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• W2100951456 abstract "Article22 January 2009free access Interaction of COP1 and UVR8 regulates UV-B-induced photomorphogenesis and stress acclimation in Arabidopsis Jean-Jacques Favory Jean-Jacques Favory Faculty of Biology, Institute of Biology II, University of Freiburg, Freiburg, Germany Centre for Biological Signalling Studies (bioss), University of Freiburg, Freiburg, Germany Search for more papers by this author Agnieszka Stec Agnieszka Stec Faculty of Biology, Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Henriette Gruber Henriette Gruber Faculty of Biology, Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Luca Rizzini Luca Rizzini Faculty of Biology, Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Attila Oravecz Attila Oravecz Faculty of Biology, Institute of Biology II, University of Freiburg, Freiburg, GermanyPresent address: Department of Cancer Biology, IGBMC, Illkirch, France Search for more papers by this author Markus Funk Markus Funk Faculty of Biology, Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Andreas Albert Andreas Albert Department of Environmental Engineering, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Catherine Cloix Catherine Cloix Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK Search for more papers by this author Gareth I Jenkins Gareth I Jenkins Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK Search for more papers by this author Edward J Oakeley Edward J Oakeley Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Harald K Seidlitz Harald K Seidlitz Department of Environmental Engineering, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Ferenc Nagy Ferenc Nagy Institute of Plant Biology, Biological Research Center, Szeged, Hungary School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author Roman Ulm Corresponding Author Roman Ulm Faculty of Biology, Institute of Biology II, University of Freiburg, Freiburg, Germany Centre for Biological Signalling Studies (bioss), University of Freiburg, Freiburg, Germany Search for more papers by this author Jean-Jacques Favory Jean-Jacques Favory Faculty of Biology, Institute of Biology II, University of Freiburg, Freiburg, Germany Centre for Biological Signalling Studies (bioss), University of Freiburg, Freiburg, Germany Search for more papers by this author Agnieszka Stec Agnieszka Stec Faculty of Biology, Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Henriette Gruber Henriette Gruber Faculty of Biology, Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Luca Rizzini Luca Rizzini Faculty of Biology, Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Attila Oravecz Attila Oravecz Faculty of Biology, Institute of Biology II, University of Freiburg, Freiburg, GermanyPresent address: Department of Cancer Biology, IGBMC, Illkirch, France Search for more papers by this author Markus Funk Markus Funk Faculty of Biology, Institute of Biology II, University of Freiburg, Freiburg, Germany Search for more papers by this author Andreas Albert Andreas Albert Department of Environmental Engineering, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Catherine Cloix Catherine Cloix Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK Search for more papers by this author Gareth I Jenkins Gareth I Jenkins Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK Search for more papers by this author Edward J Oakeley Edward J Oakeley Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Search for more papers by this author Harald K Seidlitz Harald K Seidlitz Department of Environmental Engineering, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Ferenc Nagy Ferenc Nagy Institute of Plant Biology, Biological Research Center, Szeged, Hungary School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author Roman Ulm Corresponding Author Roman Ulm Faculty of Biology, Institute of Biology II, University of Freiburg, Freiburg, Germany Centre for Biological Signalling Studies (bioss), University of Freiburg, Freiburg, Germany Search for more papers by this author Author Information Jean-Jacques Favory1,2,‡, Agnieszka Stec1,‡, Henriette Gruber1, Luca Rizzini1, Attila Oravecz1, Markus Funk1, Andreas Albert3, Catherine Cloix4, Gareth I Jenkins4, Edward J Oakeley5, Harald K Seidlitz3, Ferenc Nagy6,7 and Roman Ulm 1,2 1Faculty of Biology, Institute of Biology II, University of Freiburg, Freiburg, Germany 2Centre for Biological Signalling Studies (bioss), University of Freiburg, Freiburg, Germany 3Department of Environmental Engineering, Helmholtz Zentrum München, Neuherberg, Germany 4Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK 5Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland 6Institute of Plant Biology, Biological Research Center, Szeged, Hungary 7School of Biological Sciences, University of Edinburgh, Edinburgh, UK ‡These authors contributed equally to this work *Corresponding author. Institute of