Cellular Mechanism of Auxin-Induced Proton Excretion

项目负责人Elizabeth VanVolkenburgh博士提出研究生长素诱导的葡萄叶和烟草原生质体中质子排泄的细胞机制。本研究的目的是确定生长素如何激活生长细胞质膜中atp依赖的质子泵。这种激素刺激植物细胞扩大，在植物生命周期中起着调节发育的重要作用。据推测，该配体启动了导致泵激活、质子排泄、膜超极化、溶质积累和细胞生长的细胞事件级联。电生理技术，主要是全细胞膜片钳方法，将用于确定信号转导级联的可疑成分可能的作用，导致离子通量在质膜上的变化。细胞质内容物将与膜片移液管中的溶液平衡，溶液中含有在缺乏生长素时可能引发离子通量的物质，或可能阻断对生长素反应的物质。通过控制细胞质的pH和pCa来确定这些离子对生长素反应的影响。这些实验将为细胞质成分在生长素诱导的质子排泄中的作用提供直接证据。原生质体将从已知的生长素敏感组织，草叶胚芽和烟草叶肉中分离出来，据报道，叶肉对生长素的反应异常迅速。通过测量生长素诱导的原生质体肿胀来表征原生质体对生长素的响应性。肿胀和质子排泄之间的关系将被确定。利用微电极技术比较了生长素对烟草叶片细胞膜电位的影响。这两种细胞类型将通过膜片钳实验进一步研究，结果将证实或拒绝有关生长素对烟草原生质体作用的报道。这些实验结果将为维管植物的进一步生物技术操作奠定基础。＊＊＊

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. ***