Two distinct domains of the UVR8 photoreceptor interact with COP1 to initiate UV-B signaling in Arabidopsis

Abstract

UV-B photon reception by the Arabidopsis thaliana homodimeric UV RESISTANCE LOCUS8 (UVR8) photoreceptor leads to its monomerization and a crucial interaction with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1). Relay of the subsequent signal regulates UV-B-induced photomorphogenesis and stress acclimation. Here, we report that two separate domains of UVR8 interact with COP1: the β-propeller domain of UVR8 mediates UV-B-dependent interaction with the WD40 repeats-based predicted β-propeller domain of COP1, whereas COP1 activity is regulated by interaction through the UVR8 C-terminal C27 domain. We show not only that the C27 domain is required for UVR8 activity but also that chemically induced expression of the C27 domain is sufficient to mimic UV-B signaling. We further show, in contrast with COP1, that the WD40 repeat proteins REPRESSOR OF UV-B PHOTOMORPHOGENESIS1 (RUP1) and RUP2 interact only with the UVR8 C27 domain. This coincides with their facilitation of UVR8 reversion to the ground state by redimerization and their potential to interact with UVR8 in a UV-B-independent manner. Collectively, our results provide insight into a key mechanism of photoreceptor-mediated signaling and its negative feedback regulation.

Figure 1.

The UVR8 β-Propeller Core Fragment Lacking the C27 Domain Interacts with COP1 in a UV-B-Dependent Manner, Whereas the C-Terminal C27 Domain Alone Interacts Constitutively with COP1. (A) Schematic representation of wild-type UVR8 and different variants used in this work. UVR8 contains seven repeats of the RCC1 (Regulator of Chromosome Condensation) Pfam domain (PF00415) (Kliebenstein et al., 2002; Finn et al., 2014) and a C27 interaction domain (Cloix et al., 2012). (B) Quantitative yeast two-hybrid assays were performed in the presence (+) or absence (−) of UV-B light. Means and se from three biological replicates are shown. AD, activation domain construct; BD, binding domain construct; EV, empty vector; β-Gal., β-galactosidase; MU, Miller units. (C) Yeast two-hybrid growth assay on selective SD/-Trp/-Leu/-His medium in the presence (+UV-B) or absence (−UV-B) of UV-B irradiation. (D) Truncated UVR8-2 interacts with COP1 in planta. Coimmunoprecipitation of endogenous COP1 with UVR8 [using anti-UVR8^(1–15) antibodies] is shown in extracts from 7-d-old wild-type (Landsberg erecta [Ler]), uvr8-2, and uvr8-1 seedlings. Seedlings were either irradiated with broad-band UV-B light for 15 min (+) or not irradiated (−). IB, immunoblotting; IP, immunoprecipitation. (E) Hypocotyl lengths of 4-d-old wild-type (Ler), uvr8-2, and uvr8-1 seedlings grown under weak white light with (+UV-B) or without (−UV-B) supplemental narrow-band UV-B. Means and se are shown (n > 20).

Figure 2.

The Val-410 and Pro-411 Pair in the C27 Domain Is Critical for UVR8 Signaling. (A) Quantitative yeast two-hybrid assays were performed in the presence (+) or absence (−) of UV-B. Means and se from three biological replicates are shown. AD, activation domain construct; BD, binding domain construct; EV, empty vector; β-Gal., β-galactosidase; MU, Miller units. (B) Immunoblot analysis of UVR8 protein levels in wild-type seedlings (Wassilewskija [Ws]), uvr8-7, and four independent uvr8-7/Pro35S:UVR8^VP-AA transgenic lines (UVR8^VP-AA #10, #11, #13, and #21). (C) and (D) Images of representative individuals (C) and quantification of hypocotyl lengths (D) of 4-d-old seedlings grown under white light with (+) or without (−) supplementary UV-B light. Means and se are shown (n > 20). (E) Quantitative RT-PCR analysis of HY5 gene activation in 4-d-old seedlings in response to UV-B irradiation for 1 h. Relative expression is shown as +UV-B/−UV-B (i.e., fold change). Means and se of three biological replicates are shown. (F) The UVR8^VP-AA protein interacts with COP1 in planta. Coimmunoprecipitation of COP1 with UVR8 [using anti-UVR8^(1–15) antibodies] is shown from 7-d-old wild-type (Ws), uvr8-7/Pro35S:UVR8^VP-AA (line 11), uvr8-7/Pro35S:UVR8 (line 3), and uvr8-7 seedlings. Seedlings were irradiated with broad-band UV-B for 15 min (+) or not (−). IB, immunoblotting; IP, immunoprecipitation.

Figure 3.

The Constitutive Interaction of UVR8^W285A with COP1 Depends on the C-Terminal VP Pair. (A) Immunoblot analysis of UVR8 protein expression levels in uvr8-7 and several independent uvr8-7/Pro35S:UVR8^W285,VP-AA (W285A^VP-AA) and uvr8-7/Pro35S:UVR8^W285A (W285A) transgenic lines. (B) Hypocotyl growth in weak white light. Images of representative individuals (top panel) and quantification of hypocotyl lengths (bottom panel) of 4-d-old seedlings grown under weak white light are shown. Means and se are shown (n > 20). (C) Coimmunoprecipitation of COP1 in whole cell extracts from seedlings grown for 7 d in white light. IB, immunoblotting; IP, immunoprecipitation. (D) Quantitative yeast two-hybrid assays were performed in the presence or absence of UV-B. Means and se from three biological replicates are shown. AD, activation domain construct; BD, binding domain construct; EV, empty vector; β-Gal., β-galactosidase; MU, Miller units.

