水杨酸调控植物对镉适应的分子机理研究

Cadmium is one of the main environmental pollutants. Because of its relative mobility, persistence and wide distribution in the environment, the removal of Cd contaminants is always a hard task. Recent years, phytoremediation has been intensely investigated as a Cd contaminant restoration strategy. However, some important scientific problems are still remained to be understood, such as the material basis of plant adaption to Cd exposure. A large number of studies have demonstrated that salicylic acid (SA) plays an important role in plant adaptation to Cd by pleiotropic functions, but the molecular mechanisms of SA action need to be further evaluated. As a signal molecule, SA functions biologically in both direct and indirect patterns. The latter is embodied by complex interaction either synergistically or antagonistically with other phytohormones, which constitutes the signal network of SA. Additionally, increasing evidence has shown that a time effect is existed in SA-mediated plant adaptation to adverse stresses. Based on these properties, we speculate that the SA-mediated plant adaptation to Cd may be involved in complex biological processes, wherein a cross talk may be existed. In addition, the biological functions of SA may be dynamically changed depending on the Cd exposure time. In order to reveal these, we apply the project. Due to the significant variation of basal levels of endogenous SA in different species of plants, it becomes difficult to evaluate the role of SA in plant response to Cd by means of application of SA. However, it can be overcome by using SA-related mutants. Therefore, we will employ SA-altering Arabidopsis genotypes including high accumulation of SA such as snc1 and ssi2, constitutive deficiency of SA such as sid2, eds1 and p35S::nahG, SA signaling blockage such as npr1-1, as well as their hybridization combinants with complementation genetically or biochemically. Because proteomic method has been extensively applied to dissect complex biological phenomena, it will be used as a major technology in this project. Additionally, to analyze the regulatory role of SA in some pivotal genes expression during exposure to Cd, real-time PCR will be performed. Briefly, total proteins are separated by two-dimensional electrophoresis, and differences of protein expression abundance are identified by related software. The differentially expressed proteins are further analyzed by matrix-assisted laser desorption ionization - time of flight - mass spectrometer to obtain peptide mass fingerprints, then matched automatically to proteins in database. Based on the results obtained by proteomic study, some pivotal genes involved in plant response to Cd are scored and dissected by RT-PCR to reveal their temporal and spatial expression pattern. By these studies, we expect to reveal SA-related biological processes and their cross talk, and to provide a scientific basis to improve phytoremediation efficiency of Cd pollution soil.

外源水杨酸可显著提高植物的镉适应能力，但对其调控的分子机理尚不清楚。水杨酸的生理功能具有多效性和时效性，在植物自身防御体系中，水杨酸与其它信号物质构成协同或拮抗的信号转导网络。据此我们推测，水杨酸调控植物的镉适应机理涉及复杂的生物学过程及其相互之间的交叉对话（cross talk）。为了揭示之，本项目以多种拟南芥内源水杨酸组成型高积累突变体植株、组成型缺失突变体（或转基因）植株、信号转导阻断突变体以及它们之间遗传或生化互补的杂交组合为材料，采用蛋白组学方法（双向凝胶电泳和生物质谱）和基因表达定量检测技术（以real-time PCR为主），分析镉暴露期间蛋白质表达丰度的动态变化、差异表达蛋白质的生物学功能、关键基因的时空表达模式以及与生理生化代谢的相关性。研究结果有助于揭示水杨酸提高植物镉适应能力的分子调节机理，为通过生物技术手段提高镉污染土壤的植物修复效率提供理论依据。相关研究未见报道。

重金属Cd的植物毒性和植物对Cd的应答机制体现在植物代谢的多个方面。由于植物对Cd胁迫应答是一个复杂的生物学过程，对其机理仍缺乏足够认识。尽管水杨酸（SA）在植物对Cd胁迫应答中的调节作用已有许多报道，但其作用机理仍需要深入研究。基于此，本项目重点探讨了SA参与植物对Cd胁迫应答的可能机理，同时强调了与其它激素的交谈机制。实验材料包括：SA高积累突变体ssi和snc1、缺失突变体sid2和nahG、信号转导不敏感突变体npr1、双突变体snc1/nahG和ssi2/nahG、乙烯信号转导不敏感突变体ein2，以及双突变体npr1/ein2。主要研究内容为：（1）内源SA水平和信号转导修饰的拟南芥突变体对Cd胁迫的差异性应答；（2）突变体之间、Cd处理与对照之间的蛋白组学研究；（3）差异表达蛋白质的生物学功能；（4）具有重要生物学功能的蛋白质表达模式；（5）揭示SA和/或乙烯在植物对Cd胁迫应答过程中的调节机制。通过生理生化、蛋白组学和基因表达研究表明，SA或乙烯参与植物对Cd胁迫应答的主要生物学过程有：（1）根激素合成、信号转导及其激素之间的交叉对话。其中SA高积累增强Cd的植物毒性，而降低内源SA水平提高植物对Cd的耐受性。生长素、茉莉酸和脱落酸可能促进植物的Cd耐受性；（2）乙烯信号转导缺失突变增强了植物对Cd的耐受性，但依赖于SA信号转导的作用；（3）类黄酮生物合成与基因表达调控起重要作用；（4）细胞壁甲酯化起重要调节作用；（5）蔗糖降解和转运起正调节作用；（6）硝酸盐代谢起正调节作用；（7）转录因子和激酶参Cd胁迫应答；（8）泛素介导的蛋白质降解参与Cd胁迫应答；（9）光合作用过程参与Cd胁迫应答；（10）抗氧化防卫反应在Cd胁迫应答中起重要调节作用。上述发现有助于深入了解SA对Cd胁迫应答的调节机制，并为进一步深入研究奠定了基础，因此具有一定的理论意义和学术价值。