Location: Plant Gene Expression Center
2021 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 multiple mutant combinations to define phenotypic parameters in parallel with definition of relevant underlying molecular phenotypes of these mutants. New mutant combinations with non-Phy-Interacting transcription Factor (PIF) genes were generated for testing epistatic relationships, where appropriate. In support of Objective 2, ARS and UC Berkeley scientists have used RNA-sequencing (RNA-seq) analysis of pif-mutant combinations that indicate differential PIF-member activity based on the phenotypic data. Lines expressing epitope-tagged versions of the previously unavailable PIFs have been generated as a prelude to ChIP analysis with these PIFs. In support of Objective 3, ARS and UC Berkeley scientists have used genome-wide histone-marker (H3K4me3 and H3K9ac) assays to assess the quantitative relationship between the light- and shade-induced expression, and chromatin state (open vs. closed), of the two distinct, PIF-direct-target gene-sets exhibiting these converse expression patterns. The data show that the light-induced genes, on average, show a somewhat more rapid induction of chromatin changes than of increased expression, whereas the shade-induced genes show a more rapid increase in expression than in chromatin state. Combined, these results suggest potentially contrasting mechanisms of transcriptional regulation by light and shade.
Accomplishments
1. Light-imposed, phytochrome-mediated inhibition of transcription factor activity shapes photoperiodic growth. In normal, day-night, diurnal conditions, seedling growth is inhibited in long days (LDs), but promoted under the long nights of short days (SDs). This behavior is partly implemented by phytochrome (phy) light-receptor-imposed oscillations in the abundance of the growth-promoting, phy-interacting transcription factors (called PIFs). By combining growth- and gene-expression analysis in mutants of the different PIF-family members, ARS and University of California, Berkeley, researchers, in Albany, California, have shown that two members, PIF3 and PIF7, dominate in promoting growth during the dark hours of SD, by regulating growth-related gene expression. Collectively, the data suggest that PIF7, together with contributions from the other PIFs, 1,3,4 and 5, is required for the photoperiodic regulation of seasonal growth.
