Snustad 9604491 Technical The long-term goal of the proposed research is to understand the roles of microtubule-based components of the cytoskeleton in plant growth and development. Morphogenesis in plants is controlled primarily by cytoskeletal processes that establish the planes of cell divisions and the axes of cell elongations, and microtubule arrays that are unique to plant cells - preprophase bands and phragmoplasts. Little progress has been made towards understanding the molecular mechanisms by which these processes occur. Moreover, we still know essentially nothing about the structure or function of microtubule-associated motor proteins in plants. The specific objectives of the proposed investigations are to first, identify insertional mutations in some of the 17 characterized tubulin genes of Arabidopsis. These will be used to investigate their effects on the phenotypes of homozygous mutant plants, and to construct and study multiple mutants carrying insertions in two or more tubulin genes. The second objective is to begin to investigate the complexity of the dynein gene families in Arabidopsis and characterize them to the degree needed to screen for insertional mutations in these genes. The third objective is to identify insertional mutations in some of the dynein heavy-, intermediate- and light-chain genes. The fourth objective is to characterize the Arabidopsis gene(s) encoding proteins homologous to the 8 kD dynein light-chains of animals and protists. RNA gel blot hybridization experiments will be used to determine the pattern(s) of transcript accumulation for the 8 kD light-chain gene(s), and immunofluorescence microscopy will be used to localize the 8 kD protein(s) in plant cells, especially in germinating pollen and elongating pollen tubes. The progress of individual objectives will depend on the results of the screens for insertional mutations. When an interesting mutation is identified, it will become the focus of future experiments. The major goal will be to id entify the functions of individual gene products, not to accumulate mutations in all genes. Non Technical The shape and pattern of growth of plant cells is defined by intracellular filaments termed the cytoskeleton. The shape and orientation of individual plant cells is critical to forming the morphology of plants. The cytoskeleton is constructed from several different proteins including tubulin and dynein that are synthesized as products of a gene family. Whether different members of the tubulin and dynein gene families have different functional roles is unknown. In this research the investigators will isolate mutants in which individual genes of tubulin are disrupted to be nonfunctional and used to construct plants in which multiple tubulin genes are disrupted. These experiments will lead to a precise definition of which tubulin genes are used in specific aspects of plant development. Parallel studies will be conducted on dynein. The size and diversity of the dynein gene family will be characterized and mutants with disrupted dynein genes will be constructed. This research is important because it will examine one of the basic processes that controls the shape, size and morphology of plants. ***
