Biochemical studies of post-translational modification of nitrate transport systems in higher plants

• Project/Area Number: 14360039
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Plant nutrition/Soil science
• Research Institution: Niigata University
• Principal Investigator: SUEYOSHI Kuni Niigata University, Institute of Science and Technology, Associate Professor, 自然科学系, 助教授 (10216278)
• Co-Investigator(Kenkyū-buntansha): OHYAMA Takuji Niigata University, Institute of Science and Technology, Professor, 自然科学系, 教授 (30152268) ; OHTAKE Norikuni Niigata University, Institute of Science and Technology, Assistant, 自然科学系, 助手 (50313507)
• Project Period (FY): 2002 – 2005
• Project Status: Completed (Fiscal Year 2005)
• Budget Amount: ¥14,600,000 (Direct Cost: ¥14,600,000); Fiscal Year 2005: ¥900,000 (Direct Cost: ¥900,000); Fiscal Year 2004: ¥1,200,000 (Direct Cost: ¥1,200,000); Fiscal Year 2003: ¥6,000,000 (Direct Cost: ¥6,000,000); Fiscal Year 2002: ¥6,500,000 (Direct Cost: ¥6,500,000)
• Keywords: Anion Channel / Ion transport / Nitrate / Phosphorylation / Post-translational regulation / Transporter

Research Abstract

In higher plants, it has been known that high-affinity nitrate uptake system (HATS) was markedly decreased by supply of reduced nitrogen such as ammonium and amino acids. This repression has been thought to be post-translational regulation (phosphorylation) of high-affinity nitrate transporter (NRT2) while the evidence has not been found. In this study, we investigated the phosphorylation of NRT2 protein in barley plants. The annion channel (CLC) was expected to involve in nitrate efflux from xylem parenchyma cells. The putative phosphorylation sites were also found in C-terminal regions of CLC. We investigated the poshorylation of NRT2 and CLC in barley. The obtained results are following.
1.Phoshorylation of barley NRT2 protein.
When barley was grown in the medium containing higher nitrate concentrations, HATS activity was markedly decreased while barley NRT2(HvNRT2) protein highly accumulated. It was suggested that the HATS activity in barley was regulated at the levels of post-translation of HvNRT2. It was confirmed that the recombinant polypeptide of C-terminal region of HvNRT2 was phosphorylated in vitro by the soluble fractions prepared from barley roots. This phosphorylational activity strongly depend on Ca^<2+> and induced by medium nitrate.
The microsome fractions from barley roots were also incubated with soluble fractions in the presence of ^<32>P-γ-ATP and Ca^<2+>. The phosphorylated HvNRT2 proteins were recoverd from the reaction mixture by immuno-preciptation methods using anti-HvNRT2 antibody. The phoshorylation of HvNRT2 was also Ca^<2+>-dependent and nitrate inducible
2.Phoshorylation of barley NRT2 protein
It was confirmed that the recombinant polypeptide of C-terminal region of barley CLC was phosphorylated in vitro by the soluble fractions prepared from barley roots. This phosphorylational activity strongly depend on Ca^<2+> but not induced by medium nitrate.

Research Products

• [Journal Article] Contribution of shoots and roots to in vivo nitrate reduction in NADH-specific nitrate reductase deficient mutant seedlings of barely (Hordeum vulgare L.). (2004)
  • Author(s): Abdel-Latif, S., Kawachi, T., Fujikake, H., Ohtake, N., Ohyama, T., Sueyoshi, K.
  • Journal Title: Soil Science and Plant Nutrition 50(4) Pages: 527-535
  • Description: 「研究成果報告書概要(和文)」より