| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1999: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
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.
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