RUI: Antibodies to Flowering Plant Sperm: Development and Specificity of Epitopes

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In flowering plants, the process of fertilization underlies production of seeds and fruits. Although structural events of double fertilization have been described, the molecular mechanisms by which sperm and egg recognize each other, adhere and fuse are unknown. Plant sperm are embedded in pollen grains and move down growing pollen tubes. Near the egg, a pollen tube releases two sperm: one fuses with the egg to form the embryo, the other fuses with the central cell, adjacent to the egg, to form endosperm that supports growth of the developing embryo. A major unanswered question in plant reproduction is how sperm recognize eggs and central cells, adhere, and fuse. The goal of this proposal is to characterize molecules on sperm cell membranes. In Dr. Southworth's laboratory, three antibodies that bind to Brassica sperm cells have been identified. One anti-sperm antibody, BRSP1, has been partially characterized with regard to species specificity, presence of carbohydrate in the epitope, and molecular weight.The specific objectives of this proposal are (1) to characterize Brassica sperm membrane epitopes with regard to (a) timing of expression in the anther, (b) tissue-specific expression, (c) species-specificity of antibody binding, (d) molecular weight, and (e) location in the cell and (2) to involve undergraduates in research. Sperm and the cells leading to their development will be isolated from anthers, microspores, pollen, or pollen tubes and antibody binding will be determined by immunocytochemistry and by immunoblotting. The results will further knowledge of basic developmental mechanisms that function in plant reproduction and will lead to understanding of fertility and sterility: producing hybrids from incompatible crosses and preventing fertilization where male sterility is needed to prevent self-crossing. The identity of sperm-specific membrane molecules will be useful in future genetic studies. Undergraduate students will gain valuable insight into active research. By solving problems and assisting in attainment of goals, they will gain experience in terms of understanding the approach to research and in development of technical skills which will facilitate their development as scientists.