Ion Channel Regulation in Higher Plants

小行星9506191 植物叶片气孔关闭以减少植物在干旱等环境胁迫条件下的蒸腾水分损失。 保卫细胞响应环境刺激和胁迫条件控制气孔运动是决定作物水分利用效率和生产力的主要因素。 保卫细胞是研究高等植物早期信号转导的理想系统。 已经鉴定出几种关键的保卫细胞离子通道，它们被认为是气孔关闭的重要信号转导器和介质。 最近的数据表明，这两个Ca 2+依赖和Ca 2+非依赖的转导级联可能控制气孔关闭。 然而，在气孔关闭过程中，细胞内的信号级联信号的接收链接到离子通道的调制仍然在很大程度上是未知的。 此外，保卫细胞液泡释放离子的分子机制仍然未知，这是气孔关闭所必需的。 本研究的长期目标是表征信号级联的事件链，其整合生理刺激，如脱落酸，细胞内偶联蛋白，第二信使和离子通道，以产生气孔关闭。 为了阐明保卫细胞中的信号传导机制，我们建议使用细胞生物学、膜片钳、生物化学和遗传分析相结合的研究。 最近的研究表明，质膜上的慢阴离子通道和外向整流K+通道以及保卫细胞液泡膜上新发现的液泡K+（VK）和Ca ~（2+）激活的Ca ~（2+）-透性（SV）通道是刺激依赖的气孔关闭的主要通道。 将详细研究新发现的VK通道的调节和作用以及保卫细胞液泡中SV通道诱导的Ca 2+释放，以验证这些液泡离子通道对于信号传导和液泡离子释放至关重要的假设气孔关闭期间。 此外，这些液泡离子通道的研究与分析的限速慢阴离子通道在保卫细胞质膜将追求确定中间Ca 2+依赖和推定的Ca 2+独立的信号步骤，产生的综合反应气孔关闭。 保卫细胞液泡通道和磷酸化依赖性质膜慢阴离子通道的调节将进一步研究通过使用钙离子依赖性蛋白激酶（CDPK）的亚型。 此外，拟南芥保卫细胞信号突变体的细胞生物学特性将被追求，以阐明气孔关闭事件级联内的遗传位点的功能。 气孔调节离子脱落酸不敏感的拟南芥突变体（abi 1和abi 2）和纯化的重组ABI 1蛋白磷酸酶对保卫细胞离子通道的影响，将追求增强阿坝的敏感性和表型气孔反应，在现有的拟南芥突变体，以确定和表征额外的基因位点参与气孔和阿坝信号。 该研究将有助于理解气孔关闭信号级联反应的分子机制。 这些研究可能进一步提供重要的信息，为未来的工程策略的设计，提高作物的水分利用效率。 植物通过通常位于叶子下面的气孔进行呼吸。 然而，必须在呼吸作用和通过气孔损失水蒸气之间达到良好的平衡。为了实现这种平衡，植物对诱导气孔关闭的各种环境刺激敏感，例如二氧化碳水平升高、黑暗和植物激素脱落酸（阿坝）。 已知气孔关闭部分是由离子通过外细胞膜和包围液泡的膜中的离子通道流出引起的，液泡是构成气孔的细胞的液泡膜，保卫细胞。这个项目关注的是环境刺激如何引起通过这些通道的离子运输的量和速率的变化。 渠道已经确定。 钾从液泡通过液泡膜释放到细胞质中，在细胞质中通过外向整流钾通道释放到细胞壁中。 细胞内钙和pH值的升高刺激这种释放。 探讨了新发现的液泡钾转运蛋白的调节和作用，以及这些通道与质膜上其他通道的协调，以产生对细胞内钙信号的综合反应。 还使用分子遗传学探索信号通路的整合。 突变体的表型改变的保卫细胞响应的植物激素，阿坝，进行了分析，以确定更多的基因参与气孔开放和阿坝信号。这些信号基因之一的作用，其中一个编码蛋白磷酸酶，正在探索通过纯化蛋白质，并确定其对通道生理的直接影响。 该项目通过设计提高水利用效率的作物来应用生物技术。 ***

9506191 Schroeder Stomatal pores in leaves close to reduce transpirational water loss of plants in response to environmental stress conditions such as drought. Control of stomatal movements by guard cells in response to environmental stimuli and stress conditions is a primary factor in determining water use efficiency and productivity of crop plants. Guard cells provide an ideal system to elucidate early events in higher plant signal transduction. Several key guard cell ion channels have been identified which have been proposed to function as important signal transducers and mediators of stomatal closing. Recent data suggest that both Ca2+- dependent and Ca2+-independent transduction cascades may control stomatal closing. However, the intracellular signaling cascades which link signal reception to ion channel modulation during stomatal closing remain largely unknown. Furthermore, the molecular mechanisms have remained unknown, by which guard cell vacuoles release ions, which is essential for stomata closing. The long term goal of this research is to characterize the chain of events of the signaling cascade which integrates physiological stimuli, such as abscisic acid, intracellular coupling proteins, second messengers, and ion channels to produce stomatal closing. To elucidate signaling mechanism in guard cells, we propose studies using a combination of cell biological, patch clamp, biochemical and genetic analyses. Recent research has led to the model that slow anion channels and outward-recitifying K+ channels in the plasma membrane and newly identified vacuolar K+ (VK) and Ca2+- activated Ca2+-permeable (SV) channels in the tonoplast of guard cells are central transducers and mediators of stimulus-dependent stomatal closing. The regulation and roles of the newly identified VK channels and the proposed Ca2+-induced Ca2+ release by SV channels in guard cell vacuoles will be investigated in detail to test the hypothesis that these vacuolar ion chan nels