A comparative immunocytochemical localization study of phosphoenolpyruvate carboxylase in leaves of higher plants

The intracellular localization of phosphoenolpyruvate (PEP) carboxylase in plants belonging to the C₄, Crassulacean acid metabolism (CAM) and C₃ types was invetigated using an immunocytochemical method with an immune serum raised against the sorghum leaf enzyme. The plants studied were sorghum, maize (C₄ type), kalanchoe (CAM type), french bean, and spinach (C₃ type). In the green leaves of C₄ plants, it was shown that the carboxylase was located in the mesophyll and stomatic cells, being largely cytosolic in the mesophyll cells. Similarly, in CAM plants, the enzyme was found mainly outside the chloroplasts. In contrast, in C₃ plants, the PEP carboxylase appeared to be distributed between the cytosol and the chloroplasts of foliar parenchyma. Examination of sections from etiolated leaves showed fluorescence emission from etioplasts and cytosol for the parenchyma of french bean as well as for the bundle sheath and mesophyll of sorghum leaves. This data indicated that during the greening process photoregulation and evolution of PEP carboxylase is dependent on the tissue and on the metabolic type of the plant considered.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate     

A comparison of the kinetic properties of phosphoenolpyruvate carboxylase from guard-cell and mesophyll-cell protoplasts of Commelina communis

Some kinetic properties of partially purified phosphoenolpyruvate carboxylase (PEPCase) from guard-cell and mesophyll-cell protoplasts of Commelina communis are described. The PEPCase activity inherent to each cell type was determined and the apparent K _m (phosphoenolpyruvate) and K _i (malate) were compared. Malate sensitivity was much higher (K _i malate 0.4 mol m^–3) in the extract of guard-cell protoplasts than in that of mesophyllcell protoplasts (K _i malate 4.2 mol m^–3). The stimulation of activity by glucose-6-phosphate in the presence of malate (deinhibition) was also investigated in extracts from both cell types and was found to be similar to previously reported results with epidermal tissue. The effect of contamination of an extract of guard-cell protoplasts with mesophyll-cell protoplasts was measured in the presence and absence of malate. It was found that a small amount to mesophyll-cell contaminant appears to desensitize the malate inhibition of PEPCase from guard-cell protoplasts. It is concluded that experiments which use epidermal tissue to study guardcell PEPCase may give misleading information as a consequence of mesophyll contamination.Abbreviations Glc6P glucose-6-phosphate - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase     

Effect of LiCl on phosphoenolpyruvate carboxylase kinase and the phosphorylation of phosphoenolpyruvate carboxylase in leaf disks and leaves of <Emphasis Type="Italic">Sorghum vulgare</Emphasis>

In the present work, the effect of LiCl on phosphoenolpyruvate carboxylase kinase (PEPCase-k), C₄ phosphoenolpyruvate carboxylase (PEPCase: EC 4.1.1.31) and its phosphorylation process has been investigated in illuminated leaf disks and leaves of the C₄ plant Sorghum vulgare. Although this salt induced severe damages to older leaves, it did not significantly alter the physiological parameters (photosynthesis, transpiration rate, intercellular CO₂ concentration) of young leaves. An immunological approach was used to demonstrate that the PEPCase-k protein accumulated rapidly in illuminated leaf tissues, consistent with the increase in its catalytic activity. In vivo, LiCl was shown to strongly enhance the light effect on PEPCase-k protein content, this process being dependent on protein synthesis. In marked contrast, the salt was found to inhibit the PEPCase-k activity in reconstituted assays and to decrease the C₄ PEPCase content and phosphorylation state in LiCl treated plants. Short-term (15 min) LiCl treatment increased IP₃ levels, PPCK gene expression, and PEPCase-k accumulation. Extending the treatment (1 h) markedly decreased IP₃ and PPCK gene expression, while PEPCase-k activity was kept high. The cytosolic protein synthesis inhibitor cycloheximide (CHX), which blocked the light-dependent up-regulation of the kinase in control plants, was found not to be active on this process in preilluminated, LiCl-treated leaves. This suggested that the salt causes the kinase turnover to be altered, presumably by decreasing degradation of the corresponding polypeptide. Taken together, these results establish PEPCase-k and PEPCase phosphorylation as lithium targets in higher plants and that this salt can provide a means to investigate further the organization and functioning of the cascade controlling the activity of both enzymes.     

