PHOSPHORYLATION DEPHOSPHORYLATION STEPS ARE KEY EVENTS IN THE PHYTOCHROME-MEDIATED ENHANCEMENT OF NITRATE REDUCTASE MESSENGER-RNA LEVELS AND ENZYME-ACTIVITY IN MAIZE/

We provide evidence to show that the increase in nitrate reductase (NR ) transcript level stimulated by red light is mediated via a phosphory lation-dependent step. The light-stimulated enhancement of NR transcri pt level was significantly inhibited by H-7, a protein kinase inhibito r, whereas okadaic acid (OKA), a phosphatase inhibitor, had no effect. Phorbol myristate acetate (PMA), an activator of protein kinase C (PK C) enhanced the NR transcript level in dark-grown leaves. No correlati on between changes in NR transcript level and NR activity (NRA) was ob served. Inhibition of NRA by OKA and stimulation by H-7 indicated that NRA is increased by dephosphorylating the enzyme. We have identified a protein kinase (C type) that can phosphorylate the purified NR in vi tro without the involvement of other accessory proteins. By in vivo la belling with P-32 and immunoprecipitation of NR with NR antibodies it was found that in the presence of OKA most NR protein (NRP) was presen t in phosphorylated state, while with H-7 the reverse was seen. The re d (R) and far-red (FR) light reversible experiments suggested that phy tochrome (Pfr, an active form) stimulation of NRA is mediated by depho sphorylation of the enzyme, suggesting that Pfr regulates both NR tran scription and NRA via phosphorylation/dephosphorylation steps controll ed by separate signal transduction pathways.