Ca2+、H2O2信号转导参与亚精胺调控白三叶抗旱性机制研究

White clover (Trifolium repens L.) is currently worldwide planted as excellent legume herbage and it is also important for landscape restoration and ecological construction. However, drought has become a dominant restrictive factor for white clover production. Our preliminary studies showed that exogenous spermidine could improve drought tolerance of white clover and Ca2+ might have played an important role in signal regulation. To further such studies, this project will integrate an method of pharmacology experiment, confocal laser scanning microscopy, and high performance liquid chromatography with analyzing enzyme activities and mRNA transcription level so as to reveal the function of spermidine involved in regulating drought tolerance of white clover. The main contents are included in: (1) the pathways by which Ca2+, H2O2 signal are activated and transduced throught protein reversible phosphorylation; (2) specific genes expression for antioxidative enzyme induced by signal transduction; (3) Antioxidative enzyme activities and polyamine metabolism; (4) identifying potential feedback and synergistic reaction among Ca2+, H2O2 and protein reversible phosphorylation; and (5) analyzing the relationship among signal transduction induced by spermidine, polyamine metabolism and oxidative stress. The study results will help to reveal the relationship between polyamine and abiotic stress signal transduction in plants, to further understanding the molecular mechanism of white clover in response to drought stress and to provide a theoretical basis to enrich cultivation measures of white clover under drought stress.

白三叶为世界范围内广泛栽培的优良豆科牧草、重要的园林绿化和生态建设用草种，干旱是白三叶生产应用中的主要制约因子。我们初步研究发现，适宜浓度的外源亚精胺能明显提高白三叶抗旱性，并且Ca2+信号可能参与了调控作用。在此基础上，本项目利用药理学实验、通过激光共聚焦显微及高效液相色谱检测技术、结合酶活性和mRNA转录水平分析方法，研究亚精胺在调控白三叶抗旱性中：（1）诱发Ca2+、H2O2信号产生的来源及通过蛋白可逆磷酸化传递的途径；（2）诱导抗氧化酶基因的特异性表达；（3）对抗氧化酶活性及内源多胺含量的调节；（4）探讨Ca2+、H2O2与蛋白可逆磷酸化在调控白三叶抗旱性中可能存在的反馈或协同作用机制；（5）分析亚精胺诱导的信号传导与多胺代谢及抗氧化胁迫的相互关系。研究结果对了解多胺参与植物逆境信号转导机制及深入理解白三叶应答干旱胁迫的分子机理具有重要意义，并为丰富白三叶抗旱栽培措施提供理论依据。

受全球气候变化的影响，我国大部分地区的干旱形势日趋严峻，农作物干旱受灾面积逐年增加。白三叶是国内外建植人工草地和园林绿化的优良豆草，但在生产栽培中急需解决抗旱差这一严重制约因素。其中，有效施用外源植物生长调节剂是提高作物抗逆性的重要途径之一。多胺是一种对多种逆境胁迫具有积极调节作用的生理活性物质，但在调控植物对逆境适应性中的信号转导机制仍不清楚。本研究以广泛栽培应用的‘拉丁诺’白三叶为试材，采用药理学实验、激光扫描共聚焦显微技术、高效液相色谱方法结合酶活性和mRNA转录水平分析，研究外源亚精胺调控白三叶抗旱性中，如何诱发Ca2+、H2O2信号产生及通过蛋白质可逆磷酸化传递的途径，Ca2+、H2O2信号转导对干旱胁迫下白三叶抗氧化保护的影响以及它们在调控白三叶抗旱性中的相互关系。.主要研究结果包括：（1）在干旱胁迫下，外源亚精胺显著上调白三叶叶片SOD、CAT和POD等抗氧化酶基因的表达及其酶活，并有效促进了抗坏血酸-谷光甘肽循环代谢，从而提高白三叶的抗氧化能力；（2）添加了亚精胺的白三叶表现出积累更多的可溶性糖、甜菜碱和脯氨酸等有机渗透调节物质；（3）白三叶内源多胺水平的调节显著影响与核糖体结构和功能、氨基酸代谢、糖代谢、抗氧化防御及逆境响应相关蛋白的表达；（4）亚精胺能够分别激活Ca2+、H2O2多条信号产生途径，诱导信号产生，且这两种信号以互作的方式通过钙调蛋白和钙依赖蛋白激酶进行信号传递和信号级联放大，从而激活下游的抗氧化基因表达及其酶活；（5）外源多胺可提高内源多胺水平，变动内源CTK、GA、IAA等植物激素含量，并调节响应GA、ABA蛋白的表达，表明多胺与其它植物激素互作调控白三叶对干旱胁迫的耐受性。这些研究结果为深入开展多胺调节植物抗逆性的分子机制研究奠定了重要基础；同时亦揭示了白三叶通过合成更多的多胺以应对干旱胁迫这一适应性机制，为将多胺应用于白三叶的抗旱栽培中提供了理论依据和实践技术。