Molecular analysis of the structure and function of plant myosins

Myosin is a protein that can convert the chemical energy in ATP into mechanical energy. In plants, myosin is thought to function in actin-associated processes like cytoplasmic streaming and directed vesicle transport. My thesis project has utilized a reverse genetics approach in order to study the structure and function of plant myosin. To analyze the structure and complexity of plant myosin, four different Arabidopsis myosin cDNA clones were isolated and sequenced. Sequence analysis of these cDNA clones and three additional genomic PCR products indicated that Arabidopsis possesses a large myosin gene family that is composed of at least three distinct classes based on the overall structure of the predicted proteins (two novel classes and one previously-identified class; class V).;The expression of these myosins was analyzed by Northern blotting and RNA in situ hybridization. Northern blot analysis indicated that the four Arabidopsis myosins were differentially expressed in a variety of plant organs. RNA in situ hybridization of floral tissue sections indicated that the MYA1 and MYA3 genes were expressed in a developmentally regulated manner in distinct cells of the sepals and anther filaments. In leaves, the MYA1 and MYA3 genes were expressed in the mature trichomes, where they may function in the cytoplasmic streaming that has been visualized in these large, unicellular structures. In whole seedlings, the MYA1 gene was found to be expressed throughout the expanded cotyledons (with the exception of the vascular tissue).;To investigate the function of plant myosin, transgenic plants were generated that contained constructs designed to either repress or overexpress/ectopically express the Arabidopsis MYA1 myosin. The expression of the MYA1 gene in the different transgenic lines was found to be indistinguishable from wild type, even in plants expressing high levels of MYA1 antisense RNA. These results suggest that the expression of the MYA1 gene is tightly regulated or that changes in MYA1 expression are detrimental to Arabidopsis.