Arabidopsis FHY3 and HY5 positively mediate induction of COP1 transcription in response to photomorphogenic UV-B light

Abstract

As sessile organisms, higher plants have evolved the capacity to sense and interpret diverse light signals to modulate their development. In Arabidopsis thaliana, low-intensity and long-wavelength UV-B light is perceived as an informational signal to mediate UV-B-induced photomorphogenesis. Here, we report that the multifunctional E3 ubiquitin ligase, CONSTITUTIVE PHOTOMORPHOGENESIS1 (COP1), a known key player in UV-B photomorphogenic responses, is also a UV-B-inducible gene. Two transcription factors, FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and ELONGATED HYPOCOTYL5 (HY5), directly bind to distinct regulatory elements within the COP1 promoter, which are essential for the induction of the COP1 gene mediated by photomorphogenic UV-B signaling. Absence of FHY3 results in impaired UV-B-induced hypocotyl growth and reduced tolerance against damaging UV-B. Thus, FHY3 positively regulates UV-B-induced photomorphogenesis by directly activating COP1 transcription, while HY5 promotes COP1 expression via a positive feedback loop. Furthermore, FHY3 and HY5 physically interact with each other, and this interaction is diminished by UV-B. Together, our findings reveal that COP1 gene expression in response to photomorphogenic UV-B is controlled by a combinatorial regulation of FHY3 and HY5, and this UV-B-specific working mode of FHY3 and HY5 is distinct from that in far-red light and circadian conditions.

Figure 1.

COP1 Is Induced by UV-B at the mRNA and Protein Levels. (A) Changes in COP1 transcript levels in 4-d-old wild-type (Col) seedlings transferred from darkness (Dk) to far-red (FR), red (R), or blue (B) light conditions or from −UV-B to +UV-B and harvested at indicated time points. The transcript level at 0 h was set as 1. Error bars represent standard deviation (SD) of three biological replicates. (B) Changes in COP1 protein levels in 4-d-old wild-type (Col) seedlings transferred from −UV-B to +UV-B and harvested at indicated time points. Anti-RPN6 was used as a loading control.

Figure 2.

FHY3 and HY5 Bind to the COP1 Promoter in Vitro and in Vivo. (A) Diagram of fragments of the COP1 promoter. The adenine of the translational start codon (ATG) is designated as position +1. Orange and blue blocks indicate putative FBS and ACEs, respectively. Arrows indicate the fragments amplified in ChIP-PCR assays. The wild-type and mutated sites of the COP1 promoter subfragments are shown in uppercase and lowercase letters, respectively. (B) and (C) FHY3 (B) and HY5 (C) bind to the COP1 promoter in yeast one-hybrid assays. Empty vector expressing the AD domain alone is the negative control. WT, the wild type. (D) and (E) GST-FHY3N (D) and GST-HY5 (E) specifically bind to COP1p-FBS and COP1p-ACE3 probes respectively in EMSA assays. FP, free probes. (F) Representative results of ChIP-PCR assays. Chromatin fragments prepared from 4-d-old wild-type (Col) seedlings grown under −UV-B and +UV-B were immunoprecipitated by the polyclonal anti-FHY3 and anti-HY5 antibodies, respectively. Input, PCR reactions using the samples before immunoprecipitation. Ab, antibody. a and b correspond to the DNA fragments shown in (A). (G) FHY3 and HY5 protein levels of 4-d-old wild-type (Col) seedlings grown under −UV-B and +UV-B. Anti-RPN6 was used as a loading control. (H) qPCR analyses of the FBS-containing fragment (COP1pro-FBS) and the ACE3-containing fragment (COP1pro-ACE3) in the COP1 promoter in anti-FHY3 and anti-HY5 ChIP assays, respectively, using 4-d-old wild-type (Col) seedlings grown under −UV-B and +UV-B. The ChIP values were normalized to their respective DNA inputs. Error bars represent sd of three biological replicates.

Figure 3.

FHY3 and HY5 Target COP1 to UV-B–Dependent Transcriptional Activation. (A) The transcript levels of COP1 in 4-d-old FHY3p:FHY3-GR/fhy3-4 seedlings grown under −UV-B and then treated with 10 μM DEX before being transferred to −UV-B and +UV-B. Relative expression levels were normalized to the Mock (equal volume of ethanol) treatment. Error bars represent sd of three biological replicates. (B) Structure of the dual-luciferase reporter construct in which the firefly LUC reporter gene is driven by the wild type, FBS-mutated (mFBS), or ACE-mutated (mACE3) COP1 promoter. The REN luciferase reporter gene is controlled by the constitutive 35S promoter. The T-DNA left border and right border are indicated as LB and RB, respectively. (C) The relative LUC activity normalized to the REN activity (LUC/REN) in tobacco leave cells transiently transformed with the indicated effectors and wild-type reporter construct COP1p:LUC under −UV-B and +UV-B. Empty effector vectors were included as negative controls. (D) The LUC/REN values in tobacco leave cells transiently transformed with the indicated effectors and wild-type (WT) or mutated reporter constructs under +UV-B. Empty reporter vectors were included as negative controls. *P < 0.05 and **P < 0.01 by Student’s t test. CK, empty reporter vector. Error bars represent sd of four biological replicates. (E) The relative LUC activity normalized to the REN activity (LUC/REN) in wild-type (Col) and mutant (uvr8-6) Arabidopsis leave cells transiently transformed with the indicated effectors and wild-type reporter construct COP1p:LUC under −UV-B and +UV-B. Empty effector vectors were included as negative controls.

Figure 4.

