Phosphoenolpyruvate Carboxylase Enzyme Research Articles

Biotin concentration affects anaplerotic reactions functioning in glutamic acid production in Corynebacterium glutamicum.

The study investigates the effect of biotin concentration on the role of anaplerotic reactions catalysed by pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC) in glutamic acid production by Corynebacterium glutamicum. C. glutamicum requires biotin for its growth, and its glutamic acid production can be induced by the addition of Tween 40 or penicillin or by biotin limitation. The biotin enzyme PC and the non-biotin enzyme PEPC catalyse two anaplerotic reactions to supply oxaloacetic acid to the TCA cycle in C. glutamicum. Therefore, they are crucial for glutamic acid production in this bacterium. In this study, we investigated the contribution of each anaplerotic reaction to Tween 40- and penicillin-induced glutamic acid production using disruptants of PEPC and PC. In the presence of 20 µg l-1 biotin, which is sufficient for growth, the PEPC-catalysed anaplerotic reaction mainly contributed to Tween 40- and penicillin-induced glutamic acid production. However, when increasing biotin concentration 10-fold (i.e. 200 µg l-1), both PC- and PEPC-catalysed reactions could function in glutamic acid production. Western blotting revealed that the amount of biotin-bound PC was reduced by the addition of Tween 40 and penicillin in the presence of 20 µg l-1. However, these induction treatments did not change the amount of biotin-bound PC in the presence of 200 µg l-1 biotin. These results indicate that both anaplerotic reactions are functional during glutamic acid production in C. glutamicum and that biotin concentration mainly affects which anaplerotic reactions function during glutamic acid production.

Overexpression of phosphoenolpyruvate carboxylase kinase gene MsPPCK1 from Medicago sativa L. increased alkali tolerance of alfalfa by enhancing photosynthetic efficiency and promoting nodule development

The phosphoenolpyruvate carboxylase kinase of Medicago sativa L. (MsPPCK1) modulates the phosphorylation status and activity of the C4 pathway phosphoenolpyruvate carboxylase enzyme, which is pivotal for photosynthetic carbon assimilation in plants. This study investigated the role of MsPPCK1 in alfalfa by creating transgenic plants overexpressing MsPPCK1 under the control of the CaMV35S promoter. The enhanced alkali tolerance of transgenic plants indicated an important role of MsPPCK1 gene in regulating plant alkali tolerance. Transgenic plants exhibited heightened antioxidant activity (SOD, POD, and CAT), reduced MDA, H2O2, OFR and REC% content, increased activity of key photosynthetic enzymes (PEPC, PPDK, NADP-ME, and NADP-MDH), and enhanced photosynthetic parameters (Pn, E, Gs, and Ci). Moreover, MsPPCK1 overexpression increased the content of organic acids (oxaloacetic, malic, citric, and succinic acids) in the plants. The upregulation of MsPPCK1 under rhizobial inoculation showcased its other role in nodule development. In transgenic plants, MsDMI2, MsEnod12, and MsNODL4 expression increased, facilitating root nodule development and augmenting plant nodulation. Accelerated root nodule growth positively influences plant growth and yield and enhances alfalfa resistance to alkali stress. This study highlights the pivotal role of MsPPCK1 in fortifying plant alkali stress tolerance and improving yield, underscoring its potential as a key genetic target for developing alkali-tolerant and high-yielding alfalfa varieties.

Contributions of the anaplerotic reaction enzymes pyruvate carboxylase and phosphoenolpyruvate carboxylase to l-lysine production in Corynebacterium glutamicum

Anaplerotic reactions catalyzed by pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC) have important roles in the production of l-lysine to replenish oxaloacetic acid (OAA) in Corynebacterium glutamicum. However, the relative contributions of these enzymes to l-lysine production in C. glutamicum are not fully understood. In this study, using a parent strain (P) carrying a feedback inhibition-resistant aspartokinase with the T311I mutation, we constructed a PC gene-deleted mutant strain (PΔPC) and a PEPC gene-deleted mutant strain (PΔPEPC). Although the growth of both mutant strains was comparable to the growth of strain P, the maximum l-lysine production in strains PΔPC and PΔPEPC decreased by 14% and 49%, respectively, indicating that PEPC strongly contributed to OAA supply. l-Lysine production in strain PΔPC slightly decreased during the logarithmic phase, while production during the early stationary phase was comparable to production in strain P. By contrast, strain PΔPEPC produced l-lysine in an amount comparable to the production of strain P during the logarithmic phase; l-lysine production after the early stationary phase was completely stopped in strain PΔPEPC. These results indicate that OAA is supplied by both PC and PEPC during the logarithmic phase, while only PEPC can continuously supply OAA after the logarithmic phase.

