A UV-B-specific signaling component orchestrates plant UV protection

Abstract

UV-B radiation in sunlight has diverse effects on humans, animals, plants, and microorganisms. UV-B can cause damage to molecules and cells, and consequently organisms need to protect against and repair UV damage to survive in sunlight. In plants, low nondamaging levels of UV-B stimulate transcription of genes involved in UV-protective responses. However, remarkably little is known about the underlying mechanisms of UV-B perception and signal transduction. Here we report that Arabidopsis UV RESISTANCE LOCUS 8 (UVR8) is a UV-B-specific signaling component that orchestrates expression of a range of genes with vital UV-protective functions. Moreover, we show that UVR8 regulates expression of the transcription factor HY5 specifically when the plant is exposed to UV-B. We demonstrate that HY5 is a key effector of the UVR8 pathway, and that it is required for survival under UV-B radiation. UVR8 has sequence similarity to the eukaryotic guanine nucleotide exchange factor RCC1, but we found that it has little exchange activity. However, UVR8, like RCC1, is located principally in the nucleus and associates with chromatin via histones. Chromatin immunoprecipitation showed that UVR8 associates with chromatin in the HY5 promoter region, providing a mechanistic basis for its involvement in regulating transcription. We conclude that UVR8 defines a UV-B-specific signaling pathway in plants that orchestrates the protective gene expression responses to UV-B required for plant survival in sunlight.

Fig. 1.

UVR8 acts specifically in the UV-B regulation of CHS expression. Shown are RT-PCR measurements of CHS and control ACTIN2 transcripts (lower and upper bands, respectively, A-E). (A) Wild-type, uvr8-1, uvr8-2, and one of the F₁ progeny of uvr8-1 and uvr8-2 grown for 3 weeks in low fluence rate (25 μmol m^-2·s^-1) white light (L) and illuminated with ambient (3 μmol m^-2·s^-1) UV-B for 4 h (UB). (B) Plants grown as in A illuminated with UV-A (UA) for 6 h. (C) Four-day-old dark-grown (D) seedlings illuminated with far-red (FR) light for 6 h. (D) Plants grown as in A transferred to 7°C for 24 h. (E) Seedlings grown in darkness for 4 days with (+S) or without (-S) 2% sucrose.

Fig. 2.

HY5 is regulated by UVR8 and required for UV-B protection. (A) HY5 and control ACTIN2 transcripts measured by RT-PCR in wild-type and uvr8-2 plants grown for 3 weeks in 25 μmol m^-2·s^-1 white light (L) and illuminated with 3 μmol m^-2·s^-1 UV-B (UB) for 4 h or UV-A (UA) for 6 h. (B) HY5 and ACTIN2 transcripts (lower and upper bands, respectively) in 4-day-old dark-grown (D) seedlings illuminated with far-red (FR) light for the times indicated. (C) UV-B sensitivity assay. Wild-type, hy5-1, and uvr8-1 plants grown in 120 μmol m^-2·s^-1 white light for 12 days and then exposed to above-ambient (5 μmol m^-2·s^-1) UV-B with supplementary 40 μmol m^-2·s^-1 white light for 24 h. Plants were photographed after return to white light for 5 days.

Fig. 3.

GFP-UVR8 is present in the nucleus and associates with chromatin. (A) uvr8-2 plants expressing a 35S promoter::GFP-UVR8 fusion grown in low fluence rate white light and illuminated with 3 μmol m^-2·s^-1 UV-B for 2 h. Confocal image shows GFP fluorescence of epidermal cells. (Scale bar, 20 μm.) (B) Single cell showing the nucleus (arrowed); left, bright-field image and right, with GFP fluorescence superimposed. (Scale bars, 20 μm.) (C) Localization of fluorescence in wild-type plants expressing a control 35S promoter::GFP fusion. (Scale bar, 20 μm.) (D) Binding of E. coli-expressed GST-UVR8 to a calf thymus histone-agarose column. Western blots with anti-GST antibody showing GST-UVR8 (73 kDa) or control GST (26 kDa). Lane 1, protein applied to the columns; lane 2, unbound material that flowed through; lanes 3, 4, and 5, protein bound after a 5-min incubation, eluted with 0.1, 0.3, or 1.0 M NaCl, respectively. (E) Chromatin immunoprecipitation assay of DNA associated with GFP-UVR8. PCR of HY5 promoter (-331 to +23) and ACTIN2 DNA from 35S::GFP-UVR8 (Left) and 35S::GFP (Right) transgenic plants grown in 100 μmol m^-2·s^-1 white light and illuminated with 3 μmol m^-2·s^-1 UV-B for 4 h: lane 1, DNA immunoprecipitated by using anti-GFP antibody; lane 2, no antibody control; lane 3, input DNA before immunoprecipitation; lane 4, PCR without added DNA.