Evidence for chloroplast control of external Ca2+-induced cytosolic Ca2+ transients and stomatal closure
Evidence for chloroplast control of external Ca2+-induced cytosolic Ca2+ transients and stomatal closure
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DOI：
10.1111/j.1365-313x.2007.03390.x
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发表时间：
2008-03-01
期刊：
影响因子：
7.2
通讯作者：
Shiina, Takashi
中科院分区：
文献类型：
--
作者：
Nomura, Hironari;Komori, Teiko;Shiina, Takashi
保卫细胞叶绿体在气孔功能中的作用存在争议。通常认为气孔关闭之前保卫细胞胞内游离钙离子浓度([Ca~(2+)](Cyt))短暂升高。在此，我们提供了叶绿体在拟南芥细胞外钙([Ca+](Ext))诱导的[Ca~(2+)](Cyt)瞬变和气孔关闭中起关键作用的证据。CAS(钙敏感受体)是一种植物特有的钙结合蛋白，最初被认为是一种定位于质膜的外源钙感受器。在本研究中，我们用多种技术研究了CAS在拟南芥中的细胞内定位，包括(I)绿色荧光蛋白(GFP)融合基因表达的体内定位，(Ii)CAS的亚细胞分离和Western blotts分析，以及(Iii)类囊体膜蛋白质组的数据库分析。每种技术都产生了一致的结果。CAS主要定位于叶绿体。它是一个完整的类囊体膜蛋白，N-末端的酸性钙结合区可能暴露在膜的基质侧。T-DNA插入、CAS敲除突变体和cDNA突变互补植株的表型表明，CAS是外源Ca~(2+)诱导气孔关闭所必需的。相反，在没有外源钙离子的情况下，CAS的过表达促进了气孔的关闭。此外，利用转基因aequorin系统，我们发现在CAS基因敲除突变体中，[Ca~(2+)](Ext)诱导的[Ca~(2+)](Cyt)瞬变显著减少。我们的结果表明，类囊体膜定位的CAS在[Ca~(2+)](EXT)诱导的[Ca~(2+)](Cyt)瞬变和气孔关闭中是必不可少的。
The role of guard cell chloroplasts in stomatal function is controversial. It is usually assumed that stomatal closure is preceded by a transient increase in cytosolic free Ca2+ concentration ([Ca2+](cyt)) in the guard cells. Here, we provide the evidence that chloroplasts play a critical role in the generation of extracellular Ca2+ ([Ca2+](ext))-induced [Ca2+](cyt) transients and stomatal closure in Arabidopsis. CAS (Ca2+ sensing receptor) is a plant-specific putative Ca2+-binding protein that was originally proposed to be a plasma membrane-localized external Ca2+ sensor. In the present study, we characterized the intracellular localization of CAS in Arabidopsis with a combination of techniques, including (i) in vivo localization of green fluorescent protein (GFP) fused gene expression, (ii) subcellular fractionation and fractional analysis of CAS with Western blots, and (iii) database analysis of thylakoid membrane proteomes. Each technique produced consistent results. CAS was localized mainly to chloroplasts. It is an integral thylakoid membrane protein, and the N-terminus acidic Ca2+-binding region is likely exposed to the stromal side of the membrane. The phenotype of T-DNA insertion CAS knockout mutants and cDNA mutant-complemented plants revealed that CAS is essential for stomatal closure induced by external Ca2+. In contrast, overexpression of CAS promoted stomatal closure in the absence of externally applied Ca2+. Furthermore, using the transgenic aequorin system, we showed that [Ca2+](ext)-induced [Ca2+](cyt) transients were significantly reduced in CAS knockout mutants. Our results suggest that thylakoid membrane-localized CAS is essential for [Ca2+](ext)-induced [Ca2+](cyt) transients and stomatal closure.