uvr8 interacts with wrky36 to regulate hy5 transcription...

Articleshttps://doi.org/10.1038/s41477-017-0099-0

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1National Key Laboratory of Plant Molecular Genetics, Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China. 2Shanghai College of Life Science, University of Chinese Academy of Sciences, Shanghai, China. *e-mail: [email protected]

Ultraviolet-B light is an inherent part of sunlight, which has significant biological effects on plants. Low-level, non-dam-aging UVB serves as a photomorphogenic signal to regu-

late photomorphogenesis. For example, UVB inhibits hypocotyl growth, as well as biosynthesis and accumulation of ‘sunscreen’ pig-ments1,2. UV RESISTANCE LOCUS 8 (UVR8) is the long-sought-after UVB photoreceptor that is required for UVB responses3–5. The UVR8 protein is localized in both the cytoplasm and the nucleus, while its main activity is assumed to be nuclear. UVR8’s protein abundance is not affected by UVB, but UVB irradiation induces the nuclear accumulation of UVR8 (refs 1,6). Direct interaction between photoreceptors and their respective target proteins has been recognized as a fundamental mechanism underlying the sig-nal transduction of plant photoreceptors. Only a few proteins have been reported to physically interact with UVR8 to mediate UVB signal transduction. The E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) is a central regulator of light signalling7 that interacts with UVR8 in a UVB-dependent man-ner1,3,8,9. COP1 is required for UVB-induced nuclear accumulation of UVR8 and also UVR8-mediated UVB signalling10,11. The WD40-repeat proteins REPRESSOR OF UVB PHOTOMOPHOGENESIS 1 (RUP1) and RUP2 are negative regulators of UVB signalling12. RUP1 and RUP2 directly interact with UVR8 to mediate UVR8 redimerization so as to disrupt the UVR8–COP1 interaction13.

The basic leucine-zipper transcription factor (TF) ELONGATED HYPOCOTYL 5 (HY5) plays a very important role in de-etiolation14. HY5 and its homologue HYH mediate UVB-induced gene expression changes downstream of UVR8 (refs 15–21). HY5 was proposed to be involved in UVB signalling when its transcription was identified as UVB induced15. Both UVR8 and COP1 are required for UVB-induced HY5 transcription. Treatment with UVB induces the transcription and translation levels of HY5 in a UVR8- and COP1-dependent man-

ner1,6,16,17,22. The central role of HY5 in the UVB acclimation response is further confirmed by the UVB stress hypersensitive phenotype of the hy5 mutant1,16,21. In darkness, HY5 is a target of COP1 and gets degraded via the proteasome23. Under UVB stimulation, however, COP1 is required for the induction of HY5 expression. The UVB-inducible HY5 stabilization is probably a consequence of the UVR8–COP1 interaction1,17 and HY5 is involved in a positive feedback loop promoting COP1 expression by binding the COP1 promoter21. HY5 and HYH interact directly with a T/G-box cis-acting element of the HY5 promoter, mediating the UVB-activated HY5 transcription24.

Blue light photoreceptor cryptochromes (CRYs) interact with the TFs cryptochrome-interacting basic-helix-loop-helix 1 (CIB1) and PHYTOCHROME-INTERACTING FACTOR 4/5 (PIF4/PIF5) to regulate transcription25–29, while red/far red light photoreceptor pho-tochromes (PHYs) interact with PIFs to regulate transcription30,31. The mechanism by which UVR8 triggers UVB photomophogenic responses in the nucleus and whether UVR8 interacts with TF to directly regulate transcription are still unknown. Whether other TFs are involved in the UVB-induced HY5 transcription is also unknown. Here, we identify and characterize the Arabidopsis WRKY DNA-BINDING PROTEIN 36 (WRKY36), which physically inter-acts with UVR8 in vivo, and find that the UVR8–WRKY36 complex accumulates in nuclei in response to a photomorphogenic UVB light stimulus. WRKY36 promotes hypocotyl elongation by repressing the transcription of HY5, and WRKY36 is involved in UVB responses downstream of UVR8. Nucleus-localized UVR8 that is activated under UVB represses the DNA-binding activity of WRKY36. These results demonstrate that UVR8 can regulate gene expression in response to UVB light by directly interacting with the TF WRKY36.

UVR8 physically interacts with WRKY36. Whether UVR8 inter-acts with TFs to regulate transcription and UVB responses is still

UVR8 interacts with WRKY36 to regulate HY5 transcription and hypocotyl elongation in ArabidopsisYu Yang1,2, Tong Liang1,2, Libo Zhang1, Kai Shao1,2, Xingxing Gu1,2, Ruixin Shang1,2, Nan Shi1, Xu Li1, Peng Zhang1 and Hongtao Liu 1*

UV RESISTANCE LOCUS 8 (UVR8) is an ultraviolet-B (UVB) radiation photoreceptor that mediates light responses in plants. How plant UVR8 acts in response to UVB light is not well understood. Here, we report the identification and characteriza-tion of the Arabidopsis WRKY DNA-BINDING PROTEIN 36 (WRKY36) protein. WRKY36 interacts with UVR8 in yeast and Arabidopsis cells and it promotes hypocotyl elongation by inhibiting HY5 transcription. Inhibition of hypocotyl elongation under UVB requires the inhibition of WRKY36. WRKY36 binds to the W-box motif of the HY5 promoter to inhibit its transcription, while nuclear localized UVR8 directly interacts with WRKY36 to inhibit WRKY36–DNA binding both in vitro and in vivo, leading to the release of inhibition of HY5 transcription. These results indicate that WRKY36 is a negative regulator of HY5 and that UVB represses WRKY36 via UVR8 to promote the transcription of HY5 and photomorphogenesis. The UVR8–WRKY36 interac-tion in the nucleus represents a novel mechanism of early UVR8 signal transduction in Arabidopsis.

