Functional Analyses On Regulator Of G Protein Signaling (RGS) Proteins In Arabidopsis Thaliana

The regulator of G-protein signaling (RGS) proteins, recently identified in Arabidopsis thaliana (named as AtRGS1), has a predicted seven-transmembrane structure as well as an RGS box with GTPase-accelerating activity and thus desensitizes the G-protein-mediated signaling. The roles of AtRGS1 proteins in Arabidopsis were investigated in the present study, using seeds that carry a null mutation in the genes encoding RGS protein (AtRGS1) and the alpha subunit (AtGPA1) of the G protein in Arabidopsis, named rgs1-2 and gpa1-3 respectively as well as cell suspension, with different approaches such as genetics, pharmacology, biochemistry and molecular biology. The main results were as follows:In contrast to wild type Col-0 and gpa1-3, stratification was found not necessarily required and after-ripening process had no effect for the rgs1-2 seed germination. In addition, rgs1-2 seed germination was insensitive to glucose and sucrose. The insensitivities of rgs1-2 to glucose and sucrose were not due to a possible osmotic stress because the germination of rgs1-2 mutant seeds showed the same responses as those of gpa1-3 mutants and wild type when treated with the same concentrations of mannitol and sorbitol. The gpa1-3 seed germination was hypersensitive while rgs1-2 was less sensitive to exogenous abscisic acid (ABA). The different responses to ABA largely diminished and the inhibitory effects on seed germination by exogenous ABA and glucose were markedly alleviated when endogenous ABA biosynthesis was inhibited. Hypersensitive responses of seed germination to both glucose and ABA were also observed in the overexpressor of AtRGS1. Analysis of the active endogenous ABA levels and the expression of NCED3 and ABA2 genes showed that glucose significantly stimulated the ABA biosynthesis and increased the expression of NCED3 and ABA2 genes in germinating Col seeds, but not in rgs1-2 mutant seeds. These data suggest that AtRGS1 proteins are involved in the regulation of seed germination. The hyposensitivity of rgs1-2 mutant seed germination to glucose might be the result of the impairment of ABA biosynthesis during seed germination.Glucose and sucrose inhibited the elongation of etiolating hypocotyls of Col but had no effects on that of rgs1-2 mutant. The effect of 6% mannitol on root growth was more significant than that on seed germination, implying that the regulation mechanism of sugar on seedling growth was different to that on seed germination.The size and density of stomata of rgs1-2 was bigger and smaller than those of Col respectively, and the water loss rate detached leaves of of rgs1-2 was more rapidly than that of Col, thus rgs1-2 mutants were less drought tolerant than Col.The 35S-RGS1 overexpression system was constructed by TOPO cloning. RT-PCR analysis showed that the expression of RGS1 was enhanced in all tissues in 35S-RGS1 overexpressed transgenic plants. RGS1 overexpression affected the shapes of leaves, the development of floral buds, the elongation of stems, siliques and hypocotyls, and the time of flowering. Post-germination growth of 35S-RGS1 transgenic plants was inhibited by 1μmol L-1 ABA. RGS1 overexpression was hypersensitive to ABA in assays of root growth and confered lower water loss rate assayed with detached leaves and more drought tolerance. RT-PCR results indicated that RGS1 overexpresssion significantly stimulated the expression of NCED and ABA2, two genes encoding two key enzymes catalyzing ABA biosynthesis. Furthermore, the expression of several stress-regulated genes was either up-regulated or down-regulated in the RGS1 overexpression transgenic plants.The transgenic suspension cell culture of Col was constructed. Protein localization analysis using RGS1-GFP fusion protein technique showed that, the RGS1 protein was localized in plasma membrane and the subcellular localization of RGS1 protein was affected by ABA treatment both in protoplasts and in cells. The expression of RGS1 protein could be induced to different extent by different stress treatments.