UVR8 Interacts with WRKY36 to Regulate HY5 Transcription and Hypocotyl Elongation in Arabidopsis

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

UVR8 interacts with WRKY36 to regulate HY5 transcription and hypocotyl elongation in Arabidopsis

Yu 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 interaction in the nucleus represents a novel mechanism of early UVR8 signal transduction in Arabidopsis.

ltraviolet-B light is an inherent part of sunlight, which has ner1,6,16,17,22. The central role of HY5 in the UVB acclimation response significant biological effects on plants. Low-level, non-dam- is further confirmed by the UVB stress hypersensitive phenotype of aging UVB serves as a photomorphogenic signal to regu- the hy5 mutant1,16,21. In darkness, HY5 is a target of COP1 and gets U 23 late photomorphogenesis. For example, UVB inhibits hypocotyl degraded via the proteasome . Under UVB stimulation, however, growth, as well as biosynthesis and accumulation of ‘sunscreen’ pig- COP1 is required for the induction of HY5 expression. The UVB- ments1,2. UV RESISTANCE LOCUS 8 (UVR8) is the long-sought- inducible HY5 stabilization is probably a consequence of the UVR8– after UVB photoreceptor that is required for UVB responses3–5. The COP1 interaction1,17 and HY5 is involved in a positive feedback loop UVR8 protein is localized in both the cytoplasm and the nucleus, promoting COP1 expression by binding the COP1 promoter21. HY5 while its main activity is assumed to be nuclear. UVR8’s protein and HYH interact directly with a T/G-box cis-acting element of the abundance is not affected by UVB, but UVB irradiation induces HY5 promoter, mediating the UVB-activated HY5 transcription24. the nuclear accumulation of UVR8 (refs 1,6). Direct interaction Blue light photoreceptor cryptochromes (CRYs) interact with the between photoreceptors and their respective target proteins has TFs cryptochrome-interacting basic-helix-loop-helix 1 (CIB1) and been recognized as a fundamental mechanism underlying the sig- PHYTOCHROME-INTERACTING FACTOR 4/5 (PIF4/PIF5) to nal transduction of plant photoreceptors. Only a few proteins have regulate transcription25–29, while red/far red light photoreceptor pho- been reported to physically interact with UVR8 to mediate UVB tochromes (PHYs) interact with PIFs to regulate transcription30,31. signal transduction. The E3 ubiquitin ligase CONSTITUTIVELY The mechanism by which UVR8 triggers UVB photomophogenic PHOTOMORPHOGENIC 1 (COP1) is a central regulator of light responses in the nucleus and whether UVR8 interacts with TF to signalling7 that interacts with UVR8 in a UVB-dependent man- directly regulate transcription are still unknown. Whether other ner1,3,8,9. COP1 is required for UVB-induced nuclear accumulation TFs are involved in the UVB-induced HY5 transcription is also of UVR8 and also UVR8-mediated UVB signalling10,11. The WD40- unknown. Here, we identify and characterize the Arabidopsis WRKY repeat proteins REPRESSOR OF UVB PHOTOMOPHOGENESIS 1 DNA-BINDING PROTEIN 36 (WRKY36), which physically inter- (RUP1) and RUP2 are negative regulators of UVB signalling12. acts with UVR8 in vivo, and find that the UVR8–WRKY36 complex RUP1 and RUP2 directly interact with UVR8 to mediate UVR8 accumulates in nuclei in response to a photomorphogenic UVB light redimerization so as to disrupt the UVR8–COP1 interaction13. stimulus. WRKY36 promotes hypocotyl elongation by repressing the The basic leucine-zipper transcription factor (TF) ELONGATED transcription of HY5, and WRKY36 is involved in UVB responses HYPOCOTYL 5 (HY5) plays a very important role in de-etiolation14. downstream of UVR8. Nucleus-localized UVR8 that is activated HY5 and its homologue HYH mediate UVB-induced gene expression under UVB represses the DNA-binding activity of WRKY36. These changes downstream of UVR8 (refs 15–21). HY5 was proposed to be results demonstrate that UVR8 can regulate gene expression in involved in UVB signalling when its transcription was identified as response to UVB light by directly interacting with the TF WRKY36. UVB induced15. Both UVR8 and COP1 are required for UVB-induced HY5 transcription. Treatment with UVB induces the transcription UVR8 physically interacts with WRKY36. Whether UVR8 inter- and translation levels of HY5 in a UVR8- and COP1-dependent man- acts with TFs to regulate transcription and UVB responses is still

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]

98 Nature Plants | VOL 4 | FEBRUARY 2018 | 98–107 | www.nature.com/natureplants © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. NATURE PLAnTS Articles

unknown. To address this question, we performed a yeast-two WRKY36 messenger RNA increased by about twofold within the first hybrid screen with a library of Arabidopsis thaliana TF open read- hour of UVB light irradiation and then decreased (Supplementary ing frames32 to identify TFs that interact with A. thaliana UVR8. Fig. 1c). Interestingly, the UVB-induced WRKY36 transcription was WRKY36 was identified in this screen. In yeast cells, WRKY36 not UVR8 dependent, since UVB light induced the transcription of interacted with UVR8 in both darkness and UVB (Supplementary WRKY36 in the uvr8 mutant (Supplementary Fig. 1c). Fig. 1a). WRKY36 is a novel WRKY protein (Supplementary Fig. 1b) Next, we examined the interaction between UVR8 and whose function has not been reported previously. The Arabidopsis WRKY36 using an in vitro pull-down assay. UVR8 was expressed genome encodes more than 70 WRKY proteins33–35. WRKY36 and purified as reported before36. Dimeric UVR8 changed to belongs to the subfamily IIb33. The messenger RNA expression of monomeric UVR8 after UVB treatment (Supplementary Fig. 1d). WRKY36 is regulated by UVB light, as shown by our quantitative The Escherichia coli-expressed UVR8 interacted with the E. coli- polymerase chain reaction (qPCR) analyses. When continuous- expressed WRKY36 in a UVB-independent manner in the in white-light-grown seedlings were exposed to UVB light, the level of vitro pull-down assay (Fig. 1a), indicating that both dimeric and

a Input UVR8-IP d WRKY36 DUF WRKY

His–WRKY36 ++ + ++ + WRKY36N UVR8–His – ++ – ++ WRKY36C UVB ––+ ––+

WRKY36 UVR8

UVR8N UVR8 UVR8C

beGFP BF Merge nYFP BF Merge cCFP – cCFP 20 μm

20 m UVR8C

WRKY36–nYFP μ WRKY36C–nYFP BF Merge cCFP nYFP UVR8–cCF P

WRKY36C–nYFP BF Merge cCFP – UVR8 C UVR8–cCF P WRKY36–nYFP

cfInput Myc–IP Input GST-IP

GFPUVR8 GFPUVR8 His–WRKY36C ++ + ++ + WRKY36TAP WRKY36TAP GFP– GFP– GST–UVR8N –+ – –+ – UVR8 UVB– UVB+ UVR8 UVB– UVB+ GST–UVR8C –– + –– +

WRKY36C UVR8 UVR8N

WRKY36 UVR8C

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.

