Localization Of Regulator Of G Protein Signaling (RGS) Protein And Its Functional Analyses In Glucose Signaling In Arabidopsis Thaliana

The regulator of G-protein signaling (RGS) protein, recently identified in Arabidopsis thaliana (named as AtRGS1) has a predicted seven-transmembrane structure as well as a RGS box with GTPase-accelerating activity and thus desensitizes the G-protein-mediated signaling. The roles of AtRGS1 protein in glucose signaling of seed germination were investigated in the present study, using seeds that carry a null mutation in the genes encoding RGS protein (AtRGS1), the alpha subunit (GPA1) of the G protein, serine/threonine phosphatase and molybdenum cofactor sulfurase in Arabidopsis, named rgs1-2, gpa1-4, abi1 and aba3, respectively, with different approaches such as genetics, biochemistry and molecular biology. The main results were as follows:The 35S:RGS1 overexpression system was constructed by TOPO cloning. The transgenic suspension cell culture from Col was constructed. Protein localization analysis using RGS1-GFP fusion protein technique showed that, the RGS1 protein was localized in plasma membrane, and the N-terminal domain (7 TM domain) and C-terminal domain (RGS box) of AtRGS1 were localized in plasma membrane and nuclear, respectively, which was consistent with the predicated localization with software (TMHMM Server v. 2.0, http://www.cbs.dtu.dk/services/TMHMM/).We further investigated the expression of specific genes responding to glucose treatment using a differential display reverse transcription PCR (DDRT-PCR) approach, in combination with reverse northern blotting. Four different expressing genes were identified between Col-0 and rgs1-2, including ADP-ribosylation factor, Uridine diphosphate glycosyl transferases, threonine endopeptidase and gamma-glutamyl hydrolase. It was suggested that these four genes-encoded proteins may paly important roles in glucose signaling and their possible roles were also discussed.The roles of AtRGS1 in glucose-mediated seed germination were analyzed by comparing the different responses of seed germination of Col-0 and rgs1-2 mutant to glucose and the effects of glucose analog and a specific HXK inhibitor, N-acetyl-glucosamine (NAG). The results showed that, in contrast to wild type Col-0, rgs1-2 seed germination was insensitive to glucose, while those of gpa1-4 and 35S:RGS1 were hypersensitive to glucose. The germination of Col-0, 35S:RGS1 and rgs1-2 mutant seeds showed the same responses to mannose. The inhibition of seed germination by glucose treatment were not obviously affected by NAG, but significantly alleviated the inhibiting effect of mannose on seed germination. These data suggested that AtRGS1 protein most likely functions in an HXK-independent glucose signaling in Arabidopsis seed germination.The relationships between AtRGS1 with ABA3 and ABI1 in glucose signaling were also investigated using various single and double mutants. The seed germination of aba3rgs1-2 double mutant was less sensitive to high concentration glucose, as compared with those of single mutant, rgs1-2 and aba3, which implying that the hyposensitivity of rgs1-2 mutant seed germination to exogenous glucose might be the results of the impairment of ABA biosynthesis and the normal degradation of endogenous ABA during seed germination. The seed germination of abi1 mutant was hypersensitive to high concentration of glucose, and that of abi1rgs1-2 double mutant was insensitive to glucose.