2011 Annual Report 1a.Objectives (from AD-416) The long-term goal of this research is to define the molecular mechanisms by which the phytochrome (phy) family of photoreceptors perceive informational light signals from the environment and transduce them to photoresponsive nuclear genes, thereby controlling plant growth and development. Using genome-scale microarray-based expression profiling, we have begun to define the global transcriptional network regulated by these photoreceptors and have identified a set of rapidly light-induced or -repressed genes (primarily encoding various transcription factors) that are potential direct targets of the phytochrome signaling pathway. The specific objectives of this project plan are: 1. To identify which phy family members are responsible for signaling to each of these genes, by examining light-induced expression profiles in null mutants of each phytochrome. [NP 301, C 4, PS 4a] 2. To define the cis elements in the promoters of these genes responsible for coordinate light-regulated expression, using computational analysis coupled with targeted mutagenesis and transgenic expression of promoter::reporter fusion constructs. [NP 301, C 4, PS 4a] 3. To identify downstream targets of these genes in the light-regulated transcriptional network, using microarray-based expression profiling in knockout mutants null for each transcription factor, coupled with chromatin immuno- precipitation (ChIP) for target promoter identification and characterization. [NP 301, C 4, PS 4a] 1b.Approach (from AD-416) The specific objectives of this proposal are: (a) to identify which phys are responsible for signaling to these genes; (b) to define the cis elements in the promoters of these genes responsible for coordinate light-regulated expression; (c) to identify downstream targets of these genes in the light-regulated transcriptional network. The experimental approaches will include: (a) microarray-based expression profiling of mutants null for each phy, both to define the phy-regulated transcriptional networks and to identify rapidly light-responsive genes for targeted reverse-genetic mutagenesis; (b) computational analysis of the promoters of these genes, coupled with targeted mutagenesis and transgenic expression of promoter::reporter fusion constructs; (c) expression profiling in knockout mutants null for each transcription factor, coupled with chromatin immunoprecipitation for identification of promoters that are direct targets of these factors. REPLACING 5335-21000-027-00D (9/10). 3.Progress Report Current evidence indicates that the phytochrome (phy) signaling process involves rapid translocation of the light-activated photoreceptor into the nucleus, where it interacts with specific bHLH transcription factors, Phytochrome-Interacting-Factors (PIFs), inducing transcriptional responses in target genes. We have shown that a quadruple pif mutant (pif1pif3pif4pif5 (pifq)) exhibits a cop-like phenotype in dark-grown seedlings, indicating that these transcription factors collectively repress photomorphogenesis in post-germinative darkness, and that photoactivated phy reverses this repression by inducing rapid phosphorylation and subsequent degradation of the PIFs upon initial light exposure. Previously, using targeted and random mutagenesis, we identified binding sites on phyB and PIF3 necessary for productive signaling interactions in vivo. Recently, we have defined residues within the PIF3 protein that are targets of light-induced phosphorylation. Using genome-wide transcriptome analysis of wild-type and pifq seedlings, we have identified PIF-regulated genes that respond rapidly and reciprocally in a PIF-dependent manner, to phy photoactivation and deactivation upon initial light exposure and vegetative-shade exposure respectively. Coupled with recent ChIP-seq analysis, these data have permitted identification of potential direct targets of the phy-PIF signaling system. 4.Accomplishments 1. Integration of multiple developmental pathways using light switches. A critical factor in successful seedling establishment in crop plants is a developmental strategy that enables postgerminative seedlings emerging from buried seed to grow vigorously upward in the subterranean darkness toward the soil surface. UC Berkeley and ARS scientists in Albany, CA have discovered that a central component of the mechanism underlying this strategy is the repression, by a family of gene transcription factors called PIFs (phytochrome-interacting factors), of the converse developmental pathway, termed photomorphogenesis, familiar as the normal growth of fully green seedlings after emergence from the soil. Examining large datasets, the scientists have discovered that a number of diverse signaling pathways appear to converge on the PIF family of bHLH transcription factors as intermediates in the transduction process. The list of pathways includes ethylene signaling, thus expanding the scope of the concept further. This knowledge will help integrate future efforts to modify plant growth in a changing environment.