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Christine A. Raines - One of the best experts on this subject based on the ideXlab platform.
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inter species variation in the oligomeric states of the higher plant Calvin Cycle enzymes glyceraldehyde 3 phosphate dehydrogenase and phosphoribulokinase
Journal of Experimental Botany, 2011Co-Authors: Thomas P Howard, Julie C Lloyd, Christine A. RainesAbstract:In darkened leaves the Calvin Cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein–protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the ‘non-regulatory’ A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed.
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Comparative sequence analysis of CP12, a small protein involved in the formation of a Calvin Cycle complex in photosynthetic organisms
Photosynthesis Research, 2010Co-Authors: René Groben, Christine A. Raines, Dimitrios Kaloudas, Bernard Offmann, Stephen C. Maberly, Brigitte GonteroAbstract:CP12, a small intrinsically unstructured protein, plays an important role in the regulation of the Calvin Cycle by forming a complex with phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). An extensive search in databases revealed 129 protein sequences from, higher plants, mosses and liverworts, different groups of eukaryotic algae and cyanobacteria. CP12 was identified throughout the Plantae, apart from in the Prasinophyceae. Within the Chromalveolata, two putative CP12 proteins have been found in the genomes of the diatom Thalassiosira pseudonana and the haptophyte Emiliania huxleyi , but specific searches in further chromalveolate genomes or EST datasets did not reveal any CP12 sequences in other Prymnesiophyceae, Dinophyceae or Pelagophyceae. A species from the Euglenophyceae within the Excavata also appeared to lack CP12. Phylogenetic analysis showed a clear separation into a number of higher taxonomic clades and among different forms of CP12 in higher plants. Cyanobacteria, Chlorophyceae, Rhodophyta and Glaucophyceae, Bryophyta, and the CP12-3 forms in higher plants all form separate clades. The degree of disorder of CP12 was higher in higher plants than in the eukaryotic algae and cyanobacteria apart from the green algal class Mesostigmatophyceae, which is ancestral to the streptophytes. This suggests that CP12 has evolved to become more flexible and possibly take on more general roles. Different features of the CP12 sequences in the different taxonomic groups and their potential functions and interactions in the Calvin Cycle are discussed.
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The Calvin Cycle revisited
Photosynthesis Research, 2003Co-Authors: Christine A. RainesAbstract:The sequence of reactions in the Calvin Cycle, and the biochemical characteristics of the enzymes involved, have been known for some time. However, the extent to which any individual enzyme controls the rate of carbon fixation has been a long standing question. Over the last 10 years, antisense transgenic plants have been used as tools to address this and have revealed some unexpected findings about the Calvin Cycle. It was shown that under a range of environmental conditions, the level of Rubisco protein had little impact on the control of carbon fixation. In addition, three of the four thioredoxin regulated enzymes, FBPase, PRKase and GAPDH, had negligible control of the Cycle. Unexpectedly, non-regulated enzymes catalysing reversible reactions, aldolase and transketolase, both exerted significant control over carbon flux. Furthermore, under a range of growth conditions SBPase was shown to have a significant level of control over the Calvin Cycle. These data led to the hypothesis that increasing the amounts of these enzymes may lead to an increase in photosynthetic carbon assimilation. Remarkably, photosynthetic capacity and growth were increased in tobacco plants expressing a bifunctional SBPase/FBPase enzyme. Future work is discussed which will further our understanding of this complex and important pathway, particularly in relation to the mechanisms that regulate and co-ordinate enzyme activity.