Extremely high activities of phosphoenolpyruvate carboxylase in thermogenic tissues of Araceae

Phosphoenolpyruvate carboxylase activity in extracts of a wide range of thermogenic tissues of the Araceae was shown to be in the range 10–100 mol g^-1 fresh weight min^-1 (0.5–3.7 mol mg^-1 protein min^-1). Such high activities were not found in non-thermogenic tissues of the Araceae or in thermogenic tissues of Aristolochia brasiliensis Mart. and Zucc., Victoria amazonica Schomb. and Encephalartos barteri Carruth. During development and thermogenesis in the club of Arum maculatum L. the high activities of the carboxylase did not lead to any marked accumulation of citrate, isocitrate, 2-oxoglutarate, fumarate, malate and oxaloacetate. Clubs of Arum maculatum and of Arum italicum Miller readily fixed ¹⁴CO₂ in the dark, mostly into aspartate, malate, alanine and glutamate. Pulse and chase experiments showed that most of the fixed carbon was very rapidly metabolized to CO₂. The detailed distribution suggest that this occurred largely by decarboxylation of C-4 acids. It is suggested that thermogenic tissues of the Araceae are characterized by very high activities of phosphoenolpyruvate carboxylase, and that in vivo this leads to synthesis of C-4 acids which are promptly decarboxylated.     

Effects of temperature on the activity of phosphoenolpyruvate carboxylase and on the control of CO2 fixation in Bryophyllum fedtschenkoi

The phosphorylation state and the malate sensitivity of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in Bryophyllum fedtschenkoi Hamet et Perrier are altered by changes in the ambient temperature. These effects, in turn alter the in-vivo activity of the enzyme. Low temperature (3 °C or less), stabilizes the phosphorylated form of the enzyme, while high temperature (30 °C) promotes its dephosphorylation. The catalytic activity of the phosphorylated and dephosphorylated forms of PEPCase increases with temperature, but the apparent K _i values for malate of both forms of the enzyme decrease. Results of experiments with detached leaves maintained in darkness in normal air indicate that the changes in malate sensitivity and phosphorylation state of PEPCase with temperature are of physiological significance. When the phosphorylated form of PEPCase is stabilized by reducing the temperature of leaves 9 h after transfer to constant darkness at 15 °C, a prolonged period of CO₂ fixation follows. When leaves are maintained in constant darkness at 15 °C until CO₂ output reaches a low steady-state level and the PEPCase is dephosphorylated, reducing the temperature to 3 °C results in a further period of CO₂ fixation even though the phosphorylation state of PEPCase does not change.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase We thank the Agricultural and Food Research Council for financial support for this work.     

Purification and properties of ribulose 1,5-bisphosphate carboxylase from sunflower leaves

Extracts from sunflower leaves possess a high ribulose-1,5-bisphosphate (RuBP) carboxylase capacity but this enzyme activity is not stable. A purification procedure, developed with preservation of carboxylase activity by MgSO₄, yielded purified RuBP carboxylase with high specific activity (40 nkat mg^-1 protein). Measurement of kinetic parameters showed high Km values (RuBP, HCO ₃ ^- ) and high Vmax of the reaction catalyzed by this sunflower enzyme; the results are compared with those obtained for soybean carboxylase. Enzyme characteristics are discussed in relation to stabilization and activation procedures and to the high photosynthesis rates of this C₃ species.     