Biology II, University of Freiburg, Schänzlestrasse 1, 79104 Freiburg, Germany. Tel.: +49 761 203 2653; Fax: +49 761 203 2612; E-mail: [email protected] The EMBO Journal (2009)28:591-601https://doi.org/10.1038/emboj.2009.4 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction Sunlight is of utmost importance to plants, both as the ultimate energy source and as an environmental signal regulating growth and development. For the latter, higher plants possess several classes of photoreceptors, including the molecularly known phytochromes for the red/far-red, and cryptochromes, phototropins and members of the Zeitlupe family for the UV-A/blue part of the spectrum (e.g. Chen et al, 2004). Ultraviolet-B (UV-B; 280–315 nm) radiation is an integral part of the sunlight reaching the surface of the Earth and induces a broad range of physiological responses. The UV-B-induced photomorphogenic responses, in contrast to damage responses, are thought to be mediated by a molecularly unidentified UV-B-specific photoreceptor different from the known receptors acting in the visible part of the light spectrum (Brosche and Strid, 2003; Frohnmeyer and Staiger, 2003; Ulm and Nagy, 2005; Jenkins and Brown, 2007). Key regulatory factors involved in the UV-B-induced photomorphogenic pathway, such as the bZIP transcription factor ELONGATED HYPOCOTYL 5 (HY5), the E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) and the seven-bladed propeller protein UV RESPONSE LOCUS 8 (UVR8), have been identified and plants harbouring hy5, cop1 and uvr8 loss of function mutations display reduced tolerance to UV-B stress (Kliebenstein et al, 2002; Ulm et al, 2004; Brown et al, 2005; Oravecz et al, 2006). UVR8 was found to exclusively act in UV-B signalling, thus showing high functional specificity (Brown et al, 2005). In planta, UV-B stimulates rapid nuclear accumulation of the UVR8 protein, which seems to be required but is not sufficient for UV-B-responsive gene expression changes (Kaiserli and Jenkins, 2007). UVR8 associates constitutively with chromatin regions of several UV-B-activated genes, including the HY5 genomic locus (Brown et al, 2005; Cloix and Jenkins, 2008). Recently, it was suggested that HY5 and its homologue HYH are key effectors of the UVR8 pathway and act redundantly to control expression of most, if not all, UVR8 target genes (Brown and Jenkins, 2008). COP1 is a known repressor of photomorphogenesis in darkness as well as in light, but is a promoter of UV-B-specific responses: cop1 mutants have a light-grown phenotype in darkness, show features of enhanced photomorphogenesis in light but are deficient in UV-B photomorphogenic responses (Yi and Deng, 2005; Oravecz et al, 2006). At the molecular level, COP1 targets different photomorphogenesis-promoting transcription factors for degradation in the dark, among them HY5 (Osterlund et al, 2000; Saijo et al, 2003). Upon activation of photoreceptors by visible light, COP1 is inactivated and physically separated from HY5 by nuclear exclusion, allowing HY5 stabilization and activation of light-responsive genes (von Arnim and Deng, 1994; Yi and Deng, 2005). Light-induced, early inactivation of this E3 ligase is most likely mediated by direct interaction with active phytochromes and cryptochromes, but the precise molecular mechanism underlying this process is still unknown (Yi and Deng, 2005). However, a number of characteristics clearly distinguish COP1 function under UV-B from that in visible light signalling, including (i) promotive versus repressive function, (ii) primarily nuclear versus cytoplasmic localization, (iii) structure–function differences displayed by different cop1 alleles and (iv) independence versus dependence on accessory SPA proteins (Oravecz et al, 2006). Altogether, this set of data indicated a distinct UV-B signalling function of the multifunctional COP1 protein. Despite the ecological and economic impact of the UV-B response (e.g. Caldwell et al, 2007), very little is known about the underlying signalling mechanisms linking UV-B perception to specific photomorphogenic responses. Both UVR8 and COP1 impinge on the UV-B-mediated activation of HY5 gene expression; however, the relationship of COP1 and UVR8 UV-B-specific functions has remained unknown. Here, we show that COP1 and UVR8 proteins interact specifically in a UV-B-dependent manner in planta, suggesting that physical association between these two proteins contributes to their specific activities in UV-B signalling. This conclusion is supported by the findings that mutant alleles of COP1 or UVR8 displaying UV-B signalling deficiencies do not interact with their respective wild-type partner. Furthermore, we demonstrate the absence of UV-B-induced photomorphogenesis in uvr8 mutants at the phenotypic level and show that UVR8 overexpression, on the other hand, leads to UV-B hyperresponsiveness. As a result, uvr8 mutants are more, whereas UVR8 overexpressors are less