Regulation of phosphoenolpyruvate carboxylase in higher plants

The regulation of higher plant phosphoenolpyruvate carboxylase (PEPc) has been the focus of intensive research for many years. In CAM, C4 and C3 species the enzyme's sensitivity to feedback inhibition by malate is modulated by reversible phosphorylation of a serine residue by a calcium-independent PEPc kinase. This kinase is controlled by a circadian oscillator in CAM plants whereas in C4 and C3 plants the activity is induced by light. In all cases, PEPc kinase activity is regulated by a process involving de novo protein synthesis as opposed to a phosphorylation cascade or a second messenger. Nitrate has also been implicated in the regulation of PEPc from C3 species and the involvement of cytosolic alkalinization and calcium ion movements has been demonstrated in the signalling pathway by which light triggers phosphorylation of PEPc in C4 plants. The overall objective of this present research was to investigate the physiological roles of the phosphorylation of PEPc and the signalling pathways that bring this about. In the first part of the work, the involvement of cytosolic pH in the regulation of PEPc was studied in the CAM plant K. fedtschenkoi. An increase in cytosolic pH had no affect on the malate sensitivity of PEPc or the PEPc kinase activity. In contrast, treatment of leaf disks with 3 mM acetic acid to reduce cytosolic pH prevented the nocturnal increase in the apparent Ki of PEPc for malate, PEPc kinase activity and PEPc kinase translatable mRNA. The effect was not metabolic but was due to cytosolic acidification. Measurement of the malate content of leaf disks revealed that acetic acid treatment prevented the decarboxylation of malate. It is proposed that when the cytosolic malate concentration is above a certain threshold at the end of a day period, PEPc kinase activity is inhibited during the following night and PEPc remains in a dephosphorylated, inactive form. The effect of acid treatment was slow, requiring 9-12 hours, and was therefore not considered to be involved in CAM PEPc signal transduction. In the second part of this research aspects of PEPc regulation were studied in the model C3 species A. thaliana. The first full-length cDNA for PEPc from A. thaliana was cloned and sequenced. Northern analysis of poly A+ RNA from different A. thaliana plant tissues suggested that the cDNA encoded a root isoform of PEPc. The specific activity and malate sensitivity of PEPc were measured in a photomixotrophic cell culture of A. thaliana were measured. The phosphatase inhibitor, cantharidin, decreased the malate sensitivity of PEPc from the cell culture but light did not have any effect on the malate sensitivity of PEPc from the cell culture or A. thaliana plant tissue. Using Northern analysis of poly A+ RNA transcripts for PEPc and PEPc kinase were detected in the cell culture. PEPc transcripts were detected in root and flower/bud tissue using a conserved PEPc probe and PEPc kinase transcripts were detected in several different tissues of A. thaliana. The effects of sucrose and nitrate on the transcript levels of PEPc and PEPc kinase in A. thaliana ceil culture were studied. Transcript levels of PEPc and PEPc kinase were higher in 1% sucrose compared with 3% sucrose but no apparent trend was evident in the effect of nitrate on either PEPc or PEPc kinase. Therefore, it can be concluded that PEPc kinase is present in both A. thaliana tissue and cell culture and that the malate sensitivity of PEPc from the cell culture is regulated by reversible phosphorylation. The potential use of the A. thaliana cell culture for studies of the regulation of PEPc in C3 plants has been demonstrated.