Ion Channel Regulation in Higher Plants

干旱等环境胁迫条件下，叶片气孔闭合减少植物蒸腾水分流失。保护细胞对环境刺激和胁迫条件下气孔运动的控制是决定作物水分利用效率和生产力的主要因素。保护细胞提供了一个理想的系统来阐明高等植物信号转导的早期事件。已经确定了几个关键的保护细胞离子通道，它们是重要的信号转导器和气孔关闭介质。最近的数据表明，Ca2+依赖性和Ca2+非依赖性转导级联可能控制气孔关闭。然而，在气孔关闭期间连接信号接收和离子通道调节的细胞内信号级联在很大程度上仍然未知。此外，保护细胞液泡释放离子的分子机制仍不清楚，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一系列事件，这些事件整合了生理刺激，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。为了阐明保护细胞的信号传导机制，我们建议结合细胞生物学、膜片钳、生化和遗传分析进行研究。最近的研究表明，细胞膜上的缓慢阴离子通道和向外逆流的K+通道，以及保护细胞细胞质中新发现的液泡K+ (VK)和Ca2+激活的Ca2+-通透（SV）通道是刺激依赖性气孔关闭的中心换能器和介质。我们将详细研究新发现的VK通道和提出的SV通道在保护细胞液泡中Ca2+诱导的Ca2+释放的调控和作用，以验证这些液泡离子通道对气孔关闭过程中信号转导和液泡离子释放至关重要的假设。此外，对这些液泡离子通道的研究以及对保护细胞质膜上限速的慢阴离子通道的分析将继续进行，以确定产生气孔关闭综合响应的中间Ca2+依赖和推定的Ca2+独立信号步骤。保护细胞液泡通道和磷酸化依赖的质膜缓慢阴离子通道的调节将通过使用Ca2+依赖性蛋白激酶（CDPK）的异构体进一步研究。此外，还将对拟南芥保护细胞信号突变体的细胞生物学特性进行研究，以阐明基因位点在导致气孔关闭的一系列事件中的功能。拟南芥对脱落酸不敏感突变体（abi1和abi2）的气孔调节以及纯化的重组abi1蛋白磷酸酶对保护细胞离子通道的影响，将用于增强ABA敏感性和现有拟南芥突变体的表型气孔反应，以鉴定和表征与气孔和ABA信号通路相关的其他遗传位点。本研究将有助于进一步了解气孔关闭综合响应的信号转导级联的分子机制。这些研究可以进一步为未来提高作物水分利用效率的工程策略设计提供重要信息。植物通过被称为气孔的小开口或“嘴”来“呼吸”，气孔通常在叶子的背面。然而，在呼吸和通过气孔的水蒸气损失之间必须达到一个很好的平衡。为了达到这种平衡，植物对各种诱导气孔关闭的环境刺激非常敏感，例如二氧化碳水平升高、黑暗和植物激素脱落酸（ABA）。众所周知，气孔关闭在一定程度上是由离子通过外细胞膜和液泡周围膜的离子通道流出引起的，液泡是组成气孔的细胞的细胞质，是保卫细胞。本项目关注的是环境刺激对通过这些通道的离子传输量和速率的影响。通道已经确定。钾通过细胞质从液泡中释放出来，进入细胞质，在细胞质中通过向外矫正的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。新发现的液泡钾转运体的调节和作用被探索，以及这些通道与质膜上的其他通道的协调，以产生对细胞内钙信号的综合反应。信号通路的整合也利用分子遗传学进行了探索。分析了具有保护细胞对植物激素ABA反应改变表型的突变体，以确定更多参与气孔打开和ABA信号传导的基因。这些信号基因中的一个编码蛋白质磷酸酶，其作用正在通过纯化蛋白质和确定其对通道生理学的直接影响来探索。本项目通过设计作物植株，提高水分利用效率，进行生物技术应用。* * *

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. ***