C4 Photosynthesis Enzymes Research Articles

Overexpression of Setaria italica phosphoenolpyruvate carboxylase gene in rice positively impacts photosynthesis and agronomic traits

C4 plants have the inherent capacity to concentrate atmospheric CO2 in the vicinity of RuBisCo, thereby increasing carboxylation, and inhibiting photorespiration. Carbonic anhydrase (CA), the first enzyme of C4 photosynthesis, converts atmospheric CO2 to HCO3−, which is utilized by PEPC to produce C4 acids. Bioengineering of C4 traits into C3 crops is an attractive strategy to increase photosynthesis and water use efficiency. In the present study, we isolated the PEPC gene from the C4 plant Setaria italica and transferred it to C3 rice. Overexpression of SiPEPC resulted in a 2-6-fold increment in PEPC enzyme activity in transgenic lines with respect to non-transformed control. Photosynthetic efficiency was enhanced in transformed plants, which was associated with increased ФPSII, ETR, lower NPQ, and higher chlorophyll accumulation. Water use efficiency was increased by 16–22% in PEPC transgenic rice lines. Increased PEPC activity enhanced quantum yield and carboxylation efficiency of PEPC transgenic lines. Transgenic plants exhibited higher light saturation photosynthesis rate and lower CO2 compensation point, as compared to non-transformed control. An increase in net photosynthesis increased the yield by (23–28.9%) and biomass by (24.1–29%) in transgenic PEPC lines. Altogether, our findings indicate that overexpression of C4-specific SiPEPC enzyme is able to enhance photosynthesis and related parameters in transgenic rice.

Intensity and duration of salinity required to form adaptive response in C4 halophyte Kochia prostrata (L.) Shrad.

Plant adaptation to salinity is a highly multifaceted process, harnessing various physiological mechanisms depending on the severity and duration of salt stress. This study focuses on the effects of 4- and 10-day treatments with low (100 mM NaCl) and moderate (200 mM NaCl) salinity on growth, CO2/H2O gas exchange, stomatal apparatus performance, the efficiency of photosystems I and II (PS I and II), content of key C4 photosynthesis enzymes, and the accumulation of Na+, K+, and proline in shoots of the widespread forage C4 halophyte Kochia prostrata. Our data show that 4 days of low salinity treatment resulted in a decrease in biomass, intensity of apparent photosynthesis, and cyclic electron transport around PS I. It was accompanied by an increase in transpiration and Rubisco and PEPC contents, while the Na+ and proline contents were low in K. prostrata shoots. By the 10th day of salinity, Na+ and proline have accumulated; PS I function has stabilized, while PS II efficiency has decreased due to the enhanced non-photochemical quenching of chlorophyll fluorescence (NPQ). Thus, under low salinity conditions, Na+ accumulated slowly and the imbalance between light and dark reactions of photosynthesis was observed. These processes might be induced by an early sodium signaling wave that affects cellular pH and ion homeostasis, ultimately disturbing photosynthetic electron transport. Another adaptive reaction more “typical” of salt-tolerant species was observed at 200 mM NaCl treatment. It proceeds in two stages. First, during the first 4 days, dry biomass and apparent photosynthesis decrease, whereas stomata sensitivity and dissipation energy during dark respiration increase. In parallel, an active Na+ accumulation and a decreased K+/Na+ ratio take place. Second, by the 10th day, a fully-fledged adaptive response was formed, when growth and apparent photosynthesis stabilized and stomata closed. Decreased dissipation energy, increased WUE, stabilization of Rubisco and PEPC contents, and decreased proline content testify to the completion of the adaptation and stabilization of the physiological state of plants. The obtained results allowed us to conclude that the formation of a full-fledged salt-tolerant response common for halophytes in K. prostrata occurs by the 10th day of moderate salinity.

Integrated physiological and comparative proteomics analysis of contrasting genotypes of pearl millet reveals underlying salt-responsive mechanisms.