affected than their corresponding wild type under UV-B regimens simulating natural conditions. Results A luciferase-based genetic screen identifies novel cop1 and uvr8 mutant alleles To uncover players involved in early UV-B signalling, we screened for mutants altered in UV-B-induced expression of the HY5 gene. This was accomplished by generating an Arabidopsis line carrying a transgene consisting of the HY5 promoter fused to the firefly luciferase coding sequence (Ws/ProHY5:Luc) (Ulm et al, 2004). A number of mutants showing no UV-B induction were identified in the M2 generation after EMS mutagenesis. The identified mutants fell into two complementation groups, and we found that these constituted new cop1 and uvr8 alleles. In addition to the cop1-4 allele described before (Gln-283 to Stop) (Oravecz et al, 2006), we identified a novel allele carrying a point mutation in the region encoding the WD40 repeats of COP1, namely Gly-608 (GGA) changed to Arg (AGA). The corresponding mutant, designated as cop1-19, has a weak constitutively photomorphogenic (cop) phenotype in dark and enhanced photomorphogenesis in light, similar to cop1-4. This genetic screen also identified nine novel uvr8 alleles different from any of the previously described ones (uvr8-1 to uvr8-5) (Kliebenstein et al, 2002; Brown et al, 2005) (Supplementary Figure S1). In addition, we identified an uvr8 T-DNA insertion line from the SALK collection (uvr8-6, SALK_033468; see Supplementary Figure S1 for molecular characterization). Throughout the remainder of the work described, we used the uvr8-6 (Col) and uvr8-7 (Ws; Gln-124 to Stop)-null mutant alleles. The results were comparable for both alleles. UV-B-mediated inhibition of hypocotyl growth is absent in uvr8 mutants To increase our understanding of UVR8 function in regulating UV-B-induced photomorphogenesis, we examined UV-B-responsive hypocotyl shortening. These experiments were performed under specific UV-B irradiation conditions using white light supplemented with narrowband UV-B. Under these conditions, 4-day-old wild-type Arabidopsis seedlings are grown without any sign of damage, but display about 50% inhibition of hypocotyl growth accompanied by anthocyanin and flavonoid accumulation (Oravecz et al, 2006). Figure 1A and B and Supplementary Figure S1 show that hypocotyl growth of the uvr8 mutant seedlings, in stark contrast to wild-type seedlings, was not inhibited by UV-B. Importantly, in contrast to cop1 (Oravecz et al, 2006), the hypocotyl growth of uvr8 under visible light is not different from wild type. Thus, we conclude that uvr8 mutants are non-responsive to UV-B as a photomorphogenic signal. Moreover, these data strongly indicate that the narrowband UV-B irradiation conditions used are ideal to specifically analyse UV-B-induced photomorphogenesis and distinguish it from UV-B damage/stress responses. 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. Download figure Download PowerPoint UV-B-mediated changes in gene expression are absent in uvr8 and cop1 mutants Next to the hypocotyl phenotype, analysis of the uvr8 alleles showed that all of them are completely insensitive to UV-B concerning HY5 gene activation (data not shown). However, the same uvr8 mutants showed normal HY5 activation by red, far-red and blue light (Supplementary Figure S2). These data, together with the previous data from Brown et al (2005), indicate a UV-B-specific function of the UVR8 protein. To have a more global view on gene expression changes underlying the UV-B photomorphogenic response, we carried out Affymetrix ATH1 Genechip analysis. We investigated, parallel to wild type, the impact of the loss of UVR8 and COP1 under these low-level, narrowband (∼312 nm) UV-B conditions using the uvr8-6-null and the cop1-4 mutants. We analysed gene expression changes in 4-day-old seedlings grown under continuous light with or without supplementary UV-B in the same light field (under WG305 and WG345 cutoff, respectively). In addition, to analyse the early UV-B response (see Oravecz et al, 2006 for experimental scheme), we grew seedlings for 4 days without UV-B under a WG345 cutoff filter and then exchanged it for a WG305 cutoff filter 1 or 6 h before harvesting. These different treatments are designated as 96 h −UV-B, 1 h +UV-B, 6 h +UV-B and 96 h +UV-B. Data obtained demonstrate that in wild-type seedlings already after 1 h UV-B irradiation numerous transcripts are altered (e.g. 377 and 102 genes up- and downregulated, respectively), whereas these changes are virtually absent in the uvr8-6 and cop1-4 mutants (Figure 1C). These effects are similarly true for genes activated at 6 h +UV-B and 96 h +UV-B, as well as for genes downregulated at all time points (Figure 1C). These UV-B-activated classes include genes associated with UV-B tolerance such as photorepair of UV-B-induced DNA damage and phenylpropanoid biosynthesis to mount a sunscreen effect and their transcriptional