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unknown. To address this question, we performed a yeast-two hybrid screen with a library of Arabidopsis thaliana TF open read-ing frames32 to identify TFs that interact with A. thaliana UVR8. WRKY36 was identified in this screen. In yeast cells, WRKY36 interacted with UVR8 in both darkness and UVB (Supplementary Fig. 1a). WRKY36 is a novel WRKY protein (Supplementary Fig. 1b) whose function has not been reported previously. The Arabidopsis genome encodes more than 70 WRKY proteins33–35. WRKY36 belongs to the subfamily IIb33. The messenger RNA expression of WRKY36 is regulated by UVB light, as shown by our quantitative polymerase chain reaction (qPCR) analyses. When continuous-white-light-grown seedlings were exposed to UVB light, the level of

WRKY36 messenger RNA increased by about twofold within the first hour of UVB light irradiation and then decreased (Supplementary Fig. 1c). Interestingly, the UVB-induced WRKY36 transcription was not UVR8 dependent, since UVB light induced the transcription of WRKY36 in the uvr8 mutant (Supplementary Fig. 1c).

Next, we examined the interaction between UVR8 and WRKY36 using an in vitro pull-down assay. UVR8 was expressed and purified as reported before36. Dimeric UVR8 changed to monomeric UVR8 after UVB treatment (Supplementary Fig. 1d). The Escherichia coli-expressed UVR8 interacted with the E. coli-expressed WRKY36 in a UVB-independent manner in the in vitro pull-down assay (Fig. 1a), indicating that both dimeric and

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Fig. 1 | UVR8 physically interacts with WRKY36. a, UVR8 interacts with WRKY36, as shown by in vitro pull-down assays. His-tagged UVR8 bound to anti-UVR8 beads were mixed with His-tagged WRKY36 purified from E. coli. b, In BiFC assays, UVR8 interacts with WRKY36 in vivo under white light conditions (without UVB treatment). N. benthamiana were co-transformed with WRKY36–nYFP and cCFP, nYFP and UVR8–cCFP or WRKY36–nYFP and UVR8–cCFP. c, Co-immunoprecipitation assays using 14-day-old transgenic seedlings expressing 35 S::UVR8–GFP or 35 S::UVR8–GFP and 35 S::WRKY36–TAP treated with or without UVB for 20 min. Input: immunoblots showing the level of GFP–UVR8, WRKY36–TAP in the total protein extract. Myc-IP: immunoprecipitation products precipitated by the anti-Myc antibody. Total proteins (input) or immunoprecipitation products were probed in immunoblots with antibodies to GFP or Myc. d, Schematic representation of WRKY36 or UVR8 used in this work. WRKY36 contains an unknown domain (DUF) and a DNA-binding domain (WRKY). UVR8 contains seven repeats of a β -propeller fragment and a C terminus (including a C27 domain). e, BiFC assays of the in vivo protein interaction under white light conditions (without UVB treatment). Epidermal cells of the N. benthamiana leaf were co-transformed with WRKY36C–nYFP and cCFP or nYFP and UVR8C–cCFP or WRKY36C–nYFP or UVR8C–cCFP. f, By in vitro pull-down assays, only UVR8C interacts with WRKY36C. GST-tagged UVR8N or UVR8C bound to GST beads were mixed with His-tagged WRKY36C purified from E. coli. BF, bright field; IP, immunoprecipitation; Merge, overlay of the YFP and bright field images.

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monomeric UVR8 interact with WRKY36. UVR8 also interacted with WRKY36 in plant cells in a bimolecular fluorescence comple-mentation (BiFC) assay. Strong fluorescence was detected in the nuclei of cells co-transformed with UVR8–cCFP (carboxy termi-nal cyan fluorescent protein) and WRKY36–nYFP (amino termi-nal yellow fluorescent protein) plasmids, but no fluorescence was detected in cells transformed with cCFP and WRKY36–nYFP or UVR8–cCFP with nYFP plasmids (Fig. 1b). Both UVR8W285F (the constitutively dimeric UVR8 mutant form that is inactive regard-less of subcellular localization and UVB light conditions) and UVR8W285A (the constitutively monomeric UVR8 mutant)10 inter-acted with WRKY36 in the BiFC assay (Supplementary Fig. 1e,f), confirming that both dimeric and monomeric UVR8 could inter-act with WRKY36. A bimolecular luminescence complementation assay confirmed that UVR8 directly interacted with WRKY36 in plant cells (Supplementary Fig. 1g). UVR8 does not inter-act with WRKY72 and WRKY42—proteins related to WRKY36 (Supplementary Fig 2a). The in vivo interaction of UVR8 and WRKY36 was further confirmed by co-immunoprecipitation. WRKY36 was co-immunoprecipitated with UVR8 from tissues irradiated with and without UVB light (Fig. 1c and Supplementary Fig. 2b). These results indicate that UVR8 interacts with WRKY36 in a UVB-independent manner. Both BiFC and in vitro pull-down assays show that the carboxy (C)-terminal DNA-binding domain (WRKY36C: amino acid 191 to 388), but not the amino (N)-terminal of WRKY36 (WRKY36N: amino acid 1 to 190) interacts with the C terminus (UVR8C: amino acid 397 to 440) but not the N terminus of UVR8 (UVR8N: amino acid 1 to 396) (Fig. 1d–f and Supplementary Fig. 2c). COP1 also interacts with the C terminus of UVR89,37, and WRKY36 does not interact with COP1 (Supplementary Fig. 2d). UVR8 has the potential to bind to both WRKY36 and COP1 to repress hypocotyl elongation.

Nuclear accumulation of UVR8–WRKY36. As WRKY36 is pro-posed to be a TF, which is likely to function in the nucleus, and UVB is known to promote the nuclear accumulation of UVR81,6), there should be more UVR8–WRKY36 complex in the nucleus after UVB irradiation. Indeed, much stronger red fluorescence was detected in the nuclei of cells co-transformed with mCherry-UVR8 and green fluorescent protein (GFP)–WRKY36 plasmids irradi-ated with UVB than without UVB, and nuclear protein WRKY36 showed more co-localization with UVR8 in the nucleus with UVB treatment than without UVB treatment (Fig. 2a). The nuclear localization and protein level of WRKY36 were not significantly affected by the narrow-band UVB treatment (Supplementary Fig. 3a,b). The BiFC assay also indicated that UVB treatment pro-moted the interaction between UVR8 and WRKY36 in the nucleus where the relative fluorescence intensity of nuclei treated with UVB was about four times that of those treated without UVB (Fig. 2b,c and Supplementary Fig. 3c). We further examined whether UVB might affect the accumulation of a possible UVR8–WRKY36 com-plex in nuclei expressing tandem affinity purification (TAP)-tagged WRKY36 (WRKY36–TAP) using a co-immunoprecipitation assay. A comparable amount of WRKY36 was detected in nuclei protein extracts of seedlings treated with or without UVB light (Fig. 2d). However, due to UVB-induced nuclear accumulation of UVR8, a relatively higher level of UVR8 was detected in the samples irradi-ated with UVB than those without UVB treatment, although the total levels of UVR8 were similar (Fig. 2d). More WRKY36 was co-precipitated with UVR8 from seedlings irradiated with UVB com-pared with a non-treated control, although the total WRKY36–TAP inputs were at similar levels (Fig. 2d). These results argue strongly that UVB light stimulates accumulation of the UVR8–WRKY36 complex in nuclei. Taken together, we conclude that UVB induces the nuclear accumulation of UVR8 and promotes UVR8–WRKY36 complex formation in the nucleus.

