Phosphorylation State Of Phosphoenolpyruvate Carboxylase Research Articles

Enzymatic activity, gene expression and posttranslational modifications of photosynthetic and non-photosynthetic phosphoenolpyruvate carboxylase in ammonium-stressed sorghum plants

Sorghum plants grown with 5mM (NH4)2SO4 showed symptoms of stress, such as reduced growth and photosynthesis, leaf chlorosis, and reddish roots. Phosphoenolpyruvate carboxylase (PEPC) activity, by supplying carbon skeletons for ammonium assimilation, plays a pivotal role in tolerance to ammonium stress. This work investigated the effect of ammonium nutrition on PPC and PPCK gene expression, on PEPC activity, and on post-translational modifications (PTMs) of PEPC in leaves and roots of sorghum plants. Ammonium increased PEPC kinase (PEPCk) activity and the phosphorylation state of PEPC in leaves, both in light and in the dark, due to increased PPCK1 expression in leaves. This result resembled the effect of salinity on sorghum leaf PEPC and PEPCk, which is thought to allow a better functioning of PEPC in conditions that limit the income of reduced C. In roots, ammonium increased PEPC activity and the amount of monoubiquitinated PEPC. The first effect was related to increased PPC3 expression in roots. These results highlight the relevance of this specific isoenzyme (PPC3) in sorghum responses to ammonium stress. Although the role of monoubiquitination is not fully understood, it also increased in germinating seeds along with massive mobilization of reserves, a process in which the anaplerotic function of PEPC is of major importance.

PH and carbon supply control the expression of phosphoenolpyruvate carboxylase kinase genes in Arabidopsis thaliana

Phosphoenolpyruvate carboxylase (PEPC) is thought to play many roles in C(3) plants including the provision of biosynthetic precursors and control of pH during N assimilation. Its activity is controlled via phosphorylation catalysed by PEPC kinases, which are encoded by PPCK genes. We examined PPCK expression in response to changes in the supply of N or C, and to changes in intracellular pH, using cultured Arabidopsis cells and seedlings. The results show that expression of both PPCK1 and PPCK2 is increased by C availability, but does not respond to N availability. Expression of the two PPCK genes and the phosphorylation state of PEPC are increased in response to increasing intracellular pH. Elevated pH also reduces the repression of PPCK gene expression by P(i). Expression of phosphoenolpyruvate carboxykinase (PEPCK), which catalyses the decarboxylation of oxaloacetate, is decreased in response to increasing intracellular pH. pH homeostasis may be mediated at least partly by reciprocal changes in the expression of PPCK genes and PEPCK.

Relationships Between Quantum Yield for CO2 Assimilation, Activity of Key Enzymes and CO2 Leakiness in Amaranthus cruentus, a C4 Dicot, Grown in High or Low Light

In C(4) photosynthesis, a part of CO(2) fixed by phosphoenolpyruvate carboxylase (PEPC) leaks from the bundle-sheath cells. Because the CO(2) leak wastes ATP consumed in the C(4) cycle, the leak may decrease the efficiency of CO(2) assimilation. To examine this possibility, we studied the light dependence of CO(2) leakiness (phi), estimated by the concurrent measurements of gas exchange and carbon isotope discrimination, initial activities of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and pyruvate, orthophosphate dikinase (PPDK), the phosphorylation state of PEPC and the CO(2) assimilation rate using leaves of Amaranthus cruentus (NAD-malic enzyme subtype, dicot) plants grown in high light (HL) and low light (LL). phi was constant at photon flux densities (PFDs) >200 micromol m(-2) s(-1) and was around 0.3. At PFDs <150 micromol m(-2) s(-1), phi increased markedly as PFD decreased. At 40 micromol m(-2) s(-1), phi was 0.76 in HL and 0.55 in LL leaves, indicating that the efficiency of CO(2) assimilation at low PFD was greater in LL leaves. The activities of Rubisco and PPDK, and the phosphorylated state of PEPC all decreased as PFD decreased. Theoretical calculations with a mathematical model clearly showed that the increase in phi with decreasing PFD contributed to the decrease in the CO(2) assimilation rate. It was also shown that the 'conventional' quantum yield of photosynthesis obtained by fitting the straight line to the light response curve of the CO(2) assimilation rate at the low PFD region is seriously overestimated. Ecological implications of the increase in phi in LL are discussed.

