Plant photoreceptors and their signaling components compete for COP1 binding via VP peptide motifs

Abstract Plants sense different parts of the sun's light spectrum using distinct photoreceptors, which signal through the E3 ubiquitin ligase COP1. Here, we analyze why many COP1‐interacting transcription factors and photoreceptors harbor sequence‐divergent Val‐Pro (VP) motifs that bind COP1 with different binding affinities. Crystal structures of the VP motifs of the UV‐B photoreceptor UVR8 and the transcription factor HY5 in complex with COP1, quantitative binding assays, and reverse genetic experiments together suggest that UVR8 and HY5 compete for COP1. Photoactivation of UVR8 leads to high‐affinity cooperative binding of its VP motif and its photosensing core to COP1, preventing COP1 binding to its substrate HY5. UVR8–VP motif chimeras suggest that UV‐B signaling specificity resides in the UVR8 photoreceptor core. Different COP1–VP peptide motif complexes highlight sequence fingerprints required for COP1 targeting. The blue‐light photoreceptors CRY1 and CRY2 also compete with transcription factors for COP1 binding using similar VP motifs. Thus, our work reveals that different photoreceptors and their signaling components compete for COP1 via a conserved mechanism to control different light signaling cascades.

A Immunoblot analysis of YFP-COP1, UVR8, and actin (loading control) protein levels in lines shown in Fig 1F. Seedlings were grown for 4 days under white light. B, C Quantitative real-time PCR analysis of (B) ELIP2 and (C) CHS expression. Four-day-old seedlings grown in white light were exposed to narrowband UV-B for 2 h (+ UV-B) or not (À UV-B). Error bars represent SEM of three biological replicates. D, E Images of representative individuals (D) and quantification of hypocotyl lengths (E) of 4-day-old seedlings grown in darkness. The scale bar represents 5 mm. Violin and box plots are shown for n > 60 seedlings; upper and lower hinges correspond to the first and third quartiles; the horizontal line in the interior of the box indicates the median. F, G Quantitative real-time PCR analysis of (F) UVR8 and (G) COP1 expression in wild type (Ws), uvr8-7, cop1-4 and cop1-5/Pro 35S :YFP-COP1 (WT), cop1-5/Pro 35S :YFP-COP1 Lys422Ala , cop1-5/Pro 35S :YFP-COP1 Tyr441Ala , and cop1-5/Pro 35S :YFP-COP1 Trp467Ala seedlings grown for 4 days under white light. Error bars represent SEM of 3 biological replicates.
Source data are available online for this figure. EV3 Figure EV2. Only UV-B-activated UVR8 and mutants are able to bind COP1 in size-exclusion chromatography binding assays.
A Coomassie-stained SDS-PAGE gels from a size-exclusion chromatography binding assay between the COP1 WD40 domain and UVR8 in the presence and absence of UV-B. Purified monomeric UVR8~50 kDa. Four lM of each protein or a mix of proteins was loaded on to a Superdex 200 Increase 10/300 GL column. Indicated fractions were taken each of the size-exclusion chromatography runs and separated on a 10% SDS-PAGE gel. B Coomassie-stained SDS-PAGE gels from a size-exclusion chromatography binding assay between the COP1 WD40 domain and UVR8 12-381 in the presence and absence of UV-B. Purified monomeric UVR8 12-381~4 0 kDa. Four lM of each protein or a mix of proteins was loaded on to a Superdex 200 Increase 10/300 GL column. Indicated fractions were taken each of the size-exclusion chromatography runs and separated on a 10% SDS-PAGE gel. C Coomassie-stained SDS-PAGE gels from a size-exclusion chromatography binding assay between COP1 and UVR8 ValPro/AlaAla pre-monomerized by UV-B. Purified monomeric UVR8 ValPro/AlaAla~5 0 kDa, COP1 WD40 a smear~25-40 kDa. Four lM of each protein was loaded independently or mixed together. Indicated fractions were taken each of the size-exclusion chromatography runs and separated on a 10% SDS-PAGE gel.
Source data are available online for this figure.
◀ Figure EV4. Various VP peptides bind the COP1 WD40 domain.   Yeast 3-hybrid analysis of the COP1-HYH and COP1-HFR1 interactions in the presence of UVR8. Normalized Miller Units were calculated as a ratio of bgalactosidase activity in yeast grown under UV-B versus yeast grown without UV-B. Additionally, normalized Miller Units here are reported separately for yeast grown on media without or with 1 mM methionine, corresponding to induction (À Met) or repression (+ Met) of Met25 promoter-driven UVR8 expression, respectively. Means and SEM for three biological repetitions are shown. AD, activation domain; BD, DNA binding domain; Met, methionine.