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Fig. 2 | UVB promotes the nuclear accumulation of UVR8 as well as formation of the UVR8–WRKY36 complex. a, UVB treatment promotes UVR8 nuclear accumulation and co-localization with WRKY36 in the nucleus. N. benthamiana were co-transformed with mCherry–UVR8 and GFP–WRKY36 and treated with or without narrowband UVB (2 W m–2) for 30 min before imaging. Merge: overlay of GFP and mCherry. b, BiFC assays showing that UVB treatment promotes the formation of a UVR8–WRKY36 complex. Leaf epidermal cells of N. benthamiana were co-transformed with WRKY36–nYFP and UVR8–cCFP and treated with or without narrowband UVB (2 W m–2) for 30 min before imaging. c, The relative fluorescence intensities of nuclei and whole cells were quantified and the nucleus-to-background ratios are plotted. Error bars show the s.d. for n > 30. d, Co-immunoprecipitation assays using isolated nuclei show that there are more UVR8–WRKY36 complexes in nuclei treated with UVB than without UVB. Nuclei isolated from 14-day-old long day grown WRKY36–TAP plants treated with or without 24 h UVB were used in the co-immunoprecipitation assay. Immunoblots of total nuclear proteins (input) or immunoprecipitation products of the UVR8 antibody (α -UVR8–IP) were probed with anti-UVR8, anti-Myc or anti-H3 antibody (nuclear control).





WRKY36 and hypocotyl elongation. To determine the biologi-cal roles of WRKY36, we first checked the expression pattern of WRKY36. Analyses of β -glucuronidase (GUS) reporter expres-sion in transgenic plants expressing GUS under the control of the WRKY36 promoter demonstrated that the WRKY36 promoter was active in all tissues of the seedling, and qPCR showed that WRKY36 was expressed in mature leaves, stems and flowers (Supplementary Fig. 4a,b). We obtained transfer DNA insertion mutants from the Arabidopsis Biological Resource Center, naming them wrky36-1 and wrky36-2 (Supplementary Fig. 4c,d). Under white light condi-tions, wrky36-1 and wrky36-2 exhibited short hypocotyl phenotypes compared with the wild type (WT) (Fig. 3a,b). While UVB irra-diation inhibited hypocotyl elongation in WT plants, such inhibi-tion was compromised in uvr8 mutants (Fig. 3a–c). Interestingly, wrky36-1 and wrky36-2 mutants showed dramatic short hypocotyl phenotypes under the white light, but not under the UVB light, exhibiting less hypocotyl length difference with UVB than with-out UVB compared with the WT (the hypocotyl length ratio of wrky36-1 white + UVB/white is 0.51, for wrky36-2 it is 0.47, for the WT it is 0.36 and for uvr8 it is 0.77) (Fig. 3a–c). Transgenic plants overexpressing WRKY36 driven by the cauliflower mosaic virus 35 S promoter (Supplementary Fig. 4e) showed longer hypocotyls than the WT under white light with and without UVB (Fig. 3d–f). The hypocotyl phenotype of the wrky36-1 mutant was comple-mented when Pro35S:WRKY36–TAP was transferred into wrky36-1 (Supplementary Fig. 4f–h). WRKY72 could not interact with UVR8 and the wrky72 mutant showed no obvious hypocotyl phenotype.

The wrky36/wrky72 double mutant showed a similar hypocotyl phenotype as the wrky36 mutant (Supplementary Fig. 5). These results indicate that WRKY36 is a positive regulator of hypocotyl elongation and a negative regulator of UVB-repressed hypocotyl elongation.

WRKY36 acts downstream of UVR8. WRKY36 physically inter-acts with UVR8 and is involved in UVB-mediated hypocotyl elongation. To further study the relationship between WRKY36 and UVR8, we investigated genetic interactions between the WRKY36 and UVR8 genes. A UVR8-deficient uvr8 mutant was crossed with wrky36-1, resulting in the wrky36/uvr8 double mutant (Supplementary Fig. 6a). The long hypocotyl phenotype of uvr8 was partially suppressed in the wrky36/uvr8 mutant with UVB treatment (Fig. 4a–c and Supplementary Fig. 6b), which suggests that WRKY36 acts downstream of UVR8. In addition, as shown in Fig. 4d–e, transgenic seedlings overexpressing WRKY36–TAP showed long hypocotyl phenotypes in white light conditions, whereas the long hypocotyl phenotypes of WRKY36–TAP seedlings were repressed by UVB light. Furthermore, the suppression of the long hypocotyl phenotype of WRKY36–TAP seedlings by UVB light was UVR8 dependent because transgenic seedlings overexpressing WRKY36–TAP in the uvr8 background exhibited long hypocotyl phenotypes in both white light and white light plus UVB condi-tions (Fig. 4d–e). Transgenic seedlings expressing WRKY36–TAP and GFP–UVR8 together exhibited a phenotype similar to GFP–UVR8 seedlings under continuous white light plus UVB conditions