Salinity stress poses a significant risk to plant development and agricultural yield. Therefore, elucidation of stress-response mechanisms has become essential to identify salt-tolerance genes in plants. In the present study, two genotypes of pearl millet (Pennisetum glaucum L.) with contrasting tolerance for salinity exhibited differential morpho-physiological and proteomic responses under 150 mM NaCl. The genotype IC 325825 was shown to withstand the stress better than IP 17224. The salt-tolerance potential of IC 325825 was associated with its ability to maintain intracellular osmotic, ionic, and redox homeostasis and membrane integrity under stress. The IC 325825 genotype exhibited a higher abundance of C4 photosynthesis enzymes, efficient enzymatic and non-enzymatic antioxidant system, and lower Na+ /K+ ratio compared with IP 17224. Comparative proteomics analysis revealed greater metabolic perturbation in IP 17224 under salinity, in contrast to IC 325825 that harbored pro-active stress-responsive machinery, allowing its survival and better adaptability under salt stress. The differentially abundant proteins were in silico characterized for their functions, subcellular-localization, associated pathways, and protein-protein interaction. These proteins were mainly involved in photosynthesis/response to light stimulus, carbohydrate and energy metabolism, and stress responses. Proteomics data were validated through expression profiling of the selected genes, revealing a poor correlation between protein abundance and their relative transcript levels. This study has provided novel insights into salt adaptive mechanisms in P. glaucum, demonstrating the power of proteomics-based approaches. The critical proteins identified in the present study could be further explored as potential objects for engineering stress tolerance in salt-sensitive major crops.

Proteomic analysis of young sugarcane plants with contrasting salt tolerance.

Soil salinity affects sugarcane (Saccharum officinale L.) production in arid and semiarid climates, severely reducing productivity. This study aimed to identify differentially regulated proteins in two cultivars that differ markedly in tolerance of saline soil. Plants were grown for 30 days and then subjected to treatments of 0 and 160 mM NaCl for 15 days. The tolerant cultivar showed a 3-fold upregulation of lipid metabolising enzymes, GDSL-motif lipases, which are associated with defence to abiotic stress, and which were not upregulated in the sensitive cultivar. Lipoxygenase was 2-fold upregulated in the tolerant cultivar but not in the sensitive cultivar, as were Type III chlorophyll a/b binding proteins. Other differences were that in the sensitive cultivar, the key enzyme of C4 photosynthesis, phosphoenolpyruvate carboxylase was downregulated, along with other chloroplast enzymes. Na+ concentrations had not reached toxic concentrations in either cultivar by this time of exposure to salt, so these changes would be in response to the osmotic effect of the soil salinity, and likely be in common with plants undergoing drought stress.

Small-molecule inhibition of pyruvate phosphate dikinase targeting the nucleotide binding site.

Pyruvate phosphate dikinase (PPDK) is an essential enzyme of C4 photosynthesis in plants, catalyzing the ATP-driven conversion of pyruvate to phosphoenolpyruvate (PEP). It is further used by some bacteria and unicellular protists in the reverse, ATP-forming direction. Many weed species use C4 photosynthesis in contrast to world’s major crops, which are C3 plants. Hence inhibitors of PPDK may be used as C4-specific herbicides. By screening a library of 80 commercially available kinase inhibitors, we identified compounds derived from bisindolylmaleimide (bisindolylmaleimide IV, IC50 = 0.76 ± 0.13 μM) and indirubin (indirubin-3’-monoxime, IC50 = 4.2 ± 0.9 μM) that showed high inhibitory potency towards PPDK and are among the most effective PPDK inhibitors described today. Physiological studies on leaf tissues of a C4 model plant confirmed in vivo inhibition of C4-driven photosynthesis by these substances. Moreover, comparative docking studies of non-inhibitory bisindolylmaleimide derivatives suggest that the selectivity towards PPDK may be increased by addition of functional groups to the core structure.