The distinctive level of interaction between carbon and nitrogen metabolisms in the leaves of submerged macrophytes plays a key role in ammonium detoxification

Possible ammonium detoxification mechanisms have been proposed recently, on submerged macrophytes, evidently illustrating that glutamate dehydrogenase (GDH) plays a greater role in ammonium detoxification compared to the primary glutamine synthetase/glutamate oxaloacetate transaminase (GS/GOGAT) pathway. In the current investigation, we cultured three submerged macrophytes to extreme concentrations of [NH4+-N] of up to 50 mg/L with the aim of clarifying the interaction between carbon and nitrogen metabolisms. The activities of carboxylation enzymes pyruvate orthophosphate dikinase (PPDK) and phosphoenolpyruvate carboxylase (PEPC), in lieu of Rubisco, increased almost two-fold for ammonium tolerant species P. maackianus and M. spicatum, compared with the sensitive species P. lucens. While these enzymes are well known for their central role in CO2 fixation, their inference in conferring resistance to ammonium stress has not been well elucidated before. In this study, we demonstrate that the overproduction of PEPC and PPDK led to improved photosynthesis, better ammonium assimilation and overall ammonium detoxification in M. spicatum and P. maackianus. These findings propose likelihood for the existence of a complementary ammonium detoxification pathway that targets carbon metabolism, thus, presenting a relatively efficient linkage between nitrogen and carbon metabolisms and identify candidate species for practical restoration of fresh water resources.

The quandary of sources and sinks of CO2 efflux in tree stems-new insights and future directions.

Stem respiration (RS) substantially contributes to the return of photo assimilated carbon to the atmosphere and, thus, to the tree and ecosystem carbon balance. Stem CO2 efflux (ECO2) is often used as a proxy for RS. However, this metric has often been challenged because of the uncertain origin of CO2 emitted from the stem due to post-respiratory processes. In this Insight, we (i) describe processes affecting the quantification of RS, (ii) review common methodological approaches to quantify and model RS and (iii) develop a research agenda to fill the most relevant knowledge gaps that we identified. Dissolution, transport and accumulation of respired CO2 away from its production site, reassimilation of respired CO2 via stem photosynthesis and the enzyme phosphoenolpyruvate carboxylase, axial CO2 diffusion in the gas phase, shifts in the respiratory substrate and non-respiratory oxygen (O2) consumption are the most relevant processes causing divergence between RS and measured stem gas exchange (ECO2 or O2 influx, IO2). Two common methodological approaches to estimate RS, namely the CO2 mass balance approach and the O2 consumption technique, circumvent some of these processes but have yielded inconsistent results regarding the fate of respired CO2. Stem respiration modelling has recently progressed at the organ and tree levels. However, its implementation in large-scale models, commonly operated from a source-driven perspective, is unlikely to reflect adequate mechanisms. Finally, we propose hypotheses and approaches to advance the knowledge of the stem carbon balance, the role of sap pH on RS, the reassimilation of respired CO2, RS upscaling procedures, large-scale RS modelling and shifts in respiratory metabolism during environmental stress.

Effect of Acclimation to High Temperatures on the Mechanisms of Drought Tolerance in Species with Different Types of Photosynthesis: Sedobassia sedoides (C3–C4) and Bassia prostrata (C4-NADP)

