UV-B Promotes Rapid Nuclear Translocation of the Arabidopsis UV-B–Specific Signaling Component UVR8 and Activates Its Function in the Nucleus

The Abundance of UVR8 Is Unaffected by Different Light Qualities

UV-B stimulates expression of a range of genes via the UVR8 pathway. We therefore wanted to investigate how UV-B regulates UVR8; possibilities included alteration of the amount, localization, or posttranslational modification of the protein. In the first instance, we examined whether UV-B affected the amount of UVR8 in leaf tissue. To monitor the abundance of the protein, we obtained antipeptide antibodies that specifically recognize UVR8. As shown in Figure 1A , when an antibody to a C-terminal region of the protein is used to probe a protein gel blot of Arabidopsis leaf proteins, a strong, specific signal is observed that is absent in the uvr8-1 mutant. To examine the effects of light treatments on UVR8 abundance, plants grown in a low fluence rate of white light, equivalent to that used in previous gene expression studies (Brown et al., 2005), were exposed either to UV-B, UV-A, or red light or an increased fluence rate of white light. The amount of UVR8 was unaffected by any of these treatments (Figure 1A).

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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.

UV-B Promotes Accumulation of UVR8 in the Nucleus

We wished to examine whether the intracellular distribution of UVR8 changed following UV-B illumination. In a previous study (Brown et al., 2005), we used plants expressing a GFP-UVR8 fusion protein driven by the cauliflower mosaic virus 35S promoter to study UVR8 localization. To counter any potential problems with either the level or spatial distribution of expression from the heterologous 35S promoter, we extended the experiments using the native UVR8 promoter. As shown in Figure 2A , the UVR8 promoter was effective in driving expression of GFP-UVR8 in transgenic uvr8-1 null mutant plants. To establish that the fusion was functional, three independent lines were grown in white light lacking UV-B and then exposed to 4 h of UV-B at a fluence rate within the ambient range. This UV-B treatment induces expression of UV-protective genes via the UVR8 pathway (Brown et al., 2005). The GFP-UVR8 fusion is functional in that it complements the uvr8 mutant; UV-B–induced expression of the HY5 gene is impaired in the mutant but is restored in the transgenic lines (Figure 2B).

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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.

When plants grown in white light lacking UV-B are examined by confocal microscopy, the GFP-UVR8 fusion is detected in both the cytosol and nucleus, as can be seen in Figure 3A . However, UV-B exposure causes a marked increase in the number of nuclei displaying bright GFP-UVR8 fluorescence. By contrast, GFP alone, driven by the 35S promoter, is not present in the nucleus and does not change its localization following UV-B exposure (Figure 3B).

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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.

The change in localization of GFP-UVR8 is not accompanied by any alteration in abundance of the fusion protein. As shown in Figure 1B, exposure of the transgenic plants to various light treatments does not affect the amount of GFP-UVR8 detected on protein gel blots of leaf proteins by an anti-GFP antibody.

To quantify the change in GFP-UVR8 localization in response to UV-B, we used the fluorescent stain 4′,6-diamidino-2-phenylindole (DAPI) to identify nuclei in microscope images (Figure 3A) and then measured the percentage of those nuclei that also showed any detectable GFP fluorescence. The data in Figure 3C show that before UV-B treatment, ∼40% of nuclei have detectable GFP fluorescence, whereas after UV-B exposure, the number rises to ∼90%. Furthermore, we measured the brightness of GFP fluorescence in the nuclei. Following UV-B exposure, most of the nuclei have bright GFP fluorescence, whereas prior to UV-B treatment, a substantial proportion of the nuclei show dim GFP fluorescence (Figure 3D).

To establish whether UV-B promotes the accumulation of native UVR8 in the nucleus, we examined the abundance of the protein in cytosolic and nuclear fractions isolated from leaf tissue following UV-B exposure. Figure 4 shows that the fractions appear to be relatively free of cross-contamination, as judged by the lack of cross-reaction with antibodies to cytosolic and nuclear proteins, UDP-glucose pyrophosphorylase (UGPase) and histone H3, respectively. UVR8 is clearly more abundant in the cytosolic fraction than the nucleus, which is consistent with our calculation from confocal images that the volume of the cytosol is substantially greater than that of the nucleus in leaf epidermal cells. Following UV-B illumination, there is an increase in the amount of UVR8 detected in the nuclear protein fraction and a small decrease in the cytosolic fraction. The increase in UVR8 in the nuclear fraction is evidently not due to contamination from the cytosol.

