Intellectual merit. This project focuses on the control of pollen tube growth in flowering plants. Pollen tube growth, which is confined to the extreme apex of the tube and is fast, occupies a crucial position in plant development because it delivers the sperm cells to the egg apparatus for fertilization. The process thus is essential for sexual reproduction and also for food production because the development of the fruits, nuts, seeds and grains that we eat requires a prior fertilization event. Successful tube growth and sperm delivery can only occur if expansion at the tip, driven by high turgor pressure, is continuously balanced by deposition of new wall materials. An important observation has been the discovery that the growth rate and the underlying physiological processes oscillate, making it possible to determine which processes lead and which follow the increase in growth rate. The ultimate goal is to elucidate the mechanisms that regulate cell wall expansion. The processes that seem particularly important include secretion of new wall material, mitochondrial production of energy, actin organization, calcium influx, and pH changes. Although it is possible that a single process acts as the sole pacemaker, it seems more likely that the oscillatory behavior is an emergent property dependent on the interaction of many different processes. As a consequence a multifaceted approach will be employed to determine how parallel events interact. Firstly, the above five processes will be probed during growth reorientation. The goal is to determine which process begins first, and where in the cell it becomes evident. To perturb pollen tube growth endogenous chemotropic factors, imposed ion gradients, and elevated osmoticum will be used. A structural analysis of cytoplasmic components and cell shape will be included. Secondly, energy metabolism, specifically the oxidation/reduction of nicotinamide adenine dinucleotide phosphate (NAD(P)H), will be examined, and its effect on growth assessed. Companion studies with inhibitors and with an extracellular oxygen-selective electrode will further probe the characteristics of energy metabolism. Thirdly, the role of calcium in mitochondrial energy metabolism will be examined. Because some NAD(P)H dehydrogenases are calcium sensitive, the inter-relationship between mitochondrial calcium, cytosolic calcium, and NAD(P)H will be explored. Intellectual merit accrues from the ability of the research to elucidate structures and physiological processes that contribute to the control of pollen tube growth. The research takes advantage of the accessibility of pollen tube growth in vitro, the apical location of the growth events, and the oscillatory nature of the growth rate and underlying physiological processes. It further exploits the extensive toolkit of genetic and fluorescent probes, high resolution imaging and microelectrode techniques to decipher cause and effect relationships between interacting events. Although the pollen tube is specialized for the delivery of sperm cells, it shares mechanisms with other tip-growing cells, notably root hairs, and also very likely with diffusely growing plant cells. Information gained from this study will therefore have wide reaching relevance.
Broader impacts. The broader impacts of the research activity derive in large part from its significant educational components. The Principal Investigator has had extensive experience guiding students. In addition, he holds a close connection to a consortium of 10 universities in the northeast US involved in the recruitment and retention of under-represented minorities into graduate education. The co-Principal Investigator has worked extensively in promoting science education with inner city school students, undergraduate and graduate students, and middle school teachers. He currently directs several undergraduates in honors research. Finally, the investigators have involved a pool of research motivated undergraduates at nearby Hampshire College, including under-represented minorities in their Baldwin Scholars Program. Through these different avenues, interested undergraduate students from all ethnicities will be encouraged to participate in the research/discovery process.
