Pathways of Nitrogen Assimilation in Cowpea Nodules Studied using ¹⁵N₂ and Allopurinol

Abstract

In the presence of 0.5 millimolar aliopurinol (4-hydroxypyrazolo 13,4-dipyrimidine), an inhibitor of NAD:xanthine oxidoreductase (EC 1.23.2), intact attached nodules of cowpea ( Vigna unguiculata L. Walp. cv Vita 3) formed ['5Nlxanthine from 15N2 at rates equivalent to those of ureide synthesis, confirming the direct assimilation of fixed nitrogen into purines. Xanthine accumulated in nodules and was exported in increasing amounts in xylem of allopurinol-treated plants. Other intermediates of purine oxidation, de nvo purine synthesis, and ammonia assimilation did not increase and, over the time course of experiments (4 hours), allopurinol had no effect on nitrogenase (EC 1.7.99.2) activity. Negligible '5N-labeling of asparagine from '5N2 was observed, suggesting that the significant pool (up to 14 micromoles per gram of nodule fresh weight) of this amide in cowpea nodules was not formed directly from fixation but may have accumulated as a consequence of phloem delivery.The ureides, allantoin and allantoic acid, are the major forms of fixed nitrogen exported from nodules of a wide range of tropical legume symbioses (1). In vivo labeling studies with [14C] glycine (3) or 14CO2 (9) support the idea that allantoin is formed in nodules from purines. Consistent with these data, high activities of enzymes of de novo purine synthesis have been demonstrated in extracts from cowpea (Vigna unguiculata L. Walp.) and soybean (Glycine max L. Merr.) nodules (4), together with specific purine nucleosidase (8) and enzymes of purine oxidation (IMP:oxidoreductase, xanthine oxidoreductase, and urate oxidase) (3,6,21). Although a number of studies using labeled N2 have confirmed that currently fixed nitrogen is utilized in ureide synthesis (12-16), none has reported the recovery of purine pathway intermediates as products of fixation. As has been pointed out previously (4), the involvement of the purine pathway in the flow of N in nodules is therefore inferred rather than proven. The present study reports the recovery of ['5N] sowing, when the initially formed crown nodulation zone had reached maximum size (5) and the specific activity ofnitrogenase was greatest (7).Exposure to '5N2. The volume of nutrient solution in the containers was adjusted to 2.8 L leaving a gas space of 0.7 L in which the nodulated zone of each root system was located. Allopurinol (4-hydroxypyrazolo[3,4-d]pyrimidine) treatmentsinvolved a final concentration of0.5 mM in the nutrient solution. Plant stems were sealed to the container lid with Terostat VII (Teroson G.M.b.H., Heidelberg, FRG), and the nutrient solutions and root atmospheres were purged for 5 min with a mixture of 80% Ar:20% 02 (v/v) prior to the addition of '5N2 (99.9 atom % excess) and sealing to give a final atmosphere ofapproximately 30% N2 at 35 atom % excess '5N, 20% 02 and the balance as Ar. The five plants in a container were removed after 0.5, 1, 2 or 4 h exposure to '5N, and the root systems were plunged into liquid N2 for storage prior to extraction of nodules.Extraction of N...

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.…”

Localization of xanthine dehydrogenase in cowpea root nodules: implications for the interaction between cellular compartments during ureide biogenesis.

“…Xanthine dehydrogenase (XDH; EC 1.1.1.204) is an essential enzyme in'the biogenesis ofthe ureides allantoin and allantoic acid in cowpea and soybean nodules (1)(2)(3). The production of ureides in nodules is correlated with N2 fixation in tropical legumes ofthe tribe Phaseoleae, where up to 90% ofthe fixed nitrogen is assimilated in nodules in ureide form (4).…”

Utilization of 15NO3 − by nodulated soybean plants under conditions of root hypoxia

“…It is possible, therefore, that the 15 N present in the amino acids was not assimilated in the nodule, but in a different location and some of these labelled amino acids translocated to the nodule. In this respect, it is pertinent to observe that in other studies it was suggested that at least part of the Asn of the nodule came from a different source and not from assimilation in the nodule itself (Atkins et al 1988(Atkins et al , 1990.…”