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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.

The Nuclear Accumulation of UVR8 Is Specific to UV-B, Rapid, and Occurs at Low Fluence Rates

We wanted to examine whether the nuclear accumulation of UVR8 was specific to UV-B or occurred in response to other light qualities. Plants grown in white light lacking UV-B were exposed to either UV-B or to UV-A light to stimulate cryptochromes (Fuglevand et al., 1996) or to red light to promote formation of phytochrome Pfr. The nuclear localization of UVR8 was assayed by measuring GFP fluorescence relative to DAPI fluorescence, as described above (Figure 3C). The results (Figure 5A ) demonstrate that nuclear accumulation of UVR8 is promoted only by UV-B.

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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).

Previous studies have shown that relatively brief exposure to UV-B is sufficient to induce changes in gene expression. In Arabidopsis cells, 5 min of UV-B is sufficient to stimulate a small increase in expression of the CHS gene, which is regulated by UVR8 (Jenkins et al., 2001). We therefore wished to establish whether a brief UV-B treatment could promote the nuclear accumulation of UVR8. As shown in Figure 5B, 30 min of UV-B illumination is sufficient to produce a maximal response, with ∼90% of nuclei exhibiting GFP-UVR8 fluorescence, and as little as 10 min UV-B gives a clear increase in nuclear localization compared with the white light control. Interestingly, movement out of the nucleus is much slower, at least in darkness; a substantial amount of GFP-UVR8 was still present in nuclei 24 h after the transfer of UV-B–treated plants to darkness (Figure 5B).

We have recently found that UVR8 mediates gene expression responses to low fluence rates of UV-B (B.A. Brown and G.I. Jenkins, unpublished data). As little as 0.1 μmol m⁻² s⁻¹ UV-B, ∼1/40 of the level in full sunlight, is sufficient to initiate expression of genes concerned with UV protection, such as HY5 and CHS. It was therefore important to determine whether low fluence rates of UV-B promote the nuclear accumulation of GFP-UVR8. The results (Figure 5C) demonstrate that a clear increase in nuclear localization is observed at 0.1 μmol m⁻² s⁻¹ UV-B and a maximal response occurs at 0.5 μmol m⁻² s⁻¹ UV-B.

Accumulation of UVR8 in the Nucleus Is Correlated with Activity

The results presented in Figure 5 indicate that the nuclear accumulation of UVR8 may have functional significance in the regulation of gene expression by UV-B. To further explore this possibility, we examined whether manipulation of the localization of UVR8 affected its function. We attached a nuclear export signal (NES) at the N terminus of GFP-UVR8 and expressed the fusion in uvr8 mutant plants using the UVR8 promoter. This signal consisted of a peptide derived from the mammalian PKI protein (Wen et al., 1995) that was found to confer cytosolic localization of the N-terminal domain of phytochrome B (Matsushita et al., 2003). Several independent transgenic lines were produced. As can be seen in Figure 6A , the amount of the fusion expressed in several of these lines was similar to that of the GFP-UVR8 fusion. In transgenic plants grown in white light lacking UV-B, the NES-GFP-UVR8 fusion protein accumulated in the cytosol and was excluded from the nucleus (arrow in Figure 6B). GFP fluorescence was detected in only 1 to 2% of nuclei (Figure 6C) in contrast with the 40% accumulation seen in plants expressing GFP-UVR8 (Figure 3C). However, following UV-B illumination, the NES-GFP-UVR8 fusion accumulated in the nuclei to the same extent as the fusion lacking the NES (cf. Figures 6C and 3C). Moreover, accumulation occurred very rapidly (within 5 min) and at low fluence rates of UV-B, similar to the results with GFP-UVR8 (Figures 6C and 6D). The accumulation of the NES-GFP-UVR8 fusion in the nucleus following UV-B illumination resulted in complementation of the mutant phenotype with respect to expression of the HY5 gene, which encodes a key transcriptional target of UVR8 (Figure 6E). Similar results were found with other genes, such as CHS (data not shown).

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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).

