The long-term goal of this research is to identify and characterize the mechanisms for trafficking of plant vacuole-tonoplast membrane proteins. Chemical inhibitors of tonoplast protein trafficking and mutants with abnormal localization of a tonoplast protein were previously identified. The bioactive probes indicated that multiple pathways, including a novel Golgi-independent pathway, target vacuole-membrane proteins. This project will identify the mechanisms by which membrane proteins are targeted to the vacuole, and how these contribute to its biogenesis. The work plan will test the hypothesis that a Golgi-independent pathway engages in tonoplast protein trafficking, and will investigate a novel role for phosphoinositide lipids in plant vacuole biogenesis. The specific aims for this research are: 1) To identify the roles of IMPAIRED TONOPLAST TRAFFICKING4 (ITT4) and ITT6 genes in tonoplast-protein targeting by characterization of two mutants with defects in tonoplast protein localization. 2) To determine the role of phosphoinositide lipids in vacuole biogenesis. 3) To characterize the dynamics of the Golgi-independent pathway for tonoplast-protein targeting using real-time microscopy of live cells. Given the essential roles of vacuole membrane proteins in regulating cell turgor, and the storage potential of the vacuole, these results may have future impacts on the biology of environmental stress responses in plants and its applications to agriculture.
BROADER IMPACTS Chemical and genetic screens employed in this project will benefit the field by generating novel chemicals and mutants that will be available to the scientific community. Some of the experiments in this project are amenable for high school and undergraduate research activities, and will promote the training of students in state-of-the-art techniques in molecular biology and fluorescent high resolution live-cell imaging. The PI will use these activities to continue to engage minority high school students from the NC School of Science and Mathematics with the goal of increasing the interest of minority students in pursuing science degrees within the University. The PI's laboratory will also host visits from River Oaks Middle School students to spark the interest of these students towards science-related fields, and to convey the impacts of scientific research to the public.
