Studies on the roles of RIC1 in root development and of Phosphoinositides in actin reorganization

DoctorPlants respond to various environmental and developmental stimuli through signal transduction. Plant hormones help to coordinate growth and developmental process in response to environmental stimuli. In plant hormone signaling pathways, there are many signaling molecules such as ROP small G proteins, ROP-interactive CRIB motif containing proteins (RICs), phosphoinositides, and protein phosphatase/kinase. In case of RICs, their roles as regulators of cellular developmental processes are well known in plant leaf and pollen tube cells, but their roles in other tissues and hormone signaling are not well understood. Another signal molecule, phosphoinositides are well known as key regulators in animal cells, but their roles in plants are less well understood. In this study, I focused on the study of RIC protein and phosphoinositides as signaling molecules in hormone mediated signaling pathways.RICs are a family of effector molecules of ROP small G proteins. Previously, it is shown that RIC1 protein that binds to ROP2 and ROP6 in leaf regulates leaf pavements cell developments. In tobacco pollen tube, RIC1 binds to ROP1 and regulates pollen tube polar growth. However, it is unknown that RIC1 has a function in other tissues. In response to auxin and ABA hormone, the transcript level of RIC1 was changed. RIC1 was expressed in the roots of young seedlings, particularly in root tips and at sites of lateral root formation. Whereas auxin-responsive gene induction and the effect of auxin on root growth and lateral root formation were suppressed in the ric1 knockout, ABA-responsive gene induction and the effect of ABA on seed germination, root growth, and lateral root formation were potentiated. Thus, RIC1 positively regulates auxin responses, but negatively regulates ABA responses. Furthermore, RIC1 appears to form feedback loops that regulate each of these hormone responses and to connect auxin and ABA signal transduction pathways. I showed that RIC1 functions downstream of ROP9 and ROP10 GTPases in these pathways. Similar to ric1, null mutants of ROP9 and ROP10 exhibited altered responses to auxin and ABA in terms of gene expression, root development, and seed germination. I also observed the localization of GFP:RIC1 in root epidermal cells. Originally RIC1 was localized at the plasma membrane in WT root epidermal cells while RIC1’s plasma membrane localization was significantly reduced the in rop9, rop10 null mutants. Together, my results suggest that RIC1 is a component of the intricate signaling network that underlies auxin and ABA crosstalk. In summary, I demonstrate that RIC1, one of ROP small G proteins effector molecules, has a function as a crosstalk factor in hormonal signal transduction of plants and has upstream targets which are ROP9 and ROP10 in root developmental signaling. These finding will contribute to understand the roles of RIC and ROPs in root system under auxin and ABA. Phosphatidylinositol 3-kinases (PtdIns 3-kinases) that produce phosphatidylinositol (3,4,5) triphosphate (PtdIns(3,4,5)P3) are considered to be important regulators of actin dynamics in animal cells. In plants, neither PtdIns(3,4,5)P3 nor the enzyme that produces this lipid has been reported. One of phosphoinositides is phosphatidylinositol 3-phosphate and 4-phosphate (PtdIns3P and PtdIns4P) which are produced by Phosphatidylinositol 3-kinase and 4-kinase (PtdIns 3-kinase and 4-kinase), respectively. In previous report, PtdIns3P and PtdIns4P regulate ABA-mediated response in guard cells. PtdIns3P in plants has a function in actin dynamics instead of animal PtdIns(3,4,5)P3. Phosphatidylinositol 4-kinase (PtdIns 4-kinase) is closely associated with the actin cytoskeleton in plant cells, suggesting a role for this lipid kinase and its product phosphatidylinositol 4-phosphate (PtdIns4P) in actin-related processes. Here, I investigated whether or not PtdIns3P or PtdIns4P plays a role in actin reorganization induced by a plant hormone abscisic acid (ABA) in guard cells of day flower. ABA-induced changes in actin filaments were inhibited by LY294002 (LY) and wortmannin (WM), inhibitors of PtdIns3P and PtdIns4P synthesis. Expression of PtdIns3P- and PtdIns4P-binding domains also inhibited ABA-induced actin reorganization in a manner similar to LY and WM. These results suggest that PtdIns3P and PtdIns4P regulate actin dynamics in guard cells. Furthermore, I demonstrate that PtdIns3P exerts its effect on actin dynamics, at least in part, via generation of reactive oxygen species (ROS) in response to ABA.In my studies, I demonstrate that RIC protein functions as a crosstalk factor in root development under auxin and ABA mediated responses and phosphoinositides, PtdIns3P and PtdIns4P, can regulate ABA mediated guard cell movements via actin reorganization. These reports will help to understand more complex mechanism of responses to stimuli in other tissues of plants