Nature Plants | VOL 4 | FEBRUARY 2018 | 98–107 | www.nature.com/natureplants 99 © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. Articles NATURE PLAnTS

a GFP–WRKY36 mCherry–UVR8 Merge monomeric UVR8 interact with WRKY36. UVR8 also interacted with WRKY36 in plant cells in a bimolecular fluorescence complementation (BiFC) assay. Strong fluorescence was detected in the nuclei of cells co-transformed with UVR8–cCFP (carboxy terminal cyan fluorescent protein) and WRKY36–nYFP (amino termi- Whit e 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 interacted with WRKY36 in the BiFC assay (Supplementary Fig. 1e,f), confirming that both dimeric and monomeric UVR8 could inter- White + UVB act with WRKY36. A bimolecular luminescence complementation 40 m µ assay confirmed that UVR8 directly interacted with WRKY36 in plant cells (Supplementary Fig. 1g). UVR8 does not inter- b nYFP/ WRKY36–nYFP/ WRKY36–nYFP/ UVR8–cCFP act with WRKY72 and WRKY42—proteins related to WRKY36 UVR8–cCFP cCFP White + UVB White + UVB White White + UVB (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- 50 µm 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), cd14 Input α-UVR8-IP there should be more UVR8–WRKY36 complex in the nucleus 12 WRKY36–TAPWRKY36–TAP after UVB irradiation. Indeed, much stronger red fluorescence was 10 UVB– UVB+ UVB– UVB+ detected in the nuclei of cells co-transformed with mCherry-UVR8 8 WRKY36 and green fluorescent protein (GFP)–WRKY36 plasmids irradi- 6 ated with UVB than without UVB, and nuclear protein WRKY36 UVR8 4 showed more co-localization with UVR8 in the nucleus with UVB 2 H3 treatment than without UVB treatment (Fig. 2a). The nuclear Relative nuclear level 0 Total UVR8 localization and protein level of WRKY36 were not significantly White White + affected by the narrow-band UVB treatment (Supplementary UVB Fig. 3a,b). The BiFC assay also indicated that UVB treatment pro- moted the interaction between UVR8 and WRKY36 in the nucleus Fig. 2 | UVB promotes the nuclear accumulation of UVR8 as well as where the relative fluorescence intensity of nuclei treated with UVB formation of the UVR8–WRKY36 complex. a, UVB treatment promotes was about four times that of those treated without UVB (Fig. 2b,c UVR8 nuclear accumulation and co-localization with WRKY36 in the and Supplementary Fig. 3c). We further examined whether UVB nucleus. N. benthamiana were co-transformed with mCherry–UVR8 and might affect the accumulation of a possible UVR8–WRKY36 com- GFP–WRKY36 and treated with or without narrowband UVB (2 W m–2) for plex in nuclei expressing tandem affinity purification (TAP)-tagged 30 min before imaging. Merge: overlay of GFP and mCherry. b, BiFC assays WRKY36 (WRKY36–TAP) using a co-immunoprecipitation assay. showing that UVB treatment promotes the formation of a UVR8–WRKY36 A comparable amount of WRKY36 was detected in nuclei protein complex. Leaf epidermal cells of N. benthamiana were co-transformed with extracts of seedlings treated with or without UVB light (Fig. 2d). WRKY36–nYFP and UVR8–cCFP and treated with or without narrowband However, due to UVB-induced nuclear accumulation of UVR8, a UVB (2 W m–2) for 30 min before imaging. c, The relative fluorescence relatively higher level of UVR8 was detected in the samples irradi- intensities of nuclei and whole cells were quantified and the nucleus- ated with UVB than those without UVB treatment, although the to-background ratios are plotted. Error bars show the s.d. for n > 30. d, total levels of UVR8 were similar (Fig. 2d). More WRKY36 was co- Co-immunoprecipitation assays using isolated nuclei show that there precipitated with UVR8 from seedlings irradiated with UVB com- are more UVR8–WRKY36 complexes in nuclei treated with UVB than pared with a non-treated control, although the total WRKY36–TAP without UVB. Nuclei isolated from 14-day-old long day grown WRKY36– inputs were at similar levels (Fig. 2d). These results argue strongly TAP plants treated with or without 24 h UVB were used in the co- that UVB light stimulates accumulation of the UVR8–WRKY36 immunoprecipitation assay. Immunoblots of total nuclear proteins (input) complex in nuclei. Taken together, we conclude that UVB induces or immunoprecipitation products of the UVR8 antibody (α-UVR8–IP) were the nuclear accumulation of UVR8 and promotes UVR8–WRKY36 probed with anti-UVR8, anti-Myc or anti-H3 antibody (nuclear control). complex formation in the nucleus.

100 Nature Plants | VOL 4 | FEBRUARY 2018 | 98–107 | www.nature.com/natureplants © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. NATURE PLAnTS Articles

ab c 7 White 0.9 A A b White + UVB 0.8 6 b 0.7 C White 5 B 0.6 c c 4 0.5 a 3 0.4 1 mm a c c 0.3 2 0.2

Hypocotyl length (mm) 1

(White + UVB)/white ratio 0.1 White + UVB 0 0 2 -1 2 WT WT WT uvr8 uvr8 uvr8

wrky36-1 wrky36- wrky36 wrky36-2 wrky36-1 wrky36- deWhite f White + UVB WT 9 7 WT uvr8 uvr8 wrky36-1 *** 8 *** 6 wrky36-1 WRKY36TAP/WT#5 *** 7 *** WRKY36TAP/WT#5 WRKY36TAP/WT#6 *** WRKY36TAP/WT#6 5 *** White 6 *** 5 *** *** 4 *** 1 mm *** *** *** 4 *** *** 3 *** *** 3 *** *** 2 *** 2 *** Hypocotyl length (mm) Hypocotyl length (mm) White + UVB 1 * 1 * **

WT uvr8 0 0

wrky36-1 4 567 4567 Time (day) Time (day)

WRKY36TAP/WT#5WRKY36TAP/WT#6

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.

WRKY36 and hypocotyl elongation. To determine the biologi- The wrky36/wrky72 double mutant showed a similar hypocotyl cal roles of WRKY36, we first checked the expression pattern of phenotype as the wrky36 mutant (Supplementary Fig. 5). These WRKY36. Analyses of β-glucuronidase (GUS) reporter expres- results indicate that WRKY36 is a positive regulator of hypocotyl sion in transgenic plants expressing GUS under the control of the elongation and a negative regulator of UVB-repressed hypocotyl WRKY36 promoter demonstrated that the WRKY36 promoter was elongation. active in all tissues of the seedling, and qPCR showed that WRKY36 was expressed in mature leaves, stems and flowers (Supplementary WRKY36 acts downstream of UVR8. WRKY36 physically inter- Fig. 4a,b). We obtained transfer DNA insertion mutants from the acts with UVR8 and is involved in UVB-mediated hypocotyl Arabidopsis Biological Resource Center, naming them wrky36-1 elongation. To further study the relationship between WRKY36 and wrky36-2 (Supplementary Fig. 4c,d). Under white light condi- and UVR8, we investigated genetic interactions between the tions, wrky36-1 and wrky36-2 exhibited short hypocotyl phenotypes WRKY36 and UVR8 genes. A UVR8-deficient uvr8 mutant was compared with the wild type (WT) (Fig. 3a,b). While UVB irra- crossed with wrky36-1, resulting in the wrky36/uvr8 double mutant diation inhibited hypocotyl elongation in WT plants, such inhibi- (Supplementary Fig. 6a). The long hypocotyl phenotype of uvr8 tion was compromised in uvr8 mutants (Fig. 3a–c). Interestingly, was partially suppressed in the wrky36/uvr8 mutant with UVB wrky36-1 and wrky36-2 mutants showed dramatic short hypocotyl treatment (Fig. 4a–c and Supplementary Fig. 6b), which suggests phenotypes under the white light, but not under the UVB light, that WRKY36 acts downstream of UVR8. In addition, as shown in exhibiting less hypocotyl length difference with UVB than with- Fig. 4d–e, transgenic seedlings overexpressing WRKY36–TAP out UVB compared with the WT (the hypocotyl length ratio of showed long hypocotyl phenotypes in white light conditions, wrky36-1 white + UVB/white is 0.51, for wrky36-2 it is 0.47, for the whereas the long hypocotyl phenotypes of WRKY36–TAP seedlings WT it is 0.36 and for uvr8 it is 0.77) (Fig. 3a–c). Transgenic plants were repressed by UVB light. Furthermore, the suppression of the overexpressing WRKY36 driven by the cauliflower mosaic virus long hypocotyl phenotype of WRKY36–TAP seedlings by UVB light 35 S promoter (Supplementary Fig. 4e) showed longer hypocotyls was UVR8 dependent because transgenic seedlings overexpressing than the WT under white light with and without UVB (Fig. 3d–f). WRKY36–TAP in the uvr8 background exhibited long hypocotyl The hypocotyl phenotype of the wrky36-1 mutant was comple- phenotypes in both white light and white light plus UVB condi- mented when Pro35S:WRKY36–TAP was transferred into wrky36-1 tions (Fig. 4d–e). Transgenic seedlings expressing WRKY36–TAP (Supplementary Fig. 4f–h). WRKY72 could not interact with UVR8 and GFP–UVR8 together exhibited a phenotype similar to GFP– and the wrky72 mutant showed no obvious hypocotyl phenotype. UVR8 seedlings under continuous white light plus UVB conditions

