Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601, Japan.
Plant J. 2013 May;74(4):690-700. doi: 10.1111/tpj.12155. Epub 2013 Mar 29.
Plasma membrane-associated Ca(2+) -binding protein-2 (PCaP2) of Arabidopsis thaliana is a novel-type protein that binds to the Ca(2+) /calmodulin complex and phosphatidylinositol phosphates (PtdInsPs) as well as free Ca(2+) . Although the PCaP2 gene is predominantly expressed in root hair cells, it remains unknown how PCaP2 functions in root hair cells via binding to ligands. From biochemical analyses using purified PCaP2 and its variants, we found that the N-terminal basic domain with 23 amino acids (N23) is necessary and sufficient for binding to PtdInsPs and the Ca(2+) /calmodulin complex, and that the residual domain of PCaP2 binds to free Ca(2+) . In mutant analysis, a pcap2 knockdown line displayed longer root hairs than the wild-type. To examine the function of each domain in root hair cells, we over-expressed PCaP2 and its variants using the root hair cell-specific EXPANSIN A7 promoter. Transgenic lines over-expressing PCaP2, PCaP2(G2A) (second glycine substituted by alanine) and ∆23PCaP2 (lacking the N23 domain) exhibited abnormal branched and bulbous root hair cells, while over-expression of the N23 domain suppressed root hair emergence and elongation. The N23 domain was necessary and sufficient for the plasma membrane localization of GFP-tagged PCaP2. These results suggest that the N23 domain of PCaP2 negatively regulates root hair tip growth via processing Ca(2+) and PtdInsP signals on the plasma membrane, while the residual domain is involved in the polarization of cell expansion.
拟南芥质膜相关钙结合蛋白-2（PCaP2）是一种新型蛋白，可与钙/钙调蛋白复合物和磷脂酰肌醇磷酸（PtdInsPs）以及游离钙结合。虽然 PCaP2 基因主要在根毛细胞中表达，但尚不清楚 PCaP2 如何通过与配体结合在根毛细胞中发挥作用。通过使用纯化的 PCaP2 及其变体进行的生化分析，我们发现具有 23 个氨基酸的 N 端碱性结构域（N23）对于与 PtdInsPs 和钙/钙调蛋白复合物的结合是必需且充分的，而 PCaP2 的剩余结构域与游离钙结合。在突变分析中，pcap2 敲低系比野生型具有更长的根毛。为了研究每个结构域在根毛细胞中的功能，我们使用根毛细胞特异性 EXPANSIN A7 启动子过表达了 PCaP2 及其变体。过表达 PCaP2、PCaP2（G2A）（第二个甘氨酸被丙氨酸取代）和 ∆23PCaP2（缺失 N23 结构域）的转基因系表现出异常分支和球状根毛细胞，而过表达 N23 结构域则抑制根毛的出现和伸长。N23 结构域对于 GFP 标记的 PCaP2 的质膜定位是必需且充分的。这些结果表明，PCaP2 的 N23 结构域通过处理质膜上的钙和 PtdInsP 信号负调控根毛尖端生长，而剩余结构域参与细胞扩展的极化。
