UV-B promotes rapid nuclear translocation of the Arabidopsis UV-B specific signaling component UVR8 and activates its function in the nucleus

Abstract

Arabidopsis thaliana UV RESISTANCE LOCUS8 (UVR8) is a UV-B-specific signaling component that binds to chromatin and regulates UV protection by orchestrating expression of a range of genes. Here, we studied how UV-B regulates UVR8. We show that UV-B stimulates the nuclear accumulation of both a green fluorescent protein (GFP)-UVR8 fusion and native UVR8. Nuclear accumulation leads to UV-B induction of the HY5 gene, encoding a key transcriptional effector of the UVR8 pathway. Nuclear accumulation of UVR8 is specific to UV-B, occurs at low fluence rates, and is observed within 5 min of UV-B exposure. Attachment of a nuclear export signal (NES) to GFP-UVR8 causes cytosolic localization in the absence of UV-B. However, UV-B promotes rapid nuclear accumulation of NES-GFP-UVR8, indicating a concerted mechanism for nuclear translocation. UVR8 lacking the N-terminal 23 amino acids is impaired in nuclear translocation. Attachment of a nuclear localization signal (NLS) to UVR8 causes constitutive nuclear localization. However, NLS-GFP-UVR8 only confers HY5 gene expression following UV-B illumination, indicating that nuclear localization, although necessary for UVR8 function, is insufficient to cause expression of target genes; UV-B is additionally required to stimulate UVR8 function in the nucleus. These findings provide new insights into the mechanisms through which UV-B regulates gene expression in plants.

Figure 1.

UVR8 and GFP-UVR8 Protein Levels Are Unaffected by Different Llight Qualities. (A) Protein gel blot of total protein extracts from Arabidopsis Landsberg erecta (Ler) and uvr8-1 plants grown in 20 μmol m⁻² s⁻¹ white light (LW) and treated with either 3 μmol m⁻² s⁻¹ UV-B, 100 μmol m⁻² s⁻¹ UV-A, 100 μmol m⁻² s⁻¹ red (R), or 100 μmol m⁻² s⁻¹ white light (HW) for 4 h, probed with antibodies to UVR8 and to UGPase as a loading control. (B) Protein gel blot of total proteins from transgenic uvr8-1 plants expressing UVR8_pro:GFP-UVR8 (line 6-2) grown and treated as in (A) probed with anti-GFP antibody. Ponceau staining of ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) large subunit (rbcL) is shown as a loading control.

Figure 2.

GFP-UVR8 Expressed from the UVR8 Promoter Is Functional in Transgenic uvr8-1 Mutant Plants. (A) Protein gel blot of total protein extracts from Ler and three independent lines of transgenic uvr8-1 plants expressing UVR8_pro:GFP-UVR8 probed with an anti-UVR8 antibody. Ponceau staining of Rubisco large subunit (rbcL) is shown as a loading control. (B) RT-PCR assay of HY5 and control ACTIN2 transcripts in Ler, uvr8-1, and UVR8_pro:GFP-UVR8 lines grown in 20 μmol m⁻² s⁻¹ white light (LW) and exposed to 3 μmol m⁻² s⁻¹ UV-B for 4 h.

Figure 3.

UV-B Stimulates Nuclear Accumulation of GFP-UVR8. (A) Confocal images of GFP and DAPI fluorescence in leaf epidermal tissue of UVR8_pro:GFP-UVR8 (line 6-2) plants grown in 20 μmol m⁻² s⁻¹ white light (LW) and exposed to 3 μmol m⁻² s⁻¹ UV-B for 4 h. Bars = 20 μm. (B) Confocal images of GFP and DAPI fluorescence in leaf epidermal tissue of transgenic wild-type plants expressing GFP from the 35S promoter grown in white light (LW) and exposed to UV-B as in (A). Bars = 20 μm. (C) The percentage of nuclei identified by DAPI fluorescence showing colocalization of GFP fluorescence before (LW) and after UV-B exposure as in (A). Data are the mean ± se (n = 20 images). (D) Measurements of relative GFP-UVR8 fluorescence intensity of nuclei before (LW) and after UV-B illumination as in (A). Data are the mean ± se from three experiments each with at least 100 nuclei per treatment.