Nature Plants | VOL 4 | FEBRUARY 2018 | 98–107 | www.nature.com/natureplants 101 © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. Articles NATURE PLAnTS

a bcWhite White + UVB 8 WT uvr8 7 WT uvr8 wrky36-1 wrky36/uvr8 wrky36-1 wrky36/uvr8 7 6 6 ***

White 5 5 *** *** *** 4 *** 1 mm 4 *** *** *** 3 *** 3 *** *** *** *** *** 2 2 *** ***

Hypocotyl length (mm) *

*** Hypocotyl length (mm) 1 1 **

White + UVB 0 0 4567 4 567 WT uvr8 Time (day) Time (day) wrky36-1 wrky36/uvr8 White d e 8 f 7 White + UVB B B b 7 A A 6 6 5 b b 5 White 4 c 4 1 mm c 3 a 3 a 2 2 Hypocotyl length (mm) Hypocotyl length (mm) d d 1 1

White + UVB 0 0

WT WT uvr8 uvr8 uvr8 WT uvr8

GFPUVR8/WT WRKY36TAP/WT WRKY36TAP/uvr8 WRKY36TAP/WTWRKY36TAP/ WRKY36TAP/WT

WRKY36TAP/GFPUVR8

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.

(Fig. 4f), consistent with the notion that UVB light suppressed the biosynthesis pathway leading to anthocyanins that is regulated long hypocotyl phenotype of WRKY36–TAP seedlings in a UVR8- mainly at the transcription level in response to UVB19,38. We found dependent manner. The expression levels of WRKY36 and UVR8 that the expression of HY5 or CHS was significantly increased in genes are shown in Supplementary Fig. 6c,d. Glucocorticoid recep- wrky36-1 under white light, while the transcription of other genes tor (GR)–UVR8 (expressing UVR8 fused with a GR and YFP under was not significantly changed (Supplementary Fig. 7a), indicating the native UVR8 promoter in the uvr8 mutant background) was that the short hypocotyl phenotype of wrky36 under white light used to conditionally localize UVR8 in distinct subcellular fac- might be because of the higher expression level of HY5. To further tions11. WRKY36–TAP/uvr8 was crossed with GR–UVR8/uvr8 determine the molecular mechanisms of the UVB-insensitive hypo- to obtain transgenic plants expressing WRKY36–TAP and GR– cotyl phenotype of the wrky36 mutants, we examined the expression UVR8 in uvr8. Treatment with UVB repressed the long hypocotyl of HY5, CHS, COP1, HYH, RUP1 and RUP2 under white light with phenotype of WRKY36–TAP plus GR–UVR8/uvr8 seedlings only or without UVB light conditions. HY5, HYH, COP1 and FHY3 are when treated with dexamethasone (Supplementary Fig. 6e,f). positive regulators, while RUP1, RUP2 and BBX24 are negative regulators of UVB signalling5. Compared with the WT, the expression WRKY36 negatively regulates UVR8-responsive genes. To deter- of HY5, HYH and CHS was substantially elevated in wrky36 mutant mine molecular mechanisms of the short hypocotyl phenotype plants when plants were grown under continuous white light con- of the wrky36 mutant under white light conditions, we exam- ditions, while their expression was not obviously changed when ined the expression of HY5, CHALCONE SYNTHASE (CHS), plants were grown under continuous white light supplemented PACLOBUTRAZOL RESISTANCE 5 (PRE5), INDOLE-3-ACETIC with UVB, although UVB induced their transcription (Fig. 5a,b ACID INDUCIBLE 5 (IAA5), IAA19, HOMEODOMAIN-LEUCINE and Supplementary Fig. 7b). These results indicate that WRKY36 ZIPPER PROTEIN 2 (HAT2), SMALL AUXIN UP RNA 20 (SAUR20) represses the transcription of those genes, while UVB might induce and SAUR63, which are involved in regulating hypocotyl elongation their transcription by repressing WRKY36. We checked by qPCR or UVB responses. CHS is a key enzyme in the phenylpropanoid for the expression of HY5, HYH, CHS, COP1, RUP1 and RUP2 in

102 Nature Plants | VOL 4 | FEBRUARY 2018 | 98–107 | www.nature.com/natureplants © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. NATURE PLAnTS Articles

ab wrky36 and uvr8 single mutants and the WT when plants were 9 White White + UVB 120 White White + UVB 8 100 moved from continuous white light to UVB light. Expression 7 6 80 of HY5 and CHS was UVB induced in a UVR8-dependent manner. The UVB-induced expression of HY5, HYH, CHS, COP1, 2 60 10 RUP1 and RUP2 was more dramatic in the wrky36-1 mutant, but their expression started to decrease 2 h after UVB treatment and HY5/ACT7 CHS/ACT7 1 5 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 0 0 CHS was inhibited in the WRKY36 transgenic line (Supplementary 1 Fig. 7g,h). We hypothesized that WRKY36 might regulate hypocotyl WT uvr8 WT uvr8

