Reversible light/dark modulation of spinach leaf nitrate reductase activity involves protein phosphorylation.

Spinach (Spinacia oleracea L.) leaf nitrate reductase (NADH:NR;NADH:nitrate oxidoreductase, EC 1.6.6.1) activity was found to rapidly change during light/dark transitions. The most rapid and dramatic changes were found in a form of NR which was sensitive to inhibition by millimolar concentrations of magnesium. This form of NR predominated in leaves in the dark, but was almost completely absent from leaves incubated in the light for only 30 min. When the leaves were returned to darkness, the NR rapidly became sensitive to Mg2+ inhibition. Modulation of the overall reaction involving NADH as electron donor was also found when reduced methyl viologen was the donor (MV:NR), indicating that electron transfer had been blocked, at least in part, at or near the terminal molybdenum cofactor site. Changes in activity appear to be the result of a covalent modification that affects sensitivity of NR to inhibition by magnesium, and our results suggest that protein phosphorylation may be involved. NR was phosphorylated in vivo after feeding excised leaves [32P]Pi. The NR subunit was labeled exclusively on seryl residues in both light and dark. Tryptic peptide mapping indicated three major 32P-labeled phosphopeptide (Pp) fragments. Labeling of two of the P-peptides (designated Pp1 and 3) was generally correlated with NR activity assayed in the presence of Mg2+. In vivo, partial dephosphorylation of these sites (and activation of NR assayed with Mg2+) occurred in response to light or feeding mannose in darkness. The light effect was blocked completely by feeding okadaic acid via the transpiration stream, indicating the involvement of type 1 and/or type 2A protein phosphatases in vivo. While more detailed analysis is required to establish a causal link between the phosphorylation status of NR and sensitivity to Mg2+ inhibition, the current results are highly suggestive of one. Thus, in addition to the molecular genetic mechanisms regulating this key enzyme of nitrate assimilation, NR activity may be controlled in leaves by phosphorylation/dephosphorylation of the enzyme protein resulting from metabolic changes taking place during light/dark transitions.