The ability to respond to blue light is widespread throughout the biological kingdom. Many of these responses, including some found in bacteria, fungi, insects and plants, are believed to be mediated by flavoproteins. In spite of the fact that blue light responses were first described by Darwin over 100 years ago, no such photoreceptor has hitherto been described. This proposal is based on the recent isolation and characterization of the HY4 gene of Arabidopsis thaiiana. Mutant plants that are defective for this gene show a selective insensitivity to blue light. The sequence of the HY4 gene shows a striking similarity to that of microbial photolyases. These enzymes are a rare class of flavoproteins that repair UV-damaged DNA and are activated by the absorption of either blue or UV light; that is; they function as photoreceptors. This combination of the phenotypic characteristics of hy4 mutants and the sequence similarity to photolyases is compelling evidence that HY4 encodes a flavin-based blue light photoreceptor. This proposal is a combination of biochemical, molecular, and genetic approaches to the further characterization of the HY4 photoreceptor. Collaborative studies involving electrophysiological and biophysical approaches are also included. HY4 will be purified from transgenic tobacco plants, as well as Arabidopsis, and attempts will be made to overexpress it in heterologous expression systems in yeast and baculovirus infected insect cells. The purified protein will be used in studies aimed at determining its biochemical characteristics including the nature of the chromophores. Based on the properties of photolyases, it is believed that HY4 will function as a redox agent. The ability of HY4 to mediate blue light-dependent changes in the redox properties of isolated plasma membranes will be examined and both molecular and genetic approaches will be used to search for additional components in the photoregulatory signal transduction pathway. The ability to perceive and respond to light is universal throughout the biological kingdom and the nature of the signaling mechanisms that are associated with these responses is of wide interest. Equally important, especially in the study of human neutrophils and phagocytosis, is the means by which redox reactions impact on the activity of cellular membranes. The proposed studies, taking full advantage of the merits of both molecular and genetic analysis in Arabidopsis, will provide a significant contribution to our understanding of these important processes.

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National Institute of General Medical Sciences (NIGMS)
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Physical Biochemistry Study Section (PB)
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University of Pennsylvania
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