拟南芥对二氧化氮胁迫应答中水杨酸的调节机理

Nitrogen dioxide (NO2), one of the main traf?c-related air pollutants and precursors of photochemical smog and ground-level ozone, is currently under intensive investigation. Although visible injury due to NO2 is very rare in the ?eld where NO2 is at a ppb level, growing evidence shows that ambient NO2 has caused reduction and deterioration in crop and vegetable yield and quality in some regions on the world, especially in greenhouses where NO2 at a ppm level may induce visible injury in some vegetables. In previous studies, we found that endogenous SA and its signaling were prerequisite to improve plant tolerance to NO2 exposure,and exogenous application of SA also protect plants against NO2 damage. However, our knowledge of SA-related molecular mechanisms responsible for plant tolerance to NO2 exposure still remains largely limited. To assess the molecular mechanism of endogenous SA protecting plants against NO2 stress, proteomic studies combined with temporal and spatial expression analyses of related genes involved in plant response to NO2 are performed on SA-altering Arabidopsis mutants including high SA accumulation, SA deficiency, SA signaling blockage, as well as their hybridization combinations. The total proteins from NO2-exposed plants and their NO2-unexposed parallels are separated by two-dimensional electrophoresis, and differentially expressed proteins are analyzed by matrix-assisted laser desorption ionization - time of flight - mass spectrometer (MALDI-TOF-MS) to obtain peptide mass fingerprints, then matched automatically to proteins in database. Furthermore, the phosphorylation of selected interest proteins is analyzed by MALDI-TOF-MS combined with phosphatase hydrolysis. In addition, the temporal and spatial expression of selected proteins with important biological functions is studied by real-time PCR. To our knowledge, there are no reports related to these studies so far. Based on these studies, we intend to reveal the biological processes involved in Arabidopsis plant response to NO2 exposure, and the mechanism by which SA regulates Arabidopsis plant response to NO2 exposure. Plans to solve the key scientific problems: ⑴ protein functions involved in plant responses to NO2 exposure and regulatory role of endogenous SA in that; ⑵ the regulatory role of SA in protein phosphorylation and its biological significance in plant response to NO2 stress; and ⑶ the effect of SA on temporal and spatial expression of related genes and its biological significance in plant response to NO2 stress. Expected outcomes: ⑴ obtaining several mutants with tolerant or sensitive phenotype under NO2 stress relative to wild-type; ⑵ clarifying the regulatory role of SA in plant response to NO2 exposure from proteomics; ⑶ publishing at least 4 papers in international SCI source journal; and ⑷ training 4 or more post-graduate master students.

二氧化氮（NO2）是主要的空气污染物之一，认识植物对NO2的应答机理具有重要的科学意义与指导生产实践的价值。我们前期研究显示，内源水杨酸（SA）水平及信号转导是拟南芥耐受高浓度NO2胁迫所必需的，然而，对其分子作用机理几乎一无所知。本项目以拟南芥SA高积累和缺失突变体、信号转导阻断突变体以及相互间的遗传组合为材料，通过双向凝胶电泳和基质辅助激光解析电离时间飞行质谱分析，并结合基因表达定量分析，阐明拟南芥对NO2胁迫应答的蛋白质生物学功能，揭示SA在拟南芥对NO2胁迫应答中的调节机理，为利用植物修复环境污染提供科学依据。拟解决的关键科学问题是：植物对NO2胁迫应答的蛋白质生物学功能以及SA的调节作用、SA对蛋白质磷酸化以及某些具有重要生物学功能的蛋白质时空表达特异性的调节及其在NO2胁迫应答中的作用。到目前为止，相关的研究尚未见报道。在SCI期刊发表不少于4篇论文,并培养4名以上硕士研究生。

二氧化氮是酸雨的主要成分之一，认识植物对NO2胁迫的应答机理具有重要的科学意义与实践价值。本课题针对水杨酸、一氧化氮以及水杨酸与乙烯之间的相互作用在拟南芥植株对NO2胁迫应答中的调节机制开展了研究，从植株的形态表型、生理生化代谢、蛋白组学以及特异基因表达等层面解析了上述信号分子的作用机理。获得了以下的重要结果与关键数据。（1）内源水杨酸高积累促进植物对NO2胁迫的耐受性，但通过水杨酸羟化酶转基因在降低植株内源水杨酸的同时也逆转了其耐受性的表型；（2）内源水杨酸缺失的植株表现出较野生型更加敏感的表型；（3）拟南芥植株对NO2胁迫的应答以依赖于水杨酸信号转导和不依赖于水杨酸信号转导的方式进行；（4）水杨酸信号转导与乙烯信号转导在拟南芥对NO2胁迫应答过程中具有交叉对话作用，具体表现为二者的重要成员NPR1-1和EIN2-1的生物学功能具有重叠性；（5）在NO2暴露前一天进行一氧化氮预处理显著提高所有供试基因型（包括耐受或敏感基因型）对接下来NO2暴露的耐受性；（6）蛋白组学研究显示，在对NO2暴露应答过程中，各个供试基因型的蛋白质表达存在明显的差异；（7）综合来讲，这些差异表达的蛋白质参与以下主要的生物学过程：细胞分裂、能量代谢、细胞壁修饰、细胞程序性死亡、氧化还原动态平衡、RNA加工、硝酸盐运输与代谢、光合作用、呼吸作用、磷酸化与去磷酸化、信号转导、物质运输、防卫反应等；（8）分析了参与NO2胁迫应答的特异基因表达水平，包括硝酸盐还原酶同工酶基因NIA1和NIA2、硝酸盐转运体基因NRT1-1和NRT2-1、抗氧化防卫反应关键基因CuZnSOD和FeSOD以及水杨酸应答的标志性基因病程相关蛋白编码基因PR1。耐受基因型的上述基因表达水平明显高于野生型水平，而敏感基因型则低于野生型。一氧化氮预处理提高了上述基因的表达水平，尤其是抗氧化相关基因。已在相关SCI源刊以本课题为第一标注发表6篇论文。上述研究为认识水杨酸、一氧化氮以及水杨酸与乙烯信号转导相互作用在植物对NO2胁迫应答中的调节机理积累了资料。