Calcium-Independent Steps in Guard Cell Regulation by Abscisic Acid: The Kinase Connection

植物表皮含有称为气孔的微观孔隙，通过气孔与环境进行气体交换。通过气孔，二氧化碳被吸收用于光合作用，而水蒸气和氧气被损失。气孔开度由成对的保卫细胞调节，保卫细胞界定气孔。保卫细胞通过渗透膨胀和收缩调节气孔开度，这是由离子的吸收和有机溶质的产生（气孔开度）或离子的损失和有机溶质的释放（气孔关闭）驱动的。植物激素脱落酸（阿坝）在植物受到干旱或其他胁迫时抑制气孔开放并促进气孔关闭。几年前，PI的实验室使用生物化学方法在保卫细胞中鉴定出一种ABA激活的、钙离子非依赖性激酶（ABA激活蛋白激酶; AAPK）。这种丝氨酸/苏氨酸激酶在一分钟内被生理浓度的阿坝激活，并且在保卫细胞中检测到，但在表皮或叶肉细胞中检测不到（Li和Assmann（1996）Plant Cell 8：2359-2368）。这些特点表明，AAPK可以发挥重要作用，触发保卫细胞溶质含量的快速变化，驱动气孔关闭后阿坝exposition.This项目的要求，为相关的研究PI的当前NSF拨款MCB 98-74438，在1999年3月启动的额外资金。在MCB 98-74438资助的第一年，PI实验室成功克隆了编码AAPK的cDNA，从纯化保卫细胞蛋白的质谱分析获得的AAPK肽序列开始。PI的小组已经表明，用显性负性形式的AAPK（“AAPK（K43 A）”）对保卫细胞的生物射弹转化阻断ABA诱导的气孔关闭。PI的实验室还表明，AAPK（K43 A）抑制ABA激活一类保卫细胞阴离子通道，通过这些通道，阴离子损失通常发生在ABA诱导的气孔关闭期间。这项研究已经发表（Li et al.，（2000）Science 287：300-303）。根据这一要求寻求资金的研究是鉴定与AAPK相互作用的蛋白质。提出并优先考虑以下方法：与标记AAPK的相互作用克隆;酵母双杂交分析;免疫沉淀;和使用质谱分析，以确定当阿坝激活激酶时发生的AAPK的共价修饰。通过这些方法阐明AAPK信号转导途径将增加对植物中植物激活的细胞信号传导的理解，并且可以为气孔响应的生物技术操纵提供切入点，以在水分限制时增强ABA诱导的气孔关闭，或者在水分充足时（例如，在灌溉期间）减少ABA诱导的气孔关闭，从而减少光合作用的气孔限制。

The plant epidermis contains microscopic pores called stomata through which gas exchange with the environment occurs. Through the stomata, carbon dioxide is taken up for photosynthesis and water vapor and oxygen are lost. Stomatal apertures are regulated by pairs of guard cells which border and define the stomatal pores. Guard cells regulate stomatal apertures by osmotic swelling and shrinking, driven by uptake of ions and production of organic solutes (stomatal opening) or loss of ions and catabolism of organic solutes (stomatal closure). The plant hormone abscisic acid (ABA) inhibits stomatal opening and promotes stomatal closure when plants are droughted or otherwise stressed. A few years ago, the PI's laboratory used biochemical methods to identify in guard cells an ABA-activated, Ca2+-independent kinase (ABA-activated protein kinase; AAPK). This serine/threonine kinase is activated within one minute by physiological concentrations of ABA and is detected in guard cells but not in epidermal or mesophyll cells (Li and Assmann (1996) Plant Cell 8: 2359-2368). These characteristics suggested that AAPK could play an important role in triggering the rapid changes in guard cell solute content that drive stomatal closure upon ABA exposure.This project represents a request for additional funding for research related to that of PI's current NSF grant MCB 98-74438, initiated in March of 1999. Under the first year of funding of MCB 98-74438, the PI's laboratory succeeded in cloning the cDNA encoding AAPK, starting from AAPK peptide sequence obtained by mass spectrometric analysis of the purified guard cell protein. The PI's group has shown that biolistic transformation of guard cells with a dominant negative version of AAPK ("AAPK(K43A)") blocks ABA-induced stomatal closure. The PI's laboratory also has shown that AAPK(K43A) inhibits ABA-activation of a class of guard cell anion channels through which anion loss normally occurs during ABA-induced stomatal closure. This research has been published (Li et al., (2000) Science 287: 300-303). The research for which funding is sought under this request is the identification of proteins that interact with AAPK. The following approaches are proposed and prioritized: interaction cloning with labeled AAPK; yeast two-hybrid analysis; immunoprecipitation; and use of mass spectrometric analysis to identify the covalent modification of AAPK that occurs when ABA activates the kinase. Elucidation of the AAPK signal transduction pathway by these methods will increase understanding of hormonally-activated cellular signaling in plants and may provide an entry-point for biotechnological manipulation of stomatal responses to enhance ABA-induced stomatal closure when water is limiting, or to reduce ABA-induced stomatal closure and thus stomatal limitation of photosynthesis when water is abundantly available (e.g. during irrigation).