Phosphoenolpyruvate Carboxylase Kinase Is Controlled by a Similar Signaling Cascade in CAM and C4 Plants

In Crassulacean acid metabolism (CAM) plants, phosphoenolpyruvate carboxylase (PEPC) is subject to day-night regulatory phosphorylation of a conserved serine residue in the plant enzyme's N-terminal domain. The dark increase in PEPC-kinase (PEPC-k) activity is under control of a circadian oscillator, via the enhanced expression of the corresponding gene (1). The signaling cascade leading to PEPC-k up-regulation was investigated in leaves and mesophyll cell protoplasts of the facultative, salt-inducible CAM species, Mesembryanthemum crystallinum. Mesophyll cell protoplasts had the same PEPC-k activity as leaves from which they were prepared (i.e., high at night, low during the day). However, unlike C4 protoplasts (2), CAM protoplasts did not show marked PEPC-k up-regulation when isolated during the day and treated with a weak base such as NH4Cl. Investigations using various pharmacological reagents established the operation, in the darkened CAM leaf, of a PEPC-k cascade including the following components: a phosphoinositide-dependent phospholipase C (PI-PLC), inositol 1,4,5 P (IP3)-gated tonoplast calcium channels, and a putative Ca2+/calmodulin protein kinase. These results suggest that a similar signaling machinery is involved in both C4 (2, 3) and CAM plants to regulate PEPC-k activity, the phosphorylation state of PEPC, and, thus, carbon flux through this enzyme during CAM photosynthesis.

Metabolite Control Overrides Circadian Regulation of Phosphoenolpyruvate Carboxylase Kinase and CO(2) Fixation in Crassulacean Acid Metabolism.

Phosphoenolpyruvate carboxylase (PEPc) catalyzes the primary fixation of CO(2) in Crassulacean acid metabolism plants. Flux through the enzyme is regulated by reversible phosphorylation. PEPc kinase is controlled by changes in the level of its translatable mRNA in response to a circadian rhythm. The physiological significance of changes in the levels of PEPc-kinase-translatable mRNA and the involvement of metabolites in control of the kinase was investigated by subjecting Kalanchoë daigremontiana leaves to anaerobic conditions at night to modulate the magnitude of malate accumulation, or to a rise in temperature at night to increase the efflux of malate from vacuole to cytosol. Changes in CO(2) fixation and PEPc kinase activity reflected those in kinase mRNA. The highest rates of CO(2) fixation and levels of kinase mRNA were observed in leaves subjected to anaerobic treatment for the first half of the night and then transferred to ambient air. In leaves subjected to anaerobic treatment overnight and transferred to ambient air at the start of the day, PEPc-kinase-translatable mRNA and activity, the phosphorylation state of PEPc, and fixation of atmospheric CO(2) were significantly higher than those for control leaves for the first 3 h of the light period. A nighttime temperature increase from 19 degrees C to 27 degrees C led to a rapid reduction in kinase mRNA and activity; however, this was not observed in leaves in which malate accumulation had been prevented by anaerobic treatment. These data are consistent with the hypothesis that a high concentration of malate reduces both kinase mRNA and the accumulation of the kinase itself.

The light induction of maize phosphoenolpyruvate carboxylase kinase translatable mRNA requires transcription but not translation

We have previously demonstrated that the level of translatable mRNA for phosphoenolpyruvate carboxylase kinase in maize leaves is increased in response to light (Hartwell et al. 1996; Plant Journal10, 1071–1078). To identify the steps required for this increase, we have examined the effects of protein and RNA synthesis inhibitors. The RNA synthesis inhibitors actinomycin D and cordycepin (500 μM) strongly inhibited the light‐induced increases in kinase translatable mRNA and the apparent phosphorylation state of phosphoenolpyruvate carboxylase, as judged by its sensitivity to inhibition by L‐malate. The protein synthesis inhibitors cycloheximide and puromycin blocked the light‐induced increase in the apparent phosphorylation state of phosphoenolpyruvate carboxylase but not the increase in kinase translatable mRNA. Indeed, the amount of phosphoenolpyruvate carboxylase kinase translatable mRNA after 3 h of illumination of leaves treated with either 1 mM puromycin or 100 μM cycloheximide was double that in illuminated control leaves. Each inhibitor reduced the light‐induction of two control genes, malic enzyme and pyruvate, phosphate dikinase. Thus the light induction of phosphoenolpyruvate carboxylase kinase translatable mRNA requires RNA synthesis, but not protein synthesis.

Effect of drought stress on net CO2 uptake by Zea leaves

The net CO2 assimilation by leaves of maize (Zea mays L. cv. Adonis) plants subjected to slow or rapid dehydration decreased without changes in the total extractable activities of phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH) and malic enzyme (ME). The phosphorylation state of PEPC extracted from leaves after 2–3 h of exposure to light was not affected by water deficit, either. Moreover, when plants which had been slowly dehydrated to a leaf relative water content of about 60% were rehydrated, the net CO2 assimilation by leaves increased very rapidly without any changes in the activities of MDH, ME and PEPC or phosphorylation state of PEPC. The net CO2-dependent O2 evolution of a non-wilted leaf measured with an oxygen electrode decreased as CO2 concentration increased and was totally inhibited when the CO2 concentration was about 10%. Nevertheless, high CO2 concentrations (5–10%) counteracted most of the inhibitory effect of water deficit that developed during a slow dehydration but only counteracted a little of the inhibitory effect that developed during a rapid dehydration. In contrast to what could be observed during a rapidly developing water deficit, inhibition of leaf photosynthesis by cis-abscisic acid could be alleviated by high CO2 concentrations. These results indicate that the inhibition of leaf net CO2 uptake brought about by water deficit is mainly due to stomatal closure when a maize plant is dehydrated slowly while it is mainly due to inhibition of non-stomatal processes when a plant is rapidly dehydrated. The photosynthetic apparatus of maize leaves appears to be as resistant to drought as that of C3 plants. The non-stomatal inhibition observed in rapidly dehydrated leaves might be the result of either a down-regulation of the photosynthetic enzymes by changes in metabolite pool sizes or restricted plasmodesmatal transport between mesophyll and bundle-sheath cells.

