Negative feedback regulation of UV-B-induced photomorphogenesis and stress acclimation in Arabidopsis

Abstract

Plants respond to low levels of UV-B radiation with a coordinated photomorphogenic response that allows acclimation to this environmental stress factor. The key players in this UV-B response are COP1 (an E3 ubiquitin ligase), UVR8 (a β-propeller protein), and HY5 (a bZIP transcription factor). We have shown previously that an elevated UV-B-specific response is associated with dwarf growth, indicating the importance of balancing UV-B-specific signaling. Negative regulators of this pathway are not known, however. Here, we describe two highly related WD40-repeat proteins, REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2, that interact directly with UVR8 as potent repressors of UV-B signaling. Both genes were transcriptionally activated by UV-B in a COP1-, UVR8-, and HY5-dependent manner. rup1 rup2 double mutants showed an enhanced response to UV-B and elevated UV-B tolerance after acclimation. Overexpression of RUP2 resulted in reduced UV-B-induced photomorphogenesis and impaired acclimation, leading to hypersensitivity to UV-B stress. These results are consistent with an important regulatory role for RUP1 and RUP2, which act downstream of UVR8-COP1 in a negative feedback loop impinging on UVR8 function, balancing UV-B defense measures and plant growth.

Fig. 1.

RUP1 and RUP2 gene activation in response to UV-B depends on COP1, HY5, and UVR8. (A and B) Quantitative RT-PCR analysis of RUP1 (A) and RUP2 (B) gene activation in response to UV-B in cop1-4, hy5-215, and uvr8-6 mutants compared with WT Col. Four-day-old seedlings were irradiated with UV-B for the indicated times before harvesting. Representative data from three independent experiments are shown. Error bars represent SD of technical triplicates.

Fig. 2.

The RUP proteins interact directly with the UVR8 protein. (A) Schematic comparison of the protein domain structures of the three groups of WD40-repeat–containing repressors of photomorphogenesis (see also Fig. S3). (B) BiFC visualization of YC-RUP1 and YC-RUP2 interaction with YN-UVR8, but not with YN-COP1. A Pro_35S:CFP control plasmid was always cobombarded to identify transformed cells before the analysis of YFP fluorescence. Specific CFP and YFP filter sets were used for microscopic analysis. Differential interference contrast images are shown. (Scale bar: 10 μm.) (C) Coimmunoprecipitation of endogenous UVR8 with RUP2-YFP. Coimmunoprecipitation of proteins using anti-YFP antibodies in extracts from rup2-1/Pro_35S:RUP2-YFP transgenic seedlings. Four-day-old seedlings were irradiated with UV-B for 4 h (+UV-B) or mock-treated under a cutoff filtering out UV-B (−UV-B). An asterisk indicates a nonspecific cross-reacting band. (D) UV-B–responsive accumulation of RUP2-GFP protein expressed under its own promoter. Total protein was isolated from 4-d-old rup1 rup2/Pro_RUP2:RUP2-GFP transgenic seedlings that were irradiated with UV-B for the indicated times before harvesting. The protein gel blot was sequentially probed with anti-GFP and anti-UVR8 antibodies. (E) Coimmunoprecipitation of RUP2-GFP expressed under its own promoter with endogenous UVR8. Coimmunoprecipitation of proteins using anti-UVR8 antibodies in extracts from rup1 rup2/Pro_RUP2:RUP2-GFP transgenic seedlings and nontransgenic Col controls. Four-day-old seedlings were irradiated with UV-B for 6 h (+UV-B) or mock-treated under a cutoff filtering out UV-B (−UV-B).

Fig. 3.

RUP1 and RUP2 are repressors of UV-B–induced photomorphogenesis mediated by UVR8 and HY5. (A) Hypocotyl length of 4-d-old seedlings grown with or without supplemental UV-B. Numbers below the bars show the relative hypocotyl growth inhibition by UV-B as a percentage. Error bars represent SD (n = 30). (B) UV-B–induced flavonoid accumulation is enhanced in rup2 mutants and especially in rup1 rup2 double mutants. Error bars represent SD (n = 3). (C) Immunoblot analysis of UVR8, HY5, CHS, and actin (loading control) protein levels in 4-d-old seedlings irradiated with UV-B for the indicated times before harvesting. (D) Anthocyanin measurements of 4-d-old seedlings grown with or without supplemental UV-B showing that UV-B hypersensitivity of rup1 rup2 double mutants depends on functional UVR8 and HY5 proteins. Error bars represent SD (n = 3). (E and F) Quantitative RT-PCR analysis of HY5 (E) and CHS (F) gene activation in response to UV-B in rup1 rup2 hy5 and rup1 rup2 uvr8 triple mutants compared with WT Col, rup1 rup2, hy5-215, and uvr8-6. Four-day-old seedlings were irradiated with UV-B for the indicated times before harvesting. Error bars represent the SD of technical triplicates.

Fig. 4.

Overexpression of RUP2 represses the UV-B response. (A and B) Quantitative RT-PCR analysis of HY5 (A) and CHS (B) gene activation in response to UV-B in two independent RUP2 overexpression lines compared with WT Col and the rup2-1 single mutant. Error bars represent the SD of technical triplicates. (C) Immunoblot analysis of UVR8, HY5, CHS, and actin (loading control) protein levels. In A–C, 4-d-old seedlings were irradiated with UV-B for the indicated times before harvesting. RUP2Ox#3/#5=Pro_35S:RUP2 in rup2-1, lines 3 and 5.

Fig. 5.

The RUP proteins negatively regulate UV-B acclimation and tolerance. (A) Arabidopsis seedlings were grown for 7 d under white light (control and nonacclimated) or white light supplemented with narrowband UV-B (acclimated). Seedlings were then irradiated for 1.5 h (nonacclimated and acclimated) with broadband UV-B under a WG305 cutoff filter, or subjected to a 1.5-h mock treatment (control) under a WG345 filter (−UV-B). Treated seedlings were further grown for 7 d under standard conditions without UV-B before being photographed. (B) Identical treatment as that for A, except that the seedlings were exposed to broadband UV-B under a WG305 cutoff filter for 2 h (nonacclimated and acclimated). (C) Some 25-d-old rup1 rup2 and WT Col plants grown in sunlight simulators under realistic conditions (+UV) or with the UV portion specifically filtered out (−UV). (D) Quercetin accumulation (+UV/−UV) in 27-d-old plants grown under sun simulator conditions. Values are mean ± SD (n = 3). An asterisk indicates statistically significant differences from Col (P < 0.05, unpaired Student's t test).