regulators (see Supplementary Tables S1, S2 and S3). Most importantly, these data strongly indicate that almost all genes of the postulated UV-B photoreceptor-specific regulatory pathway(s) are dependent on functional UVR8 and COP1 proteins, supporting their major role. Overexpression of UVR8 results in an enhanced UV-B photomorphogenic response To determine whether UVR8 protein is a rate-limiting factor in the Arabidopsis UV-B response, we generated transgenic lines overexpressing UVR8 under the control of the constitutive strong CaMV35S promoter. Using western blot analysis, levels of UVR8 overexpression were estimated and two transgenic lines in which quantitative RT–PCR also detected an approximately 30-fold overexpression of UVR8 mRNA compared with wild type were used for detailed analysis (Figure 2A). In these lines, a marked UV-B photomorphogenic hypersensitivity was observed in all assays employed, including hypocotyl growth inhibition, HY5 and CHS gene activation, and anthocyanin accumulation (Figure 2B–G). Thus, we conclude that UVR8 has a rate-limiting function in the UV-B photomorphogenic pathway. 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/ProHY5:Luc; Ox no. 2/no. 3=Pro35S:UVR8 in WT, lines 2 and 3. Download figure Download PowerPoint Both COP1 and UVR8 are required for the UV-B photomorphogenic response Using quantitative RT–PCR assays, we found no detectable UV-B-mediated early activation of the endogenous HY5 and CHS genes in cop1 and uvr8 mutants (Figure 3A and B). However, it is of note that uvr8 mutants do not show any constitutively photomorphogenic phenotype, indicating normal function of COP1. Reciprocally, to analyse the UVR8 protein levels in cop1 mutants, we have generated polyclonal antibodies against a specific C-terminal peptide of UVR8. The antibody detects a single band (about 47 kDa) in wild-type cell extracts that corresponds to the expected size of the UVR8 protein (440 amino acids with predicted mass 47 kDa) and this is absent in the uvr8-6-null mutant. Importantly, levels of UVR8 protein are comparable in cop1-4, hy5-215 mutant and wild-type seedlings (Figure 3C), thereby excluding an indirect cause of their previously described UV-B phenotypes (Ulm et al, 2004; Oravecz et al, 2006). In addition, we conclude that COP1 does not affect UVR8 protein levels under standard growth conditions. Moreover, chromatin immunoprecipitation showed that UVR8 associates with the HY5 promoter region independent of COP1 (Supplementary Figure S3A). 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. Download figure Download PowerPoint The total absence of a UV-B regulatory response, for example, in HY5 and CHS gene activation, indicates that the COP1 and the UVR8 proteins function in the same genetic pathway. We thus hypothesized that COP1 and UVR8 might function together in the UV-B photomorphogenic signalling pathway. UVR8 and COP1 colocalize and interact directly in a UV-B-dependent manner To investigate whether COP1 and UVR8 proteins interact, we made use of a transient expression system in mustard (Sinapis alba), a plant with a well-established photomorphogenic response (Stolpe et al, 2005, and references therein) (Supplementary Figure S4A). We generated expression constructs of YFP–COP1 and CFP–UVR8 and delivered the corresponding plasmids into mustard hypocotyls by biolistic gene transfer. Under standard conditions without UV-B, YFP–COP1 localized to nuclear bodies in mustard hypocotyl cells (Supplementary Figure S4B), as described before for onion epidermal cells (e.g. Ang et al, 1998). In contrast, CFP–UVR8 is detected as diffuse nuclear fluorescence in the same cells. However, when the co-bombarded plants were irradiated with UV-B, also CFP–UVR8 formed nuclear bodies that largely colocalized with YFP–COP1 (Supplementary Figure S4B). This indicates that CFP–UVR8 was recruited into YFP–COP1 nuclear bodies in a UV-B-dependent manner and that these two proteins might reside in the same protein complex under UV-B specifically. To investigate whether UVR8 and COP1 are indeed directly interacting under UV-B, we used the bimolecular fluorescent complementation (BiFC) assay (Kerppola, 2006). By using this assay, we could clearly identify reconstitution of a functional YFP signal from the complementary ‘split YFP’ parts attached to the UVR8 and COP1 proteins. However, similar to the colocalization, the direct interaction of UVR8 and COP1 was again UV-B dependent (Figure 4A; Supplementary Figure S4C, right), as there was barely any YFP signal detectable when the supplementary UV-B was removed (Figure 4A; Supplementary Figure S4C, left). Importantly, we could not detect any YFP signal when empty vector controls were used in combination with YN-/YC-UVR8 and YN-/YC-COP1 (Supplementary Figure S4D). It is also of note that in sharp contrast to the UV-B-dependent interaction of COP1 with UVR8, interaction of UVR8 with itself was readily detectable independent of supplementary UV-B (Figure 4B). 