND STANDARDS Classification of Leaf Tobacco Covering Classes, Types and Groups of Grades § 30.2 Leaf... stemming, sweating or fermenting, and conditioning are not regarded as manufacturing processes. Leaf...

7 CFR 30.2 - Leaf tobacco.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 2 2014-01-01 2014-01-01 false Leaf tobacco. 30.2 Section 30.2 Agriculture... AND STANDARDS Classification of Leaf Tobacco Covering Classes, Types and Groups of Grades § 30.2 Leaf... stemming, sweating or fermenting, and conditioning are not regarded as manufacturing processes. Leaf...

7 CFR 29.3036 - Leaf surface.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 2 2013-01-01 2013-01-01 false Leaf surface. 29.3036 Section 29.3036 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Leaf surface. The smoothness or roughness of the web or lamina of a tobacco leaf. Leaf surface is...

7 CFR 30.2 - Leaf tobacco.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 2 2013-01-01 2013-01-01 false Leaf tobacco. 30.2 Section 30.2 Agriculture... AND STANDARDS Classification of Leaf Tobacco Covering Classes, Types and Groups of Grades § 30.2 Leaf... stemming, sweating or fermenting, and conditioning are not regarded as manufacturing processes. Leaf...

A Role for Mitochondrial Phosphoenolpyruvate Carboxykinase (PEPCK-M) in the Regulation of Hepatic Gluconeogenesis*

PubMed Central

Stark, Romana; Guebre-Egziabher, Fitsum; Zhao, Xiaojian; Feriod, Colleen; Dong, Jianying; Alves, Tiago C.; Ioja, Simona; Pongratz, Rebecca L.; Bhanot, Sanjay; Roden, Michael; Cline, Gary W.; Shulman, Gerald I.; Kibbey, Richard G.

2014-01-01

Synthesis of phosphoenolpyruvate (PEP) from oxaloacetate is an absolute requirement for gluconeogenesis from mitochondrial substrates. Generally, this reaction has solely been attributed to the cytosolic isoform of PEPCK (PEPCK-C), although loss of the mitochondrial isoform (PEPCK-M) has never been assessed. Despite catalyzing the same reaction, to date the only significant role reported in mammals for the mitochondrial isoform is as a glucose sensor necessary for insulin secretion. We hypothesized that this nutrient-sensing mitochondrial GTP-dependent pathway contributes importantly to gluconeogenesis. PEPCK-M was acutely silenced in gluconeogenic tissues of rats using antisense oligonucleotides both in vivo and in isolated hepatocytes. Silencing PEPCK-M lowers plasma glucose, insulin, and triglycerides, reduces white adipose, and depletes hepatic glycogen, but raises lactate. There is a switch of gluconeogenic substrate preference to glycerol that quantitatively accounts for a third of glucose production. In contrast to the severe mitochondrial deficiency characteristic of PEPCK-C knock-out livers, hepatocytes from PEPCK-M-deficient livers maintained normal oxidative function. Consistent with its predicted role, gluconeogenesis rates from hepatocytes lacking PEPCK-M are severely reduced for lactate, alanine, and glutamine, but not for pyruvate and glycerol. Thus, PEPCK-M has a direct role in fasted and fed glucose homeostasis, and this mitochondrial GTP-dependent pathway should be reconsidered for its involvement in both normal and diabetic metabolism. PMID:24497630

A role for mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) in the regulation of hepatic gluconeogenesis.

PubMed

Stark, Romana; Guebre-Egziabher, Fitsum; Zhao, Xiaojian; Feriod, Colleen; Dong, Jianying; Alves, Tiago C; Ioja, Simona; Pongratz, Rebecca L; Bhanot, Sanjay; Roden, Michael; Cline, Gary W; Shulman, Gerald I; Kibbey, Richard G

2014-03-14

Synthesis of phosphoenolpyruvate (PEP) from oxaloacetate is an absolute requirement for gluconeogenesis from mitochondrial substrates. Generally, this reaction has solely been attributed to the cytosolic isoform of PEPCK (PEPCK-C), although loss of the mitochondrial isoform (PEPCK-M) has never been assessed. Despite catalyzing the same reaction, to date the only significant role reported in mammals for the mitochondrial isoform is as a glucose sensor necessary for insulin secretion. We hypothesized that this nutrient-sensing mitochondrial GTP-dependent pathway contributes importantly to gluconeogenesis. PEPCK-M was acutely silenced in gluconeogenic tissues of rats using antisense oligonucleotides both in vivo and in isolated hepatocytes. Silencing PEPCK-M lowers plasma glucose, insulin, and triglycerides, reduces white adipose, and depletes hepatic glycogen, but raises lactate. There is a switch of gluconeogenic substrate preference to glycerol that quantitatively accounts for a third of glucose production. In contrast to the severe mitochondrial deficiency characteristic of PEPCK-C knock-out livers, hepatocytes from PEPCK-M-deficient livers maintained normal oxidative function. Consistent with its predicted role, gluconeogenesis rates from hepatocytes lacking PEPCK-M are severely reduced for lactate, alanine, and glutamine, but not for pyruvate and glycerol. Thus, PEPCK-M has a direct role in fasted and fed glucose homeostasis, and this mitochondrial GTP-dependent pathway should be reconsidered for its involvement in both normal and diabetic metabolism.

Apparent over-investment in leaf venation relaxes leaf morphological constraints on photosynthesis in arid habitats

NASA Astrophysics Data System (ADS)

de Boer, Hugo; Drake, Paul; Veneklaas, Erik

2017-04-01

The close relationship between leaf water status and stomatal conductance implies that the hydraulic architecture of leaves poses an important constraint on transpiration, specifically in arid environments with high evaporative demands. However, it remains uncertain how morphological, hydraulic and photosynthetic traits are coordinated to achieve optimal leaf functioning in arid environments. Critical is that leaf veins supply the mesophyll with water that evaporates when stomata are open to allow CO2 uptake for photosynthesis. Theoretical analyses suggest that water is optimally distributed in the mesophyll when the lateral distance between veins (dx) is equal to the distance from these veins to the epidermis (dy), expressed as dx:dy≈1. Although this theory is supported by observations on many derived angiosperms, we hypothesize that plants in arid environments may reduce dx:dy below unity owing to climate-specific functional adaptations of increased leaf thickness and increased vein density. To test our hypothesis we assembled leaf hydraulic, morphological and photosynthetic traits of 68 species from the Eucalyptus and Corymbia genera (termed eucalypts) along an aridity gradient in southwestern Australia. We inferred the potential gas exchange advantage of reducing dx beyond dy using a model that links leaf morphology and hydraulics to photosynthesis. Our observations reveal that eucalypts in arid environments have thick amphistomatous leaves with high vein densities, resulting in dx:dy ratios that range from 1.6 to 0.15 along the aridity gradient. Our model suggests that as leaves become thicker, the effect of reducing dx beyond dy is to offset the reduction in leaf gas exchange that would result from maintaining dx:dy at unity. This apparent over-investment in leaf venation may be explained from the selective pressure of aridity, under which traits associated with long leaf lifespan, high hydraulic and thermal capacitances, and high potential rates of leaf

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

PubMed

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

2009-12-24

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

In vitro antiglioma action of indomethacin is mediated via AMP-activated protein kinase/mTOR complex 1 signalling pathway.

