水稻OsDMI3与OsMKK1/OsMKK6相互作用在ABA信号转导中的功能分析

It has been shown that Ca2+/calmodulin (CaM)-dependent protein kinase (CCaMK) is required for abscisic acid (ABA)-induced antioxidant defense and for the tolerance of plants to water stress and oxidative stress. There exists a cross-talk between CCaMK and mitogen-activated protein kinase (MAPK) in ABA signaling, in which CCaMK functions upstream of MAPK to regulate the antioxidant defense systems in plants. However, the mechanisms how CCaMK induces the activation of MAPK in ABA signaling remain to be determined. In this project, based on our previous studies for this project, we intend to identify the interaction between the rice CCaMK OsDMI3 and the rice MAPK kinases (MAPKKs) OsMKK1 and OsMKK6 and to clarify whether OsMKK1 and OsMKK6 are the phosphorylated substrates of OsDMI3 in vitro and in vivo. The phosphorylation sites of OsMKK1 and OsMKK6 by OsDMI3 are also determined. Further, we want to elucidate the roles of OsDMI3-mediated phosphorylation of OsMKK1 and OsMKK6 in ABA-induced activation of OsMPKs and in ABA-regulated physiological responses in rice. This project is not only helpful to our understanding for the mechanisms how CCaMK is involved in ABA signaling and in plant response to water stress and oxidative stress, but also has an important significance for improvement of crop stress resistance by using molecular biology manipulation.

钙/钙调素依赖型蛋白激酶（CCaMK）已经被证实是脱落酸（ABA）诱导的抗氧化防护信号转导中的重要组分，增强植物对水分胁迫、氧化胁迫的耐性，且在这一过程中，促分裂原活化蛋白激酶（MAPK）作用于CCaMK的下游。然而，ABA信号转导中CCaMK是怎样调节MAPK活化的机制仍有待阐明。本项目拟在前期工作的基础上，进一步鉴定ABA作用下水稻叶片中CCaMK OsDMI3与MAPKKs OsMKK1、OsMKK6之间的互作关系，在体内外澄清OsMKK1、OsMKK6是OsDMI3磷酸化的底物，阐明其磷酸化位点，进而分析OsDMI3介导的OsMKK1、OsMKK6磷酸化在ABA诱导的OsMPKs活化中的作用以及在ABA调控的生理反应中的作用。这一研究的开展不仅有助于人们对植物CCaMK参与ABA信号转导以及植物抗逆反应的作用机制的认识，而且对于利用分子生物学手段提高作物的耐逆性具有重要的指导意义。

本项目调查了水稻Ca2+/CaM依赖型蛋白激酶OsDMI3与水稻中促分裂原活化蛋白激酶激酶OsMKK1/OsMKK6蛋白互作在ABA信号转导中的作用及其作用机制，主要研究结果如下：.（1）澄清了OsMKK1/OsMKK6是OsDMI3的互作蛋白，但在ABA信号转导中，仅仅OsMKK1的磷酸化与活化是依赖于OsDMI3的。进一步的研究发现，OsDMI3能够磷酸化OsMKK1 Thr-25，且在ABA信号转导中，这一磷酸化是OsDMI3专一的。遗传学证据揭示，OsMKK1是水稻ABA反应，包括种子萌发、根系生长以及水稻对水分胁迫与氧化胁迫的反应的正调节子，且OsDMI3介导的OsMKK1磷酸化对ABA反应中OsMKK1的功能做出了重要贡献。.（2）揭示了OsDMI3可以通过调节Na+和H+的流入量来提高水稻根系对盐碱胁迫的耐性。.（3）揭示了一个双负调节途径介导ABA诱导的OsDMI3活化机制，即在在ABA缺乏的条件下，水稻蛋白磷酸酶OsPP45通过直接脱OsDMI3磷酸化而使OsDMI3处于钝化状态，而在ABA存在下，ABA通过诱导NADPH氧化酶OsRbohB/E产生H2O2而抑制OsPP45活性，从而解除OsPP45对OsDMI3活性的抑制，进而导致ABA反应。.（4）证实了一个非典型性胚胎发生晚期丰富蛋白OsLEA5在ABA诱导的抗氧化防护中起着重要的作用。. 本项目已在国际重要学术刊物如The Plant Cell等上发表论文3篇，另有1篇论文已发布于bioRxiv。这些研究结果不仅加深了我们对ABA信号转导机理的认识，而且对于进一步利用分子手段提高农作物的抗逆性具有重要的指导意义。   .