Sucrose supply enhances phosphoenolpyruvate carboxylase phosphorylation level in in vitro Solanum tuberosum

In order to study the effect of exogenous sucrose on the phosphorylation of phosphoenolpyuruvate carboxylase (PEPC), potato plantlets (Solanum tuberosum L. cv. Kennebec) were grown on three modifications of the Murashige and Skoog (MS) medium: 3% sucrose + normal N level (1% Suc + N); 3% sucrose + normal N level (3% Suc + N); 3% sucrose + reduced N level (3% Suc – N). After 20 days of culture, PEPC phosphorylation levels were determined in leaves and roots by means of protein labelling with ³²P, and by L-malate inhibition of enzyme activity. Plants grown with 3% Suc + N had the highest phosphorylation level compared to those cultured with 3% Suc and the control (1% Suc + N). These investigations demonstrated clearly that sucrose enhanced phosphorylation. Sucrose or its metabolism may cause PEPC to become less sensitive to L-malate inhibition.     

Inhibition of phosphoenolpyruvate carboxylase from maize by 2-phosphoglycollate

The phosphoenolpyruvate carboxylase from maize leaf was strongly inhibited by 2-phosphoglycollate. The pH of the reaction did not influence the extent of inhibition by 2-phosphoglycollate. The kinetic analysis of the inhibition data by Lineweaver-Burk method showed that 2-phosphoglycollate inhibition was competitive with respect to phosphoenolpyruvate. The secondary plot of the data showed nonlinearity indicating that there may be two 2-phosphoglycollate binding sites with Ki values of 0.4 mM and 0.16 mM. The biphasic nature of the inhibition was also evident when the data were plotted using the method of Dixon. 2-phosphoglycollate inhibition was uncompetitive with respect to Mg²⁺ suggestting that it binds only to enzyme-Mg²⁺ complex.     

Cloning,sequence analysis and expression of a cDNA encoding active phosphoenolpyruvate carboxylase of the C3 plant Solanum tuberosum

A cDNA coding for phosphoenolpyruvate carboxylase (PEPC) was isolated from a cDNA library from Solanum tuberosum and the sequence of the cDNA was determined. It was inserted into a bacterial expression vector and a PEPC^- Escherichia coli mutant could be complemented by the cDNA construct. A functional fusion protein could be synthesized in E. coli. The properties of this PEPC protein clearly resembled those of typical C3 plant enzymes.     

On the cellular localization of phosphoenolpyruvate carboxylase in Sorghum leaves

The localization of phosphoenol pyruvate carboxylase (EC 4.1.1.3.1.) in the leaf cells of Sorghum vulgare was investigated by using three techniques: the conventional aqueous and non aqueous methods gave conflicting results; the immunocytochemical techniques clearly showed that the enzyme is predominantly located in the cytoplasm of mesophyll cells.Abbreviations PEP phosphoenol pyruvate - PAG polyacrylamide gel - NADP MDH NADP malate dehydrogenase - FITC fluorescein isothiocyanate - SAB serum albumine bovine - DTT dithiothreitol - MDH malate dehydrogenase - ME malic enzyme - PBS phosphate buffer saline - PAP peroxidase anti-peroxidase     

Artifacts in the assay of maize leaf phosphoenolpyruvate carboxylase activity due to its instability

When the assay of maize leaf phosphoenolpyruvate carboxylase (EC 4.1.1.31) activity is started with phosphoenolpyruvate, much lower reaction rates are obtained as compared to the enzyme-initiated reaction. The difference is due to the lability of the dilute enzyme in the absence of its substrate and is increased with incubation time in the absence of substrate or stabilizers. The activation of the enzyme by glucose-6-phosphate is overestimated with the substrate-initiated assay since a part of the apparent activation is due to stabilization of the enzymic activity by this effector during the minus-substrate preincubation. In contrast, the inhibitory effect of malate is underestimated when the reaction is started with the substrate. The enzyme-initiated assay is recommended provided that the necessary corrections for apparent activity in the absence of substrate and for inactivation during the assay at low substrate levels are made.Abbreviations DTT dithiothreitol - G-6-P glucose-6-phosphate - MDH malate dehydrogenase - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase - PVP polyvinylpyrrolidone     

