C4 Phosphoenolpyruvate Carboxylase Research Articles

Interactions between light and temperature during the in vitro as well as in situ modulation of phosphoenolpyruvate carboxylase in leaves of a C3 plant Pisum sativum L.

The C4 phosphoenolpyruvate carboxylase (PEPC) is well known for its modulation by light and temperature. We have attempted to address such coordinated effects of light and temperature on C3 PEPC in leaves of Pisum sativum. Experiments were done with dark-adapted leaf discs as well as with leaves collected at different times during the day and in each month during the year. The modulation of C3 PEPC by temperature was much stronger than that by light. The optimum temperature for in vitro activity of C3 PEPC was 25°C while 20°C was sub-optimal and 30°C to 45°C were supra-optimal. The photo-activation of PEPC was more at 25°C than at 45°C. The activation of C3 PEPC by temperature was more in the dark than in the light. In in situ conditions, a diurnal rhythm in the activity and regulatory properties of C3 PEPC was more prominent at colder than at warm temperatures. However, no consistent trends in the seasonal changes in PEPC activity/ regulatory properties were observed. Scatter plots suggested that light had a greater influence on PEPC activity while temperature exerted a much greater effect on its regulatory properties. Thus, C3 PEPC was also modulated by a natural variation in light and temperature under in situ conditions. These observations correlated well with the light and temperature requirements of C3 plants.

C4 Phosphoenolpyruvate Carboxylase: Evolution and transcriptional regulation.

Photosynthetic phosphoenolpyruvate carboxylase (PEPC) catalyses the irreversible carboxylation of phosphoenolpyruvate (PEP), producing oxaloacetate (OAA). This enzyme catalyses the first step of carbon fixation in C4 photosynthesis, contributing to the high photosynthetic efficiency of C4 plants. PEPC is also involved in replenishing tricarboxylic acid cycle intermediates, such as OAA, being involved in the C/N balance. In plants, PEPCs are classified in two types: bacterial type (BTPC) and plant-type (PTPC), which includes photosynthetic and non-photosynthetic PEPCs. During C4 evolution, photosynthetic PEPCs evolved independently. C4 PEPCs evolved to be highly expressed and active in a spatial-specific manner. Their gene expression pattern is also regulated by developmental cues, light, circadian clock as well as adverse environmental conditions. However, the gene regulatory networks controlling C4 PEPC gene expression, namely its cell-specificity, are largely unknown. Therefore, after an introduction to the evolution of PEPCs, this review aims to discuss the current knowledge regarding the transcriptional regulation of C4 PEPCs, focusing on cell-specific and developmental expression dynamics, light and circadian regulation, as well as response to abiotic stress. In conclusion, this review aims to highlight the evolution, transcriptional regulation by different signals and importance of PEPC in C4 photosynthesis and its potential as tool for crop improvement.

Shedding light on AT1G29480 of Arabidopsis thaliana-An enigmatic locus restricted to Brassicacean genomes.

A key feature of C4 Kranz anatomy is the presence of an enlarged, photosynthetically highly active bundle sheath whose cells contain large numbers of chloroplasts. With the aim to identify novel candidate regulators of C4 bundle sheath development, we performed an activation tagging screen with Arabidopsis thaliana . The reporter gene used encoded a chloroplast‐targeted GFP protein preferentially expressed in the bundle sheath, and the promoter of the C4 phosphoenolpyruvate carboxylase gene from Flaveria trinervia served as activation tag because of its activity in all chlorenchymatous tissues of A. thaliana . Primary mutants were selected based on their GFP signal intensity, and one stable mutant named kb‐1 with a significant increase in GFP fluorescence intensity was obtained. Despite the increased GFP signal, kb‐1 showed no alterations to bundle sheath anatomy. The causal locus, AT1G29480, is specific to the Brassicaceae with its second exon being conserved. Overexpression and reconstitution studies confirmed that AT1G29480, and specifically its second exon, were sufficient for the enhanced GFP phenotype, which was not dependent on translation of the locus or its parts into protein. We conclude, therefore, that the AT1G29480 locus enhances the GFP reporter gene activity via an RNA‐based mechanism.

