H+/Ca2+反向转运蛋白CAX1调控植物抗病的机理研究

Plant infections result in massive crop yield reduction and thus enhancing defense hormone salicylic acid (SA)-mediated disease resistance is very important for increasing agriculture production. Over accumulation of SA results in the increase of SAG, thus influencing plant defense. However apart from induction by SA, whether and how SAG accumulation is controlled by other mechanisms is still unknown. Previously, we found that loss function of H+/Ca2+ antiporter CAX1 leads to the more accumulation of SAG significantly and tends to accumulate less SA. Furthermore, loss function of CAX1 is more susceptible to bacterial infections. These results indicate that CAX1 might regulate plant defense by impacting SAG accumulation directly. Firstly, this project is going to analyze CAX1 expression in response to bacterial infections and how CAX1 impacts on SA marker genes expression. Secondly, SA glucosyltransferase activity will be tested by in-vitro glucosyltransferase system. Thirdly, the involvement of SA glucosyltransferases in the regulation of defense by CAX1 will be explored by genetic crossings among the loss function of CAX1 and loss function of SA glucosyltransferases with the assistance of the targeted mutation by CRISPR. This project will clarify the mechanism of CAX1 in regulating plant defense based on the new view of regulation on SAG. This will bring in the new idea for efforts to improve plant defense.

植物病原菌感染造成农作物大量减产，调控抗病激素水杨酸(SA)对提高农作物抗病有十分重要的社会意义。SA过量积累导致水杨酸葡萄糖苷（SAG）升高，继而影响植物抗病。但SAG 除受SA诱导外，如何被调控尚不清楚。本课题组预实验发现缺失H+/Ca2+反向转运蛋白CAX1的突变体 (cax1)中，SAG含量显著上升而SA含量趋向下调。进一步分析表明缺失CAX1突变体的植物抗病能力降低。这说明CAX1可能直接调控SAG积累来影响植物抗病信号传递。首先本项目将从分子水平，分析CAX1受细菌感染的表达情况以及与SA信号通路中抗病基因的关系；其次将用体外糖基转移酶体系探索CAX1如何改变SAG合成酶的活性来调节SA；第三，通过定点突变CRISPR介导的遗传杂交研究CAX1依赖于何种糖基转移酶调控SA介导植物抗病。本研究从SAG调控这个新视点为揭示CAX1调控植物抗病机理奠定基础，为提高植物抗病提供新的思路

CAXs将Ca2 +从细胞质运输到液泡中，以维持Ca2 +稳态。CAX1在耐受非生物胁迫中有重要作用，然而尚无直接证据表明CAX1在耐受生物胁迫方面有作用。我们的研究表明，CAX1能调控植物抗病。缺乏CAX1导致了对活体营养型细菌Pst-avrRpm1和死体营养型真菌B. cinerea都更抗。而且，cax1突变体中积累了更高含量的水杨酸（SA）和植物抗毒素scopoletin，分别用于抵御活体营养细菌和死体营养真菌。施加钙也增强了植物对Pst-avrRpm1和B. cinerea的抗性，这与cax1突变体类似。此外，cax1突变体在钙处理后，相对野生型显示早衰现象。且cax1突变体的早衰依赖于SID2（SA合成基因），而并不取决于SA通路的关键正调控因子NPR1。然而，与CAX1具有77％的蛋白质序列同源的CAX3并未在抗病中有作用。因此，CAX1（而非CAX3）负调控病原体防御机制和早期衰老很可能是通过操纵钙稳态来实现的。且SA作为抗病激素，寻找修饰它的糖基转移酶研究从未间断过。拟南芥UGT74F1和UGT76B1被认为是最有可能糖基化SA合成水杨酸糖苷（SAG）的体内合成酶。我们的研究显示，cax1突变中合成SAG能力比野生型Col-0有所增加。并且cax1突变体中糖基转移酶UGT76B1表达增加，而UGT74F1不变。然后，检测cax1 ugt76b1，cax1 ugt74f1，cax1 ugt76b1 ugt74f1，ugt76b1 ugt74f1，cax1，ugt76b1和ugt74f1中SA和SAG含量的变化，却发现cax1中更多SAG积累依赖于糖基转移酶UGT74F1而并不是UGT76B1。总之，CAX1在植物防御病原菌中起到关键的负调控作用，是通过影响钙稳态平衡来抑制与抗病相关代谢物（如抗病激素SA和植物保护素scopoletin）积累。而且，CAX1通过控制SA含量来抑制植物早衰。再者，缺失CAX1导致的水杨酸糖苷SAG的累积，主要是由糖基转移酶UGT74F1合成的，而并不依赖UGT76B1。