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

Plants with C4 photosynthesis are efficient in carbon assimilation and have an advantage over C3 photosynthesis. In C4 photosynthesis, the primary CO2 fixation is catalyzed by phosphoenolpyruvate carboxylase (PEPC). Here, we show that overexpression of Zea mays PEPC cDNA, under the control of 35S promoter, in Arabidopsis thaliana resulted in ~7-10 fold higher protein abundance and ~7-10 fold increase in PEPC activity in the transgenic lines than that in the vector control. We suggest that overexpression of PEPC played an anaplerotic role to increase the supply of 4-carbon carboxylic acids, which provided carbon skeletons for increased amino acid and protein synthesis. Higher protein content must have been responsible for increased metabolic processes including chlorophyll biosynthesis, photosynthesis, and respiration. Consequently, the PEPC-overexpressed transgenic plants had higher chlorophyll content, enhanced electron transport rate (ETR), lower non-photochemical quenching (NPQ) of chlorophyll a fluorescence, and a higher performance index (PI) than the vector control. Consistent with these observations, the rate of CO2 assimilation, the starch content, and the dry weight of PEPC-overexpressed plants increased by 14-18%, 10-18%, and 6.5-16%, respectively. Significantly, transgenics were tolerant to salt stress as they had increased ability to synthesize amino acids, including the osmolyte proline. NaCl (150mM)-treated transgenic plants had higher variable to maximum Chl a fluorescence (F v/F m) ratio, higher PI, higher ETR, and lower NPQ than the salt-treated vector controls. These results suggest that expression of C4 photosynthesis enzyme(s) in a C3 plant can improve its photosynthetic capacity with enhanced tolerance to salinity stress.

Structural Basis of Reversible Phosphorylation by Maize Pyruvate Orthophosphate Dikinase Regulatory Protein.

Pyruvate orthophosphate dikinase (PPDK) is one of the most important enzymes in C4 photosynthesis. PPDK regulatory protein (PDRP) regulates the inorganic phosphate-dependent activation and ADP-dependent inactivation of PPDK by reversible phosphorylation. PDRP shares no significant sequence similarity with other protein kinases or phosphatases. To investigate the molecular mechanism by which PDRP carries out its dual and competing activities, we determined the crystal structure of PDRP from maize (Zea mays). PDRP forms a compact homo-dimer in which each protomer contains two separate N-terminal (NTD) and C-terminal (CTD) domains. The CTD includes several key elements for performing both phosphorylation and dephosphorylation activities: the phosphate binding loop (P-loop) for binding the ADP and inorganic phosphate substrates, residues Lys-274 and Lys-299 for neutralizing the negative charge, and residue Asp-277 for protonating and deprotonating the target Thr residue of PPDK to promote nucleophilic attack. Surprisingly, the NTD shares the same protein fold as the CTD and also includes a putative P-loop with AMP bound but lacking enzymatic activities. Structural analysis indicated that this loop may participate in the interaction with and regulation of PPDK. The NTD has conserved intramolecular and intermolecular disulfide bonds for PDRP dimerization. Moreover, PDRP is the first structure of the domain of unknown function 299 enzyme family reported. This study provides a structural basis for understanding the catalytic mechanism of PDRP and offers a foundation for the development of selective activators or inhibitors that may regulate photosynthesis.

Phylogeny of C4-photosynthesis enzymes based on algal transcriptomic and genomic data supports an archaeal/proteobacterial origin and multiple duplication for most C4-related genes.

Both Calvin-Benson-Bassham (C3) and Hatch-Slack (C4) cycles are most important autotrophic CO2 fixation pathways on today’s Earth. C3 cycle is believed to be originated from cyanobacterial endosymbiosis. However, studies on evolution of different biochemical variants of C4 photosynthesis are limited to tracheophytes and origins of C4-cycle genes are not clear till now. Our comprehensive analyses on bioinformatics and phylogenetics of novel transcriptomic sequencing data of 21 rhodophytes and 19 Phaeophyceae marine species and public genomic data of more algae, tracheophytes, cyanobacteria, proteobacteria and archaea revealed the origin and evolution of C4 cycle-related genes. Almost all of C4-related genes were annotated in extensive algal lineages with proteobacterial or archaeal origins, except for phosphoenolpyruvate carboxykinase (PCK) and aspartate aminotransferase (AST) with both cyanobacterial and archaeal/proteobacterial origin. Notably, cyanobacteria may not possess complete C4 pathway because of the flawed annotation of pyruvate orthophosphate dikinase (PPDK) genes in public data. Most C4 cycle-related genes endured duplication and gave rise to functional differentiation and adaptation in different algal lineages. C4-related genes of NAD-ME (NAD-malic enzyme) and PCK subtypes exist in most algae and may be primitive ones, while NADP-ME (NADP-malic enzyme) subtype genes might evolve from NAD-ME subtype by gene duplication in chlorophytes and tracheophytes.

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.