Multiple conformations in solution of the maize C4-phosphoenolpyruvate carboxylase isozyme

The photosynthetic phosphoenolpyruvate carboxylase isozyme from C4 plants (PEPC-C4) has a complex allosteric regulation, involving positive cooperativity in binding the substrate phosphoenolpyruvate as well as positive and negative allosteric effectors. Besides the proposed R- and T-states, previous kinetic results suggested functionally relevant different R-states of the maize enzyme (ZmPEPC-C4) elicited by PEP or its two kinds of activators, glucose 6-phosphate or glycine. To detect these different R-state conformations, we used as conformational probes the fluorescence of 8-anilino-1-naphthalene sulfonate (ANS), near-UV circular dichroism (CD) spectroscopy, and limited proteolysis by trypsin. Phosphoenolpyruvate and malate binding caused distinct concentration-dependent fluorescence changes of ZmPEPC-C4/ANS, suggesting that they elicited conformational states different from that of the free enzyme, while glucose 6-phosphate or glycine binding did not produce fluorescence changes. Differences were also observed in the near UV CD spectra of the enzyme, free or complexed with its substrate or allosteric effectors. Additionally, differences in the trypsin-digestion fragmentation patterns, as well as in the susceptibility of the free and complexed enzyme to digestion and digestion-provoked loss of activity, provided evidence of several ZmPEPC-C4 conformations in solution elicited by the substrate and the allosteric effectors. Using the already reported ZmPEPC-C4 crystal structures and bioinformatics methods, we predicted that the most probable trypsin-cleavage sites are located in superficial flexible regions, which seems relevant for the protein dynamics underlying the function and allosteric regulation of this enzyme. Together, our findings agree with previous kinetic results, shed light on this enzyme's complex allosteric regulation, and place ZmPEPC-C4 in the growing list of allosteric enzymes possessing an ensemble of closely related R-state conformations.

A conserved C-terminal peptide of sorghum phosphoenolpyruvate carboxylase promotes its proteolysis, which is prevented by Glc-6P or the phosphorylation state of the enzyme

Main conclusionA synthetic peptide from the C-terminal end of C4-phosphoenolpyruvate carboxylase is implicated in the proteolysis of the enzyme, and Glc-6P or phosphorylation of the enzyme modulate this effect.Phosphoenolpyruvate carboxylase (PEPC) is a cytosolic, homotetrameric enzyme that performs a variety of functions in plants. Among them, it is primarily responsible for CO2 fixation in the C4 photosynthesis pathway (C4-PEPC). Here we show that proteolysis of C4-PEPC by cathepsin proteases present in a semi-purified PEPC fraction was enhanced by the presence of a synthetic peptide containing the last 19 amino acids from the C-terminal end of the PEPC subunit (pC19). Threonine (Thr)944 and Thr948 in the peptide are important requirements for the pC19 effect. C4-PEPC proteolysis in the presence of pC19 was prevented by the PEPC allosteric effector glucose 6-phosphate (Glc-6P) and by phosphorylation of the enzyme. The role of these elements in the regulation of PEPC proteolysis is discussed in relation to the physiological context.

Genetic Diversity of C4 Photosynthesis Pathway Genes in Sorghum bicolor (L.).