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computer modelling and experimental evidence for two steady states in the photosynthetic Calvin Cycle
FEBS Journal, 2001Co-Authors: Mark G Poolma, Christine A. Raines, Hulya Olce, Julie C Lloyd, David A FellAbstract:We present observations of photosynthetic carbon dioxide assimilation, and leaf starch content from genetically modified tobacco (Nicotiana tabacum) plants in which the activity of the Calvin Cycle enzyme, sedoheptulose-1,7-bisphosphatase, is reduced by an antisense construct. The measurements were made on leaves of varying ages and used to calculate the flux control coefficients of sedoheptulose-1,7-bisphosphatase over photosynthetic assimilation and starch synthesis. These calculations suggest that control coefficients for both are negative in young leaves, and positive in mature leaves. This behaviour is compared to control coefficients obtained from a detailed computer model of the Calvin Cycle. The comparison demonstrates that the experimental observations are consistent with bistable behaviour exhibited by the model, and provides the first experimental evidence that such behaviour in the Calvin Cycle occurs in vivo as well as in silico.
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new insights into the structure and function of sedoheptulose 1 7 bisphosphatase an important but neglected Calvin Cycle enzyme
Journal of Experimental Botany, 1999Co-Authors: Christine A. Raines, Julie C Lloyd, Trista A DyeAbstract:The photosynthetic carbon reduction (Calvin) Cycle is the primary pathway for carbon fixation and the enzyme sedoheptulose-1,7-bisphosphatase functions in the regenerative phase of this Cycle where it catalyses the dephosphorylation of sedoheptulose-1,7-bisphosphate. This enzyme is unique to the Calvin Cycle and has no counterpart in non-photosynthetic organisms. The isolation and sequence analysis of an SBPase clone has led to a number of investigations which have yielded interesting and novel information on this enzyme and in this paper the biochemistry and molecular biology of SBPase are reviewed. Some recent exciting developments are also reported, including the analysis of transgenic plants with reduced levels of SBPase which has shown that SBPase is a key regulator of carbon flux and mutagenesis studies which have resulted in the identification of the redox active cysteines responsible for the regulation by light of SBPase catalytic activity.
Mark Sti - One of the best experts on this subject based on the ideXlab platform.
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use of reverse phase liquid chromatography linked to tandem mass spectrometry to profile the Calvin Cycle and other metabolic intermediates in arabidopsis rosettes at different carbon dioxide concentrations
Plant Journal, 2009Co-Authors: Stephanie Arrivaul, Manuela Guenthe, Alexande Ivakov, Regina Feil, Daniel Vosloh, Joos T Van Donge, Rona Sulpice, Mark StiAbstract:Summary A platform using reverse-phase liquid chromatography coupled to tandem mass spectrometry was developed to measure 28 metabolites from photosynthetic metabolism. It was validated by comparison with authentic standards, with a requirement for distinct and clearly separated peaks, high sensitivity and repeatability in Arabidopsis rosette extracts. The recovery of authentic standards added to the plant material before extraction was 80–120%, demonstrating the reliability of the extraction and analytic procedures. Some metabolites could not be reliably measured, and were extracted and determined by other methods. Measurements of 37 metabolites in Arabidopsis rosettes after 15 min of illumination at different CO2 concentrations showed that most Calvin Cycle intermediates remain unaltered, or decrease only slightly (<30%), at compensation point CO2, whereas dedicated metabolites in end-product synthesis pathways decrease strongly. The inhibition of end-product synthesis allows high levels of metabolites to be retained in the Calvin Cycle to support a rapid Cycle with photorespiration.
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acclimation of arabidopsis leaves developing at low temperatures increasing cytoplasmic volume accompanies increased activities of enzymes in the Calvin Cycle and in the sucrose biosynthesis pathway
Plant Physiology, 1999Co-Authors: Asa Strand, Norma P A Hune, Vaugha Hurry, Stefa Henkes, Pette Gustafsso, Pe Gardestrom, Mark StiAbstract:Photosynthetic and metabolic acclimation to low growth temperatures were studied in Arabidopsis (Heynh.). Plants were grown at 23°C and then shifted to 5°C. We compared the leaves shifted to 5°C for 10 d and the new leaves developed at 5°C with the control leaves on plants that had been left at 23°C. Leaf development at 5°C resulted in the recovery of photosynthesis to rates comparable with those achieved by control leaves at 23°C. There was a shift in the partitioning of carbon from starch and toward sucrose (Suc) in leaves that developed at 5°C. The recovery of photosynthetic capacity and the redirection of carbon to Suc in these leaves were associated with coordinated increases in the activity of several Calvin-Cycle enzymes, even larger increases in the activity of key enzymes for Suc biosynthesis, and an increase in the phosphate available for metabolism. Development of leaves at 5°C also led to an increase in cytoplasmic volume and a decrease in vacuolar volume, which may provide an important mechanism for increasing the enzymes and metabolites in cold-acclimated leaves. Understanding the mechanisms underlying such structural changes during leaf development in the cold could result in novel approaches to increasing plant yield.
