Ion Channel Regulation in Higher Plants

叶片表皮的气孔调节CO2向叶片中的扩散，以进行光合固碳，并控制植物通过蒸腾作用向大气中的水分损失。保卫细胞中的信号转导机制网络整合环境刺激以调节气孔开度，从而优化植物在不同条件下的生长。气孔运动对环境胁迫条件的响应控制是决定干旱期间作物生产力和植物水分损失的重要因素。这个项目的长期目标是实现一个详细的定量了解网络的信号转导事件，调节气孔运动，使用易于操作的植物，拟南芥。保卫细胞提供了一个强大的系统，定量和时间分辨解剖的复杂分子机制介导的特异性在早期植物信号级联。 在前一个资金期间的信号转导模型已经开发，这表明，几种类型的积极和消极的调节蛋白激酶和PP 1或PP 2A型蛋白磷酸酶在介导气孔关闭的植物激素，脱落酸（阿坝）发挥核心作用。然而，编码这些蛋白激酶和PP 1/2A型磷酸酶的基因，以及它们在级联反应中的位置和功能及其下游靶点在很大程度上仍然未知。从拟南芥保卫细胞cDNA文库中，已经分离出编码受体样激酶（RLKs）、Ca 2+依赖性蛋白激酶（CDPKs）和催化PP 2A亚基的cDNA。植物中这些信号转导子的大基因家族和冗余限制了单个激酶和PP 2A的直接遗传表征。对保卫细胞表达成员的直接功能分析现在将允许确定它们的冗余和它们在信号转导中的精确功能。初步研究表明，在一个调节PP 2A亚基的插入突变导致隐性阿坝不敏感保卫细胞。此外，受体激酶的破坏废除光诱导的气孔开放，说明了所提出的研究的重要性和可行性。 本项目将检验以下假设：ABA诱导气孔关闭过程中，正、负调节蛋白激酶和PP 2As均起作用。 CDPKs控制ABA诱导的气孔关闭。PP 2As可能是ABA信号的正调节因子，也可能是阿坝信号的负调节因子。 为了测试这些，将进行以下具体目标：将鉴定插入破坏突变体等位基因，并在分离的保卫细胞RLK、CDPK和催化PP 2A基因中产生转基因阻遏系。气孔运动分析将追求的特征表型响应阿坝和光刺激的破坏突变体或双/多突变体的冗余同源CDPKs和PP 2As。具有最强气孔表型的两到三个激酶和PP 2A突变的影响将表征为对保卫细胞阴离子或K+通道的刺激诱导的调节和信号诱导的胞质Ca 2+变化。将采用已知的保卫细胞信号传导突变体进行上位性分析，以确定保卫细胞信号传导网络中事件的相对顺序。将分析选定的CDPK/PP 2A对克隆的保卫细胞离子通道的调节。此外，筛选保卫细胞表达的相互作用蛋白质将寻求一种调节剂。从这些研究中获得的知识将揭示新的基本机制，激酶和PP 2A网络控制信号转导和介导植物细胞中的信号特异性。此外，这些研究将导致阐明植物平衡CO2流入叶片和蒸腾水分损失的关键信号转导机制，并可能有助于未来的策略，操纵植物中的气体交换。

Stomatal pores in the epidermis of leaves regulate the diffusion of CO2 into leaves for photosynthetic carbon fixation and control water loss of plants via transpiration to the atmosphere. A network of signal transduction mechanisms in guard cells integrate environmental stimuli to regulate stomatal apertures for optimization of plant growth under diverse conditions. Control of stomatal movements in response to environmental stress conditions is an important factor in determining crop productivity and plant water loss during drought. The long-term goal of this project is to achieve a detailed quantitative understanding of the network of signal transduction events that regulate stomatal movements using the easily manipulated plant, Arabidopsis thaliana. Guard cells provide a powerful system for quantitative and time-resolved dissection of the complex molecular machinery mediating specificity in early plant signaling cascades. During the preceding funding period a signal transduction model has been developed, suggesting that several types of both positively- and negatively-regulating protein kinases and PP1- or PP2A-type protein phosphatases play central roles in mediating stomatal closing by the phytohormone, abscisic acid (ABA). However, the genes encoding these protein kinases and PP1/2A-type phosphatases, as well as their locations and functions within cascades and their downstream targets remain largely unknown. From an Arabidopsis guard cell cDNA libraries, the cDNAs that encoded for the receptor-like kinases (RLKs), the Ca2+-dependent protein kinases (CDPKs), and the catalytic PP2A subunit have been isolated.. The large gene families and redundancies of these signal transducers in plants have limited direct genetic characterization of individual kinases and PP2As. Direct functional analyses of guard cell-expressed members will now allow determination of their redundancies and of their precise functions in signal transduction. Preliminary studies show that an insertional mutation in a regulatory PP2A subunit causes recessive ABA insensitivity in guard cells. Furthermore, disruption of a receptor kinase abolishes light-induced stomatal opening, illustrating the importance and feasibility of the proposed research. The following hypotheses will be tested in this project: That both positively- and negatively-regulating protein kinases and PP2As function in ABA-induced stomatal closing. That CDPKs control ABA-induced stomatal closing. That specific PP2As may function either as positive or negative regulators of ABA signaling. To test these, the following specific aims will be done: Insertional disruption mutant alleles will be identified and transgenic repression lines will be generated in the isolated guard cell RLK, CDPK and catalytic PP2A genes. Stomatal movement analyses will be pursued to characterize phenotypic responses to ABA and light stimuli in disruption mutants or double/multi mutants of redundant homologous CDPKs and PP2As. Effects of two to three kinase and PP2A mutations with the strongest stomatal phenotypes will be characterized on stimulus-induced regulation of guard cell anion or K+ channels and signal-induced cytosolic Ca2+ changes. Epistasis analyses with known guard cell signaling mutants will be pursued to determine the relative sequence of events in the guard cell signaling network. Selected CDPKs/PP2As will be analyzed for modulation of cloned guard cell ion channels. Furthermore screens for guard cell-expressed interacting proteins will be pursued for one of the regulators. The knowledge gained from these studies will reveal novel fundamental mechanisms by which a network of kinases and PP2As control signal transduction and mediate signaling specificity in a plant cell. Furthermore, these studies will lead to elucidation of key signal transduction mechanisms by which plants balance CO2 influx into leaves and transpirational water loss and may contribute to future strategies for manipulating gas exchange in plants.