PubMed

Pantovic, Aleksandar; Bosnjak, Mihajlo; Arsikin, Katarina; Kosic, Milica; Mandic, Milos; Ristic, Biljana; Tosic, Jelena; Grujicic, Danica; Isakovic, Aleksandra; Micic, Nikola; Trajkovic, Vladimir; Harhaji-Trajkovic, Ljubica

2017-02-01

We investigated the role of the intracellular energy-sensing AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway in the in vitro antiglioma effect of the cyclooxygenase (COX) inhibitor indomethacin. Indomethacin was more potent than COX inhibitors diclofenac, naproxen, and ketoprofen in reducing the viability of U251 human glioma cells. Antiglioma effect of the drug was associated with p21 increase and G 2 M cell cycle arrest, as well as with oxidative stress, mitochondrial depolarization, caspase activation, and the induction of apoptosis. Indomethacin increased the phosphorylation of AMPK and its targets Raptor and acetyl-CoA carboxylase (ACC), and reduced the phosphorylation of mTOR and mTOR complex 1 (mTORC1) substrates p70S6 kinase and PRAS40 (Ser183). AMPK knockdown by RNA interference, as well as the treatment with the mTORC1 activator leucine, prevented indomethacin-mediated mTORC1 inhibition and cytotoxic action, while AMPK activators metformin and AICAR mimicked the effects of the drug. AMPK activation by indomethacin correlated with intracellular ATP depletion and increase in AMP/ATP ratio, and was apparently independent of COX inhibition or the increase in intracellular calcium. Finally, the toxicity of indomethacin towards primary human glioma cells was associated with the activation of AMPK/Raptor/ACC and subsequent suppression of mTORC1/S6K. By demonstrating the involvement of AMPK/mTORC1 pathway in the antiglioma action of indomethacin, our results support its further exploration in glioma therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Acetylcholine but not adenosine triggers preconditioning through PI3-kinase and a tyrosine kinase.

PubMed

Qin, Qining; Downey, James M; Cohen, Michael V

2003-02-01

Adenosine and acetylcholine (ACh) trigger preconditioning by different signaling pathways. The involvement of phosphatidylinositol 3-kinase (PI3-kinase), a protein tyrosine kinase, and Src family tyrosine kinase in preconditioning was evaluated in isolated rabbit hearts. Either wortmannin (PI3-kinase blocker), genistein (tyrosine kinase blocker), lavendustin A (tyrosine kinase blocker), or 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2; Src family tyrosine kinase blocker) was given for 15 min to bracket a 5-min infusion of either adenosine or ACh (trigger phase). The hearts then underwent 30 min of regional ischemia. Infarct size for ACh alone was 9.3 +/- 3.5% of the risk zone versus 34.3 +/- 4.1% in controls. All four inhibitors blocked ACh-induced protection. When wortmannin or PP2 was infused only during the 30-min ischemic period (mediator phase), ACh-induced protection was not affected (7.4 +/- 2.1% and 9.7 +/- 1.7% infarction, respectively). Adenosine-triggered protection was not blocked by any of the inhibitors. Therefore, PI3-kinase and at least one protein tyrosine kinase, probably Src kinase, are involved in the trigger phase of ACh-induced, but not adenosine-induced, preconditioning. Neither PI3-kinase nor Src kinase is a mediator of the protection of ACh.

Leaf primordium size specifies leaf width and vein number among row-type classes in barley.

PubMed

Thirulogachandar, Venkatasubbu; Alqudah, Ahmad M; Koppolu, Ravi; Rutten, Twan; Graner, Andreas; Hensel, Goetz; Kumlehn, Jochen; Bräutigam, Andrea; Sreenivasulu, Nese; Schnurbusch, Thorsten; Kuhlmann, Markus

2017-08-01

Exploring genes with impact on yield-related phenotypes is the preceding step to accomplishing crop improvements while facing a growing world population. A genome-wide association scan on leaf blade area (LA) in a worldwide spring barley collection (Hordeum vulgare L.), including 125 two- and 93 six-rowed accessions, identified a gene encoding the homeobox transcription factor, Six-rowed spike 1 (VRS1). VRS1 was previously described as a key domestication gene affecting spike development. Its mutation converts two-rowed (wild-type VRS1, only central fertile spikelets) into six-rowed spikes (mutant vrs1, fully developed fertile central and lateral spikelets). Phenotypic analyses of mutant and wild-type leaves revealed that mutants had an increased leaf width with more longitudinal veins. The observed significant increase of LA and leaf nitrogen (%) during pre-anthesis development in vrs1 mutants also implies a link between wider leaf and grain number, which was validated from the association of vrs1 locus with wider leaf and grain number. Histological and gene expression analyses indicated that VRS1 might influence the size of leaf primordia by affecting cell proliferation of leaf primordial cells. This finding was supported by the transcriptome analysis of mutant and wild-type leaf primordia where in the mutant transcriptional activation of genes related to cell proliferation was detectable. Here we show that VRS1 has an independent role on barley leaf development which might influence the grain number. © 2017 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

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

PubMed Central

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

1986-01-01

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

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

PubMed

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

2016-03-01

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

Functional reconstitution of the Mycobacterium tuberculosis long-chain acyl-CoA carboxylase from multiple acyl-CoA subunits.