Enhanced tolerance to drought in transgenic rice plants overexpressing C4 photosynthesis enzymes

Maize-specific pyruvate orthophosphate dikinase (PPDK) was overexpressed in rice independently or in combination with the maize C4-specific phosphoenolpyruvate carboxylase (PCK). The wild-type (WT) cultivar Kitaake and transgenic plants were evaluated in independent field and tank experiments. Three soil moisture treatments, well-watered (WW), moderate drought (MD) and severe drought (SD), were imposed from 9 d post-anthesis till maturity. Leaf physiological and biochemical traits, root activities, biomass, grain yield, and yield components in the untransformed WT and two transgenic rice lines (PPDK and PCK) were systematically studied. Compared with the WT, both transgenic rice lines showed increased leaf photosynthetic rate: by 20%–40% under WW, by 45%–60% under MD, and by 80%–120% under SD. The transgenic plants produced 16.1%, 20.2% and 20.0% higher grain yields than WT under the WW, MD and SD treatments, respectively. Under the same soil moisture treatments, activities of phosphoenolpyruvate carboxylase (PEPC) and carbonic anhydrase (CA) in transgenic plants were 3–5-fold higher than those in WT plants. Compared with ribulose-1,5-bisphosphate carboxylase, activities of PEPC and CA were less reduced under both MD and SD treatments. The transgenic plants also showed higher leaf water content, stomatal conductance, transpiration efficiency, and root oxidation activity and a stronger active oxygen scavenging system than the WT under all soil moisture treatments, especially MD and SD. The results suggest that drought tolerance is greatly enhanced in transgenic rice plants overexpressing C4 photosynthesis enzymes. This study was performed under natural conditions and normal planting density to evaluate yield advantages on a field basis. It may open a new avenue to drought-tolerance breeding via overexpression of C4 enzymes in rice.

Exogenous glycinebetaine and humic acid improve growth, nitrogen status, photosynthesis, and antioxidant defense system and confer tolerance to nitrogen stress in maize seedlings

Abiotic stresses, including nitrogen stress (NS), can hamper photosynthesis and cause oxidative damage to plants. Upregulation of the antioxidative defense system and photosynthesis induced by exogenous glycinebetaine (GB) and humic acid (HA) can mitigate the inhibitory effects of NS on plants. In the present investigation, the beneficial effects of exogenously applied GB and HA were examined on growth, leaf N status, photosynthesis, lipid peroxidation, and activities of some key antioxidant enzymes in the seedlings of maize cv. Zhengdan 958 (ZD958) exposed to NS. NS caused a significant reduction in total dry matter of seedlings of ZD958, but both GB and HA proved effective in mitigating this inhibition, hence, the beneficial effects of GB being more pronounced than those of HA. NS led to a considerable decrease in leaf total N and endogenous GB contents, stomatal conductance (gs), net photosynthetic rate (Pn), intercellular CO2 concentration (Ci), and activities of two key C4 photosynthesis enzymes phosphoenolpyruvate carboxylase (PEPCase) and ribulose-1,5-bisphosphate carboxylase (RuBPCase) as well as of superoxide dismutase (SOD) and peroxidase (POD). This treatment caused an increase in lipid peroxidation, but showed no effect on POD activity. Exogenous application of varying doses of GB resulted in a decrease in lipid peroxidation and Ci, and an increase in leaf total N and endogenous glycinebetaine (EGB) content, Pn, and activities of RuBPCase, PEPCase, SOD, and catalase (CAT) under NS. In contrast, application of different doses of HA resulted in a decrease in lipid peroxidation, an increase in Pn, gs, and Ci as well as SOD, CAT, and POD activities without increasing leaf total N and EGB content, and enhanced RuBPCase and PEPCase activities. The present study suggests that exogenous application of GB and HA can induce tolerance in maize plants to NS, but through the regulation of different mechanisms.

Performance of transgenic rice expressing C4 photosynthesis enzymes

Performance of transgenic rice expressing C4 photosynthesis enzymes

Introduction of genes encoding C4 photosynthesis enzymes into rice plants: physiological consequences.

