Molecular porperties and cellular function of aquaporins in the plasam and vacuolar membranes of plant

植物质体和液泡膜中水通道蛋白的分子特性和细胞功能

基本信息

批准号：
11660084
负责人：
MAESHIMA Masayoshi
金额：
$ 2.3万
依托单位：
Nagoya 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-11660084/
关键词：
Vacuolar membrane Plasma membrane Aquaporin Molecular diversity Green fluorescent protein CAM plant

项目摘要

Plant aquaporins (water channels) occur in multiple isoforms and are distributed in both plasma membrane and tonoplast. We have cloned several isoforms in plasma membranes (PAQ1, 1b, 1c, 2, 2b, and 2c) and vacuolar membranes (gamma- and delta-VM23) of radish. In this study, we investigated the time- and cell-specificity of gene expression of these aquaporins, the relationship between the aquaporin content in the membrane and membrane water permeability, and mechanism of membrane localization of each aquaporin.(1) A comprehensive investigation of the aquaporin mRNAs, including VM23, in seedlings, plants, flowers and seeds of radish showed a marked accumulation of all the mRNAs in hypocotyls and growing taproots. In some tissues, the mRNA and protein levels varied with isoforms.(2) The osmotic water permeability was determined for plasma membranes and vacuolar membranes prepared from radish and Graptopetalum (a CAM plant) with a stopped flow spectrophotometer. The water permeability of the vacuolar membrane was higher than that of the plasma membrane of both plant species. Also, the permeability of both membranes prepared from CAM plant was extremely lower than that of radish membranes. The result is consistent with the abundance of aquaporin in the membrane.(3) We expressed fusion proteins of PAQ1 and gamma-VM23 with a green fluorescent protein (GFP) in tobacco cells. PAQ-GFP protein was correctly localized on the plasma membrane and VM23-GFP was on the vacuolar membrane. Deletion of N- or C-terminal part had no effect on the localization. The results indicate that the localization signal is in the internal part of aquaporins.(4) We also tried to introduce radish aquaporins (PAQ1, PAQ2, gamma-VM23 and delta-VM23) into Kalanchoe, a CAM plant, in which the aquaporin level is very low. All the genes were detected in transgenic plants and at least PAQ2 protein was found by immunoblotting. Now, we investigate physiological properties of the transgenic Kalanchoe.

植物水通道蛋白（水通道）以多种异构体存在，分布在质膜和液泡膜中。我们在萝卜的质膜（PAQ1、1b、1c、2、2b 和 2c）和液泡膜（gamma- 和 delta-VM23）中克隆了几种亚型。在本研究中，我们研究了这些水通道蛋白基因表达的时间和细胞特异性，膜中水通道蛋白含量与膜透水性之间的关系，以及每种水通道蛋白的膜定位机制。（1）全面研究萝卜幼苗、植物、花和种子中的水通道蛋白 mRNA（包括 VM23）显示所有 mRNA 在下胚轴和生长的主根中显着积累。在一些组织中，mRNA和蛋白质水平随亚型的不同而变化。(2)用停流分光光度计测定了萝卜和笔瓣(CAM植物)制备的质膜和液泡膜的渗透水渗透率。两种植物的液泡膜的透水性均高于质膜。此外，由 CAM 植物制备的两种膜的渗透性均远低于萝卜膜。结果与膜中水通道蛋白的丰度一致。(3)在烟草细胞中表达PAQ1和γ-VM23与绿色荧光蛋白(GFP)的融合蛋白。 PAQ-GFP 蛋白正确定位在质膜上，VM23-GFP 正确定位在液泡膜上。 N-或C-末端部分的删除对定位没有影响。结果表明定位信号位于水通道蛋白的内部。(4)我们还尝试将萝卜水通道蛋白(PAQ1、PAQ2、gamma-VM23和delta-VM23)引入CAM植物长寿花中，其中水通道蛋白水平非常低。在转基因植物中检测到了所有基因，并且通过免疫印迹至少发现了PAQ2蛋白。现在，我们研究转基因长寿花的生理特性。