wrky36- wrky36-1 elongation and UVB responses by regulating the expression of HY5 cd18 WT uvr8 wrky36-1 600 and other genes. Consistent with this hypothesis, the short hypo- 16 500 cotyl phenotype of wrky36 was suppressed in wrky36/hy5 under 14 400 UVB light. Under both white light and white light plus UVB con- 12 300 10 200 ditions, wrky36/hy5 exhibited a similar long hypocotyl phenotype 8 100 to hy5 (Fig. 5e,f). WRKY36–TAP overexpression seedlings in the 6 40 CHS/ACT7 HY5/ACT7 hy5 background showed similar long hypocotyl phenotypes to hy5 4 20 2 (Fig. 5e,f). These results indicate that WRKY36 acts upstream from 0 0 HY5 to regulate hypocotyl elongation. Consistent with the notion 012346 012346 that the long hypocotyl phenotype of uvr8 was partially suppressed Time (h) Time (h) in the wrky36/uvr8 mutant with UVB treatment, the expression lev- efWhite White + UVB els of HY5 and CHS were higher in the wrky36/uvr8 mutant than in 10 C C C uvr8 (Fig. 5g,h). 1 mm 8 D A White 6 UVR8 inhibits WRKY36 binding to DNA. WRKY36 promotes c c c B d hypocotyl elongation by repressing the transcription of HY5. Could 4 a b WRKY36 directly regulate HY5 transcription? WRKY proteins pre- White + 2 33 UVB fer to bind to W boxes , which means that target genes of WRKY Hypocotyl length (mm) 0 TFs are likely to have W boxes. There are W boxes in HY5’s pro- 1 WT hy5 moter, so we analysed whether WRKY36 could bind the HY5 pro- hy5 WT hy5 hy5 moter. WRKY36 bound to the HY5 promoter fragments HY5Pb wrky36-1 wrky36- wrky36/hy5 wrky36/hy5 (−733 base pairs (bp) to −501 bp) and HY5Pa (−500 bp to −1 bp) WRKY36TAP/WT WRKY36TAP/ WRKY36TAP/WTWRKY36TAP/ that contained W boxes in yeast-one hybrid experiments (Fig. 6a gh 3.5 uvr8 6 uvr8 and Supplementary Fig. 8a,b). Further evidence supporting that wrky36/uvr8 3.0 5 wrky36/uvr8 WRKY36 directly bound the HY5 promoter came from electro- 2.5 4 phoretic mobility-shift assays using WRKY36 and UVR8 proteins 2.0 expressed in E. coli in vitro. As shown in Fig. 6b, WRKY36 bound 3 1.5 to the promoter region of HY5 ( 700 bp to 671 bp), but had much 2 − − HY5/ACT7 1.0 CHS/ACT 7 lower binding activity with the mutated W box (Supplementary 0.5 1 Fig. 8c,d). Interestingly, UVR8 could not bind to the HY5 promoter 0 0 01234 0123 fragments by itself (Fig. 6b and Supplementary Fig. 8e), but UVR8 Time (h)Time (h) inhibited the DNA-binding activity of WRKY36 especially with UVB radiation (Supplementary Fig. 8e). Next, we performed chro- Fig. 5 | WRKY36 negatively regulates the expression of HY5 downstream matin immunoprecipitation (ChIP)-qPCR to determine whether of UVR8. a,b, Quantitative reverse transcription PCR analyses of HY5 (a) WRKY36 bound to the HY5 promoter in vivo and whether UVR8 and CHS (b) expression in the WT (Col-0), uvr8 and wrky36-1. Five-day- affected the DNA-binding activity of WRKY36. WRKY36 indeed old constant-white-light-grown seedlings were transferred to UVB or kept interacted with chromatin fragments associated with the HY5 under white light for one day. The ACT7 gene was analysed as an internal genomic DNA in vivo (Fig. 6c,d and Supplementary Fig. 8f,g). control. Error bars represent the s.d. of three biological replicates. c,d, Furthermore, UVB light repressed the DNA-binding activity of Quantitative reverse transcription PCR analyses of HY5 (c) and CHS (d) WRKY36 to the HY5 promoter (Fig. 6d and Supplementary Fig. 8g). expression in the WT (Col-0), uvr8 and wrky36-1. Six-day-old constant- Since UVB light treatment dramatically inhibited the DNA bind- white-light-grown seedlings were transferred to UVB for the indicated time. ing of WRKY36, and the UVB light repression of WRKY36 DNA The ACT7 gene was analysed as an internal control. Error bars represent binding was dependent on UVR8, in the uvr8 mutant background, the s.d. of three biological replicates. e,f, Phenotypic analysis. Seedlings the UVB light treatment did not affect the DNA-binding activ- of the indicated genotypes were grown under continuous white light with ity of WRKY36 (Fig. 6e). Furthermore, the UVB light repression or without UVB light for six days. Images of the representative seedlings of WRKY36 DNA binding was dependent on nucleus-localized are shown in e. The hypocotyl lengths of the indicated genotypes were UVR8, since in the WRKY36–TAP/GR–UVR8 transgenic line (in measured and are shown in f. The error bars represent the s.d. (n > 15). The uvr8 background), the DNA-binding activity of WRKY36 was much letters ‘A’ to ‘D’ indicate statistically significant differences between the lower in plants with dexamethasone treatment than those without hypocotyl lengths of the indicated seedlings grown in the absence of UVB, dexamethasone treatment under UVB light (Supplementary Fig. 8h). as determined by Tukey’s least significant difference test (P < 0.05). The These data indicate that UVB light inhibits the DNA-binding activ- letters ‘a’ to ‘d’ indicate the same, but for seedlings grown in the presence ity of WRKY36 on the HY5 promoter through UVR8. Next, we of UVB light. g,h, qPCR results show that wrky36 partially restores the low investigated whether the transcription activity of WRKY36 on the expression of HY5 (g) and CHS (h) in uvr8. Six-day-old seedlings grown HY5 promoter was affected by UVR8. Transient transcription assays under continuous white light were transferred to UVB for the indicated in protoplasts and tobacco leaves were applied to test the activity of time. The error bars represent the s.d. of three biological replicates. WRKY36. A dual-luciferase (LUC) reporter plasmid that encodes a

Nature Plants | VOL 4 | FEBRUARY 2018 | 98–107 | www.nature.com/natureplants 103 © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. Articles NATURE PLAnTS

a 12 c P3 P2 P1 10 +1

8 –1,500 –1,000 –500 ATG 6 6 WT 4 d WRKY36TAP/WT 2 5 WRKY36TAP/WT UVB β -galactosidase activity 0 4 Vector Vector HY5Pb HY5Pb vector WRKY36 vector WRKY36 3 IP/input b Probe ++++ + 2 WRKY36 –++ – – 1 UVR8 –+–– + Cold probe –+––+ 0 P1 P2 P3 ACT7

e 5 WRKY36TAP/WT WRKY36TAP/WT UVB WRKY36TAP/uvr8 WRKY36TAP/uvr8 UVB 4

IP/input 2

1 f HY5P LUC 35S REN nos 0 P1 P2 P3 ACT7

g White White + UVB 0.5 a h 0.7 White White + UVB a A 0.6 0.4 a 0.5 a A 0.3 A A a 0.4 a b 0.2 0.3 LUC/REN B LUC/REN B 0.2 B b 0.1 B 0.1

0 0 WRKY36 –+– + WRKY36 –+–+ UVR8 ––++Dexamethasone ––+ +

i UVB– UVB+

UVR8 UVR8 UVR8 UVR8 UVR8 COP1 UVR8 RUP1/ UVR8 RUP2 COP1 UVR8 RUP1/ WRKY36 RUP2 UVR8 UVR8 UVR8 UVR8 COP1

WRKY36 UVR8 UVR8 COP1 WRKY36 HY5 UVR8 Nucleus WRKY36 HY5 UVR8 UVR8 UVR8 UVR8 Cytoplasm Nucleus Cytoplasm

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.

