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CRY1 interacts directly with HBI1 to regulate its transcriptional activity and photomorphogenesis in Arabidopsis

CRY1 interacts directly with HBI1 to regulate its transcriptional activity and photomorphogenesis... Cryptochromes (CRYs) are blue light photoreceptors that mediate various light responses in plants and animals. In Arabidopsis, there are two homologous CRYs, CRY1 and CRY2, which mediate blue light inhibition of hypocotyl elongation. It is known that CRY2 interacts with CIB1, a basic helix–loop–helix (bHLH) transcriptional factor, to regu- late transcription and floral induction. In this study, we performed yeast two-hybrid screening and identified CIB1 as a CRY1-interacting protein. Moreover, we demonstrated that CRY1 physically interacted with the close homolog of CIB1, HBI1, which is known to act downstream of brassinosteroid (BR) and gibberellin acid (GA) signaling pathways to promote hypocotyl elongation, in a blue light-dependent manner. Transgenic and genetic interaction studies showed that overexpression of HBI1 resulted in enhanced hypocotyl elongation under blue light and that HBI1 acted down- stream of CRYs to promote hypocotyl elongation. Genome-wide gene expression analysis indicated that CRYs and HBI1 antagonistically regulated the expression of genes involved in regulating cell elongation. Moreover, we dem- onstrated that CRY1–HBI1 interaction led to inhibition of HBI1’s DNA binding activity and its target gene expression. Together, our results suggest that HBI1 acts as a new CRY1-interacting protein and that the signaling mechanism of CRY1 involves repression of HBI1 transcriptional activity by direct CRY1–HBI1 interaction. Keywords: Arabidopsis, blue light, cell elongation, cryptochrome, HBI1, protein interaction, transcriptional activity. Introduction Plants on the earth are influenced by many kinds of environ- source of energy for photosynthesis but also regulatory signals for mental factors during their life cycle from seed germination to growth and development. Plants have evolved multiple photo- flowering, among which light provides not only the essential receptors to perceive light quality, quantity, and direction, which Abbreviations: 3AT, 3-amino-1,2,4-triazole;bHLH, basic helix–loop–helix; BR, brassinosteroid; BEE2, BR enhanced expression 2;CCT1, cryptochrome 1 C-terminal domain; CCT2, cryptochrome 2 C-terminal domain;CIB1, cryptochrome interacting basic helix–loop–helix 1; CIL1, CIB1-like protein 1; CNT1, cryptochrome 1 N-terminal domain; CNT2, cryptochrome 2 N-terminal domain; CO, CONSTANS; COP1, constitutively photomorphogenic 1; CRY1, cryptochrome 1; CRY2, cryp- tochrome 2; DWF4, dwarf 4; GA, gibberellin; HBI1, homolog of BEE2 interacting with IBH 1; IBH1, ILI1-binding bHLH protein 1; NB, nuclear body; PRE1, paclob- trazol resistance 1. © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 3868 | Wang et al. include the UV-B light photoreceptor UVR8, the blue light interacting basic helix–loop–helix1). It has been shown that photoreceptors cryptochromes (CRYs) and phototropins, and CRY2 interacts with CIB1 through its N-terminus to regulate the red/far-red light photoreceptors phytochromes (Cashmore the transcriptional activity of CIB1 and floral initiation (H. Liu et  al., 1999; Briggs and Christie, 2002; Quail, 2002; Rizzini et al., 2008). HBI1 (HOMOLOG OF BEE2 INTERACTING et al., 2011). Among these photoreceptors, cryptochromes play WITH IBH 1) is also a basic helix–loop–helix (bHLH) tran- critical roles not only in plants, but also in Drosophila and mam- scriptional factor that shows high amino acid sequence simi- mals. In Arabidopsis, there are two well-studied homologous larity to CIB1 and its homologs, CIL1 (CIB1 like protein CRYs, CRY1 and CRY2, which play relatively major roles in 1) and BEE2 (BR enhanced expression 2) (Bai et al., 2012a). regulating photomorphogenesis under blue light and photo- Previous studies have demonstrated that HBI1, as well as CIBs periodic flowering, respectively (Ahmad and Cashmore, 1993; (CIB4/5), CILs (CIL1/2) ,and BEEs (BEE1/2/3), have simi- Guo et al., 1998; Lin et al., 