茶树叶片液泡对氟离子区隔化及生理适应性机理研究

Tea (Camellia sinensis (L.) O. Kuntz) is a hyper-accumulator of fluoride, while the mechanism is still not entirely clear. Compartmentalization of harmful substances into vacuoles is the important physiological function for plant vacuole, of which is also the key organ to study abiotic stress and tolerance for plant. In the preliminary study, the project team has found that tea leaves could compartmentalize fluoride into vacuoles. Therefore, vacuolar compartmentation is most likely to be one of the most important mechanisms of fluoride tolerance for tea plant. On this basis, in order to analyze the key role of leaf vacuoles in the mechanism of fluoride tolerance in tea plant, the project intends to design different concentrations of fluoride stress and recovery experiments to study the characteristics of vacuolar compartmentation to fluoride using the osmoticlysis method. Then, approaches of ionomic (ICP-OES/ICP-MS) and metabolomic (UPLC-MS/GC-MS/LC-QQQ-MS) of vacuoles contained high fluoride will be carried out to explore the mechanisms of steady network regulation, and driving role of proton pump on tonoplast membrane (V-ATPase and V-PPase) in regulation will also be ascertained, so as to clarify the mechanisms of physiological adaptation of fluoride hyper-accumulation in the subcellular level. The findings will provide a positive significance for mechanism of fluoride tolerance and its physiological ecology for tea plant.

茶树是氟的超积累植物，其耐受机理尚不明确。植物液泡具有区隔化有害物质的重要生理功能，是研究植物非生物胁迫和耐受性的关键器官。项目组前期研究发现，茶树叶片可以将氟离子区隔化到液泡中。因此，液泡区隔化极有可能是茶树耐氟的重要机理之一。本项目拟在上述基础上，通过外源添加不同浓度氟胁迫和恢复实验，采用经典的渗透裂解法分离茶树叶片液泡，以叶片原生质体和液泡为研究对象，研究叶片液泡对氟离子的区隔化特征，剖析叶片液泡在茶树耐氟机理中的关键作用；随后采用离子组学（ICP-OES/ICP-MS）和代谢组学（UPLC-MS/GC-MS/LC-QQQ-MS）的分析手段阐明叶片高氟液泡的稳态平衡网络调控机理，并探明液泡膜上的质子泵（V-ATPase和V-PPase）在稳态调控中的驱动作用，从而在亚细胞水平上揭示茶树超积累氟的生理适应性调控机理。本项目研究结果对于解析茶树耐氟机理及生理生态均具有积极的意义。

茶树是氟的超积累植物，叶片是其主要的富集部位，原生质体（液泡）区隔化氟离子是茶树耐氟的重要机理之一，但是茶树叶片高氟的稳态平衡网络调控机理尚不明确。本项目采用渗透裂解法建立了高效分离茶树不同组织器官（根、茎、嫩叶和老叶）原生质体的分离纯化方法（数量>1000000个/g，活力>90%）;采用离子组学（ICP-MS）和代谢组学（LC-MS/MS，GC-MS）的分析手段阐明叶片高氟的稳态平衡网络调控机理，鉴定出主要的代谢化合物并分析出参与氟胁迫调控的代谢途径，发现小分子多肽在氟胁迫调控中具有重要的作用，筛选出特征性的挥发性代谢产物和响应离子（Na，Mo，Mn，Fe和Al等）。通过离子组学和代谢组学构建了茶树叶片响应氟胁迫的代谢调控网络，初步揭示了茶树超积累氟的生理适应性机理。项目的相关研究成果进一步丰富了茶树耐氟机理及生理生态，此外，茶树原生质体分离纯化方法的研究对于茶树遗传转化研究具有重要的意义。