Phosphoenolpyruvate carboxylase from pea root nodules

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) is essential for the assimilation of nitrogen fixed by symbiotic bacteria in the root nodules of leguminous plants. PEPC activity and the amounts of enzyme and PEPC-encoding mRNA were measured in roots and effective root nodules (Fix(+)-phenotype) from SGE and Sprint-2 wild pea lines, as well as in root nodules from mutant pea lines (SGEFix(-)-1, SGEFix(-)-2 and Sprint-2Fix(-)) incapable of fixing molecular nitrogen (Fix(-)-phenotype). During the early developmental stages of effective root nodules, prior to nitrogen fixation, PEPC activity in the nodules became 1.5-2 times higher than in roots. The highest PEPC activity was observed in nodules approaching the stage with the highest nitrogenase activity. During the formation of ineffective nodules, PEPC activity in the nodule increased slightly over root PEPC activity, but this increase was always lower than in Fix(+)-nodules. The increase in PEPC activity in effective nodules was due to the nodule-enhanced form of the enzyme. Western hybridization, with the use of polyclonal antibodies against a nodule-enhanced form of PEPC from alfalfa, revealed an increased amount of the enzyme in the developing Fix(+) pea nodules as compared to the roots and the Fix-nodules of mutant lines. The library of pea nodule cDNAs' was prepared, from which three cDNA clones of nodule-enhanced PEPC were selected. The nucleotide sequences of cDNA clones were determined from the 3'-and 5'-ends; this analysis revealed a high degree of PEPC homology with other PEPC of plant origin. Northern-hybridization analysis showed that the content of mRNA in the effective nodules was higher than in roots and ineffective nodules. Differential expression of PEPC in nodules from various mutant pea lines was found. Possible mechanisms of controlling enzyme activity in pea nodules are discussed.