C4 photosynthesis has evolved in over 60 different plant taxa and is an excellent example of convergent evolution. Plants using the C4 photosynthetic pathway have an efficiency advantage, particularly in hot and dry environments. They account for 23% of global primary production and include some of our most productive cereals. While previous genetic studies comparing phylogenetically related C3 and C4 species have elucidated the genetic diversity underpinning the C4 photosynthetic pathway, no previous studies have described the genetic diversity of the genes involved in this pathway within a C4 crop species. Enhanced understanding of the allelic diversity and selection signatures of genes in this pathway may present opportunities to improve photosynthetic efficiency, and ultimately yield, by exploiting natural variation. Here, we present the first genetic diversity survey of 8 known C4 gene families in an important C4 crop, Sorghum bicolor (L.) Moench, using sequence data of 48 genotypes covering wild and domesticated sorghum accessions. Average nucleotide diversity of C4 gene families varied more than 20-fold from the NADP-malate dehydrogenase (MDH) gene family (θπ = 0.2 × 10−3) to the pyruvate orthophosphate dikinase (PPDK) gene family (θπ = 5.21 × 10−3). Genetic diversity of C4 genes was reduced by 22.43% in cultivated sorghum compared to wild and weedy sorghum, indicating that the group of wild and weedy sorghum may constitute an untapped reservoir for alleles related to the C4 photosynthetic pathway. A SNP-level analysis identified purifying selection signals on C4 PPDK and carbonic anhydrase (CA) genes, and balancing selection signals on C4 PPDK-regulatory protein (RP) and phosphoenolpyruvate carboxylase (PEPC) genes. Allelic distribution of these C4 genes was consistent with selection signals detected. A better understanding of the genetic diversity of C4 pathway in sorghum paves the way for mining the natural allelic variation for the improvement of photosynthesis.

Structural and biochemical evidence of the glucose 6-phosphate-allosteric site of maize C4-phosphoenolpyruvate carboxylase: its importance in the overall enzyme kinetics.

Activation of phosphoenolpyruvate carboxylase (PEPC) enzymes by glucose 6-phosphate (G6P) and other phospho-sugars is of major physiological relevance. Previous kinetic, site-directed mutagenesis and crystallographic results are consistent with allosteric activation, but the existence of a G6P-allosteric site was questioned and competitive activation-in which G6P would bind to the active site eliciting the same positive homotropic effect as the substrate phosphoenolpyruvate (PEP)-was proposed. Here, we report the crystal structure of the PEPC-C4 isozyme from Zea mays with G6P well bound into the previously proposed allosteric site, unambiguously confirming its existence. To test its functionality, Asp239-which participates in a web of interactions of the protein with G6P-was changed to alanine. The D239A variant was not activated by G6P but, on the contrary, inhibited. Inhibition was also observed in the wild-type enzyme at concentrations of G6P higher than those producing activation, and probably arises from G6P binding to the active site in competition with PEP. The lower activity and cooperativity for the substrate PEP, lower activation by glycine and diminished response to malate of the D239A variant suggest that the heterotropic allosteric activation effects of free-PEP are also abolished in this variant. Together, our findings are consistent with both the existence of the G6P-allosteric site and its essentiality for the activation of PEPC enzymes by phosphorylated compounds. Furthermore, our findings suggest a central role of the G6P-allosteric site in the overall kinetics of these enzymes even in the absence of G6P or other phospho-sugars, because of its involvement in activation by free-PEP.

Drought tolerance of transgenic rice overexpressing maize C<sub>4</sub>-PEPC gene related to increased anthocyanin synthesis regulated by sucrose and calcium

