重金属胁迫下NO和ROS互作调控盐生植物应答反应的作用机理研究

Heavy metal pollution has become one of the most important ecological problems on the world scale. Stress conditions are known to induce the rapid and increased generation of nitric oxide (NO) and reactive oxygen species (ROS), which may lead to oxidative damage and severely threaten cell health. On the other hand, NO or ROS as a signal molecule is involved in growth and developmental process as well as plant responses to abiotic stresses. Moreover, in some cases, the effects of NO are the result of its interaction with ROS. Although the formation of NO and ROS has been well documented in the experiments of different plant systems, scarce information has been available on the regulatory roles of endogenous NO and ROS interactions on plant responses to abiotic stresses. Plants commonly face complex and volatile environment, and the determination of plant adaptation to different stress conditions is important to ecological reconstruction. Limonium aureum (L.) Hill and Nitraria tangutorum Bobr. are typical desert halophytes with the strong ability to resist salinity, alkalinity, drought, wind and dust, and they can efficiently fix moving sands. Drought-tolerant Longchun 27 is a wheat species widely grown in the north-west of China. In this study, these species are used to evaluate phytotoxicity mechanism and plant adaptive mechanism to heavy metal stresses by comparing seed generation, seedling growth and antioxidative responses, and so on. The changes of enzyme activities, protein content and gene expression due to NO and ROS production will be comprehensively analyzed to reveal the relationship of these two molecular generation and eco-physiological characteristics, and thus to determine the regulation function of NO and ROS on plant responses to stress conditions, through a number of control experiments combined with protein electrophoresis, Western blotting and fluorescence microscopy. On the basis of above analyses, further study mainly aims to evaluate whether or not the generation of NO and ROS influenced each other in plants, and to explore endogenous signal molecules associated with NO and ROS actions, thus to reveal the regulation approach and mechanism of NO and ROS interactions on halophyte responses to heavy metal stress. These studies aim to reveal physiological and ecological adaption mechanism of plants to heavy metal stresses, and to provide scientific basis and theoretical guidance for further investigating the roles and the regulation mechanism of NO and ROS signaling as well as for carrying out species selection of veletation ecological recovering in saline-alkali and heavy metal contamination soil.

一氧化氮（NO）和活性氧（ROS）作为胁迫因子或信号分子均参与植物生长发育和生理生态特征的调控，但它们互作调控植物对重金属胁迫应答反应的作用机理目前仍不清楚。本项目拟以西北盐生植物补血草和耐盐小麦等为材料，室内控制试验条件下，比较不同植物种子萌发、幼苗生长、抗氧化反应、渗透性调节等生理生态特征，揭示重金属的植物毒害机理及植物的适应机制；利用酶抑制剂结合凝胶电泳、Western blotting和荧光技术等手段，综合分析NO和ROS的产生、相关酶活性及基因表达的变化与生理生态特征的相关性；在此基础上，重点研究NO和ROS产生的相互影响及其互作介导植物对重金属胁迫应答反应可能的调控途径及作用机制。这些研究旨在揭示盐生植物适应重金属环境的生理生态机制及NO和ROS互作调控植物胁迫应答反应的作用机制，为西北盐碱地、重金属污染区植被生态建设过程中物种的选择提供依据和指导。

重金属、高盐等环境因子严重影响着植物的生长发育，限制着生态建设和农业可持续发展。本研究在国家自然科学基金(NO 31470464)的资助下，选取西北大面积种植的小麦和具有生态价值的西北盐生植物黄花补血草等植物为材料，主要开展了重金属或盐胁迫下植物内源一氧化氮(NO)和活性氧(ROS)的产生及其互作对植物生理生态特性的调控等方面的工作，研究发现：(1) Zn胁迫对小麦根生长的抑制是过氧化氢(H2O2)和NO相互作用的结果，胞外ROS的减少和内源NO的增加可能导致了Zn + DPI处理对小麦根生长的显著抑制；(2) NO的积累对于外源抗坏血酸(AsA)诱导的Cd耐受性和内源性AsA的产生是需要的，NO信号传导途径介导了外源AsA诱导的内源性AsA产生，二者共同作用导致小麦对Cd胁迫的耐受性；(3)Zn可以缓解铁对小麦根系生长的抑制作用，这可能与ROS产生的减少、脂质过氧化的减弱和细胞活力的增加有关；(4)不同的锌、铁和铜复合胁迫对小麦幼苗锌、铁和铜元素的吸收及根系生长表现协同作用和显著的抑制作用，且Zn + Fe + Cu处理的抑制作用最明显；在不同的Zn、Fe和Cu复合处理下，每个抗氧化酶包括总的和质外体的超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)和谷胱甘肽还原酶(GR)表现出特异性反应，其中 GR、SOD和APX等作为重要保护机制来对抗氧化损伤；(5)Fe或Cu胁迫诱导小麦根羟自由基(•OH)积累，对不同的抗氧化酶产生不同的影响，多胺氧化酶(PAO)、二胺氧化酶(DAO)和细胞壁过氧化物酶(cw-POD)可能不参与Fe或Cu胁迫诱导的ROS积累；(6)小麦幼苗根叶中内源性NO和ROS的产生相互影响，且在NO和ROS互作下小麦根叶表现出不同的抗氧化反应；盐诱导的小麦叶胞间H2O2的积累和NADPH氧化酶O2•¯的产生对总H2O2和O2•¯的积累以及DAO和PAO活性具有不同的调控作用；不同的盐浓度使小麦幼苗对Na、K和Ca吸收发生了变化，盐胁迫促进小麦根NO及质外体ROS的积累, 它们之间互作调控植物在盐胁迫响应过程中根对Na、K和Ca的吸收及其向地上部分的转运。