In a further attempt to prevent UVR8 accumulating in the nucleus, we produced transgenic uvr8 plants expressing GFP fused to an N-terminal 23–amino acid deletion of UVR8 (GFP-ΔNUVR8). The human RCC1 protein, which shares ∼50% sequence similarity with UVR8 (Kliebenstein et al., 2002), has a bipartite nuclear localization signal (NLS) toward the N terminus, and although the equivalent region of UVR8 does not have an obvious NLS, we reasoned that it might still be involved in nuclear import. Several transgenic lines were produced that expressed GFP-ΔNUVR8 (Figure 7A ). Plants grown in white light lacking UV-B accumulated the GFP-ΔNUVR8 fusion in the cytosol and nucleus (Figure 7B), at similar levels to GFP-UVR8 (Figure 7C). However, following UV-B illumination, there was only a small increase in the proportion of nuclei showing GFP fluorescence, indicating that nuclear accumulation of GFP-ΔNUVR8 was substantially impaired. We did not observe UV-B stimulation of HY5 expression following UV-B illumination in the GFP-ΔNUVR8 plants (Figure 7D) nor stimulation of CHS expression (data not shown). Nevertheless, a chromatin immunoprecipitation assay (Figure 7E) demonstrated that the GFP-ΔNUVR8 fusion was capable of binding to chromatin at the HY5 promoter, similar to GFP-UVR8 (Brown et al., 2005), indicating that it had the potential to function in transcriptional regulation of the HY5 gene.

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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.

UVR8 Function in the Nucleus Requires UV-B Illumination

We wanted to determine whether the presence of a cytoplasmic pool of UVR8 was required for the protein to be functional and, furthermore, whether constitutive localization of UVR8 in the nucleus was sufficient for its activity. To address these questions, we attached an NLS at the N terminus of GFP-UVR8 and expressed the fusion in uvr8 mutant plants via the UVR8 promoter. The NLS was a peptide from SV40 that has been shown to direct nuclear localization of the N-terminal region of phytochrome B (Matsushita et al., 2003). Several transgenic lines were produced, and the level of NLS-GFP-UVR8 was similar to that of GFP-UVR8, as shown in Figure 8A . Plants expressed NLS-GFP-UVR8 constitutively in the nucleus regardless of the light conditions (Figure 8B). The amount of the protein in the nucleus showed little change over 24 h from the start of UV-B illumination (Figure 8C). However, the NLS-GFP-UVR8 plants only showed elevated HY5 expression following UV-B illumination and not in white light lacking UV-B (Figure 8D).

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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).

UV-B Promotes Nuclear Accumulation of UVR8

In plants grown in white light lacking UV-B, GFP-UVR8 is localized in both the cytoplasm and nucleus. Although our studies have focused on leaf epidermal cells, the same distribution is observed in other tissues and organs (data not shown). Within the cytoplasm, localization appears to be principally cytosolic and not associated with any particular organelle or the plasma membrane. Two lines of evidence indicate that most of the UVR8 in the cell is in the cytoplasm. First, we estimate, from confocal images of epidermal cells, that the volume of the cytoplasm is ∼10 times that of the nucleus and, taking into account the relative brightness of GFP fluorescence in the cytoplasm and nucleus, it is evident that most of the GFP-UVR8 is in the cytoplasm. The intensity of GFP-UVR8 fluorescence in the small volume of the nucleus gives an unrealistic impression of the relative amount in the nuclear fraction. Second, the immunological detection of native UVR8 in cellular fractions (Figure 4) indicates that most of the protein is cytoplasmic. Although both these analyses are qualitative rather than quantitative, they indicate that the nuclear pool of UVR8 is relatively small, which is perhaps surprising given that the protein functions in the nucleus.

Exposure to UV-B promotes the nuclear accumulation of both GFP-tagged and native UVR8. Measurement of the colocalization of GFP and DAPI in confocal images (Figure 3C) indicates that the number of nuclei containing GFP-UVR8 rises from 40 to 90% after UV-B treatment. However, this analysis underestimates the difference in amount of GFP-UVR8 in the nuclei before and after UV-B treatment because it does not take into account the change in intensity of GFP fluorescence in the nuclei. Before UV-B illumination, most of the nuclei containing GFP have dim fluorescence, whereas after UV-B, few nuclei are in this category and ∼80% have bright fluorescence (Figure 3D). The protein gel blot analysis also indicates a strong increase in nuclear accumulation of UVR8 in response to UV-B, although the amount in the nucleus remains smaller than that in the cytoplasm.

