| The SAC9 gene in Arabidopsis thaliana encodes a protein, Sac9, which is a putative 5-phosphatase involved in phosphoinositide stress signaling pathways in plants. The SAC domain of Sac9 has sequence homology with yeast and mammalian phosphoinositide phosphatases that catalyze the hydrolysis of phosphatidylinosito14,5bisphosphate [PtdIns(4,5)P2]. The loss of the Sac9 protein in A. thaliana leads to an increase in the quantity of PtdIns(4,5)P2 in the plant. This increase in PtdIns(4,5)P 2 in the sac9-1 mutant A. thaliana likely causes plants grown under non-stress conditions to exhibit several phenotypes normally associated with stress. The focus of this thesis is the examination of the cellular phenotypes in Sac9-1 mutant plants. I observed that sac9-1 mutant plants display an increase in endocytosis, a possible increase in membrane permeability, and an overall increase in cell membrane trafficking, which sometimes correlates with stress. Phospholipase C is another enzyme that catalyzes the breakdown of PtdIns(4,5)P2; therefore, A. thaliana phospholipase C mutant plants should also have increased levels of PtdIns(4,5)P2. These plants have demonstrated cellular phenotypes similar to the sac9-1 mutant plants.; The Sac9 protein was expressed in yeast and also immunoprecipitated out of wild-type A. thaliana and incubated in a phosphatase assay with PtdIns(4,5)P2 as the substrate. Preliminary data using a malachite green assay to detect free phosphate and thin-layer chromatography suggest that the Sac9 protein has 5-phosphatase activity specific to catalyzing the hydrolysis of the 5-phosphate of PtdIns(4,5)P2. |
