Location: Plant Gene Expression Center
2019 Annual Report
Objectives
The long-term goal of this program is to define the molecular mechanisms by which the phy family of sensory photoreceptors perceive and transduce informational light signals from the environment to photoresponsive genes in regulating plant adaptational growth and development. The central goal of this proposal is to define the mechanisms by which the phys induce and modulate the Shade-Avoidance Response (SAR) in the constantly fluctuating light environment encountered in dense crop vegetational canopies. Toward this goal, the specific objectives for this funding period are:
Objective 1: Define the biological functions of the multiple Phy-Interacting transcription Factor (PIF)-family members in controlling the shade-avoidance response, including dissection of the relative contributions of the individual PIFs to this process.
Objective 2: Define the direct gene-targets of shade-active-PIF transcriptional regulation, and determine whether, and to what extent, this regulation involves differential direct targeting of rapidly shade-responsive genes by the individual PIF-family members.
Objective 3: Define the mechanism by which genes that lack apparent PIF regulation during skotomorphogenesis, but acquire PIF-dependent shade responsiveness in green seedlings (‘shade-specific’ genes), acquire this capacity.
Approach
Objective 1: To define the biological functions of the multiple Phy-Interacting transcription Factor (PIF)-family members in controlling the Shade-Avoidance Response (SAR), including dissection of the relative contributions of the individual PIFs to this process, we will perform phenotypic analyses of our array of higher order, pentuple and sextuple combinations of the pif mutants. This will enable us to define the relative quantitative contributions of the individual PIFs to the various facets of the SAR.
Objective 2: To define the direct gene-targets of shade-active-PIF transcriptional regulation, and determine whether, and to what extent, this regulation involves differential direct targeting of rapidly shade-responsive genes by the individual PIF-family members, we will analyze the global expression profiles of this set of higher-order pif-mutant combinations in response to shade, using RNA-seq, and identify the PIF-bound subset of these genes by ChIP-seq analysis. Direct targets of transcriptional regulation by the individual PIFs will be identified by integrating the RNA-seq and ChIP-seq data for each PIF as we have done for the PIF quartet in dark-grown seedlings.
Objective 3: To define the mechanism by which genes that lack apparent PIF regulation during skotomorphogenesis, but acquire PIF-dependent shade responsiveness in green seedlings (‘shade-specific’ genes), acquire this capacity, we will use ChIP-seq analysis for altered accessibility of PIF-binding sites and altered histone marks, indicative of nucleosome modifications, in response to shade. This will enable us to interrogate the genomes of light- and dark-grown Arabidopsis seedlings for differential histone-mark signatures correlated with the acquisition of PIF-dependent shade-signal responsiveness.
Progress Report
In support of Objective 1, ARS and University of California (UC) Berkeley scientists have used computational analysis of published data to identify factors that are associated with PIF (Phytochrome-Interacting Factor) transcription factors that are bound to their cis elements on direct target genes. Two of these factors are Pseudo-response Regulators 5 and 7 (PRR5 and PRR7). Mutants in these factors are being investigated for their potential effects on PIF-regulated phenotypic responses to vegetative shade.
In support of Objective 2, ARS and UC Berkeley scientists have initiated studies to determine whether the co-localization of the PRR proteins with the PIFs on the promoters of PIF target genes are functionally involved in modulating the expression of these genes. Quantitative reverse transcription PCR (RT-qPCR) analysis of known PIF-quartet-dependent, rapidly shade-responsive genes are being used, in the prr mutants, to determine whether these factors influence the expression of these genes in response to vegetative shade.
In support of Objective 3, ARS and UC Berkeley scientists are using genome-wide histone-marker assays that are providing an assessment of the quantitative relationship between the shade-induced expression and chromatin state (open versus closed) in shade-responsive and shade non-responsive genes that are direct targets of transcriptional regulation by PIF transcription factors. Initial indications are that there is strong correlation between open-chromatin markers and shade responsive expression of a subset of these genes.
Accomplishments
