Phosphoribulokinase Research Articles

Photosynthetic activity and RAPD profile of polyethylene glycol treated B. juncea L. under nitric oxide and abscisic acid application

The involvement of two extremely important signalling molecules, nitric oxide (NO) and abscisic acid (ABA) has been employed by plants to facilitate the adaptive/tolerate response during stressful conditions. However, the interactive role of exogenously applied NO and ABA is very less studied at physiological, biochemical and molecular levels. The present study therefore, evaluated the effects of individual and simultaneous addition of exogenous NO donor SNP (100μM) and ABA (10μM) on photosynthesis, Calvin-Benson cycle enzymes, S-assimilation enzymes, oxidative stress components, and genotoxicity in Brassica juncea cv. Varuna, exposed to polyethylene glycol (PEG)-induced drought stress. Results showed that a loss induced by PEG was significantly surpassed by the application of NO or/and ABA with PEG for chlorophyll content, net photosynthestic rate (Pn), internal CO2 concentration (Ci), stomatal conductance (gs), transpiration rate (Tr), maximum photosystem II (PSII) efficiency (Fv/Fm), actual PSII efficiency (ΦPSII), intrinsic PSII efficiency (Fv´/ Fm´), photochemical quenching (qP), non-photochemical quenching (NPQ), electron transport chain (ETC), ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo), glyceraldehyde-3-phosphate dehydrogenase (GapDH), phosphoribulokinase (PRK), ATP-sulfurylase (ATP-S), and serine acetyltransferase (SAT) activities. The genomic template stability (GTS) (measured as changes in RAPD profiles) was significantly affected and showed varying degrees of DNA polymorphism, highest in PEG and lowest in PEG + NO and PEG + NO + ABA. Furthermore, the changes in RAPD profiles showed consistent results when compared with various photosynthetic and oxidative parameters. Altogether, this study concluded that supplementation of individual NO and together with ABA was more effective than individual ABA in alleviating PEG-induced drought stress in B. juncea L. seedlings.

Photosynthetic Phosphoribulokinase Structures: Enzymatic Mechanisms and the Redox Regulation of the Calvin-Benson-Bassham Cycle.

The Calvin-Benson-Bassham (CBB) cycle is responsible for CO2 assimilation and carbohydrate production in oxyphototrophs. Phosphoribulokinase (PRK) is an essential enzyme of the CBB cycle in photosynthesis, catalyzing ATP-dependent conversion of ribulose-5-phosphate (Ru5P) to ribulose-1,5-bisphosphate. The oxyphototrophic PRK is redox-regulated and can be further regulated by reversible association with both glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and oxidized chloroplast protein CP12. The resulting GAPDH/CP12/PRK complex is central in the regulation of the CBB cycle; however, the PRK-CP12 interface in the recently reported cyanobacterial GAPDH/CP12/PRK structure was not well resolved, and the detailed binding mode of PRK with ATP and Ru5P remains undetermined, as only apo-form structures of PRK are currently available. Here, we report the crystal structures of cyanobacterial (Synechococcus elongatus) PRK in complex with ADP and glucose-6-phosphate and of the Arabidopsis (Arabidopsis thaliana) GAPDH/CP12/PRK complex, providing detailed information regarding the active site of PRK and the key elements essential for PRK-CP12 interaction. Our structural and biochemical results together reveal that the ATP binding site is disrupted in the oxidized PRK, whereas the Ru5P binding site is occupied by oxidized CP12 in the GAPDH/CP12/PRK complex. This structure-function study greatly advances the understanding of the reaction mechanism of PRK and the subtle regulations of redox signaling for the CBB cycle.

Structural basis of light-induced redox regulation in the Calvin–Benson cycle in cyanobacteria

Plants, algae, and cyanobacteria fix carbon dioxide to organic carbon with the Calvin-Benson (CB) cycle. Phosphoribulokinase (PRK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are essential CB-cycle enzymes that control substrate availability for the carboxylation enzyme Rubisco. PRK consumes ATP to produce the Rubisco substrate ribulose bisphosphate (RuBP). GAPDH catalyzes the reduction step of the CB cycle with NADPH to produce the sugar glyceraldehyde 3-phosphate (GAP), which is used for regeneration of RuBP and is the main exit point of the cycle. GAPDH and PRK are coregulated by the redox state of a conditionally disordered protein CP12, which forms a ternary complex with both enzymes. However, the structural basis of CB-cycle regulation by CP12 is unknown. Here, we show how CP12 modulates the activity of both GAPDH and PRK. Using thermophilic cyanobacterial homologs, we solve crystal structures of GAPDH with different cofactors and CP12 bound, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide preorders CP12 prior to binding the PRK active site, which is resolved in complex with CP12. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our structural and biochemical data explain how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation.

