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小气孔，通过气孔与环境进行气体交换。通过气孔，二氧化碳被光合作用吸收，水蒸气和氧气损失。气孔孔由一对保卫细胞调节，保卫细胞与气孔孔交界并限定气孔孔。保卫细胞通过离子的吸收和有机溶质的产生(气孔开放)或离子的丢失和有机溶质的分解代谢(气孔关闭)来调节气孔的渗透膨胀和收缩。当植物受到干旱或其他胁迫时，植物激素脱落酸(ABA)抑制气孔开放，促进气孔关闭。几年前，PI的实验室使用生化方法在保卫细胞中鉴定了一种ABA激活的、不依赖于钙的激酶(ABA激活的蛋白激酶；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，并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)植物细胞8：2359-2368)。这些特征表明，AAPK可能在触发保卫细胞溶质含量的快速变化中发挥重要作用，这些变化导致气孔在脱落酸胁迫下关闭。该项目代表着对与PI目前的国家科学基金拨款MCB98-74438相关的研究的额外资金的请求，该基金于1999年3月启动。在MCB 98-74438第一年的资助下，PI的实验室从纯化的保卫细胞蛋白的质谱分析获得的AAPK多肽序列开始，成功地克隆了编码AAPK的c DNA。PI的小组已经证明，保卫细胞与显性的AAPK(“AAPK(K43A)”)的生物转化阻断了ABA诱导的气孔关闭。PI的实验室还表明，AAPK(K43A)抑制了一类保卫细胞阴离子通道的ABA激活，在ABA诱导的气孔关闭过程中，通常通过该通道发生负离子丢失。这项研究已经发表(Li等人，(2000)科学287：300-303)。根据这一要求寻求资助的研究是鉴定与AAPK相互作用的蛋白质。以下方法被提出并优先考虑：与标记的AAPK的相互作用克隆；酵母双杂交分析；免疫沉淀；以及使用质谱分析来鉴定当ABA激活该激酶时发生的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激素激活的细胞信号转导的理解，并可能为生物技术操纵气孔反应提供一个切入点，以增强ABA诱导的气孔关闭，或在水分充足时(如灌溉期间)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).