Institute for Molecular Plant Physiology and Biophysics, University of Wuerzburg, 97082 Wuerzburg, Germany.
Proc Natl Acad Sci U S A. 2013 May 14;110(20):8296-301. doi: 10.1073/pnas.1211667110. Epub 2013 Apr 29.
The phytohormone abscisic acid (ABA) plays a key role in the plant response to drought stress. Hence, ABA-dependent gene transcription and ion transport is regulated by a variety of protein kinases and phosphatases. However, the nature of the membrane-delimited ABA signal transduction steps remains largely unknown. To gain insight into plasma membrane-bound ABA signaling, we identified sterol-dependent proteins associated with detergent resistant membranes from Arabidopsis thaliana mesophyll cells. Among those, we detected the central ABA signaling phosphatase ABI1 (abscisic-acid insensitive 1) and the calcium-dependent protein kinase 21 (CPK21). Using fluorescence microscopy, we found these proteins to localize in membrane nanodomains, as observed by colocalization with the nanodomain marker remorin Arabidopsis thaliana remorin 1.3 (AtRem 1.3). After transient coexpression, CPK21 interacted with SLAH3 [slow anion channel 1 (SLAC1) homolog 3] and activated this anion channel. Upon CPK21 stimulation, SLAH3 exhibited the hallmark properties of S-type anion channels. Coexpression of SLAH3/CPK21 with ABI1, however, prevented proper nanodomain localization of the SLAH3/CPK21 protein complex, and as a result anion channel activation failed. FRET studies revealed enhanced interaction of SLAH3 and CPK21 within the plasma membrane in response to ABA and thus confirmed our initial observations. Interestingly, the ABA-induced SLAH3/CPK21 interaction was modulated by ABI1 and the ABA receptor RCAR1/PYL9 [regulatory components of ABA receptor 1/PYR1 (pyrabactin resistance 1)-like protein 9]. We therefore propose that ABA signaling via inhibition of ABI1 modulates the apparent association of a signaling and transport complex within membrane domains that is necessary for phosphorylation and activation of the S-type anion channel SLAH3 by CPK21.
植物激素脱落酸（ABA）在植物对干旱胁迫的反应中起着关键作用。因此，ABA 依赖性基因转录和离子运输受多种蛋白激酶和磷酸酶调节。然而，膜限定的 ABA 信号转导步骤的性质在很大程度上仍然未知。为了深入了解质膜结合的 ABA 信号，我们从拟南芥叶肉细胞中鉴定出与固醇相关的与去污剂抗性膜相关的蛋白。其中，我们检测到中央 ABA 信号磷酸酶 ABI1（ABA 不敏感 1）和钙依赖性蛋白激酶 21（CPK21）。通过荧光显微镜，我们发现这些蛋白定位于膜纳米域中，如与纳米域标记物拟南芥 remorin 1.3（AtRem 1.3）的共定位所观察到的那样。瞬时共表达后，CPK21 与 SLAH3 [慢阴离子通道 1（SLAC1）同源物 3]相互作用并激活该阴离子通道。在 CPK21 刺激下，SLAH3 表现出 S 型阴离子通道的标志性特性。然而，SLAH3/CPK21 与 ABI1 的共表达阻止了 SLAH3/CPK21 蛋白复合物的适当纳米域定位，结果阴离子通道激活失败。FRET 研究表明，ABA 响应增强了质膜中 SLAH3 和 CPK21 之间的相互作用，从而证实了我们的初步观察结果。有趣的是，ABI1 和 ABA 受体 RCAR1/PYL9 [调节 ABA 受体 1/拟南芥 PYR1（吡喃醇抗药性 1 样蛋白 9）的组成部分]调节 ABA 诱导的 SLAH3/CPK21 相互作用。因此，我们提出，通过抑制 ABI1 的 ABA 信号调节了信号和运输复合物在膜域中的表观关联，这对于 CPK21 对 S 型阴离子通道 SLAH3 的磷酸化和激活是必要的。