Transgenic rice plants expressing the maize phosphoeno/pyruvate carboxylase (PEPC) and pyruvate, orthophosphate dikinase (PPDK) exhibit a higher photosynthetic capacity (up to 35%) than untransformed plants. The increased photosynthetic capacity in these plants is mainly associated with an enhanced stomatal conductance and a higher internal CO2 concentration. Plants simultaneously expressing high levels of both enzymes also have a higher photosynthetic capacity. The results suggest that both PEPC and PPDK play a key role in organic acid metabolism in the guard cells to regulate stomatal opening. Under photoinhibitory and photooxidative conditions, PEPC transgenic rice plants are capable of maintaining a higher photosynthetic rate, a higher photosynthetic quantum yield by PSII and a higher capacity to dissipate excess energy photochemically and non-photochemically than untransformed plants. Preliminary data from field trials show that relative to untransformed plants, the grain yield is about 10-20% higher in selected PEPC and 30-35% higher in PPDK transgenic rice plants, due to increased tiller number. Taken together, these results suggest that introduction of C4 photosynthesis enzymes into rice has a good potential to enhance its tolerance to stress, photosynthetic capacity and yield.

Performance of transgenic rice expressing C4 photosynthesis enzymes

Performance of transgenic rice expressing C4 photosynthesis enzymes

Effect of hypermethylation of CCWGG sequences in DNA of Mesembryanthemum crystallinum plants on their adaptation to salt stress

Under salt stress conditions, the level of CpNpG-methylation (N is any nucleoside) of the nuclear genome of the facultative halophyte Mesembryanthemum crystallinum in the CCWGG sequences (W = A or T) increases two-fold and is coupled with hypermethylation of satellite DNA on switching-over of C3-photosynthesis to the crassulacean acid metabolism (CAM) pathway of carbon dioxide assimilation. The methylation pattern of the CCWGG sequences is not changed in both the 5'-promoter region of the gene of phosphoenolpyruvate carboxylase, the key enzyme of C4-photosynthesis and CAM, and in the nuclear ribosomal DNA. Thus, a specific CpNpG-hypermethylation of satellite DNA has been found under conditions of expression of a new metabolic program. The functional role of the CpNpG-hypermethylation of satellite DNA is probably associated with formation of a specialized chromatin structure simultaneously regulating expression of a large number of genes in the cells of M. crystallinum plants on their adaptation to salt stress and switching-over to CAM metabolism.

Photosynthetic characteristics of PEPC transgenic rice

With the rapid development of molecular biological technology in the 1990s, the transfer of foreign genes into crops has become a routine, making it possible to introduce genes that encode the C4 photosynthesis enzyme into C3 plants. Ku et al (1999) were the first to successfully introduce the intact gene of maize phospho- enol pyruvate carboxylase (PEPC), which is the key enzyme in the C4 photosynthesis pathway in maize, into a C3 crop such as rice. They obtained plants that express a high level of photosynthetic activity, even exceeding that in maize. We thus propagated and identified transgenic rice plants in every generation and are now in the 7 th generation.

The characteristics of CO2 assimilation of photosynthesis and chlorophyll fluorescence in transgenic PEPC rice

With PEPC, PPDK, NADP-ME and PEPC+ PPDK transgenic and untransformed rice (Orysa sativa L.), the activities of related C4 photosynthesis enzymes, the chlorophyll fluorescence parameters, CO2 exchange and other physiological indexes were compared, in which the physiological characteristics of PEPC transgenic rice were mainly studied. The results were as follows: ( i ) The activities of PEPC in PEPC transgenic rice were 20-fold higher than those in untransformed rice; the light-saturation photosynthetic rates and the carboxylation efficiency of PEPC transgenic rice were increased by 55% and 50% more than those of untransformed rice, respectively, while the CO2 compensation point decreased by 27%. (ii) The PS II photochemical efficiency (F v/F m) and photochemical quenching (q p) of transgenic PEPC rice decreased less in comparison with those of untransformed rice after the treatment with high light intensity (3 h) or methyl viologen (MV), a photooxidative reagent, which demonstrated that the tolerance of PEPC transgenic rice to photoinhibition and photooxidation was enhanced. (iii) Under the condition of high light intensity, the activity of RuBPCase in PEPC transgenic rice did not obviously vary while the activity induced of carbonic anhydrase (CA) in PEPC transgenic rice increased by 1.8 fold. These results would provide some beneficial enlightment for revealing the mechanism of high photosynthetic efficiency and breeding with high photosynthetic efficiency in rice.