PubMed

Bazet Lyonnet, Bernardo; Diacovich, Lautaro; Gago, Gabriela; Spina, Lucie; Bardou, Fabienne; Lemassu, Anne; Quémard, Annaïk; Gramajo, Hugo

2017-04-01

Mycobacterium tuberculosis produces a large number of structurally diverse lipids that have been implicated in the pathogenicity, persistence and antibiotic resistance of this organism. Most building blocks involved in the biosynthesis of all these lipids are generated by acyl-CoA carboxylases whose subunit composition and physiological roles have not yet been clearly established. Inconclusive data in the literature refer to the exact protein composition and substrate specificity of the enzyme complex that produces the long-chain α-carboxy-acyl-CoAs, which are substrates involved in the last step of condensation mediated by the polyketide synthase 13 to synthesize mature mycolic acids. Here we have successfully reconstituted the long-chain acyl-CoA carboxylase (LCC) complex from its purified components, the α subunit (AccA3), the ε subunit (AccE5) and the two β subunits (AccD4 and AccD5), and demonstrated that the four subunits are essential for its activity. Furthermore, we also showed by substrate competition experiments and the use of a specific inhibitor that the AccD5 subunit's role in the carboxylation of the long acyl-CoAs, as part of the LCC complex, was structural rather than catalytic. Moreover, AccD5 was also able to carboxylate its natural substrates, acetyl-CoA and propionyl-CoA, in the context of the LCC enzyme complex. Thus, the supercomplex formed by these four subunits has the potential to generate the main substrates, malonyl-CoA, methylmalonyl-CoA and α-carboxy-C 24-26 -CoA, used as condensing units for the biosynthesis of all the lipids present in this pathogen. © 2017 Federation of European Biochemical Societies.

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

DOEpatents

Houtz, Robert L.

1999-01-01

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

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

DOEpatents

Houtz, R.L.

1999-02-02

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

Body weight management effect of burdock (Arctium lappa L.) root is associated with the activation of AMP-activated protein kinase in human HepG2 cells.

PubMed

Kuo, Daih-Huang; Hung, Ming-Chi; Hung, Chao-Ming; Liu, Li-Min; Chen, Fu-An; Shieh, Po-Chuen; Ho, Chi-Tang; Way, Tzong-Der

2012-10-01

Burdock (Arcticum lappa L.) root is used in folk medicine and also as a vegetable in Asian countries. In the present study, burdock root treatment significantly reduced body weight in rats. To evaluate the bioactive compounds, we successively extracted the burdock root with ethanol (AL-1), and fractionated it with n-hexane (AL-2), ethyl acetate (AL-3), n-butanol (AL-4), and water (AL-5). Among these fractions, AL-2 contained components with the most effective hypolipidemic potential in human hepatoma HepG2 cells. AL-2 decreased the expression of fatty acid synthase (FASN) and inhibited the activity of acetyl-coenzyme A carboxylase (ACC) by stimulating AMP-activated protein kinase (AMPK) through the LKB1 pathway. Three active compounds were identified from the AL-2, namely α-linolenic acid, methyl α-linolenate, and methyl oleate. These results suggest that burdock root is expected to be useful for body weight management. Copyright © 2012 Elsevier Ltd. All rights reserved.

Changes in small angle X-ray scattering parameters observed upon ligand binding to rabbit muscle pyruvate kinase are not correlated with allosteric transitions†

PubMed Central

Fenton, Aron W.; Williams, Rachel; Trewhella, Jill

2010-01-01

Protein fluorescence and small-angle X-ray scattering (SAXS) have been used to monitor effector affinity and conformational changes previously associated with allosteric regulation in rabbit muscle pyruvate kinase (M1-PYK). In the absence of substrate (phosphoenolpyruvate; PEP), SAXS-monitored conformational changes in M1-PYK elicited by the binding of phenylalanine (an allosteric inhibitor that reduces the affinity of M1-PYK for PEP) are similar to those observed upon binding of alanine or 2-aminobutyric acid. Under the current assay conditions, these small amino acids bind to the protein, but elicit a minimal change in the affinity of the protein for PEP. Therefore, if changes in scattering signatures represent cleft closure via domain rotation as previously interpreted, it can be concluded that these motions are not sufficient to elicit allosteric inhibition. Additionally, although PEP has similar affinities for the free enzyme and the M1-PYK/small-amino-acid complexes (i.e. the small amino acids have minimal allosteric effects), PEP binding elicits different changes in the SAXS signature of the free enzyme vs. the M1-PYK/small-amino-acid complexes. PMID:20712377

Leaf-rolling in maize crops: from leaf scoring to canopy-level measurements for phenotyping

PubMed Central

Madec, Simon; Irfan, Kamran; Lopez, Jeremy; Comar, Alexis; Hemmerlé, Matthieu; Dutartre, Dan; Praud, Sebastien; Tixier, Marie Helene

2018-01-01

Abstract Leaf rolling in maize crops is one of the main plant reactions to water stress that can be visually scored in the field. However, leaf-scoring techniques do not meet the high-throughput requirements needed by breeders for efficient phenotyping. Consequently, this study investigated the relationship between leaf-rolling scores and changes in canopy structure that can be determined by high-throughput remote-sensing techniques. Experiments were conducted in 2015 and 2016 on maize genotypes subjected to water stress. Leaf-rolling was scored visually over the whole day around the flowering stage. Concurrent digital hemispherical photographs were taken to evaluate the impact of leaf-rolling on canopy structure using the computed fraction of intercepted diffuse photosynthetically active radiation, FIPARdif. The results showed that leaves started to roll due to water stress around 09:00 h and leaf-rolling reached its maximum around 15:00 h (the photoperiod was about 05:00–20:00 h). In contrast, plants maintained under well-watered conditions did not show any significant rolling during the same day. A canopy-level index of rolling (CLIR) is proposed to quantify the diurnal changes in canopy structure induced by leaf-rolling. It normalizes for the differences in FIPARdif between genotypes observed in the early morning when leaves are unrolled, as well as for yearly effects linked to environmental conditions. Leaf-level rolling score was very strongly correlated with changes in canopy structure as described by the CLIR (r2=0.86, n=370). The daily time course of rolling was characterized using the amplitude of variation, and the rate and the timing of development computed at both the leaf and canopy levels. Results obtained from eight genotypes common between the two years of experiments showed that the amplitude of variation of the CLIR was the more repeatable trait (Spearman coefficient ρ=0.62) as compared to the rate (ρ=0.29) and the timing of development (ρ=0

Apparent Overinvestment in Leaf Venation Relaxes Leaf Morphological Constraints on Photosynthesis in Arid Habitats.

PubMed

de Boer, Hugo J; Drake, Paul L; Wendt, Erin; Price, Charles A; Schulze, Ernst-Detlef; Turner, Neil C; Nicolle, Dean; Veneklaas, Erik J

2016-12-01

Leaf veins supply the mesophyll with water that evaporates when stomata are open to allow CO 2 uptake for photosynthesis. Theoretical analyses suggest that water is optimally distributed in the mesophyll when the lateral distance between veins (d x ) is equal to the distance from these veins to the epidermis (d y ), expressed as d x :d y ≈ 1. Although this theory is supported by observations of many derived angiosperms, we hypothesize that plants in arid environments may reduce d x :d y below unity owing to climate-specific functional adaptations of increased leaf thickness and increased vein density. To test our hypothesis, we assembled leaf hydraulic, morphological, and photosynthetic traits of 68 species from the Eucalyptus and Corymbia genera (termed eucalypts) along an aridity gradient in southwestern Australia. We inferred the potential gas-exchange advantage of reducing d x beyond d y using a model that links leaf morphology and hydraulics to photosynthesis. Our observations reveal that eucalypts in arid environments have thick amphistomatous leaves with high vein densities, resulting in d x :d y ratios that range from 1.6 to 0.15 along the aridity gradient. Our model suggests that, as leaves become thicker, the effect of reducing d x beyond d y is to offset the reduction in leaf gas exchange that would result from maintaining d x :d y at unity. This apparent overinvestment in leaf venation may be explained from the selective pressure of aridity, under which traits associated with long leaf life span, high hydraulic and thermal capacitances, and high potential rates of leaf water transport confer a competitive advantage. © 2016 American Society of Plant Biologists. All Rights Reserved.

