Location: Plant Gene Expression Center
2020 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
Under Objective 1, ARS and University of California (UC) Berkeley scientists have investigated mutants in Pseudo-Response Regulators 5 and 7 (PRR5 and PRR7) for their effects on PIF-regulated phenotypic responses to vegetative shade. They have shown that the PIFs and PRRs conversely modulate seedling responsiveness to vegetative shade.
In support of Objective 2, ARS and UC Berkeley scientists have generated mutants in PRR5 and PRR7, and used real-time quantitative reverse transcription (RT-qPCR) analysis of known Phytochrome Interacting Factor (PIF) quintet (PIFs 1,3,4, 5 and 7)-dependent, rapidly shade-responsive genes in these prr mutants, to determine whether the PRRs influence the expression of these genes in response to vegetative shade. The data show that the PRRs repress the PIF-induced expression of those genes.
In support of Objective 3, ARS and UC Berkeley scientists have used genome-wide histone-marker (H3K4me3) assays to assess the quantitative relationship between the shade-induced expression and chromatin state (open vs. closed) of shade-responsive and shade non-responsive genes that are direct targets of transcriptional regulation by the PIF transcription factors. The data show that the shade responsive expression acquired by a subset of ‘shade-specific’ genes is strongly correlated with an increase in H3K4me3 levels, indicative of shade-induced chromatin remodeling.
Accomplishments
1. Central clock components modulate plant shade avoidance by directly repressing transcriptional activity of PIF proteins. Plants use phytochrome photoreceptors to monitor for the presence of competitors for photosynthetically active sunlight. They do so by sensing near-proximity or direct shade (“shade signals”) from neighboring vegetation. ARS scientists in Albany, California, and colleagues at University of California, Berkeley, have defined the functional role and molecular mechanism of action of Pseudo-Response Regulator proteins (PRRs) (integral components of the plant central circadian oscillator) in regulating growth responses to such signals. These PRRs have been shown to bind, intranuclearly, directly to members of the Phytochrome Interacting Factor (PIF) family of transcription factors, repressing their capacity to transcriptionally activate a subset of their Direct Target Genes. These findings show that the plant clock communicates with the genome using direct, physical interaction of multiple central oscillator components with the transcriptional regulatory machinery of the cell.
Review Publications
Zhang, Y., Pfeiffer, A., Tepperman, J.M., Dalton-Roesler, J., Leivar, P., Quail, P. 2020. Central clock components modulate plant shade avoidance by directly repressing transcriptional activation activity of PIF proteins. Proceedings of the National Academy of Sciences. 117(6):3261-3269. https://doi.org/10.1073/pnas.1918317117.
Leivar, P., Martin, G., Soy, J., Dalton-Roesler, J., Quail, P., Monte, E. 2020. Phytochrome-imposed inhibition of PIF7 activity shapes photoperiodic growth in Arabidopsis together with PIF1, 3, 4 and 5. Physiologia Plantarum. 169(3):452-466. https://doi.org/10.1111/ppl.13123.
