Modulation of nitrate reductase activity in cucumber (Cucumis sativus) roots

Abstract

Nitrate reductase (NR) (EC 1.6.6.1) activity and NR activation state, i.e. activity in the presence of Mg(2+) relative to activity in the absence of Mg(2+), in cucumber (Cucumis sativus) leaves increased in the light and decreased in the dark. In contrast to leaves, NR activation state in the roots did not show light/dark-dependent changes. Root NR was activated by anoxia or by addition of uncoupler (CCCP) or mannose. These treatments decreased ATP levels in root tissue. On the contrary, high oxygen supply promoted some NR inactivation. When an extract from anoxic roots was preincubated with ATP, NR was gradually inactivated. Subsequent addition of 5'-AMP resulted in a remarkable reactivation of the enzyme. NR extracted from hyperoxygenated roots was activated by preincubation with 5'-AMP, and the process was reversed by ATP. These results suggest the participation of adenine nucleotides on the in vivo modulation of NR activity in cucumber roots. NR was activated in vivo by cellular acidification and inactivated by alkalinisation. The acid-induced activation of NR was greatly prevented by okadaic acid, a protein phosphatase inhibitor. Our data indicate that, as in barley roots, anoxia, uncouplers, and mannose feeding activate cucumber root NR, at least partly, by enhancing NR dephosphorylation via a decrease in the internal level of ATP and a concomitant cellular acidification.