Crystal structure of phosphoribulokinase from Synechococcus sp. strain PCC 6301.

Phosphoribulokinase (PRK) catalyses the ATP-dependent phosphorylation of ribulose 5-phosphate to give ribulose 1,5-bisphosphate. Regulation of this reaction in response to light controls carbon fixation during photosynthesis. Here, the crystal structure of PRK from the cyanobacterium Synechococcus sp. strain PCC 6301 is presented. The enzyme is dimeric and has an α/β-fold with an 18-stranded β-sheet at its core. Interestingly, a disulfide bond is found between Cys40 and the P-loop residue Cys18, revealing the structural basis for the redox inactivation of PRK activity. A second disulfide bond appears to rigidify the dimer interface and may thereby contribute to regulation by the adaptor protein CP12 and glyceraldehyde-3-phosphate dehydrogenase.

Arabidopsis and Chlamydomonas phosphoribulokinase crystal structures complete the redox structural proteome of the Calvin–Benson cycle

In land plants and algae, the Calvin-Benson (CB) cycle takes place in the chloroplast, a specialized organelle in which photosynthesis occurs. Thioredoxins (TRXs) are small ubiquitous proteins, known to harmonize the two stages of photosynthesis through a thiol-based mechanism. Among the 11 enzymes of the CB cycle, the TRX target phosphoribulokinase (PRK) has yet to be characterized at the atomic scale. To accomplish this goal, we determined the crystal structures of PRK from two model species: the green alga Chlamydomonas reinhardtii (CrPRK) and the land plant Arabidopsis thaliana (AtPRK). PRK is an elongated homodimer characterized by a large central β-sheet of 18 strands, extending between two catalytic sites positioned at its edges. The electrostatic surface potential of the catalytic cavity has both a positive region suitable for binding the phosphate groups of substrates and an exposed negative region to attract positively charged TRX-f. In the catalytic cavity, the regulatory cysteines are 13 Å apart and connected by a flexible region exclusive to photosynthetic eukaryotes-the clamp loop-which is believed to be essential for oxidation-induced structural rearrangements. Structural comparisons with prokaryotic and evolutionarily older PRKs revealed that both AtPRK and CrPRK have a strongly reduced dimer interface and an increased number of random-coiled regions, suggesting that a general loss in structural rigidity correlates with gains in TRX sensitivity during the molecular evolution of PRKs in eukaryotes.

The chloroplast 2-cysteine peroxiredoxin functions as thioredoxin oxidase in redox regulation of chloroplast metabolism.

Thiol-dependent redox regulation controls central processes in plant cells including photosynthesis. Thioredoxins reductively activate, for example, Calvin-Benson cycle enzymes. However, the mechanism of oxidative inactivation is unknown despite its importance for efficient regulation. Here, the abundant 2-cysteine peroxiredoxin (2-CysPrx), but not its site-directed variants, mediates rapid inactivation of reductively activated fructose-1,6-bisphosphatase and NADPH-dependent malate dehydrogenase (MDH) in the presence of the proper thioredoxins. Deactivation of phosphoribulokinase (PRK) and MDH was compromised in 2cysprxAB mutant plants upon light/dark transition compared to wildtype. The decisive role of 2-CysPrx in regulating photosynthesis was evident from reoxidation kinetics of ferredoxin upon darkening of intact leaves since its half time decreased 3.5-times in 2cysprxAB. The disadvantage of inefficient deactivation turned into an advantage in fluctuating light. Physiological parameters like MDH and PRK inactivation, photosynthetic kinetics and response to fluctuating light fully recovered in 2cysprxAB mutants complemented with 2-CysPrxA underlining the significance of 2-CysPrx. The results show that the 2-CysPrx serves as electron sink in the thiol network important to oxidize reductively activated proteins and represents the missing link in the reversal of thioredoxin-dependent regulation.