The crystal structure of Toxoplasma gondii pyruvate kinase 1.

PubMed

Bakszt, Rebecca; Wernimont, Amy; Allali-Hassani, Abdellah; Mok, Man Wai; Hills, Tanya; Hui, Raymond; Pizarro, Juan C

2010-09-14

Pyruvate kinase (PK), which catalyzes the final step in glycolysis converting phosphoenolpyruvate to pyruvate, is a central metabolic regulator in most organisms. Consequently PK represents an attractive therapeutic target in cancer and human pathogens, like Apicomplexans. The phylum Aplicomplexa, a group of exclusively parasitic organisms, includes the genera Plasmodium, Cryptosporidium and Toxoplasma, the etiological agents of malaria, cryptosporidiosis and toxoplasmosis respectively. Toxoplasma gondii infection causes a mild illness and is a very common infection affecting nearly one third of the world's population. We have determined the crystal structure of the PK1 enzyme from T. gondii, with the B domain in the open and closed conformations. We have also characterized its enzymatic activity and confirmed glucose-6-phosphate as its allosteric activator. This is the first description of a PK enzyme in a closed inactive conformation without any bound substrate. Comparison of the two tetrameric TgPK1 structures indicates a reorientation of the monomers with a concomitant change in the buried surface among adjacent monomers. The change in the buried surface was associated with significant B domain movements in one of the interacting monomers. We hypothesize that a loop in the interface between the A and B domains plays an important role linking the position of the B domain to the buried surface among monomers through two α-helices. The proposed model links the catalytic cycle of the enzyme with its domain movements and highlights the contribution of the interface between adjacent subunits. In addition, an unusual ordered conformation was observed in one of the allosteric binding domains and it is related to a specific apicomplexan insertion. The sequence and structural particularity would explain the atypical activation by a mono-phosphorylated sugar. The sum of peculiarities raises this enzyme as an emerging target for drug discovery.

The Crystal Structure of Toxoplasma gondii Pyruvate Kinase 1

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

Bakszt, R.; Wernimont, A; Allali-Hassani, A

Pyruvate kinase (PK), which catalyzes the final step in glycolysis converting phosphoenolpyruvate to pyruvate, is a central metabolic regulator in most organisms. Consequently PK represents an attractive therapeutic target in cancer and human pathogens, like Apicomplexans. The phylum Aplicomplexa, a group of exclusively parasitic organisms, includes the genera Plasmodium, Cryptosporidium and Toxoplasma, the etiological agents of malaria, cryptosporidiosis and toxoplasmosis respectively. Toxoplasma gondii infection causes a mild illness and is a very common infection affecting nearly one third of the world's population. We have determined the crystal structure of the PK1 enzyme from T. gondii, with the B domain inmore » the open and closed conformations. We have also characterized its enzymatic activity and confirmed glucose-6-phosphate as its allosteric activator. This is the first description of a PK enzyme in a closed inactive conformation without any bound substrate. Comparison of the two tetrameric TgPK1 structures indicates a reorientation of the monomers with a concomitant change in the buried surface among adjacent monomers. The change in the buried surface was associated with significant B domain movements in one of the interacting monomers. We hypothesize that a loop in the interface between the A and B domains plays an important role linking the position of the B domain to the buried surface among monomers through two {alpha}-helices. The proposed model links the catalytic cycle of the enzyme with its domain movements and highlights the contribution of the interface between adjacent subunits. In addition, an unusual ordered conformation was observed in one of the allosteric binding domains and it is related to a specific apicomplexan insertion. The sequence and structural particularity would explain the atypical activation by a mono-phosphorylated sugar. The sum of peculiarities raises this enzyme as an emerging target for drug discovery.« less

Scaling up stomatal conductance from leaf to canopy using a dual-leaf model for estimating crop evapotranspiration.

PubMed

Ding, Risheng; Kang, Shaozhong; Du, Taisheng; Hao, Xinmei; Zhang, Yanqun

2014-01-01

The dual-source Shuttleworth-Wallace model has been widely used to estimate and partition crop evapotranspiration (λET). Canopy stomatal conductance (Gsc), an essential parameter of the model, is often calculated by scaling up leaf stomatal conductance, considering the canopy as one single leaf in a so-called "big-leaf" model. However, Gsc can be overestimated or underestimated depending on leaf area index level in the big-leaf model, due to a non-linear stomatal response to light. A dual-leaf model, scaling up Gsc from leaf to canopy, was developed in this study. The non-linear stomata-light relationship was incorporated by dividing the canopy into sunlit and shaded fractions and calculating each fraction separately according to absorbed irradiances. The model includes: (1) the absorbed irradiance, determined by separately integrating the sunlit and shaded leaves with consideration of both beam and diffuse radiation; (2) leaf area for the sunlit and shaded fractions; and (3) a leaf conductance model that accounts for the response of stomata to PAR, vapor pressure deficit and available soil water. In contrast to the significant errors of Gsc in the big-leaf model, the predicted Gsc using the dual-leaf model had a high degree of data-model agreement; the slope of the linear regression between daytime predictions and measurements was 1.01 (R2 = 0.98), with RMSE of 0.6120 mm s-1 for four clear-sky days in different growth stages. The estimates of half-hourly λET using the dual-source dual-leaf model (DSDL) agreed well with measurements and the error was within 5% during two growing seasons of maize with differing hydrometeorological and management strategies. Moreover, the estimates of soil evaporation using the DSDL model closely matched actual measurements. Our results indicate that the DSDL model can produce more accurate estimation of Gsc and λET, compared to the big-leaf model, and thus is an effective alternative approach for estimating and partitioning λET.

Alkyl isothiocyanates suppress epidermal growth factor receptor kinase activity but augment tyrosine kinase activity.

