茶树叶片质膜H+-ATPase CsA1和CsA7在干旱胁迫与复水处理下调控钾稳态的功能研究

Drought stress seriously limits the growth and development of tea plants, thus affecting tea yield and quality. Illustrating the intrinsic mechanisms of drought resistance is beneficial for breeding the drought-resistant tea plant to cope with drought stress. This study was focused on the youngest first tea leaf which is the most important tissues for tea industry. Tea plants were treated by PEG and rewatering to mimic drought and rehydration condition. We found that drought stress inhibited plasma membrane H+-ATPase activities and induced net H+ influx, which led to membrane potential depolarization and induced a massive of K+ efflux/loss in tea mesophyll cells. Rehydration treatment up-regulated plasma membrane H+-ATPase activities and induced net H+ efflux, leading to membrane potential hyperpolarization and thus reducing K+ loss. In addition, the expression level of plasma membrane H+-ATPase protein and the transcriptional level of CsA1 and CsA7 were consistent with the change of plasma membrane H+-ATPase activities in the first leaf under drought stress and rehydration treatment. We hypothesized that CsA1 and CsA7 might be of importance for regulating potassium homeostasis in tea mosephyll cells in response to drought stress and rehydration treatment. In addition, homologous genes associated with CsA7 in tea plants were not explored in previous studies focusing on drought resistance genes. Therefore, to further confirm the functions of CsA1 and CsA7 in drought stress and rehydration treatment, CsA1 and CsA7 overexpressing lines of tobacco will be constructed and tested. Besides, the possible interaction between 14-3-3 protein and plasma membrane H+-ATPase CsA1 and CsA7 will be investigated by yeast two-hybrid and bimolecular fluorescence complementation techniques. Taken together, our study will be useful for revealing the molecular mechanism of the role of plasma membrane H+-ATPase CsA1 and CsA7 on regulating potassium homeostasis in tea plants under drought stress and rehydration treatment. Our work will be potentially beneficial for screening and breeding drought stress tolerant tea varieties.

旱害严重影响茶树生长发育，造成茶叶减产和品质下降。揭示茶树抗旱机理，对培育耐旱茶树品种，应对干旱胁迫具有重要的理论意义。申请人以茶树一叶为研究对象，通过PEG和复水模拟干旱和补水灌溉。在干旱胁迫和复水处理下，茶树叶片质膜H+-ATPase介导膜电位调控叶片钾含量，表现出先减少再升高的趋势；叶片质膜H+-ATPase CsA1 和CsA7的转录和蛋白表达水平也呈现出相同的趋势，推测二者可能参与调控茶树叶片钾稳态对干旱和复水响应。前人研究其它植物耐旱机制时并未报道与茶树CsA7相关的同源基因。项目拟通过烟草异源体系，揭示茶树叶片H+-ATPase CsA1和CsA7 在干旱胁迫和复水处理下调控钾稳态的生理功能；采用酵母双杂交和双分子荧光互补，分析与上游蛋白14-3-3的互作方式。研究结果将阐明CsA1和CsA7在茶树响应干旱胁迫和复水处理中的作用机制，为筛选和培育耐旱茶树品种提供候选基因。

茶树属多年生常绿经济作物，干旱对茶树产量和品质有着显著的负面影响，因此探究茶树抗旱机理具有重要的产业意义。本项目首先在安徽农业大学农翠园选择12种不同茶树品种，通过PEG模拟干旱实验，筛选耐旱型茶树品种台茶12和敏感型品种福云6号；通过检测茶树叶肉细胞K含量和K离子流，发现K滞留和叶片鲜重呈显著正相关和MDA呈显著负相关。同时，通过 RNA-seq 技术分析外向K通道和内向K通道的表达模式，明确钾在茶树抗旱中的作用。选择不同抗性的茶树品种，比较茶树叶肉细胞质膜H+-ATPase与叶肉细胞K滞留的关系，发现抗性品种在干旱胁迫下叶肉细胞质子泵活性显著高于敏感品种，且较高的质子泵活性维系较高的膜电位，进而保持着较高的K滞留能力。最后，通过构建转基因拟南芥，验证超表达质膜H+-ATPase可显著提高K滞留能力，进而提高茶树抗旱性。这些研究结果将揭示茶树叶肉细胞质膜H+-ATPase抵御干旱胁迫的的生理机制，为茶树抗旱提供候选基因提供理论基础。