The perception of informational light signals by the five-membered phytochrome (phy) family of sensory photoreceptors (phyA through phyE) initiates an intracellular transduction process that culminates in the altered expression of nuclear genes that direct growth and developmental responses, termed photomorphogenesis, appropriate to the prevailing environment. The long-term goal of this program is to define the cellular, molecular and biochemical mechanisms by which this process occurs. Current data indicate that the transduction process involves rapid translocation of the light-activated photoreceptor molecule from the cytoplasm to the nucleus, where it interacts physically with a subset of members of the bHLH transcription- factor family, termed phytochrome-interacting factors (PIFs), inducing transcriptional responses in target genes. Recent evidence shows that members of the PIF family collectively repress photomorphogenesis in young dark-grown seedlings, and that photoactivated phy reverses this repression by inducing rapid degradation of the PIF molecules upon initial exposure to light. This process involves rapid, phy-induced phosphorylation of the interacting bHLH protein, followed by degradation via the ubiquitin proteasome system. Despite these advances, several central questions remain, including the identity of the protein kinase responsible for PIF phosphorylation and the primary target genes of the phy-PIF signaling pathway. We propose to address these deficiencies here, using Arabidopsis as a model system. The specific objectives of this proposal are: (a) To define the functions of the different PIF-family members in controlling early, post- germinative seedling development;(b) To dissect the molecular and biochemical transactions occurring at the phy-PIF signaling interface, including molecular identification of the protein kinase component(s) responsible for phy-induced phosphorylation of the PIF proteins;and (c) To define the transcriptional interface between the phy-interacting bHLH factors and their target genes. The experimental approaches will include: (a) Genetic screens and reverse-genetic analyses of pif-mutant combinations to dissect out the differential and redundant functions of the family members and to identify additional factors;(b) Molecular-genetic functional analyses of targeted, site-specific, missense mutants of the phy and PIF proteins in transgenic plants to define residues necessary for interaction, phosphorylation and ubiquitylation in the cell;(c) Protein-interaction screens, including a yeast """"""""tribrid"""""""" screen and mass-spectrometry of affinity-purified complexes from Arabidopsis, directed at identifying components that associate with either phy and/or PIF proteins before, during and after the light- induced signaling transaction between the two molecules in vivo;and (d) Integrated genome-wide expression profiling and chromatin-immunoprecipitation (ChIP) analyses aimed at identifying direct, primary targets of the PIF-bHLH transcription factors in the phy-regulated transcriptional network.
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, by defining the fundamental mechanism by which a sensory photoreceptor transduces perceived light signals from the environment to the transcriptional network that it controls. Successful execution of this program will provide mechanistic insight into the functioning of a nuclear-localized signaling hub where multiple, related, activated receptors converge to directly communicate their signaling information to multiple, related transcription factors that are responsible for regulating the primary transcriptional network in the response pathway.
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