Construction Of AtRGS1Signaling Network In Glucose And Abscisic Acid Cascade Pathway

The mechanism of RGS1in glucose and abscisic acid signaling were investigated in the present study using a series of genotypes including wildtype Col, rgsl-2and gpal-3mutants, and transgenic plants of RGS1/GPA1constructed with E320K, G220A and Q222L fragment. The main results were as follows:The overexpression and complementing expression system were constructed by TOPO cloning. The leave development including the lamina length and width, and the petiole length were affected by RGS1and the null mutation in the gene encoding GPA1resulted in rounder leaf, longer spike and higher aerial height, compared to wildtype Col. There was no significant difference between the phonetype of AtRGSl/rgs and E320K/rgs, while the the morphological trait of rosette leaves in transgenic plants complementing expression in gpa or overexpression in Col of GPA1, G220A and Q222L were not different from that of gpal-3, which indicating that the G protein components were involed in the arabidopsis morphological development, and the effects of RGS1and GPA1on rosette were differently.The seed germination and hypocotyl and root growth of arabidopsi were inhibited by6%glucose. The gemination of E320Klrgs, rgs1-2and AtRGS1-N/rgs were insensitive to glucose, that of AtRGS1/Col was hypersensitive to glucose, and that of E320K/Co1, AtRGS1/rgs and AtRGS1-C/rgs showed the sme responses as that of wild type Col.The inhibition effect of glucose on the seed germination and root development of AtGPA1/Col and Q222L/Col was not significant, while those of gpal-3and G220A/gpa were hypersensitive to glucose, which indicating that both Ga-GTP and Ga-GDP were involved in the repose of GPA1to glucose and there existed in both dependent and independent pathways in the reponse of RGS1to glucose.Compared to Col, the gemination and root growth of E320K/Col, AtRGS1/rgs and AtRGS1-C/rgs, AtGPA1/Col, Q222L/Col were insensitive, and those of AtRGS1/Col.gpa1-3and G220A/gpa were hypersensitive to ABA.The results of soil drought treatment and water loss of detached leaves showed that Col, rgsl-2and E320K/rgs were less drought tolerant than AtRGSl/Col, E320K/Col and AtRGS1/rgs. gpal-3, G220A/Col and G220A/gpa were less tolerant than AtGPA1/Col, Q222L/Col, AtGPAl/gpa and Q222L/gpa。Compare to Col, the stomatal aperture of AtRGS1/Col was lower than than of Col, and there was no difference between that E320K/Col and, which indicating that the mutantation/overexpression of RGS1showed insensitive/sensitive to the effects of ABA on stomatal opening and closing, and the regulation of RGS1on stomata depended on the G protein signaling.RT-PCR analysis showed that the expression of ARF and NCED3in RGS1, RGS-C and GPA1overexpression transgenic platns. The difference of the expression of P5CS, AtHK1and MAPK3in different genotypes was significantly when treated with6%glucose, implying that these three genes involved in RGSl mediated glucose signaling. The interaction between RGS-C and ARF was verified by yeast two hybrid assay.Based on previous and prsent results, we constructed the cascade pathway of AtRGS1singal in glucose and ABA. When cell percepted the extracelluar signal, glucose or ABA. the RGS1-box was sepreted from RGS1-7TM, and transported into cell with the help of ARF, which resulted in alternation of the expression of MAPK3/AtHK1/P5CS/NCED3/AOO3related to ABA and glucose signal. When the signaling was teminated, the RGS-C was back to cell membrane and recombinated with RGS-N.