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. 2022 Feb 21;34(5):2038–2055. doi: 10.1093/plcell/koac064

Activation and negative feedback regulation of SlHY5 transcription by the SlBBX20/21–SlHY5 transcription factor module in UV-B signaling

Guoqian Yang 1, Chunli Zhang 2,3,4, Huaxi Dong 5, Xiaorui Liu 6, Huicong Guo 7, Boqin Tong 8, Fang Fang 9, Yiyang Zhao 10, Yunji Yu 11, Yue Liu 12, Li Lin 13,14,15, Ruohe Yin 16,17,18,✉,†
PMCID: PMC9048894  PMID: 35188198

Abstract

In tomato (Solanum lycopersicum) and other plants, the photoreceptor UV-RESISTANCE LOCUS 8 regulates plant UV-B photomorphogenesis by modulating the transcription of many genes, the majority of which depends on the transcription factor ELONGATED HYPOCOTYL 5 (HY5). HY5 transcription is induced and then rapidly attenuated by UV-B. However, neither the transcription factors that activate HY5 transcription nor the mechanism for its attenuation during UV-B signaling is known. Here, we report that the tomato B-BOX (BBX) transcription factors SlBBX20 and SlBBX21 interact with SlHY5 and bind to the SlHY5 promoter to activate its transcription. UV-B-induced SlHY5 expression and SlHY5-controlled UV-B responses are normal in slbbx20 and slbbx21 single mutants, but strongly compromised in the slbbx20 slbbx21 double mutant. Surprisingly, UV-B responses are also compromised in lines overexpressing SlBBX20 or SlBBX21. Both SlHY5 and SlBBX20 bind to G-box1 in the SlHY5 promoter. SlHY5 outcompetes SlBBX20 for binding to the SlHY5 promoter in vitro, and inhibits the association of SlBBX20 with the SlHY5 promoter in vivo. Overexpressing 35S:SlHY5-FLAG in the WT background inhibits UV-B-induced endogenous SlHY5 expression. Together, our results reveal the critical role of the SlBBX20/21-SlHY5 module in activating the expression of SlHY5, the gene product of which inhibits its own gene transcription under UV-B, forming an autoregulatory negative feedback loop that balances SlHY5 transcription in plants.

Tomato transcription factor module BBX20/21-HY5 activates HY5 transcription, and HY5 protein inhibits its own gene transcription, forming an autoregulatory negative feedback loop in UV-B signaling.

Introduction

Light is a key environmental factor affecting plant growth and development. Plants respond to light signals via multiple photoreceptor-controlled signaling pathways. The phytochrome photoreceptors are primarily responsible for red and far-red light signaling, cryptochrome and phototropin photoreceptors are responsible for blue light signaling, and UV-RESISTANCE LOCUS 8 (UVR8) is responsible for UV-B (UV-B; 280–315 nm) signaling. UVR8 regulates multiple aspects of plant growth and development in response to UV-B, including seedling de-etiolation, leaf and root development, stomatal density and aperture, phototropism, and flowering time (Jenkins, 2009; Liang et al., 2019; Podolec et al., 2021). In addition, the UVR8 pathway promotes acclimatory responses and plant tolerance to UV-B (Kliebenstein et al., 2002; Brown et al., 2005; Favory et al., 2009; Rai et al., 2019; Liang et al., 2020). UVR8 regulates UV-B photomorphogenesis and survival through the transcriptional regulation of a broad set of genes (Jenkins, 2017; Yin and Ulm, 2017).

UVR8 homodimers perceive UV-B photons, which leads to the dissociation of UVR8 homodimers into monomers to initiate the light-mediated developmental program known as photomorphogenesis (Rizzini et al., 2011; Christie et al., 2012; Wu et al., 2012). The WD40 proteins REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2 are important negative regulators of UVR8-mediated UV-B signaling (Gruber et al., 2010). UV-B-activated UVR8 monomers can reassociate to form inactive dimers, which is accelerated by RUP1 and RUP2 (Heijde and Ulm, 2013; Heilmann and Jenkins, 2013). UV-B induces RUP1 and RUP2 transcription via the UVR8 pathway, thus forming a negative feedback loop that coordinates the activity of UVR8 (Gruber et al., 2010).

Although it is well established that UVR8 mediates massive transcriptional changes in response to UV-B, the relevant mechanisms are poorly understood. UVR8 is distributed in both the cytoplasm and the nucleus in seedlings grown in white light (Kaiserli and Jenkins, 2007). UV-B promotes the nuclear translocation of UVR8 and its accumulation in the nucleus (Kaiserli and Jenkins, 2007), which requires the E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) (Qian et al., 2016; Yin et al., 2016). Both UV-B-induced gene transcriptional changes and UV-B photomorphogenesis are compromised in cop1 mutants (Oravecz et al., 2006; Favory et al., 2009). Several proteins have been identified as contributing to the transcriptional regulation downstream of UVR8, including the transcription factor ELONGATED HYPOCOTYL 5 (HY5), which plays major roles (Ulm et al., 2004; Brown et al., 2005; Favory et al., 2009). HY5 together with its close homolog HY5 HOMOLOG (HYH) mediate the transcriptional regulation of most UV-B target genes (Brown and Jenkins, 2008; Stracke et al., 2010). HY5 was shown to bind to cis-regulatory sequences including the G-box in the promoters of its target genes (Lee et al., 2007). HY5 lacks a transactivation domain (AD) and requires cofactors to regulate gene transcription (Oyama et al., 1997). Several HY5 cofactors have been identified, including PICKLE, CALMODULIN7 (CAM7), BBX20/21/22, the COLD-REGULATED (COR) proteins COR27 and COR28, and the SWR1 complex core subunitC6 (SWC6) and ACTIN-RELATED PROTEIN 6 (ARP6; Jing et al., 2013; Abbas et al., 2014; Xu et al., 2016; Bursch et al., 2020; Li et al., 2020; Zhu et al., 2020; Mao et al., 2021). All these proteins physically interact with HY5 to regulate transcription in visible light. Whether these HY5 cofactors regulate gene transcription under UV-B is unknown.

In addition to the HY5/HYH-dependent pathway, UVR8 can regulate transcription by directly interacting with transcription factors including BRI1-EMS-SUPPRESSOR 1 (BES1), BES1-INTERACTING MYC-LIKE1, WRKY DNA-BINDING PROTEIN 36 (WRKY36), MYB73, MYB77, and MYB13 to regulate gene expression (Liang et al., 2018; Yang et al., 2018, 2019; Qian et al., 2020). UVR8 was shown to interact with the de novo DNA methyltransferase DOMAINS REARRANGED METHYLTRANSFERASE 2, inhibiting its activity and promoting transcriptional de-repression (Jiang et al., 2021). Moreover, chromatin immunoprecipitation (ChIP) experiments provided evidence that UVR8 binds to the genomic regions of some but not all UVR8 target genes, possibly via binding to histone H2B (Brown et al., 2005; Cloix and Jenkins, 2008). The association between UVR8 and chromatin has been debated and its functional significance in UV-B signaling remains to be fully investigated (Binkert et al., 2016; Jenkins, 2017).

HY5 is extensively investigated in photomorphogenesis. HY5 mutants show defects in the inhibition of hypocotyl elongation in all light conditions, indicating that HY5 acts downstream of multiple photoreceptors to promote photomorphogenesis (Oyama et al., 1997; Ulm et al., 2004). As the key transcription factor in the UVR8 pathway, HY5 accumulation is tightly regulated at both transcriptional and posttranscriptional levels. The cop1 mutants exhibit light grown phenotypes including open cotyledons and short hypocotyls in the dark (Deng et al.,1992). Later research revealed that COP1 targets HY5 for degradation in the dark (Osterlund et al., 2000). COP1 is a critical negative regulator of red light, far-red light, and blue light signaling pathways (Lau and Deng 2012). UV-B induces the interaction of UVR8 with COP1, which leads to COP1 inactivation and HY5 stabilization (Favory et al., 2009; Huang et al., 2013; Yin et al., 2015; Lau et al., 2019; Lin et al., 2020). HY5 binds to its own promoter (Abbas et al., 2014), a binding that is further enhanced by UV-B (Binkert et al., 2014). HY5 is necessary but not sufficient for the activation of its own transcription under both white light and UV-B (Abbas et al., 2014; Binkert et al., 2014; Burko et al., 2020). The transcription factor that acts together with HY5 to promote HY5 transcription in the UV-B pathway is currently unknown. Moreover, transcription of HY5 is induced and then rapidly attenuated by UV-B in plants (Ulm et al., 2004; Brown et al., 2005; Favory et al., 2009), suggesting the existence of negative regulations on UV-B-induced HY5 transcription.

