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

Abstract

Products of the nodule cytosol in vivo dark [14C]C02 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 CO2 fixation products was compared in effective (fix+) nodules infected by a wild-type Rhizobium leguminosarum (MNF 300), and ineffective (fix-) nodules of the R. Ieguminosarum mutant MNF 3080. The latter has a defect in the dicarboxylic acid transport system of the bacterial membrane.The 14C incorporation from ['4C]CO2 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 14C 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 14C was contained in the amino acid fraction of the cytosol in both symbioses. More than 70% of the 14C 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 14C labeling of malate and aspartate from nodule dark (14CJCO2 fixation is consistent with the role of phosphoenolpyruvate carboxiase in nodule dark CO2 fixation. Bacteroids from the effective wild-type symbiosis accumulated sevenfold more 14C than did the dicarboxylic acid transport defective bacteroids. The bacteroids of the effective MNF 300 symbiosis contained the largest proportion of the incorporated 14C in the organic acids, whereas ineffective MNF 3080 bacteroids mainly contained 14C in the amino acid fraction. In both symbioses a larger proportion of the bacteroid 14C 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 14C 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 CO2 fixation in the supply of the bacteroids with dicarboxylic acids.Symbiotic N2 fixation in legume root nodules is fueled by carbon sources supplied by the host plant. Substantial evi-'This research was initiated via a fellowship to L.

Labeling of Carbon Pools in Bradyrhizobium japonicum and Rhizobium leguminosarum bv viciae Bacteroids following Incubation of Intact Nodules with 14CO2

“…In pea nodule cytosol, the rate of asparagine labeling exceeded that of aspartate. Although this might be consistent with pea being an amide transporter, it disagrees with the data of Rosendahl et al (23), who found 72% of the label in amino acids in the pea cytosol in aspartate. Considering the eight labeled amino acids in our samples of pea nodules (aspartate, asparagine, glutamate, glutamine, alanine, glycine, serine, and proline), proportional distribution of label after 6 min showed 39% of the total dpm in asparagine, 21 % in aspartate, and 15% in glycine.…”

“…These facts combined with the established importance of dicarboxylic acids in bacteroid carbon nutrition make this an attractive approach for the study of carbon metabolism in intact nodules. The approach has been employed by others, but, in those studies, bacteroids after a single 5-min incubation were isolated by a method requiring about 25 min, during which time labeling pattems in bacteroids could change (23).…”

Routes of pyruvate synthesis in phosphorus‐deficient lupin roots and nodules

“…However, since LP supply did not affect the level of nodular pyruvate that was synthesized from PEPc-derived malate, this implies that malate may have been used for other reactions. Under these conditions it is likely that malate was a source for bacterial respiration inside the nodules (Rosendahl et al, 1990). It has been proposed that the low oxygen concentration in nodules would favour malate, rather than pyruvate as the main end product of glycolysis (Vance & Heichel, 1991).…”

“…Furthermore, PEPc provides a portion of the C for both malate and aspartate synthesis (Romanov et al ., 1985). There is consensus that dicarboxylic acids are the major source of C for bacteroids in the symbiosome (Streeter, 1987;Rosendahl et al ., 1990). In addition, malate is an important bacteroid respiratory fuel because of the high concentration of this metabolite in the nodules (Vance et al ., 1985;Rosendahl et al ., 1990).…”

“…There is consensus that dicarboxylic acids are the major source of C for bacteroids in the symbiosome (Streeter, 1987;Rosendahl et al ., 1990). In addition, malate is an important bacteroid respiratory fuel because of the high concentration of this metabolite in the nodules (Vance et al ., 1985;Rosendahl et al ., 1990). A recent study of the P i -starved model legume, Lotus japonicus found that the concerted actions of PEPc and MDH are upregulated under P deficiency (Keller, 2003).…”