Evidence for a UV-B–Stimulated Nuclear Translocation Mechanism

The nuclear accumulation of UVR8 occurs without any change in the total amount of the protein. UV-B, and indeed other light qualities, do not affect the abundance of either the GFP-UVR8 fusion (Figure 1B) or the native protein (Figure 1A), consistent with UVR8 transcript measurements (Kliebenstein et al., 2002). Hence, the simplest explanation of the nuclear accumulation of UVR8 is that UV-B promotes a redistribution of UVR8 within the cell by stimulating movement from the cytoplasm into the nucleus. An alternative possibility is that UVR8 continually cycles between the cytoplasm and nucleus and that UV-B inhibits translocation into the cytosol, thereby causing nuclear accumulation. However, such a mechanism is insufficient to explain all the data, in particular the results with the NES-GFP-UVR8 fusion. The NES-GFP-UVR8 fusion is excluded from the nucleus in white light lacking UV-B but accumulates in the nucleus following UV-B exposure (Figure 6C). An inhibition of nuclear export by UV-B does not explain these observations, and the most logical interpretation is that UV-B actively stimulates nuclear accumulation of the NES-GFP-UVR8 fusion.

Another possible explanation of the nuclear accumulation of UVR8 in response to UV-B is that UV-B does not promote movement, but instead both inhibits degradation of the nuclear pool and stimulates destruction of the cytosolic pool without affecting the net amount of the protein. Again, the results with the NES-GFP-UVR8 fusion do not support this possibility because there does not appear to be a nuclear pool of NES-GFP-UVR8 prior to UV-B illumination; therefore, inhibition of its degradation could not lead to accumulation. One could argue that the NES does not prevent nuclear import in the absence of UV-B, and its presence leads to complete destruction of nuclear NES-GFP-UVR8, which is then prevented following UV-B illumination. However, such a mechanism seems very unlikely. Experiments with the NLS-GFP-UVR8 fusion also do not support the idea of UV-B preventing nuclear destruction of UVR8. The fusion was clearly present in the nucleus prior to UV-B illumination (Figure 8C) and thus was not being destroyed. Moreover, there was no change in amount even 24 h after the start of UV-B illumination (Figure 8D), in contrast with the increase predicted by the inhibition of destruction model.

Thus, taken together, our data indicate that UV-B actively promotes translocation of a fraction of the cytoplasmic pool of UVR8 into the nucleus. This conclusion is consistent with the observed redistribution of GFP-UVR8 and native UVR8 in the absence of any net change in abundance of the protein. Furthermore, the observation that UV-B overcomes the ability of the NES to retain GFP-UVR8 in the cytoplasm points to a concerted mechanism for nuclear import of UVR8 in response to UV-B. As shown in Figure 5, this mechanism is specific to UV-B and able to drive rapid nuclear accumulation of GFP-UVR8. There is no precedent for such a mechanism in plant cells. Although there is recent evidence that COP1 accumulates in the nucleus in response to UV-B, the response is very slow and takes ∼24 h (Oravecz et al., 2006). Quite rapid nuclear translocation of proteins in response to UV-B has been reported in mammalian cells, for instance, for the Nrf2 transcription factor (Kannan and Jaiswal, 2006) and Fyn protein kinase (He et al., 2005), but the mechanism is unknown. In plants, the most comparable response to that reported here is the rapid nuclear accumulation of phytochrome Pfr in response to red (phyB) or red and far-red wavelengths (phyA) (Kircher et al., 1999; Yamaguchi et al., 1999).

The UV-B–specific nuclear translocation mechanism could regulate the localization of additional proteins, but no information is available on this point. In addition, we have very little information on the mechanism of nuclear translocation. The data presented in Figure 7C indicate that the N-terminal 23 amino acids of UVR8 are required for efficient nuclear translocation, as deletion of this region reduces import by ∼70%. There is no cluster of basic amino acids characteristic of a classic NLS in this region. Thus, we speculate that the N-terminal region binds proteins that facilitate nuclear import specifically in response to UV-B. Evidence that some proteins enter the nucleus by binding to other proteins containing an NLS has been obtained in experiments with mammalian cells (e.g., Dostie et al., 2000; Kong et al., 2000), and in plants, the nuclear accumulation of phyA is regulated through interaction with FHY1 and FHL (Hiltbrunner et al., 2006).