Chloroplast Protein 12 Expression Alters Growth and Chilling Tolerance in Tropical Forage Stylosanthes guianensis (Aublet) Sw

Stylosanthes guianensis (Aublet) Sw. is a tropical forage legume with soil acidity tolerance and excellent adaptation to infertile soils, but sensitive to chilling. To understand the molecular responses of S. guianensis to chilling, differentially expressed genes between a chilling tolerant mutant 7–1 and the wild type were identified using suppression subtractive hybridization, and eight of them were confirmed and the regulation pattern were analyzed using quantitative reverse transcription PCR (qRT-PCR). Chloroplast protein 12 (CP12) functions to regulate the Calvin cycle by forming a ternary complex with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK). SgCP12 transcript was induced by chilling in both plants, and higher levels were observed in 7–1 than in the wild type, implying a potential role of SgCP12 in chilling tolerance. To confirm this, transgenic S. guianensis plants over-expressing or down-regulating SgCP12 were generated, respectively. Higher Fv/Fm and survival rate and lower ion leakage were observed in transgenic plants overexpressing SgCP12 as compared with the wild type after chilling treatment, while lower Fv/Fm and survival rate and higher ion leakage were found in SgCP12 antisense plants. SgCP12 overexpression plants showed promoted growth with increased plant height and fresh weight, while the antisense plants exhibited reduced growth with decreased plant height and fresh weight as compared with the wild type. The results indicated that regulation of SgCP12 expression alters plant growth and chilling tolerance in S. guianensis. In addition, higher levels of net photosynthetic rate (Pn), GAPDH and PRK activities were observed in SgCP12 overexpression transgenic plants, while lower levels in antisense plants than in the wild type under both control and chilling conditions, indicating that altered activities of GAPDH and PRK were associated with the changed Pn in transgenic S. guianensis. Our results suggest that SgCP12 regulates GAPDH and PRK activities, Pn, and chilling tolerance in S. guianensis.

The Proteome Response of Salt-Sensitive Rapeseed (Brassica napus L.) Genotype to Salt Stress

Productivity of rapeseed (Brassica napus L.), the third most important oilseed crop, was reduced more than other crops under the salt stress higher than the threshold. Thus, breeding, especially at seedling stage, seems necessary. Plants under salt stress, by synthesis of essential metabolites, specific structural proteins or enzymes of metabolic pathways deal with the stress. To identify the molecular mechanisms of salt responsiveness in rapeseed, ‘Option500’ a salt-sensitive genotype was exposed to 0, 150, and 300mM NaCl during the seedling stage. An increase in proline and the Na+ content of leaf and a reduction in shoot dry weight, plant height, K+ content and K+/Na+ ratio were observed. Protein expression changes were examined by two-dimensional electrophoresis (2-DE). Out of 110 protein spots identified by 2-DE gels, 37 spots showed significant abundant changes based on induction factor (IF), and 7 spots were recognized significantly at 5% probability level, which 1 and 6 spots were up and down-regulated, respectively. By using LC-MS/MS mass spectrometry analysis, proteins were identified which are involved in energy production and photosynthesis. Activity of enzymes involved in energy production decreased under stress, while the abundance of Phosphoribulokinase (PRK) -an important enzyme in the pentose phosphate pathway- increased.

Nitric oxide is involved in abscisic acid-induced photosynthesis and antioxidant system of tall fescue seedlings response to low-light stress

Abscisic acid (ABA) and nitric oxide (NO) are both extremely important signalling molecules employed by plants to mediate plant tolerance under abiotic stresses. In the present study, the role of NO in ABA-induced photosynthesis and antioxidant system in leaves of tall fescue (Festuca arundinacea Schreb.) seedlings was investigated. Low-light (LL) stress increased intercellular CO2 concentration (Ci), as well as decreased plant height, leaf width, tiller number, biomass, chlorophyll content, net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), the maximum quantum yield of photosystem photochemistry (Fv/Fm), actual photochemical efficiency of photosystem II (ΦPSII), photochemical quenching (qP), the apparent electron transport rate (ETR), enzymatic activity of ribulose-1,5-bisphosphate carboxylase (Rubisco) and glyceraldehyde phosphate dehydrogenase (GADPH) and phosphoribulokinase (PRK), which were substantially alleviated by exogenous ABA application. Moreover, application of these leaves with exogenous ABA resulted in reduced LL stress induced ion leakage and reduced contents of MDA, H2O2 and superoxide radicals O2•-, as well as higher activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX). Expression of genes coding antioxidant enzymes increased in tall fescue in the presence of ABA under LL stress. These responses could be reversed by treatment with fluridone (the ABA biosynthesis inhibitor). The application of ABA elevated NO level, enhanced nitric oxide synthase (NOS)-like activity and up-regulated the tall fescue nitric oxide synthase-associated gene, NITRIC OXIDE ASSOCIATED 1 (FaNOA1) expression level. In addition, neither exogenous NO nor specific NO scavenger 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) or NO synthase (NOS) specific inhibitor Nω-nitro-L-arginine (LNNA) treatments had no effect on endogenous ABA concentration and ABA receptors transcript levels (FaPYR1 and FaPYL1), implying that the protective effect of ABA under LL stress might be mediated by NO. These results indicated that NO was involved in the ABA-mediated LL tolerance via regulating the antioxidant defense system and keeping a high level of photosynthetic rate in tall fescue.

