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Molecular biological study on bule light signaling system in stomatal guard cells.

气孔保卫细胞蓝光信号系统的分子生物学研究。

基本信息

批准号：
06454015
负责人：
SHIMAZAKI Ken-ichiro
金额：
$ 4.61万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06454015/
关键词：
stomata blue light effect proton pump signal transduction phosphorylation dephosphorylation protein kinase protein phosphatas 光情報伝達系プロテインキナーゼプロテインフォスファターゼ Guard cell Stomata Ilne light response Ca^<2+>H^+-ATPane Proton p

项目摘要

Molecular mechanisms in blue light response of stomata were investigated regarding protein phosphorylation and dephosphorylation in guard cell protoplasts from Vicia faba. Blue light-dependent proton pumping has been shown to be inhibited by myosin light chain kinase (MLCK) inhibitor ML-7, suggesting that MLCk or MLCK-like protein may be involved in this stomatal response. We found that the antibody against MLCK from chicken gizzard reacted with guard cells proteins with molecular masses of 32 and 17kD.cDNA library of Vicia guard cells was immunoscreened using this polyclonal antibody. Two cDNA clones, VFPK1 and VFPK2, encoding putative protein kinases, were isolated from the library, and their nucleotide sequences were determined. The deduced amino acid sequences of VFPK1 and VFPK2 contain 271 and 219 amino acid residues, respectively, and are 88% identical at overlapping amino acid sequence level. The amino acid sequence revealed that they conserve invariant amino acid residues of kinase domain, and exhibited slight similarity to known protein kinases (<17%). Fusion protein of VFPK1 with glutathione-S-transferase was expressed in E.coli and the protein had protein kinase activity. These results suggest that VFPK1 cDNA encodes new protein kinase. However, the function and role in stomatal responses should be elucidated.Furthermore, we found protein phosphorylation of 80 kD protein in guard cells in response to blue light. The blue light-dependent protein phosphorylation was inhibited by MLCK inhibitor, supporting the notion that MLCK-like protein might be involved in blue light response of stomatal guard cells.

以蚕豆保卫细胞原生质体为材料，从蛋白质磷酸化和去磷酸化两个方面研究了气孔蓝光响应的分子机制。蓝光依赖的质子泵已被证明是由肌球蛋白轻链激酶（MLCK）抑制剂ML-7抑制，这表明MLCk或MLCK样蛋白可能参与了气孔反应。结果表明，抗MLCK抗体能与32和17 kD的保卫细胞蛋白发生反应，并利用该抗体对蚕豆保卫细胞cDNA文库进行了免疫筛选。两个cDNA克隆，VFPK 1和VFPK 2，编码推定的蛋白激酶，从文库中分离，并确定其核苷酸序列。推导的VFPK 1和VFPK 2的氨基酸序列分别含有271和219个氨基酸残基，在重叠氨基酸序列水平上的同源性为88%。氨基酸序列分析表明，它们保守了激酶结构域的恒定氨基酸残基，与已知的蛋白激酶有轻微的相似性（<17%）。在大肠杆菌中表达了VFPK 1与谷胱甘肽-S-转移酶的融合蛋白，该蛋白具有蛋白激酶活性。这些结果表明，VFPK 1 cDNA编码新的蛋白激酶。此外，我们还发现保卫细胞中80 kD蛋白质在蓝光下发生磷酸化反应。MLCK抑制剂可以抑制蓝光依赖的蛋白磷酸化，支持MLCK样蛋白可能参与气孔保卫细胞的蓝光反应的观点。