Nuclear Accumulation of UVR8 Correlates with Function

UVR8 is required for the UV-B–induced expression of a set of genes concerned with UV protection (Brown et al., 2005). The data presented here show that the nuclear accumulation of UVR8 is directly related to its role in regulating gene expression. UVR8 nuclear accumulation is stimulated specifically by UV-B rather than other light qualities, consistent with its UV-B–specific function. In addition, the rapid accumulation of UVR8 in the nucleus correlates with previous observations that brief exposure to UV-B is sufficient to initiate expression of genes regulated by UVR8, such as CHS (Jenkins et al., 2001). Moreover, the nuclear accumulation of UVR8 occurs over the range of fluence rates that promote gene expression responses mediated by UVR8 (B.A. Brown and G.I. Jenkins, unpublished data). Experiments with the NES-GFP-UVR8 line show that significant nuclear accumulation occurs after 5 min of UV-B exposure and at a low fluence rate (Figures 6D and 6E). The NES-GFP-UVR8 line is evidently a very useful tool for further characterizing the nuclear translocation response because negligible accumulation is observed in the absence of UV-B.

The ability of GFP-UVR8 to complement the uvr8 mutant phenotype allowed us to investigate directly the function of UVR8 in the regulation of gene expression. In these experiments, we monitored HY5 transcript accumulation because UVR8 controls HY5 expression specifically in UV-B and HY5 effects transcription of most, if not all, UVR8 regulated genes (Brown et al., 2005; B.A. Brown and G.I. Jenkins, unpublished data). HY5 is required for UV protection, and the hy5 mutant is just as sensitive to UV-B as uvr8 (Brown et al., 2005). Thus, by assaying HY5 expression in the transgenic lines, we are able to relate UVR8 localization to plant UV protection.

Our previous report that GFP-UVR8 binds to chromatin in the region of genes it regulates (Brown et al., 2005) provides strong evidence that nuclear localization is required for UVR8 function. Thus, it would not be surprising if any treatment that impaired the nuclear accumulation of UVR8 resulted in a loss of function. Transgenic plants expressing GFP-ΔNUVR8 have impaired nuclear translocation of the fusion in response to UV-B and lack UV-B induction of HY5 expression. Although the greatly reduced amount of GFP-ΔNUVR8 in the nucleus probably reduced the capacity for HY5 expression, it may not account for the complete absence of HY5 induction in response to UV-B. It is possible that the N-terminal region of UVR8 is required for its maximal activity as well as its efficient nuclear translocation. However, GFP-ΔNUVR8 retains the capacity to bind to chromatin at the HY5 promoter (Figure 7E), indicating that it may be functional.

When UVR8 is constitutively localized in the nucleus using an NLS, it is able to direct HY5 expression in response to UV-B illumination. Furthermore, the results with the NLS-GFP-UVR8 fusion show that the maintenance of a cytoplasmic pool of UVR8 is not essential for its function in stimulating transcription. So what is the purpose of the cytoplasmic pool and of nuclear translocation? A possible explanation is that the nuclear activity of UVR8 results in turnover of the protein and nuclear translocation is required to replenish the nuclear pool. Although the amount of NLS-GFP-UVR8 remains roughly constant over 24 h of UV-B illumination, turnover and replacement may still be occurring. Synthesis of the NLS-GFP-UVR8 fusion will continue during UV-B illumination, but the protein will be directly imported into the nucleus rather than accumulate in the cytoplasm. Thus, the cytoplasmic pool may simply serve as a buffer to regulate the level of nuclear import in relation to nuclear activity. Alternatively, it is possible that cytoplasmic UVR8 has an undiscovered function. Hence, further research is needed to examine turnover of the protein and function of the cytoplasmic pool.

UV-B Illumination Is Necessary for UVR8 Function in the Nucleus

Although the presence of UVR8 in the nucleus is required for its function, nuclear localization is not sufficient for function. The GFP-UVR8 fusion is present in the nucleus in white light lacking UV-B, but minimal HY5 expression is observed (Figure 2A). Furthermore, the results in Figure 8D show that constitutive localization of the NLS-GFP-UVR8 fusion in the nucleus is insufficient to promote HY5 expression in the absence of UV-B. We therefore conclude that UV-B illumination is required for the nuclear activity of UVR8. This may involve direct activation of UVR8 in some way or the activation of some other component that acts in the UVR8 pathway to stimulate transcription. The results with the NLS-GFP-UVR8 fusion show that the process of UVR8 nuclear import itself does not lead to UVR8 activation. Figure 9 summarizes our model of UVR8 action and regulation. It shows that UV-B illumination both stimulates the movement of a fraction of the cellular pool of UVR8 into the nucleus and is required for UVR8 function in the nucleus.