1998). Photomorphogenesis is one lar functions and work to promote hypocotyl elongation in of the critical and best studied light-controlled processes in Arabidopsis, respectively (Friedrichsen et  al., 2002; Bai et  al., Arabidopsis, which is characterized by shortened hypocotyls, 2012a; Ikeda et  al., 2012). It has been demonstrated that expanded cotyledons, and accumulation of chlorophyll under ILI1 BINDING bHLH PROTEIN1 (IBH1) interacts with light, and cryptochromes and phytochromes are shown to play HBI1 to inhibit HBI1 transcriptional activity and hypocotyl critical roles in regulating this process (Pepper et  al., 1994; elongation by repressing the DNA binding ability of HBI1, McNellis and Deng, 1995; Cashmore et  al., 1999; Deng and and that the inhibitory effects of IBH1 on HBI1 transcrip- Quail, 1999; Quail, 2002; Lin and Shalitin, 2003). Furthermore, tional activity is counteracted by PRE1 (PACLOBTRAZOL both CRY1 and CRY2 have been shown to entrain the circa- RESISTANCE1) through its direct interaction with IBH1 dian clock and mediate blue light induction of stomatal open- (Bai et  al., 2012a). Whether CRY1 interacts with CIB1 and ing and development (Somers et  al., 1998; Mao et  al., 2005; its homologs through its N-terminus to regulate hypocotyl Kang et al., 2009). In Drosophila, cryptochrome acts in the input elongation remains unknown. pathway to entrain the circadian clock (Emery et al., 1998) and, In this study, we sought potential CRY1 N-terminus in mammals, cryptochromes serve as integral components of (CNT1)-interacting proteins through yeast two-hybrid the central oscillator of the circadian clock (Kume et al., 1999). screening, and identified CIB1 as a candidate. We confirmed In migratory birds, cryptochromes act as the magnetoreceptors by yeast two-hybrid and protein co-localization assays that to perceive magnetic fields and navigate during long-distance CNT1 interacts with CIB1 and its homologs BEE2 and CIL1. migration (Gegear et al., 2010). Furthermore, we demonstrated by yeast two-hybrid, pull- Cryptochomes are structurally divided into an N-terminal down, protein co-localization, and co-immunoprecipitation domain that shares high sequence similarity to photolyases, and (co-IP) assays that CRY1 interacted with HBI1 in a blue light- a distinguishing C-terminal extension that is absent in pho- dependent manner. By making transgenic lines in which HBI1 tolyases (Sancar, 1994; Cashmore et al., 1999; Lin and Shalitin, function was either enhanced or repressed, we demonstrated 2003). It has been shown that the C-terminal domains of that HBI1 acted to regulate hypocotyl elongation positively Arabidopsis CRY1 and CRY2 mediate CRY1/2 signaling under blue, red, and far-red light, respectively. Moreover, by through direct interactions with COP1 (Yang et  al., 2000, creating transgenic lines in which HBI1 function was sup- 2001; Wang et  al., 2001), a RING-finger E3 ubiquitin ligase pressed in the cry1cry2 mutant background, we demonstrated (Deng et  al., 1992) that interacts with and targets the deg- that HBI1 acted downstream of CRY1 and CRY2 to regu- radation of a set of transcription factors, such as HY5/HYH late hypocotyl elongation under blue light. We demonstrated and CONSTANS (CO), to regulate photomorphogenesis and by genome-wide gene expression studies that CRY1/2 and flowering under long days, respectively (Putterill et  al., 1995; HBI1 acted antagonistically to regulate the expression of a set Oyama et  al., 1997; Osterlund et  al., 2000; Lee et  al., 2007; of genes related to cell elongation. We also showed by EMSA Jang et  al., 2008; L.J. Liu et  al., 2008). Moreover, CRY1 and and dual luciferase (Dual-LUC) assays that CRY1 inhibited CRY2 also interact with the COP1 enhancer, SPA1 (Lian the transcriptional activity of HBI1 through its N-terminus. et al., 2011; Liu et al., 2011; Zuo et al., 2011), which interacts This study therefore reveals a new mechanism of CRY1 sign- with COP1 to enhance its E3 ligase activity (Seo et al., 2003). aling, which involves direct blue light-dependent interaction The outcome of interactions of CRY1/CRY2 with COP1/ with the transcriptional factor