Isolation of a cDNA for nodule-enhanced phosphoenolpyruvate carboxylase from pea and its expression in effective and plant-determined ineffective pea nodules

Abstract

A cDNA clone for phosphoenolpyruvate carboxylase (PEPC) was isolated from nodules of pea (Pisum sativum L.). The levels of activity, the PEPC polypeptide and the mRNA for PEPC in effective nodules and in ineffective nodules induced on the pea mutant E135 (sym 13) were compared to evaluate the regulation of PEPC activity in pea nodules. The cDNA was 3 222 bp long and contained an open reading frame of 2 901 bp, capable of encoding a polypeptide of 967 amino acids. The deduced amino acid sequence was similar to those of other plant PEPCs. The expression of this gene was enhanced in nitrogen-fixing effective pea nodules. The activity of PEPC in the effective nodules increased during development of the plants and the activity was maintained until senescence. This increase was associated with increased levels of the 110 kDa polypeptide and the mRNA for PEPC. By contrast, roots always had lower levels of activity, the polypeptide and the mRNA for PEPC than effective nodules. In ineffective E135 nodules, the activity of PEPC increased similarly to that in effective nodules, but then it decreased to the level in roots after 2.5 weeks post-planting. However, the levels of the 110 kDa polypeptide and the mRNA for PEPC in ineffective nodules were higher than those in roots, even though ineffective nodules had reduced levels of the polypeptide and mRNA for PEPC as compared with those in effective nodules. These findings indicate that after nodule initiation, the activity of PEPC in pea nodules may be regulated at the transcriptional level and after translation by a putative factor(s) that governs the effectiveness of the nodules.