9601087 Hepler Pollen tube growth is the process that delivers the male gametes to the egg apparatus and thus is essential for sexual reproduction in higher plants. Understanding the mechanisms addresses issues of both economic and fundamental importance. Recent work reveals that growing pollen tubes posses a steep, tip-focused gradient of intracellular free calcium (Ca), and a pronounced tip-directed influx of extracellular Ca, of which both are essential for growth. In addition the gradient fluctuates in magnitude, being correlated with changes in the growth rate. Ca thus appears to occupy a pivotal position in the control of pollen tube growth. Specifically, Ca may stimulate the occurrence and define the location of vesicle exocytosis required for cell wall formation and cell elongation. Ca might also control the formation of the cytoskeleton, which is responsible for transporting the vesicles to the apical region of the tube. We know much less about the role of hydrogen ions (H), but it is plausible that local gradients in pH exist and that they are essential for growth and development. It is a goal of the proposed work to elucidate the organization, activity and interaction of the Ca gradient, the putative H gradient, and cytoskeletal elements, and to understand how these factors contribute to the polarity and growth of the pollen tube. Particular emphasis will be given to events as they occur in living pollen tubes using intracellular microinjection as a means for introducing specific fluorescent analogs, probes and indicator dyes. Intracellular Ca, for example, will be analyzed by fluorescence ratiometric ion imaging on pollen tubes loaded with the indicator dye, fura-2 dextran, while H can be detected with BCECF-dextran or SNAFL-dextran. Through rapid, sequential image acquisition, Ca or H levels will be directly correlated with growth. Similarly, in cells injected with low amounts of fluorescent phalloidin, specific for F-actin, it will be possible to observe the relati onship between growth, cytoplasmic streaming and the organization of the microfilaments. The live cell studies will be augmented by those using immunofluorescence and immunogold to spatially localize key proteins, e.g., myosin, in an attempt to further elucidate function. The proposed studies address basic aspects of pollen tube growth, particularly within the context of the live cell. Whereas we know the existence of certain fundamental processes, e.g., tip-focused Ca entry, and structural complexes, e.g., the actin microfilaments, we understand much less about how these are integrated within the cell, and how they interact to produce the highly polarized and rapidly elongating pollen tube. The proposed work attempts to bridge this gap and thus to provide a new level of understanding that can apply not only to tip growing cells, but also to plant cells in general. ***