Figure 4.

UV-B Stimulates Nuclear Accumulation of Native UVR8. Protein gel blot of cytosolic and nuclear fractions (20 and 30 μg protein, respectively) of Ler plants grown in 20 μmol m⁻² s⁻¹ white light (LW) and treated with 3 μmol m⁻² s⁻¹ UV-B for 4 h probed with anti-UVR8, anti-UGPase, and anti-histone H3 antibodies.

Figure 5.

Spectral Specificity, Kinetics, and Fluence Rate Dependence of GFP-UVR8 Nuclear Accumulation. (A) The percentage of nuclei identified by DAPI fluorescence showing colocalization of GFP fluorescence in UVR8_pro:GFP-UVR8 (line 6-2) plants grown in 20 μmol m⁻² s⁻¹ white light (LW) and exposed to either 3 μmol m⁻² s⁻¹ UV-B, 100 μmol m⁻² s⁻¹ red, or 100 μmol m⁻² s⁻¹ UV-A light for 4 h. (B) Nuclear GFP/DAPI colocalization in UVR8_pro:GFP-UVR8 (line 6-2) plants grown in white light (LW) as in (A) and exposed to UV-B (3 μmol m⁻² s⁻¹) for 10 min, 30 min, 1 h, 4 h, or 4 h then transferred to darkness (D) for 24 h. (C) Nuclear GFP/DAPI colocalization in UVR8_pro:GFP-UVR8 (line 6-2) plants grown in white light (LW) as in (A) and exposed to different fluence rates of UV-B (0.1, 0.3, 0.5, 1, and 3 μmol m⁻² s⁻¹) for 4 h. Data for all graphs are the mean ± se (n = 20 images).

Figure 6.

UV-B Induces Nuclear Accumulation of the NES-GFP-UVR8 Fusion. (A) Protein gel blot of total protein extracts from UVR8_pro:GFP-UVR8 plants (line 6-2), uvr8-1, and three independent lines of uvr8-1 plants expressing NES-GFP-UVR8 from the UVR8 promoter probed with an anti-GFP antibody. Ponceau staining of Rubisco large subunit (rbcL) is shown as a loading control. (B) Confocal images of GFP and DAPI fluorescence in leaf epidermal tissue of UVR8_pro:NES-GFP-UVR8 transgenic plants (line 14-5) grown in 20 μmol m⁻² s⁻¹ white light (LW) and exposed to 3 μmol m⁻² s⁻¹ UV-B for 4 h. The arrow indicates a nucleus with no GFP fluorescence. Bars = 20 μm. (C) The percentage of nuclei identified by DAPI fluorescence showing colocalization of GFP fluorescence in UVR8_pro:NES-GFP-UVR8 plants (line 14-5) grown in white light (LW) as in (B) and exposed to UV-B (3 μmol m⁻² s⁻¹) for 5 min, 30 min, 1 h, 4 h, or 4 h then transferred to darkness for 24 h. Data are the mean ± se (n = 20 images). (D) Nuclear GFP/DAPI colocalization in UVR8_pro:NES-GFP-UVR8 plants (line 14-5) grown in white light (LW) as in (B) and exposed to different fluence rates of UV-B (0.1, 0.3, 0.5, 1, and 3 μmol m⁻² s⁻¹) for 4 h. Data are the mean ± se (n = 20 images). (E) RT-PCR assay of HY5 and control ACTIN2 transcripts in Ler, uvr8-1, and UVR8_pro:NES-GFP-UVR8 lines grown in white light (LW) and exposed to UV-B as in (B).

