Jacob A. MayfieldGraduate Student
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Research Summary: The focus of my thesis research has been communication between cells, at the level of individual molecules and specialized cellular structures. Without the benefit of mating behavior, plants have evolved a complex system of cell signaling. Each pollen grain carries not only the information necessary to complete fertilization in a dry environment, but also a tag that allows female stigma cells to discriminate the gametes of other plant species. The specificity of the pollen-stigma interaction, the unique biophysical mechanics surrounding cellular communication in a dry environment and the extensive list of genetic tools available for Arabidopsis led me to the laboratory of Dr. Daphne Preuss. Communication with the stigma requires an intact pollen coat, a lipid- and protein-rich extracellular matrix. Genetic lesions that drastically reduce pollen coat material are sterile. However, individual pollen coat components had been recalcitrant to further analysis: genetic screens for sterile plants identified only pleiotropic mutations, and the high lipid content prevented easy biochemical purification. I began my investigation by developing a scheme that allowed purification and concentration of pollen coat proteins in sufficient quantity for microsequence. I then sought mutations that disrupted individual pollen coat proteins. One such mutation, grp17-1, provides great insight into pollen coat function. Unlike all previous pollen coat mutations, the grp17-1 lesion eliminates only the GRP17 protein, allowing its specific function to be addressed. In the absence of this lipid-binding oleosin protein, interaction with the stigma was less efficient and the overall fitness of the pollen compromised. Analysis of other, similar pollen coat oleosins is ongoing. The pollen coat is remarkable not only for its contents, but also its method of function. After adhesion to a compatible stigma, the pollen coat mobilizes to the point of contact and facilitates rapid transfer of water from the stigma to the dehydrated pollen grain. My study of the molecules regulating communication has expanded into a study of how pollen coat proteins and lipids combine to aid this process. This coalescence of distinct molecules to perform striking cellular and developmental processes is where my interests lie. Within my own thesis, I have started experiments aimed at correlating the biophysical properties of the pollen coat with the lipid and protein composition. By approaching the pollen coat as a whole, as well as the sum of individual components, I have contributed to the understanding of how plants have adapted their cellular machinery to allow communication.
Technical Skills: Growth, culture and genetic manipulation of Arabidopsis,
Drosophila,
E.
coli and Agrobacterium. Molecular biology techniques including
cloning, vector construction, library construction and screening, DNA sequencing,
Southern blot, Northern blot, Western blot, PCR, RT-PCR, and chromosome
walking. Microscopy techniques including light microscopy, immunofluorescence,
transmission electron microscopy, tissue preparation and sectioning. Biochemical
techniques including SDS-PAGE, protein purification, and immunoaffinity
purification. Genetic techniques including recombinant inbred mapping,
marker segregation analysis and plant tetrad analysis.
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