| 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.Specific structure determines its complex functions.The roles of two domains of AtRGS1 proteins in D-Glucose and ABA signaling in Arabidopsis were investigated in the present study, using seeds that carries a null mutation in the AtRGS1 gene and overexpress the genes encoding two domains of AtRGS1, named rgs1-2, 35S:RGS1-N and 35S:RGS1-C, respectively, with different approaches such as biochemistry and molecular biology. The main results were as follows:The 35S:RGS1 overexpression system was constructed by TOPO cloning. N-terminal and C-terminal cDNA of AtRGS1 was first cloned into TOPO vector respectively, then subcloned into binary vector that carrying CaMV 35S promoter and GFP reporter. Fusion genes 35S:RGS1-N-GFP and 35S:RGS1-C-GFP were introduced into rgs1-2 plant by Agrobacterium-mediated Transformation. Trans-35S:RGS1-N and trans-35S:RGS1-C plants were screened by resistance selection, observation of root fluorescence and identification of GFP. The acquired transgenic plants were used as materials for physiological analysis.The roles of two domains of AtRGS1 in D-Glucose and ABA signaling were analyzed by comparing the different responses of four genetypes to D-Glucose and ABA in seed germination and primary root growth. Comparing their responses to glucose, mannose 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 35S:RGS1-N and 35S:RGS1-C were hypersensitive to glucose. But they showed the same responses to mannose. NAG didn't obviously affect the inhibitory effect of glucose on seed germination, but significantly alleviated the effect of mannose. These data suggest that two domains of AtRGS1 protein most likely functions in an HXK-independent glucose signaling in Arabidopsis seed germination. Four genetypes show the same tendency of seed germination to ABA as glucose. ABA and D-Glucose had additive effect on seed germination of four genetypes. When endogenous ABA biosynthesis was inhibited by Fluridone, the inhibitory effect of glucose on seed germination were markedly alleviated, implying that glucose inhibites seed germination through increasing content of endogenous ABA.Root growth of four genetypes showed significant differences. Comparing to Col and rgs1-2, primary root growth of 35S:RGS1-N and 35S:RGS1-C had remarkable short roots. Low concentration of glucose and ABA promoted primary root growth, and four genetypes always keep the same growth tendency. These data suggest that mutation of AtRGS1 and overexpression of AtRGS1-N and AtRGS1-C alter primary root growth of Arabidopsis seedlings and responses to glucose and ABA.A significant difference in drought tolerance was observed among four genotypes investigated. Transgenic plants 35S:RGS1-N and 35S:RGS1-C showed the highest tolerant to soil drying among four genotypes. Water loss assay with the detached leaves indicated that Col and rgs1-2 lost water more rapidly than those of 35S:RGS1-N and 35S:RGS1-C. Under soil drying condition, the leaf water potential of all four genotypes decreased gradually with the progress of soil drying. However, the leaf water potential of 35S:RGS1-N and 35S:RGS1-C were always higher than that of col and rgs1-2. Accordingly, the leaf ABA content of 35S:RGS1-N and 35S:RGS1-C were significantly higher than col and rgs1-2.According to the above results and other reports, it is suggested that both N-terminal and C-terminal domain play an important role in glucose and ABA signaling and both proteins may involve in the signal transduction of drought stress.
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