Metabolic regulation and gene expression of root phosphoenolpyruvate carboxylase by different nitrogen sources

Alfalfa (Medicago sativa L.) N-sufficient plants were fed 1·5 mM N in the form of NO₃⁻, NH₄⁺ or NO₃⁻ in conjunction with NH₄⁺, or were N-deprived for 2 weeks. The specific activity of phosphoenolpyruvate carboxylase (PEPC) from the non-nodulated roots of N-sufficient plants was increased in comparison with that of N-deprived plants. The PEPC value was highest with NO₃⁻ nutrition, lowest with NH₄⁺ and intermediate in plants that were fed mixed salts. The protein was more abundant in NO₃⁻-fed plants than in either NH₄⁺- or N mixed-fed plants. Nitrogen starvation decreased the level of PEPC mRNA, and nitrate was the N form that most stimulated PEPC gene expression. The malate content was significantly lower in NO₃⁻-deprived than in NO₃⁻-sufficient plants. Root malate accumulation was high in NO₃⁻-fed plants, but decreased significantly in plants that were fed with NH₄⁺. The effect of malate on the desalted enzyme was also investigated. Root PEPC was not very sensitive to malate and PEPC activity was inhibited only by very high concentrations of malate. Asparagine and glutamine enhanced PEPC activity markedly in NO₃⁻-fed plants, but failed to affect plants that were either treated with other N types or N starved. Glutamate and citrate inhibited PEPC activity only at optimal pH. N-nutrition also influenced root nitrate and ammonium accumulation. Nitrate accumulated in the roots of NO₃⁻- and (NO₃⁻ + NH₄⁺)-fed plants, but was undetectable in those administered NH₄⁺. Both the nitrate and the ammonium contents were significantly reduced in NO₃⁻- and (NO₃⁻ + NH₄⁺)-starved plants. Root accumulation of free amino acids was strongly influenced by the type of N administered. It was highest in NH₄⁺-fed plants and the most abundant amides were asparagine and glutamine. It was concluded that root PEPC from alfalfa plants is N regulated and that nitrate exerts a strong influence on the PEPC enzyme by enhancing both PEPC gene expression and activity.     

Immunocytochemical study of phosphoenolpyruvate carboxylase in nodulated Alnus glutinosa

The activities of phosphoenolpyruvate carboxylase (PEP carboxylase, EC 4.1.1.3.1) have been investigated in various organs of young nodulated Alnus glutinosa. The root nodules exhibited the highest specific enzyme activity when compared with the one in roots and leaves. Furthermore, in the root nodules the PEP carboxylase was predominantly localized in the cytosol of the large cortical cells containing the endophyte vesicles.Abbreviations PEP carboxylase phosphoenolpyruvate carboxylase - MDH malate dehydrogenase - PVP polyvinylpyrrolidone - PBS phosphate buffer saline     

Effect of freezing and thawing on the distribution of lysosomal hydrolases in leaves of Solanum tuberosum L.

Density-gradient ultracentrifugation techniques showed that freezing and thawing of potato leaves resulted in a change in the density of subcellular particles containing acid phosphatase and acid ribonuclease (RNase). Gel filtration experiments were used to characterise the molecular forms of the hydrolases associated with the various cell fractions. Freezing and thawing promoted a release of a portion of the complement of acid phosphatase and RNase from the lysosomes to the supernatant fluid fraction of cell homogenates. The freezing treatment appeared to activate latent lysosomal RNase.     