The effect of drought on the morphophysiological, biochemical, and molecular genetic parameters of plants Sedobassia sedoides (Pall.) Freitag G. Kadereit with an intermediate C3–C4-type of photosynthesis and Bassia prostrata (L.) A.J. Scott with a C4-NADP type of photosynthesis grown at different temperatures (25 and 30°C) was studied. A decrease in the biomass, water content, and effective quantum yield (ΦPSII) of PSII, as well as an increase in the expression of the psbA gene encoding the PSII D1 protein under the action of drought, was observed in both species regardless of the growing temperature. Both species showed a decrease in the content of photosynthetic enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC) under drought conditions at 25°С, which was accompanied by a significant increase in the expression of the rbcL and PPDK genes in S. sedoides. Acclimation of S. sedoides plants to elevated temperatures led to an increase in the activity of cyclic electron transport around PSI, to mitigation of the negative effect of drought on the light reactions of photosynthesis (reduction in NPQ) and the content of the PEPC enzyme, as well as to a shift in the ionic balance caused by a decrease in the potassium content. B. prostrata showed greater drought resistance and was characterized by greater thermolability of photosynthetic enzymes, changes in the content and ratio of which allowed this species to maintain growth in drought conditions at different temperatures.

Sage leaf rock rose water extract: a bio-solution for enhancing the growth and salt stress resistance of sorghum plants.

Sorghum bicolor, a versatile cereal grain, holds significant agronomic importance globally and plays a crucial role in addressing food insecurity. However, salinity, a major abiotic stress, poses a threat to food production by reducing soil fertility and hindering plant growth and yield. In this study, we investigated the potential of Cistus salviifolius water extract (CSE) in mitigating salt stress in sorghum plants. Salt stress severely impacted plant growth, biomass, and chlorophyll production, and reduced indole-3-acetic acid (IAA) levels, which negatively affected plant development. Salt stress also led to the buildup of reactive oxygen species (ROS), hence, resulting in oxidative harm to sorghum plants and also affecting their carbon and nitrogen metabolism. On the other hand, CSE treatments increased IAA and chlorophyll content which promoted growth under stress. Furthermore, this extract exhibited strong ROS scavenging capacity and safeguarded plants against oxidative stress by enhancing the activities of antioxidant enzymes (superoxide dismutase, glutathione peroxidase, glutathione-S-transferase, and glutathione reductase) and increasing the production of osmolytes. Additionally, CSE treatments enhanced the activities of carbon/nitrogen enzymes (phosphoenolpyruvate carboxylase, malate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase, and glutamine synthase), promoting energy synthesis and crop growth. This led to a significant increase in sorghum growth in salted soil with the highest rise recorded for 5mg/L of CSE (an increase of 48.23% and 158.36% in length and weight compared to the salt control), which highlights this extract's potential as a biostimulant to enhance crop tolerance to salinity and contribute to sustainable agriculture.

Protein-Protein Interactions and Quantitative Phosphoproteomic Analysis Reveal Potential Mitochondrial Substrates of Protein Phosphatase 2A-B'ζ Holoenzyme.

Protein phosphatase 2A (PP2A) is a heterotrimeric conserved serine/threonine phosphatase complex that includes catalytic, scaffolding, and regulatory subunits. The 3 A subunits, 17 B subunits, and 5 C subunits that are encoded by the Arabidopsis genome allow 255 possible PP2A holoenzyme combinations. The regulatory subunits are crucial for substrate specificity and PP2A complex localization and are classified into the B, B', and B" non-related families in land plants. In Arabidopsis, the close homologs B'η, B'θ, B'γ, and B'ζ are further classified into a subfamily of B' called B'η. Previous studies have suggested that mitochondrial targeted PP2A subunits (B'ζ) play a role in energy metabolism and plant innate immunity. Potentially, the PP2A-B'ζ holoenzyme is involved in the regulation of the mitochondrial succinate/fumarate translocator, and it may affect the enzymes involved in energy metabolism. To investigate this hypothesis, the interactions between PP2A-B'ζ and the enzymes involved in the mitochondrial energy flow were investigated using bimolecular fluorescence complementation in tobacco and onion cells. Interactions were confirmed between the B'ζ subunit and the Krebs cycle proteins succinate/fumarate translocator (mSFC1), malate dehydrogenase (mMDH2), and aconitase (ACO3). Additional putative interacting candidates were deduced by comparing the enriched phosphoproteomes of wild type and B'ζ mutants: the mitochondrial regulator Arabidopsis pentatricopeptide repeat 6 (PPR6) and the two metabolic enzymes phosphoenolpyruvate carboxylase (PPC3) and phosphoenolpyruvate carboxykinase (PCK1). Overall, this study identifies potential PP2A substrates and highlights the role of PP2A in regulating energy metabolism in mitochondria.