Tomato (Solanum lycopersicum) is widely used for photomorphogenic research (Kendrick et al., 1997; Liu et al., 2004; Jones et al., 2012; Llorente et al., 2016; Fantini 2019; Guo et al., 2021). Early studies have uncovered multiple key mutants in photomorphogenesis (Kendrick et al., 1997; Jones et al., 2012). Tomato cryptochrome 1a (CRY1a) and CRY2 regulate multiple agronomic traits such as flowering time and fruit quality (Fantini et al., 2019). Tomato phytochrome B promotes iron uptake dependent on transcription factor HY5 (SlHY5) in the light (Guo et al., 2021). Our previous work characterized several key UV-B signaling components including tomato UVR8 (SlUVR8) and SlRUP (Liu et al., 2020; Zhang et al., 2021). In this work, we provide evidence that SlBBX20 and SlBBX21 interact with SlHY5 and act together with SlHY5 to directly activate SlHY5 transcription under UV-B. In turn, the resulting accumulating SlHY5 outcompetes SlBBX20 and SlBBX21 for binding to G-box1 in the SlHY5 promoter to attenuate HY5 transcription, forming a negative feedback loop. Consistent with this, UV-B-induced SlHY5 expression is compromised in lines overexpressing SlBBX20, SlBBX21, or SlHY5, which have high basal levels of SlHY5 prior to UV-B radiation.

Results

SlHY5 transcription is transiently induced by UV-B

We showed previously that UV-B rapidly induces SlHY5 transcription through SlUVR8 (Liu et al., 2020). To test the dynamic expression of SlHY5 under UV-B, we exposed tomato seedlings to UV-B for 1–24 h. SlHY5 transcript levels were induced by UV-B, peaking after 1 h and then declined rapidly in wild-type (WT) seedlings (Figure 1). SlHY5 transcript levels were not induced by UV-B in the sluvr8 mutant (Figure 1). This observation suggests that SlHY5 transcription is negatively regulated shortly after its induction by UV-B through the SlUVR8 pathway.

Figure 1.

Figure 1

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Transcript of SlHY5 is transiently induced by UV-B in tomato. RT-qPCR analysis of SlHY5 expression. WT, slrup, and sluvr8 tomato seedlings were grown in white light for 4 days and then treated with white light supplemented with UV-B for the indicated periods. Data were normalized against samples from seedlings grown under white light at time zero for the respective genotype. The SlACTIN gene serves as an internal control. Means and sd (Standard Deviation) from three biological replicates are shown (n = 3). Asterisks indicate significant differences (*P < 0.05; **P < 0.01; Student’s t test).

Similar to its Arabidopsis counterparts, SlRUP is a major negative regulator of UV-B signaling by promoting the reassociation of active SlUVR8 monomers to form inactive homodimers (Zhang et al., 2021). We, therefore, tested whether SlRUP contributed to the dynamic expression of SlHY5 under UV-B. Overall, UV-B-induced SlHY5 transcription was higher in the slrup mutant than in WT (Figure 1). Notably, the attenuation of UV-B-induced SlHY5 transcription was delayed by 1–3 h in the slrup mutant relative to WT (Figure 1). We concluded that SlRUP participates in, but is not sufficient for the negative regulation of SlHY5 transcription in response to UV-B. Unknown factors are involved in the negative regulation of UV-B-induced SlHY5 transcription, particularly after prolonged UV-B treatment.

SlBBX20 and SlBBX21 interact with SlHY5

Without an AD, SlHY5 needs cofactors to promote target gene expression, including its own. To identify such cofactors, we performed a yeast two-hybrid (Y2H) screen with a library derived from tomato seedlings, with SlHY5 as bait. We isolated multiple clones including a gene encoding zinc finger transcription factor SlBBX20 during this screen (Supplemental Table S1). The tomato genome encodes at least 29 members of the BBX gene family (Chu et al., 2016). Phylogenetic analysis revealed that SlBBX21 is a homolog of SlBBX20 within subfamily IV (Chu et al., 2016), with 54% sequence identity and 68% sequence similarity (Emboss Water version 6.6.0). Both SlBBX20 and SlBBX21 contain two B-box domains (Figure 2A).

Figure 2.

Figure 2

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SlBBX20 interacts with SlHY5 in vitro and in vivo. A, Schematic diagram of various constructs of SlBBX20, SlBBX21, and SlHY5 used in (B). Numbers indicate the amino acid positions in the respective sequences of SlBBX20, SlBBX21, and SlHY5. The conserved BBOX domain and bZIP domain (black boxes) are shown. B, Y2H assays for the interactions between SlBBX20/SlBBX21 and SlHY5. Yeast growth on SD/–Trp, –Leu, –His solid medium is shown. EV designates EV. BD designates DNA-binding domain vector. See also Supplemental Figure S2, A and B. C and D, Co-IP assays showing the interaction of SlBBX20/21 with SlHY5 in N. benthamiana leaves. Anti-HA and anti-FLAG antibodies were used for immunoprecipitation in (C) and (D), respectively. E and F, BiFC assay showing the interaction of SlBBX20/21 with SlHY5 in N. benthamiana leaves. SlHY5N75-YFPc and SlBBX20△N100-YFPn or SlBBX21△N100-YFPn were used as negative controls. DAPI: DAPI staining was used to indicate the nucleus. Merge: merged images of YFP channel, DAPI staining, and bright field. Images were taken using a confocal microscopy. Bar = 25 µm for (E) and (F).

We first determined whether the transcript levels of SlBBX20 and SlBBX21 and those of the encoded proteins were regulated by UV-B. SlBBX21 transcript levels did not change in response to UV-B, while SlBBX20 transcripts accumulated only after long periods of UV-B exposure of at least 12 h, and in a SlUVR8- and SlHY5-dependent manner (Supplemental Figure 1, A and B). Whereas UV-B does not alter SlBBX20 protein levels, it promotes SlBBX21 accumulation in the respective overexpression lines (Supplemental Figure 1, C and D).

Similar to Arabidopsis HY5, SlHY5 harbors an N-terminal COP1 binding motif (amino acids 1–75) and a bZIP (basic leucine zipper) domain (amino acids 76–145) (Figure 2A). The bZIP domain of SlHY5 is responsible for its interaction with SlBBX20 and SlBBX21 in Y2H assays (Figure 2B;  Supplemental Figure 2, A and C). No autoactivations were detected in various negative controls including empty vector (EV) controls (Figure 2B). Moreover, mapping of the interaction interface demonstrated that the second B-box domain of SlBBX20 is critical for interaction with SlHY5 (Figure 2B;  Supplemental Figure 2A).

Aspartate residues in B-box domains are critical for their coordination with zinc cations, and mutating them to alanine may disrupt the structure of the corresponding B-box domain (Massiah et al., 2007; Crocco and Botto, 2013). To further delineate the interaction domain between SlBBX20 and SlHY5, we mutated individual aspartate residues in the first (D20) or second B-box domains (D73 and D82) of SlBBX20 to alanine (Supplemental Figure 2D). While SlBBX20D20A interacted normally with SlHY5, the SlBBX20D73A and SlBBX20D82A point mutants did not (Figure 2B;  Supplemental Figure 2B). These observations reinforced the conclusion that the second B-box domain of SlBBX20 is responsible for its interaction with SlHY5.

In vitro pull-down assays showed that recombinant SlHY5 associates with maltose-binding protein (MBP)-SlBBX20, but not MBP alone (Supplemental Figure 2E). In plant cells of Nicotiana benthamiana leaves, SlBBX20 and SlHY5 largely colocalized in the nucleus (Supplemental Figure 2F), in agreement with the previously reported nuclear localization of SlBBX20 (Xiong et al., 2019). Co-immunoprecipitation (Co-IP) assays demonstrated the existence of SlBBX20/SlHY5 and SlBBX21/SlHY5 protein complexes in N. benthamiana leaves (Figure 2, C and D). With bimolecular fluorescence complementation (BiFC) assays, we showed that both SlBBX20 and SlBBX21 interact directly with SlHY5 in plants (Figure 2E and F; Supplemental Figure 2G). Taken together, we conclude that SlBBX20 and SlBBX21 interact with SlHY5 in vitro and in planta.