are crucial for signal transduction and vacuolar ion release during stomatal closure. Furthermore, studies of these vacuolar ion channels together with analyses of rate-limiting slow anion channels in guard cell plasma membrane will be pursued to determine intermediate Ca2+-dependent and putative Ca2+- independent signaling steps which produce the integrated response of stomatal closing. Regulation of guard cell vacuole channels and phosphorylation-dependent plasma membrane slow anion channels will further be studied by using isoforms of the Ca2+-dependent protein kinase (CDPK). In addition, cell biological characterization of guard cell signaling mutants in Arabidopsis will be pursued to elucidate the function of genetic loci within the cascade of events which produced stomatal closing. Stomatal regulation ion abscisic acid-insensitive Arabidopsis mutants (abi1 and abi2) and effects of the purified recombinant ABI1 protein phsophatase on guard cell ion channels will be pursued for enhanced ABA sensitivity and for phenotypic stomatal responses in existing Arabidopsis mutants to identify and characterize additional genetic loci involved in stomatal and ABA signaling. The proposed research will contribute significantly to the understanding of the molecular mechanisms of the signal transduction cascade which produces the integrated response of stomatal closing. These studies may further provide important information for the design of strategies for future engineering of improved water use efficiency in crop plants. %%% Plants "breathe" through tiny openings or "mouths" called stomata which are often found on the underside of leaves. However, a fine balance must be achieved between respiration and loss of water vapor through the stomata. To achieve this balance, the plant is sensitive to a wide variety of environmental stimuli which induce stomatal closure, such as elevated carbon dioxide levels, darkness and the phytohormone abscisic acid (ABA). It is known that stomatal closing is, in part, brought about by ion efflux through ion channels in the outer cell membrane and the membrane which surrounds the vacuole, the tonoplast of the cells which comprise the stomate, the guard cells. This project is concerned with the way the environment stimuli bring about changes in the amount and rate of ion transport through these channels. The channels have been identified. Potassium is released from the vacuole through the tonoplast, into the cytoplasm where it is released into the cell wall via an outward-rectified potassium channel. A rise in intracellular calcium and pH stimulates this release. The regulation and role of the newly-identified vacuolar potassium transporter is explored, as is the coordination of these channels with others at the plasma membrane to produce an integrated response to intracellular calcium signals. The integration of the signaling pathways is also explored using molecular genetics. Mutants which have the phenotype of an altered guard cell response to the phytohormone, ABA, are analyzed in order to identify more genes involved in stomatal opening and ABA signaling. The role of one of these signaling genes, one which encodes a protein phosphatase, is being explored by purifying the protein and determining its direct effect on channel physiology. This project has biotechnical application through the design of crop plants which have improved water use efficiency. ***