Structural and functional insights into the unique CBS–CP12 fusion protein family in cyanobacteria

Cyanobacteria are important photosynthetic organisms inhabiting a range of dynamic environments. This phylum is distinctive among photosynthetic organisms in containing genes encoding uncharacterized cystathionine β-synthase (CBS)-chloroplast protein (CP12) fusion proteins. These consist of two domains, each recognized as stand-alone photosynthetic regulators with different functions described in cyanobacteria (CP12) and plants (CP12 and CBSX). Here we show that CBS-CP12 fusion proteins are encoded in distinct gene neighborhoods, several unrelated to photosynthesis. Most frequently, CBS-CP12 genes are in a gene cluster with thioredoxin A (TrxA), which is prevalent in bloom-forming, marine symbiotic, and benthic mat cyanobacteria. Focusing on a CBS-CP12 from Microcystis aeruginosa PCC 7806 encoded in a gene cluster with TrxA, we reveal that the domain fusion led to the formation of a hexameric protein. We show that the CP12 domain is essential for hexamerization and contains an ordered, previously structurally uncharacterized N-terminal region. We provide evidence that CBS-CP12, while combining properties of both regulatory domains, behaves different from CP12 and plant CBSX. It does not form a ternary complex with phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase. Instead, CBS-CP12 decreases the activity of PRK in an AMP-dependent manner. We propose that the novel domain architecture and oligomeric state of CBS-CP12 expand its regulatory function beyond those of CP12 in cyanobacteria.

Interaction between adenylate kinase 3 and glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii.

Adenylate kinase (EC 2.7.4.3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.13), phosphoribulokinase (PRK, EC 2.7.1.19).

Optimizing anaerobic growth rate and fermentation kinetics in Saccharomyces cerevisiae strains expressing Calvin-cycle enzymes for improved ethanol yield

BackgroundReduction or elimination of by-product formation is of immediate economic relevance in fermentation processes for industrial bioethanol production with the yeast Saccharomyces cerevisiae. Anaerobic cultures of wild-type S. cerevisiae require formation of glycerol to maintain the intracellular NADH/NAD+ balance. Previously, functional expression of the Calvin-cycle enzymes ribulose-1,5-bisphosphate carboxylase (RuBisCO) and phosphoribulokinase (PRK) in S. cerevisiae was shown to enable reoxidation of NADH with CO2 as electron acceptor. In slow-growing cultures, this engineering strategy strongly decreased the glycerol yield, while increasing the ethanol yield on sugar. The present study explores engineering strategies to improve rates of growth and alcoholic fermentation in yeast strains that functionally express RuBisCO and PRK, while maximizing the positive impact on the ethanol yield.ResultsMulti-copy integration of a bacterial-RuBisCO expression cassette was combined with expression of the Escherichia coli GroEL/GroES chaperones and expression of PRK from the anaerobically inducible DAN1 promoter. In anaerobic, glucose-grown bioreactor batch cultures, the resulting S. cerevisiae strain showed a 31% lower glycerol yield and a 31% lower specific growth rate than a non-engineered reference strain. Growth of the engineered strain in anaerobic, glucose-limited chemostat cultures revealed a negative correlation between its specific growth rate and the contribution of the Calvin-cycle enzymes to redox homeostasis. Additional deletion of GPD2, which encodes an isoenzyme of NAD+-dependent glycerol-3-phosphate dehydrogenase, combined with overexpression of the structural genes for enzymes of the non-oxidative pentose-phosphate pathway, yielded a CO2-reducing strain that grew at the same rate as a non-engineered reference strain in anaerobic bioreactor batch cultures, while exhibiting a 86% lower glycerol yield and a 15% higher ethanol yield.ConclusionsThe metabolic engineering strategy presented here enables an almost complete elimination of glycerol production in anaerobic, glucose-grown batch cultures of S. cerevisiae, with an associated increase in ethanol yield, while retaining near wild-type growth rates and a capacity for glycerol formation under osmotic stress. Using current genome-editing techniques, the required genetic modifications can be introduced in one or a few transformations. Evaluation of this concept in industrial strains and conditions is therefore a realistic next step towards its implementation for improving the efficiency of first- and second-generation bioethanol production.