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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.

The site of UV-B perception in the above processes is uncertain. At present, there is no evidence from studies of the heterologously expressed protein that UVR8 itself acts as a UV-B photoreceptor. The experiments with the NLS-GFP-UVR8 fusion show that UV-B illumination is required for UVR8 function in the nucleus, but that does not necessarily mean that UV-B is detected in the nucleus. It is conceivable that cytoplasmic UV-B perception directs the rapid nuclear translocation of an unidentified component that is required for activation of the UVR8 pathway in the nucleus. However, it is not clear that nuclear translocation involves cytoplasmic UV-B perception because a signal could emanate from the nucleus as a result of UV-B photoreception that activates rapid nuclear translocation. Thus, the nature and site of UV-B photoreception remain a mystery.

Plant Materials and Treatments

Seeds of wild-type Arabidopsis thaliana ecotype Ler were obtained from the Nottingham Arabidopsis Stock Centre. The uvr8-1 mutant allele in the Ler background (Kliebenstein et al., 2002) was obtained from Dan Kliebenstein (University of California, Davis). All the transgenic lines were produced in the uvr8-1 mutant.

For chromatin immunoprecipitation assays, subcellular localization analyses, and most of the protein studies, seeds were surface sterilized and sown on filter paper on 0.8% agar plates containing half-strength Murashige and Skoog (MS) salts. Plates were cold treated in the dark at 4°C for 48 h and plants grown for 12 d at 21°C under 20 μmol m⁻² s⁻¹ constant white light (Osram warm white fluorescent tubes). Plants were given light treatments as described in the figure legends using light sources for UV-B, UV-A, or red light described by Wade et al. (2001). The UV-B source (UV-B-313 fluorescent tubes; Q-Panel) was covered with cellulose acetate, which was changed every 24 h. This source emits maximally at 313 nm and has no emission below 290 nm. It emits very low levels of UV-A and blue light that are insufficient to induce CHS expression (Christie and Jenkins, 1996). Fluence rates were measured either using a Skye RS232 meter (Skye Instruments) equipped with a Quantum sensor or SKU 430 sensor (280 to 315 nm) or using a Macam spectroradiometer (model SR9910; Macam Photometrics).

For RT-PCR analysis of transgenic lines, cellular fractionation experiments, and some of the protein studies, plants were grown on compost for 3 weeks under 20 μmol m⁻² s⁻¹ constant white light at 21°C and then treated with 3 μmol m⁻² s⁻¹ UV-B for 4 h.

Plasmid Constructs and Transformation

To generate gene fusions driven by the native UVR8 promoter, the –1426 to +163 genomic sequence upstream of the UVR8 coding sequence was PCR amplified using the following primers: forward, 5′-GTATATAGTACTTCCAATGGC-3′; reverse, 5′-ATCACAGTTGCAGTTTTCACA-3′. This promoter fragment replaced the cauliflower mosaic virus 35S promoter upstream of GFP in the pEZR(K)-LC vector (Brown et al., 2005) at restriction sites 5′ SacI and 3′ HindIII. UVR8 and ΔNUVR8 (UVR8 lacking the first 23 amino acids) cDNA sequences were PCR amplified and cloned at restriction sites 5′ EcoRI and 3′ SalI in the modified pEZR(K)-LC vector. The NLS and NES sequences used for the generation of NLS-GFP-UVR8 and NES-GFP-UVR8 constructs were PCR synthesized as described by Matsushita et al. (2003). DNA sequencing confirmed that each fusion had been made correctly.

The fusions were introduced into uvr8-1 mutant plants by Agrobacterium tumefaciens–mediated transformation using the floral dip method (Bechtold and Pelletier, 1998). T1 seed from transformed plants was selected on 0.8% agar plates containing half-strength MS salts and 75 μg/mL of kanamycin. T2 generation plants exhibiting 3:1 segregation were selected.

At least four independent homozygous T3 lines were tested for protein expression, localization studies, and complementation of gene expression. Similar results were obtained from all lines expressing a particular fusion even though data are presented from only one line.

