铜胁迫下NO介导番茄植物螯合肽的解毒机制研究

Copper is an essential micronutrient for plant growth, also as a heavy metal element. Phytochelatins (PCs) are the main thiol polypeptides rich in homocysteine, have the unique detoxification function to heavy metal. During the past researches we found exogenous nitric oxide significantly enhenced the tolerance of tomato exposed to copper stress, and the V-H+-ATPase and V-H+-PPase located in tonoplast and Cu-PCs complex reacted sensitively, while still unknown to detoxification pathway mediated by nitric oxide. Here, the nutrient solution will be used to cultivate tomato seedlings under copper stress, and sodium nitroprusside (SNP) provides exogenous nitric oxide. The key enzymes PCS, GSHS and γ-ECS participating the synthetic pathway of PCs and the corresponding substrates and the middle products are going to be analysized, which could manifest key reaction or step of PCs synthesis mediated by nitric oxide under copper stress; Mark copper ions with specific fluorescence probe in nutrient solution, the dynamic change of Cu-PCs complex inside and outside tonoplast will be detected real-time through laser confocal microscope, combining with the the dynamic analysis of V-H+-ATPase and V-H+-PPase in tonoplast, the corresponding relations between the distribution characteristics of Cu-PCs complex in cell and the master enzyme in tonoplast will be discussed; To take DAF-FMDA as fluorescent probes for nitric oxide, the dynamic change of nitric oxide in organs can be determined through laser confocal microscope, combining with the related analysis of proline, NH4+,glutaminc acid, arginine,polyamine and so on, we expect to find out the possible sources or metabolic pathway closely related PCs or GSH mediated by nitric oxide. From the synthetizing, chelating and spatial orientation of PCs pathway in cell,tissue and organ, the detoxification mechanism of PCs pathway mediated by nitric oxide under copper stress will be promising to be revealed soon.

铜是植物生长发育必需的营养元素，也是重金属元素。植物螯合肽（PCs）是一类富含半胱氨酸的小分子多肽，具有独特的重金属解毒功能。研究发现外源NO能够显著增强番茄耐铜性，液泡膜H+-ATPase、H+-PPase和Cu络合物响应剧烈，而对其解毒机制尚缺乏研究。本项目拟采用营养液培养番茄模拟铜胁迫，硝普钠提供外源NO，检测PCs合成途径的限速酶PCS、GSHS、γ-ECS活性及相应底物、产物的动态变化，并分析其与耐铜性的关系，探讨NO介导的耐铜机制中PCs途径的关键响应因子；以罗丹明特异荧光探针标记铜，激光共聚焦显微镜监测液泡内外Cu-PCs的动态变化，结合液泡膜ATPase、PPase活性分析，探讨Cu-PCs微域分布特性与液泡膜功能蛋白的关系；检测NO的微域分布特性及脯氨酸、NH4+、多胺等动态变化，综合PCs代谢和空间定位的系统分析，可望探明铜胁迫下NO介导PCs的解毒机制。

铜是植物必需微量元素，也是重金属元素。镉是生物毒性最强的重金属。植物螯合肽（PCs）,谷胱甘肽（GSH），金属硫蛋白（MT），抗坏血酸（AsA）是重金属的重要解毒途径。健康土壤里，植物体内此类代谢物含量较低，外界逆境刺激能诱导其高效合成。番茄是广泛食用的蔬菜，在城市郊区、设施栽培中常遇到不同程度的重金属胁迫，研究外源物质介导的解毒途径，对于指导污染土壤进行生产及利用常规作物进行生物修复具有重要的理论和实践意义。本项目利用营养液水培模式，研究铜、镉单一胁迫或复合胁迫下，番茄植株在器官、细胞、离子形态、代谢途径等方面对外源NO的响应。结果如下：.（1）优化根系构型和超微结构 外施NO可显著增加Cu胁迫下番茄植株的生物量、株高和茎粗，提高根系活力，改善根系总长度、根平均直径、根总表面积和根总体积，维持其组织结构的稳定。.（2）离子区隔化 外源NO增强Cu胁迫下番茄液泡的隔离和细胞壁的固持，细胞器Cu含量降低，减轻过多Cu对胞质的伤害；.（3）外源NO协调离子平衡，降低Cu的生物毒性 外源NO调控番茄幼苗各器官细胞及亚细胞Fe、Zn、Mn的合理分布，提高根系固定态含量，降低水溶态铜含量与比例，以减轻铜的生物毒性。.（4）根系分泌物的响应 外源NO能显著提高番茄幼苗根系和叶片植物螯合肽、叶片氨基酸、根系分泌物（果糖、葡萄糖和蔗糖）含量；降低根系氨基酸、有机酸（草酸、苹果酸、柠檬酸和琥珀酸）的分泌。.（5）AsA-GSH-PCs解毒途径 外源NO介导Cu胁迫下番茄AsA-GSH循环，并通过调节AsA/DHA、GSH/GSSH含量和比值的变化减轻氧化胁迫，外源NO可能启动了某些信号机制，通过激活或增强GSH-PCs合成途径中酶促和非酶促系统，使GSH-PCS代谢向PCs方向进行。.（6）精氨酸-NO代谢途径 Cu胁迫下外源NO调节番茄根系和叶片精氨酸脱羧酶、鸟氨酸脱羧酶及一氧化氮合酶活性，使L-精氨酸代谢向着NO合成方向进行；Cu胁迫下外源NO可促进L-精氨酸合成，而作为多胺、NO的合成前体，精氨酸含量升高会间接促进PA、NO合成，从而提高番茄Cu耐性。.（7）铜、镉互作效应 外源NO促进MT合成，复合胁迫下，外源NO可能启动了叶片对胁迫的应答机制，在缓解胁迫中起主导作用。番茄对Cu的耐性显著高于Cd。Cu+Cd复合胁迫下，Cu2+可部分替代Cd2+，以降低Cd2+的生物毒性。