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观孔隙，通过气孔与环境进行气体交换。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。保护细胞通过渗透膨胀和收缩来调节气孔开度，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。当植物经历干旱或其他胁迫时，植物激素脱落酸（ABA）抑制气孔打开并促进关闭。ABA对保护细胞离子运输途径的影响包括抑制钾摄取通道和激活介导钾和阴离子外排的通道。ABA作用的一个途径似乎利用细胞质钙水平的升高作为信号传导步骤，实验中细胞质钙浓度的升高可以模拟ABA对钾摄取通道的抑制和阴离子通道的激活。然而，其他数据也指向ABA的不依赖钙的作用模式，这是本研究的重点。在ABA不引起胞质钙水平增加的情况下，利用同时全细胞膜片钳分析钾电流和用吲哚-1染料进行共聚焦比例细胞质钙成像，观察到ABA仍然抑制钾摄取通道。电生理和成像实验旨在测试细胞质pH的改变是否在该途径中发挥信号作用，以及该途径是否与细胞内ABA受体相关。此外，由于最近在蚕豆保护细胞中获得了aba激活的钙独立激酶（aba激活蛋白激酶；AAPK）的证据，因此将评估磷酸化/去磷酸化在该途径中的作用。这种丝氨酸/苏氨酸激酶在一分钟内被生理水平的ABA激活，并且在保护细胞中检测到，但在表皮或叶肉细胞中检测不到。利用最近获得的AAPK肽序列探针，在蚕豆和拟南芥中鉴定AAPK cDNA和基因组克隆。免疫定位将用于确定AAPK的亚细胞定位。将评估T-DNA插入系在AAPK中的“敲除”；此外，AAPK过表达或过低表达的转基因拟南芥植株也将获得。这些植物将在不同的生长条件下评估AAPK水平改变对表型的影响。此外，来自这些植物的保护细胞将受到详细的膜片钳分析，以确定ABA对通道活性的调节是否被改变。保护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，保护细胞aba反应途径中的其他基因，即拟南芥中的ABI1和ABI2（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对保护细胞aba反应基因（即AAPK）的特定操作，对植物功能具有潜在的有用影响。例如，人们可以假设AAPK的过度表达会导致更耐旱的植物。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.