PHOSPHOENOLPYRUVATE CARBOXYLASE FROM PEA ROOT-NODULES

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) is essential for t he assimilation of nitrogen fixed by symbiotic bacteria in the root no dules of leguminous plants. PEPC activity and the amounts of enzyme an d PEPC-encoding mRNA were measured in roots and effective root nodules (Fix(+)-phenotype) from SGE and Sprint-2 wild pea lines, as well as i n root nodules from mutant pea lines (SGEFix(-)-1, SGEFix(-)-2 and Spr int-2Fix(-)) incapable of fixing molecular nitrogen (Fix(-)-phenotype) . During the early developmental stages of effective root nodules, pri or to nitrogen fixation, PEPC activity in the nodules became 1.5-2 tim es higher than in roots. The highest PEPC activity was observed in nod ules approaching the stage with the highest nitrogenase activity. Duri ng the formation of ineffective nodules, PEPC activity in the nodule i ncreased slightly over root PEPC activity, but this increase was alway s lower than in Fix(+)-nodules. The increase in PEPC activity in effec tive nodules was due to the nodule-enhanced form of the enzyme. Wester n hybridization, with the use of polyclonal antibodies against a nodul e-enhanced form of PEPC from alfalfa, revealed an increased amount of the enzyme in the developing Fix(+) pea nodules as compared to the roo ts and the Fix-nodules of mutant lines. The library of pea nodule cDNA s' 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 anal ysis showed that the content of mRNA in the effective nodules was high er than in roots and ineffective nodules. Differential expression of P EPC in nodules from various mutant pea lines was found. Possible mecha nisms of controlling enzyme activity in pea nodules are discussed.