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Fig. 3 | WRKY36 is involved in UVB-controlled hypocotyl elongation. a,b, Phenotypic analysis. Seedlings of indicated genotypes were grown in the presence of white light or white light plus UVB. Images of the representative six-day-old seedlings are shown in a. The hypocotyl lengths of the indicated genotypes were measured and are shown in b. Error bars indicate s.d. (n > 15). The letters ‘A’ to ‘C’ indicate statistically significant differences between the hypocotyl lengths of the indicated seedlings grown in the absence of UVB, as determined by Tukey’s least significant difference test (P < 0.05). The letters ‘a’ to ‘c’ indicate the same, but for seedlings grown in the presence of UVB light. c, Hypocotyl length ratios (white + UVB-to-white) of the quantified hypocotyl lengths in a and b. Error bars indicate s.d. (n = 3). The letters ‘a’ to ‘c’ indicate statistically significant differences by Tukey’s least significant difference test (P < 0.05). d–f, Phenotypic analysis. Seedlings of the indicated genotypes were grown in the presence of white light or white light plus UVB. Images of representative six-day-old seedlings are shown in d. The hypocotyl lengths of the indicated genotypes were measured and are shown in e (white) and f (white + UVB). Error bars indicate s.d. (n > 15). The asterisks indicate significant differences by Tukey’s least significant difference test (P < 0.05): *P < 0.05; **P < 0.01; ***P < 0.001.





(Fig. 4f), consistent with the notion that UVB light suppressed the long hypocotyl phenotype of WRKY36–TAP seedlings in a UVR8-dependent manner. The expression levels of WRKY36 and UVR8 genes are shown in Supplementary Fig. 6c,d. Glucocorticoid recep-tor (GR)–UVR8 (expressing UVR8 fused with a GR and YFP under the native UVR8 promoter in the uvr8 mutant background) was used to conditionally localize UVR8 in distinct subcellular fac-tions11. WRKY36–TAP/uvr8 was crossed with GR–UVR8/uvr8 to obtain transgenic plants expressing WRKY36–TAP and GR–UVR8 in uvr8. Treatment with UVB repressed the long hypocotyl phenotype of WRKY36–TAP plus GR–UVR8/uvr8 seedlings only when treated with dexamethasone (Supplementary Fig. 6e,f).

WRKY36 negatively regulates UVR8-responsive genes. To deter-mine molecular mechanisms of the short hypocotyl phenotype of the wrky36 mutant under white light conditions, we exam-ined the expression of HY5, CHALCONE SYNTHASE (CHS), PACLOBUTRAZOL RESISTANCE 5 (PRE5), INDOLE-3-ACETIC ACID INDUCIBLE 5 (IAA5), IAA19, HOMEODOMAIN-LEUCINE ZIPPER PROTEIN 2 (HAT2), SMALL AUXIN UP RNA 20 (SAUR20) and SAUR63, which are involved in regulating hypocotyl elongation or UVB responses. CHS is a key enzyme in the phenylpropanoid

biosynthesis pathway leading to anthocyanins that is regulated mainly at the transcription level in response to UVB19,38. We found that the expression of HY5 or CHS was significantly increased in wrky36-1 under white light, while the transcription of other genes was not significantly changed (Supplementary Fig. 7a), indicating that the short hypocotyl phenotype of wrky36 under white light might be because of the higher expression level of HY5. To further determine the molecular mechanisms of the UVB-insensitive hypo-cotyl phenotype of the wrky36 mutants, we examined the expression of HY5, CHS, COP1, HYH, RUP1 and RUP2 under white light with or without UVB light conditions. HY5, HYH, COP1 and FHY3 are positive regulators, while RUP1, RUP2 and BBX24 are negative reg-ulators of UVB signalling5. Compared with the WT, the expression of HY5, HYH and CHS was substantially elevated in wrky36 mutant plants when plants were grown under continuous white light con-ditions, while their expression was not obviously changed when plants were grown under continuous white light supplemented with UVB, although UVB induced their transcription (Fig. 5a,b and Supplementary Fig. 7b). These results indicate that WRKY36 represses the transcription of those genes, while UVB might induce their transcription by repressing WRKY36. We checked by qPCR for the expression of HY5, HYH, CHS, COP1, RUP1 and RUP2 in

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Fig. 4 | WRKY36 regulates UVB-controlled hypocotyl elongation downstream of UVR8. a–c, wrky36 partially complements the long hypocotyl phenotype of uvr8 under UVB light. Seedlings of the indicated genotypes were grown in continuous white light or white plus UVB light for 7 days and measurements were made every day from day 4 to day 7. Images of the representative seedlings are shown in a. The hypocotyl lengths of the indicated genotypes were measured and are shown in b (white light) and c (white plus UVB light). The error bars represent s.d. (n > 15). The asterisks indicate significant differences by Tukey’s least significant difference test (P < 0.05). d,e, Phenotypic analysis. Seedlings of the indicated genotypes were grown in the presence of white light or white light plus UVB. Images of the representative six-day-old seedlings are shown in d. The hypocotyl lengths of the indicated genotypes were measured and are shown in e. The error bars represent s.d. (n > 15). The letters ‘A’ and ‘B’ indicate statistically significant differences between the hypocotyl lengths of the indicated seedlings grown in the absence of UVB, as determined by Tukey’s least significant difference test (P < 0.05). The letters ‘a’ to ‘c’ indicate the same, but for seedlings grown in the presence of UVB light. f, WRKY36 regulates hypocotyl elongation in a UVR8-dependent manner. Seedlings of the indicated genotypes were grown in the presence of white light plus UVB for six days. The hypocotyl lengths of the indicated genotypes were measured and are shown. The error bars represent s.d. (n > 15). The letters ‘a’ to ‘c’ indicate statistically significant differences by Tukey’s least significant difference test (P < 0.05): *P < 0.05; **P < 0.01; ***P < 0.001.





wrky36 and uvr8 single mutants and the WT when plants were moved from continuous white light to UVB light. Expression of HY5 and CHS was UVB induced in a UVR8-dependent man-ner. The UVB-induced expression of HY5, HYH, CHS, COP1, RUP1 and RUP2 was more dramatic in the wrky36-1 mutant, but their expression started to decrease 2 h after UVB treatment and reached a similar level as in the WT around 6 h after UVB treatment (Fig. 5c,d and Supplementary Fig. 7c–f). The expression of HY5 and CHS was inhibited in the WRKY36 transgenic line (Supplementary Fig. 7g,h). We hypothesized that WRKY36 might regulate hypocotyl elongation and UVB responses by regulating the expression of HY5 and other genes. Consistent with this hypothesis, the short hypo-cotyl phenotype of wrky36 was suppressed in wrky36/hy5 under UVB light. Under both white light and white light plus UVB con-ditions, wrky36/hy5 exhibited a similar long hypocotyl phenotype to hy5 (Fig. 5e,f). WRKY36–TAP overexpression seedlings in the hy5 background showed similar long hypocotyl phenotypes to hy5 (Fig. 5e,f). These results indicate that WRKY36 acts upstream from HY5 to regulate hypocotyl elongation. Consistent with the notion that the long hypocotyl phenotype of uvr8 was partially suppressed in the wrky36/uvr8 mutant with UVB treatment, the expression lev-els of HY5 and CHS were higher in the wrky36/uvr8 mutant than in uvr8 (Fig. 5g,h).