104 Nature Plants | VOL 4 | FEBRUARY 2018 | 98–107 | www.nature.com/natureplants © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. NATURE PLAnTS Articles

firefly LUC gene driven by a varied HY5 promoter (HY5PA: –500 bp of WRKY36 to the HY5 promoter in vivo to promote HY5 tran- to −1 bp; HY5PB: −700 bp to −1 bp; HY5PC: −2,000 bp to −1 bp) scription and inhibit hypocotyl elongation, which establishes a new (Supplementary Fig. 9a) and a Renilla luciferase gene driven by the UVB signalling pathway. WRKY36 is a novel UVR8-interacting constitutive 35 S promoter were used in the assays (Fig. 6f)25,27,28. protein and UVR8–WRKY36–HY5 is a novel UVB signalling path- The HY5PA–LUC, HY5PB–LUC or HY5PC–LUC reporter was way. Interestingly, as WRKY36, COP1 and the RUP proteins all bind transiently expressed in tobacco leaves together with either UVR8 to the C-terminal region of UVR8, it is possible that they compete to or WRKY36, or both. The expression level of all three was about bind to UVR8 and form different protein complexes. two fold lower when WRKY36 was expressed with or without UVB HY5 is required for UVB-induced gene transcription and is the light (Fig. 6g and Supplementary Fig. 9c–e). WRKY36 did not major TF required downstream of UVR815–21). HY5 and HYH can affect the expression of HY5PA-m-LUC, in which the W box was bind to the cis-regulatory element T/GHY5-box on the HY5 promoter mutated (Supplementary Fig. 9b,c). Furthermore, UVR8 repressed to activate its own transcription24. It has been reported that three cis- the transcription activity of WRKY36 only under UVB light. UVR8 regulatory elements mediate the transcription activation of HY5— itself did not significantly affect the transcription of the HY5PA/ namely, an ACG-box, a T/G box and an E-box, with the ACG-box PB/PC–LUC construct and the expression level of HY5PA/PB/ functioning as a light-induced HY5 repression element24. It was PC–LUC was approximately 1.5 to 2-fold higher when WRKY36 proposed that an unknown TF bound to this ACG-box to repress was combined with UVR8 than when only WRKY36 was infil- HY5’s transcription in continuous visible light, while in response to trated with UVB treatment, although the same volume of WRKY36 UVB light, a positive regulator with a higher affinity for the ACG Agrobacteria cells was used (Fig. 6g and Supplementary Fig. 9d,e). motif might compete with this repressing transcription regulator or UVR8W285A repressed the transcription activity of WRKY36 even the repressor might be degraded24. Here, we show that a novel pro- without UVB treatment, while UVR8W285F could not repress the tein, WRKY36, binds to the W-box of the HY5 promoter. WRKY36 transcription activity of WRKY36 with UVB (Supplementary represses HY5 expression in continuous visible light and, in response Fig. 9f). The HY5PA–LUC reporter was transiently expressed in to UVB light, UVR8 interacts with WRKY36 and represses the bind- GR–UVR8 protoplasts with or without WRKY36. Treatment with ing of WRKY36 to the HY5 promoter to promote HY5 transcription UVB repressed the transcription activity of WRKY36 in GR–UVR8 (Fig. 6i). The ACG-box and W-box are different cis-elements. There only when induced with dexamethasone (Fig. 6h). The HY5PA– was an ACG-box (−300 bp to −294 bp) and a W-box (−185 bp to LUC reporter was also transiently expressed in the WT and uvr8 −180 bp) close to each other in the HY5 promoter. mutant protoplasts with or without WRKY36 and/or UVR8. The UVR8 physically interacts with WRKY36 in the nucleus to expression level of HY5PA–LUC was approximately twofold higher inhibit WRKY36’s DNA-binding activity. UVR8 can thus regulate when WRKY36 and UVR8 were expressed together in the WT gene expression in response to UVB light by directly interacting (Supplementary Fig. 9g) and uvr8 mutant (Supplementary Fig. 9h) with TFs. The interactions between CRYs and CIBs/PIFs are blue protoplast than when only WRKY36 was expressed with UVB light dependent and the interactions between PHYs and PIFs are treatment (Supplementary Fig. 9g,h), while the expression level of red light dependent25–29. While the interaction between UVR8 and HY5PA–LUC was approximately twofold higher with UVB treat- WRKY36 is not UVB dependent, UVB-light-triggered nuclear ment than without it in the WT protoplast (Supplementary Fig. 9g). localization of UVR8 modulates the UVB control of WRKY36 DNA Taken together, these data indicate that UVR8 interacts with binding in the nucleus, which also explains why nucleus localization WRKY36 to suppress the DNA binding and transcription activity of is required for the function of UVR8. CRYs form complexes with WRKY36 in a UVB-light-dependent manner. PIFs to repress the transcription activity of PIFs28,29; similarly, PHYA also associates with regulatory regions in the genome48, while PHYB Discussion inhibits PIF1 and PIF3 by releasing them from their DNA targets49. A fundamental mechanism of the signal transduction of the pho- UVR8 interacts with WRKY36 to inhibit its DNA-binding activity, toreceptors is protein–protein interactions between photorecep- revealing that plant photoreceptors can regulate transcription via tors and their interacting proteins. It has been reported that red/ multiple mechanisms. far red light photoreceptor phytochromes interact with multiple Taken together, our data support a model in which WRKY36 acts proteins to modulate phytochrome function and regulation, such as as a transcription repressor of HY5, UVB light induces the nuclear several bHLH (basic helix loop helix) TFs (PIF proteins), a nucleo- localization of UVR8, and nuclear-localized UVR8 interacts with side diphosphate kinase (NDPK2, NUCLEOSIDE DIPHOSPHATE WRKY36 to prevent it from binding DNA. Thereafter, HY5 and KINASE 2), protein phosphotases (PAPPs, PHYTOCHROME HYH promote the transcription of HY5 (Fig. 6i). ASSOCIATED PROTEIN PHOSPHATASE), a response regulator (ARR4, ARABIDOPSIS RESPONSE REGULATOR 4) and PKS1 30,39–45 Methods (PHYTOCHROME KINASE SUBSTRATE 1) . Blue light pho- Plant materials and growth conditions. Te Columbia ecotype of A. thaliana toreceptor cryptochromes interact with the TFs CIB1 and PIF4, was used. Transfer DNA insertion mutants uvr8-61) (SALK_033468), wrky36-1 COP1, blue-light inhibitor of cryptochromes 1 (BIC1) and pho- (SALK_204285), wrky36-2 (CS108822), hy5 (SALK_096651) and wrky72 toregulatory protein kinases (PPKs) to regulate light responses (SALK_145765) were obtained from the Arabidopsis Biological Resource Center. 25–29,46,47 Te uvr8/wrky36, hy5/wrky36 and wrky36/wrky72 double mutants were prepared and transcription . Only a few proteins have been reported by genetic crossing, and their identities were verifed by genotyping. Te full-length to physically interact with UVR8 to mediate UVB signal trans- UVR8 and WRKY36 coding sequences were cloned into pEarly-104 (Arabidopsis duction. COP1 interacts with UVR8 to mediate UVB-induced Biological Resource Center) or pCambia1300 (Cambia), bearing either GFP nuclear accumulation of UVR8 and UVR8-mediated UVB sig- (Pro35S::GFP–UVR8) or Myc–His–Flag (Pro35S::WRKY36–TAP). Col-0 was nalling10,11. The WD40-repeat proteins REPRESSOR OF UVB transformed with Pro35S::GFP–UVR8 and Pro35S:WRKY36–TAP. WTs were transformed and for every transformation, more than ten independent transgenic PHOTOMOPHOGENESIS (RUP1) and RUP2 directly interact lines with a single copy of the transgene were generated. Phenotypes of transgenic with UVR8 to mediate UVR8 redimerization, thereby disrupt- plants were verifed in at least three independent transgenic lines. qPCRs were ing the UVR8–COP1 interaction12,13. Whether UVR8 physically performed to verify overexpression of the transgenes. WRKY36–TAP/uvr8, interacts with TFs to regulate transcription and UVB responses WRKY36–TAP/hy5, WRKY36–TAP/GFP–UVR8 and WRKY36–TAP/GR–UVR8 was not known. Here, we identify and characterize the UVR8- were prepared by genetic crossing. Seeds were sterilized in 10% bleach, placed on 1/2 Murshige and Skoog interacting protein WRKY36. WRKY36 interacts with UVR8 in a medium containing 0.8% agar and 1% sucrose, and stratified for 4 days at 4 °C UVB-independent manner, it interacts with both monomeric and in the dark before being transferred to white light (Philips TLD18W/54-765; dimeric UVR8. Our results show that UVR8 represses the binding 6 μmol m−2 s−1; measured using an ILT1400 Radiometer Photometer) or white

