NRT1.1磷酸化修饰调控植物侧根发育的分子细胞学机制研究

Nitrate, the most important nitrogen form in soils, is an essential nitrogen source for higher plants. Plants are able to modulate root development allowing them to cope with nitrate fluctuation in the soil and maintain growth despite changes in external nitrogen availability. NRT1.1, a dual affinity nitrate transporter/sensor in Arabidopsis, could affect lateral root development through its nitrate-dependent auxin transport activity and its nitrate-regulated downstream signaling transduction under uneven nitrate distribution. It is well known the phosphorylation of NRT1.1 at T101 is critical for the functional switch of nitrate transport modes and different nitrate response levels. However, the role of phosphorylation of NRT1.1 at T101 in its regulation of Arabidopsis lateral root development remains to be elucidated. .Recently, we found that the phosphorylation of NRT1.1 at T101 was important for its nitrate-dependent auxin transport activity and its nitrate-regulated downstream signaling transduction，thereby functioning in Arabidopsis lateral root development at whole plant level. To further understand the molecular cytology regulatory mechanism of NRT1.1 phosphorylation in nitrate-regulated lateral root development, we plan to investigate the effect of NRT1.1 phosphorylationon on its endocytic trafficking and dynamics behavior in lateral root cell combining VA-TIRFM, FCCS, FLIM-FRET, LSCM and biochemical techniques: (1) the endocytosis extent of different phosphorylated forms of NRT1.1; (2) the intracellular target, endotytosis pathway and endocytic route of different phosphorylated forms of NRT1.1; (3) the effect of unphosphorylated NRT1.1 endocytosis on nitrate-regulated signal transduction; (4) the spatial distribution, oligomeric states and movement of different phosphorylated forms of NRT1.1 on the plasma membrane. Our results would provide new insight into the role of NRT1.1 phosphorylation on its nitrate-dependent auxin transport activity and its nitrate-regulated downstream signaling transduction involved in Arabidopsis lateral root development. Thus, this concept can help to understand the crosstalk between nutrition and development of plants, and can be used to design plant for sustainable agriculture.

不同浓度硝酸根环境中，硝酸根转运和感受蛋白NRT1.1可通过转运生长素和调控下游信号转导等方式调节侧根发育。T101磷酸化可调控NRT1.1转运和感受硝酸根的能力，但其磷酸化是否影响NRT1.1调控的侧根发育及机制尚不明确。.前期工作表明：T101磷酸化同样参与NRT1.1转运生长素和调控下游信号转导进而影响侧根发育的过程。本项目将利用多种新型光学成像技术，辅以生化和分子生物学手段，展开以下研究：（1）分析磷酸化与NRT1.1在侧根细胞胞吞程度的相关性；（2）探究磷酸化对NRT1.1在侧根细胞的胞吞途径和去向的影响机制；（3）明确去磷酸化NRT1.1的胞吞对其介导的下游信号转导的调控作用；（4）比较不同磷酸化状态NRT1.1在侧根细胞质膜上的分布、寡聚和运动规律；从而阐释磷酸化调控的NRT1.1胞吞循环和膜上动态参与其介导的生长素转运和下游信号转导进而调控硝酸根影响的侧根发育的作用机理。

本论文利用多种新型光学成像技术和数据分析方法，辅以恰当的生化和分子生物学手段，对不同磷酸化状态NRT1.1蛋白调控拟南芥侧根生长发育的作用机制进行了研究。研究结果表明：在低浓度硝酸根环境中，少部分磷酸化NRT1.1蛋白通过Clathrin介导的胞吞途径胞吞进入细胞，大部分磷酸化的NRT1.1蛋白驻留在膜上，其中多数以单体形式定位在膜筏微区，高效转运生长素；而在高浓度硝酸根环境中，大部分去磷酸化的NRT1.1蛋白经Clathrin介导的胞吞途径和膜筏微区介导的胞吞途径胞吞进入细胞，并经晚期内含体进入液泡降解，其胞吞可激活ANR1基因的表达，少部分去磷酸化的NRT1.1蛋白驻留在膜上，其中多数以二聚体形式定位在非筏区，转运生长素效率较低。上述研究结果加深了对NRT1.1蛋白介导的硝酸根调控拟南芥侧根发育分子机制的理解，拓展了对硝态氮影响植物根系形态建成的认识。