Regulation of the Calvin-Benson-Bassham cycle in the enigmatic diatoms: biochemical and evolutionary variations on an original theme.

In Plantae, the Calvin-Benson-Bassham (CBB) cycle is highly regulated and most of its enzymes have been thoroughly studied. Since diatoms arose as a result of secondary endosymbiosis with one or more Plantae ancestors, their precise evolutionary history is enigmatic and complex resulting in biochemical variations on the original CBB cycle theme. The Rubisco Michaelis constant for CO2 is higher in diatoms than land plants and the nuclear-encoded Rubisco activase in Plantae is replaced by an analogous chloroplast-encoded CbbX (Calvin-Benson-Bassham protein X) in diatoms. In the CBB cycle reduction phase, phosphoglycerate kinase in diatoms is redox-regulated and similar to that in red algae; however, glyceraldehyde phosphate dehydrogenase (GAPDH) is not redox-regulated, unlike in Plantae. The phosphoribulokinase (PRK)-GAPDH-CP12 complex found in many photosynthetic organisms has not yet been found in diatoms, but a ferredoxin-NADP reductase (FNR)-GAPDH-CP12 complex has been found in one species. In the CBB cycle regeneration phase, sedoheptulose 1,7-bisphosphatase and PRK are not redox-regulated in diatoms, unlike in Plantae. Regulation at the transcriptional level seems to be important in diatoms. CBB cycle enzyme properties appear to be variable among diatoms, but this view relies on results from a few model species: a greater range of diatoms need to be studied to test this.This article is part of the themed issue 'The peculiar carbon metabolism in diatoms'.

Arabidopsis CP12 mutants have reduced levels of phosphoribulokinase and impaired function of the Calvin-Benson cycle.

CP12 is a small, redox-sensitive protein, the most detailed understanding of which is the thioredoxin-mediated regulation of the Calvin-Benson cycle, where it facilitates the formation of a complex between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) in response to changes in light intensity. In most organisms, CP12 proteins are encoded by small multigene families, where the importance of each individual CP12 gene in vivo has not yet been reported. We used Arabidopsis thaliana T-DNA mutants and RNAi transgenic lines with reduced levels of CP12 transcript to determine the relative importance of each of the CP12 genes. We found that single cp12-1, cp12-2, and cp12-3 mutants do not develop a severe photosynthetic or growth phenotype. In contrast, reductions of both CP12-1 and CP12-2 transcripts lead to reductions in photosynthetic capacity and to slower growth and reduced seed yield. No clear phenotype for CP12-3 was evident. Additionally, the levels of PRK protein are reduced in the cp12-1, cp12-1/2, and multiple mutants. Our results suggest that there is functional redundancy between CP12-1 and CP12-2 in Arabidopsis where these proteins have a role in determining the level of PRK in mature leaves and hence photosynthetic capacity.

A RuBisCO-mediated carbon metabolic pathway in methanogenic archaea

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

Assessment of photosynthesis regulation in mixotrophically cultured microalga Chlorella sorokiniana

Mixotrophic growth of microalgae offers great potential as an efficient strategy for biofuel production. In this study, photosynthetic regulation of mixotrophically cultured Chlorella sorokiniana cells was systematically evaluated. Mixotrophic cells in the exponential growth phase showed the highest photosynthetic activity, where maximum photosynthetic O2 evolution was approximately 3- and 4-fold higher than cells in the same phase grown photoautotrophically in 1% CO2 (in air) and air, respectively. Additionally, characteristic chlorophyll fluorescence parameters demonstrated that no limitation in electron transport downstream of PSII was detected in mixotrophic cells. Up-regulation of photosynthetic activity was associated with high total ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) carboxylase activity and expression level of phosphoribulokinase (PRK). After 3days, photosynthetic O2 evolution of mixotrophic cells that went to the stationary phase, was strongly reduced, with reduced photochemical efficiency and reorganization of the PSII complex. Simultaneously, enzymatic activity for Rubisco carboxylase and mRNA levels of Rubisco and PRK diminished. Importantly, there was almost no non-photochemical quenching for mixotrophic cells, whether grown in log or stationary phase. A decline in the quantum efficiency of PSII and an oxidized plastoquinone pool (PQ pool) was observed under N-depleted conditions during mixotrophic growth. These results demonstrate that photosynthesis is regulated differently in mixotrophically cultured C. sorokiniana cells than in cells grown under photoautotrophic conditions, with a particularly strong impact by nitrogen levels in the cells.