Nature Plants | VOL 4 | FEBRUARY 2018 | 98–107 | www.nature.com/natureplants 105 © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. Articles NATURE PLAnTS light plus UVB (Philips TL20W/01RS narrowband UVB tubes; 2 W m–2; measured Tag (Takara) was used for the qPCR reaction on the MX3000 System (Stratagene). using a LUYOR-340 UV Light Meter) modulated by 300 nm transmission cutoff The level of ACTIN7 messenger RNA expression (AT5G09810) was used as the filters (ZJB300; Yongxing Information Sensing Technology) or 340 nm cutoff filters internal control. Quantitative reverse transcription PCR data for each sample were (ZJB340; Yongxing Information Sensing Technology). normalized to the respective ACT7 expression level. The complementary DNAs were amplified following denaturation using 40-cycle programmes (95 °C for 5 s Yeast-two hybrid and yeast-one hybrid assays. The coding sequences of UVR8 and 60 °C for 20 s per cycle). Biological replicates represented three independent were fused in-frame with the GAL4 DNA binding domain of the bait vector experiments involving about 30 seedlings per experiment. Three technical pDest32 (Clontech) and transformed into Y187. The library of 1362 TFs (in vector replicates were performed for each experiment. 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), Electrophoretic mobility-shift assays. Two methods were used to perform suspended in synthetic defined–Trp–Leu medium and incubated overnight. The electrophoretic mobility-shift assays. One was as described25,27,28. His–UVR8 and interactions were tested by galactosidase assays. His–WRKY36 fusion proteins were expressed and purified from E. coil (BL21). HY5 promoter fragments (−733 bp to −501 bp and −500 bp to −1bp) were The double-stranded DNA (Supplementary Table 1) was labelled with digoxigenin cloned into the pLACZi (LacZi reporter) destination vector and transformed by terminal transferase according to the manufacturer’s instructions (DIG Gel Shift into YM4271 yeast strains. WRKY36 was cloned into the pDest22 vector and Kit; Roche). Then, 100 ng of total protein was added in each binding reaction. transformed into EGY48 yeast strains. YM4271 yeast strains were mated with In the other method, for the probe, the synthetic complementary EGY48 yeast strains (in 6 h yeast extract peptone dextrose media), suspended in oligonucleotides of the HY5 promoter were annealed and cloned to a T-vector. The synthetic defined–Trp–Ura medium and incubated overnight 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). Bimolecular luminescence complementation assay. UVR8 or WRKY36 was BiFC, co-localization ChIP and accession numbers have been reported fused to the C or N terminus of firefly luciferase and transformed to Agrobacterium previously28 and are listed in the Supplementary Methods. 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 Life Sciences Reporting Summary. Further information on experimental design is luciferin and 0.01% Triton X-100). Images were captured using a charge-coupled available in the Life Sciences Reporting Summary. device camera 5 min later. Data availability. The data that support the findings of this study are available In vitro pull-down. The in vitro pull-down protein–protein interaction assay from the corresponding author upon request. 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 Received: 1 August 2017; Accepted: 27 December 2017; (amino acid 191–388 ) were cloned into pCold TF to generate His–TF–WRKY36, Published online: 29 January 2018 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)– References UVR8N and GST–UVR8C. These proteins were expressed and purified from 1. Favory, J. J. et al. Interaction of COP1 and UVR8 regulates UV-B-induced E. coli BL21. UVR8–His was incubated with the His–TF–WRKY36 under white photomorphogenesis and stress acclimation in Arabidopsis. EMBO J. 28, light (Philips TLD18W/54-765; 6 mol m−2 s−1; measured using an ILT1400 μ 591–601 (2009). Radiometer Photometer) or white light plus UVB (Philips TL20W/01RS 2. Jenkins, G. I. Signal transduction in responses to UV-B radiation. Annu. Rev. narrowband UVB tubes; 2 W m−2; measured using a LUYOR-340 UV Light Meter) Plant Biol. 60, 407–431 (2009). for 30 min. Anti-UVR8 antibodies were used to pull down the protein complexes, 3. Rizzini, L. et al. Perception of UV-B by the Arabidopsis UVR8 protein. Science and unbound proteins were removed via washing. The bound proteins were 332, 103–106 (2011). eluted and analysed using an immunoblot probed with anti-His antibody (MBL; 4. Jenkins, G. I. Structure and function of the UV-B photoreceptor UVR8. #M136-3). Samples were boiled before sodium dodecyl sulfate polyacrylamide gel Curr. Opin. Struct. Biol. 29, 52–57 (2014). electrophoresis (UVR8 dimers turned to be monomers after heat denaturation). 5. Tilbrook, K. et al. Te UVR8 UV-B photoreceptor: perception, signaling and GST–UVR8N or GST–UVR8C was incubated with the His–TF–WRKY36N or response. Arab. Book 11, e0164 (2013). His–TF–WRKY36C and GST beads were used to pull down the protein complexes. 6. Kaiserli, E. & Jenkins, G. I. UV-B promotes rapid nuclear translocation of the The proteins were eluted and analysed using an immunoblot probed with anti-His Arabidopsis UV-B specifc signaling component UVR8 and activates its or anti-GST antibody (Abmart; #M20007). UVR8 antibody is a polyclonal antibody function in the nucleus. Plant Cell. 19, 2662–2673 (2007). made by Youke using the reported peptide1. 7. Yi, C. & Deng, X. W. COP1—from plant photomorphogenesis to mammalian tumorigenesis. Trends Cell. Biol. 15, 618–625 (2005). Nuclear fractionation. Nuclear fractionation was performed as described 8. Huang, X., Yang, P., Ouyang, X., Chen, L. & Deng, X. W. Photoactivated 10,26 previously with modifications. Fourteen-day-old seedlings were collected, UVR8–COP1 module determines photomorphogenic UV-B signaling output ground in liquid nitrogen, homogenized in extraction buffer (20 mM Tris/HCl, in Arabidopsis. PLoS Genet. 10, e1004218 (2014). pH 7.4, 25% (vol/vol) glycerol, 20 mM KCl, 2 mM EDTA, 2.5 mM MgCl2, 250 mM 9. Cloix, C. et al. C-terminal region of the UV-B photoreceptor UVR8 initiates sucrose, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride and 1× complete signaling through interaction with the COP1 protein. Proc. Natl Acad. Sci. protease inhibitor cocktail (Roche)). Total protein extracts were filtered through USA 109, 16366–16370 (2012). three layers of Miracloth. After centrifugation at 1,500g for 10 min at 4 °C, the 10. Yin, R., Skvortsova, M. Y., Loubery, S. & Ulm, R. COP1 is required for pellet was washed twice with nuclei resuspension Triton buffer (20 mM Tris/HCl, UV-B-induced nuclear accumulation of the UVR8 photoreceptor. Proc. Natl pH 7.4, 25% glycerol, 2.5 mM MgCl2 and 0.2% Triton X-100) and then was used in Acad. Sci. USA 113, E4415–E4422 (2016). co-immunoprecipitation. 11. Qian, C. et al. Dual-source nuclear monomers of UV-B light receptor direct photomorphogenesis in Arabidopsis. Mol. Plant 9, 1671–1674 (2016). Co-immunoprecipitation. The co-immunoprecipitation procedure has been 12. Gruber, H. et al. Negative feedback regulation of UV-B-induced described previously25,27,28. Briefly, 14-day-old GFP–UVR8 and WRKY36–TAP/ photomorphogenesis and stress acclimation in Arabidopsis. Proc. Natl Acad. GFP–UVR8 seedlings were grown in long-day conditions, moved to continuous Sci. USA 107, 20132–20137 (2010). white light for 1 day before treatment with or without UVB (2 W m–2) for 20 min, 13. Heijde, M. & Ulm, R. Reversion of the Arabidopsis UV-B photoreceptor then ground in liquid nitrogen, homogenized in binding buffer (20 mM Hepes pH UVR8 to the homodimeric ground state. Proc. Natl Acad. Sci. USA 110, 7.5, 40 mM KCl, 1 mM EDTA, 1% Triton X-100 and 1 mM phenylmethylsulfonyl 1113–1118 (2013). fluoride) and incubated at 4 °C for 5 min. They then went through a 1 ml syringe 14. Jiao, Y., Lau, O. S. & Deng, X. W. Light-regulated transcriptional networks in twice (with a metal needle) to promote nucleus lysis and were centrifuged at higher plants. Nat. Rev. Genet. 8, 217–230 (2007). 14,000 g for 10 min. The supernatant was mixed with 35 μl of anti-c-Myc Affinity 15. Ulm, R. et al. Genome-wide analysis of gene expression reveals function of Gel (Sigma–Aldrich; #E6654), incubated at 4 °C for 30 min and washed twice with the bZIP transcription factor HY5 in the UV-B response of Arabidopsis. washing buffer (20 mM Hepes pH 7.5, 40 mM KCl, 1 mM EDTA and 0.1% Triton Proc. Natl Acad. Sci. USA 101, 1397–1402 (2004). X-100). The bound proteins were eluted from the affinity beads with 4× sodium 16. Brown, B. A. et al. A UV-B-specifc signaling component orchestrates plant dodecyl sulfate polyacrylamide gel electrophoresis sample buffer and analysed by UV protection. Proc. Natl Acad. Sci. USA 102, 18225–18230 (2005). immunoblot using anti-Myc (Millipore; #05-724), GFP (Abicode; #M0802-3a) or 17. Oravecz, A. et al. CONSTITUTIVELY PHOTOMORPHOGENIC1 is required H3 (Sigma–Aldrich; #9289) antibodies. for the UV-B response in Arabidopsis. Plant Cell. 18, 1975–1990 (2006). 18. Brown, B. A. & Jenkins, G. I. UV-B signaling pathways with diferent Messenger RNA expression analyses. Total RNAs were isolated using the RNAiso fuence-rate response profles are distinguished in mature Arabidopsis leaf Plus (Takara). Complementary DNA was synthesized from 500 ng of total RNA tissue by requirement for UVR8, HY5, and HYH. Plant Physiol. 146, using a PrimeScript RT Reagent Kit with gDNA Eraser (Takara). SYBR Premix Ex 576–588 (2008).

