Functional Analysis On AtRhoGDI1That Is Involved In Arabidopsis Cell Morphogenesis And The ABA Signal Transduction

Plant RhoGTPases (ROPs) involve in diverse aspects during plant development. Regulations of ROPs in morphogenesis of leaf epidermal cells through modulating cytoskeleton dynamics in Arabidopsis have been characterized. As one of pivotal regulators for ROPs activities, the role of RhoGDIs (RhoGTPase GDP dissociation inhibitors, GDIs) that might be critical in the process remains elusive. GDIs are considered to regulate GDP/GTP and membrane/cytosol cycling of RhoGTPases. GDIs have active contributions to targeting RhoGTPase proteins to specific subcellular localizations and signaling pathways. In plant cells, however, the regulatory mechanism of GDIs is poorly understood. How GDIs interact with candidate displacement factors, and how GDIs are modified at post-translational level are unknown. Although AtRhoGDI1(GDI1) has been reported to regulate root hair growth and pollen tube elongation, little is known about the regulation of GDI1in other developmental aspects and signaling pathway, such as in development of seedlings and leaf epidermal cells as well as in the ABA signal transduction. More important, the phosphorylation mechanism on plant GDI1has poorly understood. In this work, we analyzed the role of Arabidopsis GDI1in development of seedlings and mophorgenesis of leaf epidermal cells. We analyzed the mechanism that modulates GDI1activity and found out that the calcium-dependent protein kinase3, CPK3is able to phosphorylate GDI1. Phosphorylation of GDI1by CPK3is essential for the ROP signaling. Moreover, we characterized the role of GDI1in responding to ABA in guard cells, and provided evidences in which the negative regulation of GDI1in the guard cell ABA signaling is associated to the spatial-temporal dynamic change of actin cytoskeleton. We further attested the linkage of ABA-responsive protein complex (including ABA receptor PYR1, ABI1, SnRK2.6) and the activity of GDI1in vitro. Our data indicated that ABA promotes activation of SnRK2.6, in turn to triger phosphorylation of GDI1by activating SnRK2.6.The significance and advances in our work are described in below.1. Identified function of GDI1in seedling development and leaf pavement cells morphogenesis, through analyzing gdil-1mutant and GDI1-overexpression transgenic plants (GDI1-14).2. Analyzed the spatial-temporal interaction between GDI1and ROP2or ROP6respectively, through pull-down, BiFC and FRET imaging assays. Analyzed that GDI1could interact with ROP6significantly at the lobing region in cytoplasm of pavement cells. In contrast, the interaction between GDI1and ROP2was detected in the manner of uniform distribution in the PM and cell cortex region. These results suggest that GDI1may have spatial interactions with individual ROPs in order to regulate patterning pavement cells.3. Determined correlations of cytoskeleton dynamics of actin MFs and microtubule MTs with activities of GDI1-regulated ROPs. Overexpression of GDI1produced actin bundles and disorganized MTs in pavement cells.4. Characterized genetic cross-lines (hybrid lines) of GDI1ROP6-WT, GDI1CA-rop6, GDI1DN-rop6. Observed that GDI1-14plants possessed similar phenotypes to those shown in CA-rop6transgenic plants. Notably, ROP6-WT and DN-rop6could rescue abnormal development of seedlings and pavement cells in GDI1-14background, suggesting that GDI1might play positive role in the ROP-modulated developmental processes in Arabidopsis.5. Significantly, we identified three phosphorylation sites which were not revealed before. These phosphorylation sites (Ser45/Ser48/Thr52A) are critical for phosphorylation of Arabidopsis GDI1. Mutations at these amino acids could partially abrogate GDI1function, leading to irregular development of cotyledens (such as, production of three cotyledons, single or fused cotyledon).6. Remarkably, determined that CPK3(member of calcium-dependent protein kinases family) is able to phosphorylate GDI1protein. Thus, it might be a mechanism converging the calcium-mediated signaling and ROP activation.7. Confirmed that gdil-1and its allele scn1mutants are insentitive to the ABA treatment, suggesting the role of GDI1in the ABA-induced stomatal closure. Contrarily, plants with GDI1-overexpression (GDI1) turned to be hypersensitive to ABA.The ABA-responsive hypersensitivity shown in guard cells of GDI1-overexpression plants can be recovered in the CA-rop6GDI1hybrid line.8. Through the living cell time lapse observation with confocal microscopy, we recorded the rearrangement of actin filaments in guard cells while they were responding to ABA treatment. Analyzed with3D reconstruction, we found out that the actin filaments in open guard cells were not only organized as radial array but also surrounded the cell entirely.9. Actin filaments in guard cells of mutant scn1-1were less affected by ABA. In most of WT guard cells, MFs were in the transverse radial array format and gradually turned to longitudinal bundles in responding to ABA. However, in scnl-1most of the guard cells still have radial arrayed MFs after ABA treatment.10. Acting as a positive regulator in the ABA signal transduction pathway, kinase SnRK2.6is able to phosphorylate GDI1, which was confirmed in the in vitro kinase assay. SnRK2.6phosphorylated GDI1possessed higher dissociation inhibitor activity, resulting enhanced inhibitory effect to the activity of ROP6.11. ABA treatment stimulated phosphorylation of GDI1by SnRK2.6. In vitro assay suggested that, after ABA treatment, ABA receptor PYR1recruit ABU (PP2C) and promote activation of SnRK2.6, thus, phosphorylation of GDI1is incurred.