High level expression of C4 photosynthesis enzymes in transgenic rice

In C4 plants, the C4 photosynthetic pathway confers a number of advantages, including enhanced photosynthetic capacity and high nitrogen and water use efficiencies. It has long been anticipated that the transfer of C4 trait to C3 plants could improve the photosynthetic performance of C3 plants. The recent progress in molecular engineering enabled us to express C4 enzymes at high levels and in desired locations in the leaves of C3 plants. Phosphoenolpyruvate carboxylase (PEPC) and pyruvate, orthophosphate dikinase (PPDK) can be expressed at high levels in rice leaves by introduction of the intact maize C4-specific genes. In transgenic rice plants carrying the intact maize gene, the level of transcript per copy of the maize gene in the leaves was comparable to that in maize, and the maize gene was expressed in an organ-specific manner as in maize, suggesting that the maize C4-specific genes for PEPC and PPDK behave in a quantitatively and qualitatively similar way in both maize and transgenic rice plants. NADP-malic enzyme (NADP-ME) can be expressed at high levels in rice leaves by introduction of cDNA for the maize C4-specific NADP-ME fused to the rice Cab promoter. Although high level expression of a single C4 enzyme did not improve photosynthetic performance of rice plant, we observed changes in some traits of rice plants, probably through enhancement of housekeeping functions of the endogenous C4 enzymes.

Genomic structure and expression of the pyruvate, orthophosphate dikinase gene of the dicotyledonous C4 plant Flaveria trinervia (Asteraceae)

Pyruvate orthophosphate dikinase (PPDK) is a key enzyme of C4 photosynthesis providing the acceptor molecule for the primary CO2 fixation in the mesophyll cells. Here we present the isolation and characterisation of the corresponding gene (termed pdk) from the C4 plant Flaveria trinervia (Asteraceae). Southern analysis indicates that in contrast to maize pdk sequences in F. trinervia are present as single copy. Sequence analysis of the entire gene reveals that its coding sequence is identical to the previous isolated PPDK-cDNA from this species. The gene spans about 13 kb and consists of 21 exons, it thus contains two additional exons compared to the maize gene. As in maize, a long intervening sequence of 6.1 kb is positioned at the boundary of the transit peptide segment and the mature protein region. Pdk transcripts accumulate abundantly in leaves, but are also detectable in stems and roots. While the leaf and stem transcripts are 3.4 kb in size and encode the chloroplastic PPDK isoform, a 3.0 kb transcript lacking the region encoding the plastidic transit peptide accumulates in roots. Thus two different transcripts can be produced from a single pdk gene most likely by use of alternative promoters and not by alternative splicing. The accumulation of the 3.4 kb transcript is under light control. Darkening leads to a drastic depletion of this transcript in both leaves and stems. Instead, the 3.0 kb transit peptide-lacking pdk transcript accumulates, but only in stems and roots, not in leaves.

Kinetic Analysis of the Non-Phosphorylated, in Vitro Phosphorylated, and Phosphorylation-Site-Mutant (Asp8) Forms of Intact Recombinant C4 Phosphoenolpyruvate Carboxylase from Sorghum

Steady-state kinetic analyses were performed on the non-phosphorylated, in vitro phosphorylated and phosphorylation-site mutant (Ser8-->Asp) forms of purified recombinant sorghum C4 phosphoenolpyruvate (P-pyruvate) carboxylase (EC 4.1.1.31) containing an intact N-terminus. Significant differences in certain kinetic parameters were observed between these three enzyme forms when activity was assayed at a suboptimal but near-physiological pH (7.3), but not at optimal pH (8.0). Most notably, at pH 7.3 the apparent Ki for the negative allosteric effector L-malate was 0.17 mM, 1.2 mM and 0.45 mM while the apparent Ka for the positive allosteric effector glucose 6-phosphate (Glc6P) at 1 mM P-pyruvate was 1.3 mM, 0.28 mM and 0.45 mM for the dephosphorylated, phosphorylated and mutant forms of the enzyme, respectively. These and related kinetic analyses at pH 7.3 show that phosphorylation of C4 P-pyruvate carboxylase near its N-terminus has a relatively minor effect on V and Km (total P-pyruvate) but has a dramatic effect on the extent of activation by Glc6P, type of inhibition by L-malate and, most especially, Ka (Glc6P) and Ki (L-malate). Thus, regulatory phosphorylation profoundly influences the interactive allosteric properties of this cytosolic C4-photosynthesis enzyme.