In order to reveal the role of sucrose (Suc) in early drought response in plants, transgenic rice (Oryza sativa L.) plants overexpressing the maize (Zea mays L.) C4-phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) gene (C4-pepc) (PC) and their untransformed wild type (WT) were used under 12 % (m/v) polyethylene glycol 6000 to simulate drought conditions. The results showed that PC has higher relative water content, the increased Suc content, and anthocyanin accumulation than WT during PEG treatment. By spraying 1 % Suc and 1 % Suc non-metabolic analog, turanose, on these plants, Suc in PC leaves increased anthocyanin content and Ca2+ content. Further experiments using the Ca2+ chelator (EGTA), Ca2+ channel antagonist (ruthenium red), and abscisic acid inhibitor (nordihydroguaiaretic acid), showed that, in PC plants, Suc content is closely related to the expression of sucrose nonfermenting-1-related protein kinases 2 (SnRK2s) such as SAPK8, SAPK9, and SAPK10 via abscisic acid, and the SnRK3 such as SnRK3.1, SnRK 3.4, and SnRK3.21 via Ca2+ and calcineurin B-like as well. Furthermore, the target genes associated with anthocyanin synthesis phenylalanine ammonia lyase, chalcone isomerase, chalcone synthase, flavonoid-3-hydrogenase, flavonoid-3'-hydrogenase, dihydroflavonone reductase, and anthocyanin synthase, their regulated genes basic helix-loop-helix (bHLH) proteins OsB1 and OsB2, R2R3-MYB transcription factor OsC1, and some transcription factors (constitutively photomorphogenic 1, elongated hypocotyl 5, and purple acid phosphatase gene 2) in PC plants also increased via Suc and Ca2+ during the PEG treatment. Some Suc tranporter genes OsSUT1 and OsSUT5 in PC lines during PEG treatment further showed an enhancement for the function of the signal of Suc. Thereby, increasing anthocyanin biosynthesis via Suc and Ca2+ signaling cascade is one of the important mechanisms on drought tolerance in the PC lines.

Expression Profiles and Post-Translational Modifications of Phosphoenolpyruvate Carboxylase Isozymes of Bienertia sinuspersici during Leaf Development

Phosphoenolpyruvate carboxylase (PEPC) is the primary acceptor of carbon dioxide in C4 photosynthesis. Three isozymes of PEPC: PEPC1, PEPC2, and PEPC3, in the single cell, C4 plant Bienertiasinuspersici Akhani were determined in this study. Differential expressions of these isoforms were observed at different stages of leaf development. Amino acid sequence alignment revealed that PEPC1, PEPC2, and PEPC3 had characteristics of C3-C4 PEPC type, C4 PEPC, and C3 PEPC, respectively. Gene expression profiles showed that PEPC3 was highly expressed in young leaves while PEPC1 and PEPC2 were more expressed in intermediate and mature leaves, respectively. Using immunoblotting, we also assessed tissue-specific expression and post-translational modifications (PTMs) of PEPC in B. sinuspersici. During leaf maturation, phosphorylation at Ser 11 and monoubiquitination at Lys 629 were highly increased. The phosphorylation pattern on PEPC was diurnally regulated but monoubiquitination was not. Moreover, dimorphic chloroplasts from B. sinuspersici chlorenchyma cell of mature leaf were isolated to study spatial localization of PEPC by PTM. Monoubiquitinated PEPC were specifically interacting with peripheral chloroplasts. These results indicated that phosphorylation and monoubiquitination on PEPC were mutually exclusive as well as affecting spatial localization in single cell C4 plants.

Enhanced photorespiration in transgenic rice over-expressing maize C4 phosphoenolpyruvate carboxylase gene contributes to alleviating low nitrogen stress

The objective of this study was to reveal the physiological and molecular mechanisms of low-nitrogen (N) tolerance in transgenic plant lines containing C4 phosphoenolpyruvate carboxylase (C4-PEPC) gene. The transgenic rice lines only over-expressing the maize C4-PEPC) (PC) and their untransformed wild type, Kitaake (WT), were used in this study. At different N levels, the dry weight, total N content, carbon and N levels, photorespiration-related enzymatic activities, gene expression levels and photorespiration-related product accumulations were measured, as were the transgenic lines' agronomic traits. The PC line, having lower total N and higher soluble sugar contents, was more tolerant to low-N stress than WT, which was consistent with its higher PEPC and lower N-assimilation-related enzyme activity levels. The photosynthetic parameters, enzymatic activity levels, transcripts and products related to photorespiration in PC were also greater than in WT under low-N conditions. This study showed that increased carbon levels in transgenic rice lines overexpressing C4-PEPC could help regulate the photorespiratory pathway under low-N conditions, conferring low-N tolerance and a higher grain yield per plant.

