Blue Light Photoreception and Guard Cell Function

9723605 Zeiger这项研究建议集中在保卫细胞中蓝光的感知和转导，以及它在气孔功能中的作用。保卫细胞感知叶片环境中的内部和外部信号，并将它们整合到控制叶片气体交换的调节气孔中。保卫细胞的蓝光反应属于蓝光反应的一个大家族，蓝光反应是植物适应和适应环境的核心。在高等植物中介导蓝光感应的发色团(S)的身份尚不清楚。最近对两个拟南芥突变体hy4和nph1的研究为黄素蛋白介导蓝光接受提供了证据。然而，据报道，这两个突变体对蓝光都有正常的气孔反应，这表明保卫细胞可能有一个明显的蓝光感受器。最近的研究发现，类胡萝卜素玉米黄质可能是保卫细胞中的蓝光感受器。保卫细胞对蓝光的敏感性随着玉米黄质含量的增加而增加，二硫苏糖醇(DTT)抑制玉米黄质的形成，抑制蓝光依赖的气孔开放，但不改变保卫细胞光合作用刺激的气孔开放。在拟议的实验中，将表征野生型拟南芥叶片保卫细胞的蓝光反应，并与玉米黄质缺失突变体npq1进行比较。该突变体的紫黄质脱环氧合酶有缺陷，不能积累玉米黄质。由于去环氧酶基因似乎是一个单拷贝基因，预计npq1保卫细胞也将缺乏玉米黄质。我们最近证实，来自npq1的保卫细胞不含玉米黄质。用完整的野生型和npq1叶片进行的气体交换实验将被用来确定拟南芥气孔对蓝光是否有典型的反应，以及这种反应是否因npq1中缺乏玉米黄质而改变。酶清洗的表皮中的保卫细胞将被用来研究分离的保卫细胞中气孔对蓝光的反应。初步实验刚刚表明，在红光背景下，npq1保卫细胞无法对蓝光做出反应。如果详细的研究支持这一初步资助，该突变体将被用于测试玉米黄质形成的抑制剂DTT是否可以模拟npq1的遗传损伤。其他实验将研究钙离子作为第二信使的可能作用，将保卫细胞叶绿体的蓝光传感传递到保卫细胞质膜的H+ATPase。整个细胞结构的膜片钳实验将被用来测量保卫细胞质膜上蓝光诱导的电流，在一系列被证明改变保卫细胞质子泵活性的细胞内钙离子浓度范围内。分离的保卫细胞叶绿体将被用来测试蓝光是否引起叶绿体被膜上的钙离子流动，这可能解释体内胞质钙离子浓度的变化。我们还将研究表达钙指示剂aequorin的转基因拟南芥是否可以用来表征叶绿体和细胞质之间的钙通量。这项拟议的研究对于确定保卫细胞中的蓝光感受器和气孔对蓝光的反应突变体以及详细描述重要的蓝光反应的感觉转导级联具有重要的潜力。植物能感知控制生长、定向和发育的几种波长的光。蓝光感知以及如何发出信号来调节下游效应一直存在争议。争论的核心是什么色素吸收了感知到的光，并将信息传递到下游。齐格博士已经获得了玉米黄质合成的突变体，Mu突变体表现出的效果表明，这种色素负责感知蓝光。获得这一奖项后，齐格博士将利用突变植物和野生植物来分析玉米黄质蓝光如何通过保卫细胞的开放反应来调节叶片中的气体交换。其他研究将集中在玉米黄质光接收如何诱导细胞内钙离子流动，从而介导细胞内信号。这项研究很重要，因为它解决了植物如何通过对环境中的光做出反应来发挥功能的最基本的方面之一。***

9723605 Zeiger This research proposal focuses on blue light perception and transduction in guard cells, and its role in stomatal function. Guard cells sense internal and external signals in the leaf environment and integrate them into regulated stomatal apertures that control leaf gas exchange. The blue light response of guard cells belongs to a large family of blue light responses which are central to plant adaptations and acclimations to their environment. The identity of the chromophore(s) mediating blue light sensing in higher plants remains unknown. Recent work with hy4 and nph1, two Arabidopsis mutants for blue light- dependent inhibition of stem elongation and phototropism, has provided evidence for a flavoprotein mediating blue light photoreception. Both mutants however are reported to have normal stomatal responses to blue light, indicating that guard cells might have a distinct blue light photoreceptor. Recent studies have identified the carotenoid, zeaxanthin, as a putative blue light photoreceptor in guard cells. The blue light sensitivity of guard cells increases with their zeaxanthin content, and inhibition of zeaxanthin formation by dithiothreitol (DTT), inhibits blue light-dependent stomatal opening, without altering opening stimulated by guard cell photosynthesis.. In the proposed experiments, the blue light responses of guard cells from wild type Arabidopsis leaves will be characterized, and compared with that of the zeaxanthin-less mutant, npq1. This mutant has a defective violaxanthin de-epoxidase and is unable to accumulate zeaxanthin. Since the de-epoxidase gene appears to be a single copy gene, it is anticipated that npq1 guard cells will also lack zeaxanthin. We have recently confirmed that guard cells from npq1 are devoid of zeaxanthin. Gas exchange experiments with intact wild type and npq1 leaves will be used to establish whether Arabidopsis stomata have a typical response to blue light and whether the response has been altered by the lack of zeaxanthin in npq1. Guard cells in enzymatically cleaned epidermis will be used to study the stomatal response to blue light in isolated guard cells. Initial experiments have just shown that npq1 guard cells fail to open in response to blue light under a red light background. If detailed studies support this initial funding, the mutant will be used to test whether the inhibitor of zeaxanthin formation, DTT can mimic the genetic lesion in npq1. Other experiments will investigate a possible role of Ca2+ as a second messenger transducing blue light sensing at the guard cell chloroplast to a H+ ATPase at the guard cell plasma membrane. Patch clamp experiments in the whole cell configuration will be used to measure blue light-induced electrical currents at the guard cell plasma membrane, over a range of intracellular Ca2+ concentrations shown to alter proton pumping activity in guard cells. Isolated guard cell chloroplasts will be used to test whether blue light elicits Ca2+ fluxes across the chloroplast envelope that could account for in vivo changes in cytosolic Ca2+ concentrations. We will also investigate whether transgenic Arabidopsis plants expressing the Ca2+ indicator, aequorin, can be used to characterize Ca2+ fluxes between the chloroplast and the cytosol. The proposed research has significant potential for the conclusive identification of a blue light photoreceptor in guard cells and a mutant for stomatal responses to blue light, and for the detailed characterization of the sensory transducing cascade of an important blue light response. Plants perceive light in several wavelengths that control growth, orientation and development. Blue light perception and how it is signaled to mediate downstream effects has been a controversy. At the heart of the controversy is what pigment absorbs the perceived light and transmits the information downstream. Dr. Zeiger has obtained mutants of the pigment zeaxanthin synthesis and the mu tants exhibit effects that indicate that this pigment is responsible for perceiving blue light. With this award Dr. Zeiger will use mutant and wild type plants to analyze how zeaxanthin blue light photoreception signals opening response of guard cells mediating gas exchange in leaves. Other studies will focus on how zeaxanthin photoreception induces calcium fluxes in cells that mediate intracellular signaling. This research is important because it addresses one of the most basic aspects of how plants function by responding to light in its environment. ***