C-terminal region of the UV-B photoreceptor UVR8 initiates signaling through interaction with the COP1 protein

Abstract

UV-B light initiates photomorphogenic responses in plants. Arabidopsis UV RESISTANCE LOCUS8 (UVR8) specifically mediates these responses by functioning as a UV-B photoreceptor. UV-B exposure converts UVR8 from a dimer to a monomer, stimulates the rapid accumulation of UVR8 in the nucleus, where it binds to chromatin, and induces interaction of UVR8 with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), which functions with UVR8 to control photomorphogenic UV-B responses. Although the crystal structure of UVR8 reveals the basis of photoreception, it does not show how UVR8 initiates signaling through interaction with COP1. Here we report that a region of 27 amino acids from the C terminus of UVR8 (C27) mediates the interaction with COP1. The C27 region is necessary for UVR8 function in the regulation of gene expression and hypocotyl growth suppression in Arabidopsis. However, UVR8 lacking C27 still undergoes UV-B-induced monomerization in both yeast and plant protein extracts, accumulates in the nucleus in response to UV-B, and interacts with chromatin at the UVR8-regulated ELONGATED HYPOCOTYL5 (HY5) gene. The UV-B-dependent interaction of UVR8 and COP1 is reproduced in yeast cells and we show that C27 is both necessary and sufficient for the interaction of UVR8 with the WD40 domain of COP1. Furthermore, we show that C27 interacts in yeast with the REPRESSOR OF UV-B PHOTOMORPHOGENESIS proteins, RUP1 and RUP2, which are negative regulators of UVR8 function. Hence the C27 region has a key role in UVR8 function.

Fig. 1.

The GFP–ΔC27UVR8 fusion does not functionally complement transgenic uvr8-1 plants. (A) The GFP–ΔC27UVR8 fusion protein lacking UVR8 amino acids 397–423 coupled to the UVR8 promoter. (B) Western blot of whole cell extracts from uvr8-1 plants expressing UVR8_pro:GFP–UVR8 or UVR8_pro:GFP–ΔC27UVR8 (lines 4.3, 11.2, and 7.2) probed with anti-GFP antibody. Ponceau staining of Rubisco large subunit (rbcL) is shown as a loading control. (C) RT-PCR assays of HY5 and control ACTIN2 transcripts in Ler, uvr8-1, uvr8-1/UVR8_pro:GFP–ΔC27UVR8 (lines 4.3, 11.2, and 7.2) and uvr8-1/UVR8_pro:GFP–UVR8 (line 6.2) (13) plants grown under 20 μmol m⁻² s⁻¹ white light (−) and exposed to 3 μmol m⁻² s⁻¹ broadband UV-B for 4 h (+). (D) Hypocotyl lengths (±SE, n = 10) for 4-d-old wild-type Ler, wild-type Ws, uvr8-1, uvr8-1/UVR8_pro:GFP–UVR8, uvr8-1/UVR8_pro:GFP–ΔC27UVR8 (line 4.3), and hy5-ks50 hyh (Ws background) plants grown in 1.5 μmol m⁻² s⁻¹ white light (−UV-B) supplemented with 1.5 μmol m⁻² s⁻¹ narrowband UV-B (+UV-B).

Fig. 2.

The C27 region of UVR8 is not required for chromatin association, UV-B–induced monomerization or nuclear accumulation. (A) Chromatin immunoprecipitation assays of DNA associated with GFP–UVR8 or GFP–ΔC27UVR8. PCR of the HY5 promoter (−331 to +23) and control ACTIN2 DNA from uvr8-1/UVR8_pro:GFP–UVR8 and uvr8-1/UVR8_pro:GFP–ΔC27UVR8 (line 11.2) plants grown under 20 μmol m⁻² s⁻¹ white light and exposed to 3 μmol m⁻² s⁻¹ broadband UV-B for 4 h. IN, input DNA before immunoprecipitation; GFP, DNA immunoprecipitated by anti-GFP antibody; mock, no antibody control. (B) Western blots of whole cell extracts from yeast expressing HA-UVR8 (Left) or HA-ΔC27UVR8 (Right) probed with anti-HA antibody. The yeast extracts were treated (+) or not (−) with 1 μmol m⁻² s⁻¹ narrowband UV-B for the times shown. Samples were run on an 8% native gel. The UVR8 dimer (D) and monomer (M) are indicated. (C) Western blot of whole cell extracts from uvr8-1/UVR8_pro:GFP–UVR8 and uvr8-1/UVR8_pro:GFP–ΔC27UVR8 (line 11.2) plants probed with anti-GFP antibody. Extracts were treated (+) or not (−) with 4 μmol m⁻² s⁻¹ narrowband UV-B for 30 min before SDS-loading buffer was added. Samples were then run on a 7.5% SDS/PAGE gel without boiling. Ponceau staining of Rubisco large subunit (rbcL) is shown as a loading control. The UVR8 dimer (D) and monomer (M) are indicated. (D) Confocal images of GFP fluorescence in leaf epidermal tissue of uvr8-1/UVR8_pro:GFP–ΔC27UVR8 (line 11.2) plants grown under 20 μmol m⁻² s⁻¹ white light (−UV-B) and exposed to 3 μmol m⁻² s⁻¹ broadband UV-B for 4 h (+UV-B). (Scale bars, 20 μm.) (E) Percentage of nuclei identified by DAPI fluorescence in uvr8-1/UVR8_pro:GFP–ΔC27UVR8 (line 11.2) plants showing colocalization of GFP fluorescence under 20 μmol m⁻² s⁻¹ white light (−UV-B) and following exposure to 3 μmol m⁻² s⁻¹ broadband UV-B for 4 h (+UV-B). Data are the mean ± SE (n = 20 images).

Fig. 3.

The C27 region of UVR8 is necessary and sufficient for interaction with both the WD40 region of COP1 and RUP proteins. (A) The ΔC27UVR8 protein does not interact with COP1 in plants. Whole cell extracts were obtained from uvr8-1/UVR8_pro:GFP–UVR8 and uvr8-1/UVR8_pro:GFP–ΔC27UVR8 (line 4.3) plants treated (+) or not (−) with 3 μmol m⁻² s⁻¹ narrowband UV-B. Coimmunoprecipitation assays were carried out under the same illumination conditions. Input samples (15 μg, IN) and eluates (IP) were fractionated by SDS/PAGE and Western blots were probed with anti-COP1 and anti-GFP antibodies. (B) Yeast two-hybrid plasmids containing a DNA binding domain (BD) and an activation domain (AD) fused to the proteins indicated were cotransformed in yeast, which were then spotted on fully selective media plates. All colonies grew on nonselective media (not shown). As controls, yeast were cotransformed with plasmids containing mammalian p53 and antigen T (positive control) or no inserts (−, negative control). ΔC27UVR8 lacks amino acids 397–423 of UVR8 (Fig. 1A). Construct C27–UVR8 contains only these 27 amino acids of UVR8. WD40 corresponds to the 341 C-terminal amino acids of COP1 (334–675). Yeast were left to grow in darkness (−) or under 0.1 μmol m⁻² s⁻¹ narrowband UV-B (+). (C) Yeast two-hybrid assay undertaken as in B with RUP1 and RUP2. (D) Yeast two-hybrid assay undertaken as in B except that yeast were spotted onto plates in a serial dilution.