Effects of the maize C4 phosphoenolpyruvate carboxylase (ZmPEPC) gene on nitrogen assimilation in transgenic wheat

Nitrogen (N) is the primary limiting factor for crop growth, development, and productivity. Transgenic technology is a straightforward strategy for improving N assimilation in crops. The present study assessed the effects of maize C4 phosphoenolpyruvate carboxylase (ZmPEPC) gene overexpression on N assimilation in three independent transgenic lines and wild-type (WT) wheat (Triticum aestivum L.). The transgenic wheat lines depicted ZmPEPC overexpression and higher PEPC enzyme activity relative to that in the WT. The leaves of the transgenic wheat lines subjected to low N treatment showed an increase in ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) expression, content, and carboxylase activity. The transgenic wheat lines also depicted an upregulation of genes associated with the anaplerotic pathway for the TCA cycle, suggesting that more carbon (C) skeleton material is being allocated for N assimilation under low N conditions. Furthermore, ZmPEPC expression in transgenic wheat lines induced the upregulated of genes associated primary N metabolism, including TaNR, TaGS2, TaGOGAT, TaAspAT, and TaASN1. The average total free amino acid content in the transgenic wheat lines was 48.18% higher than that in the WT, and asparagine (Asn), glutamine (Gln), aspartic acid (Asp), and serine (Ser) were also markedly enhanced. In addition, elementary analysis showed that N and C content, and the biomass of the transgenic wheat lines increased with low N treatment. Yield trait analysis indicated that ZmPEPC overexpression improved grain yield by increasing 1000-grain weight. In conclusion, ZmPEPC overexpression in wheat could modulate C metabolism, significantly improve N assimilation, enhances growth, and improves yield under low N conditions.

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

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 C4-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 H2O2 than PC under the 2-h PEG 6000 treatment, indicating greater damages in WT. Results from pretreatments with a Ca2+ chelator and/or antagonist showed that the regulation of the early drought response in PC was Ca2+-dependent. The NO and H2O2 levels in PC lines were also up-regulated via Ca2+ signals, indicating that Ca2+ in PC lines also reacted upstream of NO and H2O2. 2-h drought treatment increased the transcripts of CPK9 and CPK4 in PC via positive up-regulation of Ca2+. 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 Ca2+ signals in PC. PEPC activity, expressions of C4-pepc, and the antioxidant enzyme activities in PC lines were up-regulated via Ca2+. These results indicated that Ca2+ 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.

Salinity promotes opposite patterns of carbonylation and nitrosylation of C4 phosphoenolpyruvate carboxylase in sorghum leaves.

Carbonylation inactivates sorghum C 4 PEPCase while nitrosylation has little impact on its activity but holds back carbonylation. This interplay could be important to preserve photosynthetic C4 PEPCase activity in salinity. Previous work had shown that nitric acid (NO) increased phosphoenolpyruvate carboxylase kinase (PEPCase-k) activity, promoting the phosphorylation of phosphoenolpyruvate carboxylase (PEPCase) in sorghum leaves (Monreal et al. in Planta 238:859-869, 2013b). The present work investigates the effect of NO on C4 PEPCase in sorghum leaves and its interplay with carbonylation, an oxidative modification frequently observed under salt stress. The PEPCase of sorghum leaves could be carbonylated in vitro and in vivo, and this post-translational modification (PTM) was accompanied by a loss of its activity. Similarly, PEPCase could be S-nitrosylated in vitro and in vivo, and this PTM had little impact on its activity. The S-nitrosylated PEPCase showed increased resistance towards subsequent carbonylation, both in vitro and in vivo. Under salt shock, carbonylation of PEPCase increased in parallel with decreased S-nitrosylation of the enzyme. Subsequent increase of S-nitrosylation was accompanied by decreased carbonylation. Taken together, the results suggest that S-nitrosylation could contribute to maintain C4 PEPCase activity in stressed sorghum plants. Thus, salt-induced NO synthesis would be protecting photosynthetic PEPCase activity from oxidative inactivation while promoting its phosphorylation, which will guarantee its optimal functioning in suboptimal conditions.