PubMed

Nomura, Takahiro; Uehara, Yoshimasa; Kawajiri, Hiroo; Ryoyama, Kazuo; Yamori, Takao; Fuke, Yoko

2009-10-01

We have reported the in vitro and in vivo anticancer activities of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) derived from a Japanese spice, wasabi. In order to obtain some clues about the mechanism of the anticancer activity, we have studied the effect of alkyl isothiocyanates (MITCs) on protein kinase activities. The anti-autophosphorylation activity of MITCs with respect to the epidermal growth factor (EGF)-stimulated receptor kinase of A431 epidermoid carcinoma cells was examined by incorporation of radioactive ATP into an acid-insoluble fraction. Their anti-phosphorylation activity with respect to the non-receptor protein kinase was analyzed by a standard SDS-PAGE method. All the tested MITCs interfered with the EGF-stimulated receptor kinase activity in a dose-dependent manner, although their effects were less than 1/10 of that of erbstatin in microg/ml. On the other hand, the MITCs did not interfere with non-receptor kinases (kinase A, kinase C, tyrosine kinase and calmodulin dependent kinase III), but enhanced non-receptor tyrosine kinase. A possible anticancer mechanism of MITCs may involve the suppression of EGF receptor kinase activity and augmentation of non-receptor PTK.

Seasonality of Leaf Carbon Isotopic Composition and Leaf Water Isotopic Enrichment in a Mixed Evergreen Forest in Southern California

NASA Astrophysics Data System (ADS)

Santiago, L. S.; Sickman, J. O.; Goulden, M.; DeVan, C.; Pasquini, S. C.; Pivovaroff, A. L.

2011-12-01

Leaf carbon isotopic composition and leaf water isotopic enrichment reflect physiological processes and are important for linking local and regional scale processes to global patterns. We investigated how seasonality affects the isotopic composition of bulk leaf carbon, leaf sugar carbon, and leaf water hydrogen under a Mediterranean climate. Leaf and stem samples were collected monthly from four tree species (Calocedrus decurrens, Pinus lambertiana, Pinus ponderosa, and Quercus chrysolepis) at the James San Jacinto Mountain Reserve in southern California. Mean monthly bulk leaf carbon isotopic composition varied from -34.5 % in P. ponderosa to -24.7 % in P. lambertiana and became more depleted in 13C from the spring to the summer. Mean monthly leaf sugar varied from -29.3 % in P. ponderosa to -21.8 % in P. lambertiana and was enriched in 13C during the winter, spring and autumn, but depleted during the mid-summer. Leaf water hydrogen isotopic composition was 28.4 to 68.8 % more enriched in deuterium than source water and this enrichment was greater as seasonal drought progressed. These data indicate that leaf carbon and leaf water hydrogen isotopic composition provide sensitive measures that connect plant physiological processes to short-term climatic variability.

7 CFR 28.464 - Leaf Grade 4.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 2 2013-01-01 2013-01-01 false Leaf Grade 4. 28.464 Section 28.464 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.464 Leaf Grade 4. Leaf Grade 4 is leaf which is within the range represented by...

7 CFR 28.465 - Leaf Grade 5.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 2 2011-01-01 2011-01-01 false Leaf Grade 5. 28.465 Section 28.465 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.465 Leaf Grade 5. Leaf Grade 5 is leaf which is within the range represented by...

7 CFR 28.467 - Leaf Grade 7.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 2 2014-01-01 2014-01-01 false Leaf Grade 7. 28.467 Section 28.467 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.467 Leaf Grade 7. Leaf Grade 7 is leaf which is within the range represented by...

7 CFR 28.463 - Leaf Grade 3.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 2 2013-01-01 2013-01-01 false Leaf Grade 3. 28.463 Section 28.463 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.463 Leaf Grade 3. Leaf Grade 3 is leaf which is within the range represented by...

7 CFR 28.462 - Leaf Grade 2.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 2 2012-01-01 2012-01-01 false Leaf Grade 2. 28.462 Section 28.462 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.462 Leaf Grade 2. Leaf Grade 2 is leaf which is within the range represented by...

7 CFR 28.462 - Leaf Grade 2.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 2 2011-01-01 2011-01-01 false Leaf Grade 2. 28.462 Section 28.462 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.462 Leaf Grade 2. Leaf Grade 2 is leaf which is within the range represented by...

7 CFR 28.465 - Leaf Grade 5.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false Leaf Grade 5. 28.465 Section 28.465 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.465 Leaf Grade 5. Leaf Grade 5 is leaf which is within the range represented by...

7 CFR 28.464 - Leaf Grade 4.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 2 2014-01-01 2014-01-01 false Leaf Grade 4. 28.464 Section 28.464 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.464 Leaf Grade 4. Leaf Grade 4 is leaf which is within the range represented by...

7 CFR 28.462 - Leaf Grade 2.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false Leaf Grade 2. 28.462 Section 28.462 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.462 Leaf Grade 2. Leaf Grade 2 is leaf which is within the range represented by...

7 CFR 28.465 - Leaf Grade 5.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 2 2013-01-01 2013-01-01 false Leaf Grade 5. 28.465 Section 28.465 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.465 Leaf Grade 5. Leaf Grade 5 is leaf which is within the range represented by...

7 CFR 28.463 - Leaf Grade 3.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 2 2011-01-01 2011-01-01 false Leaf Grade 3. 28.463 Section 28.463 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.463 Leaf Grade 3. Leaf Grade 3 is leaf which is within the range represented by...

7 CFR 28.462 - Leaf Grade 2.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 2 2013-01-01 2013-01-01 false Leaf Grade 2. 28.462 Section 28.462 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.462 Leaf Grade 2. Leaf Grade 2 is leaf which is within the range represented by...

7 CFR 28.464 - Leaf Grade 4.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 2 2011-01-01 2011-01-01 false Leaf Grade 4. 28.464 Section 28.464 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.464 Leaf Grade 4. Leaf Grade 4 is leaf which is within the range represented by...

7 CFR 28.463 - Leaf Grade 3.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false Leaf Grade 3. 28.463 Section 28.463 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.463 Leaf Grade 3. Leaf Grade 3 is leaf which is within the range represented by...

7 CFR 28.466 - Leaf Grade 6.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 2 2012-01-01 2012-01-01 false Leaf Grade 6. 28.466 Section 28.466 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.466 Leaf Grade 6. Leaf Grade 6 is leaf which is within the range represented by...

7 CFR 28.461 - Leaf Grade 1.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 2 2011-01-01 2011-01-01 false Leaf Grade 1. 28.461 Section 28.461 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.461 Leaf Grade 1. Leaf Grade 1 is leaf which is within the range represented by...

7 CFR 28.461 - Leaf Grade 1.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false Leaf Grade 1. 28.461 Section 28.461 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.461 Leaf Grade 1. Leaf Grade 1 is leaf which is within the range represented by...

