Phosphoenolpyruvate carboxylase kinase in higher plants

Phosphoenolpyruvate carboxylase (PEPc) is a ubiquitous enzyme in higher plants. Several isoforms of PEPc exist in plants. They carry out a variety of roles including the anapleurotic role of re-supplying the tricarboxylic acid cycle with four- carbon intermediates that are depleted by aminoacid biosynthesis. PEPc is also involved in processes requiring four-carbon acid accumulation, such as ripening. In C4 and CAM plants, PEPc has a further photosynthetic role in performing the primary fixation of CO2. PEPc is regulated by reversible phosphorylation, which modulates the allosteric properties of the enzyme. The phosphorylated form is less sensitive to negative feedback inhibition by malate. The phosphorylation state of PEPc is primarily controlled by the activity of its kinase, phosphoenolpyruvate carboxylase kinase (PPCK). This Ca2+-independent kinase, with minimal N- or C-terminal extensions is in turn dependent upon de novo protein synthesis for its activity. This activity is controlled by a circadian oscillator in CAM plants and induction by light in C4 and C3 plants. The aim of this work was to identify and characterise PPCK in C3 and C4 plants. A partial maize PPCK was identified by est database mining. A full-length clone was obtained by RACE PCR and the cDNA clone was sequenced, revealing high similarity to other known PPCKs. To prove function, the cDNA clone was in vitro transcribed and translated and shown to phosphorylate PEPc. The gene expression was then studied in mature leaves by RT-PCR. Analysis revealed that this PPCK was not upregulated in the light. It is therefore thought not to be the photo synthetic relevant PPCK but a separate isoform with a possible house-keeping role in vivo. Several other ests from the maize database show similarity to PPCK and are in the process of being investigated by members of our lab. A similar database mining approach revealed two putative PPCK in the tomato database. Full-length genomic and cDNA clones were generated and sequenced. The cDNA clones were tested for PPCK activity as the maize PPCK was. The first of these, LePPCK1, showed PPCK activity. It bears high similarity to other PPCKs genes with a small 3' end intron. The second, LePPCK2, appears to be a novel PPCK gene. RT-PCR analysis and sequencing show that a second intron exists in this gene that is subject to alternative splicing. This intron, near the middle of the gene, allows the introduction of a premature stop codon into the open reading frame, truncating the kinase, thereby preventing PPCK activity. Correct splicing of this intron though. allows PPCK activity. This is demonstrated in a similar fashion to the aforementioned maize PPCK. Expression analysis of these genes was undertaken by semi-quantitative RT-PCR analysis over a range of tissues and compartments of the tomato plant. This revealed that LePPCK1 is expressed at similar rates across tissues suggesting that its role is anapleurotic. The expression of LePPCK2 is induced in post-breaker fruit. In combination, the alternative splicing of the pre-mRNA appears to be dependent upon tissue type and compartment type as suggested by semi-quantitative RT-PCR transcript ratios and transcripts in red-ripe fruit compartments. Furthermore, these types of PPCK genes appear to exist in other Solanaceae plants, suggesting a PPCK sub-family. A phylogenetic analysis of all known PPCKs (and ones recently identified by myself and members of our lab) is also undertaken. This reveals a number of interesting traits. Perhaps the most interesting is the above average GC content of monocot PPCKs of ~70%. Collectively these results indicate that monocots and dicots probably hold multigene PPCK families