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ribulose 1 5 bisphosphate carboxylase oxygenase other Calvin Cycle enzymes and chlorophyll decrease when glucose is supplied to mature spinach leaves via the transpiration stream
Planta, 1991Co-Authors: Anne Krapp, W P Quick, Mark StiAbstract:The inhibition of photosynthesis after supplying glucose to detached leaves of spinach (Spinacia oleracea L.) was used as a model system to search for mechanisms which potentially contribute to the “sink” regulation of photosynthesis. Detached leaves were supplied with 50 mM glucose or water for 7 d through the transpiration stream, holding the leaves in low irradiance (16 μmol photons · m−2 · s−1) and a Cycle of 9 h light/15 h darkness to prevent any endogenous accumulation of carbohydrate. Leaves supplied with water only showed marginal changes of photosynthesis, respiration, enzyme levels or metabolites. When leaves were supplied with 50 mM glucose, photosynthesis was gradually inhibited over several days. The inhibition was most marked when photosynthesis was measured in saturating irradiance and ambient CO2, less marked in saturating irradiance and saturating CO2, and least marked in limiting irradiance. There was a gradual loss of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) protein, fructose-1,6-bisphosphatase, NADP-glyceraldehyde-3-phosphate dehydrogenase and chlorophyll. The inhibition of photosynthesis was accompanied by a large decrease of glycerate-3-phosphate, an increase of triose-phosphates and fructose-1,6-bisphospate, and a small decrease of ribulose-1,5-bisphosphate. The stromal NADPH/NADP ratio increased (as indicated by increased activation of NADP-malate dehydrogenase), and the ATP/ADP ratio increased. Chlorophyll-fluorescence analysis indicated that thylakoid energisation was increased, and that the acceptor side of photosystem II was more reduced. Similar results were obtained when glucose was supplied by floating leaf discs in low irradiance on glucose solution, and when detached spinach leaves were held in high light to produce an endogenous accumulation of carbohydrate. Feeding glucose also led to an increased rate of respiration. This was not accompanied by any changes of pyruvate kinase, phosphofructokinase, or pyrophosphate: fructose-6-phosphate phosphotransferase activity. There was a decrease of phosphoenolpyruvate, glycerate-3-phosphate and glycerate-2-phosphate, an increase of pyruvate and triose-phosphates, and an increased ATP/ADP ratio. These results show (i) that accumulation of carbohydrate can inhibit photosynthesis via a long-term mechanism involving a decrease of Rubisco and other Calvin-Cycle enzymes and (ii) that respiration is stimulated due to an unknown mechanism, which increases the utilisation of phosphoenolpyruvate.
