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. ***
