9304953 Hepler Pollen tube elongation delivers the male gametes to the egg apparatus and is thus essential for sexual reproduction in plants. Within this highly polarized cell, vesicles are produced throughout and transported by cytoplasmic streaming to the apical region. By a process that is not well understood, these vesicles fuse specifically at the tip, providing new wall material that supports unidirectional elongation. Recent work has shown that a growing pollen tube of Llium has a sharply defined gradient in free calcium focused at the extreme apex of the tube. The further observation that dissipation of this calcium gradient leads to cessation of growth, and that reinitiation of growth is accompanied by its reformation provide evidence that the gradient is essential for tip growth. The purpose of this research is to analyze the relationship between the calcium gradient and tip extension in detail. Using indicator dyes that have been microinjected into pollen tubes the spatial profile of the calcium gradient will be examined in living cells and directly correlated with the rate of tube elongation. Cells will then be experimentally treated with a variety of agents including calcium buffers, channel agonists, antagonists, osmotic modulators, and growth inhibitors to allow a careful exploration of the relationship between the position and magnitude of the calcium gradient with the modulated growth behavior, and with the underlying cell structure and patterns of cytoplasmic streaming. The position and activity of calmodulin will also be studied in living cells using confocal laser scanning microscopy and fluorescence anisotropy. Finally, the relationship between the calcium gradient and the actin cytoskeleton will be examined in living pollen tubes using confocal microscopic analysis of cells that have been microinjected with fluorescent actin monomer. By correlating the calcium gradient with these several factors, a new and greater understanding of the mechanism of pollen tube elongation should result. %%% In higher plants, the male reproductive cells (gametes) are carried in pollen grains. For fertilization to take place, the pollen must deliver the male gametes to the egg apparatus within a flower by producing a tube, along which the male gametes move toward the egg. The pollen tube is the fastest growing of all plant cells. As it grows, it becomes several thousand times longer than it is wide. Because of its importance to plant reproduction and its dramatic growth characteristics, the mechanisms regulating pollen tube are important topics of research. The goal of this project is to understand the role of calcium ions in pollen tube growth. Sophisticated biophysical and structural techniques will be used to study the gradient of internal calcium along a growing pollen tube. The results should determine whether the calcium gradient plays a role in the organization and movement of the sub-cellular structures involved in pollen tube growth. ***
