The vacuole is an essential organelle in plants and has major implications for human health. In fact, an important portion of our diet, including the essential amino acids found in beans and corn, comes from molecules stored in the vacuole. The integrity of the vacuolar membrane is important for the storage of metabolites and hormones, the sequestration of ions and the maintenance of cellular turgor. To improve plant stress tolerance and ultimately increase the nutritional value of plants for human consumption, it is essential that we understand how to regulate vacuolar membrane biogenesis.
The long-term goal of this research is to identify and characterize the trafficking machinery for vacuolar membrane proteins. An integrated strategy of genetic, biochemical and cell biological approaches will be utilized to identify and characterize mutations and chemical inhibitors that disrupt the trafficking of membrane proteins to the vacuole. The specific aims are to: 1) Identify mutants impaired in the targeting of membrane proteins. 2) Identify small inhibitors that induce mis-localization of vacuolar membrane proteins. Both mutants and inhibitors will uncouple the distinct targeting pathways of membrane proteins to the vacuole. This research will have a major impact in the understanding of vacuole biogenesis and the ability to improve plants with enhanced vacuolar content.
The broader impacts of this project include the training of minority undergraduate and graduate students in state-of-the-art techniques in molecular biology and microscopy. In addition, a teacher and minority high-school students from the Wake County public schools system will be engaged as part of this research program.
Intellectual Merit The plant vacuole is the major storage compartment for proteins, hormones, metabolites and ions in plant cells. The biogenesis, maintenance and function of this essential organelle have large implications for overall plant health and the tolerance of plants to environmental stresses. The vacuole biogenesis and maintenance depends on constant trafficking of membrane proteins to the vacuole. Many of these proteins are transporters of metabolites and ions, and critical for cellular homeostasis. However, the mechanisms for trafficking of these important proteins to the vacuolar membrane remain unknown. The long-term goal of this research is to characterize the targeting of membrane proteins to the vacuole in the model plant Arabidopsis. The aims of this award were to: 1) Characterizing three mutants that are hypersensitive to a small chemical inhibitor; 2) Identify mutants impaired in the targeting of tonoplast proteins using a new dual-fluorescent marker line; 3) to identify novel chemical inhibitors of tonoplast-protein trafficking. The project included the development of a novel screen using dual-label marker line carrying two compatible fluorescent protein fusions. Mutants with trafficking defects of abnormal vacuole morphology were identified. These mutants are informative because they point to specific compartments or protein complexes involved in the targeting of vacuolar membrane proteins in plants. In addition, novel small molecules that inhibit the delivery of membrane proteins to the vacuole were identified. One inhibitor was identified that can specifically block the trafficking pathway for two vacuolar membrane proteins of the tonoplast intrinsic family. This compound is being used to characterize the subcellular compartments involved in targeting of membrane transporters to the vacuole. The different sensitivities of tonoplast intrinsic proteins to our drug and to Brefeldin A, a well characterized inhibitor of the ER to Golgi pathway, demonstrated that members of this family traffic either in a Golgi-dependent or independent pathway. Studies of protein trafficking have important implications for biotechnology because successful expression of gene products in transgenic crops is dependent on their targeting to the appropriate compartment where they can be active. Our inhibitors can be used in cell biology research as specific pharmacological tools for interrogating the function of specific pathways in plant cells. Broader Impacts This project contributed to the training of three graduate students in live-cell fluorescence microscopy, molecular biology and biochemistry. Four undergraduates, including two REU students, actively participated in our research efforts, and gained experience in plant cell biology. In addition, three groups of minority students from the North Carolina School of Science and Mathematics participated in a three-day immersion in plant research in our lab. Each year, one group of students from River Oaks Middle School visualized the morphology of plant organs and cellular organelles under different microscopes including a confocal microscope from the Cellular and Molecular Imaging Facility at NCSU.