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-COP1H69Y under UV-B. (A–E) A Pro35S: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. Download figure Download PowerPoint Single amino-acid changes in COP1 and UVR8 proteins impair UV-B signalling function and also abrogate direct interaction with their partner proteins We further investigated whether mutant alleles of COP1 and UVR8 are still able to interact with their corresponding wild-type partner or whether COP1–UVR8 interaction correlates with a functional UV-B response. A number of uvr8 mutants express mutant UVR8 proteins at about wild-type level (Supplementary Figure S1B). This is of note as the mutants with single amino-acid changes in UVR8 displayed absence of the UV-B response, apparently identical to the null alleles (e.g. uvr8-1 and uvr8-6). Thus, we have tested interaction of UVR8G145S (corresponding to uvr8-15) and UVR8G202R (corresponding to uvr8-9) with wild-type COP1 and found that these non-functional UVR8 alleles were not capable of interacting with COP1 anymore (Figure 4C). By using the COP1N282 (corresponding to cop1-4) truncation and COP1G608R (corresponding to cop1-19) protein, we found that it is the WD40 repeats of COP1 that are important for interaction with UVR8 (Figure 4D). In contrast to cop1-4 and cop1-19, the cop1eid6 mutant is still able to respond to UV-B (Oravecz et al, 2006), despite their comparable enhanced photomorphogenic phenotype in visible light (Dieterle et al, 2003). In agreement, we found that the corresponding COP1H69Y protein, mutated in a conserved histidine residue of the RING finger domain, still interacts with UVR8 under UV-B (Figure 4E). Thus, we conclude that functional UVR8 and COP1 are required for direct interaction with their wild-type partner protein. YFP–COP1 and UVR8 are co-immunoprecipitated from UV-B-treated seedlings To further investigate COP1–UVR8 interaction in planta, we performed co-immunoprecipitation experiments. To do this, we have generated transgenic lines constitutively expressing YFP-tagged COP1 in cop1-4 mutants, which led to complementation of the cop1-4 UV-B response (Oravecz et al, 2006). In agreement with the BiFC data, endogenous UVR8 protein was co-immunoprecipitated with YFP–COP1 from cop1-4/Pro35S:YFP-COP1 under UV-B specifically (Figure 5A). In contrast, no co-immunoprecipitation of UVR8 was found under conditions devoid of UV-B or from control plants not expressing YFP–COP1 (Figure 5A). Similarly, no protein cross-reacting with our anti-UVR8 antibodies was detected in the YFP control pull downs from plants expressing YFP–COP1 in a cop1 uvr8 double mutant background (cop1-4 uvr8-6/Pro35S:YFP-COP1) (Figure 5A). It should also be pointed out that YFP–COP1 protein levels are stabilized under UV-B and that this effect is dependent on the presence of UVR8 protein (Figure 5A). Notwithstanding this, we could detect co-immunoprecipitation of UVR8 with YFP–COP1 as early as 5 min after UV-B irradiation, when YFP–COP1 levels are not yet elevated (Figure 5B). Thus, we conclude that COP1 and UVR8 interact in vivo in a specific, rather rapid and UV-B-dependent manner. 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/Pro35S:YFP-COP1 and cop1-4 uvr8-6/Pro35S: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/Pro35S:YFP-COP1 seedlings exposed to UV-B for the indicated times. Download figure Download PowerPoint UV-B-induced photomorphogenesis is required for UV-B acclimation and survival in sunlight Altogether, our and published data predict an important role of the UVR8/COP1-mediated UV-B photomorphogenic pathway in UV-B acclimation and tolerance. To further support this notion and provide a physiological demonstration of UV-B acclimation, we combined weak narrowband UV-B exposure with subsequent broadband UV-B stress. Exposure of wild-type seedlings for 7 days to narrowband UV-B that activates photomorphogenic responses resulted in tolerance to a subsequent broadband UV-B stress treatment (Figure 6A). This acclimation effect was absent in uvr8 mutants and enhanced in UVR8 overexpressor lines (Figure 6A). Similarly, cop1-4 mutants were impaired in their acclimation response, whereas the cop1eid6 displayed higher UV-B stress tolerance after acclimation (Supplementary Figure S5). This is in good agreement with the previously demonstrated absence and presence of UV-B photomorphogenic response in cop1-4 and cop1eid6 alleles, respectively (Oravecz et al, 2006). Thus, weak photomorphogenic UV-B promotes plant survival under higher fluence rates of UV-B in a UVR8- and COP1-dependent manner. 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" @default.
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• W2100951456 title "Interaction of COP1 and UVR8 regulates UV-B-induced photomorphogenesis and stress acclimation in Arabidopsis" @default.
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