UVR8 inhibits WRKY36 binding to DNA. WRKY36 promotes hypocotyl elongation by repressing the transcription of HY5. Could WRKY36 directly regulate HY5 transcription? WRKY proteins pre-fer to bind to W boxes33, which means that target genes of WRKY TFs are likely to have W boxes. There are W boxes in HY5’s pro-moter, so we analysed whether WRKY36 could bind the HY5 pro-moter. WRKY36 bound to the HY5 promoter fragments HY5Pb (− 733 base pairs (bp) to − 501 bp) and HY5Pa (− 500 bp to − 1 bp) that contained W boxes in yeast-one hybrid experiments (Fig. 6a and Supplementary Fig. 8a,b). Further evidence supporting that WRKY36 directly bound the HY5 promoter came from electro-phoretic mobility-shift assays using WRKY36 and UVR8 proteins expressed in E. coli in vitro. As shown in Fig. 6b, WRKY36 bound to the promoter region of HY5 (− 700 bp to − 671 bp), but had much lower binding activity with the mutated W box (Supplementary Fig. 8c,d). Interestingly, UVR8 could not bind to the HY5 promoter fragments by itself (Fig. 6b and Supplementary Fig. 8e), but UVR8 inhibited the DNA-binding activity of WRKY36 especially with UVB radiation (Supplementary Fig. 8e). Next, we performed chro-matin immunoprecipitation (ChIP)-qPCR to determine whether WRKY36 bound to the HY5 promoter in vivo and whether UVR8 affected the DNA-binding activity of WRKY36. WRKY36 indeed interacted with chromatin fragments associated with the HY5 genomic DNA in vivo (Fig. 6c,d and Supplementary Fig. 8f,g). Furthermore, UVB light repressed the DNA-binding activity of WRKY36 to the HY5 promoter (Fig. 6d and Supplementary Fig. 8g). Since UVB light treatment dramatically inhibited the DNA bind-ing of WRKY36, and the UVB light repression of WRKY36 DNA binding was dependent on UVR8, in the uvr8 mutant background, the UVB light treatment did not affect the DNA-binding activ-ity of WRKY36 (Fig. 6e). Furthermore, the UVB light repression of WRKY36 DNA binding was dependent on nucleus-localized UVR8, since in the WRKY36–TAP/GR–UVR8 transgenic line (in uvr8 background), the DNA-binding activity of WRKY36 was much lower in plants with dexamethasone treatment than those without dexamethasone treatment under UVB light (Supplementary Fig. 8h). These data indicate that UVB light inhibits the DNA-binding activ-ity of WRKY36 on the HY5 promoter through UVR8. Next, we investigated whether the transcription activity of WRKY36 on the HY5 promoter was affected by UVR8. Transient transcription assays in protoplasts and tobacco leaves were applied to test the activity of WRKY36. A dual-luciferase (LUC) reporter plasmid that encodes a

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Fig. 5 | WRKY36 negatively regulates the expression of HY5 downstream of UVR8. a,b, Quantitative reverse transcription PCR analyses of HY5 (a) and CHS (b) expression in the WT (Col-0), uvr8 and wrky36-1. Five-day-old constant-white-light-grown seedlings were transferred to UVB or kept under white light for one day. The ACT7 gene was analysed as an internal control. Error bars represent the s.d. of three biological replicates. c,d, Quantitative reverse transcription PCR analyses of HY5 (c) and CHS (d) expression in the WT (Col-0), uvr8 and wrky36-1. Six-day-old constant-white-light-grown seedlings were transferred to UVB for the indicated time. The ACT7 gene was analysed as an internal control. Error bars represent the s.d. of three biological replicates. e,f, Phenotypic analysis. Seedlings of the indicated genotypes were grown under continuous white light with or without UVB light for six days. Images of the representative seedlings are shown in e. The hypocotyl lengths of the indicated genotypes were measured and are shown in f. The error bars represent the s.d. (n > 15). The letters ‘A’ to ‘D’ indicate statistically significant differences between the hypocotyl lengths of the indicated seedlings grown in the absence of UVB, as determined by Tukey’s least significant difference test (P < 0.05). The letters ‘a’ to ‘d’ indicate the same, but for seedlings grown in the presence of UVB light. g,h, qPCR results show that wrky36 partially restores the low expression of HY5 (g) and CHS (h) in uvr8. Six-day-old seedlings grown under continuous white light were transferred to UVB for the indicated time. The error bars represent the s.d. of three biological replicates.