HBI1 to regulate its transcrip- SPA1 is disruption of the COP1–SPA1 core complex, thus sta- tional activity and photomorphogenesis. bilizing HY5/CO proteins and promoting their accumulation. The N-terminal domain of CRY1 and CRY2 mediate CRY dimerization (Sang et al., 2005; Yu et al., 2007), and it is shown Materials and methods that CRY2 dimerization is inhibited by Blue-light Inhibitor of Plant materials and growth conditions Cryptochromes 1 (BIC1) (Q. Wang et al., 2016). Recently, the Arabidopsis thaliana ecotype Columbia (Col-0) was used as the wild-type CRY1 N-terminus has been shown to mediate CRY1 signal- (WT) control. cry1cry2, phyA-211, and phyB-9 mutants, and transgenic ing independent of the CRY1 C-terminus (He et  al., 2015). lines overexpressing Myc-CRY1 (CRYI-OX), Myc-CRY2 (CRY2-OX), However, the underlying mechnism is not well understood. and GUS-CCT1 (GUS-CCT1) were described previously (Yang et  al., The discovery showing direct cryptochrome regulation of 2000; Mao et al., 2005; Sang et al., 2005). HBI1-related transgenic plants transcription comes from screening for CRY2-interacting fac- were generated using corresponding constructs by an in planta method (Bechtold and Pelletier, 1998). For phenotype analyses of seedlings, seeds tors, which leads to the identification of CIB1 (cryptochrome Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 Regulation of photomorphogenesis by CRY1–HBI1 interaction | 3869 were sown on half-strength Murashige and Skoog (MS) medium plus (~0.2 μg) of prey proteins of His-TF, His-TF–CNT1, and His-TF–CCT1 1% sucrose with 0.8% agar and were cold treated for 4 d at 4 °C. After in a final volume of 1 ml at 4 °C for 0.5 h. Then the beads were washed exposure to white light for 12  h to promote germination, plates with with TBST three times, dissolved in 30 μl of 2× SDS loading buffer, and seeds were transferred to appropriate light conditions for 4 d at 22 °C. subjected to western blot or Coomassie Brilliant Blue staining. For adult plants, seeds were sown as above and grown under continuous For the semi-in vivo pull-down assay with Arabidopsis protein extracts, white light for 7 d, then transferred to soil to continue growing for the CRY1-OX seedlings grown in soil for ~2 weeks under white light were time indicated. kept in darkness for 2 d before they were transferred to red light (30 μmol –2 –1 –2 –1 –2 –1 m s ), blue light (50 μmol m s ), far-red light (10 μmol m s ), or continuing darkness for 3 h. These seedlings were quickly frozen by liquid Plasmid construction nitrogen and homogenized with TBST buffer containing 1 mM protein- ase inhibitor (Pefabloc and Cocktail, Roche). The supernatant adjusted The constructs expressing COP1 in yeast, and cLUC-Flag, CRY1–yel- to contain the same amount of total protein quantified by Bradford assay low fluorescent protein (YFP), CRY2–YFP, β-glucuronidase (GUS)– (Biorad) was infused to tubes containing MBP–HBI1-conjugated beads, nuclear localization signal (NLS)–YFP, and CNT1–NLS–YFP in incubated for 30 min at 4 °C, and washed three times with 1 ml of TBST plants were described previously (Lian et  al., 2011; He et  al., 2015; Xu buffer each time, then the precipitates were eluted into 30 μl of 2× SDS et  al., 2016). PCR-amplified fragments encoding HBI1, HBI1– ERF- loading buffer and subjected to western blot analysis with anti-Myc anti- associated amphiphilic repression (EAR), CNT1–NLS, NLS–CCT1, body (Millipore). CIB1, CNT2–NLS, NLS–CCT2, CRY2, BEE2, CIL1, CO, and NLS– GUS–CCT1 were cloned into the multiple cloning sites (MCS) of pHB- MCS-3×Flag, pHB-6×Myc-MCS, pGBKT7, pGADT7, pHB-MCS-YFP Protein co-localization assay [cyan fluorescent protein (CFP)], and pHB-YFP (CFP)-MCS, respect- ively. All of the constructs and primers used in this study are listed in Agrobacterium tumefaciens strain GV3101 harboring the constructs express- Supplementary Table S1 at JXB online. ing the studied proteins or p19 plasmid were diluted by MS liquid medium to OD =0.6 individually and treated with 200 μM acetosyrin- gone and 10 mM MES (pH 5.6) for 3 h at room temperature. Then the Analyses of RNA-Seq data mixture of Agrobacterium harboring