PIF3 regulates anthocyanin biosynthesis in an HY5‐dependent manner with both factors directly binding anthocyanin biosynthetic gene promoters in Arabidopsis

Abstract

SummaryPhytochromes are red/far-red light receptors that regulate various light responses by initiating the transcriptional cascades responsible for changing the expression patterns of 10-30% of the entire plant transcriptome. Several transcription factors that are thought to participate in this process have been identified, but the functional relationships among them have not yet been fully elucidated. Here we investigated the functional relationship between two such transcription factors, PIF3 and HY5, and their effects on anthocyanin biosynthesis. Our results revealed that PIF3 and HY5 do not regulate each other at either the transcriptional or the protein levels in continuous light conditions, suggesting that they are not directly linked within phytochrome-mediated signaling. We found that both PIF3 and HY5 positively regulate anthocyanin biosynthesis by activating the transcription of the same anthocyanin biosynthetic genes, but the positive effects of PIF3 required functional HY5. Chromatin immunoprecipitation analyses indicated that both PIF3 and HY5 regulate anthocyanin biosynthetic gene expression by directly binding to different regions of the gene promoters in vivo. Additional experiments revealed that PIF3 bound the promoters regardless of light and HY5. Collectively, these data show that PIF3 and HY5 regulate anthocyanin biosynthesis by simultaneously binding anthocyanin biosynthetic gene promoters at separate sequence elements.

Sugar-Hormone Cross-Talk in Anthocyanin Biosynthesis

“…Anthocyanin biosynthesis is mainly regulated at the transcriptional level via a set of transcription factors including basic helix-loop-helix (bHLH), Leu-zipper, MADS-box, R2R3-MYB, WD40, WIP and WRKY factors (Marles et al, 2003). In Arabidopsis, the phytochrome (PHY)-interacting transcription factor 3 (PIF3), a bHLH protein, interacts directly with PHYs and positively regulates anthocyanin biosynthesis (Kim et al, 2006;Shin et al, 2007). LONG HYPOCOTYL5 (HY5), a Leu-zipper transcription factor (TF), serves as a point of convergence for phytochrome (PHY) and cryptochrome (CRY) signalings (Gyula et al, 2003), functioning as a positive regulator of anthocyanin biosynthesis (Chattopadhyay et al, 1998).…”

“…LONG HYPOCOTYL5 (HY5), a Leu-zipper transcription factor (TF), serves as a point of convergence for phytochrome (PHY) and cryptochrome (CRY) signalings (Gyula et al, 2003), functioning as a positive regulator of anthocyanin biosynthesis (Chattopadhyay et al, 1998). It binds directly to the promoters of early biosynthesis genes (EBGs) such as chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H) and flavonoid 3′-hydroxylase (F3′H), which are common to different flavonoid subpathways, and late biosynthesis genes (LBGs) such as dihydroflavonol 4-reductase (DFR), leucoanthocyanidin oxygenase (LDOX), anthocyanidin reductase (ANR) and UDP-glucose: flavonoid 3-O-glucosyltransferase (UF3GT) (Lee et al, 2007;Shin et al, 2007). Transcription factors from the R2R3-MYB, bHLH and WD40 classes interact to form MBW regulatory complexes (Borevitz et al, 2000;Gonzalez et al, 2008;Tohge et al, 2005;Zhang et al, 2003).…”

DNA-Binding Study Identifies C-Box and Hybrid C/G-Box or C/A-Box Motifs as High-Affinity Binding Sites for STF1 and LONG HYPOCOTYL5 Proteins

“…One of the best characterized bZIP factors thought to play a role in photomorphogenic seedling development and hormone signaling in Arabidopsis (Arabidopsis thaliana) is LONG HYPOCOTYL5 (HY5) (Oyama et al, 1997;Ang et al, 1998;Holm et al, 2002;Cluis et al, 2004). The function of HY5 in photomorphogenesis is well illustrated in hy5 mutant seedlings, which have defects in light inhibition of hypocotyl elongation, in light-induced chlorophyll, and in anthocyanin accumulation (Oyama et al, 1997;Sibout et al, 2006;Shin et al, 2007). The model for light-signaling pathways in photomorphogenic development includes the photoreceptor phytochromes, the ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), and the positive signaling component HY5 (Deng et al, 1992;Ang and Deng, 1994;Holm et al, 2002).…”

“…An in vitro binding analysis of STF1 and HY5 demonstrate that these bZIP proteins preferentially recognize C-, hybrid C/G-, and C/A-box motifs over G-box motifs. The in vitro analysis corresponds with earlier in vivo and functional analyses that identified the predicted locations of the HY5 binding sites in the promoters of anthocyanin biosynthetic genes (Hartmann et al, 2005;Shin et al, 2007). It also helps explain the abundance (approximately 3,900) of in vivo HY5 targets in the Arabidopsis genome identified by coupled chromatin immunoprecipitation and DNA chip hybridization (ChIP-chip; Lee et al, 2007).…”

Phytochrome‐mediated inhibition of shade avoidance involves degradation of growth‐promoting bHLH transcription factors

“…In its light-activated state (Pfr), phyB can physically interact with a group of related bHLH transcription factors known as phytochrome interacting proteins (PIFs), suggesting a short signal transduction cascade from light sensing to regulated gene expression (Jiao et al, 2007;Khanna et al, 2004;Martinez-Garcia et al, 2000). Members of the PIF family control phytochrome responses including seed germination, seedling de-etiolation and shade avoidance (Huq and Quail, 2002;Huq et al, 2004;Kim et al, 2003;Oh et al, 2004;Penfield et al, 2005;Salter et al, 2003;Shin et al, 2007). Interaction between phyB and the PIFs is mediated by the active phytochrome binding (APB) domain that is present at the N-terminus of these proteins (Khanna et al, 2004).…”