De Novo Purine Synthesis in Nitrogen-Fixing Nodules of Cowpea (Vigna unguiculata [L.] Walp.) and Soybean (Glycine max [L.] Merr.)

Abstract

Partially purified, cell-free extracts from nodules of cowpea (Vigna unguiculata L. Walp. cv. Caloona) and soybean (Glycine mar L. Merr. cv. Bragg) showed high rates of de novo purine nucleotide and purine base synthesis. Activity increased with rates of nitrogen fixation and ureide export during development of cowpea plants; maximum rates (equivalent to 1.2 micromoles N2 per hour per gram fresh nodule) being similar to those of maximum nitrogen fixation (1-2 micromoles N2 per hour per gram fresh nodule). Extracts from actively fixing nodules of a symbiosis not producing ureides, Lupinus albus L. cv. Ultra, showed rates of de novo purine synthesis 0.1% to 0.5% those of cowpea and soybean. Most (70-90%) of the activity was associated with the particulate components of the nodule, but up to 50% was released from this fraction by osmotic shock. The accumulated end products with particulate fractions were inosine monophosphate and aminoimidazole carboxamide ribonucleotide. Further metabolism to purine bases and ureides was restricted to the soluble fraction of the nodule extract. High rates of inosine monophosphate synthesis were supported by glutamine as amide donor, lower rates (10-20%) by ammonia, and negligible rates with asparagine as substrate.The ureides, allantoin and allantoic acid, are formed as major products of nitrogen fixation in a wide range of tropical legumes (8,14). Studies with cell-free extracts of cowpea and soybean nodules have demonstrated that these compounds may be formed from oxidation of purine bases and nucleotides (2,5,22,23) and that this activity is associated with the plant cell cytosol of the central, infected tissue of the nodule (5). A pathway for de novo synthesis of purine nucleotides has been suggested (3) as the source of these purines with currently fixed nitrogen being utilized from the amino groups of glycine and aspartate and the amide group of glutamine. While [1 Clglycine supplied to nodule slices was more effectively metabolized to ureides than was [14Cjglucose or acetate (5), consistent with an active purine pathway, the level of labeling was relatively low. Furthermore, of the eleven enzymes likely to be involved in purine biosynthesis (6)

Nitrate Inhibition of Legume Nodule Growth and Activity

“…That is, most of the accumulation of amino N and ureide N was in the cytosol and not in the bacteroids. This would be the expected result since these compounds are synthesized in the cytosol (1,2,4,5,8). Ureide concentrations in the range from 8 to 12 mm can be estimated in nodules supplied with very high nitrate.…”

Effects of Short-Term N2 Deficiency on N Metabolism in Legume Nodules

“…Other enzymes which also utilize ammonia as a substrate, namely glutamate oxidoreductase and the bacteroid-located alanine oxidoreductase (6) …”

Reexamination of the Intracellular Localization of de Novo Purine Synthesis in Cowpea Nodules

“…The ureides are translocated in xylem to the host and constitute the major source of N for plant growth. Studies based on the use of stable and radioactive isotopes (Atkins et al, 1982(Atkins et al, , 1988, on the application of specific metabolic inhibitors (Fujihara and Yamaguchi, 1978;Atkins et al, 1980), and on the isolation and characterization of critical enzymes (Atkins, 1991) have established the broad features of the metabolic pathway of ureide synthesis. Ammonia is assimilated in the infected plant cell by Gln synthetase and GOGAT, providing both amide and amino-N to de novo purine synthesis.…”

“…The rates of purine synthesis demonstrated in cell-free extracts of cowpea nodules are high enough to account for the rates of ureide synthesis occurring during N fixation (Atkins et al, 1982). However, much lower rates of synthesis, typically 10 to 20% for cell fractions and <10% for plastids in the case of cowpea (Shelp et al, 1983), were recovered from isolated infected and uninfected cells and their organelles separated on SUC density gradients (Boland and Schubert, 1982;Shelp et al, 1983).…”