Ion Channel Regulation in Higher Plants

叶片表皮的气孔调节CO2向叶片的扩散进行光合固碳，并通过蒸腾作用控制植物向大气的水分流失。保护细胞的信号转导机制网络整合环境刺激调节气孔开度，以优化植物在不同条件下的生长。在干旱条件下，控制气孔运动对环境胁迫的响应是决定作物生产力和植物水分流失的重要因素。该项目的长期目标是利用易于操纵的植物拟南芥，对调节气孔运动的信号转导事件网络进行详细的定量了解。保护细胞提供了一个强大的系统，可以定量和时间分辨地解剖复杂的分子机制，介导早期植物信号级联反应的特异性。在之前的资助期间，已经建立了一个信号转导模型，表明几种正向和负向调节的蛋白激酶和PP1-或pp2a型蛋白磷酸酶在介导植物激素脱落酸（ABA）的气孔关闭中起核心作用。然而，编码这些蛋白激酶和pp1 / 2a型磷酸酶的基因，以及它们在级联中的位置和功能及其下游靶标在很大程度上仍然未知。从拟南芥保护细胞cDNA文库中分离出了受体样激酶（RLKs）、Ca2+依赖性蛋白激酶（CDPKs）和催化PP2A亚基的编码cDNA。这些信号转导在植物中的大基因家族和冗余限制了对单个激酶和PP2As的直接遗传表征。保护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，调控PP2A亚基的插入突变导致保护细胞中隐性ABA不敏感。此外，受体激酶的破坏会消除光诱导的气孔打开，这说明了所提出研究的重要性和可行性。本项目将验证以下假设：正、负调节蛋白激酶和PP2As在aba诱导的气孔关闭中都起作用。CDPKs控制aba诱导的气孔关闭。特异的PP2As可作为ABA信号的正调控或负调控。为了验证这些，将完成以下具体目标：鉴定插入中断突变等位基因，并在分离的保护细胞RLK， CDPK和催化PP2A基因中产生转基因抑制系。将对冗余同源CDPKs和PP2As的破坏突变体或双/多突变体进行气孔运动分析，以表征对ABA和光刺激的表型反应。具有最强气孔表型的2到3个激酶和PP2A突变将被表征为刺激诱导的保护细胞阴离子或K+通道的调节以及信号诱导的细胞质Ca2+变化。将对已知的保护细胞信号突变体进行上位性分析，以确定保护细胞信号网络中事件的相对顺序。将分析选定的CDPKs/PP2As对克隆保护细胞离子通道的调制作用。此外，保护细胞表达的相互作用蛋白的筛选将用于其中一种调节因子。从这些研究中获得的知识将揭示植物细胞中激酶和PP2As网络控制信号转导和介导信号特异性的新的基本机制。此外，这些研究将有助于阐明植物平衡二氧化碳流入叶片和蒸腾水分损失的关键信号转导机制，并可能有助于未来操纵植物气体交换的策略。

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.