7 CFR 28.461 - Leaf Grade 1.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 2 2012-01-01 2012-01-01 false Leaf Grade 1. 28.461 Section 28.461 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.461 Leaf Grade 1. Leaf Grade 1 is leaf which is within the range represented by...

7 CFR 28.465 - Leaf Grade 5.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 2 2012-01-01 2012-01-01 false Leaf Grade 5. 28.465 Section 28.465 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.465 Leaf Grade 5. Leaf Grade 5 is leaf which is within the range represented by...

7 CFR 28.463 - Leaf Grade 3.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 2 2012-01-01 2012-01-01 false Leaf Grade 3. 28.463 Section 28.463 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.463 Leaf Grade 3. Leaf Grade 3 is leaf which is within the range represented by...

7 CFR 28.467 - Leaf Grade 7.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 2 2011-01-01 2011-01-01 false Leaf Grade 7. 28.467 Section 28.467 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.467 Leaf Grade 7. Leaf Grade 7 is leaf which is within the range represented by...

7 CFR 28.467 - Leaf Grade 7.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 2 2013-01-01 2013-01-01 false Leaf Grade 7. 28.467 Section 28.467 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.467 Leaf Grade 7. Leaf Grade 7 is leaf which is within the range represented by...

7 CFR 28.466 - Leaf Grade 6.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 2 2014-01-01 2014-01-01 false Leaf Grade 6. 28.466 Section 28.466 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.466 Leaf Grade 6. Leaf Grade 6 is leaf which is within the range represented by...

7 CFR 28.465 - Leaf Grade 5.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 2 2014-01-01 2014-01-01 false Leaf Grade 5. 28.465 Section 28.465 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.465 Leaf Grade 5. Leaf Grade 5 is leaf which is within the range represented by...

7 CFR 28.462 - Leaf Grade 2.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 2 2014-01-01 2014-01-01 false Leaf Grade 2. 28.462 Section 28.462 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.462 Leaf Grade 2. Leaf Grade 2 is leaf which is within the range represented by...

7 CFR 28.463 - Leaf Grade 3.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 2 2014-01-01 2014-01-01 false Leaf Grade 3. 28.463 Section 28.463 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.463 Leaf Grade 3. Leaf Grade 3 is leaf which is within the range represented by...

7 CFR 28.464 - Leaf Grade 4.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 2 2012-01-01 2012-01-01 false Leaf Grade 4. 28.464 Section 28.464 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.464 Leaf Grade 4. Leaf Grade 4 is leaf which is within the range represented by...

7 CFR 28.461 - Leaf Grade 1.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 2 2013-01-01 2013-01-01 false Leaf Grade 1. 28.461 Section 28.461 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.461 Leaf Grade 1. Leaf Grade 1 is leaf which is within the range represented by...

7 CFR 28.466 - Leaf Grade 6.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false Leaf Grade 6. 28.466 Section 28.466 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.466 Leaf Grade 6. Leaf Grade 6 is leaf which is within the range represented by...

7 CFR 28.464 - Leaf Grade 4.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false Leaf Grade 4. 28.464 Section 28.464 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.464 Leaf Grade 4. Leaf Grade 4 is leaf which is within the range represented by...

7 CFR 28.466 - Leaf Grade 6.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 2 2011-01-01 2011-01-01 false Leaf Grade 6. 28.466 Section 28.466 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.466 Leaf Grade 6. Leaf Grade 6 is leaf which is within the range represented by...

7 CFR 28.466 - Leaf Grade 6.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 2 2013-01-01 2013-01-01 false Leaf Grade 6. 28.466 Section 28.466 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.466 Leaf Grade 6. Leaf Grade 6 is leaf which is within the range represented by...

7 CFR 28.467 - Leaf Grade 7.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 2 2012-01-01 2012-01-01 false Leaf Grade 7. 28.467 Section 28.467 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.467 Leaf Grade 7. Leaf Grade 7 is leaf which is within the range represented by...

7 CFR 28.461 - Leaf Grade 1.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 2 2014-01-01 2014-01-01 false Leaf Grade 1. 28.461 Section 28.461 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.461 Leaf Grade 1. Leaf Grade 1 is leaf which is within the range represented by...

7 CFR 28.467 - Leaf Grade 7.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false Leaf Grade 7. 28.467 Section 28.467 Agriculture..., TESTING, AND STANDARDS Standards Official Cotton Standards of the United States for the Leaf Grade of American Upland Cotton § 28.467 Leaf Grade 7. Leaf Grade 7 is leaf which is within the range represented by...

Biotin increases glucokinase expression via soluble guanylate cyclase/protein kinase G, adenosine triphosphate production and autocrine action of insulin in pancreatic rat islets.

PubMed

Vilches-Flores, Alonso; Tovar, Armando R; Marin-Hernandez, Alvaro; Rojas-Ochoa, Alberto; Fernandez-Mejia, Cristina

2010-07-01

Besides its role as a carboxylase prosthetic group, biotin has important effects on gene expression. However, the molecular mechanisms through which biotin exerts these effects are largely unknown. We previously found that biotin increases pancreatic glucokinase expression. We have now explored the mechanisms underlying this effect. Pancreatic islets from Wistar rats were treated with biotin, in the presence or absence of different types of inhibitors. Glucokinase mRNA and 18s rRNA abundance were determined by real-time PCR. Adenosine triphosphate (ATP) content was analyzed by fluorometry. Biotin treatment increased glucokinase mRNA abundance approximately one fold after 2 h; the effect was sustained up to 24 h. Inhibition of soluble guanylate cyclase or protein kinase G (PKG) signalling suppressed biotin-induced glucokinase expression. The cascade of events downstream of PKG in biotin-mediated gene transcription is not known. We found that inhibition of insulin secretion with diazoxide or nifedipine prevented biotin-stimulated glucokinase mRNA increase. Biotin treatment increased islet ATP content (control: 4.68+/-0.28; biotin treated: 6.62+/-0.26 pmol/islet) at 30 min. Inhibition of PKG activity suppressed the effects of biotin on ATP content. Insulin antibodies or inhibitors of phosphoinositol-3-kinase/Akt insulin signalling pathway prevented biotin-induced glucokinase expression. The nucleotide 8-Br-cGMP mimicked the biotin effects. We propose that the induction of pancreatic glucokinase mRNA by biotin involves guanylate cyclase and PKG activation, which leads to an increase in ATP content. This induces insulin secretion via ATP-sensitive potassium channels. Autocrine insulin, in turn, activates phosphoinositol-3-kinase/Akt signalling. Our results offer new insights into the pathways that participate in biotin-mediated gene expression. (c) 2010 Elsevier Inc. All rights reserved.

