Ca2+/calmodulin (CaM)-dependent protein kinase (CCaMK) is an important positive regulator of abscisic acid (ABA) and abiotic stress signaling in plants and is believed to act upstream of mitogen-activated protein kinase (MAPK) in ABA signaling. However, it is unclear how CCaMK activates MAPK in ABA signaling. Here, we show that OsDMI3, a rice (Oryza sativa) CCaMK, directly interacts with and phosphorylates OsMKK1, a MAPK kinase (MKK) in rice, in vitro and in vivo. OsDMI3 was found to directly phosphorylate Thr-25 in the N-terminus of OsMKK1, and this Thr-25 phosphorylation is OsDMI3-specific in ABA signaling. The activation of OsMKK1 and its downstream kinase OsMPK1 is dependent on Thr-25 phosphorylation of OsMKK1 in ABA signaling. Moreover, ABA treatment also induces the phosphorylation in the activation loop of OsMKK1, and the two phosphorylations in the N-terminus and in the activation loop are independent. Further analyses revealed that OsDMI3-mediated phosphorylation of OsMKK1 positively regulates ABA responses in seed germination, root growth, and tolerance to both water stress and oxidative stress. Our results indicate that OsMKK1 is a direct target of OsDMI3, and OsDMI3-mediated phosphorylation of OsMKK1 plays an important role in the activation of MAPK cascade and ABA signaling.

中文翻译：

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水稻钙/钙调蛋白依赖性蛋白激酶直接磷酸化丝裂原活化的蛋白激酶激酶，以调节脱落酸的响应。

Ca2 + /钙调蛋白（CaM）依赖性蛋白激酶（CCaMK）是植物中脱落酸（ABA）和非生物胁迫信号的重要正调节剂，并且据信在ABA信号中是促分裂原活化蛋白激酶（MAPK）的上游。但是，尚不清楚CCaMK如何激活ABA信号传导中的MAPK。在这里，我们显示了水稻OsDMI3，一种水稻（Oryza sativa）CCaMK，可在体内和体外直接与水稻中的MAPK激酶（MKK）的OsMKK1相互作用并使其磷酸化。发现OsDMI3直接在OsMKK1的N端磷酸化Thr-25，并且该Thr-25磷酸化在ABA信号传导中是OsDMI3特异性的。OsMKK1及其下游激酶OsMPK1的激活取决于ABA信号转导中OsMKK1的Thr-25磷酸化。此外，ABA处理还会在OsMKK1的激活环中诱导磷酸化，N末端和激活环中的两个磷酸化是独立的。进一步的分析表明，OsDMI3介导的OsMKK1磷酸化在种子萌发，根系生长以及对水分胁迫和氧化胁迫的耐受性中正调控ABA反应。我们的结果表明，OsMKK1是OsDMI3的直接靶标，并且OsDMI3介导的OsMKK1磷酸化在激活MAPK级联和ABA信号传导中起重要作用。