A vacuolar Na+/H+ antiporter gene, IbNHX2, enhances salt and drought tolerance in transgenic sweetpotato

Plant vacuolar Na+/H+ antiporters (NHX) play a critical role in adaption to abiotic stresses by compartmentalizing Na+ into vacuoles from the cytosol. In this study, a vacuolar Na+/H+ antiporter gene, named IbNHX2, was isolated and characterized from salt-tolerant sweetpotato (Ipomoea batatas (L.) Lam.) line ND98. IbNHX2 consisted of 542 amino acid residues with a conserved binding domain ‘FFIYLLPPI’ for amiloride and a cation/H+ exchanger domain, and shared a high amino acid identity (73.72–96.13%) with the identified vacuolar Na+/H+ antiporters in other plant species. The genomic DNA of IbNHX2 contained 14 exons and 13 introns. Expression of IbNHX2 was induced by abscisic acid (ABA), NaCl and polyethylene glycol (PEG). Its overexpression significantly enhanced salt and drought tolerance in the transgenic sweetpotato. An significant increase of proline content and superoxide dismutase (SOD) and photosynthesis activities and significant reduction of malonaldehyde (MDA) and H2O2 content were found in the transgenic sweetpotato plants. Up-regulation of the stress-responsive genes encoding pyrroline-5-carboxylate synthase (P5CS), SOD, catalase (CAT), zeaxanthinepoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED), aldehyde oxidase (AO), late embryogenesis abundant protein (LEA), psbA and phosphoribulokinase (PRK) in the transgenic plants was also found under salt and drought stresses. The overall results demonstrate the explicit role of IbNHX2 in conferring salt and drought tolerance of sweetpotato. The IbNHX2 gene has the potential to be used for improving salt and drought tolerance of plants.

Overexpression of IbMIPS1 gene enhances salt tolerance in transgenic sweetpotato

AbstractMyo-inositol-1-phosphate synthase (MIPS) is a key rate limiting enzyme in the de novo biosynthesis of myo-inositol in plants. In the present study, the IbMIPS1 gene was introduced into sweetpotato cultivar Xushu 18 and the transgenic plants exhibited significantly enhanced salt tolerance compared with the wild-type (WT). Overexpression of IbMIPS1 up-regulated the salt stress responsive genes, including myo-inositol monophosphatase (MIPP), pyrroline-5-carboxylate synthase (P5CS), pyrroline-5-carboxylate reductase (P5CR), psbA, phosphoribulokinase (PRK), and superoxide dismutase (SOD) genes, under salt stress. Inositol and proline content, SOD and photosynthesis activities were significantly increased, whereas malonaldehyde (MDA) and H2O2 contents were significantly decreased in the transgenic plants. These findings suggest that the IbMIPS1 gene may enhance salt tolerance of sweetpotato by regulating the expression of salt stress responsive genes, increasing the content of inositol and proline and enhancing the activity of photosynthesis.

Disrupting Calvin cycle phosphoribulokinase activity in Rhodopseudomonas palustris increases the H2 yield and specific production rate proportionately

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.

Unravelling the shape and structural assembly of the photosynthetic GAPDH-CP12-PRK complex from Arabidopsis thaliana by small-angle X-ray scattering analysis.

Oxygenic photosynthetic organisms produce sugars through the Calvin-Benson cycle, a metabolism that is tightly linked to the light reactions of photosynthesis and is regulated by different mechanisms, including the formation of protein complexes. Two enzymes of the cycle, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), form a supramolecular complex with the regulatory protein CP12 with the formula (GAPDH-CP122-PRK)2, in which both enzyme activities are transiently inhibited during the night. Small-angle X-ray scattering analysis performed on both the GAPDH-CP12-PRK complex and its components, GAPDH-CP12 and PRK, from Arabidopsis thaliana showed that (i) PRK has an elongated, bent and screwed shape, (ii) the oxidized N-terminal region of CP12 that is not embedded in the GAPDH-CP12 complex prefers a compact conformation and (iii) the interaction of PRK with the N-terminal region of CP12 favours the approach of two GAPDH tetramers. The interaction between the GAPDH tetramers may contribute to the overall stabilization of the GAPDH-CP12-PRK complex, the structure of which is presented here for the first time.