title: Phosphorylation/dephosphorylation steps are key events in the phytochrome-mediated enhancement of nitrate reductase mRNA levels and enzyme activity in maize.
abstract: We provide evidence to show that the increase in nitrate reductase (NR) transcript level stimulated by red light is mediated via a phosphorylation-dependent step. The light-stimulated enhancement of NR transcript level was significantly inhibited by H-7, a protein kinase inhibitor, whereas okadaic acid (OKA), a phosphatase inhibitor, had no effect. Phorbol myristate acetate (PMA), an activator of protein kinase C (PKC) enhanced the NR transcript level in dark-grown leaves. No correlation between changes in NR transcript level and NR activity (NRA) was observed. 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 vitro without the involvement of other accessory proteins. By in vivo labelling with 32P and immunoprecipitation of NR with NR antibodies it was found that in the presence of OKA most NR protein (NRP) was present in phosphorylated state, while with H-7 the reverse was seen. The red (R) and far-red (FR) light reversible experiments suggested that phytochrome (Pfr, an active form) stimulation of NRA is mediated by dephosphorylation of the enzyme, suggesting that Pfr regulates both NR transcription and NRA via phosphorylation/dephosphorylation steps controlled by separate signal transduction pathways.
authors.affiliation: Molecular Plant Physiology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
authors.forename: M R
authors.initials: MR
authors.lastname: Chandok
authors.forename: S K
authors.initials: SK
authors.lastname: Sopory
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D003624:Darkness; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004988:Ethers, Cyclic / Q000494:pharmacology; D018506:Gene Expression Regulation, Plant / Q000502:physiology* / Q000528:radiation effects; D007546:Isoquinolines / Q000494:pharmacology; D008027:Light; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D010834:Phytochrome / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D018749:RNA, Plant / Q000032:analysis; D015398:Signal Transduction / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics
chemical_list: D004791:Enzyme Inhibitors; D004988:Ethers, Cyclic; D007546:Isoquinolines; D010879:Piperazines; D012333:RNA, Messenger; D018749:RNA, Plant; D010834:Phytochrome; D019319:Okadaic Acid; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D009565:Nitrate Reductases; D050901:Nitrate Reductase; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D013755:Tetradecanoylphorbol Acetate