Products of Dark CO2 Fixation in Pea Root Nodules Support Bacteroid Metabolism

Products of the nodule cytosol in vivo dark [(14)C]CO(2) fixation were detected in the plant cytosol as well as in the bacteroids of pea (Pisum sativum L. cv "Bodil") nodules. The distribution of the metabolites of the dark CO(2) fixation products was compared in effective (fix(+)) nodules infected by a wild-type Rhizobium leguminosarum (MNF 300), and ineffective (fix(-)) nodules of the R. leguminosarum mutant MNF 3080. The latter has a defect in the dicarboxylic acid transport system of the bacterial membrane. The (14)C incorporation from [(14)C]CO(2) was about threefold greater in the wild-type nodules than in the mutant nodules. Similarly, in wild-type nodules the in vitro phosphoenolpyruvate carboxylase activity was substantially greater than that of the mutant. Almost 90% of the (14)C label in the cytosol was found in organic acids in both symbioses. Malate comprised about half of the total cytosol organic acid content on a molar basis, and more than 70% of the cytosol radioactivity in the organic acid fraction was detected in malate in both symbioses. Most of the remaining (14)C was contained in the amino acid fraction of the cytosol in both symbioses. More than 70% of the (14)C label found in the amino acids of the cytosol was incorporated in aspartate, which on a molar basis comprised only about 1% of the total amino acid pool in the cytosol. The extensive (14)C labeling of malate and aspartate from nodule dark [(14)C]CO(2) fixation is consistent with the role of phosphoenolpyruvate carboxlase in nodule dark CO(2) fixation. Bacteroids from the effective wild-type symbiosis accumulated sevenfold more (14)C than did the dicarboxylic acid transport defective bacteroids. The bacteroids of the effective MNF 300 symbiosis contained the largest proportion of the incorporated (14)C in the organic acids, whereas ineffective MNF 3080 bacteroids mainly contained (14)C in the amino acid fraction. In both symbioses a larger proportion of the bacteroid (14)C label was detected in malate and aspartate than their corresponding proportions of the organic acids and amino acids on a molar basis. The proportion of (14)C label in succinate, 2-oxogultarate, citrate, and fumarate in the bacteroids of the wild type greatly exceeded that of the dicarboxylate uptake mutant. The results indicate a central role for nodule cytosol dark CO(2) fixation in the supply of the bacteroids with dicarboxylic acids.