Interaction of COP1 and UVR8 regulates UV-B-induced photomorphogenesis and stress acclimation in Arabidopsis

Abstract

The ultraviolet-B (UV-B) portion of the solar radiation functions as an environmental signal for which plants have evolved specific and sensitive UV-B perception systems. The UV-B-specific UV RESPONSE LOCUS 8 (UVR8) and the multifunctional E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) are key regulators of the UV-B response. We show here that uvr8-null mutants are deficient in UV-B-induced photomorphogenesis and hypersensitive to UV-B stress, whereas overexpression of UVR8 results in enhanced UV-B photomorphogenesis, acclimation and tolerance to UV-B stress. By using sun simulators, we provide evidence at the physiological level that UV-B acclimation mediated by the UV-B-specific photoregulatory pathway is indeed required for survival in sunlight. At the molecular level, we demonstrate that the wild type but not the mutant UVR8 and COP1 proteins directly interact in a UV-B-dependent, rapid manner in planta. These data collectively suggest that UV-B-specific interaction of COP1 and UVR8 in the nucleus is a very early step in signalling and responsible for the plant's coordinated response to UV-B ensuring UV-B acclimation and protection in the natural environment.

Figure 1

Absence of UV-B-induced hypocotyl growth inhibition and gene expression changes in uvr8 and cop1 mutants. (A, B) Wild type (Ws) and uvr8-7 mutant were grown under white light with or without supplementary narrowband UV-B. Here, 4-day-old seedlings were photographed and their hypocotyl length was measured. Error bars represent s.d. (n=30). (C) Venn diagrams showing the number of genes classified as responding to narrowband UV-B (⩾2-fold) in uvr8-6, cop1-4 and wild type (Col) and their overlap. The corresponding gene lists can be found in Supplementary Tables S1, S2 and S3.

Figure 2

UVR8 protein amount is rate limiting for UV-B-induced photomorphogenesis. (A) Quantitative RT–PCR data showing overexpression of UVR8 but no effect on COP1 expression in lines Ox nos. 2 and 3 compared with wild type. (B) Hypocotyl length measurements of 4-day-old seedlings grown with or without supplemental UV-B. Error bars represent s.d. (n=30). (C) Luciferase assays visualizing HY5 promoter activation in response to UV-B in UVR8 overexpression lines nos. 2 and 3 compared with wild type. Error bars represent s.e. (n=30). (D, E) Quantitative RT–PCR analysis of HY5 and CHS gene activation in response to UV-B in UVR8 overexpressor lines compared with wild type. (F) Immunoblot analysis of UVR8, CHS and actin (loading control) protein levels in 4-day-old seedlings grown with or without supplementary UV-B. (G) Anthocyanin accumulation of 4-day-old seedlings grown with or without supplemental UV-B. Error bars represent s.d. (n=3). (A–G) WT=Ws/Pro_HY5:Luc; Ox no. 2/no. 3=Pro_35S:UVR8 in WT, lines 2 and 3.

Figure 3

UV-B-induced HY5 and CHS gene activation strictly requires UVR8 and COP1. (A, B) Quantitative RT–PCR of HY5 and CHS gene activation in response to UV-B in cop1-4 and uvr8-6 compared with wild type (Col). Error bars represent s.d. of triplicate. (C) Immunoblot analysis with anti-UVR8 and anti-actin (loading control) antibodies on protein extracts from 4-day-old mutant and wild-type seedlings.

Figure 4

Wild-type UVR8 and COP1 proteins interact directly in a UV-B-dependent manner, but not the mutant versions that are impaired in UV-B signalling. (A) Direct interaction of YN-COP1 with YC-UVR8 under UV-B. (B) BiFC visualization of UVR8 dimerization independent of UV-B. (C) No interaction of mutant UVR8 proteins with wild-type COP1 under UV-B detectable by BiFC. (D) No interaction of mutant COP1 proteins with wild-type UVR8 under UV-B. (C, D) No YFP signal was detected in at least 20 CFP positive cells and in two independent repetitions. (E) Direct interaction of YN-UVR8 with YC-COP1^H69Y under UV-B. (A–E) A Pro_35S:CFP control plasmid was always co-bombarded to identify transformed cells prior to the analysis of YFP fluorescence. Specific CFP and YFP filter sets were used for microscopic analysis. DIC (differential interference contrast images) are shown. Bars=10 μm.