Site-directed mutagenesis of serine 158 demonstrates its role in spinach leaf sucrose-phosphate synthase modulation

NASA Technical Reports Server (NTRS)

Toroser, D.; McMichael, R. Jr; Krause, K. P.; Kurreck, J.; Sonnewald, U.; Stitt, M.; Huber, S. C.; Davies, E. (Principal Investigator)

1999-01-01

Site-directed mutagenesis of spinach sucrose-phosphate synthase (SPS) was performed to investigate the role of Ser158 in the modulation of spinach leaf SPS. Tobacco plants expressing the spinach wild-type (WT), S158A, S158T and S157F/S158E SPS transgenes were produced. Expression of transgenes appeared not to reduce expression of the tobacco host SPS. SPS activity in the WT and the S158T SPS transgenics showed light/dark modulation, whereas the S158A and S157F/S158E mutants were not similarly light/dark modulated: the S158A mutant enzyme was not inactivated in the dark, and the S157F/S158E was not activated in the light. The inability to modulate the activity of the S158A mutant enzyme by protein phosphorylation was demonstrated in vitro. The WT spinach enzyme immunopurified from dark transgenic tobacco leaves had a low initial activation state, and could be activated by PP2A and subsequently inactivated by SPS-kinase plus ATP. Rapid purification of the S158A mutant enzyme from dark leaves of transgenic plants using spinach-specific monoclonal antibodies yielded enzyme that had a high initial activation state, and pre-incubation with leaf PP2A or ATP plus SPS-kinase (the PKIII enzyme) caused little modulation of activity. The results demonstrate the regulatory significance of Ser158 as the major site responsible for dark inactivation of spinach SPS in vivo, and indicate that the significance of phosphorylation is the introduction of a negative charge at the Ser158 position.

Isolation and identification of peanut leaf proteins regulated by water stress.

PubMed

Akkasaeng, Chutipong; Tantisuwichwong, Napaporn; Chairam, Issariya; Prakrongrak, Narumon; Jogloy, Sanun; Pathanothai, Aran

2007-05-15

Water deficits trigger signaling cascades leading to modulation of protein expression in plant tissues. Identification of peanut leaf proteins regulated by water stress provides some insights of cellular and molecular response of peanut plants to drought stress. Peanut variety Khon Kaen 4, a water-stress sensitive variety, was grown in a growth chamber under controlled environment. Water stress was imposed on day 30 after seedling emergence by withholding watering peanut plants for 6 days as compared to plants adequately supplied with water. Total protein were prepared from a leaflet of fully expanded leaf on the main stem. Proteins were separated in duplicated gels using two-dimensional gel electrophoresis and visualized by silver nitrate staining. Image analysis was performed using ImageMaster 2D Platinum 5.0 to determine proteins regulated by water stress. Molecular mass and isoelectric point of each regulated protein were used in database queries for protein identification. One protein was induced under water stress and the homologous protein was identified as Serine/threonine-protein phosphatase PP 1. Five proteins were down-regulated by water deficit. The homologous proteins were chaperone protein DNAJ, auxin-responsive protein IAA29, peroxidase 43, caffeoyl-CoA O-methyltransferase and SNF1-related protein kinase regulatory subunit beta-2. Down-regulated proteins may be associated with sensitivity of the peanut variety to water stress.

Map-based cloning and functional analysis of YGL8, which controls leaf colour in rice (Oryza sativa).

PubMed

Zhu, Xiaoyan; Guo, Shuang; Wang, Zhongwei; Du, Qing; Xing, Yadi; Zhang, Tianquan; Shen, Wenqiang; Sang, Xianchun; Ling, Yinghua; He, Guanghua

2016-06-13

As the indispensable part of plant, leaf blade mainly functions as the production workshops where organic substance is produced by photosynthesis. Leaf colour mutation is a genetic phenomenon that has a high frequency and is easily identified. The mutations always exhibit negative impact on the development of plants in any of the different stages of growth. Up to now, numerous genes involved in leaf colour mutations have been cloned. In this study, a yellow-green leaf mutant, yellow-green leaf 8 (ygl8), with stable genetic phenotype, has been screened out in the progeny of an excellent indica restorer line Jinhui 10 with seeds treated by EMS. The levels of Chl a, Chl b and total chlorophyll were significantly lower in ygl8 than those in the WT throughout the whole growth period, while no clear change was noted in the Chl a/b ratio. Transmission electron microscopy demonstrated that the lamellae were clearly intumescent and intricately stacked in ygl8. Furthermore, compared with those of the WT, the stomatal conductance, intercellular CO2 concentration, photosynthetic rate and transpiration rate of ylg8 were all significantly lower. Map-based cloning results showed that Loc_Os01g73450, encoding a chloroplast-targeted UMP kinase, corresponded to Ygl8 and played an important role in regulating leaf colour in rice (Oryza sativa). Complementation of ygl8 with the WT DNA sequence of Loc_Os01g73450 led to restoration of the normal phenotype, and transgenic RNA interference plants showed a yellow-green colour. Analysis of the spatial and temporal expression of Ygl8 indicated that it was highly expressed in leaf blades and weakly expressed in other tissues. qRT-PCR also showed that the expression levels of the major Photosystem I core subunits plastome-encoded PsaA, PsaB and PsbC were significantly reduced in ygl8. The expression levels of nuclear-encoded gene involved in Chl biosynthesis HEMC, HEME, and PORA were also decreased when compared with the wild-type. Independent

Is leaf dry matter content a better predictor of soil fertility than specific leaf area?

PubMed Central

Hodgson, J. G.; Montserrat-Martí, G.; Charles, M.; Jones, G.; Wilson, P.; Shipley, B.; Sharafi, M.; Cerabolini, B. E. L.; Cornelissen, J. H. C.; Band, S. R.; Bogard, A.; Castro-Díez, P.; Guerrero-Campo, J.; Palmer, C.; Pérez-Rontomé, M. C.; Carter, G.; Hynd, A.; Romo-Díez, A.; de Torres Espuny, L.; Royo Pla, F.