SlBBX20 and SlBBX21 most likely act upstream of SlHY5 to promote UV-B photomorphogenesis

We asked whether SlBBX20 and SlBBX21 were involved in photomorphogenesis upon illumination with white light or UV-B. We generated slbbx20 and slbbx21 mutant lines by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based gene editing. We obtained two slbbx20 mutant alleles carrying deletions (of 2 and 80 bp, respectively) (Supplemental Figure 3A). For slbbx21, we obtained two alleles harboring a 103-bp deletion and a 1-bp insertion, respectively (Supplemental Figure 3B). We generated a slbbx20 slbbx21 double mutant by genetic crossing of slbbx20 (80-bp deleted) and slbbx21 (103-bp deleted) to assess redundancy. Hypocotyl elongation is a typical assay used to quantify seedling photomorphogenesis. The hypocotyl lengths of slbbx20 and slbbx21 single mutants were of WT levels in both white light and UV-B, whereas the slbbx20 slbbx21 double mutant developed significantly longer hypocotyls than WT in white light (Figure 3, A and B). The inhibition of hypocotyl elongation caused by UV-B was strongly reduced in the slbbx20 slbbx21 double mutant, suggesting that SlBBX20 and SlBBX21 play redundant roles (Figure 3, A and B).

Figure 3.

Figure 3

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SlBBX20 and SlBBX21 probably act upstream of SlHY5 to promote UV-B signaling. A, Representative images of tomato seedlings grown for 4 days under white light (−UV-B) or white light supplemented with UV-B (+UV-B). B, Quantification of hypocotyl length of seedlings grown under white light or white light supplemented with UV-B. Mean and sd are shown (n ≥ 15). Statistically significant differences were determined by one-way ANOVA (analysis of variance), followed by Tukey’s test (P < 0.05). C, RT-qPCR analysis of SlHY5 expression in 4-day-old tomato seedlings grown under white light or white light supplemented with UV-B for 3 h. Mean and sd from three biological replicates are shown (n = 3). SlACTIN served as a reference gene. Data were normalized against WT sample grown under white light. Asterisks indicate significant differences (*P < 0.05; **P < 0.01; ns, nonsignificant; Student’s t test).

To dissect the genetic interaction of SlBBX20 and SlBBX21 with SlHY5, we crossed the slbbx20 slbbx21 double mutant with the slhy5 mutant to generate a slbbx20 slbbx21 slhy5 triple mutant. Hypocotyl elongation of the slbbx20 slbbx21 slhy5 triple mutant was similar to that of the slhy5 mutant, suggesting that SlBBX20/21 act upstream of SlHY5 to promote photomorphogenesis under both white light and UV-B (Figure 3, A and B). We next generated a slbbx20 slbbx21 sluvr8 triple mutant by genetic crossing. The slbbx20 slbbx21 sluvr8 triple mutant was hyposensitive to UV-B, suggesting that SlBBX20 and SlBBX21 regulate UV-B photomorphogenesis in a SlUVR8-dependent manner (Figure 3, A and B).

We measured SlHY5 transcript levels in response to UV-B as a marker of UVR8-dependent UV-B signaling (Figure 3C). Indeed, the induction of SlHY5 transcription by UV-B was strongly compromised in the slbbx20 slbbx21 double mutant (Figure 3C). We conclude that SlBBX20 and SlBBX21 most likely act upstream of SlHY5 to promote UV-B signaling in the SlUVR8 pathway.

SlBBX20 and SlBBX21 bind to the SlHY5 promoter to activate SlHY5 transcription

Although SlBBX20 and SlBBX21 appear to promote SlHY5 transcription under UV-B, the underlying mechanism is unknown. We tested whether SlBBX20 (Figure 4) and SlBBX21 (Supplemental Figure S4) bind to the SlHY5 promoter. We divided the SlHY5 promoter into two fragments, designated D (Distal) and P (Proximal), with the P fragment containing the two G-box elements: G-box1, located 232–238 bp, and G-box2, located 683- to 689-bp upstream of the translational start codon (the first nucleotide of the ATG start codon being designated as 1), respectively (Figure 4A). Yeast one-hybrid (Y1H) analyses indicated that SlBBX20 binds to the P fragment, containing the two G-box elements (Figure 4B).

Figure 4.

Figure 4

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SlBBX20 binds to the SlHY5 promoter and promotes its expression. A, Illustration of the genomic organization of the SlHY5 promoter. The first nucleotide of the translational start codon is designated as 1. Dark circles indicate G-box elements. Numbers above green lines indicate the span of qPCR amplicons. D indicates distal and P indicates proximal positions. B, Y1H analysis for the interaction of SlBBX20 with the SlHY5 promoter. Yeast growth on SD/–Trp–Ura solid medium is shown. Promoter P fragment contains both G-box1 and G-box2. C, ChIP assay for the binding of SlBBX20 to the SlHY5 promoter. Four-day-old WT and SlBBX20-OX (SlBBX20-FLAG) seedlings were sampled. The level of binding was calculated as the ratio between IP and Input. Mean and sd are shown (n = 3). No antibody was used in the mock treatment. Asterisk represents statistically significant difference to the corresponding Mock samples as determined by Student’s t test (**P < 0.01). The experiment was performed 2 times independently with similar results and one representative result is shown here. D and E, EMSAs showing the interaction of SlBBX20 (D) and the second B-box domain of SlBBX20, namely B-box2, (E) with the G-box1 of the SlHY5 promoter. Competitor, nonlabeled WT G-box1 probe. Mutant Competitor, nonlabeled mutant-type G-box1 probe. F, Dual-LUC assays for the effect of SlBBX20 on the activity of the SlHY5 promoter in N. benthamiana leaves. Schematic representation of SlHY5 promoter-driven dual-LUC reporter plasmids and two effector plasmids. LUC activity values normalized to REN. The activation value of SlHY5pro:Luc with EV was set at 1. Mean and sd  from four technical replicates are shown. Statistical tests were performed by one-way ANOVA, followed by Tukey’s test (P < 0.01). The experiment was performed 3 times independently with similar results and one representative result is shown here.

We observed constitutive transcriptional activation of the one-hybrid when driven by the D fragment of the SlHY5 promoter, even in the absence of SlBBX20, preventing a clear interpretation (Figure 4B). We turned to ChIP followed by quantitative PCR (polymerase chain reaction) to determine whether SlBBX20 binds to the SlHY5 promoter in tomato seedlings overexpressing SlBBX20 (35S:SlBBX20-FLAG). After ChIP, we used four primer pairs targeting different regions of the SlHY5 promoter, centered around G-box1 or G-box2, or spanning the intervals from –1,915 to –1,811 bp and from –1,698 to –1,592 bp for qPCR analyses (Figure 4A). Only G-box1 was enriched by immunoprecipitation with the anti-FLAG antibody from SlBBX20-FLAG seedlings, indicating that SlBBX20 binds specifically to G-box1 of the SlHY5 promoter in plants (Figure 4C).

We then performed Electrophoretic Mobility Shift Assays (EMSAs) and found that recombinant MBP-SlBBX20 protein bound to the G-box1 in the SlHY5 promoter (Figure 4D), as did the isolated B-box2 domain of SlBBX20 (Figure 4E). Moreover, with Y1H assays and EMSAs, we found that SlBBX21 also binds to the G-box1 element in the SlHY5 promoter (Supplemental Figure 4, A–C). We tested whether SlBBX20 and SlBBX21 can directly activate transcription from the SlHY5 promoter with transient expression assays in N. benthamiana leaves. Luciferase (LUC) activity derived from SlHY5pro:LUC was approximately seven- and five-fold higher than the control when SlBBX20 and SlBBX21, respectively, were co-infiltrated with the reporter constructs in N. benthamiana leaves (Figure 4F;  Supplemental Figure 4, D and E). Thus, both SlBBX20 and SlBBX21 can bind to and activate the SlHY5 promoter.

SlBBX20 and SlHY5 act together to promote UV-B signaling

Next, we tested whether SlHY5 is required for the SlBBX20-mediated activation of SlHY5 expression and photomorphogenesis. We generated slhy5 SlBBX20-FLAG by genetic crossing. Although expression of SlHY5 was elevated in the SlBBX20 overexpression line, SlHY5 transcript levels diminished to ˂10% of WT levels in the slhy5 SlBBX20-FLAG line, which resembles SlHY5 transcript levels in slhy5 (Figure 5A), suggesting that SlBBX20 requires SlHY5 to promote SlHY5 transcription in white light. UV-B failed to induce the expression of SlHY5 in either slhy5 or slhy5 SlBBX20-FLAG (Figure 5A). Like slhy5 mutant, the slhy5 SlBBX20-FLAG line developed long hypocotyls when grown in white light regardless of UV-B supplementation (Figure 5B). Therefore, SlBBX20 requires SlHY5 to induce SlHY5 transcription and photomorphogenesis in both white light and UV-B.

Figure 5.

