9316947 VanVolkenburgh The PI, Dr. Elizabeth Van Volkenburgh proposes to investigate the cellular mechanism of auxin-induced proton excretion in protoplasts from Avena coleoptiles and tobacco plants. The goal of this research is to determine how the hormone, auxin, activates the ATP-dependent proton-pump in plasma membranes of growing cells. This hormone stimulates cell enlargement in plants, and plays a fundamental role in regulating development during the plants life cycle. It is hypothesized that this ligand initiates cascades of cellular events that lead to pump activation, proton excretion, membrane hyperpolarization, solute accumulation and cell growth. Electrophysiological techniques, primarily whole cell patch clamp methods will be used to determine the possible roles of suspected components of signal transduction cascades leading to changes in ion flux across the plasma membrane. Cytoplasmic contents will be equilibrated with the solution in the patch pipette containing either substances that might initiate ion flux in the absence of auxin, or substances that might block the response to auxin. The pH and pCa of the cytoplasm will be manipulated to determine the effect of these ions of the auxin response. These experiments will provide direct evidence concerning the role of cytoplasmic components in auxin induced proton excretion. %%% Protoplasts will be isolated from the known auxin-sensitive tissue, the Avena coleoptile, and from tobacco mesophyll which reportedly shows an unusually rapid response to auxin. Protoplasts will be characterized for responsiveness to auxin by measurement of auxin-induced protoplasts swelling. The relationship between swelling and proton excretion will be determined. The effect of auxin on the membrane potential of tobacco leaf cells will be compared to that of coleoptiles using microelectrode techniques. Both cell types will be investigated further with patch clamp experiments, and the results will confirm or reject the reports of auxin effects on tobacco protoplasts. The results of these experiments will serve as foundation for further biotechnological manipulation of vascular plants. ***
