The goal for the present proposal is to understand the molecular mechanism underlying pollen tube elongation. Pollen tube elongation is a process central to plant sexual reproduction. After pollen is deposited on the receptive stigmatic surface of the female reproductive organ pistil, the male gametes are transported by pollen tube elongation to the ovary to fertilize the egg cell. Pollen tube elongation is a directed outgrowth from the pollen grain on the stigmatic surface toward the ovary which is located at the basal end of the pistil, hundreds to thousand times the diameter of pollen grains away. Pollen tube elongation is often compared to axon outgrowth in animal systems since it is a directed cell movement based on a tip growth process in the extracellular matrix of pistillate tissues. Similar to understanding the mechanism underlying axon outgrowth, studies on pollen tube elongation face the same challenges of the difficulty to decipher among mechanisms that are based on specific chemotactic attractants, or to surface structure adhesion, or to a lack of deliberate attraction and pollen tubes follow a path of least resistance. We have isolated and characterized the first protein component (TTS protein) and its cDNAs from pistillate tissues of tobacco which can promote pollen tube elongation and display properties suggesting it may contribute to the overall pistil mechanism of guiding pollen tubes towards the ovary. TTS protein is an arabinogalactan protein specifically located in the intercellular matrix of the stylar transmitting tissue, a specialized medium through which pollen tubes elongate from the stigma to the ovary. Our results show that TTS protein attains its pollen tube growth promotion activity by adhering to the surface and to the growth tips of pollen tubes which uptake and hydrolyze the sugar residues on these proteins. TTS proteins also display a gradient of increasing glycosylation levels from the stigmatic to the ovarian end of the transmitting tissue, the same direction as pollen tube growth. The proposed research focuses specifically on elucidating the molecular mechanism underlying the interactions between TTS protein and pollen tubes, on determining the contribution of its pollen tube surface adhesion properties and the mobilization of its sugar components by pollen tubes to its activity in promoting pollen tube elongation, and on examining the functional significance of the gradient of glycosylation levels associated with this protein. A combination of molecular, cellular, biochemical and transgenic approaches will be used. The biochemical parameters involved in the interaction between TTS protein and pollen tubes will be determined. The structure-functional relationship of TTS protein will be examined by generating modified TTS proteins and determining how these changes affect their pollen tube growth promoting activity and their interactions with pollen tubes. The participation of TTS proteins in the direction of pollen tube elongation will also be examined in vivo and in vitro. The proposed experiments together should yield a clear understanding of how a major transmitting tissue protein mediates its functions and demonstrate whether a sugar_gradient associated with this protein is chemotactically significant in directional pollen tube elongation. Understanding pollen tube elongation should extend our knowledge on cell migration in general.
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