Effects of Citric Acid and Humic-like Substances on Yield, Enzyme Activities, and Expression of Genes Involved in Iron Uptake in Tomato Plants

Iron (Fe) deficiency is a common abiotic stress on plants growing in calcareous soils where low organic matter content, high carbonate–bicarbonate concentration, and high pH precipitate Fe in unavailable forms. Enzymatic activity is a mechanism for plants to access soil nutrients; enzymes such as H+-ATPase, phosphoenolpyruvate carboxylase (PEPC), and the intracellular enzyme ferric reduction oxidase (FRO) are involved in Fe absorption. The effects of the application of citric acid (CA) and humic-like substances (HLS) on the yield, H+-ATPase, PEPC, and FRO enzyme activity, and expression of LeHA1, LePEPC1, and LeFRO1 genes in tomato plants grown under calcareous soil were studied. CA and HLS improved the SPAD units and increased the number of harvested fruits and yield per plant. Temporary alterations in enzyme activity, which reduced PEPC and FRO activity in roots, were documented. In leaf tissue, CA resulted in lower expression of LeHA1 and LePEPC1 and the induction of LeFRO1 expression, whereas HLS application resulted in higher expression of LePEPC1 and LeFRO1. In roots, LeHA1 expression increased with HLS, whereas LePEPC1 and LeFRO1 showed lower expression with CA and HLS, respectively. The application of CA and HLS through a nutrient solution in combination with Fe-chelate can improve Fe nutrition in tomato plants potted in calcareous soil by inducing temporal alterations in PEPC and FRO enzyme activity and LeFRO1 and LeHA1 gene expression.

Loading and co-solvent-triggered release of okanin, a C4 plant key enzyme inhibitor, into/from functional microgels

The constantly growing world population leads to increasing demands for food, which challenges modern agriculture manifold. Pests, such as weeds, require the application of agrochemicals to increase crop yield. Due to the environmental impact of these potentially hazardous chemicals, the demand for more efficient formulations is increasing. Promising formulations consist of easily adaptable carriers from which controllable stimuli release the agrochemicals. Here, we investigated poly(N–vinylcaprolactam) (pVCL)-based microgels as a potential carrier for okanin, an inhibitor of the C4 plant key enzyme phosphoenolpyruvate carboxylase, by combining experiments, molecular simulations, and free energy computations. Dynamic light scattering, scanning transmission electron and atomic force microscopy revealed that pVCL microgels collapse and rigidify upon the loading of okanin. The simulations identified loosely adsorbed okanin and tightly bound okanin mediating inter-chain crosslinks. With increasing okanin concentration, stacking interactions of okanin occur with adsorbed and bound okanin. These findings can explain the experimentally observed collapse and the rigidification of the microgels. Based on the atomistic insights, two poly(N–vinylcaprolactam–co–glycidyl methacrylate) microgels were synthesized, for which a doubled loading capacity of okanin was found. Finally, we investigated the triggered release of okanin using the addition of green solvents as a stimulus both in vitro and in planta. This work establishes a basis for the further optimization of pVCL-based microgels as a carrier for the delivery of polyphenolic agrochemicals.

Enhancement of Carbon Assimilates and Macronutrients in Legumes under Elevated CO2 Concentration

