The perception of informational light signals by the five-member phytochrome (phy) family of sensory photoreceptors (phyA to phyE) initiates an intracellular transduction process that culminatesin the altered expression of nuclear genes that direct adaptational responses appropriate to the prevailing environment. The long-term goal of this research program is to define the molecular, cellular and biochemicalmechanismsby which this process occurs. The available data indicate that phy signaling involves rapid light-activated translocation of the photoreceptor molecule from the cytoplasm to the nucleus where it interacts physically with signaling partners, that include members of the bHLH family of transcription factors, resulting in transcriptional regulation of target genes. Recent evidence has resulted in a paradigm shift regarding the potential mechanisminvolvedin the signaling transaction. The data suggest that for at least two of these bHLH factors, PIF1and PIF3, phy binding induces their rapid degradation, possibly via the ubiquitin proteosome system (UPS). However, the molecularbasis and regulatoryconsequences of this phenomenonare yet to be fully defined. We propose to address these deficiencies using phyA, the best characterized and experimentallymost tractable member of the family. The specific objectives of this proposal are:(a) to identify and characterizemolecular components in phyA signal transduction;(b) to define the molecular mechanism of phyA-induced degradation of the bHLH transcription factors, PIF1and PIF3;and (c) to define the primary target genes and transcriptionalnetworks that implement the phyA-induced gene expression program. The experimental approaches will include: (a) cloning of components identified in genetic screens for phyA signaling intermediates in Arabidopsis;(b) yeast two-hybrid screens for potential primary phy signaling partners;(c) molecular-genetic and biochemical studies to examine potential UPS involvement in PIF1and PIF3 degradation, to identify the potential E3 ubiquitin ligase involved, and to define the mechanism of phy signal transfer;(d) microarray-based expression profiling of phyA-signaling-defective mutants to identify early-response genes and to map signal channeling through the phyA-reglated transcriptional network;and (e) chromatin immunoprecipitation to identify promoters that are potential direct targets of the photoreceptor and/or its bHLH signaling partners. Understandingthe full spectrum of molecularand cellularmechanisms by whicheukaryotic 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 significantlyto this goal.
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