An efficient method for the determination of K m values for HCO 3 - of phosphoenolpyruvate carboxylase

One of the most serious problems in obtaining estimates of the K _m values for HCO ₃ ^- of phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) by measurement of initial rates at varying HCO ₃ ^- is the impossibility of completely excluding any contaminating HCO ₃ ^- . A method is proposed which has no need for the careful control of HCO ₃ ^- /CO₂ contamination. The kinetic data are obtained by the evaluation of progress curves of HCO ₃ ^- consumption. The method is discussed and the K _m values for HCO ₃ ^- of PEPCase from several C₄-species are presented.Abbreviations C₃, C₄ assimilated CO₂ initially found in 3-phosphoglycerate (C₃) or malate and aspartate (C₄) - OAA oxaloacetic acid - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase     

Control of photosynthesis by the carbohydrate level in leaves of the C4 plant Amaranthus edulis L.

Photosynthesis was studied in relation to the carbohydrate status in intact leaves of the C₄ plant Amaranthus edulis. The rate of leaf net CO₂ assimilation, stomatal conductance and intercellular partial pressure of CO₂ remained constant or showed little decline towards the end of an 8-h period of illumination in ambient air (340 bar CO₂, 21% O₂). When sucrose export from the leaf was inhibited by applying a 4-h cold-block treatment (1°C) to the petiole, the rate of photosynthesis rapidly decreased with time. After the removal of the cold block from the petiole, further reduction in photosynthetic rate occurred, and there was no recovery in the subsequent light period. Although stomatal conductance declined with time, intercellular CO₂ partial pressure remained relatively constant, indicating that the inhibition of photosynthesis was not primarily caused by changes in stomatal aperture. Analysis of the leaf carbohydrate status showed a five- to sixfold increase in the soluble sugar fraction (mainly sucrose) in comparison with the untreated controls, whereas the starch content was the same. Leaf osmotic potential increased significantly with the accumulation of soluble sugars upon petiole chilling, and leaf water potential became slightly more negative. After 14 h recovery in the dark, photosynthesis returned to its initial maximum value within 1 h of illumination, and this was associated with a decline in leaf carbohydrate levels overnight. These data show that, in Amaranthus edulis, depression in photosynthesis when translocation is impaired is closely related to the accumulation of soluble sugars (sucrose) in source leaves, indicating feedback control of C₄ photosynthesis. Possible mechanisms by which sucrose accumulation in the leaf may affect the rate of photosynthesis are discussed with regard to the leaf anatomy of C₄ plants.Abbreviations and symbols A net CO₂ assimilation rate - Ci intercellular CO₂ partial pressure - PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate - water potential - osmotic pressure     

Induction of a Crassulacean acid like metabolism in the C4 succulent plant,Portulaca oleracea L.: physiological and morphological changes are accompanied by specific modifications in phosphoenolpyruvate carboxylase

The induction of a Crassulacean acid like metabolism (CAM) was evidenced after 21–23 days of drought stress in the C₄ succulent plant Portulaca oleracea L. by changes in the CO₂ exchange pattern, in malic acid content and in titratable acidity during the day–night cycle. Light microscopy studies also revealed differences in the leaf structure after the drought treatment. Following the induction of the CAM-like metabolism, the regulatory properties of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), the enzyme responsible for the diurnal fixation of CO₂ in C₄ plants but nocturnal in CAM plants, were studied. The enzyme from stressed plants showed different kinetic properties with respect to controls, notably its lack of cooperativity, higher sensitivity to L-malate inhibition, higher PEP affinity and lower enzyme content on a protein basis. In both conditions, PEPC's subunit mass was 110 kDa, although changes in the isoelectric point and electrophoretic mobility of the native enzyme were observed. In vivo phosphorylation and native isoelectrofocusing studies indicated variations in the phosphorylation status of the enzyme of samples collected during the night and day, which was clearly different for the control and stressed groups of plants. The results presented suggest that PEPC activity and regulation are modified upon drought stress treatment in a way that allows P. oleracea to perform a CAM-like metabolism. This revised version was published online in August 2006 with corrections to the Cover Date.     

