Phytochrome A: Structure/Function and Signaling Pathways

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The phytochromes (phyA to phyE) are a family of unique sensory photoreceptors that regulate expression of developmentally important genes in response to informational light signals from the environment. The long-term goal of this research program is to define the molecular and cellular mechanisms by which these sensory molecules perceive, interpret and transduce light signals to photoresponsive nuclear genes. Recent evidence from this and other laboratories has resulted in a paradigm shift in concepts regarding the potential intracellular pathways and mechanisms utilized in this signaling process. The data suggest that one pathway involves light- activated translocation of phy molecules from the cytoplasm to the nucleus, followed by specific interaction with a promoter- bound basic helix-loop-helix (bHLH)-class transcription factor (PIF3), and consequent transcriptional activation of target genes. In addition, oligonucleotide microarray-based expression profiling suggests that these target genes may include a master set of transcriptional-regulator genes that orchestrate expression in various branches of a phyA-regulated transcriptional network. Despite this progress, definitive evidence of the postulated direct involvement of phy molecules in transcriptional regulation and the possible mechanisms involved are lacking, and the components and circuitry comprising primary phy-regulated transcriptional networks remain to be fully defined. We propose to address these deficiencies using phyA, the best characterized and experimentally most tractable member of the family. The specific objectives of this proposal are: (a) to identify and characterize molecular components involved in phyA signal transduction, with particular focus on those specific to the phyA pathway; (b) to explore the molecular basis of transcriptional regulation by phyA, given its established interaction with the bHLH factor PIF3; (c) to explore the biochemical mechanism of phyA signal transfer to PIF3; and (d) to map the primary transcriptional network that mediates phyA- regulated seedling deetiolation. The experimental approaches will include: (a) cloning and molecular characterization of components identified in genetic screens for phyA-specific signaling intermediates; (b) molecular cloning and characterization of additional phyA-interacting proteins using a novel yeast two-hybrid screen; (c) transcriptional activation assays in plant cells transformed with phyA-or PIF3-fusion proteins artificially targeted to reporter-gene promoters via fused DNA-binding domains; (d) enzymatic assays with recombinant wild-type and signaling-compromised mutant phyA proteins to determine whether protein kinase activity toward PIF3 detected biochemically in phyA preparations is correlated with phyA signaling activity in vivo; and (e) comprehensive oligonucleotide microarray-based expression profiling of wild-type and phyA- signaling-defective Arabidopsis mutants. Understanding the full spectrum of molecular and cellular mechanisms by which eukaryotic cells perceive and transduce extracellular informational signals remains a central goal of biomedical research. The experimental system and strategies proposed here have the potential to contribute significantly to this goal.