Impact of elevated carbon dioxide (Free Air Carbon dioxide Enrichment) was studied on the plant chlorophyll, plant growth, plant macronutrients, total starch, total carbohydrate and the activity of phosphoenol pyruvate carboxylase (Pep C) enzymatic assays in leaves, pods and seeds in leguminous plant Cyamopsis tetragonoloba. Plants of Cyamopsis tetragonoloba (C3) were exposed to different atmospheric CO2 concentrations 420 ± 20 ppm (ambient) and 550 ± 20ppm (elevated). An average increase in the plant total chlorophyll (+39.17%), total starch (+43.73%, +25.44% and +26.35% in leaves, pods, and seeds), sucrose (+69.77%, +22.27% and +33.77% in leaves, pods and seed) and total carbohydrate (+58.88%, +30.54% and +28.38 % in leaves, pods and seeds) content were found in plant grown under elevated condition when compared to ambient counterpart. More over overall plant growth (+40% height and +25% biomass) increased in [CO2] concentration. Plant total nitrogen (N) content decreased (−12.55% in leaves) under the elevated condition where as total phosphorus (P) decreased (−3.15% in leaves) along with total potassium (K) (−46.63% in leaves). In soil, total potassium (+60.23%) and phosphorous (+48.88%) were found to increase with (−16%) decrease in soil nitrogen content. In seed total nitrogen content increased (+18.15%) on an average with no significant change in total potassium and phosphorus content under e[CO2]. Phosphoenolpyruvate carboxylase enzyme (Pep C) (+139.5% in leaves) activity and total organic carbon (TOC) (+19.12%, +17.85% in leaves and seeds) increases in elevated concentration thus promoting and indicating higher photosynthesis via enhanced CO2 fixation. Thus our studies showed that [CO2] positively promotes sugars, carbohydrates synthesis, translocation and partitioning in plant tissues and enhanced macronutrients level in leaves and seeds tissue which is contradictory to other C3 plants. Thus [CO2] works as the boon for Cyamopsisvarieties and the seeds are nutritionally rich, healthy, balanced in proteins and carbohydrates (C/N) and so these varieties have future implication for industrial use in the agricultural country like India.

Salt stress induces Kranz anatomy and expression of C4 photosynthetic enzymes in the amphibious sedge Eleocharis vivipara

Eleocharis vivipara Link is a unique amphibious leafless plant of the Cyperaceae. The terrestrial form develops culms with Kranz anatomy and C4-like traits, while the submerged form does culms with non-Kranz anatomy and C3 traits. The submerged form develops new culms with C4-like mode when exposed to air or exogenous abscisic acid. In this study, we investigated whether salt stress (0.05-0.3M NaCl) has a similar effect. When the submerged form was grown for one month in solutions of 0.1M NaCl and more, culm growth was strongly suppressed. However, these plants slowly developed new culms that had Kranz anatomy with chloroplast-abundant Kranz bundle sheath cells. Although the culms of the submerged form had only few stomata, culms grown in the NaCl solution had many stomata. The NaCl-grown culms also accumulated large amounts of C4 photosynthetic enzymes (phosphoenolpyruvate carboxylase and pyruvate Pi dikinase), and the cellular localization patterns of these enzymes and ribulose 1,5-bisphosphate carboxylase/oxygenase were similar to those in terrestrial culms. Accumulation of C4 enzymes increased in mature culms of the submerged form (with non-Kranz anatomy) when exposed to 0.2M NaCl solution for one week. These results suggest that salt stress induces development of Kranz anatomy and expression of C4 photosynthetic enzymes in the submerged C3 form of E. vivipara, whereas the anatomical and biochemical traits of C4 photosynthesis appear to be regulated independently.

Comparing the Effects of N and P Deficiency on Physiology and Growth for Fast- and Slow-Growing Provenances of Fraxinus mandshurica

With the continuous increase in atmospheric carbon dioxide emissions, nitrogen (N) and phosphorus (P) as mineral elements increasingly restrict plant growth. To explore the effect of deficiency of P and N on growth and physiology, Fraxinus mandshurica (hereafter “F. mandshurica”) Rupr. annual seedlings of Wuchang (WC) provenance with fast growth and Dailing (DL) provenance with slow growth were treated with complete nutrition or starvation of N (N-), P (P-) or both elements (NP-). Although P- and N- increased the use efficiency of P (PUE) and N (NUE), respectively, they reduced the leaf area, chlorophyll content and activities of N assimilation enzymes (NR, GS, GOGAT), which decreased the dry weight and P or N amount. The free amino acid content and activities of Phosphoenolpyruvate carboxylase (PEPC) and acid phosphatase enzymes were reduced by N-. The transcript levels of NRT2.1, NRT2.4, NRT2.5, NRT2.7, AVT1, AAP3, NIA2, PHT1-3, PHT1-4 and PHT2-1 in roots were increased, but those of NRT2.1, NRT2.4, NRT2.5, PHT1-3, PHT1-4, PHT2-1 and AAP3 in leaves were reduced by P-. WC was significantly greater than DL under P- in dry weight, C amount, N amount, leaf area, PUE, NUE, which related to greater chlorophyll content, PEPC enzyme activity, N assimilation enzyme activities, and transcript levels of N and P transporter genes in roots and foliage, indicating a greater ability of WC to absorb, transport and utilize N and P under P-. WC was also greater than DL under N- in terms of the above indicators except the transcript levels of N and P assimilation genes, but most of the indicators did not reach a significant level, indicating that WC might be more tolerant to N- than DL, which requires further verification. In summary, WC was identified as a P-efficient provenance, as the growth rate was greater for the genetic type with high than low tolerance to P-.