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Fig. 6 | UVR8 inhibits the DNa-binding activity of WRKY36. a, ß-galactosidase assays of yeast cells harbouring the indicated constructs. The results indicate that WRKY36 binds to the HY5 promoter in the yeast-one hybrid system. b, An electrophoretic mobility-shift assay shows that WRKY36 binds to the HY5 promoter in vitro. A cold probe was added as a competitor. c, Diagram depicting the putative promoter of HY5. d, ChIP-qPCR results show that WRKY36 binds to the HY5 promoter and UVB inhibits the binding activity of WRKY36. WT or transgenic seedlings expressing 35 S::WRKY36–TAP treated with or without 5 h of UVB were used. The error bars represent the s.d. of three biological replicates. e, ChIP-qPCR results showing that UVB inhibits the DNA-binding activity of WRKY36 in a UVR8-dependent manner. ChIP-qPCR assays were performed using WRKY36–TAP/WT and WRKY36–TAP/uvr8 transgenic plants treated with or without 5 h of UVB. The error bars represent the s.d. of three biological replicates. f, Structure of the HY5 promoter-driven dual-luciferase (LUC) reporter gene. The HY5 promoter, 35 S promoter, REN luciferase (REN), firefly LUC and nopaline sythase terminator (nos) are indicated. g, Relative reporter activity (LUC/REN) in plants with different effector expressions. Tobacco leaves were transfected with the reporter (HY5PB) and the effectors (only WRKY36, only UVR8 or both WRKY36 and UVR8). The error bars represent the s.d. of three biological repeats. h, The Arabidopsis protoplast GR–UVR8 (YFP–GR–UVR8/uvr8) was transfected with reporter DNA (HY5PA) with or without WRKY36 DNA. The error bars represent the s.d. of three biological replicates. In g and h, the letters ‘A’ and ‘B’ indicate statistically significant differences between LUC-to-REN ratios without UVB light, as determined by Tukey’s least significant difference test (P < 0.05). The letters ‘a’ and ‘b’ indicate the same, but for UVB light treatment. i, Hypothetical model depicting how UVR8 acts with WRKY36 to regulate transcription and photomorphogenesis in Arabidopsis. The model hypothesizes that in the absence of UVB light, UVR8 mainly localizes in the cytoplasm, while WRKY36 is localized in the nucleus where it inhibits the transcription of HY5 and promotes hypocotyl elongation. In the presence of UVB, UVR8 accumulates in the nucleus and interacts with nuclear WRKY36 to inhibit its DNA-binding activity. As a result, UVB promotes the transcription of HY5 and inhibits hypocotyl elongation. IP, immunoprecipitation.





firefly LUC gene driven by a varied HY5 promoter (HY5PA: –500 bp to − 1 bp; HY5PB: − 700 bp to − 1 bp; HY5PC: − 2,000 bp to − 1 bp) (Supplementary Fig. 9a) and a Renilla luciferase gene driven by the constitutive 35 S promoter were used in the assays (Fig. 6f)25,27,28. The HY5PA–LUC, HY5PB–LUC or HY5PC–LUC reporter was transiently expressed in tobacco leaves together with either UVR8 or WRKY36, or both. The expression level of all three was about two fold lower when WRKY36 was expressed with or without UVB light (Fig. 6g and Supplementary Fig. 9c–e). WRKY36 did not affect the expression of HY5PA-m-LUC, in which the W box was mutated (Supplementary Fig. 9b,c). Furthermore, UVR8 repressed the transcription activity of WRKY36 only under UVB light. UVR8 itself did not significantly affect the transcription of the HY5PA/PB/PC–LUC construct and the expression level of HY5PA/PB/PC–LUC was approximately 1.5 to 2-fold higher when WRKY36 was combined with UVR8 than when only WRKY36 was infil-trated with UVB treatment, although the same volume of WRKY36 Agrobacteria cells was used (Fig. 6g and Supplementary Fig. 9d,e). UVR8W285A repressed the transcription activity of WRKY36 even without UVB treatment, while UVR8W285F could not repress the transcription activity of WRKY36 with UVB (Supplementary Fig. 9f). The HY5PA–LUC reporter was transiently expressed in GR–UVR8 protoplasts with or without WRKY36. Treatment with UVB repressed the transcription activity of WRKY36 in GR–UVR8 only when induced with dexamethasone (Fig. 6h). The HY5PA–LUC reporter was also transiently expressed in the WT and uvr8 mutant protoplasts with or without WRKY36 and/or UVR8. The expression level of HY5PA–LUC was approximately twofold higher when WRKY36 and UVR8 were expressed together in the WT (Supplementary Fig. 9g) and uvr8 mutant (Supplementary Fig. 9h) protoplast than when only WRKY36 was expressed with UVB treatment (Supplementary Fig. 9g,h), while the expression level of HY5PA–LUC was approximately twofold higher with UVB treat-ment than without it in the WT protoplast (Supplementary Fig. 9g). Taken together, these data indicate that UVR8 interacts with WRKY36 to suppress the DNA binding and transcription activity of WRKY36 in a UVB-light-dependent manner.

DiscussionA fundamental mechanism of the signal transduction of the pho-toreceptors is protein–protein interactions between photorecep-tors and their interacting proteins. It has been reported that red/far red light photoreceptor phytochromes interact with multiple proteins to modulate phytochrome function and regulation, such as several bHLH (basic helix loop helix) TFs (PIF proteins), a nucleo-side diphosphate kinase (NDPK2, NUCLEOSIDE DIPHOSPHATE KINASE 2), protein phosphotases (PAPPs, PHYTOCHROME ASSOCIATED PROTEIN PHOSPHATASE), a response regulator (ARR4, ARABIDOPSIS RESPONSE REGULATOR 4) and PKS1 (PHYTOCHROME KINASE SUBSTRATE 1)30,39–45. Blue light pho-toreceptor cryptochromes interact with the TFs CIB1 and PIF4, COP1, blue-light inhibitor of cryptochromes 1 (BIC1) and pho-toregulatory protein kinases (PPKs) to regulate light responses and transcription25–29,46,47. Only a few proteins have been reported to physically interact with UVR8 to mediate UVB signal trans-duction. COP1 interacts with UVR8 to mediate UVB-induced nuclear accumulation of UVR8 and UVR8-mediated UVB sig-nalling10,11. The WD40-repeat proteins REPRESSOR OF UVB PHOTOMOPHOGENESIS (RUP1) and RUP2 directly interact with UVR8 to mediate UVR8 redimerization, thereby disrupt-ing the UVR8–COP1 interaction12,13. Whether UVR8 physically interacts with TFs to regulate transcription and UVB responses was not known. Here, we identify and characterize the UVR8-interacting protein WRKY36. WRKY36 interacts with UVR8 in a UVB-independent manner, it interacts with both monomeric and dimeric UVR8. Our results show that UVR8 represses the binding

of WRKY36 to the HY5 promoter in vivo to promote HY5 tran-scription and inhibit hypocotyl elongation, which establishes a new UVB signalling pathway. WRKY36 is a novel UVR8-interacting protein and UVR8–WRKY36–HY5 is a novel UVB signalling path-way. Interestingly, as WRKY36, COP1 and the RUP proteins all bind to the C-terminal region of UVR8, it is possible that they compete to bind to UVR8 and form different protein complexes.