干旱条件下外源蔗糖对高表达玉米C4型pepc水稻种子萌发的影响

为揭示外源蔗糖参与干旱胁迫下高表达转玉米C₄ 型磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvate carboxylase, PEPC)基因(C₄ pepc)水稻(简称：PC)种子萌发的生理机制,该研究以 PC及其未转基因野生型受体‘Kitaake’(简称：WT)的种子为材料,研究外施不同浓度蔗糖联合模拟干旱(10% PEG 6000)处理下,其种子发芽参数、总可溶性糖及可溶性蛋白含量、蔗糖非发酵1 (sucrose nonfermenting 1, SNF1)相关蛋白激酶(SNF1 related protein kinase 1s, SnRK1s)基因以及PEPC基因表达等参数的变化。结果表明：(1)PEG 6000模拟干旱处理均显著抑制两材料发芽,但明显促进胚根的生长;外施蔗糖则呈现浓度效应,高浓度蔗糖(>150 mmol·L^-1)进一步加剧了干旱对发芽的抑制效应,而低浓度(<30 mmol·L^-1)则可缓解干旱的抑制,但与WT(<30 mmol·L^-1)相比,促进PC水稻萌发的外施蔗糖浓度(<6 mmol·L^-1)更低,且各处理的发芽表现与其α 淀粉酶活性的动态表现一致。(2)与WT相比,外施3 mmol·L^-1蔗糖联合干旱处理下,显著提高了PC种子的发芽率,且伴随PC内源蔗糖含量、总可溶糖和可溶性蛋白含量显著增加;且外施3 mmol·L^-1蔗糖使PC中内源C₃ pepc基因表达下调,而外源导入C₄ pepc基因表达显著增加。(3)与WT相比,干旱处理下外施3 mmol·L^-1蔗糖,PC的糖信号相关基因SnRKs亚家族基因(包括SnRK1s：OsK1a OsK24 OsK35和SnRK2s:SAPK6)的表达也显著增加。研究发现,外施低浓度蔗糖通过上调PC水稻种子中可溶性糖和可溶性蛋白含量,增强SnRK1s亚家族基因和外源C₄ pepc基因的表达,提高了α 淀粉酶活性,从而缓解了干旱胁迫对PC种子萌发的抑制。     

Expression of a phosphoenolpyruvate carboxylase promoter from Mesembryanthemum crystallinum is not salt-inducible in mature transgenic tobacco

The 5 flanking region of a salt-stress-inducible, CAM-specific phosphoenolpyruvate carboxylase (PEPC) gene from the facultative halophyte Mesembryanthemum crystallinum, was fused to the -glucuronidase (GUS) reporter gene and introduced into Nicotiana tabacum SR1. The Ppc1 promoter displayed high levels of expression in transgenic tobacco quantitatively and qualitatively similar to a full-length 35S CaMV-GUS construct. Histochemical assays revealed that the full-length Ppc1-GUS fusions expressed GUS activity in all tissues except in root tips. While tobacco is capable of utilizing the Ppc1 cis-acting regulatory regions from M. crystallinum to yield high levels of constitutive expression, this glycophyte fails to direct a stress-inducible pattern of gene expression typical of this promoter in its native, facultative halophytic host.     

Production inEscherichia coli of activeSorghum phosphoenolpyruvate carboxylase which can be phosphorylated

Phosphoenolpyruvate carboxylase (PEPC)-deficient mutants ofEscherichia coli have been complemented with a plasmid bearing a full-length cDNA encoding the C4-type form ofSorghum leaf PEPC. Transformed cells grew on minimal medium. Two clones were selected which produce a functional and full-sized enzyme protein as determined by activity assays, immunochemical behavior and SDS-PAGE. In addition, regulatory phosphorylation of immunopurified recombinant PEPC was observed when the enzyme was incubated with a partially purified plant PEPC kinase. These results establish thatE. coli cells produce a genuine, phosphate-free, higher-plant PEPC. Application of immunoadsorbtion chromatography to bacterial extracts makes it possible to prepare highly pure protein available for biochemical studies.     

Immunocytochemical localization of phosphoenolpyruvate carboxylase and photosynthetic gas-exchange characteristics in ears of Triticum durum Desf.