the constructs expressing CFP and YFP fusion proteins, and p19 plasmid with a volume ratio of 1:1:1 were The RNA sequencing (RNA-Seq) data indicating the genes regu- introduced into tobacco (Nicotiana benthamiana) leaf epidermal cells by lated by HBI1 and CRYs have the accession numbers GSE53078 and infiltration. After incubation for 2 d or 3 d in dim light, leaves were col- GSE58552, respectively (Fan et al., 2014; He et al., 2015). A Venn diagram lected for confocal microscopic examination (Leica TCS SP5II confocal was generated in Venny (http://bioinfogp.cnb.csic.es/tools/venny/index. laser scanning microscope). html). A heatmap was generated with hierarchical clustering analysis by the MeV 4.7 software. Gene Ontology (GO) analysis was performed in agriGO v2.0 (http://systemsbiology.cau.edu.cn/agriGOv2/) (Tian et al., Co-immunoprecipitation assay 2017), with TAIR10 being as the reference and hypergeometric statistical test method and complete GO type. Tobacco leaves were infiltrated with a mixture of Agrobacterium culture (OD =0.6) harboring the constructs expressing p19, HBI1-Flag, and YFP fusion proteins or p19, CNT1–NLS–YFP, and Flag-tagged fusion Quantitative real-time PCR proteins, with a volume ratio of 1:1:1 and then kept in dim light for 3 d as for co-localization assays. Samples from tobacco leaves were collected We used WT, cry1cry2, CRY1-OX, CRY2-OX, HBI1-EAR-OX/cry1cry2, and homogenized in TBST containing proteinase inhibitors. After cen- and HBI1-OX seedlings for quantitative real-time PCR (qRT-PCR). trifugation, protein supernatant was incubated with 10 μl of protein G Seeds were sown as above and then subjected to white light treatment for magnetic beads (Invitrogen) [which had been incubated with 1  μl of 12 h to promote germination. The plates with seeds were kept in darkness anti-Flag antibody (Sigma) overnight at 4 °C] for 1 h at 4 °C. The immu- for 4 d before being exposed to blue light irradiation. Total RNAs were noprecipitate was washed three times with TBST and eluted with 0.5 μg isolated according to the manufacturer’s instructions (TIANGEN). The –1 μl 3×Flag peptide (Sigma) in 30 μl of TBST. A 24 μl aliquot of eluate detailed method of qRT-PCR was described previously (Zhang et  al. was resuspended with 6 μl of 5× SDS loading buffer, boiled for 5 min, 2014). PP2A was used as an internal control. The primers used are listed and then subjected to immunoblot analysis with anti-Flag (Sigma) and in Supplementary Table S1. anti-green fluorescent protein (GFP; Abmart) antibodies. Yeast two-hybrid assay Hypocotyl measurement Yeast two-hybrid assay was performed according to the user manual Seedlings were photographed, and then the digital photographs were for the Yeastmaker Yeast Transformation System (Clontech Laboratories, utilized to measure hypocotyl length through Image J software (http:// Mountain View, CA, USA). Combinations containing the fragments fused rsbweb.nih.gov/ij/). in pGBKT7 and pGADT7 vectors were co-transformed into the AH109 strain via the PEG/LiAc transformation procedure. At 3 d after trans- formation, yeast cells were spread on selective media with 3-amino-1,2,4- Western blotting triazole (3AT) in continuous blue light or darkness for 3 d. Arabidopsis seeds of different genotypes were grown on half-strength MS medium under continuous white light for 5 d. Seedlings with differ- Pull-down assay ent light treatments were collected and homogenized in TBST described above, and the protein supernatant, after quantification by Bradford For in vitro pull-down assay, the fragments encoding CNT1, CCT1, and assay (BioRad), was subjected to immunoblot analysis with the related HBI1 were each cloned into pCold-trigger factor (TF) or pMAL-c2X antibody. vector. The fusion proteins were expressed in Escherichia coli (Rosetta) and purified according to the manufacturer’s protocol (QIAGEN and NEB). Maltose-binding protein (MBP)–HBI1 was first incubated with EMSA amylose–magnetic beads (NEB) in TBST buffer (50 mM Tris–HCl, pH 7.5, 150  mM NaCl, 0.2% Triton X-100, and 10% glycerol) containing The proteins used in this experiment were expressed and purified as 1 mM proteinase