Figure 5

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SlBBX20 and SlHY5 act together to promote SlHY5 expression and UV-B signaling. A, RT-qPCR analysis for the expression of SlHY5. Seedlings were grown under white light (−UV-B) or white light supplemented with UV-B for 2 h. Relative expression was calculated against the expression level in the WT under white light, which was designated as 1. The expression under +UV-B was divided by that under −UV-B to obtain fold change. Mean and sd from three biological samples are shown. Asterisk represents statistically significant difference to WT (−UV-B) as determined by Student’s t test (**P < 0.01). B, Representative and quantification of hypocotyl length for seedlings grown under white light (−) or white light supplemented with UV-B (+) for 4 days. Mean and sd are shown (n = 15). C, Dual-LUC assay for the activity of the SlHY5 promoter in N. benthamiana leaves. D20A, aspartate 20 in SlBBX20 substituted with alanine. DTripleA, three aspartates including 20, 73, and 82, substituted with alanine. In (B) and (C), statistical tests were performed by one-way ANOVA, followed by Tukey’s test (P < 0.01). Mean and sd from four technical replicates are shown. From (A) to (C), each experiment was performed 3 times independently with similar results and data from one experiment are shown.

Next, we tested whether the protein–protein interaction between SlBBX20 and SlHY5 is important for the activation of SlHY5 transcription by the SlBBX20–SlHY5 module. Y2H analyses revealed that an alanine substitution of the key aspartate residues in the second B-box domain (SlBBX20D73A and SlBBX20D82A), but not in the first B-box domain (SlBBX20D20A), of SlBBX20 abolishes its interaction with SlHY5 (Figure 2B;  Supplemental Figure 2, B and D). Again, in contrast to a construct encoding SlBBX20D20A, constructs encoding SlBBX20D73A or SlBBX20D82A essentially lost the ability to activate SlHY5 transcription in transient infiltration assays in N. benthamiana leaves (Figure 5C). We speculate that SlBBX20 acts together with NbHY5 to promote SlHY5 transcription in N. benthamiana leaves. Thus, the physical interaction between SlBBX20 and SlHY5 appears to be critical for the activation of SlHY5 transcription. Notably, infiltration of SlHY5 failed to induce SlHY5 transcription, possibly due to the negative effect of excessive SlHY5 protein on its own gene transcription (see below the autoregulatory negative feedback loop).

UV-B inhibits the association of SlBBX20 to the SlHY5 promoter through SlHY5

Arabidopsis HY5 binds to its own promoter (Abbas et al., 2014; Binkert et al., 2014). Similarly, we found that SlHY5 binds to the G-box1 within the SlHY5 promoter in both Y1H assays and EMSAs (Supplemental Figure S5). Indeed, recombinant MBP-SlHY5 bound to a 40-bp DNA fragment (from –255 bp to –215 bp) containing G-box1, as demonstrated by the formation of a specific DNA–protein complex (Supplemental Figure 5B, lane 2). We noticed multiple bands for the SlHY5/DNA complex, a pattern that was previously reported (Nawkar et al., 2017). Excess unlabeled DNA probes (cold probes) competed with labeled probes for binding to MBP-SlHY5 (Supplemental Figure 5B, lanes 3 and 4). We conclude that SlHY5 associates with G-box1 in the SlHY5 promoter in vitro.

Next, we performed ChIP-qPCR assays to investigate the binding site of SlHY5 within its own promoter in tomato seedlings with an anti-SlHY5 antibody. After ChIP, we performed qPCR with primer pairs targeting different regions of the SlHY5 promoter: G-box1, G-box2, and the fragment from −1,915 to −1,811 bp, used previously for ChIP-qPCR in the SlBBX20-FLAG line. We established that SlHY5 only binds to G-box1 in the SlHY5 promoter (Figure 6A). Moreover, the association of SlHY5 to its own promoter was enhanced by UV-B treatment (Figure 6A).

Figure 6.

Figure 6

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UV-B inhibits the binding of SlBBX20 to the SlHY5 promoter in a SlHY5-dependent manner. A, ChIP-qPCR assay for the binding of SlHY5 to its own gene promoter under white light and white light supplemented with UV-B. Primer pairs used here are the same as those used for CHIP-qPCR with the SlBBX20 overexpression line in Figure 4C. The level of binding was calculated as the ratio between IP and Input. Mean and sd are shown (n = 3). No antibody was used in the mock control. Asterisk represents statistically significant difference to white light treatment (−UV-B) as determined by Student’s t test (*P < 0.05). B, ChIP-qPCR assay for the effect of SlHY5 on the binding of SlBBX20 to G-box1 in the promoter of SlHY5. Seedlings were either grown under white light or white light supplemented with UV-B for 3 h. The binding level was calculated as the ratio of IP to Input. Asterisk indicates significant difference as determined by Student’s t test (P < 0.05). Data shown are representative of three biological replicates. Mean and sd from three technical replicates are shown. C, EMSA for the competitive binding between SlHY5 and SlBBX20-B-box2 to the promoter of SlHY5. 1× to 3× represent increasing protein input, 1× indicates 0.5 μg of SlHY5, and 1.5 μg of SlBBX20-B-box2 fusion protein. Sequence of the G-box1-probe is shown in Figure 4D.

Our results above showed that UV-B promotes the association of SlHY5 to G-box1 in its own promoter, the same SlHY5 promoter fragment that is bound by SlBBX20 (Figure 4C). We, therefore, tested whether the association of SlBBX20 to G-box1 in the SlHY5 promoter was affected by UV-B exposure. In sharp contrast to SlHY5, UV-B inhibited the binding of SlBBX20 to G-box1 within the SlHY5 promoter (Figure 6B). Moreover, UV-B-inhibition of SlBBX20 binding to the SlHY5 promoter is dependent on SlHY5, as the association of SlBBX20 to G-box1 in the SlHY5 promoter was not inhibited by UV-B in the slhy5 SlBBX20-FLAG (Figure 6B). Notably, the binding of SlBBX20 to the SlHY5 promoter increased in the slhy5 SlBBX20-FLAG line irrespective of UV-B supplementation (Figure 6B).

Since UV-B promoted the association of SlHY5 with the SlHY5 promoter, but repressed the association of SlBBX20 with the SlHY5 promoter through SlHY5, we postulated that SlHY5 might compete with SlBBX20 for binding to the SlHY5 promoter. To test this hypothesis, we first performed EMSAs to assess the association of SlHY5 and SlBBX20 to G-box1 in the SlHY5 promoter and the possible binding competition between the two transcription factors. However, the protein–DNA complexes corresponding to SlBBX20-Gbox1, SlHY5-Gbox1, and SlHY5△N75- Gbox1 all migrated to similar positions in PAGE (polyacrylamide gel electrophoresis) during EMSAs, making it not possible to detect competition between SlHY5/SlHY5△N75 and full-length SlBBX20 (Supplemental Figure S6). Therefore, we used the B-box2 domain of SlBBX20, which can bind to the SlHY5 promoter and migrates faster than full-length SlBBX20 by PAGE (Figure 4E), for competitive binding in EMSAs. Both MBP-SlBBX20-B-box2 and MBP-SlHY5 fusion proteins exhibited dose-dependent binding to G-box1 in the SlHY5 promoter (Figure 6C, lanes1–6). Notably, the detected SlHY5–DNA complex (0.5 µg SlHY5) appeared to be much stronger than the SlBBX20-B-box2–DNA complex (1.5 µg SlBBX20-B-box2), suggesting that SlHY5 has a higher affinity for the SlHY5 promoter than does SlBBX20-B-box2 (Figure 6C). By adding an increasing amount of SlHY5 to a fixed amount of SlBBX20-B-box2, the binding of MBP-SlHY5 to DNA increased while the binding of MBP-SlBBX20-B-box2 to DNA gradually decreased, indicating that SlHY5 outcompetes SlBBX20 for binding to the SlHY5 promoter in vitro (Figure 6C).

