拟南芥钙依赖型蛋白激酶CPK32响应低氮胁迫的分子机制研究

Nitrate is not only one of the main absorbed nitrogen nutritions in the soil, but also acts as an important signaling molecular for the plants. Nitrate, sensed promptly and specifically by the plant cells, regulates a set of genes governing growth and morphology of the plants through the complex nitrate signaling network. In the recent years, protein phosphorylation which provides a major regulatory mechanism has become a hot topic in nitrate signaling research. In the prior period of the project, T-DNA insertion lines corresponding to Arabidopsis thaliana calcium-dependent protein kinase (CPK) family genes were tested for their sensitivity to the low nitrate stress treatment. Compared to the wild type plants, the mutant cpk32 showed retarded leaf yellowing, more total nitrogen content, stronger root nitrate influx and more nitrate concentration storage in shoots, after transferring to the low concentration nitrate medium. Some published in-vitro experiments data have shown that the transcription factor ABRE binding factor 4 (ABF4) could be phosphated by CPK32. And in the prior experiments, the mutant abf4 showed similarly retarded leaf yellowing after transferring to the low concentration nitrate medium, indicating that ABF4 might act as the downstream target protein regulated by CPK32 in response to low nitrate stess. Therefore, this project will try to elucidate the mechanism of CPK32 in response to low nitrate stress through modulating the transcription factor ABF4 activity by phosphorylation from physiology, biochemistry and genetics.

硝酸根离子是土壤中植物吸收氮营养的主要形式之一，也是重要的信号分子。硝酸根离子能够迅速地、特异地被植物细胞所感知，通过硝酸根离子信号网络调控一系列基因表达，影响植物的生长和形态。作为一种重要的调控方式，蛋白质磷酸化修饰在硝酸根离子信号中的作用成为近年来的研究热点。本项目前期对拟南芥钙依赖型蛋白激酶家族CPKs基因T-DNA插入突变体进行了低氮胁迫表型观测，发现cpk32突变体在低氮条件下叶片发黄延迟、总氮含量更高、硝酸根离子内流速率更大，冠部硝酸根离子含量更高。已报道的体外实验数据显示转录因子ABF4能够被CPK32磷酸化。而本项目前期发现，在低氮培养基上abf4突变体出现和cpk32类似的叶片发黄延迟表型，推测ABF4可能作为CPK32的下游蛋白参与低氮胁迫信号转导。因而，本项目将从生理学、生物化学和遗传学的角度，深入研究CPK32通过磷酸化调控ABF4活性响应低氮胁迫的信号转导机制。

铵离子是土壤中植物吸收氮营养的主要形式之一。拟南芥从土壤中吸收铵主要依赖一类特异的铵转运体蛋白AMTs。这类蛋白属于Amt/Mep/Rh超家族，广泛存在于细菌、真菌和动植物中，负责铵吸收转运。AMTs能够在转录水平响应外界氮浓度变化，调节植物的铵吸收能力。蛋白质磷酸化修饰作为一种转录后水平调控方式，在植物铵吸收转运调控过程中也发挥重要作用。目前已有报道AMT蛋白C末端一个保守的苏氨酸残基的磷酸化与其转运活性的丧失密切相关。本项目利用反向遗传学的方法，发现了拟南芥钙依赖型蛋白激酶CPK32参与植株铵吸收的正向调控过程。并且系统全面地从生理表型和分子生化机理层面阐释了CPK32响应外界氮饥饿信号，通过磷酸化作用修饰下游AMT来调控其转运活性的分子机制。进一步，本项目对AMT受到CPK32磷酸化的作用位点进行了深入研究。通过生化生理实验证明了AMT蛋白C末端一个新的丝氨酸残基磷酸化位点，这个位点受到CPK32的磷酸化后能够正向调节AMT活性。该新位点与已报道的保守的苏氨酸残基都位于AMT蛋白C末端，但功能相反，揭示了植物体内磷酸化调节的复杂性。