Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channnel SLAH3

Analysis of Arabidopsis nanodomains shows that Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channnel SLAH3. 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 AtRem 1.3. After transient coexpression, CPK21 interacted with SLAH3 (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. 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. Data processing and bioinformatics: MS result peak files were run on a Mascot daemon using the Mascot algorithm (version 2.2; Matrix Science) with TAIR v9 protein database, trypsin/P as protease. Allowed fixed modification was carbamidomethylation (C), and variable modifications were oxidized methionines (N) and pyroglutamic acid (pyro-Glu at N-terminal Q). Peptide and fragment mass tolerance were set to +-1.5 Da for the ion trap and +-0.2 Da for the Quad-TOF, maximum missed cleavages to two, and only singly, doubly, and triply charged ions were analyzed. After manual inspection, protein identifications with at least three unique identified peptides were automatically approved. Protein identifications based on two unique identified were only regarded if the two unique identified peptides displayed an ions score above the Mascot significance threshold for P < 0.05 (Mascot score >40 for the ion trap and >32 for the QUAD-TOF). Detailed information for the identified proteins was queried from the Uniprot consortium database (www.uniprot.org) and The Arabidopsis Information Center (TAIR: www.arabidopsis.org). The functional classification was assigned manually according to the annotations found in these two databases. Two independent biological replicates were analyzed: first setup of DRM isolation was performed with the detergents TritonX-100 and Brij-98 in-solution and in-gel digested. The second setup for DRM isolation was only performed with the detergent Triton X-100 and in-gel digested but subjected to methylcyclodextrin treatment to identify sterol-dependent lipid nanodomain proteins. For the proteomic analysis of DRMs data sets from both experimental setups were analyzed; the MCD treatment was only analyzed for the second setup. Protein amounts were estimated only for the MCD treatment using the Exponentially Modified Protein Abundance Index, which correlates with the number of observed and observable peptides per protein.

本研究针对拟南芥膜纳米域（nanodomains）开展分析，结果表明拟南芥纳米域限定的脱落酸（abscisic acid, ABA）信号通路可调控阴离子通道SLAH3（SLAC1 homolog 3）。植物激素脱落酸（ABA）在植物应对干旱胁迫的过程中发挥关键作用，因此ABA依赖的基因转录与离子转运过程受到多种蛋白激酶和磷酸酶的调控。然而，膜限定的ABA信号转导步骤的本质目前仍在很大程度上未明确。为深入探究质膜结合型ABA信号传导机制，我们从拟南芥（Arabidopsis thaliana）叶肉细胞中分离出与去垢剂抗性膜（detergent resistant membranes, DRM）相关的固醇依赖性蛋白。在这些蛋白中，我们检测到ABA信号通路核心磷酸酶ABI1（abscisic-acid insensitive 1）以及钙依赖性蛋白激酶21（calcium-dependent protein kinase 21, CPK21）。通过荧光显微镜观察，我们发现这两类蛋白定位于膜纳米域，这一结果可通过与纳米域标记物remorin AtRem1.3的共定位得到验证。在瞬时共表达实验中，CPK21可与SLAH3相互作用并激活该阴离子通道。经CPK21激活后，SLAH3表现出S型阴离子通道的典型特征。然而，当将SLAH3/CPK21与ABI1共表达时，会阻碍SLAH3/CPK21蛋白复合物在膜纳米域中的正常定位，进而导致阴离子通道激活失败。荧光共振能量转移（fluorescence resonance energy transfer, FRET）实验显示，在ABA刺激下，质膜内SLAH3与CPK21的相互作用增强，从而验证了我们的初始观测结果。有趣的是，ABA诱导的SLAH3/CPK21相互作用可被ABI1与ABA受体RCAR1/PYL9所调控。因此我们提出，ABA信号通过抑制ABI1，调控膜域内信号转导与转运复合物的表观结合状态，而该复合物是CPK21磷酸化并激活S型阴离子通道SLAH3所必需的。
数据处理与生物信息学分析：质谱结果峰文件通过Mascot算法（版本2.2；Matrix Science公司）在Mascot守护进程上运行分析，所用数据库为TAIR v9蛋白数据库，蛋白酶选用胰蛋白酶P（trypsin/P）。允许的固定修饰为半胱氨酸氨基甲酰化（carbamidomethylation, C），可变修饰为甲硫氨酸氧化（oxidized methionines, M）以及N端谷氨酰胺形成的焦谷氨酸（pyroglutamic acid, pyro-Glu at N-terminal Q）。肽段与碎片离子的质量容差设置为：离子阱模式±1.5 Da，四极杆-飞行时间质谱（Quad-TOF）模式±0.2 Da；最大漏切位点数为2；仅分析单电荷、双电荷与三电荷离子。经人工校验后，至少包含3条唯一鉴定肽段的蛋白鉴定结果将被自动认可；基于2条唯一鉴定肽段的蛋白鉴定结果，仅当两条肽段的离子得分均满足Mascot显著性阈值（P<0.05，离子阱模式下Mascot得分>40，Quad-TOF模式下>32）时方可被采纳。已鉴定蛋白的详细信息从UniProt联盟数据库（www.uniprot.org）与拟南芥信息资源中心（The Arabidopsis Information Resource, TAIR；www.arabidopsis.org）查询获取。功能分类根据上述两个数据库的注释信息手动完成。本研究设置了两组独立生物学重复：第一组去垢剂抗性膜分离实验分别使用TritonX-100与Brij-98两种去垢剂，样品分别进行溶液内酶解与胶内酶解；第二组去垢剂抗性膜分离实验仅使用Triton X-100进行处理，样品仅进行胶内酶解，但经过甲基-β-环糊精（methylcyclodextrin, MCD）处理，以鉴定固醇依赖性脂质纳米域蛋白。针对两组实验的去垢剂抗性膜蛋白质组学分析均基于对应数据集展开，其中MCD处理相关数据仅在第二组实验中进行分析。蛋白质相对定量仅针对MCD处理样本，采用指数修饰蛋白丰度指数（Exponentially Modified Protein Abundance Index, emPAI）进行估算，该指数与每个蛋白的已观测及可观测肽段数量呈正相关。