106 Nature Plants | VOL 4 | FEBRUARY 2018 | 98–107 | www.nature.com/natureplants © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. NATURE PLAnTS Articles

19. Stracke, R. et al. Te Arabidopsis bZIP transcription factor HY5 regulates 39. Sweere, U. et al. Interaction of the response regulator ARR4 with expression of the PFG1/MYB12 gene in response to light and ultraviolet-B phytochrome B in modulating red light signaling. Science 294, radiation. Plant Cell. Environ. 33, 88–103 (2010). 1108–1111 (2001). 20. Feher, B. et al. Functional interaction of the circadian clock and UV 40. Huq, E. et al. PHYTOCHROME-INTERACTING FACTOR 1 is a critical RESISTANCE LOCUS 8-controlled UV-B signaling pathways in Arabidopsis bHLH regulator of chlorophyll biosynthesis. Science 305, 1937–1941 (2004). thaliana. Plant J. 67, 37–48 (2011). 41. Quail, P. H. Photosensory perception and signalling in plant cells: new 21. Huang, X. et al. Arabidopsis FHY3 and HY5 positively mediate induction of paradigms? Curr. Opin. Cell. Biol. 14, 180–188 (2002). COP1 transcription in response to photomorphogenic UV-B light. Plant Cell. 42. Al-Sady, B., Kikis, E. A., Monte, E. & Quail, P. H. Mechanistic duality of 24, 4590–4606 (2012). transcription factor function in phytochrome signaling. Proc. Natl Acad. Sci. 22. Brown, B. A., Headland, L. R. & Jenkins, G. I. UV-B action spectrum for USA 105, 2232–2237 (2008). UVR8-mediated HY5 transcript accumulation in Arabidopsis. Photochem. 43. Choi, G. et al. Phytochrome signalling is mediated through nucleoside Photobiol. 85, 1147–1155 (2009). diphosphate kinase 2. Nature 401, 610–613 (1999). 23. Osterlund, M. T., Hardtke, C. S., Wei, N. & Deng, X. W. Targeted 44. Ryu, J. S. et al. Phytochrome-specifc type 5 phosphatase controls light signal destabilization of HY5 during light-regulated development of Arabidopsis. fux by enhancing phytochrome stability and afnity for a signal transducer. Nature 405, 462–466 (2000). Cell 120, 395–406 (2005). 24. Binkert, M. et al. UV-B-responsive association of the Arabidopsis bZIP 45. Fankhauser, C. & Chory, J. Light receptor kinases in plants! Curr. Biol. 9, transcription factor ELONGATED HYPOCOTYL5 with target genes, R123–R126 (1999). including its own promoter. Plant Cell. 26, 4200–4213 (2014). 46. Wang, Q. et al. Photoactivation and inactivation of Arabidopsis cryptochrome 25. Liu, H. et al. Photoexcited CRY2 interacts with CIB1 to regulate transcription 2. Science 354, 343–347 (2016). and foral initiation in Arabidopsis. Science 322, 1535–1539 (2008). 47. Liu, Q. et al. Molecular basis for blue light-dependent phosphorylation of 26. Liu, H. et al. Arabidopsis CRY2 and ZTL mediate blue-light regulation of the Arabidopsis cryptochrome 2. Nat. Commun. 8, 15234 (2017). transcription factor CIB1 by distinct mechanisms. Proc. Natl Acad. Sci. USA 48. Park, E. et al. Phytochrome B inhibits binding of phytochrome-interacting 110, 17582–17587 (2013). factors to their target promoters. Plant J. 72, 537–546 (2012). 27. Liu, Y., Li, X., Li, K., Liu, H. & Lin, C. Multiple bHLH proteins form 49. Chen, F. et al. Arabidopsis phytochrome A directly targets numerous heterodimers to mediate CRY2-dependent regulation of fowering-time in promoters for individualized modulation of genes in a wide range of Arabidopsis. PLoS Genet. 9, e1003861 (2013). pathways. Plant Cell. 26, 1949–1966 (2014). 28. Ma, D. et al. Cryptochrome 1 interacts with PIF4 to regulate high temperature-mediated hypocotyl elongation in response to blue light. Proc. Natl Acad. Sci. USA 113, 224–229 (2016). Acknowledgements 29. Pedmale, U. V. et al. Cryptochromes interact directly with PIFs to control We thank X. Huang, Y. Shi, R. P. Hellens and Q. Hu for materials and technical assistance. plant growth in limiting blue light. Cell 164, 233–245 (2016). We thank G. A. Gomez for copyediting the manuscript. This work is supported in part 30. Ni, M., Tepperman, J. M. & Quail, P. H. PIF3, a phytochrome-interacting by the National Key Research and Development Program of China (2017YFA 0503800), factor necessary for normal photoinduced signal transduction, is a novel National Natural Science Foundation of China (31730009, 31721001, 31670282 and basic helix-loop-helix protein. Cell 95, 657–667 (1998). 31670307) and Strategic Priority Research Program ‘Molecular Mechanism of Plant 31. Leivar, P. & Quail, P. H. PIFs: pivotal components in a cellular signaling hub. Growth and Development’ (XDPB04). Trends Plant Sci. 16, 19–28 (2011). 32. Castrillo, G. et al. Speeding cis–trans regulation discovery by phylogenomic Author contributions analyses coupled with screenings of an arrayed library of Arabidopsis Y.Y. and H.L. conceived the project. Y.Y. performed most of the experiments. L.Z., transcription factors. PLoS ONE 6, e21524 (2011). X.G., R.S. and N.S. made some constructs. T.L., X.L. and P.Z. provided materials. K.S. 33. Eulgem, T., Rushton, P. J., Robatzek, S. & Somssich, I. E. Te WRKY performed some protein expression in E. coli. Y.Y. and H.L. analysed the data and wrote superfamily of plant transcription factors. Trends Plant Sci. 5, 199–206 (2000). the manuscript. 34. Riechmann, J. L. et al. Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science 290, 2105–2110 (2000). 35. Czechowski, T., Bari, R. P., Stitt, M., Scheible, W. R. & Udvardi, M. K. Competing interests Real-time RT-PCR profling of over 1400 Arabidopsis transcription factors: The authors declare no competing financial interests. unprecedented sensitivity reveals novel root- and shoot-specifc genes. Plant J. Cell. Mol. Biol. 38, 366–379 (2004). 36. Wu, D. et al. Structural basis of ultraviolet-B perception by UVR8. Nature Additional information 484, 214–219 (2012). Supplementary information accompanies this paper at https://doi.org/10.1038/s41477- 37. Yin, R., Arongaus, A. B., Binkert, M. & Ulm, R. Two distinct domains of the 017-0099-0. UVR8 photoreceptor interact with COP1 to initiate UV-B signaling in Reprints and permissions information is available at www.nature.com/reprints. Arabidopsis. Plant Cell. 27, 202–213 (2015). 38. Kreuzaler, F., Ragg, H., Fautz, E., Kuhn, D. N. & Hahlbrock, K. UV-induction Correspondence and requests for materials should be addressed to H.L. of chalcone synthase mRNA in cell suspension cultures of Petroselinum Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in hortense. Proc. Natl Acad. Sci. USA 80, 2591–2593 (1983). published maps and institutional affiliations.