Figure 5

UV-B-dependent co-immunoprecipitation of UVR8 with YFP–COP1. (A) Co-immunoprecipitation of proteins using anti-YFP antibodies in extracts from wild-type (Col), cop1-4, cop1-4/Pro_35S:YFP-COP1 and cop1-4 uvr8-6/Pro_35S:YFP-COP1 transgenic seedlings. Here, 6-day-old seedlings were UV-B irradiated for 24 h (+UV-B) or mock treated under a cutoff filtering out UV-B (−UV-B). ^*A nonspecific cross-reacting band. (B) Early UV-B-dependent interaction detected by co-immunoprecipitation of UVR8 with YFP–COP1 from 5-day-old cop1-4/Pro_35S:YFP-COP1 seedlings exposed to UV-B for the indicated times.

Figure 6

UVR8-dependent acclimation to UV-B and its importance for survival under simulated sunlight. (A) Arabidopsis seedlings were grown for 7 days under white light (a, b, c; non-acclimated) or white light supplemented with narrowband UV-B (d, e, f; acclimated). Seedlings were then irradiated for 1 h (b, e) and 2 h (c, f) with broadband UV-B under a WG305 cutoff filter, or subjected to a 2 h mock treatment (a, d) under a WG345 filter (−UV-B). Treated seedlings were further grown for 7 days under standard conditions without UV-B before the picture was taken. (B) Here, 25-day-old plants grown in sunlight simulators under realistic conditions (+UV) or with the UV portion specifically filtered out (−UV). (C) Close up of 27-day-old single plants grown under +UV conditions. WT=Ws/Pro_HY5:Luc+, Ox no. 2/no. 3=Pro_35S:UVR8 in WT, lines 2 and 3.

Figure 7

COP1-mediated degradation of HY5 is inhibited under UV-B. (A) WT (Col) and cop1-4 mutant seedlings were grown for 4 days under white light supplemented with narrowband UV-B (4d+UV). Then the seedlings were either left under UV-B or the cutoff filter was exchanged to filter out UV-B (−UV-B) for 5, 10, 15 and 25 h before samples were analysed. (B) WT (Col) and a complemented hy5 transgenic line constitutively expressing HY5 (hy5-215/Pro_35S:HY5) were grown for 4 days under white light supplemented with narrowband UV-B (4d+UV). Then the seedlings were either left under UV-B (+UV), UV-B was filtered out (−UV) or they were transferred to darkness (D) for the indicated times. (A, B) Protein gel blots were sequentially probed with anti-HY5 and anti-actin (loading control) antibodies.

Figure 8

Working model of COP1 and UVR8 function in the UV-B photoregulatory pathway. Left panel: under white light (WL), active photoreceptors partially inhibit COP1, which balances the response by repressing light signalling through degradation of HY5, HYH and other positive regulators of photomorphogenesis. A portion of UVR8 is constitutively associated with chromatin, for example, at the HY5 promoter region. A yet unidentified protein X represses HY5 transcription, possibly through keeping UVR8 inactive. Right panel: under supplementary UV-B (WL+UV-B), the specific perception by a UV-B photoreceptor (PR) results in rapid UVR8–COP1 interaction. This interaction is very closely linked to the UV-B PR function and confers UV-B-specific function to COP1, changing its substrate specificity away from HY5/HYH and functionally related proteins towards repressor protein X. Degradation of X then allows UVR8-mediated activation of genes, including HY5, that confers UV acclimation and protection.