Kobayashi Yuhko, Murata Michiharu, Minami Hideyuki, Yamamoto Shuhei, Kagaya Yasuaki, Hobo Tokunori, Yamamoto Akiko, Hattori Tsukaho
Bioscience and Biotechnology Center, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan.
Plant J. 2005 Dec;44(6):939-49. doi: 10.1111/j.1365-313X.2005.02583.x.
The plant hormone abscisic acid (ABA) induces gene expression via the ABA-response element (ABRE) present in the promoters of ABA-regulated genes. A group of bZIP proteins have been identified as ABRE-binding factors (ABFs) that activate transcription through this cis element. A rice ABF, TRAB1, has been shown to be activated via ABA-dependent phosphorylation. While a large number of signalling factors have been identified that are involved in stomatal regulation by ABA, relatively less is known about the ABA-signalling pathway that leads to gene expression. We have shown recently that three members of the rice SnRK2 protein kinase family, SAPK8, SAPK9 and SAPK10, are activated by ABA signal as well as by hyperosmotic stress. Here we show that transient overexpression in cultured cell protoplasts of these ABA-activated SnRK2 protein kinases leads to the activation of an ABRE-regulated promoter, suggesting that these kinases are involved in the gene-regulation pathway of ABA signalling. We further show several lines of evidence that these ABA-activated SnRK2 protein kinases directly phosphorylate TRAB1 in response to ABA. Kinetic analysis of SAPK10 activation and TRAB1 phosphorylation indicated that the latter immediately followed the former. TRAB1 was found to be phosphorylated not only in response to ABA, but also in response to hyperosmotic stress, which was interpreted as the consequence of phosphorylation of TRAB1 by hyperosmotically activated SAPKs. Physical interaction between TRAB1 and SAPK10 in vivo was demonstrated by a co-immunoprecipitation experiment. Finally, TRAB1 was phosphorylated in vitro by the ABA-activated SnRK2 protein kinases at Ser102, which is phosphorylated in vivo in response to ABA and is critical for the activation function.
植物激素脱落酸（ABA）通过存在于ABA调控基因启动子中的ABA反应元件（ABRE）诱导基因表达。一组bZIP蛋白已被鉴定为ABRE结合因子（ABF），它们通过这个顺式元件激活转录。一种水稻ABF，即TRAB1，已被证明可通过ABA依赖的磷酸化作用被激活。虽然已经鉴定出大量参与ABA介导的气孔调节的信号因子，但对于导致基因表达的ABA信号通路却知之甚少。我们最近发现，水稻SnRK2蛋白激酶家族的三个成员，即SAPK8、SAPK9和SAPK10，可被ABA信号以及高渗胁迫激活。在此我们表明，在培养细胞原生质体中瞬时过表达这些被ABA激活的SnRK2蛋白激酶会导致ABRE调控的启动子被激活，这表明这些激酶参与了ABA信号的基因调控途径。我们进一步展示了几条证据，表明这些被ABA激活的SnRK2蛋白激酶在响应ABA时直接磷酸化TRAB1。对SAPK10激活和TRAB1磷酸化的动力学分析表明，后者紧随前者之后。发现TRAB1不仅在响应ABA时被磷酸化，而且在响应高渗胁迫时也被磷酸化，这被解释为高渗激活的SAPK对TRAB1磷酸化的结果。体内TRAB1与SAPK10之间的物理相互作用通过免疫共沉淀实验得以证明。最后，在体外，被ABA激活的SnRK2蛋白激酶在Ser102位点对TRAB1进行磷酸化，该位点在体内响应ABA时被磷酸化，并且对于激活功能至关重要。
