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

气生植物器官的表皮含有称为气孔的微孔，通过气孔与环境进行气体交换。 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，这在很大程度上是由钾离子和阴离子的吸收或损失驱动的。 植物激素脱落酸（阿坝）抑制气孔开放，并促进关闭时，植物经历干旱或其他压力。 ABA对保卫细胞离子转运途径的影响包括抑制钾离子吸收通道和激活介导钾离子和阴离子流出的通道。 阿坝作用的一个途径似乎利用细胞溶质钙水平的升高作为信号步骤，并且细胞溶质钙浓度的实验升高可以模拟ABA对钾摄取通道的抑制和阴离子通道的激活。 然而，其他数据点钙独立模式的阿坝的行动，以及，这形成了本研究的重点。 利用全细胞膜片钳分析的钾电流和共聚焦比率细胞溶质钙成像与Indo-1染料的条件下，阿坝不会引起细胞溶质钙水平的增加，它已被观察到，阿坝仍然抑制钾摄取通道。 电生理学和成像实验的目的是测试是否在胞质pH值的变化在这一途径中发挥信号作用，以及这一途径是否与细胞内的阿坝受体。 此外，磷酸化/去磷酸化的途径中的作用将进行评估，因为最近获得的证据ABA激活，钙非依赖性激酶（ABA激活蛋白激酶; AAPK）在蚕豆保卫细胞。 这种丝氨酸/苏氨酸激酶在一分钟内被生理水平的阿坝激活，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。 利用最近获得的基于AAPK肽序列的探针，将在蚕豆和拟南芥中鉴定AAPK cDNA和基因组克隆。将利用免疫定位来精确定位AAPK的亚细胞定位。 将评估T-DNA插入系中AAPK的“敲除”;此外，将产生过表达或低表达AAPK的转基因拟南芥植物。 这些植物将在一系列不同的生长条件下评估AAPK水平改变的表型效应。 此外，将对这些植物的保卫细胞进行详细的膜片钳分析，以确定阿坝对通道活性的调节是否改变。 保卫细胞是调节光合作用二氧化碳吸收和蒸腾水分损失的重要控制点，因此，代表了生物技术操纵以改变植物水分利用效率的潜在目标。 一段时间以来，人们已经知道，保卫细胞ABA反应途径中的其他基因，即拟南芥中的ABI 1和ABI 2（编码磷酸酶）的改变，会导致植物枯萎，但这些基因也参与其他细胞类型的发育途径。 这项研究的结果应该允许特定的操纵保卫细胞ABA反应基因（即AAPK），对植物功能具有潜在的有用影响。 例如，可以假设AAPK的过表达将导致更耐旱的植物。 这些假设是可以检验的。

The epidermes of aerial plant organs contain microscopic pores called stomata through which gas exchange with the environment occurs. Stomatal apertures are regulated by pairs of guard cells that border and define the stomatal pores. Guard cells regulate stomatal apertures by osmotic swelling and shrinking, driven in large part by uptake or loss of potassium ions and anions. The plant hormone abscisic acid (ABA) inhibits stomatal opening and promotes closure when plants experience drought or are otherwise stressed. ABA effects on guard-cell ion-transport pathways include inhibition of potassium uptake channels and activation of channels mediating potassium and anion efflux. One pathway of ABA action appears to utilize elevation of cytosolic calcium levels as a signaling step, and experimental elevation of cytosolic calcium concentration can mimic ABA inhibition of potassium uptake channels and activation of anion channels. However, other data point to calcium independent modes of ABA action as well, which forms the focus of this research. Utilizing simultaneous whole-cell patch clamp analysis of potassium currents and confocal ratiometric cytosolic calcium imaging with Indo-1 dye under conditions where ABA does not evoke an increase in cytosolic calcium levels, it has been observed that ABA still inhibits the potassium uptake channels. Electrophysiological and imaging experiments are designed to test whether alterations in cytosolic pH play a signaling role in this pathway, and whether this pathway is linked to an intracellular ABA receptor. In addition, the role of phosphorylation/dephosphorylation in the pathway will be evaluated, because of evidence recently obtained for an ABA-activated, calcium independent kinase (ABA-activated protein kinase; AAPK) in guard cells of Vicia faba. This serine/threonine kinase is activated within one minute by physiological levels of ABA, and is detected in guard cells, but not epidermal or mesophyll cells. Utilizing probes based on AAPK peptide sequence recently obtained, AAPK cDNA and genomic clones will be identified in Vicia faba and Arabidopsis thaliana. Immunolocalization will be utilized to pinpoint the subcellular localization of AAPK. T-DNA insertion lines will be evaluated for "knockouts" in AAPK; in addition, transgenic Arabidopsis plants over- or under-expressing AAPK will be produced. These plants will be evaluated under a battery of different growth conditions for phenotype effects of altered AAPK levels. In addition, guard cells from these plants will be subjected to a detailed patch clamp analysis to determine whether ABA regulation of channel activity is altered. Guard cells are a vital control point in the regulation of photosynthetic carbon dioxide uptake and transpirational water loss, and, accordingly, represent potential targets for biotechnological manipulations to alter plant water use efficiency. It has been known for some time that alteration of other genes in the ABA-response pathways of guard cells, namely ABI1 and ABI2 in Arabidopsis (which encodes phosphatases), results in wilty plants, but these genes are also involved in developmental pathways in other cell types. The results of this research should allow the specific manipulation of a guard-cell ABA-response gene (namely AAPK), with potentially useful effects on plant function. For example, one could hypothesize that overexpression of AAPK would result in more drought-tolerant plants. Such hypotheses are testable.