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

在高等植物中，已经知道高亲和力硝酸盐吸收系统（HATS）被还原性氮如铵和氨基酸的供应显著降低。这种抑制作用被认为是高亲和力硝酸盐转运蛋白（NRT 2）的翻译后调节（磷酸化），但尚未发现证据。annion通道（CLC）可能参与木质部薄壁细胞的硝酸盐外流。在CLC的C-末端区域也发现了推测的磷酸化位点。我们研究了大麦中NRT 2和CLC的磷酸化。主要结果如下：1.大麦NRT 2蛋白的磷酸化：当大麦生长在硝酸盐浓度较高的培养基中时，HATS活性显著降低，而大麦NRT 2（HvNRT 2）蛋白大量积累。这表明大麦中的HATS活性在HvNRT 2翻译后水平上受到调节。结果表明，大麦根可溶性组分可使HvNRT 2蛋白C-末端重组多肽磷酸化。这种磷酸化活性强烈依赖于Ca^&lt;2 +&gt;，并可被硝酸盐诱导<32>。通过使用抗HvNRT 2抗体的免疫沉淀方法从反应混合物中回收磷酸化的HvNRT 2蛋白。HvNRT 2的磷酸化也是Ca^&lt;2+&gt;依赖的和硝酸盐诱导的。2.大麦NRT 2蛋白的磷酸化证实了大麦CLC C-末端区域的重组多肽在体外被从大麦根中制备的可溶性组分磷酸化。这种磷酸化活性强烈依赖于Ca^&lt;2+&gt;，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.