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

在高等植物中，高亲和硝酸盐吸收系统（HATS）因供给还原性氮（如铵和氨基酸）而显著降低。这种抑制一直被认为是高亲和硝酸盐转运体（NRT2）的翻译后调控（磷酸化），但尚未发现证据。在本研究中，我们研究了大麦植物NRT2蛋白的磷酸化。阴离子通道（CLC）被认为参与了木质部薄壁细胞的硝酸盐外排。在CLC的c端区域也发现了可能的磷酸化位点。我们研究了大麦中NRT2的磷酸化和CLC。得到的结果如下：大麦NRT2蛋白的磷酸化。当大麦在硝酸盐浓度较高的培养基中生长时，HATS活性显著降低，而大麦NRT2（HvNRT2）蛋白大量积累。结果表明，HvNRT2翻译后水平调控了大麦中HATS的活性。证实了HvNRT2 c端重组多肽在体外被大麦根的可溶性组分磷酸化。这种磷酸化活性强烈依赖于Ca^<2+>，并由培养基硝酸盐诱导。在^<32>P-γ-ATP和Ca^<2+>存在下，将大麦根微粒体与可溶性组分一起培养。用抗HvNRT2抗体免疫沉淀法从反应混合物中回收磷酸化的HvNRT2蛋白。HvNRT2的磷酸化也是Ca^<2+>依赖性和硝酸盐诱导的2。大麦NRT2蛋白磷酸化证实了大麦CLC c端重组多肽在体外被大麦根制备的可溶性组分磷酸化。这种磷酸化活性强烈依赖于Ca^<2+>，但不受培养基硝酸盐的诱导。

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 inducible2.Phoshorylation of barley NRT2 proteinIt 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.