Nature Plants | VOL 4 | FEBRUARY 2018 | 98–107 | www.nature.com/natureplants 107 © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. nature research | life sciences reporting summary

Corresponding author(s): Hongtao Liu Initial submission Revised version Final submission Life Sciences Reporting Summary Nature Research wishes to improve the reproducibility of the work that we publish. This form is intended for publication with all accepted life science papers and provides structure for consistency and transparency in reporting. Every life science submission will use this form; some list items might not apply to an individual manuscript, but all fields must be completed for clarity. For further information on the points included in this form, see Reporting Life Sciences Research. For further information on Nature Research policies, including our data availability policy, see Authors & Referees and the Editorial Policy Checklist.

` Experimental design 1. Sample size Describe how sample size was determined. For mRNA expression, CO-IP, ChIP, each sample is a group of arabidopsis seedlings (about 0.5 g to 2 g). For dual-LUC assay, one sample is a piece of tobacco leaf (got with a hole punch) about 0.01g or a group of protoplasts (around 40000 protoplasts) 2. Data exclusions Describe any data exclusions. No data was excluded from the analysis. 3. Replication Describe whether the experimental findings were Each experiment was performed at least three times with similar results. reliably reproduced. 4. Randomization Describe how samples/organisms/participants were Seedlings were allocated to groups randomly. allocated into experimental groups. 5. Blinding Describe whether the investigators were blinded to Describe the extent of blinding used during data acquisition and analysis. If blinding group allocation during data collection and/or analysis. was not possible, describe why OR explain why blinding was not relevant to your study. Note: all studies involving animals and/or human research participants must disclose whether blinding and randomization were used.

6. Statistical parameters For all figures and tables that use statistical methods, confirm that the following items are present in relevant figure legends (or in the Methods section if additional space is needed). n/a Confirmed

The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement (animals, litters, cultures, etc.) A description of how samples were collected, noting whether measurements were taken from distinct samples or whether the same sample was measured repeatedly A statement indicating how many times each experiment was replicated The statistical test(s) used and whether they are one- or two-sided (note: only common tests should be described solely by name; more complex techniques should be described in the Methods section) A description of any assumptions or corrections, such as an adjustment for multiple comparisons

The test results (e.g. P values) given as exact values whenever possible and with confidence intervals noted June 2017 A clear description of statistics including central tendency (e.g. median, mean) and variation (e.g. standard deviation, interquartile range) Clearly defined error bars

See the web collection on statistics for biologists for further resources and guidance.

1 ` Software nature research | life sciences reporting summary Policy information about availability of computer code 7. Software Describe the software used to analyze the data in this excel study.

For manuscripts utilizing custom algorithms or software that are central to the paper but not yet described in the published literature, software must be made available to editors and reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). Nature Methods guidance for providing algorithms and software for publication provides further information on this topic.

` Materials and reagents Policy information about availability of materials 8. Materials availability Indicate whether there are restrictions on availability of N0 unique materials or if these materials are only available for distribution by a for-profit company. 9. Antibodies Describe the antibodies used and how they were validated anti Myc (Millipore, #05-724) and GFP (Abicode, #M0802-3a) antibodies were for use in the system under study (i.e. assay and species). validated by using Wild type plant which has not those tagged proteins expressing as a control. H3 (Sigma-Aldrich, #9289) antibody were verified by using cytoplasm protein as a control. anti-His antibody (MBL, #M136-3), anti-GST antibody (Abmart, #M20007) were validated using E.coli total proteins without His tagged or GST tagged protein expressing. UVR8 antibody was a polyclonal antibody made by Youke Company (Shanghai, China) using the reported peptide. UVR8 antibody was validated using total proteins extracted from uvr8 mutant as a control. 10. Eukaryotic cell lines a. State the source of each eukaryotic cell line used. Provide information on cell line source(s) OR state that no eukaryotic cell lines were used.

b. Describe the method of cell line authentication used. Describe the authentication procedures for each cell line used OR declare that none of the cell lines used have been authenticated OR state that no eukaryotic cell lines were used.

c. Report whether the cell lines were tested for Confirm that all cell lines tested negative for mycoplasma contamination OR mycoplasma contamination. describe the results of the testing for mycoplasma contamination OR declare that the cell lines were not tested for mycoplasma contamination OR state that no eukaryotic cell lines were used.

d. If any of the cell lines used are listed in the database Provide a rationale for the use of commonly misidentified cell lines OR state that no of commonly misidentified cell lines maintained by commonly misidentified cell lines were used. ICLAC, provide a scientific rationale for their use.

` Animals and human research participants Policy information about studies involving animals; when reporting animal research, follow the ARRIVE guidelines 11. Description of research animals Provide details on animals and/or animal-derived For laboratory animals, report species, strain, sex and age OR for animals observed materials used in the study. in or captured from the field, report species, sex and age where possible OR state that no animals were used.

Policy information about studies involving human research participants 12. Description of human research participants June 2017 Describe the covariate-relevant population Provide all relevant information on human research participants, such as age, characteristics of the human research participants. gender, genotypic information, past and current diagnosis and treatment categories, etc. OR state that the study did not involve human research participants.