The Light-Dependent Transduction Pathway Controlling the Regulatory Phosphorylation of C4 Phosphoenolpyruvate Carboxylase in Protoplasts from Digitaria sanguinalis.

Phosphoenolpyruvate carboxylase (PEPC) was characterized in extracts from C4 mesophyll protoplasts isolated from Digitaria sanguinalis leaves and shown to display the structural, functional, and regulatory properties typical of a C4 PEPC. In situ increases in the apparent phosphorylation state of the enzyme and the activity of its Ca2+-independent protein-serine kinase were induced by light plus NH4Cl or methylamine. The photosynthesis-related metabolite 3-phosphoglycerate (3-PGA) was used as a substitute for the weak base in these experiments. The early effects of light plus the weak base or 3-PGA treatment were alkalinization of protoplast cytosolic pH, shown by fluorescence cytometry, and calcium mobilization from vacuoles, as suggested by the use of the calcium channel blockers TMB-8 and verapamil. The increases in PEPC kinase activity and the apparent phosphorylation state of PEPC also were blocked in situ by the electron transport and ATP synthesis inhibitors DCMU and gramicidin, respectively, the calcium/calmodulin antagonists W7, W5, and compound 48/80, and the cytosolic protein synthesis inhibitor cycloheximide. These results suggest that the production of ATP and/or NADPH by the illuminated mesophyll chloroplast is required for the activation of the transduction pathway, which presumably includes an upstream Ca2+-dependent protein kinase and a cytosolic protein synthesis event. The collective data support the view that the C4 PEPC light transduction pathway is contained entirely within the mesophyll cell and imply cross-talk between the mesophyll and bundle sheath cells in the form of the photosynthetic metabolite 3-PGA.

In Vivo Regulatory Phosphorylation of Soybean Nodule Phosphoenolpyruvate Carboxylase

In this report we provide evidence that cytosolic phosphoenolpyruvate carboxylase (PEPC) in soybean (Glycine max L.) root nodules is regulated in vivo by a seryl-phosphorylation cycle, as with the C4, Crassulacean acid metabolism, and C3 leaf isoforms. Pretreatment of parent plants by stem girdling for 5 or 14 h caused a significant decrease in the apparent phosphorylation state of nodule PEPC, as indicated by the 50% inhibition constant (L-malate) and specific activity values assayed at suboptimal conditions, whereas short-term darkness alone was without effect. However, extended (26 h) darkness led to the formation of a relatively dephosphorylated nodule PEPC, an effect that was reversed by illuminating the darkened plants for 3 h. This reversal of the apparent phosphorylation state in the light was prevented by concomitant stem girdling. In contrast, the optimal activity of nodule PEPC and its protein level showed little or no change in all pretreated plants. These results suggest that the phosphorylation state of PEPC in soybean root nodules is possibly modulated by photosynthate transported recently from the shoots. In situ [32P]orthophosphate labeling, immunoprecipitation, and phosphoamino acid analyses confirmed directly that PEPC in detached intact soybean nodules is phosphorylated on a serine residue(s).

Persistent circadian rhythms in the phosphorylation state of phosphoenolpyruvate carboxylase from Bryophyllum fedtschenkoi leaves and in its sensitivity to inhibition by malate

Phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPCase) from Bryophyllum fedtschenkoi leaves has previously been shown to exist in two forms in vivo. During the night the enzyme is phosphorylated and relatively insensitive to feedback inhibition by malate whereas during the day the enzyme is dephosphorylated and more sensitive to inhibition by malate. These properties of PEPCase have now been investigated in leaves maintained under constant conditions of temperature and lighting. When leaves were maintained in continuous darkness and CO2-free air at 15°C, PEPCase exhibited a persistent circadian rhythm of interconversion between the two forms. There was a good correlation between periods during which the leaves were fixing respiratory CO2 and periods during which PEPCase was in the form normally observed at night. When leaves were maintained in continuous light and normal air at 15°C, starting at the end of a night or the end of a day, a circadian rhythm of net uptake of CO2 was observed. Only when these constant conditions were applied at the end of a day was a circadian rhythm of interconversions between the two forms of PEPCase observed and the rhythms of enzyme interconversion and CO2 uptake did not correlate in phase or period.