9724047 Yang Tip-growing pollen tubes grow directionally toward the ovules to delivery sperms to the embryo sac and thus are critical for sexual reproduction in higher plants. Pollen tubes also present an attractive model system for the study of polarized growth, partly because they are haploid and can be easily cultured. The long-term goal of this project is to elucidate molecular pathways that govern polarized cell growth in pollen tubes. We have identified a rho-type small GTP-binding protein, Rop1, that preferentially expressed in pollen and pollen tubes and is localized to the apex of pea and Arabidopsis pollen tubes. Antibody microinjection experiments indicate that Rop1 plays a pivotal role in pollen tube growth likely by modulating Ca2+-dependent exocytosis. The goal of this proposed work is to confirm the role of Rop1 in polarized tube growth using a reverse genetics approach and to investigate underlying molecular and cellular mechanisms for the function of Rop1 in pollen tubes. The role of Rop1 will be genetically tested using transgenic Arabidopsis plants that express dominant positive and negative mutant Rop1 alleles under the control of a pollen tube-specific promoter. The transgenic pollen tubes will be analyzed for changes in tip growth and in vivo guidance. The transgenic pollen tubes expressing Rop1 dominant mutant alleles will also be analyzed for changes in the behavior of secretory vesicles using TEM and the rapid freeze and freeze substitution technique and for changes in the influx of extracellular Ca2+ and the tip-focused intracellular Ca2+-gradients using a Ca2+-selective vibrating probe and ratiometric ion imaging. These careful analyses will test the notion that Rop1 controls polarized cell growth by modulating Ca2+ signaling-dependent polarized exocytosis. To further understand the underlying mechanism for Rop1-mediated polarized growth, Rop1 effectors will be identified by cloning genes encoding Rop1 partners using interactive cloning techniques such as the yeast two-hybrid method. Rop1 effectors are expected to interact specifically with GTP-bound Rop1 (dominant positive mutant) but not with GDP-bound Rop1 (dominant negative mutant). These studies will establish a solid foundation for our long-term goal of elucidating molecular pathways that lead to directional pollen tube growth. This work may also generate a broad impact on understanding the molecular basis of polarized growth in higher organisms as well as produce useful information for manipulating sexual reproduction in higher plants, such as engineering male sterility for hybrid production. The polar growth of pollen tubes is an interesting and significant process in which a germinating pollen tube extends in a single direction to mediate fertilization and reproduction in plants. The PI has identified a protein (ROP1, GTP-binding protein) involved in the signal transduction system that appears to control the polar growth of pollen tubes through calcium fluxes. With this preliminary data as a base the PI will use mutants of ROP1 to examine the function of the protein. Using light microscopy assays the role of ROP1 in inducing a calcium fluxes will be visualized and correlated with electron microscopy of the intracellular events that are required to extend the pollen tube. ROP1 must interact with other proteins in cascade that transmits signals through the cell. Proteins that interact with ROP1 will be identified and characterized. This research is of fundamental importance in describing a critical process in the reproduction of plants. Practical applications of the research include modifying plant reproductive growth to create superior plants. ***
