Identification and expression analysis of phosphoenolpyruvate carboxylase (PEPC) and PEPC kinase genes in C3 plants

Sullivan, Jonathan Stuart (2004) Identification and expression analysis of phosphoenolpyruvate carboxylase (PEPC) and PEPC kinase genes in C3 plants. PhD thesis, University of Glasgow.

Abstract

Phosphoenolpyruvate carboxylase (PEPc) is a cytosolic enzyme that plays a wide range of roles in different tissues of higher plants. A photosynthetic isoform of PEPc catalyses the primary fixation of CO2 in C4 and CAM plants. Other isoforms are thought to be responsible for different functions, including the replenishment of tricarboxylic acid cycle intermediates, pH control, and the provision of malate in guard cells, developing fruit and legume root nodules. PEPc is an allosteric enzyme, inhibited by malate and activate by glucose 6- phosphate. Superimposed on this is control by reversible phosphorylation of a single serine residue close to the N-terminus of the protein. Phosphorylation modulates the allosteric properties of the enzyme, with the phosphorylated form being less sensitive to malate inhibition. The phosphorylation state of PEPc is controlled largely at the level of expression of PEPc kinase (PPCK) genes. The aim of this work was to identify and characterise PEPC and PPCK genes in C3 plants, particularly in the leguminous plant soybean {Glycine max). In legume root nodules, PEPc plays a central role in the metabolism that allows fixation of atmospheric N2 by bacteroids. It provides the C4 dicarboxylates that are required by the bacteroids for energy generation and also the carbon skeletons that are needed for the subsequent assimilation of ammonium into amino acids. In soybean one PEPc gene (GmPEPCT) is highly and relatively specifically expressed in nodules. To test the hypothesis that PEPc kinase expression would show a similar pattern, the expression of both PPCK and PEPC genes were analysed in soybean. Soybean contains at least four PPCK genes. The genomic and cDNA sequences of these genes were determined, and the function of the gene products demonstrated by in vitro expression and enzyme assays. For two of these genes, GmPPCK2 and GmPPCKS, transcript abundance is highest in nodules and is markedly influenced by supply of photosynthate from the shoots. One gene, GmPPCK4, is under robust circadian control in leaves but not in roots. Its transcript abundance peaks in the latter stages of subjective day. This is the first report of a C3 PPCK gene that is controlled in this manner. The expression patterns of five PEPC genes, including one encoding a bacterial-type PEPc lacking the phosphorylation site of the "plant" PEPcs, were also investigated. The PEPC expression patterns do not match those of any of the PPCK genes, arguing against the existence of specific PEPc-PEPc kinase expression partners. Collectively, the results demonstrate that the PEPC and PPCK gene families in soybean are significantly more complex than previously understood. Another role of PEPc is the production of organic acids in developing fruit. Previous work had identified two PPCK genes in tomato (Lycopersicon esculentum), one of which (LePPCK2) contained a novel second intron that exhibits alternative splicing. The correctly spliced transcript encodes a functional PEPc kinase, whereas unspliced or incorrectly spliced transcripts encode a truncated, inactive protein. The relative abundance of the transcripts varies in different tissues, both in wild-type plants and in the greenflesh tomato mutant. A PPCK2 gene was identified in tobacco (Nicotiana tabacum), also exhibiting alternative splicing and producing various amounts of the transcript in different tissues. Similar sequences are also present in potato {Solanum tuberosum) and aubergine {Solarium melongena). The data suggest that the alternative splicing of PPCK2 transcripts is functionally significant. The release of the rice {Oryza sativa) genome sequence allowed the identification of three putative PPCK genes. The sequence of one of these genes (OsPPCK2) was unusual in containing three in frame methionine residues at the 5' end, potentially allowing the production of a larger PEPc kinase protein. The functionality of these genes was assessed, but the results were unable to provide unequivocal evidence demonstrating the phosphorylation of PEPc by the Tong' form ofOsPPCK2. Overall the data presented in this thesis reveal a novel picture of the transcriptional control of PEPc kinase in C3 plants.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Plant sciences.
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Jenkins, Prof. Gareth I. and Nimmo, Prof. Hugh G.
• Date of Award: 2004
• Depositing User: Enlighten Team
• Unique ID: glathesis:2004-71434
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 10 May 2019 10:49
• Last Modified: 05 Aug 2021 15:43
• URI: https://theses.gla.ac.uk/id/eprint/71434

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