Figure 7.

The GFP-ΔNUVR8 Fusion Protein Is Impaired in UV-B–Specific Nuclear Accumulation and Fails to Complement Transgenic uvr8-1 Plants. (A) Protein gel blot of total protein extracts from UVR8_pro:GFP-UVR8 (line 6-2), uvr8-1, and three independent lines of uvr8-1 plants expressing GFP-ΔNUVR8 from the UVR8 promoter probed with an anti-GFP antibody. Ponceau staining of Rubisco large subunit (rbcL) is shown as a loading control. (B) Confocal images of GFP and DAPI fluorescence in leaf epidermal tissue of UVR8_pro:GFP-ΔNUVR8 plants (line 8-2) grown in 20 μmol m⁻² s⁻¹ white light (LW) and exposed to 3 μmol m⁻² s⁻¹ UV-B for 4 h. Bars = 20 μm. (C) The percentage of nuclei identified by DAPI fluorescence showing colocalization of GFP fluorescence before (LW) and after UV-B exposure of UVR8_pro:GFP-ΔNUVR8 (line 8-2) plants as in (B). Data are the mean ± se (n = 20 images). (D) RT-PCR assay of HY5 and control ACTIN2 transcripts in Ler, uvr8-1, and UVR8_pro:GFP-ΔNUVR8 lines grown in white light (LW) and exposed to UV-B as in (B). (E) Chromatin immunoprecipitation assay of DNA associated with GFP-UVR8 or GFP-ΔNUVR8. PCR of HY5 promoter (−331 to +23) and control ACTIN2 DNA from UVR8_pro:GFP-UVR8 (line 6-2) (top panel) and UVR8_pro:GFP-ΔNUVR8 (line 8-2) (bottom panel) transgenic plants grown in white light and exposed to UV-B as in (B). Lane 1, input DNA before immunoprecipitation; lane 2, DNA immunoprecipitated by anti-GFP antibody; lane 3, DNA immunoprecipitated by anti-UVR8 antibody; lane 4, no antibody control.

Figure 8.

The NLS-GFP-UVR8 Fusion Protein Is Constitutively Nuclear but Requires UV-B for Function. (A) Protein gel blot of total protein extracts from UVR8_pro:GFP-UVR8 (line 6-2), uvr8-1, and three independent lines of uvr8-1 plants expressing NLS-GFP-UVR8 from the UVR8 promoter probed with an anti-GFP antibody. Ponceau staining of Rubisco large subunit (rbcL) is shown as a loading control. (B) Confocal image of GFP fluorescence in leaf epidermal tissue of UVR8_pro:NLS-GFP-UVR8 (line 1-5) plants grown in 20 μmol m⁻² s⁻¹ white light (LW) and exposed to 3 μmol m⁻² s⁻¹ UV-B for 4 h. Bars = 20 μm. (C) Top panel: protein gel blot of total protein extracts (5 μg) from UVR8_pro:NLS-GFP-UVR8 (line 1-5) plants grown in white light (LW) as in (B) and exposed to 3 μmol m⁻² s⁻¹ UV-B for 4 or 24 h probed with an anti-GFP antibody. Ponceau staining of Rubisco large subunit (rbcL) is shown as a loading control. Bottom panel: protein gel blot as above, showing increased amounts of protein (LW sample) loaded on the gel. (D) RT-PCR assay of HY5 and control ACTIN2 transcripts in Ler, uvr8-1, and UVR8_pro:NLS-GFP-UVR8 lines grown in white light (LW) and exposed to UV-B as in (B).

Figure 9.

Model Showing the Dual Role of UV-B in Regulating UVR8. UV-B stimulates both the nuclear translocation of a fraction of the cytoplasmic UVR8 pool (drawn larger than the nuclear pool) and the function of UVR8 in the nucleus, leading to increased transcription of genes, such as HY5, that confer UV protection.