Phytochrome is a regulatory master switch that controls gene expression in response to fight signals from the environment. The long term goal of the proposed research is to define the molecular mechanism by which this regulation is exerted. The specific objectives of this proposal are: (a) to identify sequence and structural features of the phytochrome A molecule responsible for its regulatory action; and (b) to define regulatory elements in the multidomain phyA gene promoter Arabidopsis responsible for differential expression from multiple transcription start sites. The experimental approaches will include: (a) biochemical analysis to assess the possibility that Avena phytochrome A has protein kinase activity similar to prokaryotic sensor proteins; (b) overexpression of mutagenized Avena phytochrome A in transgenic Arabidopsis and microorganisms to identify structurally and functionally important domains and residues; (c) microinjection of mutagenized phytochrome A into phytochrome-deficient transgenic Arabidopsis cells carrying phytochrome-responsive promoter-reporter fusion constructs to identify functionally critical sequences in the photoreceptor by transient expression assay; (d) production of homogeneous phytochrome A preparations by overexpression in microorganisms for X-ray crystallographic analysis; and (e) deletion and sequence substitution analysis of the Arabidopsis phyA promoter in transgenic Arabidopsis to delineate cis-acting DNA sequences involved in the regulation of this complex promoter. The significance of this research is that it will help define the mechanism by which a central regulatory molecule controls gene expression. Because phytochrome appears as yet to have no molecular counterpart in other eukaryotes, elucidation of this mechanism has the potential to broaden our understanding of the strategies used by eukaryotic cells to regulate gene expression in response to environmental stimuli.
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