Evolution of C4 phosphoenolpyruvate carboxylase in the genus Alternanthera: gene families and the enzymatic characteristics of the C4 isozyme and its orthologues in C3 and C3/C4 Alternantheras

Phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.3) is a key enzyme of C₄ photosynthesis. It has evolved from ancestral non-photosynthetic (C₃) isoforms and thereby changed its kinetic and regulatory properties. We are interested in understanding the molecular changes, as the C₄ PEPCases were adapted to their new function in C₄ photosynthesis and have therefore analysed the PEPCase genes of various Alternanthera species. We isolated PEPCase cDNAs from the C₄ plant Alternanthera pungens H.B.K., the C₃/C₄ intermediate plant A. tenella Colla, and the C₃ plant A. sessilis (L.) R.Br. and investigated the kinetic properties of the corresponding recombinant PEPCase proteins and their phylogenetic relationships. The three PEPCases are most likely derived from orthologous gene classes named ppcA. The affinity constant for the substrate phosphoenolpyruvate (K _0.5 PEP) and the degree of activation by glucose-6-phosphate classified the enzyme from A. pungens (C₄) as a C₄ PEPCase isoform. In contrast, both the PEPCases from A. sessilis (C₃) and A. tenella (C₃/C₄) were found to be typical C₃ PEPCase isozymes. The C₄ characteristics of the PEPCase of A. pungens were accompanied by the presence of the C₄-invariant serine residue at position 775 reinforcing that a serine at this position is essential for being a C₄ PEPCase (Svensson et al. 2003). Genomic Southern blot experiments and sequence analysis of the 3′ untranslated regions of these genes indicated the existence of PEPCase multigene family in all three plants which can be grouped into three classes named ppcA, ppcB and ppcC.     

Light/dark modulation of phosphoenolpyruvate carboxylase in C3 and C4 species

In this report, the effects of light on the activity and allosteric properties of phosphoenolpyruvate (PEP) carboxylase were examined in newly matured leaves of several C₃ and C₄ species. Illumination of previously darkened leaves increased the enzyme activity 1.1 to 1.3 fold in C₃ species and 1.4 to 2.3 fold in C₄ species, when assayed under suboptimal conditions (pH 7) without allosteric effectors. The sensitivities of PEP carboxylase to the allosteric effectors malate and glucose-6-phosphate were markedly different between C₃ and C₄ species. In the presence of 5 mM malate, the activity of the enzyme extracted from illuminated leaves was 3 to 10 fold higher than that from darkened leaves in C₄ species due to reduced malate inhibition of the enzyme from illuminated leaves, whereas it increased only slightly in C₃ species. The Ki(malate) for the enzyme increased about 3 fold by illumination in C₄ species, but increased only slightly in C₃ species. Also, the addition of the positive effector glucose-6-phosphate provided much greater protection against malate inhibition of the enzyme from C₄ species than C₃ species. Feeding nitrate to excised leaves of nitrogen deficient plants enhanced the degree of light activation of PEP carboxylase in the C₄ species maize, but had little or no effect in the C₃ species wheat. These results suggest that post-translational modification by light affects the activity and allosteric properties of PEP carboxylase to a much greater extend in C₄ than in C₃ species.     

Sequence analysis of cDNA encoding phosphoenolpyruvate carboxylase from cultured tobacco cells

The complete nucleotide sequence of cDNA encoding phosphoenolpyruvate carboxylase (PEPCase) from cultured tobacco (a C₃ plant) cells was determined and the deduced amino acid sequence was compared with those of PEPCases from other higher plants.     

The relationship between contents of photosynthetic metabolites and the rate of photosynthetic carbon assimilation in leaves of Amaranthus edulis L.