Differential Expression, Tissue-Specific Distribution, and Posttranslational Controls of Phosphoenolpyruvate Carboxylase.

Phosphoenolpyruvate carboxylase (PEPC) is a ubiquitous cytosolic enzyme, which is crucial for plant carbon metabolism. PEPC participates in photosynthesis by catalyzing the initial fixation of atmospheric CO2 and is abundant in both C4 and crassulacean acid metabolism leaves. PEPC is differentially expressed at different stages of plant development, mostly in leaves, but also in developing seeds. PEPC is known to show tissue-specific distribution in leaves and in other plant organs, such as roots, stems, and flowers. Plant PEPC undergoes reversible phosphorylation and monoubiquitination, which are posttranslational modifications playing important roles in regulatory processes and in protein localization. Phosphorylation activates the PEPC enzyme, making it more sensitive to glucose-6-phosphate and less sensitive to malate or aspartate. PEPC phosphorylation is known to be diurnally regulated and delicately changed in response to various environmental stimuli, in addition to light. PEPCs belong to a small gene family encoding several plant-type and distantly related bacterial-type PEPCs. This paper provides a minireview of the general information on PEPCs in both C4 and C3 plants.

Kinetic variation in grass phosphoenolpyruvate carboxylases provides opportunity to enhance C4 photosynthetic efficiency.

The high rates of photosynthesis and the carbon-concentrating mechanism (CCM) in C4 plants are initiated by the enzyme phosphoenolpyruvate (PEP) carboxylase (PEPC). The flow of inorganic carbon into the CCM of C4 plants is driven by PEPC's affinity for bicarbonate (KHCO3 ), which can be rate limiting when atmospheric CO2 availability is restricted due to low stomatal conductance. We hypothesize that natural variation in KHCO3 across C4 plants is driven by specific amino acid substitutions to impact rates of C4 photosynthesis under environments such as drought that restrict stomatal conductance. To test this hypothesis, we measured KHCO3 from 20 C4 grasses to compare kinetic properties with specific amino acid substitutions. There was nearly a twofold range in KHCO3 across these C4 grasses (24.3±1.5 to 46.3±2.4μm), which significantly impacts modeled rates of C4 photosynthesis. Additionally, molecular engineering of a low-HCO3- affinity PEPC identified key domains that confer variation in KHCO3 . This study advances our understanding of PEPC kinetics and builds the foundation for engineering increased-HCO3- affinity and C4 photosynthetic efficiency in important C4 crops.

Increased [CO2] Causes Changes in Physiological and Genetic Responses in C4 Crops: A Brief Review.

Climate change not only worries government representatives and organizations, but also attracts the attention of the scientific community in different contexts. In agriculture specifically, the cultivation and productivity of crops such as sugarcane, maize, and sorghum are influenced by several environmental factors. The effects of high atmospheric concentration of carbon dioxide ([CO2]) have been the subject of research investigating the growth and development of C4 plants. Therefore, this brief review presents some of the physiological and genetic changes in economically important C4 plants following exposure periods of increased [CO2] levels. In the short term, with high [CO2], C4 plants change photosynthetic metabolism and carbohydrate production. The photosynthetic apparatus is initially improved, and some responses, such as stomatal conductance and transpiration rate, are normally maintained throughout the exposure. Protein-encoding genes related to photosynthesis, such as the enzyme phosphoenolpyruvate carboxylase, to sucrose accumulation and to biomass growth and are differentially regulated by [CO2] increase and can variably participate owing to the C4 species and/or other internal and external factors interfering in plant development. Despite the consensus among some studies, mainly on physiological changes, further studies are still necessary to identify the molecular mechanisms modulated under this condition. In addition, considering future scenarios, the combined effects of high environmental and [CO2] stresses need to be investigated so that the responses of maize, sugarcane, and sorghum are better understood.