Protein Extraction and Protein Gel Blot Analysis

Total protein was extracted by grinding plants in microextraction buffer (20 mM HEPES, pH 7.8, 450 mM NaCl, 50 mM NaF, 200 μM EDTA, 500 μM PMSF, 1 mM DTT, and 25% glycerol with one protease inhibitor mix tablet [Complete Mini; Roche] added per 10 mL of buffer). Homogenization was followed by a freeze-thaw treatment (10 s on dry ice followed by 10 s at 37°C) repeated three times. The homogenate was centrifuged for 10 min at 16,100g in a microcentrifuge at 4°C, and the supernatant was collected. Nuclear and cytosolic fractions were obtained and used for protein extraction as described by Cho et al. (2006). The protein concentration of the total, nuclear, and cytosolic fractions was determined by a Bradford assay (Bio-Rad), and equal amounts of protein were loaded on a 10% SDS-PAGE gel. Protein was transferred onto a nitrocellulose membrane (Bio-Rad) by protein gel blotting. The membrane was blocked to remove nonspecific binding using 8% milk powder in TBS-T (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% [v/v] Triton X-100). A polyclonal antibody made against a C-terminal peptide of UVR8 (VPDETGLTDGSSKGN) and an anti-GFP monoclonal antibody (632375; Clontech) were used to detect UVR8 and GFP-UVR8, respectively. These proteins appeared as doublets in some protein gel blots, but the banding pattern was not consistent and the reason is unknown. Cytosolic UGPase and nuclear histone H3 were detected using polyclonal antibodies (anti-UGPase AS05086 [AgriSera] and antihistone H3, 07-441 [Upstate]). Antibodies were used at 1:3000 dilution and detected using secondary anti-rabbit or anti-mouse antibodies conjugated to horseradish peroxidase (Promega). To visualize the abundant Rubisco large subunit as a loading control, blots were stained with ponceau S (0.1% [w/v] in 1% [v/v] acetic acid). The data shown are representative of at least three independent experiments.

RNA Extraction and RT-PCR Analysis

Total RNA was extracted from leaf tissue by the RNeasy plant mini kit (Qiagen) according to the manufacturer's instructions. cDNA synthesis and RT-PCR of HY5 and control ACTIN2 transcripts were performed as described by Brown et al. (2005). For each transcript, amplification was assayed over a range of cycle numbers to select optimal conditions for visualization of the PCR product and quantification. Transcript levels in different RNA samples were compared using cycle numbers within the linear range of amplification.

Chromatin Immunoprecipitation

Chromatin isolation and chromatin immunoprecipitation were performed using the method of Gendrel et al. (2002) as described by Brown et al. (2005). Immunoprecipitation was performed using either a polyclonal anti-GFP antibody (Invitrogen A-11122) at a dilution of 1/100 or the anti-UVR8 antibody at a dilution of 1/500. The immunoprecipitated DNA was used in PCR reactions to amplify fragments from the HY5 gene promoter (−331 to +23) or control ACTIN2 gene as described by Brown et al. (2005).

Subcellular Localization Analysis

Transgenic plants were grown on half-strength MS agar plates for 12 d and given the light treatments described in the figure legends. To visualize nuclei, plants were incubated with 50 μg/mL of DAPI (Invitrogen Molecular Probes) for 15 min. The subcellular localization of GFP and DAPI was visualized by a confocal laser scanning microscope (Zeiss LSM510) under water with a ×40 objective. GFP and DAPI were excited using an argon laser at 488 nm and a UV laser at 395 nm, respectively. GFP emission was collected between 505 and 530 nm to avoid crosstalk with chloroplast autofluorescence. The same microscope settings for GFP and DAPI detection were used before and after UV-B illumination. To measure the relative brightness of nuclear GFP fluorescence shown in Figure 3D, we used the region of interest function of the Zeiss LSM510 as described by Trotman et al. (2007). Relative fluorescence values were recorded for >100 nuclei for each treatment, and means were calculated from three independent experiments. For the measurements of percentage of GFP/DAPI colocalization, each nucleus identified by DAPI fluorescence was examined for GFP fluorescence; if any level of GFP fluorescence was detected, the nucleus was scored as showing colocalization, so the relative intensity of nuclear GFP fluorescence did not influence the measurement. Approximately 20 separate images containing at least 20 cells from six different plants were analyzed for each light treatment, time point, or fluence rate. Colocalization analysis was performed on three independent transgenic lines. The data shown are representative of at least three independent experiments.

Accession Number

The Arabidopsis Genome Initiative locus identifier for UVR8 is At5g63860.