The relationship between the gas-exchange characteristics of attached leaves of Amaranthus edulis L. and the contents of photosynthetic intermediates was examined in response to changing irradiance and intercellular partial pressure of CO₂. After determination of the rate of CO₂ assimilation at known intercellular CO₂ pressure and irradiance, the leaf was freeze-clamped and the contents of ribulose-1,5-bisphosphate, glycerate-3-phosphate, fructose-1,6-bisphosphate, glucose-6-phosphate, fructose-6-phosphate, triose phosphates, phosphoenolpyruvate, pyruvate, oxaloacetate, aspartate, alanine, malate and glutamate were measured. A comparison between the sizes of metabolite pools and theoretical calculations of metabolite gradients required for transport between the mesophyll and the bundle-sheath cells showed that aspartate, alanine, glycerate-3-phosphate and triose phosphates were present in sufficient quantities to support transport by diffusion, whereas pyruvate and oxaloacetate were not likely to contribute appreciably to the flux of carbon between the two cell types. The amounts of ribulose-1,5-bisphosphate were high at low intercellular partial pressures of CO₂, and fell rapidly as the CO₂-assimilation rate increased with increasing intercellular partial pressures of CO₂, indicating that bundle-sheath CO₂ concentrations fell at low intercellular partial pressures of CO₂. In contrast, the amount of phosphoenolpyruvate and of C₄-cycle intermediates declined at low intercellular partial pressures of CO₂. This behaviour is discussed in relation to the co-ordination of carbon assimilation between the Calvin and C₄ cycles.Abbreviations PEP phosphoenolpyruvate - PGA glycerate-3-phosphate - p _i intercellular CO₂ pressure - RuBP ribulose-1,5-bisphosphate - triose-P triose phosphates     

The phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum: molecular cloning, nucleotide sequence, and expression

Summary The ppc gene of Corynebacterium glutamicum encoding phosphoenolpyruvate (PEP) carboxylase was isolated by complementation of a ppc mutant of Escherichia coli using a cosmid gene bank of chromosomal c. glutamicum DNA. By subsequent subcloning into the plasmid pUC8 and deletion analysis, the ppc gene could be located on a 3.3 kb SalI fragment. This fragment was able to complement the E. coli ppc mutant and conferred PEP carboxylase activity to the mutant. The complete nucleotide sequence of the ppc gene including 5 and 3 flanking regions has been determined and the primary structure of PEP carboxylase was deduced. The sequence predicts a 919 residue protein product (molecular weight of 103154) which shows 34% similarity with the respective E. coli enzyme. Present address: Institut für Biotechnologie 1 der Kernforschungsanlage, Postfach 1913, D-5170 Jülich, Federal Republic of Germany     

Sequence-specific interactions of a maize factor with a GC-rich repeat in the phosphoenolpyruvate carboxylase gene

Summary A plant nuclear protein PEP-I, which binds specifically to the promoter region of the phosphoenolpyruvate carboxylase (PEPC) gene, was identified. Methylation interference analysis and DNA binding assays using synthetic oligonucleotides revealed that PEP-I binds to GC-rich elements. These elements are directly repeated sequences in the promoter region of the PEPC gene and we have suggested that they may be cis-regulatory element of this gene. The consensus sequence of the element is CCCTCTCCACATCC and the CTCC is essential for binding of PEP-I. PEP-I is present in the nuclear extracts of green leaves, where the PEPC gene is expressed. However, no binding was detected in tissues where the PEPC gene is not expressed in vivo, such as roots or etiolated leaves. Thus, PEP-1 is the first factor identified in plants which has different binding activity in light-grown compared with dark-grown tissue. PEP-I binding is also tissue-specific, suggesting that PEP-1 may function to coordinate PEPC gene expression with respect to light and tissue specificity. This report describes the identification and characterization of the sequences required for PEP-1 binding.     

Evolution of carboxylating enzymes involved in paramylon synthesis (phosphoenolpyruvate carboxylase and carboxykinase) in heterotrophically grown Euglena gracilis

Heterotrophically grown Euglena synthesize grains of paramylon, its reserve carbohydrate, in a vesicular complex of mitochondrial origin. A CO₂ fixation activity in dark grown Euglena was demonstrated in the mitochondria via paramylon. At the beginning of the exponential phase of growth, the activity of phosphoenolpyruvate carboxykinase increases before the augmentation of paramylon.At the end of the exponential phase, the activity of this enzyme decreases, and low residual levels persist in the transition and stationary phases of growth. The activity of phosphoenolpyruvate carboxylase evolves inversely during the heterotrophic growth of the algae in succinate- or a lactate-containing medium. A compartmentalized scheme of carbon metabolism in mitochondria is presented.Abbreviations PEP phosphoenolpyruvate - OAA oxaloacetate - PGA phosphoglyceric acid     

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