Expression Of RACK1 Protein Of Arabidopsis And Rice In Pichia Pastoris And Its Possible Functions In Regulating Stomatal Movement

Receptor for Activated C Kinase 1 (RACK1) is a tryptophan-aspartic acid-domain (WD40) repeat protein. Compelling evidence supports the notion that RACK1 is a versatile scaffold protein which binds numerous signaling molecules from diverse signal transduction pathways and plays critical roles in multiple developmental processes in mammals. High homologous proteins to mammal RACK1 have been identified in higher plants and are proposed to play regulatory roles in multiple developmental processes and in the responses to hormone,drought stress etc.Stomata are considered as a highly developed model system for characterizing early signal transduction mechanism in plants. Complicated signal trasduction network exists in the processes of stomatal opening and closure, which includes ABA-, G-protein- and NO-mediated signal transduction pathways and their cross-talk.In this study, in virtue of Pichia pastoris expression system, AtRACK1/OsRACK1 were cloned and integrated into Pichia pastoris GS115 genome successfully.With the induction of methanol, AtRACK1/OsRACK1 protein has been effectively secreted and expressed. Moreover, by using corresponding Arabidopsis single and double mutants, i.e., gpa1-4, agb1-2, rack1a-1, athk1, atnos1,αβ36, rack1a-1gpa1-4, rack1a-1agb1-2, athk1rack1a-1 and their wild type Col or WT, we studied the roles of RACK1 and related proteins in ABA-mediated stomoatal movement and in the regulation of stomatal development. The major results are showed as following:(1)ABA induced NO synthesis in guard cells of wild type Col and WT. GPA1 and AGB1 may mediate constitutive or inducible NO synthesis with different mechanisms. GPA1, AGB1 and RACK1 had positive regulating roles in ABA-induced NO biosynthesis in guard cells. ABA-induced NO synthesis partially depended on NOS1 and AtHK1.(2)Exogenous ABA and NO can induce stomatal closure in Arabidopsis wild type. GPA1, AGB1 and RACK1 were not the necessary components in the process of ABA–induced stomatal closure. NO treatment had no stimulating effect on stomatal closing in gpa1-4 and agb1-2 mutants, but had a significant effect on stomatal closing in gpa1-4 and agb1-2 double mutants, indicating that GPA1 and AGB1 jointly mediate NO inducing stomatal closure.(3)Exogenous ABA and NO can inhibit stomatal opening in Arabidopsis wild type. GPA1 played a key role in ABA-inhibited stomatal opening. RACK1 and AtHK1 may not play the key roles in ABA signalling. GPA1, AGB1 and RACK1 were not the necessary components in NO-mediated signalling pathway.(4)The stomatal densities of rack1a-1, rack1a-1agb1-2 and rack1a-1gpa1-4 mutants were significantly lower than that of Col, while the stomatal densities of athk1rack1a-1 and athk1 were higher than that of WT.(5)Great differences were observed in stomatal morphology among different genotypes. As compared with those of Col, WT and athk1rack1a-1, which the guard cell surface was almost smooth, gpa1-4, the surface of epidermal cells around guard cells of agb1-2 and rack1a-1 mutants was wave-like shape. The stomata of rack1a-1gpa1-4 and rack1a-1agb1-2 double mutants were obviously protuberant.According to above results obtained, it was suggested that RACK1 was involved in ABA-induced NO biosynthesis in guard cells, but had no roles both in ABA-induced stomatal closure and in ABA-inhibited stomatal opening.