拟南芥Ca2+-ATPase ACA8基因响应低温胁迫的分子机制

Cold stress is one of the most significant abiotic stresses of plants, affecting both plant growth and yield potential of agricultural crops. The researches on the mechanism of plants in response to cold stress have important scientific significance and application value. Ca2+ signalling is essential for plant responses to low temperature. Influx mechanisms have received considerable attention because their regulation is of primary importance to initiate a Ca2+ signal. By contrast, less consideration has been given to the role of efflux systems in Ca2+ signalling. In previous research, we found that the expression of ACA8 was quickly responded to low temperature. ACA8 participates in the cold-acclimation response by negatively modulating [Ca2+]cyt and controlling the induction of CBFs and downstream genes, probably by ensuring the proper control of Ca2+ homeostasis in response to low temperature. In this project, we choose the wild-type, the insertion mutant and the rescued line to study the mechanism of ACA8 in response to cold stress. And then we analyze (1) the impact of Ca2+ signalling and Ca2+-ATPase activity by the disruption of ACA8, (2) conducting the transcriptome sequencing to identify potential downstream genes and pathways of ACA8, (3) using the Pulldown-MS to screen interacting proteins of ACA8, and (4) verifying the interactions in vivo and determining the domains of interaction. Finally, we want to reveal the molecular mechanism of ACA8 in response to cold stress and fill gaps in understanding the role of efflux systems in Ca2+ signalling and provide a new candidate gene for improving crop freezing tolerance.

低温是植物遭受最普遍的环境胁迫，严重损害作物生长并导致减产，研究植物响应低温胁迫的机制具有重要科学意义和运用价值。Ca2+信号是植物响应低温的主要形式，产生Ca2+信号的内向转运机制得到较多关注，但外向转运知之甚少。研究发现，Ca2+外向转运的Ca2+-ATPase ACA8表达受低温激烈诱导，负调节CBFs及下游一系列冷响应基因的表达。为研究ACA8调节植物响应低温的途径，本项目拟以野生型、缺失型和回补型拟南芥为研究对象：1）分析低温下ACA8缺失对Ca2+信号及质膜Ca2+-ATPase活性的影响；2）通过转录组测序筛选ACA8调节的下游基因和信号通路；3）利用融合蛋白沉淀技术钓取ACA8的互作蛋白；4）进行体内互作验证，确定互作结构域。由此探明ACA8调节植物响应低温的机制，为认识Ca2+外向转运系统对Ca2+信号调节的分子机理提供新知识，为改良作物低温抗性提供新线索。

低温胁迫是限制植物生长和发育的重要环境因子。细胞内部对低温做出响应的首要反应就是细胞质中钙离子浓度的增加。低温胁迫下胞质内的Ca2+信号是由Ca2+流入和Ca2+输出系统共同决定的。在本项目中，我们研究发现细胞质膜定位的拟南芥Ca2+-ATPase ACA8通过调节Ca2+信号影响植物的抗冻性能。首先，ACA8是Ca2+输出系统中最显著的冷响应基因。ACA8基因启动子区含有多个常见的低温响应元件及ABA响应元件，也暗示ACA8基因的转录调控受到低温和ABA信号的调节。其次，在aca8-1突变体中植株的总钙含量降低了，但在正常生长条件下其表型与野生型植株没有显著差异。在经历寒害和冻害处理后，aca8-1突变株与野生型相比，表现出较强的抗寒性和抗冻性，并且抗性的增加并不依赖于冷驯过程。此外，渗透调节和抗氧化系统活性的改变并不是aca8-1突变株抗性增加的主要原因，但ACA8基因缺失会明显改变细胞内Ca2+分布和Ca2+流动。通过基因转录表达检测，我们发现aca8-1突变株表现出较强的低温抗性与低温信号通路中的大多数关键基因的表达上调直接相关，还与其膜系统的稳定性较好相关。若在aca8-1突变株中过表达ACA8基因则所有观察的表型都恢复至与野生型类似。因此，本研究揭示了ACA8参与拟南芥抗冻性调节的作用机制，ACA8通过负调控细胞内的钙信号和ABA在低温信号通路中发挥重要作用 。