inhibitor (Pefabloc, Roche) at 4 °C for 4 h, then the described above prior to being quantified by comparison with the BSA amylose–magnetic beads affiliated with MBP–HBI1 serving as bait was standard, then 1.5 μg of MBP and 0.5 μg of MBP–HBI1 were utilized. washed with TBST three times and incubated with the same amounts Biotin-labeled probes were commercially synthesized and annealed. The Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 3870 | Wang et al. binding reaction was carried out in 20 μl of reaction buffer [10× Binding prompted us to look for potential CNT1-interacting proteins. –1 buffer, 100 mM MgCl , 1 μg μl poly(dI dC), 1% NP-40] with 20 fM To this end, we carried out GAL4 yeast two-hybrid screening probe and purified proteins according to the manufacturer’s instructions against CNT1 and identified 15 clones encoding CIB1, a bHLH (Thermo Scientific). The reactions were resolved by 6% native PAGE at transcriptional factor with which CRY2 interacts through its 4 °C after 15 min incubation at room temperature, and then the DNA– N-terminus to regulate photoperiodic flowering (H. Liu et al., protein complexes were transferred to a nylon membrane. Biotin-labeled probes were detected by horseradish peroxidase (HRP)-conjugated 2008). To confirm the interaction of CNT1 with CIB1, we streptavidin and visualized with an ECL detection kit. performed GAL4 yeast two-hybrid assays by co-transforming yeast cells with the bait construct expressing CNT1 fused to the GAL4 DNA-binding domain (BD) tagged by an NLS sequence Dual-LUC assay (Fig. 1A), together with a prey construct expressing the GAL4 An ~2  kb fragment upstream of the start codon of Exp16 was ampli- fied and cloned into pGreen0800 vector, which expresses Renilla lucif- activation domain (AD) fused to CIB1 (Fig.  1B). The results erase (REN) driven by the 35S promoter serving as an internal reference showed that CNT1 interacted with CIB1 (Fig.  1C). We then and firefly luciferase (LUC) driven by the Exp16 promoter serving as a performed an in vivo protein co-localization assay by making reporter. Agrobacterium culture (OD =0.6) harboring the related con- constructs expressing CNT1 fused to the NLS followed by YFP, structs was mixed with the volume ratio indicated in Fig.  8F, G and CIB1, and its homologs BEE2 and CIL1 fused to cyan CFP Supplementary Fig. S7; the lack of any effector in all groups was supple- mented with liquid MS medium to the same volume. The mixture was (Fig.  1D). As shown in Fig.  1E, expression of CNT1–NLS– introduced into tobacco leaves by infiltration and kept in dim white light YFP produced nuclear bodies (NBs) in tobacco leaf epidermal for 3 d, and then the samples were collected for measurements of lucif- cells, whereas expression of CFP–CIB1, CFP–BEE2, or CIL1– erase activity using commercial reagents according to the manufacturer’s CFP did not. However, when co-expressed with CNT1–NLS– instructions (Promega). YFP, CFP–CIB1, CFP–BEE2, and CIL1–CFP proteins were co-localized to the same NBs as CNT1–NLS–YFP, indicating interactions of CNT1 with these proteins. Results The CRY1 N-terminus interacts with CIB1 and its CNT1 physically interacts with HBI1 in yeast cells and homologs BEE2 and CIL1 in yeast and tobacco cells in vitro Our previous demonstration that CNT1 is able to mediate Among the CIBs and CIB-like proteins, HBI1 is preferen- CRY1 signaling independent of its C-terminus (He et al., 2015) tially expressed in hypocotyl and cotyledon, and acts as a Fig. 1. CNT1 physically interacts with CIB1, BEE2, and CIL1 in yeast and tobacco cells. (A) Bait protein in yeast two-hybrid assay. CNT1 is fused with the GAL4-binding domain (BD) and nuclear localization signal (NLS) sequence. (B) Prey protein in yeast two-hybrid assay. CIB1 is fused with the GAL4 activation domain (AD). (C) Analysis of CNT1–CIB1 interaction in yeast cells. In this and other figures, all vector combinations are given as bait/prey. Yeast cells co-expressing CNT1/CIB1 were grown on basic (SD-W-L) or selective media with 10 mM 3AT (SD-W-L-H-A+3AT) in darkness for 3 d. (D) Schematic diagram of constructs expressing CNT1 fused to the NLS sequence and yellow fluorescent protein (YFP) and constructs expressing CIB1, BEE2, and CIL1 fused to cyan fluorescent protein (CFP). (E) Co-localization assay showing that CNT1 and these homologous proteins, CIB1, BEE2, and CIL1, localize together to the same nuclear bodies (NBs) in tobacco cells. Dic, differential interference contrast. Scale bars=5 μm. Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 Regulation of photomorphogenesis by CRY1–HBI1 interaction | 3871 positive regulator of cell elongation downstream of the BR CRY1 interacts with HBI1 through its N-terminus in (brassinosteroid) and GA (gibberellin acid) signaling pathways plant cells through the PREs (PACLOBTRAZOL RESISTANCE1)– With the demonstration that CNT1 interacts with HBI1 in IBH1 (ILI1 BINDING bHLH PROTEIN1)–HBI1 module yeast cells and in an in vitro pull-down assay, we asked whether (Bai et al. 2012a). We therefore focused on exploring whether CNT1 might interact with HBI1 in vivo. To test this possibility, CRY1 might interact directly with HBI1 through CNT1 we first performed a protein co-localization assay in tobacco to regulate hypocotyl elongation. To test this possibility, we cells using a construct expressing HBI1 tagged by CFP (Fig. 3A) first performed a yeast two-hybrid assay with bait constructs and a construct expressing CNT1–NLS–YFP (Fig.  1D). The expressing CNT1–NLS (Fig. 1A) and the CRY1 C-terminus results showed that, CO–CFP (Fig.  3A) and CNT1–NLS– fused to an NLS (NLS–CCT1) (Fig. 2A), and prey constructs YFP expressed in the same tobacco cells were not merged expressing the full-length HBI1 (Fig.  2B) and COP1 (Lian together and localized to different NBs, while CNT1–NLS– et al., 2011), respectively. As shown in Fig. 2C, CNT1 inter- YFP and HBI1–CFP proteins were co-localized to the same acted with HBI1 in both darkness and blue light, whereas NBs (Fig. 3B), indicating a possible interaction of CNT1 with CCT1 did not interact with HBI1 in either darkness or blue HBI1. Next, we performed a co-IP assay with tobacco leaves light but interacted with the positive control, COP1 (Yang transiently co-expressing CNT1–NLS–YFP, YFP–NLS–GUS– et  al., 2001). These results indicated that CNT1 interacted CCT1, or GUS–NLS–YFP with Flag-tagged HBI1 proteins with HBI1 in yeast cells. We then performed an in vitro pull- to confirm the interaction of CNT1 with HBI1. The results down assay to confirm the interaction of CNT1 with HBI1 showed that CNT1 was immunoprecipitated by HBI1, but not using CNT1 and CCT1 polypeptides tagged by His-tagged by CCT1 or the control protein GUS (Fig. 3C), further indicat- TF, and HBI1 protein tagged by MBP expressed in E. coli. The ing an interaction of CNT1 with HBI1. Moreover, co-IP assay results showed that CNT1 was pulled-down by HBI1, but with tobacco leaves transiently co-expressing Flag-tagged HBI1 CCT1 was not (Fig.  2D). These results indicate that CNT1 or cLUC (the C-terminus of firefly luciferase) with CNT1– rather than CCT1 might mediate the interaction of CRY1 NLS–YFP confirmed that the binding of CNT1 to HBI1 is with HBI1. Fig. 2. CNT1 physically interacts with HBI1 in yeast cells and in vitro. (A) Bait protein. CCT1 is fused with the GAL4-binding domain (BD) and NLS sequence. (B) Prey protein. HBI1 is fused with the GAL4 activation domain (AD). (C) HBI1 interacts with CNT1 rather than CCT1 in yeast cells. Yeast cells –2 –1 co-expressing CNT1/HBI1 were grown on basic (SD-W-L) or selective media with 10 mM 3AT (SD-W-L-H-A+3AT) in continuous 30 μmol m s blue light (BL) or darkness (DK) for 3 d at the top or cells co-expressing CCT1/HBI1 were grown on basic (SD-W-L) or selective media with 2 mM 3AT (SD-W- L-H-A+3AT) in darkness for 3 d at the bottom. (D) MBP pull-down assay showing the interaction of HBI1 with CNT1 rather than CCT1. CBB Staining denotes Coomassie Brilliant Blue staining. His-TF was used as negative control. All these experiments were independently repeated three times. Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 3872 | Wang et al. Fig. 3. CRY1 physically interacts with HBI1 through its N-terminus in a blue light-dependent manner. (A) Schematic diagram of constructs expressing HBI1 and the negative control CO fused to CFP. (B) Co-localization assay of studied proteins in tobacco cells. CNT1 and HBI1 localize together to the same NBs, whereas CNT1 and CO localize to different NBs. Dic, differential interference contrast. Scale bars=5 μm. (C) Co-IP assay showing interaction of CNT1 with HBI1. YFP-tagged CNT1, CCT1, and the negative control, GUS protein, were co-expressed with Flag-tagged HBI1 in tobacco leaves. The immunoprecipitates were detected by anti-GFP and anti-Flag antibodies. A single asterisk and double asterisks denote a non-specific band and the heavy chain of IgG, respectively. (D) Co-IP assay showing that CNT1 interacts specifically with HBI1. Flag-tagged HBI1 and the negative control, cLUC protein, were co-expressed with YFP-tagged CNT1 in tobacco leaves. The immunoprecipitates were detected by anti-Flag and anti-GFP antibodies. A single asterisk and double asterisks denote a non-specific band and the heavy chain of IgG, respectively. (E) Semi-in vivo pull-down assay showing the blue light-specific interaction of CRY1 with HBI1. MBP–HBI1 served as bait. Myc-CRY1-containing protein extracts from CRY1-OX seedlings that were adapted to darkness –2 –1 –2 –1 –2 –1 (DK) or exposed to 50 μmol m s blue (BL), 30 μmol m s red (RL), or 10 μmol m s far-red (FRL) light for 3 h served as preys. CBB Staining denotes Coomassie Brilliant Blue staining. All experiments above were independently repeated at least three times. specific (Fig.  3D). Furthermore, we performed a semi-in vivo whereas CRY2 regulates this process mainly under low inten- pull-down assay using MBP–HBI1 fusion protein as a bait, and sity of blue light (Lin et  al., 1998). Given the previous dem- Myc-CRY1-containing protein extracts as preys, which were onstration that the signaling mechanism of both CRY1 and prepared from Arabidopsis seedlings overexpressing Myc-CRY1 CRY2 involves direct interactions with COP1, SPAs, and PIFs (CRY1-OX) adapted in the dark and exposed to blue, red, and (Wang et al., 2001; Yang et al., 2001; Lian et al., 2011; Liu et al., far-red light. The results showed that MBP–HBI1 pulled-down 2011; Ma et  al., 2016; Pedmale et  al., 2016), we first exam- Myc-CRY1 from CRY1-OX seedlings exposed to blue light, ined whether CRY2 might also interact with HBI1 through but not from those either adapted in darkness or exposed to red yeast two-hybrid assay using the bait construct expressing the or far-red light (Fig. 3E). These results indicate that CRY1 inter- full-length CRY2 (Fig. 4A) and the prey construct expressing acts with HBI1 in a blue light-dependent manner. HBI1 (Fig. 2B). The results showed that the full-length CRY2 interacted with HBI1 in the dark and in blue light (Fig. 4B). To determine further whether the CRY2 N-terminus or CRY2 interacts with HBI1 through its N-terminus C-terminus (designated as CNT2 and CCT2) might medi- It has been demonstrated that CRY1 regulates hypocotyl ate the interaction of CRY2 with HBI1, we made bait con- elongation under both low and high intensity of blue light, structs expressing CNT2–NLS and NLS–CCT2, respectively Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 Regulation of photomorphogenesis by CRY1–HBI1 interaction | 3873 Fig. 4. CRY2 interacts with HBI1 through its N-terminus in yeast and tobacco cells. (A) Bait proteins. All proteins, CNT2, CCT2, and CRY2, are fused with the GAL4 binding domain (BD), among which CNT2 and CCT2 are also tagged with the NLS sequence. (B) The physical interaction of CRY2 and HBI1 analyzed in yeast cells. Yeast cells co-expressing CRY2/HBI1 were grown on basic (SD-W-L) or selective media with 5 mM 3AT (SD-W-L-H+3AT) –2 –1 in continuous 30 μmol m s blue light (BL) or darkness (DK) for 3 d. (C) The physical interaction of CNT2 and HBI1 analyzed in yeast cells. Yeast cells –2 –1 co-expressing CNT2/HBI1 were grown on basic (SD-W-L) or selective media with 5 mM 3AT (SD-W-L-H+3AT) in continuous 30 μmol m s blue light (BL) or darkness (DK) for 3 d at the top or cells co-expressing CCT2/HBI1 were grown on basic (SD-W-L) or selective media with 2 mM 3AT (SD-W-L- H+3AT) in darkness for 3 d at the bottom. (D) Schematic