干旱胁迫下NO对小麦幼苗D1蛋白磷酸化的调节作用

Photosynthesis is one of the most physiological processes to be very sensitive to abiotic stresses. D1 protein in PSII system is the most sensitive protein in response to abiotic stresses, and its phosphorylation has been reported to regulate the D1 turnover, which can enhance photosynthetic ability to abiotic stresses. Drought stress has become a key ecological factor to inhibit crop yields in the world. Our previous results indicated that drought destroyed the dynamic balance of D1 protein, however, exogenous application of nitric oxide (NO) remarkably increased the expression levels of D1 protein and enhanced photosynthetic capacity. Thus, it is speculated that this may be related to phosphorylation of D1 protein controlled by NO. To verify this speculation, in this study, NO donor and NO specific scavenger will be applied to analyze D1 phosphorylation expression and photosynthetic capacity to reveal whether NO improves photosynthetic capacity through the phosphorylation of D1 protein in drought stressed wheat seedlings. Moreover, biological mass spectrometry will be used to detect the phosphorylation sites of D1 protein in thylakoid membrane, and vitro test will be used to verify whether D1 protein is the substrates of STN8 by measuring thylakoid membrane protein phosphorylation. Finally, transcription and protein levels of STN8, and NO signal and its subcellular localization in plants treated with NO donor and NO specific scavenger will be also measured to explore whether STN8 participates in the regulation of D1 protein phosphorylation induced by NO under drought stress. The present study will help to further explore the molecular mechanism of NO for improving abiotic tolerance in higher plants.

光合作用是作物对环境变化很敏感的生理过程，D1是光系统II（PSII）中最敏感的一种蛋白，逆境条件下可通过磷酸化调节D1蛋白的周转，从而提高光合作用的适应性。申请人前期研究发现，干旱胁迫破坏了D1蛋白的动态平衡，外源一氧化氮（NO）能调节D1蛋白的合成，进而提高光合性能。由此推测NO提高逆境胁迫下植物的光合作用可能与其调节D1蛋白磷酸化有关。为了证明这一推测，本项目拟通过免疫印迹技术，测定NO供体处理后，干旱胁迫下小麦幼苗叶片内磷酸化D1 蛋白的表达和光合特性，并利用生物质谱技术，测定D1磷酸化的作用位点；其次，利用体外类囊体膜蛋白质磷酸化试验，确定STN8激酶直接作用底物是否为D1蛋白；最后，通过分析NO供体和NO专一清除剂处理后STN8的转录和蛋白水平的表达及NO信号的变化和定位，以明确NO对D1蛋白磷酸化的调控是否需要STN8的参与。通过本研究可进一步揭示NO提高植物抗逆性的分子机制

一氧化氮（NO）作为一种新型的植物生长调节物质，它在调控植物生长、发育与逆境胁迫等一系列代谢活动中发挥了重要功能，但以往有关其研究主要集中在生理机制方面，而有关其提高植物这些代谢活动的分子机制报道甚少。为了探究NO提高小麦植株抗旱性的分子机制，项目主持人利用相对和绝对定量同位素标记（iTRAQ）的磷酸化蛋白质组学方法，辨析了NO供体（硝普钠，SNP）处理后小麦叶片总蛋白丰度的变化及其相应的蛋白磷酸化位点。利用此方法，项目主持人辨析出148个蛋白的丰度发生了显著变化（>1.50, p <0.05），这些蛋白涉及了植物的光合作用、碳水化合物代谢、信号转导、胁迫防御等一系列代谢活动，其中涉及光合作用的磷酸化蛋白数量最多，以STN7/STN8激酶尤甚，表明NO提高小麦幼苗的抗旱性与光合作用蛋白尤其是STN7/STN8激酶的磷酸化密切相关。为了验证这一结果，项目主持人克隆了小麦NO合成关键酶基因（TaNOS），采用大麦条纹花叶病毒诱导的基因沉默方法（BSMV-VIGS），检测了BSMV-VIGS-TaNOS沉默植株在干旱胁迫后的表型、生理生化及STN7/STN8激酶表达量的变化，结果发现TaNOS基因沉默植株对干旱胁迫的敏感性显著高于对照，同时光合能力、蛋白激酶活性与TaSTN7和TaSTN8基因表达量均显著降低，这些结果负向验证了NO提高小麦的抗旱性与光合过程中STN7/STN8激酶的磷酸化密切相关。项目主持人进一步检测了田间应用NO对小麦抗旱性的影响，并开发出相关的专利产品。