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sink regulation of photosynthetic metabolism in transgenic tobacco plants expressing yeast invertase in their cell wall involves a decrease of the Calvin Cycle enzymes and an increase of glycolytic enzymes
Planta, 1991Co-Authors: Mark Sti, A Von Schaewe, Lotha WillmitzeAbstract:Leaves on transgenic tobacco plants expressing yeast-derived invertase in the apoplast develop clearly demarcated green and bleached sectors when they mature. The green areas contain low levels of soluble sugars and starch which are turned over on a daily basis, and have high rates of photosynthesis and low rates of respiration. The pale areas accumulate carbohydrate, photosynthesis is inhibited, and respiration increases. This provides a model system to investigate the “sink” regulation of photosynthetic metabolism by accumulating carbohydrate. The inhibition of photosynthesis is accompanied by a decrease of ribulose-1,5-bisphosphate and glycerate-3-phosphate, and an increase of triosephosphate and fructose-1,6-bisphosphate. The extracted activities of ribulose-1,5-bisphosphate carboxylase, fructose-1, 6-bisphosphatase and NADP-glyeraldehyde-3-phosphate dehydrogenase decreased. The activity of sucrose-phosphate synthase remained high or increased, an increased portion of the photosynthate was partitioned into soluble sugars rather than starch, and the pale areas showed few or no oscillations during transitions between darkness and saturating light in saturating CO2. The increased rate of respiration was accompanied by an increased level of hexose-phosphates, triose-phosphates and fructose-1,6-bisphosphate while glycerate-3-phosphate and phosphoenolpyruvate decreased and pyruvate increased. The activities of pyruvate kinase, phosphofructokinase and pyrophosphate: fructose-6-phosphate phosphotransferase increased two- to four-fold. We conclude that an increased level of carbohydrate leads to a decreased level of Calvin-Cycle enzymes and, thence, to an inhibition of photosynthesis. It also leads to an increased level of glycolytic enzymes and, thence, to a stimulation of respiration. These changes of enzymes are more important in middle- or long-term adjustments to high carbohydrate levels in the leaf than fine regulation due to depletion of inorganic phosphate or high levels of phosphorylated metabolites.
William Marti - One of the best experts on this subject based on the ideXlab platform.
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the Calvin Cycle and its regulation
2000Co-Authors: William Marti, Renate Scheibe, Claus SchnarrenbergeAbstract:The Calvin Cycle is the starting point of carbon metabolism in higher plants. It is a typically eubacterial pathway, as comparative biochemistry of all of its enzymes from prokaryotes and eukaryotes has revealed. The structural basis of Calvin Cycle function is reviewed with an attempt at a balanced consideration of biochemical and molecular findings. The structural diversity of prokaryotic enzymes is emphasized, since the genes encoding the pathway in eukaryotes have all been inherited by plants from prokaryotes through endosymbiosis. Curiously, the enzymes that constitute the pathway in different organisms are of ten structurally unrelated—what is conserved in evolution is merely the set of substrate conversions, not the enzymes that catalyze them. Some of the structural and regulatory properties of the enzymes were present in the antecedents of organelles, but others were newly acquired at the eukaryotic level. The expression of Calvin Cycle genes is regulated by a wide spectrum of factors, though the molecular details of the regulation have yet to be unraveled. Findings that suggest the existence of multienzyme-like Calvin Cycle complexes are summarized. The molecular basis of redox-modulated light regulation through the thioredoxin system and its importance for flexible control of the pathway under varying conditions is illustrated. Expression of Calvin Cycle enzymes in response to external or internal stimuli is briefly reviewed, as are newer findings from the expression of antisense constructs of Calvin Cycle enzymes in transgenic plants.
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chloroplast class i and class ii aldolases are bifunctional for fructose 1 6 biphosphate and sedoheptulose 1 7 biphosphate cleavage in the Calvin Cycle
FEBS Letters, 1999Co-Authors: Anke Flechne, William Marti, Wolfgang Gross, Claus SchnarrenbergeAbstract:Class I and class II aldolases are products of two evolutionary non-related gene families. The cytosol and chloroplast enzymes of higher plants are of the class I type, the latter being bifunctional for fructose-1,6- and sedoheptulose-1,7-P2 in the Calvin Cycle. Recently, class II aldolases were detected for the cytosol and chloroplasts of the lower alga Cyanophora paradoxa. The respective chloroplast enzyme has been shown here to be also bifunctional for fructose-1,6- and sedoheptulose-1,7-P2. Kinetics, also including fructose-1-P, were determined for all these enzymes. Apparently, aldolases are multifunctional enzymes, irrespective of their class I or class II type.
