Functional characterization of cell polarity regulators in arabidopsis stomatal development

Cell polarity plays an important role in a wide range of biological processes in plant growth and development. Cell polarity is manifested as the asymmetric distribution of molecules, for example, proteins and lipids, at the plasma membrane and/or inside of a cell. In Arabidopsis stomatal development, cell polarity underlies asymmetric cell division (ACD) that generates both physical asymmetry and cell-fate asymmetry. The polarity protein BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL) controls stomatal asymmetric cell division. However, the cellular machinery by which this intrinsic polarity site is established remains unknown.In the first Chapter, we summarize a few polarized proteins that have been characterized in plants and we review recent advances towards understanding the molecular mechanism for them to polarize at the plasma membrane. Multiple mechanisms, including membrane trafficking, cytoskeletal activities, cell wall integrity, protein phosphorylation, and so forth define the polarized plasma membrane domains. Recent discoveries suggest that the polar positioning of the proteo-lipid membrane domain may instruct the formation of polarity complexes in plants. We highlight the factors and regulators for their functions in establishing the membrane asymmetries in plant development. Furthermore, we discuss a few outstanding questions to be addressed to better understand the mechanisms by which cell polarity is regulated in plants. In the second Chapter, we identify the plant-specific PH, RCC1 and FYVE (PRAF) proteins as BASL physical partners and mutating four PRAF members leads to defects in BASL polarization. Members of PRAF proteins are polarized in stomatal lineage cells in a BASL-dependent manner. Developmental defects of the praf mutants phenocopy those of the gnom mutants. GNOM is an activator of the conserved Arf GTPases and plays important roles in membrane trafficking. We further find PRAF physically interacts with GNOM in vitro and in vivo. Thus, we propose that the positive feedback of BASL and PRAF at the plasma membrane and the connected function of PRAF and GNOM in endosomal trafficking establish intrinsic cell polarity in the Arabidopsis stomatal lineage. In the third Chapter, we characterized the four PRAF genes (PRAF4, 5, 8, and 9) that are broadly expressed in the vegetative tissues in Arabidopsis. Genetic results revealed that both genetic redundancy and specificity exist among the four PRAF members. In addition, each PRAF protein contains five functionally annotated domains. Domain deletion experiments showed that BRX domain is necessary to convey the biological outputs of PRAFs’ function. Through genetic engineering of the PRAF proteins at the subcellular level, we show that PRAFs function at both the plasma membrane and the endosomes. Interestingly, PRAF was found to show polarity at the cell cortex independent of BASL and possibly involves the binding with Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2),through its PH domain. Taken together, our results reveal that PRAFs integrates multiple regulators, to provide a mechanism that promotes cell polarity during stomatal lineage ACDs.Ph.D.Includes bibliographical reference