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

植物水通道蛋白（水通道）有多种同工型，分布在质膜和细胞质中。我们在萝卜的质膜（PAQ1, 1b, 1c, 2, 2b和2c）和液泡膜（γ -和δ - vm23）中克隆了几个同种异构体。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的关系，以及每种水通道蛋白的膜定位机制。(1)对萝卜幼苗、植株、花和种子中包括VM23在内的水通道蛋白mrna的综合研究表明，所有mrna在下胚轴和生长主根中都有明显的积累。在一些组织中，mRNA和蛋白水平随同种异构体而变化。(2)用停流分光光度计测定了萝卜和葡萄叶的质膜和液泡膜的渗透性。两种植物液泡膜的透水性均高于质膜。此外，CAM植物制备的两种膜的透性都极低于萝卜膜。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在液泡膜上。N端或c端部分的缺失对定位没有影响。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， γ - vm23和δ - vm23）引入到CAM植物kalanche中，该植物的水通道蛋白水平很低。所有基因均在转基因植物中检测到，免疫印迹法至少发现了PAQ2蛋白。现在，我们研究转基因甘蓝的生理特性。

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.