2011-01-01

Background and Aims Specific leaf area (SLA), a key element of the ‘worldwide leaf economics spectrum’, is the preferred ‘soft’ plant trait for assessing soil fertility. SLA is a function of leaf dry matter content (LDMC) and leaf thickness (LT). The first, LDMC, defines leaf construction costs and can be used instead of SLA. However, LT identifies shade at its lowest extreme and succulence at its highest, and is not related to soil fertility. Why then is SLA more frequently used as a predictor of soil fertility than LDMC? Methods SLA, LDMC and LT were measured and leaf density (LD) estimated for almost 2000 species, and the capacity of LD to predict LDMC was examined, as was the relative contribution of LDMC and LT to the expression of SLA. Subsequently, the relationships between SLA, LDMC and LT with respect to soil fertility and shade were described. Key Results Although LD is strongly related to LDMC, and LDMC and LT each contribute equally to the expression of SLA, the exact relationships differ between ecological groupings. LDMC predicts leaf nitrogen content and soil fertility but, because LT primarily varies with light intensity, SLA increases in response to both increased shade and increased fertility. Conclusions Gradients of soil fertility are frequently also gradients of biomass accumulation with reduced irradiance lower in the canopy. Therefore, SLA, which includes both fertility and shade components, may often discriminate better between communities or treatments than LDMC. However, LDMC should always be the preferred trait for assessing gradients of soil fertility uncoupled from shade. Nevertheless, because leaves multitask, individual leaf traits do not necessarily exhibit exact functional equivalence between species. In consequence, rather than using a single stand-alone predictor, multivariate analyses using several leaf traits is recommended. PMID:21948627

Is leaf dry matter content a better predictor of soil fertility than specific leaf area?

PubMed

Hodgson, J G; Montserrat-Martí, G; Charles, M; Jones, G; Wilson, P; Shipley, B; Sharafi, M; Cerabolini, B E L; Cornelissen, J H C; Band, S R; Bogard, A; Castro-Díez, P; Guerrero-Campo, J; Palmer, C; Pérez-Rontomé, M C; Carter, G; Hynd, A; Romo-Díez, A; de Torres Espuny, L; Royo Pla, F

2011-11-01

Specific leaf area (SLA), a key element of the 'worldwide leaf economics spectrum', is the preferred 'soft' plant trait for assessing soil fertility. SLA is a function of leaf dry matter content (LDMC) and leaf thickness (LT). The first, LDMC, defines leaf construction costs and can be used instead of SLA. However, LT identifies shade at its lowest extreme and succulence at its highest, and is not related to soil fertility. Why then is SLA more frequently used as a predictor of soil fertility than LDMC? SLA, LDMC and LT were measured and leaf density (LD) estimated for almost 2000 species, and the capacity of LD to predict LDMC was examined, as was the relative contribution of LDMC and LT to the expression of SLA. Subsequently, the relationships between SLA, LDMC and LT with respect to soil fertility and shade were described. Although LD is strongly related to LDMC, and LDMC and LT each contribute equally to the expression of SLA, the exact relationships differ between ecological groupings. LDMC predicts leaf nitrogen content and soil fertility but, because LT primarily varies with light intensity, SLA increases in response to both increased shade and increased fertility. Gradients of soil fertility are frequently also gradients of biomass accumulation with reduced irradiance lower in the canopy. Therefore, SLA, which includes both fertility and shade components, may often discriminate better between communities or treatments than LDMC. However, LDMC should always be the preferred trait for assessing gradients of soil fertility uncoupled from shade. Nevertheless, because leaves multitask, individual leaf traits do not necessarily exhibit exact functional equivalence between species. In consequence, rather than using a single stand-alone predictor, multivariate analyses using several leaf traits is recommended.

The Ω-loop lid domain of phosphoenolpyruvate carboxykinase is essential for catalytic function

PubMed Central

Johnson, Troy A.; Holyoak, Todd

2012-01-01

Phosphoenolpyruvate carboxykinase (PEPCK) is an essential metabolic enzyme operating in the gluconeogenesis and glyceroneogenesis pathways. Recent studies have demonstrated that the enzyme contains a mobile active site lid domain that transitions between an open/disorded conformation to a closed/ordered conformation as the enzyme progresses through the catalytic cycle. The understanding of how this mobile domain functions in catalysis is incomplete. Previous studies show that the closure of the lid domain stabilizes the reaction intermediate and protects the reactive intermediate from spurious protonation and thus contributes to the fidelity of the enzyme. In order to more fully investigate the roles of the lid domain in PEPCK function we created three mutations that replaced the 11-residue lid domain with one, two or three glycine residues. Kinetic analysis of the mutant enzymes demonstrates that none of the enzyme constructs exhibit any measurable kinetic activity resulting in a decrease in the catalytic parameters by at least 106. Structural characterization of the mutants in complexes representing the catalytic cycle suggest that the inactivity is due to a role for the lid domain in the formation of the fully closed state of the enzyme that is required for catalytic function. In the absence of the lid domain, the enzyme is unable to achieve the fully closed state and is rendered inactive despite possessing all of the residues and substrates required for catalytic function. This work demonstrates how enzyme catalytic function can be abolished through the alteration of conformational equilibria despite all elements required for chemical conversion of substrates to products remaining intact. PMID:23127136

7 CFR 28.471 - Below Leaf Grade Cotton.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 2 2013-01-01 2013-01-01 false Below Leaf Grade Cotton. 28.471 Section 28.471... REGULATIONS COTTON CLASSING, TESTING, AND STANDARDS Standards Below Leaf Grade Cotton § 28.471 Below Leaf Grade Cotton. Below leaf grade cotton is American Upland cotton which is lower in leaf grade than Leaf...

7 CFR 28.471 - Below Leaf Grade Cotton.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false Below Leaf Grade Cotton. 28.471 Section 28.471... REGULATIONS COTTON CLASSING, TESTING, AND STANDARDS Standards Below Leaf Grade Cotton § 28.471 Below Leaf Grade Cotton. Below leaf grade cotton is American Upland cotton which is lower in leaf grade than Leaf...

7 CFR 28.471 - Below Leaf Grade Cotton.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 2 2011-01-01 2011-01-01 false Below Leaf Grade Cotton. 28.471 Section 28.471... REGULATIONS COTTON CLASSING, TESTING, AND STANDARDS Standards Below Leaf Grade Cotton § 28.471 Below Leaf Grade Cotton. Below leaf grade cotton is American Upland cotton which is lower in leaf grade than Leaf...

7 CFR 28.471 - Below Leaf Grade Cotton.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 2 2014-01-01 2014-01-01 false Below Leaf Grade Cotton. 28.471 Section 28.471... REGULATIONS COTTON CLASSING, TESTING, AND STANDARDS Standards Below Leaf Grade Cotton § 28.471 Below Leaf Grade Cotton. Below leaf grade cotton is American Upland cotton which is lower in leaf grade than Leaf...

7 CFR 28.471 - Below Leaf Grade Cotton.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 2 2012-01-01 2012-01-01 false Below Leaf Grade Cotton. 28.471 Section 28.471... REGULATIONS COTTON CLASSING, TESTING, AND STANDARDS Standards Below Leaf Grade Cotton § 28.471 Below Leaf Grade Cotton. Below leaf grade cotton is American Upland cotton which is lower in leaf grade than Leaf...

Scaling Up Stomatal Conductance from Leaf to Canopy Using a Dual-Leaf Model for Estimating Crop Evapotranspiration