Physiological characteristics and metabolomics of transgenic wheat containing the maize C4 phosphoenolpyruvate carboxylase (PEPC) gene under high temperature stress

In this paper, two transgenic wheat lines, PC27 and PC51, containing the maize PEPC gene and its wild-type (WT) were used as experimental material to study the effects of high temperature on their photosynthetic physiological characteristics and metabolome. The results showed that transgenic wheat lines had higher photosynthetic rate (P n) than WT under non-stress treatment (NT) and high temperature stress treatment (HT), and more significantly under HT. The change trends of F v/F m, Ф PSII, and q P were similar to P n, whereas that of non-photochemical quenching (NPQ) was the opposite. Compared with WT, no differences in chlorophyll content between the transgenic wheat and WT were observed under NT, but two transgenic lines had relatively higher contents than WT under HT. The change trends of Chlorophyll a/b radio, the decreased values of F m, Wk, and Vj, and the activity of the antioxidant enzyme were consistent with the chlorophyll content. Compared with WT, transgenic wheat lines exhibited lower rate of superoxide anion production, H2O2 and malondialdehyde content under HT, and no significant differences were observed under NT. The expression pattern of the ZmPEPC gene and wheat endogenous photosynthesis-related genes were in agreement with that of P n. Compared with WT, about 13 different metabolites including one organic acid, six amino acids, four sugars, and two polyols were identified under NT; 25 different metabolites including six organic acids, 12 amino acids, four sugars, and three polyols were identified under HT. Collectively, our results indicate that ZmPEPC gene can enhance photochemical and antioxidant enzyme activity, upregulate the expression of photosynthesis-related genes, delay degradation of chlorophyll, change contents of proline and other metabolites in wheat, and ultimately improves its heat tolerance.

Chalcone-based Selective Inhibitors of a C4 Plant Key Enzyme as Novel Potential Herbicides.

Weeds are a challenge for global food production due to their rapidly evolving resistance against herbicides. We have identified chalcones as selective inhibitors of phosphoenolpyruvate carboxylase (PEPC), a key enzyme for carbon fixation and biomass increase in the C4 photosynthetic pathway of many of the world’s most damaging weeds. In contrast, many of the most important crop plants use C3 photosynthesis. Here, we show that 2′,3′,4′,3,4-Pentahydroxychalcone (IC50 = 600 nM) and 2′,3′,4′-Trihydroxychalcone (IC50 = 4.2 μM) are potent inhibitors of C4 PEPC but do not affect C3 PEPC at a same concentration range (selectivity factor: 15–45). Binding and modeling studies indicate that the active compounds bind at the same site as malate/aspartate, the natural feedback inhibitors of the C4 pathway. At the whole plant level, both substances showed pronounced growth-inhibitory effects on the C4 weed Amaranthus retroflexus, while there were no measurable effects on oilseed rape, a C3 plant. Growth of selected soil bacteria was not affected by these substances. Our chalcone compounds are the most potent and selective C4 PEPC inhibitors known to date. They offer a novel approach to combat C4 weeds based on a hitherto unexplored mode of allosteric inhibition of a C4 plant key enzyme.

Temperature response of bundle-sheath conductance in maize leaves.