The presence and distribution of phosphoenolpyruvate carboxylase (PEPCase) in the glumes and immature grains of durum wheat (Triticum durum Desf.) were studied by electron-microscopical immunolabeling of PEPCase with polyclonal antibodies followed by protein A-gold. Plants were grown under mediterranean field conditions and samples were obtained two weeks after anthesis. In the kernels, high gold label was associated with the unstained areas of the protein bodies of aleurone cells, whereas labeling in the cytoplasm and chloroplasts of the pericarp was slight, although significantly above the background. In the glumes, high gold label was only located in cytoplasmic granules (vesicles) of the mesophyll cells, although labeling in the cytoplasm and chloroplasts was also significantly above the background. These observations in immature kernels and glumes are in accordance with the anaplerotic role of this enzyme, as evidenced in C₃ plants. Measurements of apparent photosynthesis and its O₂ dependence and CO₂ compensation concentration were made on ears and flag leaves of durum wheat. In addition, an analog of phosphoenolpyruvate, 3,3-dichloro-2-dihydroxy-phosphinoylmethyl-2-propenoate, was used to inhibit PEPCase and, thereby, to assess the contribution of the PEPCase to photosynthesis in detached ears. There was no effect of the inhibitor on the apparent photosynthesis of ears. Whereas inhibition of apparent photosynthesis by 210 mL · L^–1 O₂ in flag leaves was typical of C₃ species, inhibition in ears was even greater. The CO₂ compensation concentrations in different ear parts were similar to or higher than in flag leaves and the O₂ dependence was also comparable (about 70%). Therefore, gas-exchange data give further support to the assumption that a C₄ cycle is absent or limited to very low rates in ears of durum wheat.Abbreviations and Symbol AP apparent photosynthesis - BSA bovine serum albumin - DCDP 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate - PBS phosphate-buffered saline - PEP phosphoenolpyruvate - PEPCase PEP carboxylase - CO₂ compensation concentration We thank Dr. Sam Sun (University of Hawaii) for kindly providing the PEPCase antibodies and Drs. C.L.D. Jenkins and M.D. Hatch (Division of Plant Industry, CSIRO, Canberra, Australia) for supplying DCDP for these experiments. We are also grateful to Dr. Robert Bargalló and Mrs. Ana Ribero (Servei de Microscopia Electronica, Universitat de Barcelona) for technical assistance in electron-microscopy studies. Participation of Drs. J.L. Araus and J. Bort in this work was supported by the Research Project of CICYT AGF92-0301, Spain.     

Purification and molecular and kinetic properties of phosphoenolpyruvate carboxylase from Amaranthus viridis L. leaves

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified 43-fold from Amaranthus viridis leaves by using a combination of ammonium-sulphate fractionation, chromatography on O-(diethylaminoethyl)-cellulose and hydroxylapatite, and filtration through Sepharose 6B. The purified enzyme had a specific activity of 17.1 mol·(mg protein)^-1·min^-1 and migrated as a single band of relative molecular weight 100000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A homotetrameric structure was determined for the native enzyme. Phosphoenolpyruvate carboxylase from Zea mays L. and A. viridis showed partial identity in Ouchterlony two-dimensional diffusion. Isoelectric focusing showed a band at pI 6.2. K_m values for phosphoenolpyruvate and bicarbonate were 0.29 and 0.17 mM, respectively, at pH 8.0. The activation constant (K_a) for Mg²⁺ was 0.87 mM at the same pH. The carboxylase was activated by glucose-6-phosphate and inhibited by several organic acids of three to five carbon atoms. The kinetic and structural properties of phosphoenolpyruvate carboxylase from A. viridis leaves are similar to those of the enzyme from Zea mays leaves.Abbreviations MW molecular weight - PEP (Case) phosphoenolpyruvate (carboxylase) - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Effects of altered phosphoenolpyruvate carboxylase activities on transgenic C3 plant Solanum tuberosum

