Protein phosphorylation and 14-3-3 protein in volved in blue-light response of stomata

蛋白质磷酸化和14-3-3蛋白质参与气孔蓝光反应

• 批准号: 11640650
• 负责人: SHIMAZAKI Kenichiro
• 金额: $ 2.5万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11640650/
• 关键词: Stomata; 14-3-3 protein; blue light response; H^+-ATPase; protein phosphosylation; 孔辺細胞; 青色光反応; 14-3-3タンパク質; リン酸化; ソラマメ; 青色光; H^+-ATPase

The plasma membrane H^+-ATPase is activated by blue light with concomitant binding of the 14-3-3 protein to the C-terminus in guard cells. Since several isoforms of the 14-3-3 protein are expressed in plants, we determined which isoform (s) bound to the H^+-ATPase in vivo. Four cDNA clones (vf14-3-3a, vf14-3-3b, vf14-3-3c and vf14-3-3d) encoding 14-3-3 proteins were isolated from Vicia guard cells. Northern analysis revealed that mRNAs encoding vf14-3-3a and vf14-3-3b proteins were expressed predominantly in guard cells. The 14-3-3 protein that bound to the H^+-ATPase in guard cells had the same molecular mass as the recombinant vf14-3-3a protein. The H^+-ATPase immunoprecipitated from mesophyll cell protoplasts (MCPs), which had been stimulated by fusicoccin, co-precipitated with the 32.5-kDa 14-3-3 protein, although three 14-3-3 isoproteins were found in MCPs. Digestions of the bound 14-3-3 protein and recombinant vf14-3-3a with CNBr gave the identical migration profiles on SDS-PAGE, but that of vf14-3-3b gave a different profile. Mass profiling of trypsin-digested 14-3-3 protein bound to the H^+-ATPase gave the predicted peptide masses of vf14-3-3a. Far Western analysis revealed that the H^+-ATPase had a higher affinity for vf14-3-3a than for vf14-3-3b. These results suggest that the 14-3-3 protein that bound to the plasma membrane H^+-ATPase in vivo is vf14-3-3a and that it may play a key role in the activation of H^+-ATPase in guard cells.

蓝光激活质膜H^+-ATP酶，伴随着14-3-3蛋白与保卫细胞C端的结合。由于14-3-3蛋白的几种异构体在植物中表达，我们确定了哪种异构体在体内与H^+-ATP酶结合。从野豌豆保卫细胞中分离到4个编码14-3-3蛋白的cDNA克隆（vf 14 -3- 3a、vf 14 -3-3b、vf 14 -3-3c和vf 14 -3-3d）。北方分析显示编码vf 14 -3-3a和vf 14 -3-3b蛋白的mRNA主要在保卫细胞中表达。与保卫细胞中H^+-ATPase结合的14-3-3蛋白与重组vf 14 -3-3a蛋白具有相同的分子量。从叶肉细胞原生质体（MCP）中免疫沉淀的H^+-ATP酶，在被梭孢菌素刺激后，与32.5-kDa的14-3-3蛋白共沉淀，尽管在MCP中发现了三种14-3-3同工蛋白。结合的14-3-3蛋白和重组vf 14 -3-3a经溴化氰双酶切后，在SDS-PAGE上显示出相同的迁移曲线，而vf 14 -3-3b的迁移曲线则不同。用胰蛋白酶消化与H^+-ATP酶结合的14-3-3蛋白质，得到了预测的vf 14 -3-3a肽质量。远Western分析表明，H^+-ATPase对vf 14 -3-3a的亲和力高于vf 14 -3-3b。这些结果表明，在体内与质膜H^+-ATP酶结合的14-3-3蛋白是vf 14 -3-3a，它可能在保卫细胞H^+-ATP酶的激活中起关键作用。

项目成果

Kinoshita,T. and Shimazaki,K.: "Characterization of cytosolic cyclophilin from guard cells of Vicia faba L"Plant&Cell Physiol.. 40. 53-59 (1999)

木下，T.

Kinoshita,T.and Shimazaki K.: "Analysis of the phosphorylation level in guard-cell plasma membrane H^+-ATPase in response to fusicoccin."Plant Cell Physiology.. (in press). (2001)

Kinoshita,T. 和 Shimazaki K.：“分析保卫细胞质膜 H+ -ATP 酶响应梭菌素的磷酸化水平。”植物细胞生理学..（出版中）。

Assmann,S,M. and Shimazaki,K.: "The multisensory guard cell : Stomatal responses to blue light and abscisic acid"Plant Physiol.. 119. 809-815 (1999)

阿斯曼，S，M。

Kinoshita,T.and Shimazakik: "Blue light activates the plasma membrane H^+-ATPase by phosphorylation of the C-terminus in stomatal guard cells."EMBO J.. 18. 5548-5558 (1999)

Kinoshita,T. 和 Shimazakik：“蓝光通过气孔保卫细胞中 C 末端的磷酸化来激活质膜 H+ -ATP 酶。”EMBO J.. 18. 5548-5558 (1999)

Emi, T et al.: "Specific binding of vf14-3-3a isoform to the plasma membrane H^+-ATPase in response to blue light and fusicoccin in guard cells of broad bean."Plant Physiol. 125. 1115-1125 (2001)

Emi, T 等人：“蚕豆保卫细胞中 vf14-3-3a 同工型与质膜 H2-ATP 酶响应蓝光和梭菌素的特异性结合。”植物生理学。