Figure 4.

Induced Expression of UVR8^C44 Leads to HY5 Transcript Accumulation in Arabidopsis Seedlings in the Absence of UV-B. (A) Immunoblot analysis of YFP protein accumulation induced by β-estradiol in Arabidopsis seedlings. uvr8-7/XVE:YFP alone (YFP) or uvr8-7/XVE:UVR8^C44-UVR8^C44-NLS-UVR8^C44-2A-YFP (3XC44-2A-YFP) was treated for 5 h with 50 μM 17-β-estradiol in DMSO (Estr.) or with DMSO only. Extracts were probed with anti-YFP (top panel) or anti-actin (bottom panel) antibody. Untreated controls (−) were included. 2A encodes a cotranslational cleavage site. (B) Quantitative real-time PCR analysis of HY5 mRNA accumulation in Arabidopsis seedlings. Seedlings were either treated for 5 h with 50 μM 17-β-estradiol in DMSO or with DMSO only or were left untreated (−) Data were normalized against untreated samples without treatment at time 0. Means and se are shown (n = 3).

Figure 5.

Seedlings Stably Expressing UVR8^C44 Show a Constitutive Photomorphogenic Phenotype. (A) UVR8 protein levels in uvr8-7, wild-type (Wassilewskija [Ws]), YFP-NLS-C44-expression line 1, 13, and 15, and YFP-YFP-expression line (far right) seedlings. (B) and (C) Hypocotyl growth in weak white light. Images of representative individuals (B) and quantification of hypocotyl lengths (C) of 4-d-old seedlings grown under white light are shown. Means and se are shown (n > 20). (D) Transcript levels of CHS in uvr8-7/Pro35S:YFP-NLS-UVR8^C44 transgenic seedlings (lines 1, 13, and 15) compared with a uvr8-7/Pro35S:YFP-YFP line (YFP-YFP) as measured by quantitative RT-PCR analysis. Error bars represent se in triplicate biological measurements. (E) Coimmunoprecipitation of COP1 in protein extracts from uvr8-7, uvr8-7/Pro35S:YFP-YFP, and uvr8-7/Pro35S:YFP-NLS-UVR8^C44 transgenic seedlings grown for 7 d in white light. IB, immunoblotting; IP, immunoprecipitation.

Figure 6.

UVR8 Interacts with RUP1 and RUP2 Only via Its C27 Domain. (A) and (B) Yeast two-hybrid growth assay on selective SD/-Trp/-Leu/-His medium in the presence (+) or absence (−) of UV-B light. AD, activation domain construct; BD, binding domain construct; EV, empty vector. (C) and (D) Quantitative yeast two-hybrid assays were performed in the presence or absence of UV-B light. Means and se from three biological replicates are shown. β-Gal., β-galactosidase; MU, Miller units. (E) Dimerization of UVR8 in 7-d-old wild-type (Wassilewskija [Ws]) and uvr8-7/Pro35S:UVR8^VP-AA line 11 seedlings. Seedlings were either left unirradiated (−UV) or irradiated with broad-band UV-B light for 15 min (+UV) to induce UVR8 monomerization before being subjected to a recovery period in white light for 0.5 to 4 h. Asterisks indicate nonspecific cross-reacting bands.

Figure 7.

Working Model of the UVR8 Photocycle. In response to UV-B irradiation, UVR8 homodimers dissociate instantly, which allows the UVR8 seven-bladed β-propeller domain to interact with the COP1 WD40 domain (a structurally related seven-bladed β-propeller). The activated UVR8 monomer then also binds to the COP1 WD40 domain with its C27 domain (indicated by the pink crescent labeled C) and initiates UV-B signaling. Release of the C27 domain from structural constraints in the UV-B light-activated UVR8 is thought to allow its interaction with COP1. The activated UVR8-COP1 signaling pathway induces RUP1 and RUP2 expression, forming a negative feedback loop. RUP1 and RUP2 are WD40-repeat proteins that are phylogenetically and structurally related to COP1. They interact solely with the C27 domain of UVR8 and facilitate UVR8 redimerization and disruption of the UVR8-COP1 interaction. Note that no stable RUP1/RUP2-UVR8-COP1 complex is known, but it is assumed here to occur transiently when RUP1 and RUP2 attach to the C27 domain of UVR8 while UVR8 and COP1 still interact via their β-propeller surfaces. In contrast with COP1, RUP1 and RUP2 proteins are still able to interact with the C27 domain in the inactive homodimeric UVR8. The COP1-interacting SPA proteins as well as the homodimeric constitution of COP1 are omitted from the model. cc, coiled coil; RING, Really Interesting New Gene; WD40, WD40 repeat domain.