HY5 is required for UVB-induced gene transcription and is the major TF required downstream of UVR815–21). HY5 and HYH can bind to the cis-regulatory element T/GHY5-box on the HY5 promoter to activate its own transcription24. It has been reported that three cis-regulatory elements mediate the transcription activation of HY5—namely, an ACG-box, a T/G box and an E-box, with the ACG-box functioning as a light-induced HY5 repression element24. It was proposed that an unknown TF bound to this ACG-box to repress HY5’s transcription in continuous visible light, while in response to UVB light, a positive regulator with a higher affinity for the ACG motif might compete with this repressing transcription regulator or the repressor might be degraded24. Here, we show that a novel pro-tein, WRKY36, binds to the W-box of the HY5 promoter. WRKY36 represses HY5 expression in continuous visible light and, in response to UVB light, UVR8 interacts with WRKY36 and represses the bind-ing of WRKY36 to the HY5 promoter to promote HY5 transcription (Fig. 6i). The ACG-box and W-box are different cis-elements. There was an ACG-box (− 300 bp to − 294 bp) and a W-box (− 185 bp to − 180 bp) close to each other in the HY5 promoter.

UVR8 physically interacts with WRKY36 in the nucleus to inhibit WRKY36’s DNA-binding activity. UVR8 can thus regulate gene expression in response to UVB light by directly interacting with TFs. The interactions between CRYs and CIBs/PIFs are blue light dependent and the interactions between PHYs and PIFs are red light dependent25–29. While the interaction between UVR8 and WRKY36 is not UVB dependent, UVB-light-triggered nuclear localization of UVR8 modulates the UVB control of WRKY36 DNA binding in the nucleus, which also explains why nucleus localization is required for the function of UVR8. CRYs form complexes with PIFs to repress the transcription activity of PIFs28,29; similarly, PHYA also associates with regulatory regions in the genome48, while PHYB inhibits PIF1 and PIF3 by releasing them from their DNA targets49. UVR8 interacts with WRKY36 to inhibit its DNA-binding activity, revealing that plant photoreceptors can regulate transcription via multiple mechanisms.

Taken together, our data support a model in which WRKY36 acts as a transcription repressor of HY5, UVB light induces the nuclear localization of UVR8, and nuclear-localized UVR8 interacts with WRKY36 to prevent it from binding DNA. Thereafter, HY5 and HYH promote the transcription of HY5 (Fig. 6i).

MethodsPlant materials and growth conditions. The Columbia ecotype of A. thaliana was used. Transfer DNA insertion mutants uvr8-61) (SALK_033468), wrky36-1 (SALK_204285), wrky36-2 (CS108822), hy5 (SALK_096651) and wrky72 (SALK_145765) were obtained from the Arabidopsis Biological Resource Center. The uvr8/wrky36, hy5/wrky36 and wrky36/wrky72 double mutants were prepared by genetic crossing, and their identities were verified by genotyping. The full-length UVR8 and WRKY36 coding sequences were cloned into pEarly-104 (Arabidopsis Biological Resource Center) or pCambia1300 (Cambia), bearing either GFP (Pro35S::GFP–UVR8) or Myc–His–Flag (Pro35S::WRKY36–TAP). Col-0 was transformed with Pro35S::GFP–UVR8 and Pro35S:WRKY36–TAP. WTs were transformed and for every transformation, more than ten independent transgenic lines with a single copy of the transgene were generated. Phenotypes of transgenic plants were verified in at least three independent transgenic lines. qPCRs were performed to verify overexpression of the transgenes. WRKY36–TAP/uvr8, WRKY36–TAP/hy5, WRKY36–TAP/GFP–UVR8 and WRKY36–TAP/GR–UVR8 were prepared by genetic crossing.

Seeds were sterilized in 10% bleach, placed on 1/2 Murshige and Skoog medium containing 0.8% agar and 1% sucrose, and stratified for 4 days at 4 °C in the dark before being transferred to white light (Philips TLD18W/54-765; 6 μ mol m−2 s−1; measured using an ILT1400 Radiometer Photometer) or white





light plus UVB (Philips TL20W/01RS narrowband UVB tubes; 2 W m–2; measured using a LUYOR-340 UV Light Meter) modulated by 300 nm transmission cutoff filters (ZJB300; Yongxing Information Sensing Technology) or 340 nm cutoff filters (ZJB340; Yongxing Information Sensing Technology).

Yeast-two hybrid and yeast-one hybrid assays. The coding sequences of UVR8 were fused in-frame with the GAL4 DNA binding domain of the bait vector pDest32 (Clontech) and transformed into Y187. The library of 1362 TFs (in vector pDest22) was in the YM4271 yeast strain (Clontech). Y187 yeast strains were mated with YM4271 yeast strains (in 6 h yeast extract peptone dextrose media), suspended in synthetic defined–Trp–Leu medium and incubated overnight. The interactions were tested by galactosidase assays.

HY5 promoter fragments (− 733 bp to − 501 bp and − 500 bp to − 1bp) were cloned into the pLACZi (LacZi reporter) destination vector and transformed into YM4271 yeast strains. WRKY36 was cloned into the pDest22 vector and transformed into EGY48 yeast strains. YM4271 yeast strains were mated with EGY48 yeast strains (in 6 h yeast extract peptone dextrose media), suspended in synthetic defined–Trp–Ura medium and incubated overnight

Bimolecular luminescence complementation assay. UVR8 or WRKY36 was fused to the C or N terminus of firefly luciferase and transformed to Agrobacterium strain GV3101. Nicotiana benthamiana plants were left under long day white light for 3 days after infiltration and were infiltrated with luciferin solution (1 mM luciferin and 0.01% Triton X-100). Images were captured using a charge-coupled device camera 5 min later.

