Developmental and Biochemical Regulation of ‘Constitutive’ Nitrate Reductase Activity in Leaves of Nodulating Soybean

The developmental profile of ‘constitutive’ nitrate reductase activity (cNRA) in leaves of soybean (Glycine max (L.) cv. Bragg) plants at different ages is described. The youngest leaves had most cNRA and the activity dropped off as a newer leaf developed above it. Each leaf had its distinct active period of in vivo cNRA. This pattern was different in urea-grown and symbiotically-grown plants (inoculated with Bradyrhizobium japonicum strain USDA 110), where the latter had no detectable in vivo cNRA in older leaves. Urea-grown plants maintained considerable in vivo NRA in such older leaves. When symbiotically-grown plants had their nodules removed, in vivo cNRA reappeared in older leaves within 1 d of removal, nearly reaching levels of young leaves at 3 d after nodule excision. Allantoic acid (ALL), one of the known transport ureides of soybeans, was implicated as a possible signal molecule from nodules to leaves. Allantoic acid (100 μM) inhibited in vitro c1 NRA significantly, with 400 μM ALL resulting in complete inhibition. In contrast, allantoin (ALN) had no inhibitive effect on NRA. Inhibition of c1NRA by ALL was by a competitive process, judging from Lineweaver-Burke plots against nitrate. Kinetics showed a constant Vmax of around 105 nmol NO2 mg−1 protein h−1 and a Km for nitrate of 15 mM, which increased to 60 mM in the presence of 200 μM allantoic acid. Non-specific (ionic and pH-related) influences were eliminated. Allantoic acid also had a slight stimulating effect of in vitro NRA (up about 25% at 400 μM). These findings suggest that c1NRA may be involved in ureide metabolism, rather than in vivo nitrate metabolism.