PIPE Regulates Leaf Epidermis Cell Morphogenesis By ADFx In Arabidopsis

Pavement cells are the most abundant epidermal cell type, which is important for studying cell division, polarity maintenance, fate determination and morphogenesis. Pavement cells have been the ideal model for studying cell fate decision and morphogenesis in plants.From an ethyl methane sulfonate (EMS)-mutagenized Arabidopsis population, a mutant named pipe with loss of functional protein phosphatase was isolated. The pavement cell in pipe mutant exhibited significant defects in cell shape, and reduced lobe number and lobe length. We observed pipe mutant displayed abnormal microfilament assembly of cytoskeleton structure. By treatment with Lat-B on GFP-FABD2/Col and GFP-FABD2/pipe, we observed that microfilament dynamic updates in the pipe was slower than that in Col. By yeast two-hybrid assay, ADFx was isolated from an Arabidopsis flower cDNA library using PIPE as the bait protein. But ADFx didn’t interact with the mutant protein of pipe. These results suggested that ADFx may interact directly with PIPE. Moreover, pull-down assay further proved that PIPE and ADFx had directly interaction in vitro. Genetic analysis found that the double mutant adfx pipe partially rescued the defects of the pipe pavement cells, which suggesting that PIPE might be upstream to ADFx in the microfilament assembly signaling pathway.Furthermore, to determine that the abnormal pavement cells in pipe is due to increased pADFx but not different ADFx protein expression, we are going to analyze the ADFx expression and phosphorylated state by construction of pADFx::ADFx::GFP/Coland pADFx::ADFx::GFP /pipe transgenic plants. The pipe might alter the distribution of phosphorylated and unphosphorylated forms of ADFx in the pipe pavement cells.Through site-directed mutagenesis to mimic the phosphorylated state (S6D) and unphosphorylated state (S6A) of ADFx, we are constructing 35S::ADFxS6D::GFP/Col and 35S::ADFxS6A::GFP/pipe transgenic plants. These results will demonstrat that PIPE regulates the microfilament dynamic updates by ADFx phosphoregulation, which further involved in the morphogenesis of pavement cells.