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

植物表皮包含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，而水蒸气和氧气则被消耗掉。气孔的开度是由气孔边缘和限定气孔的保护细胞对调节的。保护细胞通过吸收离子和产生有机溶质（气孔打开）或离子损失和有机溶质分解代谢（气孔关闭）驱动的渗透性膨胀和收缩来调节气孔开度。植物激素脱落酸（ABA）在植物遭受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的实验室使用生化方法在保护细胞中鉴定了aba激活的Ca2+独立激酶（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ABA激活，在保护细胞中检测到，但在表皮或叶肉细胞中检测不到（Li和Assmann (1996) Plant Cell 8: 2359-2368）。这些特征表明，AAPK可能在触发ABA暴露后保护细胞溶质含量的快速变化，从而驱动气孔关闭方面发挥重要作用。本项目为PI目前的NSF基金MCB 98-74438的相关研究申请额外资金，该基金于1999年3月启动。在MCB 98-74438资助的第一年，PI实验室成功克隆了编码AAPK的cDNA，从纯化的保卫细胞蛋白的质谱分析获得的AAPK肽序列开始。PI的团队已经证明，具有AAPK显性阴性版本的保护细胞的生物学转化（“AAPK（K43A）”）阻断了aba诱导的气孔关闭。PI的实验室还表明，AAPK（K43A）抑制aba激活一类保护细胞阴离子通道，在aba诱导的气孔关闭过程中，阴离子损失通常通过这些通道发生。这项研究已经发表（Li et al., (2000) Science 287: 300-303）。在此请求下寻求资助的研究是鉴定与AAPK相互作用的蛋白质。提出并优先考虑以下方法：与标记的AAPK互作克隆；酵母双杂交分析；免疫沉淀反应;并使用质谱分析来鉴定ABA激活AAPK时发生的共价修饰。通过这些方法阐明AAPK信号转导通路将增加对植物中激素激活的细胞信号传导的理解，并可能为生物技术操纵气孔反应提供一个起点，以增强水分有限时aba诱导的气孔关闭，或减少水分充足时aba诱导的气孔关闭，从而减少光合作用的气孔限制（例如在灌溉期间）。

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).