Nagata Chisako, Miwa Chika, Tanaka Natsuki, Kato Mariko, Suito Momoe, Tsuchihira Ayako, Sato Yori, Segami Shoji, Maeshima Masayoshi
Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601, Japan.
Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto, 611-0011, Japan.
J Plant Res. 2016 May;129(3):539-50. doi: 10.1007/s10265-016-0787-2. Epub 2016 Mar 15.
The Ca(2+)-binding protein-1 (PCaP1) of Arabidopsis thaliana is a new type protein that binds to phosphatidylinositol phosphates and Ca(2+)-calmodulin complex as well as free Ca(2+). Although biochemical properties, such as binding to ligands and N-myristoylation, have been revealed, the intracellular localization, tissue and cell specificity, integrity of membrane association and physiological roles of PCaP1 are unknown. We investigated the tissue and intracellular distribution of PCaP1 by using transgenic lines expressing PCaP1 linked with a green fluorescence protein (GFP) at the carboxyl terminus of PCaP1. GFP fluorescence was obviously detected in most tissues including root, stem, leaf and flower. In these tissues, PCaP1-GFP signal was observed predominantly in the plasma membrane even under physiological stress conditions but not in other organelles. The fluorescence was detected in the cytosol when the 25-residue N-terminal sequence was deleted from PCaP1 indicating essential contribution of N-myristoylation to the plasma membrane anchoring. Fluorescence intensity of PCaP1-GFP in roots was slightly decreased in seedlings grown in medium supplemented with high concentrations of iron for 1 week and increased in those grown with copper. In stomatal guard cells, PCaP1-GFP was strictly, specifically localized to the plasma membrane at the epidermal-cell side but not at the pore side. A T-DNA insertion mutant line of PCaP1 did not show marked phenotype in a life cycle except for well growth under high CO2 conditions. However, stomata of the mutant line did not close entirely even in high osmolarity, which usually induces stomata closure. These results suggest that PCaP1 is involved in the stomatal movement, especially closure process, in leaves and response to excessive copper in root and leaf as a mineral nutrient as a physiological role.
拟南芥的钙离子结合蛋白1（PCaP1）是一种新型蛋白质，它能与磷脂酰肌醇磷酸、钙离子-钙调蛋白复合物以及游离钙离子结合。尽管已经揭示了其与配体结合和N-肉豆蔻酰化等生化特性，但PCaP1的细胞内定位、组织和细胞特异性、膜结合完整性以及生理作用尚不清楚。我们通过使用在PCaP1羧基末端与绿色荧光蛋白（GFP）相连的PCaP1转基因系，研究了PCaP1的组织和细胞内分布。在包括根、茎、叶和花在内的大多数组织中都明显检测到了GFP荧光。在这些组织中，即使在生理胁迫条件下，PCaP1-GFP信号也主要在质膜中观察到，而在其他细胞器中未观察到。当从PCaP1中删除25个残基的N端序列时，在细胞质中检测到荧光，这表明N-肉豆蔻酰化对质膜锚定起着重要作用。在补充高浓度铁1周的培养基中生长的幼苗中，根中PCaP1-GFP的荧光强度略有降低，而在补充铜的幼苗中则增加。在气孔保卫细胞中，PCaP1-GFP严格、特异性地定位于表皮细胞一侧的质膜，而不是孔一侧。PCaP1的T-DNA插入突变体株系在生命周期中除了在高二氧化碳条件下生长良好外，没有表现出明显的表型。然而，即使在通常诱导气孔关闭的高渗透压下，突变体株系的气孔也不会完全关闭。这些结果表明，PCaP1参与叶片中的气孔运动，特别是关闭过程，并且作为一种生理作用，参与根和叶对过量铜作为矿质营养的响应。