In vitro pull-down. The in vitro pull-down protein–protein interaction assay was modified from what has been described previously25,27,28. The full-length coding sequences of WRKY36 or WRKY36N (amino acid 1–190 ) and WRKY36C (amino acid 191–388 ) were cloned into pCold TF to generate His–TF–WRKY36, His–TF–WRKY36N and His–TF–WRKY36C. pET29b–UVR8 (UVR8–His) has been reported previously36. UVR8N (amino acid 1–396 ) and UVR8C (amino acid 397–440) were cloned into pGEX4T to generate GST(Glutathione S-transferase)–UVR8N and GST–UVR8C. These proteins were expressed and purified from E. coli BL21. UVR8–His was incubated with the His–TF–WRKY36 under white light (Philips TLD18W/54-765; 6 μ mol m−2 s−1; measured using an ILT1400 Radiometer Photometer) or white light plus UVB (Philips TL20W/01RS narrowband UVB tubes; 2 W m−2; measured using a LUYOR-340 UV Light Meter) for 30 min. Anti-UVR8 antibodies were used to pull down the protein complexes, and unbound proteins were removed via washing. The bound proteins were eluted and analysed using an immunoblot probed with anti-His antibody (MBL; #M136-3). Samples were boiled before sodium dodecyl sulfate polyacrylamide gel electrophoresis (UVR8 dimers turned to be monomers after heat denaturation). GST–UVR8N or GST–UVR8C was incubated with the His–TF–WRKY36N or His–TF–WRKY36C and GST beads were used to pull down the protein complexes. The proteins were eluted and analysed using an immunoblot probed with anti-His or anti-GST antibody (Abmart; #M20007). UVR8 antibody is a polyclonal antibody made by Youke using the reported peptide1.

Nuclear fractionation. Nuclear fractionation was performed as described previously10,26 with modifications. Fourteen-day-old seedlings were collected, ground in liquid nitrogen, homogenized in extraction buffer (20 mM Tris/HCl, pH 7.4, 25% (vol/vol) glycerol, 20 mM KCl, 2 mM EDTA, 2.5 mM MgCl2, 250 mM sucrose, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride and 1× complete protease inhibitor cocktail (Roche)). Total protein extracts were filtered through three layers of Miracloth. After centrifugation at 1,500g for 10 min at 4 °C, the pellet was washed twice with nuclei resuspension Triton buffer (20 mM Tris/HCl, pH 7.4, 25% glycerol, 2.5 mM MgCl2 and 0.2% Triton X-100) and then was used in co-immunoprecipitation.

Co-immunoprecipitation. The co-immunoprecipitation procedure has been described previously25,27,28. Briefly, 14-day-old GFP–UVR8 and WRKY36–TAP/GFP–UVR8 seedlings were grown in long-day conditions, moved to continuous white light for 1 day before treatment with or without UVB (2 W m–2) for 20 min, then ground in liquid nitrogen, homogenized in binding buffer (20 mM Hepes pH 7.5, 40 mM KCl, 1 mM EDTA, 1% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride) and incubated at 4 °C for 5 min. They then went through a 1 ml syringe twice (with a metal needle) to promote nucleus lysis and were centrifuged at 14,000 g for 10 min. The supernatant was mixed with 35 μ l of anti-c-Myc Affinity Gel (Sigma–Aldrich; #E6654), incubated at 4 °C for 30 min and washed twice with washing buffer (20 mM Hepes pH 7.5, 40 mM KCl, 1 mM EDTA and 0.1% Triton X-100). The bound proteins were eluted from the affinity beads with 4× sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer and analysed by immunoblot using anti-Myc (Millipore; #05-724), GFP (Abicode; #M0802-3a) or H3 (Sigma–Aldrich; #9289) antibodies.

Messenger RNA expression analyses. Total RNAs were isolated using the RNAiso Plus (Takara). Complementary DNA was synthesized from 500 ng of total RNA using a PrimeScript RT Reagent Kit with gDNA Eraser (Takara). SYBR Premix Ex

Tag (Takara) was used for the qPCR reaction on the MX3000 System (Stratagene). The level of ACTIN7 messenger RNA expression (AT5G09810) was used as the internal control. Quantitative reverse transcription PCR data for each sample were normalized to the respective ACT7 expression level. The complementary DNAs were amplified following denaturation using 40-cycle programmes (95 °C for 5 s and 60 °C for 20 s per cycle). Biological replicates represented three independent experiments involving about 30 seedlings per experiment. Three technical replicates were performed for each experiment.

Electrophoretic mobility-shift assays. Two methods were used to perform electrophoretic mobility-shift assays. One was as described25,27,28. His–UVR8 and His–WRKY36 fusion proteins were expressed and purified from E. coil (BL21). The double-stranded DNA (Supplementary Table 1) was labelled with digoxigenin by terminal transferase according to the manufacturer’s instructions (DIG Gel Shift Kit; Roche). Then, 100 ng of total protein was added in each binding reaction.

In the other method, for the probe, the synthetic complementary oligonucleotides of the HY5 promoter were annealed and cloned to a T-vector. The probe was then PCR amplified using Cy5-labelled M13 primer pairs. Cy5-labelled DNA on the gel was then detected with the Starion FLA-9000 (FujiFilm).

BiFC, co-localization ChIP and accession numbers have been reported previously28 and are listed in the Supplementary Methods.

Life Sciences Reporting Summary. Further information on experimental design is available in the Life Sciences Reporting Summary.

Data availability. The data that support the findings of this study are available from the corresponding author upon request.

Received: 1 August 2017; Accepted: 27 December 2017; Published online: 29 January 2018

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acknowledgementsWe thank X. Huang, Y. Shi, R. P. Hellens and Q. Hu for materials and technical assistance. We thank G. A. Gomez for copyediting the manuscript. This work is supported in part by the National Key Research and Development Program of China (2017YFA 0503800), National Natural Science Foundation of China (31730009, 31721001, 31670282 and 31670307) and Strategic Priority Research Program ‘Molecular Mechanism of Plant Growth and Development’ (XDPB04).

author contributionsY.Y. and H.L. conceived the project. Y.Y. performed most of the experiments. L.Z., X.G., R.S. and N.S. made some constructs. T.L., X.L. and P.Z. provided materials. K.S. performed some protein expression in E. coli. Y.Y. and H.L. analysed the data and wrote the manuscript.

Competing interestsThe authors declare no competing financial interests.

additional informationSupplementary information accompanies this paper at https://doi.org/10.1038/s41477-017-0099-0.

Reprints and permissions information is available at www.nature.com/reprints.

Correspondence and requests for materials should be addressed to H.L.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


https://doi.org/10.1038/s41477-017-0099-0

https://doi.org/10.1038/s41477-017-0099-0

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uvr8 interacts with wrky36 to regulate hy5 transcription...

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