A Partial C4 Photosynthetic Biochemical Pathway in Rice

Introduction of a C4 photosynthetic pathway into C3 rice (Oryza sativa) requires installation of a biochemical pump that concentrates CO2 at the site of carboxylation in modified bundle sheath cells. To investigate the feasibility of this, we generated a quadruple line that simultaneously accumulates four of the core C4 photosynthetic enzymes from the NADP-malic enzyme subtype, phosphoenolpyruvate carboxylase (ZmPEPC), NADP-malate dehydrogenase (ZmNADP-MDH), NADP-malic enzyme (ZmNADP-ME), and pyruvate phosphate dikinase (ZmPPDK). This led to enhanced enzyme activity and mild phenotypic perturbations but was largely neutral in its effects on photosynthetic rate. Measurements of the flux of 13CO2 through photosynthetic metabolism revealed a significant increase in the incorporation of 13C into malate, consistent with increased fixation of 13CO2 via PEP carboxylase in lines expressing the maize PEPC enzyme. However, there was no significant differences in labeling of 3-phosphoglycerate (3PGA) indicating that there was no carbon flux through NADP-ME into the Calvin-Benson cycle. There was also no significant difference in labeling of phosphoenolpyruvate (PEP) indicating that there was no carbon flux through PPDK. Crossing the quadruple line with a line with reduced glycine decarboxylase H-protein (OsGDCH) abundance led to a photosynthetic phenotype characteristic of the reduced OsGDCH line and higher labeling of malate, aspartate and citrate than in the quintuple line. There was evidence of 13C labeling of aspartate indicating 13CO2 fixation into oxaloacetate by PEPC and conversion to aspartate by the endogenous aspartate aminotransferase activity. While Kranz anatomy or other anatomical modifications have not yet been installed in these plants to enable a fully functional C4 cycle, these results demonstrate for the first-time a partial flux through the carboxylation phase of NADP-ME C4 metabolism in transgenic rice containing two of the key metabolic steps in the C4 pathway.

Does Exogenous Application of Melatonin Ameliorate Lead Toxicity in Eruca vesicaria Plants?

MELATONIN (N-acetyl-5-methoxytryptamine) is receiving a considerable interest due to its prospected role in alleviating the heavy metals stress in plants. Thus a step toward the profitable use of melatonin (ME) in agriculture, the present study investigated the exogenous application of ME in Eurica vesicaria grown in contaminated soil amended with lead nitrate Pb(NO3)2 to produce Pb levels of 350mg/kg. Melatonin was applied by two methods: priming of seeds for 12hrs [0.0μM (P0); 25μM (P1) and 50μM (P2)] and by foliar spray at seedling foliage [0.0μM (F0); 25μM (F1) and 50μM (F2)].Rocket plants affected by Pb toxicity exhibited significant reduction in the growth characteristics (fresh and dry weights of shoot and root, and leaf area/plant), photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids), photosynthetic activity (14CO2-assimilation), and photosynthetic enzymes (phosphoenolpyruvate carboxylase and ribulose-1,5-bisphosphate-carboxylase/oxygenase). Meanwhile, exogenous application of ME appeared to ameliorate toxic effects of lead by improving the above criteria as compared to the control or Pb treated plants. These results showed that ME can promote the growth of Eurica vesicaria growing in Pb-contaminated soil by stimulating the photosynthetic activity, and the foliar spray method is the optimum at a dose of ME (50μM).

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.

Enhanced Production of Malic Acid by Co-localization of Phosphoenolpyruvate Carboxylase and Malate Dehydrogenase Using Synthetic Protein Scaffold in Escherichia coli

To produce malic acid from non-oxidative pathway route in Escherichia coli using two key enzymes and synthetic scaffold complex. E. coli was engineered to produce malic acid from glucose by co-localization of two key enzymes phosphoenolpyruvate carboxylase (Ppc) and malate dehydrogenase (MdhA) with synthetic scaffold complex. Scaffold plasmid has produced the maximum concentration of 3.51 g/L malic acid from 10 g/L glucose in 48 h of culture. pH 5.5 and temperature 30°C were optimum for malic acid production without any engineering of competing metabolic pathways. E. coli mutant strains and different concentrations of glucose also tested. When 50 g/L glucose was used as substrate, 20.4 g/L of malic acid was produced.