FHY3 Is Involved in UV-B–Induced Photomorphogenesis at the Seedling Stage. (A) Phenotypes of 4-d-old wild-type (No-0), fhy3-4, and 3FLAG-FHY3-3HA/fhy3-4 seedlings grown under −UV-B and +UV-B. Quantitative analysis of hypocotyl length is presented as percentage of −UV-B. Error bars represent sd of three biological replicates. Bar = 1 mm. (B) Quantitative analysis of anthocyanin accumulation of the seedlings in (A). Error bars represent sd of three biological replicates. (C) to (F) The transcript levels of UV-B–induced marker genes ELIP2 (C), At4g15480 (D), DREB2A (E), and HY5 (F) in 4-d-old wild-type (No-0) and fhy3-4 seedlings grown under −UV-B to +UV-B. The transcript level in No-0 under −UV-B was set as 1. Error bars represent sd of three biological replicates. (G) to (J) The transcript levels of UV-B–induced marker genes ELIP2 (G), At4g15480 (H), DREB2A (I), and HY5 (J) in 4-d-old wild-type (No-0) and fhy3-4 seedlings transferred from −UV-B to +UV-B and harvested at indicated time points. The transcript level at 0 h in No-0 was set as 1. Error bars represent sd of three biological replicates. [See online article for color version of this figure.]

Figure 5.

FHY3 and HY5 Are Required for Tolerance to Damaging UV-B. Wild-type (No-0 and Ws), fhy3-4, and hy5-ks50 seedlings were grown under white light supplemented with photomorphogenic UV-B for 12 d and then were irradiated without (−UV-B) or with (+UV-B) damaging broadband UV-B for 12 h. Bar = 5 mm.

Figure 6.

FHY3 and HY5 Are Required for Accumulation of COP1 Transcripts and Proteins under Photomorphogenic UV-B. (A) and (B) Changes in CHS (A) and COP1 (B) transcript levels in 4-d-old wild-type (No-0) and fhy3-4 seedlings transferred from −UV-B to +UV-B and harvested at indicated time points. The transcript level at 0 h in No-0 was set as 1. Error bars represent sd of three biological replicates. (C) COP1 protein levels in 4-d-old wild-type (No-0) and fhy3-4 seedlings transferred from −UV-B to +UV-B and harvested at indicated time points. Anti-RPN6 was used as a loading control. (D) and (E) CHS (D) and COP1 (E) transcript levels in 4-d-old wild-type (Ws) and hy5-ks50 seedlings transferred from −UV-B to +UV-B and harvested at the indicated time points. The transcript level at 0 h in Ws was set as 1. Error bars represent sd of three biological replicates. (F) COP1 protein levels in 4-d-old wild-type (Ws) and hy5-ks50 seedlings transferred from −UV-B to +UV-B and harvested at indicated time points. Anti-RPN6 was used as a loading control.

Figure 7.

COP1, FHY3, and HY5 Genetically Interact with Each Other under Photomorphogenic UV-B. (A) and (B) Phenotypes (A) and CHS transcript levels (B) of 4-d-old wild-type (No-0), fhy3-4, COP1OE, and COP1OE/fhy3-4 seedlings grown under −UV-B and +UV-B. (C) and (D) Phenotypes (C) and the CHS transcript levels (D) of 4-d-old wild-type (Ws and No-0), hy5-ks50, GUS-COP1/cop1-5, and GUS-COP1/hy5-ks50 seedlings grown under −UV-B and +UV-B. (E) and (F) Phenotypes (E) and the CHS transcript levels (F) of 4-d-old wild-type (Col), fhy3-1, hy5-215, and fhy3-1 hy5-215 seedlings grown under −UV-B and +UV-B. Quantitative analysis of hypocotyl length is presented as percentage of −UV-B. The transcript level in the wild type under -UV-B was set as 1. Error bars represent sd of three biological replicates. Bars = 1 mm. [See online article for color version of this figure.]

Figure 8.

Photomorphogenic UV-B Affects the Functional Interaction between FHY3 and HY5. (A) The interaction between FHY3 and HY5 under −UV-B and +UV-B in LCI assays. Error bars represent sd of four biological replicates. (B) FHY3 and HY5 protein levels in 4-d-old wild-type (Col), fhy3-1, and hy5-215 seedlings grown under +UV-B. Anti-RPN6 was used as a loading control. Band intensities of FHY3, HY5, and RPN6 in Col seedlings were set to 100. Relative band intensities were then calculated and are indicated by numbers below blots. (C) and (D) qPCR analysis of the FBS-containing fragment (COP1pro-FBS) in the COP1 promoter in anti-FHY3 ChIP assays (C) and the ACE3-containing fragment (COP1pro-ACE3) in the COP1 promoter in anti-HY5 ChIP assays (D) using 4-d-old wild-type (Col), hy5-215, and fhy3-1 seedlings grown under +UV-B. The ChIP values were normalized to their respective DNA inputs. Error bars represent sd of three biological replicates. [See online article for color version of this figure.]

Figure 9.

A Model for the Functional Interaction of COP1, FHY3, and HY5 in UV-B–Induced Photomorphogenesis. Under photomorphogenic UV-B, UVR8 monomerizes and interacts with COP1, triggering downstream signaling, including HY5-regulated gene expression, which is repressed by RUPs. COP1 is a UV-B–inducible gene. FHY3 and HY5 bind to the FBS motif and the ACE element in the COP1 promoter, respectively, and activate COP1 transcription. This transcriptional modulation ensures more active UVR8-COP1-HY5 core pathway. Red arrows, positive regulation in the core pathway; blue arrows, positive regulation in UV-B–mediated gene induction; black bar, negative regulation.