A small bundle-sheath conductance (g bs) is essential for the C4 CO2-concentrating mechanism to suppress photorespiration effectively. To predict the productivity of C4 crops accurately under global warming, it is necessary to examine whether and how g bs responds to temperature. We investigated the temperature response of g bs in maize by fitting a C4 photosynthesis model to combined gas exchange and chlorophyll fluorescence measurements of irradiance and CO2 response curves at 21% and 2% O2 within the range of 13.5-39 °C. The analysis was based on reported kinetic constants of C4 Rubisco and phosphoenolpyruvate carboxylase and temperature responses of C3 mesophyll conductance (g m). The estimates of g bs varied greatly with leaf temperature. The temperature response of g bs was well described by the peaked Arrhenius equation, with the optimum temperature being ~34 °C. The assumed temperature responses of g m had only a slight impact on the temperature response of g bs In contrast, using extreme values of some enzyme kinetic constants changed the shape of the response, from the peaked optimum response to the non-peaked Arrhenius pattern. Further studies are needed to confirm such an Arrhenius response pattern from independent measurement techniques and to assess whether it is common across C4 species.

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

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.

Response of photosynthesis to short-term drought stress in rice seedlings overexpressing C<sub>4</sub> phosphoenolpyruvate carboxylase from maize and millet

Abiotic stresses induce phosphoenolpyruvate carboxylase (PEPC) expression in C3 plants which suggests PEPC function in plant adaptation to stresses. Here, we studied the response of photosynthesis to short-term drought stress in rice seedlings overexpressing C4 PEPC from maize and millet. The transgenic lines exhibited 1.2–5.5 fold of PEPC activities than the wild type before the treatment, while 1.5–8.5 fold after five or ten days of water deficit. Net photosynthetic rate (P N) declined less during the water stress and recovered more after rewatering in the transgenic lines. These changes were accompanied with changes in the stomatal conductance (g s). The lower decrease in P N and g s resulted in significantly higher intrinsic water use efficiency in the transgenic rice lines after ten days of water withdrawal. There were no significant differences between the wild type and transgenic lines in maximum photochemical efficiency of PSII and photochemical quenching. The nonphotochemical quenching and the quantum efficiency of PSII maintained both higher in transgenic lines than those in the wild type during drought stress. This indicated that the transgenic lines could dissipate more excess energy to heat to protect PSII. Our result suggested that the increased PEPC activities in rice could alleviate the decrease of photosynthesis during short-term drought stress.

Enhanced drought tolerance in transgenic rice over-expressing of maize C4 phosphoenolpyruvate carboxylase gene via NO and Ca2+

We determined the effects of endogenous nitric oxide and Ca2+ on photosynthesis and gene expression in transgenic rice plants (PC) over-expressing the maize C4pepc gene, which encodes phosphoenolpyruvate carboxylase (PEPC) under drought. In this study, seedlings were subjected to PEG 6000 treatments using PC and wild type (WT; Kitaake). The results showed that, compared with WT, PC had higher relative water content (RWC) and net photosynthetic rate (Pn) under drought. During a 2-day re-watering treatment, Pn recovered faster in PC than in WT. Further analyses showed that, under the drought treatment, the amount of endogenous hydrogen peroxide (H2O2) increased in WT mainly via NADPH oxidase. While in PC, the endogenous nitric oxide (NO) content increased via nitrate reductase and nitric oxide synthase on day 2 of the drought treatment and day 1 of the re-watering treatment. After 2 days of drought treatment, PC also showed higher PEPC activity, calcium content, phospholipase D (PLD) activity, C4-pepc and NAC6 transcript levels, and protein kinase activity as compared with PC without treatment. These changes did not occur in WT. Correlation analysis also proved NO associated with these indicators in PC. Based on these results, there was a particular molecular mechanism of drought tolerance in PC. The mechanism is related to the signaling processes via NO and Ca2+ involving the protein kinase and the transcription factor, resulted in up-regulation of PEPC activity and its gene expression, such as C4pepc. Some genes encode antioxidant system, cu/zn-sod as well, which promote antioxidant system to clear MDA and superoxide anion radical, thereby conferring drought tolerance.

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

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.