Functional characterization of Arabidopsis thaliana ROP GTPase activating proteins during cell division and pavement cell morphogenesis

Cell division and cell expansion are fundamentally important processes of plant development. Although, many proteins have been functionally characterized and implicated in those processes, there is still a lot to investigate. In plant cell division, the future division site is marked by the preprophase band, a belt of cortical microtubules. In the final phase of cell division, called cytokinesis, serves the position of the preprophase band as fusion site of the cell plate. The centrifugal growing cell plate is guided by the expanding phragmoplast, a set of bipolar microtubules, from the centre towards the cell periphery and connects at the fusion site with the parental cell wall. However, the preprophase band is disassembled before metaphase. Prior to this, proteins such as the PHRAGMOPLAST ORIENTING KINESIN 1 (POK1) are recruited to the preprophase band to establish the division site and mark the future division plane throughout mitosis and cytokinesis. In the present thesis, we identified two Arabidopsis thaliana pleckstrin homology domain containing GTPase activating proteins, PHGAP1 and PHGAP2, as interaction partners of POK1, a core component of the cortical division site. Unlike POK1, which is only present in dividing cells, PHGAPs were localized at the plasma membrane and in the cytoplasm in interphase cells and started to accumulate in a POK-dependent manner at the division site during metaphase, where they remained till the end of cytokinesis. Further experiments revealed that the PHGAP carboxyl-terminus, containing coiled coil domain, was required for division site accumulation and function. Additionally, interactions studies and PHGAP phospho-mutant analysis suggested that the timely localization of PHGAPs at the division site was phospho-regulated. Overlapping expression patterns of PHGAP1 and PHGAP2 and a lack of single mutant phenotypes pointed to redundant functions. Analysis of phgap1 phgap2 dividing double mutant cells revealed that preprophase bands and consequently phragmoplasts were misaligned, implicating a function of PHGAPs in preprophase band orientation. In general, PHGAPs are known to mediate Rho of plant (ROP) inactivation by stimulating the guanosine triphosphate (GTP) hydrolysis function of these signal proteins. In vivo interaction studies uncovered that PHGAPs were able to interact with several ROPs, providing the first evidence that not only PHGAPs but also ROP activity might play a role during root cell division. The second part of this thesis describes functional aspects of PHGAP1 and PHGAP2 mediated cell polarity during pavement cell shape establishment. Two antagonistic ROP signal transduction pathways are responsible for their characteristic jigsaw puzzle form, involving the establishment of cell polarity and thereby determining growth direction. The distinct polarized lobe and indentation regions of epidermis cells, where drastically reduced in phgap1 phgap2 double mutants, which pointed to miss regulation of the above mentioned ROP activity in phgap1 phgap2. Investigations revealed interaction of PHGAP2 with ROP2 and increased ROP2 activity in phgap1 phgap2 double mutants. These data indicated that PHGAPs stimulate ROP2 GTP-hydrolysis, leading in ROP inactivation during pavement cell development. Analysis of localization pattern uncovered that both PHGAPs accumulate in a microtubule-dependent manner on the anticlinal face in indentation regions. The amount of PHGAP accumulation in indentation regions increased with their development, which correlates with an increasing surface that has to be free of ROP2 activity to maintain cell polarity. Also in leaf pavement cells was the carboxyl-terminal domain of PHGAPs necessary for anticlinal face localization in indentation regions. A point mutation in the PHGAP GTPase activating domain abolished PHGAP function completely, but had no effect on the distinct localization pattern. Collectively, the data of this thesis describe significant roles for PHGAPs on the mechanisms orchestrating cell division and cell polarity establishment that promote cellular morphogenesis