Phosphoenolpyruvate carboxylase (PEPC) genes from Corynebacterium glutamicum (cppc), Escherichia coli (eppc) or Flaveria trinervia (fppc) were transferred to Solanum tuberosum. Plant regenerants producing foreign PEPC were identified by Western blot analysis. Maximum PEPC activities measured in eppc and fppc plants grown in the greenhouse were doubled compared to control plants. For cppc a transgenic plant line could be selected which exhibited a fourfold increase in PEPC activity. In the presence of acetyl-CoA, a known activator of the procaryotic PEPC, a sixfold higher activity level was observed. In cppc plants grown in axenic culture PEPC activities were even higher. There was a 6-fold or 12-fold increase in the PEPC activities compared to the controls measured in the absence or presence of acetyl-CoA, respectively. Comparable results were obtained by transient expression in Nicotiana tabacum protoplasts. PEPC of C. glutamicum (PEPC C.g.) in S. tuberosum leaf extracts displays its characteristic K _m(PEP) value. Plant growth was examined with plants showing high expression of PEPC and, moreover, with a plant cell line expressing and antisense S. tuberosum (anti-sppc) gene. In axenic culture the growth rate of a cppc plant cell line was appreciably diminished, whereas growth rates of an anti-sppc line were similar or slightly higher than in controls. Malate levels were increased in cppc plants and decreased in antisense plants. There were no significant differences in photosynthetic electron transport or steady state CO₂ assimilation between control plants and transformants overexpressing PEPC C.g. or anti-sppc plants. However, a prolonged dark treatment resulted in a delayed induction of photosynthetic electron transport in plants with less PEPC. Rates of CO₂ release in the dark determined after a 45 min illumination period at a high proton flux density were considerably enhanced in cppc plants and slightly diminished in anti-sppc plants. When CO₂ assimilation rates were corrected for estimated rates of mitochondrial respiration in the light, the electron requirement for CO₂ assimilation determined in low CO₂ was slightly lower in transformants with higher PEPC, whereas transformants with decreased PEPC exhibited an appreciably elevated electron requirement. The CO₂ compensation point remained unchanged in plants (cppc) with high PEPC activity, but might be increased in an antisense plant cell line. Stomatal opening was delayed in antisense plants, but was accelerated in plants overexpressing PEPC C.g. compared to the controls.Abbreviations CO₂ compensation point - CO₂ quantum efficiency of CO₂ assimilation - _PSII quantum efficiency of photosystem II electron transport - A CO₂ assimilation rate - C_i intercellular CO₂ concentration; e, electron - PFD photon flux density - Q_A primary quinone electron acceptor of photosystem II - Q_N non-photochemical chlorophyll a fluorescence quenching - q_P photochemical chlorophyll a fluorescence quenching     

Properties of phosphoenolpyruvate carboxylase in rapidly prepared,desalted leaf extracts of the Crassulacean acid metabolism plant Mesembryanthemum crystallinum L.

Properties of phosphoenolpyruvate (PEP) carboxylase, obtained from leaves of Mesembryanthemum crystallinum L. performing Crassulacean acid metabolism (CAM), were determined at frequent time points during a 12-h light/12-h dark cycle. Leaf extracts were rapidly desalted and PEP carboxylase activity as a function of PEP concentration, malate concentration, and pH was measured within 2 min after homogenization of the tissue. Maximum velocity of PEP carboxylase was similar in the light and dark at pH 7.5 and pH 8.0. However, PEP carboxylase had as much as a 12-fold lower K _m for PEP and as much as a 20-fold higher K _i for malate during the dark than during the light periods, the magnitude of these differences being dependent on the assay pH. Assuming that enzyme properties immediately after isolation reflect the approximate state of the enzyme in vivo, these differences in enzyme properties reduce the potential for CO₂ fixation via PEP carboxylase in the light. A small decrease in cytoplasmic pH in the light would greatly magnify the above differences in day/night properties of PEP carboxylase, because the sensitivity of PEP carboxylase to inhibition by malate increased with decreasing pH. Properties of PEP carboxylase were also studied in plants exposed to short-term perturbations of the normal 12-h light/12-h dark cycle (e.g., prolonged light period, prolonged dark period). Under all light/dark regimes, there was a close correlation between change in properties of PEP carboxylase and changes of the tissue from acidification to deacidification, and vice versa. Changes in properties of PEP carboxylase were not merely light/dark phenomena because they were also observed in plants exposed to continuous light or dark. the data indicate that, during CAM, PEP carboxylase exists in two stages which differ in their capacity for net malate synthesis. The physiologically-active state is distinguished by a low K _m for PEP and a high K _i for malate and favors malate synthesis. The physiologically-inactive state has a high K _m for PEP and a low K _i for malate and exists during periods of deacidification and other periods lacking synthesis of malic acid.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPC PEP carboxylase - RuBP ribulose 1,5-bisphosphate - RH relative humidity     

Light regulation of phosphoenolpyruvate carboxylase in barley mesophyll protoplasts is modulated by protein synthesis and calcium, and not necessarily correlated with phosphoenolpyruvate carboxylase kinase activity