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the evolution of the Calvin Cycle from prokaryotic to eukaryotic chromosomes a case study of functional redundancy in ancient pathways through endosymbiosis
Current Genetics, 1997Co-Authors: William Marti, Claus SchnarrenbergeAbstract:The evolutionary histories of the 12 enzymes that catalyze the reactions of the Calvin Cycle in higher-plant chloroplasts are summarized. They are shown to be encoded by a mixture of nuclear genes of cyanobacterial and proteobacterial origin. Moreover, where cytosolic isoforms of these enzymes are found they are almost invariably encoded by genes of clearly endosymbiont origin. We infer that endosymbiosis resulted in functional redundancy that was eliminated through differential gene loss, with intruding eubacterial genes repeatedly replacing pre-existing nuclear counterparts to which they were either functionally or structurally homologous. Our findings fail to support the `product-specificity corollary', which predicts re-targeting of nuclear-encoded gene products to the organelle from whose genome they originated. Rather it would appear that the enzymes of central carbohydrate metabolism have evolved novel targeting possibilities regardless of their origins. Our findings suggest a new hypothesis to explain organelle genome persistence, based on the testable idea that some organelle-encoded gene products might be toxic when present in the cytosol or other inappropriate cellular compartments.
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molecular characterization of transketolase ec 2 2 1 1 active in the Calvin Cycle of spinach chloroplasts
Plant Molecular Biology, 1996Co-Authors: Anke Flechne, Claus Schnarrenberge, Uta Dresse, Pete Westhoff, Katri Henze, William MartiAbstract:A cDNA encoding the Calvin Cycle enzyme transketolase (TKL; EC 2.2.1.1) was isolated from Sorghum bicolor via subtractive differential hybridization, and used to isolate several full-length cDNA clones for this enzyme from spinach. Functional identity of the encoded mature subunit was shown by an 8.6-fold increase of TKL activity upon induction of Escherichia coli cells that overexpress the spinach TKL subunit under the control of the bacteriophage T7 promoter. Chloroplast localization of the cloned enzyme is shown by processing of the in vitro synthesized precursor upon uptake by isolated chloroplasts. Southern blot-analysis suggests that TKL is encoded by a single gene in the spinach genome. TKL proteins of both higher-plant chloroplasts and the cytosol of non-photosynthetic eukaryotes are found to be unexpectedly similar to eubacterial homologues, suggesting a possible eubacterial origin of these nuclear genes. Chloroplast TKL is the last of the demonstrably chloroplast-localized Calvin Cycle enzymes to have been cloned and thus completes the isolation of gene probes for all enzymes of the pathway in higher plants.
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cloning of the amphibolic Calvin Cycle oppp enzyme d ribulose 5 phosphate 3 epimerase ec 5 1 3 1 from spinach chloroplasts functional and evolutionary aspects
Plant Molecular Biology, 1995Co-Authors: Ulrich Nowitzki, Claus Schnarrenberge, Pete Westhoff, Katri Henze, Ralf Wyrich, William MartiAbstract:Exploiting the differential expression of genes for Calvin Cycle enzymes in bundle-sheath and mesophyll cells of the C4 plant Sorghum bicolor L., we isolated via subtractive hybridization a molecular probe for the Calvin Cycle enzyme d-ribulose-5-phosphate 3-epimerase (R5P3E) (EC 5.1.3.1), with the help of which several full-size cDNAs were isolated from spinach. Functional identity of the encoded mature subunit was shown by R5P3E activity found in affinity-purified glutatione S-transferase fusions expressed in Escherichia coli and by three-fold increase of R5P3E activity upon induction of E. coli overexpressing the spinach subunit under the control of the bacteriophage T7 promoter, demonstrating that we have cloned the first functional ribulose-5-phosphate 3-epimerase from any eukaryotic source. The chloroplast enzyme from spinach shares about 50% amino acid identity with its homologues from the Calvin Cycle operons of the autotrophic purple bacteria Alcaligenes eutrophus and Rhodospirillum rubrum. A R5P3E-related eubacterial gene family was identified which arose through ancient duplications in prokaryotic chromosomes, three R5P3E-related genes of yet unknown function have persisted to the present within the E. coli genome. A gene phylogeny reveals that spinach R5P3E is more similar to eubacterial homologues than to the yeast sequence, suggesting a eubacterial origin for this plant nuclear gene.
James Mckinlay - One of the best experts on this subject based on the ideXlab platform.
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reductive tricarboxylic acid Cycle enzymes and reductive amino acid synthesis pathways contribute to electron balance in a rhodospirillum rubrum Calvin Cycle mutant
Microbiology, 2020Co-Authors: Alexandra L Mccully, Mauree C Onyeziri, Eah Lasarre, Jennife R Gliessma, James MckinlayAbstract:Purple non-sulfur bacteria (PNSB) use light for energy and organic substrates for carbon and electrons when growing photoheterotrophically. This lifestyle generates more reduced electron carriers than are required for biosynthesis, even during consumption of some of the most oxidized organic substrates like malate and fumarate. Reduced electron carriers not used in biosynthesis must still be oxidized for photoheterotrophic growth to occur. Diverse PNSB commonly rely on the CO2-fixing Calvin Cycle to oxidize reduced electron carriers. Some PNSB also produce H2 or reduce terminal electron acceptors as alternatives to the Calvin Cycle. Rhodospirillum rubrum Calvin-Cycle mutants defy this trend by growing phototrophically on malate or fumarate without H2 production or access to terminal electron acceptors. We used 13C-tracer experiments to examine how a Rs. rubrum Calvin-Cycle mutant maintains electron balance under such conditions. We detected the reversal of some tricarboxylic acid Cycle enzymes, carrying reductive flux from malate or fumarate to αKG. This pathway and the reductive synthesis of αKG-derived amino acids are likely important for electron balance, as supplementing the growth medium with αKG-derived amino acids prevented Rs. rubrum Calvin-Cycle-mutant growth unless a terminal electron acceptor was provided. Flux estimates also suggested that the Calvin-Cycle mutant preferentially synthesized isoleucine using the reductive threonine-dependent pathway instead of the less-reductive citramalate-dependent pathway. Collectively, our results suggest that alternative biosynthetic pathways can contribute to electron balance within the constraints of a relatively constant biomass composition.
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the reverse tca Cycle and reductive amino acid synthesis pathways contribute to electron balance in a rhodospirillum rubrum Calvin Cycle mutant
bioRxiv, 2019Co-Authors: Alexandra L Mccully, Mauree C Onyeziri, Eah Lasarre, Jennife R Gliessma, James MckinlayAbstract:Abstract Purple nonsulfur bacteria (PNSB) use light for energy and organic substrates for carbon and electrons when growing photoheterotrophically. This lifestyle generates more reduced electron carriers than are required for biosynthesis. It is essential that this excess reducing power be oxidized for photoheterotrophic growth to occur. Diverse PNSB commonly rely on the CO2-fixing Calvin Cycle to oxidize excess reducing power. Some PNSB additionally utilize H2 production or reduction of electron acceptors, such as dimethylsulfoxide, as alternative reductive pathways to the Calvin Cycle. Rhodospirillum rubrum Calvin Cycle mutants defy this trend by growing phototrophically on relatively oxidized substrates like malate and fumarate without H2 production or access to electron acceptors. How Rs. rubrum Calvin Cycle mutants maintain electron balance under these conditions was unknown. Here, using 13C-tracer experiments and physiological assays, we found that Rs. rubrum Calvin Cycle mutants use a reductive arm of the tricarboxylic acid Cycle when growing phototrophically on malate and fumarate. The reductive synthesis of amino acids stemming from α-ketoglutarate is also likely important for electron balance, as supplementing the growth medium with α-ketoglutarate-derived amino acids prevented Rs. rubrum Calvin Cycle mutant growth unless dimethylsulfoxide was provided as an electron acceptor. Fluxes estimated from 13C-tracer experiments also suggested the preferential use of a reductive isoleucine synthesis pathway when the Calvin Cycle was genetically inactivated; however, this pathway was not essential for growth of a Calvin Cycle mutant. Importance The lifestyle by which PNSB use organic carbon and light for energy comes with a challenge in managing electrons. Excess electrons from the organic substrates can be coupled to the assimilation of CO2 in the Calvin Cycle, avoiding a buildup of reduced electron carriers that would halt metabolism. As an exception, Rs. rubrum can grow without the Calvin Cycle when provided with light and relatively oxidized substrates. By tracking stable isotopes in a Rs. rubrum Calvin Cycle mutant, we observed the reversal of an arm of the tricarboxylic acid Cycle, feeding electron-requiring amino acid synthesis pathways. Providing the mutant with these amino acids prevented growth, suggesting that their synthesis is required for electron balance. Our results highlight the contribution of biosynthetic reactions to electron balance and the metabolic diversity that exists between PNSB, as most PNSB cannot grow without the Calvin Cycle under the conditions used in this study.
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disrupting Calvin Cycle phosphoribulokinase activity in rhodopseudomonas palustris increases the h 2 yield and specific production rate proportionately
International Journal of Hydrogen Energy, 2016Co-Authors: Alexandra L Mccully, James MckinlayAbstract:Abstract Anoxygenic phototrophs, like Rhodopseudomonas palustris, can convert light energy and electrons from organic waste into H2 gas, a potential biofuel. During phototrophic growth on organic compounds, the CO2-fixing Calvin Cycle competes against H2 production for electrons. Here we address why genetically disrupting the CO2-fixing enzyme, ribulose 1,5-bisphosphate carboxylase (Rubisco), increases the H2 yield but not the specific H2 production rate. We hypothesized that remaining upstream phosphoribulokinase (PRK) activity negatively impacts growth and thereby the specific H2 production rate, likely due to the accumulation of ribulose-1,5-bisphosphate, the substrate for Rubisco. In agreement with our hypothesis, deletion of PRK resulted in proportional increases to both the H2 yield and the specific production rate. Thus, even though Rubsico is traditionally a more common target to eliminate Calvin Cycle activity we propose PRK as a favorable alternative to avoid undesirable pleiotropic effects.
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Calvin Cycle mutants of photoheterotrophic purple nonsulfur bacteria fail to grow due to an electron imbalance rather than toxic metabolite accumulation
Journal of Bacteriology, 2014Co-Authors: Gina C Gordo, James MckinlayAbstract:ABSTRACT Purple nonsulfur bacteria grow photoheterotrophically by using light for energy and organic compounds for carbon and electrons. Disrupting the activity of the CO 2 -fixing Calvin Cycle enzyme, ribulose 1,5-bisphosphate carboxylase (RubisCO), prevents photoheterotrophic growth unless an electron acceptor is provided or if cells can dispose of electrons as H 2 . Such observations led to the long-standing model wherein the Calvin Cycle is necessary during photoheterotrophic growth to maintain a pool of oxidized electron carriers. This model was recently challenged with an alternative model wherein disrupting RubisCO activity prevents photoheterotrophic growth due to the accumulation of toxic ribulose-1,5-bisphosphate (RuBP) (D. Wang, Y. Zhang, E. L. Pohlmann, J. Li, and G. P. Roberts, J. Bacteriol. 193:3293-3303, 2011, http://dx.doi.org/10.1128/JB.00265-11). Here, we confirm that RuBP accumulation can impede the growth of Rhodospirillum rubrum (Rs. rubrum) and Rhodopseudomonas palustris (Rp. palustris) RubisCO-deficient (ΔRubisCO) mutants under conditions where electron carrier oxidation is coupled to H 2 production. However, we also demonstrate that Rs. rubrum and Rp. palustris Calvin Cycle phosphoribulokinase mutants that cannot produce RuBP cannot grow photoheterotrophically on succinate unless an electron acceptor is provided or H 2 production is permitted. Thus, the Calvin Cycle is still needed to oxidize electron carriers even in the absence of toxic RuBP. Surprisingly, Calvin Cycle mutants of Rs. rubrum, but not of Rp. palustris, grew photoheterotrophically on malate without electron acceptors or H 2 production. The mechanism by which Rs. rubrum grows under these conditions remains to be elucidated.
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Calvin Cycle flux pathway constraints and substrate oxidation state together determine the h2 biofuel yield in photoheterotrophic bacteria
Mbio, 2011Co-Authors: James Mckinlay, Caroline S HarwoodAbstract:ABSTRACT Hydrogen gas (H 2 ) is a possible future transportation fuel that can be produced by anoxygenic phototrophic bacteria via nitrogenase. The electrons for H 2 are usually derived from organic compounds. Thus, one would expect more H 2 to be produced when anoxygenic phototrophs are supplied with increasingly reduced (electron-rich) organic compounds. However, the H 2 yield does not always differ according to the substrate oxidation state. To understand other factors that influence the H 2 yield, we determined metabolic fluxes in Rhodopseudomonas palustris grown on 13 C-labeled fumarate, succinate, acetate, and butyrate (in order from most oxidized to most reduced). The flux maps revealed that the H 2 yield was influenced by two main factors in addition to substrate oxidation state. The first factor was the route that a substrate took to biosynthetic precursors. For example, succinate took a different route to acetyl-coenzyme A (CoA) than acetate. As a result, R. palustris generated similar amounts of reducing equivalents and similar amounts of H 2 from both succinate and acetate, even though succinate is more oxidized than acetate. The second factor affecting the H 2 yield was the amount of Calvin Cycle flux competing for electrons. When nitrogenase was active, electrons were diverted away from the Calvin Cycle towards H 2 , but to various extents, depending on the substrate. When Calvin Cycle flux was blocked, the H 2 yield increased during growth on all substrates. In general, this increase in H 2 yield could be predicted from the initial Calvin Cycle flux. IMPORTANCE Photoheterotrophic bacteria, like Rhodopseudomonas palustris, obtain energy from light and carbon from organic compounds during anaerobic growth. Cells can naturally produce the biofuel H 2 as a way of disposing of excess electrons. Unexpectedly, feeding cells organic compounds with more electrons does not necessarily result in more H 2 . Despite repeated observations over the last 40 years, the reasons for this discrepancy have remained unclear. In this paper, we identified two metabolic factors that influence the H 2 yield, (i) the route taken to make biosynthetic precursors and (ii) the amount of CO 2 -fixing Calvin Cycle flux that competes against H 2 production for electrons. We show that the H 2 yield can be improved on all substrates by using a strain that is incapable of Calvin Cycle flux. We also contributed quantitative knowledge to the long-standing question of why photoheterotrophs must produce H 2 or fix CO 2 even on relatively oxidized substrates.
Xizhen Ai - One of the best experts on this subject based on the ideXlab platform.
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changes in sbpase activity influence photosynthetic capacity growth and tolerance to chilling stress in transgenic tomato plants
Scientific Reports, 2016Co-Authors: Fei Ding, Meiling Wang, Shuoxin Zhang, Xizhen AiAbstract:Sedoheptulose-1, 7-bisphosphatase (SBPase) is an important enzyme involved in photosynthetic carbon fixation in the Calvin Cycle. Here, we report the impact of changes in SBPase activity on photosynthesis, growth and development, and chilling tolerance in SBPase antisense and sense transgenic tomato (Solanum lycopersicum) plants. In transgenic plants with increased SBPase activity, photosynthetic rates were increased and in parallel an increase in sucrose and starch accumulation was evident. Total biomass and leaf area were increased in SBPase sense plants, while they were reduced in SBPase antisense plants compared with equivalent wild-type tomato plants. Under chilling stress, when compared with plants with decreased SBPase activity, tomato plants with increased SBPase activity were found to be more chilling tolerant as indicated by reduced electrolyte leakage, increased photosynthetic capacity, and elevated RuBP regeneration rate and quantum efficiency of photosystem II. Collectively, our data suggest that higher level of SBPase activity gives an advantage to photosynthesis, growth and chilling tolerance in tomato plants. This work also provides a case study that an individual enzyme in the Calvin Cycle may serve as a useful target for genetic engineering to improve production and stress tolerance in crops.
