RAPID MODULATION OF NITRATE REDUCTASE IN PEA ROOTS

The regulatory properties of nitrate reductase (NR; EC 1.6.6.1) in roo t extracts from hydroponically grown pea (Pisum sativum L. cv. Kleine Rheinlanderin) plants were examined and compared with known properties of NR from spinach and pea leaves. Nitrate-reductase activity (NRA) e xtracted from pea roots decreased slowly when plants were kept in the dark, or when illuminated plants were detopped, with a half-time of ab out 4 h (= slow modulation in vivo). In contrast, the half-time for th e dark-inactivation of NR from pea leaves was only 10 min. However, wh en root tip segments were transferred from aerobic to anaerobic condit ions or vice versa, changes in NRA were as rapid as in leaves (= rapid modulation in vivo). Nitrate-reductase activity was low when extracte d from roots kept in solutions flushed with air or pure oxygen, and hi gh in nitrogen. Okadaic acid, a specific inhibitor of type-1 and type- 2A protein phosphatases, totally prevented the in vivo activation by a naerobiosis of NR, indicating that rapid activation of root NR involve d protein dephosphorylation. Under aerobic conditions, the low NRA in roots was also rapidly increased by incubating the roots with either u ncouplers or mannose. Under these conditions, and also under anaerobio sis, ATP levels in roots were much lower than in aerated control roots . Thus, whenever ATP levels in roots were artificially decreased, NRA increased rapidly. The highly active NR extracted from anaerobic roots could be partially inactivated in vitro by preincubation of desalted root extracts with MgATP (2 mM), with a half-time of about 20 min. It was reactivated by subsequently incubating the extracts with excess AM P (2 mM). Thus, pea root NR shares many of the previously described pr operties of NR from spinach leaves, suggesting that the root enzyme, l ike the leaf enzyme, can be rapidly modulated, probably by reversible protein phosphorylation/dephosphorylation.