The regulation of phosphoenolpyruvate carboxylase (PEPCase, EC. 4.1.1.31) and PEPCase kinase was investigated using barley (Hordeum vulgare L.) mesophyll protoplasts. Incubation of protoplasts in the light resulted in a reduction in the sensitivity of PEPCase to the inhibitor L-malate; PEPCase from protoplasts incubated in the light for 1 h was inhibited 48±2% by 2mM malate, whereas the enzyme from protoplasts incubated for 1 h in the dark was inhibited by 67±2%. Light-induced reduction of sensitivity of PEPCase to malate was decreased by cycloheximide (CHM), indicating the involvement of protein synthesis. The PEPCase kinase in protoplasts increased with time after isolation in darkness, and increased still further following light treatment. The increase in kinase activity in the light was sensitive to CHM. When protoplasts were illuminated in the presence of EGTA and the calcium ionophore A23187 to reduce intracellular Ca²⁺, the reduction in the senstivity of PEPCase to malate was enhanced, though no more PEPCase kinase activity was detected than in protoplasts illuminated in the absence of EGTA and A23187. Incubation with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) had no effect on the light-induced reduction of sensitivity of PEPCase to malate inhibition or on light-activation of PEPCase kinase. These results indicate that there is a constitutive PEPCase kinase activity in C₃ leaf tissue, that there is another kinase which is light-activated in a CHMsensitive way, that the sensitivity of PEPCase to its inhibitor may not always be correlated with apparent PEPCase kinase actvity, and that PEPCase and PEPCase kinase are regulated in a different manner in C₃ protoplasts than in C₄ protoplasts or leaf tissue.Abbreviations CAM Crassulacean acid metabolism - Chl chlorophyll - CHM cycloheximide - DCMU 3-(3,4-dichloro-phenyl)-1,1-dimethylurea - PEP phosphoenolpyruvate - PEPCase PEP carboxylase     

Tree stem phosphoenolpyruvate carboxylase (PEPC): lack of biochemical and localization evidence for a C4-like photosynthesis system

Here, the kinetic properties and immunolocalization of phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in young stems of Fagus sylvatica were investigated. The aim of the study was to test the hypothesis that there is a C4-like photosynthesis system in the stems of this C3 tree species. The activity, optimal pH and L-malate sensitivity of PEPC, and the Michaelis-Menten constant (Km) for phosphoenolpyruvate (PEP), were measured in protein extracts from current-year stems and leaves. A gel blot experiment and immunolocalization studies were performed to examine the isozyme complexity of PEPC and the tissue distribution of PEPC and Rubisco in stems. Leaf and stem PEPCs exhibited similar, classical values characteristic of C3 PEPCs, with an optimal pH of c. 7.8, a Km for PEP of c. 0.3 mM and a IC50 for L-malate (the L-malate concentration that inhibits 50% of PEPC activity at the Km for PEP) of c. 0.1 mM. Western blot analysis showed the presence of two PEPC subunits (molecular mass c. 110 kDa) both in leaves and in stems. Immunogold labelling did not reveal any differential localization of PEPC and Rubisco, neither between nor inside cells. This study suggests that C4-type photosynthesis does not occur in stems of F. sylvatica and underlines the importance of PEPC in nonphotosynthetic carbon fixation by most stem tissues (fixation of respired CO2 and fixation via the anaplerotic pathway).     

Properties of phosphoenolpyruvate carboxylase in desalted extracts from isolated guard-cell protoplasts

Properties of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) obtained from isolated guard-cell protoplasts of Vicia faba L. were determined following rapidly desalting of the extract on a Sephadex G 25 column. The activity of PEP carboxylase was measured as a function of PEP and malate concentration, pH and K⁺ concentration within 2–3 min after homogenization of the guard-cell protoplasts. The activity of this enzyme was stimulated by PEP concentrations of 0.1 to 0.75 mM and by K⁺ ions (12 mM), but inhibited by PEP concentrations above 1 mM and by malate. Changes in the K_m(PEP) and V_max values with increasing malate concentrations (2.5 and 5 mM) indicate that the malate level, varying in relation to the physiological state of guard cells, plays an important role in regulating the properties of phosphoenolpyruvate carboxylase.Abbreviations CAM Crassulacean acid metabolism - GCP guard-cell protoplast - PEP phosphoenolpyruvate Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday