A Quartet of PIF bHLH Factors Provides a Transcriptionally Centered Signaling Hub That Regulates Seedling Morphogenesis through Differential Expression-Patterning of Shared Target Genes in Arabidopsis

Abstract

Dark-grown seedlings exhibit skotomorphogenic development. Genetic and molecular evidence indicates that a quartet of Arabidopsis Phytochrome (phy)-Interacting bHLH Factors (PIF1, 3, 4, and 5) are critically necessary to maintaining this developmental state and that light activation of phy induces a switch to photomorphogenic development by inducing rapid degradation of the PIFs. Here, using integrated ChIP–seq and RNA–seq analyses, we have identified genes that are direct targets of PIF3 transcriptional regulation, exerted by sequence-specific binding to G-box (CACGTG) or PBE-box (CACATG) motifs in the target promoters genome-wide. In addition, expression analysis of selected genes in this set, in all triple pif-mutant combinations, provides evidence that the PIF quartet members collaborate to generate an expression pattern that is the product of a mosaic of differential transcriptional responsiveness of individual genes to the different PIFs and of differential regulatory activity of individual PIFs toward the different genes. Together with prior evidence that all four PIFs can bind to G-boxes, the data suggest that this collective activity may be exerted via shared occupancy of binding sites in target promoters.

The Arabidopsis ZINC FINGER PROTEIN3 Interferes with Abscisic Acid and Light Signaling in Seed Germination and Plant Development

“…Thus, we propose that ZFP3 is a positive component of the red light signaling pathway(s). PIF transcription factors are negative regulators of red light signaling and promote scotomorphogenesis in dark-grown seedlings, controlling the expression of a large set of target genes (Leivar et al, 2012;Zhang et al, 2013). While transcript levels of PIF genes were not altered in ZFP3ox seedlings, many genes that were repressed or derepressed by ZFP3 overexpression were downand up-regulated in the quadruple pif1,3,4,5 mutant, respectively (Table III; Supplemental Data Sets S2-S5; Leivar et al, 2012).…”

PIFs: Systems Integrators in Plant Development

“…RNA-seq studies allow the identification of PIFq-regulated genes with full genome coverage, such as the identification of 2025 genes that are misregulated in the pifq mutant in the dark (Zhang et al, 2013), nearly twice the number of genes identified by microarray analysis under equivalent growth conditions (Leivar et al, 2009). ChIP-seq technology has been used to define the genomic occupancy of PIF3 (Zhang et al, 2013) and PIF4 in etiolated seedlings and of PIF5 in shade (Hornitschek et al, 2012) (see below). These studies have defined high confidence binding sites in the genome for each of these PIFs and the associated genes, including 828 genes for PIF3, 4363 genes for PIF4, and 1218 genes for PIF5.…”

“…These studies have defined high confidence binding sites in the genome for each of these PIFs and the associated genes, including 828 genes for PIF3, 4363 genes for PIF4, and 1218 genes for PIF5. The defined binding sites for these PIFs are located predominantly in promoter regions of target genes and are strongly enriched in the DNA motif G-box (CACGTG) and the E-box variant (CACATG and CATGTG) that has been called the PBE-box (for PIF binding E-box), suggesting that these PIFs have similar sequence recognition requirements in terms of DNA binding (Hornitschek et al, 2012;Oh et al, 2012;Zhang et al, 2013). In vitro assays have shown specific binding to the G-box for all the PIFs tested (PIF1, PIF3, PIF4, PIF5, and PIF7) (Martínez-García et al, 2000;Huq and Quail, 2002;Leivar et al, 2008a;Moon et al, 2008;Hornitschek et al, 2009) and to the PBE-box for PIF1, PIF3, and PIF4 (Kim et al, 2008;Hornitschek et al, 2012;Zhang et al, 2013).…”

“…By merging the list of PIF3 promoter-bound genes with the list of PIFq-regulated genes in etiolated seedlings, Zhang et al (2013) identified 128 genes as potential direct targets of transcriptional regulation by PIF3. Expression analysis of a subset of these genes in triple pif-mutant combinations compared with pifq show different degrees of regulation by all PIFq members (from marginal to robust), resulting in a mosaic of differential transcriptional responsiveness of individual genes to the different PIFs and of differential regulatory activity of individual PIFs toward the different genes (Zhang et al, 2013).…”

“…Expression analysis of a subset of these genes in triple pif-mutant combinations compared with pifq show different degrees of regulation by all PIFq members (from marginal to robust), resulting in a mosaic of differential transcriptional responsiveness of individual genes to the different PIFs and of differential regulatory activity of individual PIFs toward the different genes (Zhang et al, 2013). Because a majority of these genes are also bound by PIF4 and/or PIF5 (Zhang et al, 2013), the data suggest that the collective transcriptional activity of PIFq members may be exerted via shared occupancy of binding sites in target promoters.…”