杨树响应对羟基苯甲酸胁迫中的氧化还原调控机制解析

The declined land capacity of continuous cropping plantation is a worldwide problem, which seriously affects the sustainable management of plantation. Our previous study revealed that p-hydroxybenzoic acid (pHBA), a principal component of allelochemicals in populus, is an important factor that leads to the decline of the productivity of continuous cropping populus, and the redox regulations play important roles in pHBA response in poplar. However, the redox regulation mechanism in response to pHBA is still unclear so far. In this study, Populus trichocarpa is to be used as material, and a recently developed high-throughput quantitative redox proteomics approach termed OxiTRAQ and a combined GC/MS and LC/MS/MS metabolomic platform, combining with the analyses of plant physiological state and the metabolism of active oxygen, will be applied for identifying the variant proteins whose thiols undergo reversible oxidative modifications and the variant metabolisms in Populus subjected to pHBA stress and oxidative stress. In addition, a combined analysis of redox proteomic, metabolomic, and our previous transcriptomic data will be performed to systematic analyse the mechanism and network of redox regulation under pHBA stress. Furthermore, functions of some key redox-sensitive proteins will be verified by transferring to populus using point mutation and CRISPR/Cas9 technique. These studies will reveal the role of redox regulation mechanism in pHBA stress response in Populus. In this study, the mechanism of populus response to pHBA stress will be discussed from systems biology research, especially the post-translational modification level, and these will offer new detailed information and clues underlying the complex physiological and ecological mechanism of productivity decline in continuous cropping plantation.

连作人工林地力衰退是一个世界性的问题，严重影响人工林可持续经营。本课题组前期研究发现，对羟基苯甲酸（pHBA）是导致连作杨树生产力下降的重要因素，且氧化还原调控在杨树响应pHBA胁迫过程中起重要作用，但其调控机制尚不清楚。本项目拟以毛果杨（Populus trichocarpa）为材料，利用氧化还原蛋白质组学技术与代谢组学技术，结合对植株生理状态和活性氧代谢的分析，探讨杨树响应pHBA胁迫和氧化胁迫过程中可逆氧化还原敏感蛋白质表达丰度与修饰状态和代谢物的变化，并对氧化还原蛋白质组、代谢组以及我们已有的转录组数据进行联合分析，系统分析pHBA胁迫下的氧化还原调控机制和网络；进一步对重要的氧化还原敏感蛋白进行功能验证，深入探讨氧化还原调控在杨树响应pHBA胁迫中的作用。应用这些系统生物学方法探讨杨树响应pHBA胁迫的机制，将为阐明连作人工林生产力衰退的生理生态学机制提供证据与线索。

杨树连作人工林大量积累pHBA是导致其生产力下降的重要因素之一，并且活性氧清除/平衡机制以及氧化还原稳态在杨树响应pHBA胁迫中起重要的作用，然而，植物在响应pHBA胁迫条件下的氧化还原调控机制目前仍然是空白。本项目以84K杨（Populus alba × Populus glandulosa ‘84K’）为实验材料，利用蛋白质组学、氧化还原蛋白质组学与代谢组学技术，结合对植株生理和活性氧代谢的分析，探讨了杨树响应pHBA胁迫和H2O2胁迫中可逆氧化还原敏感蛋白质表达丰度与修饰状态和代谢物的变化，系统分析了pHBA胁迫下的氧化还原调控机制和网络。（1）通过光合生理生化分析，pHBA和H2O2处理后，野生型杨树的光合作用能力显著降低，植物体内出现氧化胁迫和渗透胁迫，并且明确了pHBA和H2O2处理对杨树幼苗生理生化指标有相似影响的浓度和时间点。（2）采用定量蛋白质组学技术分析鉴定了杨树响应pHBA和H2O2的差异表达蛋白，获得杨树叶片响应pHBA胁迫的蛋白质组模型，并推断杨树的pHBA响应机制比H2O2响应机制更复杂。（3）基于iodoTMT标记的氧化还原蛋白质组学数据，结合之前的定量蛋白质组学分析结果，构建了杨树响应pHBA和H2O2诱导的内源氧化应激的氧化还原调控网络。在pHBA和H2O2胁迫诱导的氧化应激中，Cys位点的可逆氧化作用在杨树酶活性和其他蛋白质稳定性的调节开关中起主要作用。（4）基于广靶代谢组学技术分析了鉴定了杨树响应pHBA胁迫的差异代谢物。（5）由于pHBA和H2O2胁迫会触发MAPK信号通路相关蛋白的丰度和氧化还原态发生变化，分析了过量表达PeMPK15基因的转基因杨树在pHBA胁迫条件下的光合生理特性，明确了该基因在pHBA胁迫条件下具有较高的维持氧化还原稳态的能力；进一步通过广靶代谢组学技术分析了过量表达PeMPK15基因的转基因杨树在pHBA胁迫条件下的代谢组学特征，为将来开展PeMPK15基因参与pHBA胁迫条件下杨树的氧化还原调控机制研究提供了基础数据。研究成果对深入理解连作人工林地力衰退机理具有重要意义，同时，对于通过遗传育种途径提高杨树连作人工林长期生产力提供重要的分子生物学基础。