SlBBX20 and SlBBX21 overexpression lines exhibit attenuated UV-B responses

SlHY5 transcript levels were elevated in SlBBX20/21 overexpression lines grown in white light (Figures 5, A, 7, A, and 8, A). Since SlHY5 inhibits the binding of SlBBX20 to the SlHY5 promoter (Figure 6, B and C), we reasoned that UV-B-induced SlHY5 expression might be attenuated in SlBBX20/21 overexpression lines. Indeed, the induction of SlHY5 expression by UV-B was strongly reduced in SlBBX20/21 overexpression lines, already in the first hours of UV-B treatment in comparison to WT (Figures 7, A and 8, A). Next, we directly tested the inhibitory effect of SlHY5 on the activation of SlHY5 transcription by SlBBX20 or SlBBX21 with dual-LUC assays in N. benthamiana leaves. The addition of SlHY5 repressed the transcriptional activity of SlBBX20 and SlBBX21 on the SlHY5 promoter (Figures 7, B and 8, B). Moreover, the SlBBX20 and SlBBX21 overexpression lines developed shorter hypocotyls relative to the WT in white light (Figures 7, C and 8, C), consistent with elevated SlHY5 expression. The inhibition of hypocotyl elongation by UV-B was strongly compromised in the SlBBX20 and SlBBX21 overexpression lines (Figures 7, C, D, 8, C, and D). Taken together, the SlBBX20/21 overexpression lines are hyposensitive to UV-B, reinforcing the conclusion that elevated SlHY5 represses its own transcription via a negative feedback loop.

Figure 7.

Figure 7

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UV-B responses are compromised in SlBBX20 overexpression lines. A, RT-qPCR analysis of SlHY5 expression. WT and SlBBX20-FLAG tomato seedlings were treated with UV-B for the indicated periods. Relative quantification was calculated against the expression level under white light (0 h) in WT. Mean and sd from three biological samples are shown (n = 3). Asterisk represents statistically significant difference to WT treated with UV-B for the indicated periods as determined by Student’s t test (P < 0.01). B, Dual-LUC assays for the effect of SlBBX20 and SlHY5 on the activity of the SlHY5 promoter in N. benthamiana leaves. SlHY5 promoter-driven dual-LUC reporter plasmids and effector plasmids used in the assay were the same as those used in the analysis shown in Figure 4F. An equal amount of SlBBX20 and SlHY5 was added in the SlBBX20 + SlHY5 treatment. The activation value of SlHY5pro:Luc with EV was set at 1. Mean and sd are shown (n = 4). Asterisk indicates significant difference as determined by Student’s t test (P < 0.01). C, Hypocotyl lengths of tomato seedlings. WT and three different SlBBX20-FLAG lines were grown under white light or white light supplemented with UV-B for 4 days. Mean and sd are shown (n = 15). D, Relative hypocotyl length (+UV-B/−UV-B). The calculation was based on the absolute hypocotyl length obtained from (C). In (C), statistically significant differences were determined by one-way ANOVA, followed by Tukey’s test (P < 0.05). In (D), asterisk represents statistically significant difference to WT as determined by Student’s t test (P < 0.05). From (A) to (D), each experiment was performed at least twice independently with similar results and data from one experiment are shown.

Figure 8.

Figure 8

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UV-B responses are attenuated in SlBBX21 overexpression lines. A, RT-qPCR analysis for SlHY5 expression. WT and SlBBX21-FLAG were treated with UV-B for the indicated periods. Relative quantification was calculated against the expression level under white light (0 h) in WT. Mean and sd from three biological samples are shown (n = 3). Asterisk represents statistically significant difference to WT treated with UV-B for the indicated periods as determined by Student’s t test (P < 0.01). B, Dual-LUC assays for the effects of SlBBX21 and SlHY5 on the activity of the SlHY5 promoter in N. benthamiana leaves. An equal amount of SlBBX21 and SlHY5 was added in the SlBBX21 + SlHY5 treatment. Mean and sd from three biological replicates are shown. Asterisks indicate significant differences (P < 0.01); Student’s t test. C, Hypocotyl lengths of tomato seedlings. Seeds of WT and two different SlBBX21-FLAG lines were grown under white light or white light supplemented with UV-B for 4 days. Mean and sd are shown (n = 15). Multiple comparisons were calculated by one-way ANOVA followed by Tukey’s hoc tests (P < 0.05). Different letters indicate statistically significant differences. D, Relative hypocotyl length (+UV-B/−UV-B). The calculation was based on the absolute hypocotyl length obtained in (C). Mean and sd are shown (n = 15). **P < 0.01; Student’s t test. From (A–D), each experiment was performed at least twice independently with similar results and data from one experiment are shown.

Overexpression of SlHY5 under WT background inhibits UV-B-induced expression of endogenous SlHY5

To directly test the autoregulatory negative feedback loop of SlHY5, we generated lines overexpressing 35S:SlHY5-FLAG in the WT tomato background (Figure 9, A and B). Expression of SlHY5 is >30-fold higher than in WT as measured with primers targeting the SlHY5 gene body in RT-qPCR assays (Figure 9B). A primer pair spanning the SlHY5 gene body and its 5′-UTR (5′ untranslated region) was used to specifically measure the transcript of endogenous SlHY5 in 35S:SlHY5-FLAG/WT lines in RT-qPCR assays (Figures 9A). Notably, under white light, transcript levels of endogenous SlHY5 were already diminished by the exogenous overexpression of this transcription factor (Figure 9C). The induction of endogenous SlHY5 expression by UV-B in 35S:SlHY5-FLAG/WT lines was strongly inhibited in relation to WT (Figure 9D). To test whether the SlHY5 autoregulatory negative feedback loop is also present in other plant species, we performed similar experiments, with a previously established Arabidopsis HY5 overexpression line in the WT background (Chen et al., 2021). Again, overexpression of 35S:AtHY5-MYC has a strong inhibitory effect on the expression of endogenous AtHY5 under both white light and UV-B (Figure 9, E–G). We conclude that excessive HY5 inhibits its own gene expression under both white light and UV-B in different plant species.

Figure 9.

Figure 9

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Overexpression of SlHY5 in the WT background inhibits endogenous SlHY5 expression under UV-B in tomato. A, Illustration of the locations of primer pairs in SlHY5 and Arabidopsis HY5 (AtHY5). Black arrows indicate primer pairs targeting the exon regions of SlHY5 and AtHY5, while blue arrows indicate primer pairs spanning the 5′-UTR region and the first exon of SlHY5 and AtHY5. B and C, RT-qPCR analysis of the expression of both 35S:SlHY5-FLAG and endogenous SlHY5 with primers targeting the SlHY5 gene body (B) and specifically endogenous SlHY5 with primers spanning the 5′-UTR region (C). D, Induction of endogenous SlHY5 expression is inhibited in 35S:SlHY5-FLAG/WT tomato lines. Tomato seedlings were treated with UV-B for the indicated periods. E and F, RT-qPCR analysis of the expression of both 35S:AtHY5-MYC and endogenous AtHY5 with primers targeting the AtHY5 gene body (E) and specifically endogenous AtHY5 with primers spanning the 5′-UTR region (F). G, Induction of endogenous AtHY5 expression is inhibited in 35S:AtHY5-MYC/Col-0. Arabidopsis seedlings were treated with UV-B for the indicated periods. Data shown from (B–G) are from three biological samples. Mean and sd are shown (n = 3). From (B–G), asterisk represents statistically significant difference to AC/Col-0 as determined by Student’s t test (*P < 0.05; **P < 0.01). H, Proposed working model depicting how the SlBBX20/21-SlHY5 module mediates the activation and negative feedback regulation of SlHY5 transcription under UV-B.

Discussion

SlHY5 is implicated in an autoregulatory negative feedback regulation of its own transcription during UV-B signaling

HY5 is a key transcription factor acting downstream of multiple photoreceptors, including the UV-B photoreceptor UVR8, and its transcription is transiently induced by UV-B. HY5 lacks a clear transcriptional AD and HY5 is necessary but not sufficient for the activation of its own transcription (Abbas et al., 2014; Binkert et al., 2014; Burko et al., 2020; Bursch et al., 2020). In this work, we showed that tomato SlBBX20 and SlBBX21 interact with SlHY5 and bind to the SlHY5 promoter to activate its transcription in response to both white light and UV-B. The functional significance of SlBBX20 and SlBBX21 is reflected by the compromised SlHY5 expression and UV-B responses displayed by the tomato slbbx20 slbbx21 double mutant (Figure 3).

Like many other signaling pathways, turning off UV-B signaling is as important as turning it on, as sustained activation of UV-B signaling can severely inhibit plant growth. Indeed, prolonged UV-B exposure reduces SlHY5 transcript levels, indicating the existence of negative feedback mechanisms impinging on SlHY5 transcription (Figure 1). We demonstrated that the SlBBX20/21-SlHY5 transcription module activates SlHY5 transcription, leading to an accumulation of HY5 that in turn inhibits SlHY5 transcription, forming an autoregulatory negative feedback loop. First, ChIP-qPCR and EMSA experiments showed that both SlBBX20/21 and SlHY5 associate with the same G-box element in the SlHY5 promoter (Figures 4 and 6; Supplemental Figures S4–S6). UV-B promoted the association of SlHY5 to its promoter, but inhibited the association of SlBBX20 to the SlHY5 promoter, the latter being dependent on SlHY5 (Figure 6). EMSAs showed that SlHY5 can outcompete SlBBX20 for association to the SlHY5 promoter (Figure 6). Second, adding excessive SlHY5 blocked the transcriptional activation of the SlHY5 promoter by SlBBX20 and SlBBX21 in transient expression assays (Figures 7, B and 8, B). Third, UV-B-induced SlHY5 transcription and photomorphogenesis were strongly compromised in the SlBBX20 and SlBBX21 overexpression lines, which accumulate higher basal levels of SlHY5 transcripts prior to UV-B exposure (Figures 7 and 8). Lastly, overexpression of SlHY5 (AtHY5) in the WT background impairs UV-B-induced SlHY5 (AtHY5) expression in the respective plant species (Figure 9), substantiating the conclusion that SlHY5 (AtHY5) is involved in an autoregulatory negative feedback loop in UV-B signaling in different plant species (Figure 9H).

Several SlBBX proteins closely related to SlBBX20/21 may participate in UV-B signaling

Although SlBBX20 and SlBBX21 are not completely essential for UV-B-induced SlHY5 expression, shown by the fact that about 40% of the expression was retained in the slbbx20 slbbx21 double mutant compared to the WT, the data suggest that these two SlBBXs are major contributors in this process and that other SIBBXs likely are of lesser importance (Figure 3C). The SlBBX subfamily IV, to which SlBBX20 and SlBBX21 belong, comprises other members including SlBBX18, 19, and 22–24 with high sequence similarities (Chu et al., 2016; Supplemental Figure S8). Genetic evidence in Arabidopsis demonstrated that BBX20/21/22 act as HY5 cofactors to regulate the transcription of some HY5 target genes in white light (Fan et al., 2012; Bursch et al., 2020). BBX21 was also shown to bind to the HY5 promoter and promotes its transcription in visible light (Xu et al., 2016). Notably, the bbx20 bbx21 bbx22 triple mutant affected only ∼15% of all known HY5 target genes (Bursch et al., 2020), clearly pointing to additional but unknown HY5 cofactors in Arabidopsis. In fact, many HY5-interacting factors, including CAM7, COR27, COR28, SWC6, and ARP6, have been identified, and HY5 may act together with some of these cofactors as a function of growth conditions and developmental stages. It will be interesting to determine whether other members of the SlBBX subfamily IV are also involved in UV-B signaling. Importantly, based on the strongly reduced SlHY5 transcription and UV-B signaling observed in the slbbx20 slbbx21 double mutant, SlBBX20 and SlBBX21 play key roles in the regulation of SlHY5 expression and UV-B photomorphogenesis.

SlBBX20 forms a complex with SlHY5 to activate SlHY5 transcription

As with their Arabidopsis counterparts BBX20 and BBX21, a putative transcriptional AD is also present in SlBBX20 and SlBBX21 (Supplemental Figure S8; Bursch et al., 2020). We demonstrated that SlBBX20/21 interact with SlHY5, thus providing the transcriptional AD (Oyama et al., 1997; Abbas et al., 2014; Binkert et al., 2014; Burko et al., 2020; Bursch et al., 2020). UV-B-induced SlHY5 transcription is compromised in both the slhy5 single and the slbbx20 slbbx21 double mutant, suggesting that SlBBX20/21 form a complex with SlHY5 to activate SlHY5 transcription. The second B-Box domain of SlBBX20 was responsible for the interaction with SlHY5. Mutations in key residues within the second B-Box domain abolished the SlBBX20–SlHY5 interaction and the transcriptional activation of the SlHY5 promoter, supporting the notion that the SlBBX20–SlHY5 complex is important for function.

The interaction between these transcription factors and DNA is further complicated by the fact that SlHY5 can also bind to the same cis-element within the SlHY5 promoter as SlBBX20. Arabidopsis BBX21 exhibits a similar domain organization as tomato SlBBX20 and SlBBX21. The second B-Box domain of BBX21 was shown to be required for binding to and activating transcription from the HY5 promoter (Xu et al., 2018). Here, we further showed that the second B-Box domain of SlBBX20 is not only necessary, but also sufficient for binding to the relevant cis-element in EMSAs.

In Arabidopsis, both BBX20/21 and HY5 can bind to the promoters of MYB12 and FLAVANONE 3-HYDROXYLASE (F3H; Lee et al., 2007; Stracke et al., 2010; Bursch et al., 2020). However, the binding of BBX20 and BBX21 to these promoters was previously shown to be reduced in the hy5 mutant relative to the WT, suggesting that HY5 is partly required for the association of BBX20/21 with DNA (Bursch et al., 2020). However, we demonstrated that the binding of SlBBX20 to the SlHY5 promoter does not require SlHY5 in tomato. Moreover, the binding of SlBBX20 to the SlHY5 promoter in slhy5 SlBBX20-FLAG lines was higher than in SlBBX20 overexpression lines. It remains to be tested whether BBX20/21 and HY5 bind to different cis elements in the promoters of MYB12 and F3H. It appears that HY5 can have contrasting effects on the binding of BBX20/21 to different gene promoters.

SlBBX20 was shown to be ubiquitinated and degraded via the 26S proteasome pathway (Xiong et al., 2019). A well conserved VP (valine-proline) motif is present in several COP1-interacting proteins (Holm et al., 2001; Cloix et al., 2012; Lau et al., 2019). Consistent with the absence of a VP motif in SlBBX20, no interaction between SlBBX20 and SlCOP1 was detected (Xiong et al., 2019). However, SlBBX20 does interact with DE-ETIOLATED 1 (SlDET1). Silencing of SlDET1 leads to the accumulation of SlBBX20 in tomato (Xiong et al., 2019). Thus, the protein stability of SlBBX20 is regulated by the E3 ligase complex Cullin 4–DAMAGED DNA-BINDING PROTEIN 1–DET1 (CUL4–DDB1–DET1) in tomato. It will be interesting to test whether SlDET1 participates in UV-B signaling by affecting SlBBX20 protein stability in tomato. Intriguingly, under our conditions, SlBBX20–FLAG fusion protein levels were not obviously altered by UV-B (Supplemental Figure S1C). In contrast to SlBBX20, a VP motif is present in SlBBX21, raising the possibility that SlBBX21 protein stability might be regulated by SlCOP1. Indeed, SlBBX21-FLAG protein levels were obviously promoted by UV-B, consistent with the notion that UV-B inactivates SlCOP1 (Supplemental Figure S1D). Notably, available slcop1 mutant alleles do not show typical cop-like phenotypes (Jones et al., 2012). Future work is needed for the functional characterization of SlCOP1 in photomorphogenesis.

Two key negative feedback loops in the SlUVR8 signaling pathway

In agriculture, long hypocotyls can result in decreased survival rates during planting. Tomato slhy5 mutants developed long hypocotyls (Liu et al., 2004; Wang et al., 2021) (this work). Indeed, the survival rate of slhy5 decreased in comparison to the WT during transplantation to the field (Wang et al., 2021). Adverse environmental conditions, including dim light and elevated ambient temperatures, can increase hypocotyl elongation. The effects of nondamaging levels of photomorphogenic UV-B on hypocotyl shortening may provide an effective measure to promote seedling fitness and increase survival rates. It is therefore important to dissect the molecular mechanisms underlying the regulation of seedling photomorphogenesis for crops.

SlRUP is a negative regulator that inactivates the photoreceptor SlUVR8 by accelerating its conversion from active monomers to inactive homodimers (Zhang et al., 2021). Transcription of SlRUP is induced by UV-B, forming a negative feedback loop to adjust SlUVR8 activity (Zhang et al., 2021). This UVR8–RUP feedback loop has been elegantly previously demonstrated in Arabidopsis (Heijde and Ulm, 2013). In the slrup mutant, the attenuation of SlHY5 transcription under prolonged UV-B exposure is delayed but still apparent. Thus, inactivation of photoreceptor SlUVR8 by SlRUP is not sufficient for the termination of downstream transcriptional events. Here, we characterized the roles of the SlBBX20/21–SlHY5 module in activating SlHY5 transcription and forming an autoregulatory negative feedback loop to adjust SlHY5 transcription to maintain appropriate UV-B photomorphogenesis. We propose a working model in which the SlUVR8–SlRUP and SlBBX20/21–SlHY5 negative feedback loops contribute to the early and later attenuation of SlHY5 transcription, respectively, to ensure balanced UV-B photomorphogenesis (Figure 9H).

Materials and methods

Plant materials and growth conditions

Tomato mutants and overexpression lines used in this study were generated in the Ailsa Craig WT background. Tomato plants were grown in a growth chamber with a long day photoperiod (16/8 h) and temperature of 24°C/21°C (day/night). Seeds were surface sterilized with 75% ethanol and sown on half-strength Murashige and Skoog (1/2 MS) medium supplemented with 0.8% Agar and 1% sucrose. For photomorphogenic experiment, seedlings were grown under white light (3.6 mmol/m2/s) or white light supplemented with narrow band UV-B (PhilipsTL20W/01RS; 1.5 mmol/m2/s).

The coding sequences of SlBBX20, SlBBX21, and SlHY5 were amplified and cloned into the pMV2 vector (Xiong et al., 2019), to generate 35S:SlBBX20-FLAG, 35S:SlBBX21-FLAG and 35S:SlHY5-FLAG for overexpression. The constructs were transformed into cotyledons using Agrobacterium tumefaciens. Transgenic plants were screened on 1/2 MS medium supplemented with kanamycin (100 ng/μL). Three SlBBX20 overexpression lines (#001, #004, and #A) and two SlBBX21 overexpression lines (#1 and #2) were used for further analyses. The slbbx20 and slbbx21 mutants were generated with similar approaches as described previously (Liu et al., 2020). Briefly, in the CRISPR/Cas9 binary vectors (pTX) that were used, the target sequence was driven by the tomato U6 promoter and Cas9 by a 2x35S promoter (Xiong et al., 2019). Two single-guide RNAs for each gene were used (Supplemental Data Set 1). Double and triple mutants were obtained by genetic crossing with slbbx20 #2, slbbx21 #2, SlBBX20-Flag #A, SlBBX21-Flag #1, sluvr8 #1 , and slhy5 #1 (Zhang et al., 2021). The mutations were confirmed by PCR and sequencing.

Y2H assay

SlHY5, SlHY5N75, and SlHY5ΔN75 fragments were cloned into the pGBKT7 vector (Clontech, Mountain View, CA, USA). SlBBX20, SlBBX21, and versions of these genes with various deletion fragments and point mutations were cloned into the pGADT7 vector (Clontech, USA). The bait and prey constructs were introduced into yeast strain AH109 and cultured on synthetic dropout SD/–Trp/–Leu medium (Yeast Protocols Handbook, Clontech). Colonies were spotted on SD/–His/–Leu/–Trp solid medium for selection of interactions.

Pull-down assay

Pull-down assays were performed as described with some modifications (Seo et al., 2004). The coding sequence of SlBBX20 was cloned into the pMAL-c5x vector to generate MBP designates maltose-binding protein (MBP-SlBBX20). The coding sequence of SlHY5 was cloned into the pET32a (+) vector to generate a C-terminal 6×His fusion. For pull-down experiments, 1.5 µg of MBP or MBP-SlBBX20 prey proteins were incubated with 1.5 µg of SlHY5-His bait proteins in 250-μL MBP column binding buffer (50-mM Tris–HCl, pH 7.5, 100-mM NaCl, 0.2% (v/v) glycerol, 0.1% (v/v) Triton X-100, 1-mM EDTA (Ethylenediaminetetraacetic acid), pH 8.0, 1-mM PMSF (phenylmethylsulfonyl fluoride), 0.1% (v/v) protease inhibitor cocktail, and Nonidet P-40) containing 30-μL Amylose Magnetic Beads for 2 h at 4°C. Beads were washed 4 times with the same buffer after incubation. The proteins were eluted from the beads by boiling in 50 μL 2× SDS (sodium dodecyl sulfate) buffer. Anti-His (Abmart, Shanghai, China; M30111, 1:3,000) and anti-MBP (Proteintech, Rosemont, IL, USA; 66003-1, 1:5,000) antibodies were used for detection. The primers are listed in Supplemental Data Set 1.

Colocalization assay

SlBBX20 and SlHY5 were fused to the C terminus of GFP in the pHellsgate8 vector (Biofeng Inc., Arlington, SD, USA) and N-terminus of mCherry in the 35S:mCherry vector (BioVector Inc., San Diego, CA, USA), respectively, and then transformed into Agrobacterium. Agrobacteria harboring the GFP-SlBBX20 and SlHY5-mCherry constructs at a ratio of 1:1 were introduced into N. benthamiana leaves by infiltration. Fluorescence signals were visualized and imaged using a Leica SP8 confocal microscope in sequential scanning mode. GFP was excited at a wavelength of 488 nm, with an emission wavelength of 520–580 nm, mCherry was excited at 561 nm, with an emission wavelength of 610–630 nm.

BiFC assay

The cDNA fragments of SlBBX20/21 were cloned individually into pXY104 (Xu et al., 2019) carrying fragments encoding the C-terminal half of YFP (SlBBX20/21-cYFP), while SlHY5 was cloned into pXY106 (Xu et al., 2019) carrying fragments encoding the N-terminal half of YFP (SlHY5-nYFP). Then, SlBBX20/21-cYFP and SlHY5-nYFP were transformed into Agrobacterium. SlBBX20/21-cYFP and SlHY5-nYFP were mixed at a ratio of 1:1 and infiltrated into N. benthamiana leaves. SlBBX20-YFPn/SlHY5N75-YFPc, SlBBX20-YFPn/SlUVR8-YFPc, SlBBX21-YFPn/SlHY5N75-YFPc, SlBBX21-YFPn/SlUVR8-YFPc, SlBBX20△N100-YFPn/SlHY5-YFPc, SlBBX21△N100-YFPn/SlHY5-YFPc, and SlUVR8-YFPn/SlHY5-YFPc were used as negative controls. A confocal laser scanning microscope (Leica TCS SP8) was used to detect YFP fluorescence signals.

Co-IP assays

Full length CDS of SlBBX20 and SlBBX21 were cloned individually into the pBTEX-Flag vector (Xiong et al., 2019). Nicotiana benthamiana leaves were infiltrated with Agrobacterium harboring these constructs and those expressing SlHY5-HA or GFP-HA. Total protein was extracted from N. benthamiana leaves using extraction buffer containing 50-mM Tris–HCl, pH 7.5, 150-mM NaCl, 0.2% Triton X-100, 1×Protease Inhibitor Cocktail (Roche, Basel, Switzerland), and 50-μM MG132 (a proteasome inhibitor). The extracts were centrifuged at 13,000 g at 4°C for 30 min, and supernatants were used for immunoprecipitation. For co-IP assays of SlBBX20 and SlHY5, equal amounts of total protein were extracted with 1 mL of lysis buffer from N. benthamiana leaves co-infiltrated with SlBBX20-Flag and SlHY5-HA or GFP-HA (negative control). The protein extracts were then incubated with 10-μL anti-HA antibody and 20-μL Protein A Agarose beads (Invitrogen, Waltham, MA, USA; 15918) at 4°C. For SlBBX21 and SlHY5, Flag-tagged SlBBX21 were co-infiltrated with SlHY5-HA or GFP-HA in N. benthamiana leaves. Equal amounts of protein supernatants extracted with 1 mL of lysis buffer from N. benthamiana leaves co-infiltrated with SlBBX21 and SlHY5 or GFP were incubated with 20-μL anti-Flag beads (MBL, M185-10) at 4°C for 2 h. All immunoprecipitates were washed 4 times with lysis buffer, and then eluted with 30-μL 2 × SDS loading buffer before being subjected to immunoblot analysis with anti-HA (Abmart, M20003M, 1:5,000) or anti-Flag (Sigma, St Louis, MO, USA; A8592, 1:5,000) antibodies. Anti-mouse (Proteintech, SA00001, 1:5,000) was used as a secondary antibody.

Hypocotyl measurement

Hypocotyl lengths of 4-day-old seedlings were measured using ImageJ software (http://imagej.nih.gov/ij/index.html).

Y1H assay

SlHY5 promoter (−61 to −2,000 bp) or different truncated fragments containing G-box1, G-box2 or no G-box were cloned into the placZ vector (Xie et al., 2021) as the bait. The coding sequences of SlBBX20 and SlBBX21 were fused with the GAL4 AD in the pB42AD vector as the prey. The pB42AD effectors and placZ reporters were co-transformed into yeast strain EGY48, and cultured on SD/–Trp/–Ura solid medium. The positive clones were picked and cultured on 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside medium in the dark.

Dual-LUC assay

Full-length CDS of SlBBX20, SlBBX21, and SlHY5 were cloned into the PHB vector (Xie et al., 2021). The SlHY5 promoter (−61 to −2,000 bp) was cloned into pGreen0800-LUC to drive expression of firefly LUC. The PHB plasmids were transformed into GV3101 directly, while pGreenII 0800-LUC plasmids were each transformed into GV3101 with the help of the pSoup19 plasmid (Xie et al., 2021). Then, the reporter and effector strain were mixed 1:1 and used to infiltrate the leaves of 3-week-old N. benthamiana plants. After infiltration, leaf discs were collected to assay of the activities of LUC and renilla LUC with the Dual-LUC Reporter Assay System on a GLO-MAX 20/20 luminometer (Promega, Madison, WI, USA).

RNA extraction and RT-qPCR

Total RNA was extracted from 4-day-old tomato seedlings using RNeasy Plant Mini Kits (Omega, Knoxville, TN, USA). Total RNA (1 μg) was reverse transcribed into cDNA using the RT Master Mix cDNA synthesis system (Takara, Tokyo, Japan) according to the manufacturer’s instructions. The cDNA was diluted 1:10 for quantitative PCR in an CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using the qPCR SYBR Green Master Mix (Takara, Japan). SlACTIN served as a reference gene.

ChIP-qPCR

ChIP assays were performed as described previously (Binkert et al., 2014). In brief, 4-day-old WT, slhy5, SlBBX20-FLAG, or slhy5 SlBBX20-FLAG seedlings were cross-linked using 1% formaldehyde for 20 minutes and de-crosslinked for 5 min by adding 2.5-M glycine. Chromatin was isolated before being sonicated into 200- to 500-bp fragments. Then immunoprecipitation was performed with anti-FLAG (Sigma-Aldrich, St Louis, MO, USA; A8592, 1:500) and/or polyclonal anti-SlHY5 antibody (Made by Jiayuanbio with full length of SlHY5) with a dilution of 1:500 overnight. Protein A beads (Invitrogen) were added to IP the DNA fragments binded to antibody for 2 h. Then the beads were washed thoroughly and reverse cross-linked at 65°C overnight. The eluted products were purified to a final volume of 50 μL for qPCR analysis. The data were analyzed using the percentage of input method (Haring et al., 2007).

EMSA

EMSA was performed using a Light Shift Chemiluminescent EMSA kit (Thermo Fisher Scientific, Waltham, MA, USA) with biotin-labeled probes. About 40-bp SlHY5 promoter sub-fragments containing G-box1 (or mutated G-box1) were synthesized as single-stranded oligonucleotides (5′-End DNA biotinylated) and annealed to obtain a double-stranded probe (Supplemental Data Set 1). Competitive probes were unlabeled with biotin. Full-length SlBBX20, SlBBX21, and SlHY5 coding sequence were cloned into the pMAL-c5x vector and expressed in BL21 cells with induction of 0.1-mM isopropyl β-d-1-thiogalactopyranoside under 28°C for 12 h. Unfused MBP and MBP fusion proteins were purified with Amylose Magnetic Beads (NEB, Ipswich, MA, USA). The probes (20 fmol) were incubated with 500 ng to 5 μg MBP, MBP-SlHY5, or MBP-SlBBX20/MBP-SlBBX21 in buffer containing 10 mM Tris–HCl, pH 7.5, 0.05% Nonidet P-40, 10 mM MgCl2, 5% (v/v) glycerol, and 0.1-μg/mL poly (dI·dC) for 20 min at room temperature. An aliquot of 5-μL DNA loading dye was added to the reaction system before running a 6% native polyacrylamide gel in 0.5× TBE buffer. The gels were then electroblotted to Hybond positively charged nylon membranes (Millipore, Burlington, MA, USA) for 40 min and cross-linked for 30 min under a 302 nm UV bulb. Then the labeled probes on the membrane were detected following the manufacturer’s protocols.

Statistical analysis

Statistics used in this article are indicated in the respective figure legends and further statistical analyses results are included in Supplemental Data Set 2.

Accession numbers

Sequence data from this article can be found in the Sol Genomics Network databases (https://solgenomics.net/) under the following accession numbers: SlBBX20 (Solyc01g110180), SlBBX21 (Solyc12g089240), SlUVR8 (Solyc05g018615), SlHY5 (Solyc08g061130), SlRUP (Solyc11g005190), and SlACTIN (Solyc03g078400).

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1. Expression of SlBBX20 and SlBBX21 in response to UV-B.

Supplemental Figure S2. SlBBX20 and SlBBX21 interact with SlHY5 in vitro and in vivo.

Supplemental Figure S3. Generation of the slbbx20 and slbbx21 single mutants by CRISPR gene editing.

Supplemental Figure S4. SlBBX21 binds to and promotes the activity of the SlHY5 promoter.

Supplemental Figure S5. SlHY5 binds to its own gene promoter.

Supplemental Figure S6. Similar migration of protein/DNA complexes in EMSAs.

Supplemental Figure S7. Immunoblotting for the detection of SlBBX20-FLAG and SlBBX21–FLAG fusion proteins in the respective tomato overexpression lines.

Supplemental Figure S8. Multiple protein sequence alignment of BBX proteins belonging to subfamily IV (SlBBX18-24).

Supplemental Table S1. SlHY5 Y2H library screening results.

Supplemental Data Set 1. Primers used in this study.

Supplemental Data Set 2. Results of statistical analyses.

Supplementary Material

koac064_supplementary_data
Click here for additional data file. (1.2MB, zip)

Acknowledgments

We are grateful to Dr Roman Ulm for critical reading of the manuscript, to Dr Taotao Wang for insightful discussions.

Funding

This study was supported by the National Key Research and Development Program of China (2018YFD1000800), the National Natural Science Foundation of China (32102459, 31870261, and 32170246), a Startup Fund for Young Faculty at SJTU (21X010500765), Zhiyuan Scholar Program at SJTU to Y.Y and R.Y, Natural Science Foundation of Shanghai (22ZR1431300), and the Medicine and Engineering Interdisciplinary Research Fund of SJTU (YG2021ZD07).

Conflict of interest statement: The authors declare no conflict of interest.

Contributor Information

Guoqian Yang, Shanghai Cooperative Innovation Center for Modern Seed Industry/School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.

Chunli Zhang, Shanghai Cooperative Innovation Center for Modern Seed Industry/School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China; Key Laboratory of Urban Agriculture Ministry of Agriculture, Shanghai Jiao Tong University, Shanghai 200240, China; Joint Center for Single Cell Biology, Shanghai Jiao Tong University, Shanghai 200240, China.

Huaxi Dong, Shanghai Cooperative Innovation Center for Modern Seed Industry/School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.

Xiaorui Liu, Shanghai Cooperative Innovation Center for Modern Seed Industry/School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.

Huicong Guo, Shanghai Cooperative Innovation Center for Modern Seed Industry/School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.

Boqin Tong, Shanghai Cooperative Innovation Center for Modern Seed Industry/School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.

Fang Fang, Shanghai Cooperative Innovation Center for Modern Seed Industry/School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.

Yiyang Zhao, Shanghai Cooperative Innovation Center for Modern Seed Industry/School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.

Yunji Yu, Zhiyuan College, Shanghai Jiao Tong University, Shanghai 200240, China.

Yue Liu, Shanghai Cooperative Innovation Center for Modern Seed Industry/School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.

Li Lin, Shanghai Cooperative Innovation Center for Modern Seed Industry/School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China; Key Laboratory of Urban Agriculture Ministry of Agriculture, Shanghai Jiao Tong University, Shanghai 200240, China; Joint Center for Single Cell Biology, Shanghai Jiao Tong University, Shanghai 200240, China.

Ruohe Yin, Shanghai Cooperative Innovation Center for Modern Seed Industry/School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China; Key Laboratory of Urban Agriculture Ministry of Agriculture, Shanghai Jiao Tong University, Shanghai 200240, China; Joint Center for Single Cell Biology, Shanghai Jiao Tong University, Shanghai 200240, China.

 

R.Y. and G.Y. conceived the project. G.Y. performed most of the experiments. C.Z. generated the slhy5 mutants and contributed to Figure 9, A–G. H.D. generated slbbx21 mutants and SlBBX21 overexpression lines. X.L. generated the SlHY5-FLAG lines. H.G. contributed Figure 1 and Supplemental Figure S1, A and B. B.T. contributed Supplemental Figure S1C. F.F. contributed Supplemental Figure S2F and Y.L. contributed Figure 5A. Y.Y. participated in the analysis of the AtHY5-MYC lines. Y.Z. contributed analytic tools. G.Y., L.L., and R.Y. designed the experiments, analyzed the data, and wrote the manuscript.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell) is Ruohe Yin (ruohe.yin@sjtu.edu.cn).

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