Phosphoenolpyruvate carboxylase in developing seeds of Vicia faba L.: Geneexpression and metabolic regulation

To analyze the role of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.3 1) during seed development, two cDNA clones encoding two isoforms of PEPCas e were isolated from a seed-specific library of Vicia faba. The two sequenc es (VfPEPCase1 and VfPEPCase2) have a sequence identity of 82 and 89% on th e nucleotide and amino acid levels. The VfPEPCase1 mRNA was found to be pre dominantly expressed in roots and developing cotyledons whereas the VfPEPCa se2 mRNA was more abundant in green and maternal tissues. In the cotyledons , PEPCase mRNAs accumulated from early to mid cotyledon stage and decreased thereafter. The PEPCase activity increased continuously during cotyledon d evelopment. The enzyme was strongly activated by glucose-6-phosphate, but n ot by glucose, fructose or sucrose. Asparagine was weakly activating wherea s malate, aspartate and glutamate were inhibitory. The inhibitors became le ss effective with increasing pH. Aspartate was a much stronger inhibitor of cotyledonary PEPCase than glutamate at both pH 7.0 and 7.5. The sensitivit y of PEPCase to malate inhibition decreased from early to mid cotyledon sta ge at a time when storage proteins are synthesized. This indicates activati on on the protein level, possibly by protein phosphorylation. Nitrogen star vation in the presence of hexoses but not sucrose decreased mRNA levels of VfPEPCase1 and enzyme activity, indicating control on the mRNA level by bot h carbon and nitrogen. it is concluded that in developing cotyledons PEPCas e is probably important for the synthesis of organic acids to provide carbo n skeletons for amino acid synthesis.