diagram of constructs expressing CNT2 tagged by the NLS sequence and CRY2 fused to YFP. (E) Co-localization assay showing that HBI1 co-localizes in the same NBs with CNT2 and CRY2 in tobacco cells. Dic, differential interference contrast. Scale bars=5μm. (Fig. 4A), and performed a yeast two-hybrid assay again with and light signaling pathways to promote hypocotyl elongation these bait constructs and the prey constructs expressing HBI1 (Bai et  al., 2012a). Our demonstration that CRY1 physically and COP1. As shown in Fig. 4C, CNT2 interacted with HBI1 interacts with HBI1 suggests that CRY1-mediated blue light in blue light, but not in darkness, whereas CCT2 did not, but inhibition of hypocotyl elongation might proceed, at least in interacted with the positive control COP1 (Wang et al., 2001). part, through HBI1. To define a role for HBI1 in regulating Moreover, we performed a protein co-localization study to hypocotyl elongation under blue light, we first generated trans- confirm the interactions of CRY2 and CNT2 with HBI1 genic lines overexpressing HBI1 tagged with Flag (HBI1-OX) in tobacco cells using the constructs expressing CRY2–YFP (Fig.  5A, B), in which the expression of HBI1-Flag fusion and CNT2–NLS–YFP (Fig.  4D), and the construct express- protein was detected by western blot (Fig. 5C), and analyzed ing HBI1–CFP (Fig. 3A), respectively. The results showed that the hypocotyl elongation phenotype in blue light. The results HBI1 was localized to the same NBs of CRY2 and CNT2 showed that these lines exhibited a significantly taller hypoco- (Fig. 4E), indicating interactions of CRY2/CNT2 with HBI1. tyl phenotype than the WT under blue light (Fig. 5B, D). We Taken together, these results suggest that, like CRY1, CRY2 then made a construct expressing Myc-HBI1 fused to the EAR interacts with HBI1 through its N-terminus. motif (HBI1-EAR) (Fig. 5E), which is shown to inactivate the function of the native HBI1 and its close homologs (Bai et al., 2012a; Liu et al., 2013), and generated transgenic lines overex- HBI1 positively regulates hypocotyl elongation under pressing Myc-HBI1–EAR (HBI1-EAR-OX). Analysis of the blue, red, and far-red light hypocotyl phenotype demonstrated that these lines had sig- It has been demonstrated that HBI1 functions in a triantag- nificantly shorter hypocotyls than the WT under blue light, onistic cascade system downstream of BR, GA, temperature and that the severity of this shortened hypocotyl phenotype Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 3874 | Wang et al. Fig. 5. HBI1 acts as a positive regulator of hypocotyl elongation under blue light. (A and E) Schematic diagram depicting the constructs expressing fusion proteins of HBI1-Flag and Myc-HBI1–EAR used for transformation of WT plants. (B and F) Seedling phenotypes of HBI1-OX and HBI1-EAR-OX –2 –1 transgenic plants grown on half-strength MS medium under 2 μmol m s blue light for 5 d. Scale bars=5 mm. (C) Western blot analysis showing HBI1-Flag fusion protein expression in the corresponding lines shown in (B). An asterisk denotes a non-specific band recognized by the antibody, which served as a loading control. HBI1/* denotes the relative band intensities of HBI1-Flag normalized to the non-specific band and presented relative to that in HBI1-OX #2 set at unity. (D) Statistical analysis of hypocotyl length of seedlings shown in (B). Data are presented as means ±SD. Asterisks denote a significant difference between the indicated lines and the WT (t-test, P<0.001), n=30. (G) Western blot analysis showing Myc-HBI1–EAR fusion protein expression in the corresponding lines shown in (F). Actin served as a loading control. HBI1-EAR/Actin indicates the relative band intensities of Myc-HBI1– EAR normalized to Actin and presented relative to that in HBI1-EAR-OX #3 set at unity. (H) Statistical analysis of hypocotyl length of seedlings shown in (F). Data are presented as means ±SD. Asterisks denote a significant difference between the indicated lines and the WT (t-test, P<0.001), n=30. was positively correlated with the HBI1–EAR protein levels promote hypocotyl elongation preferentially under low fluence (Fig. 5F–H), confirming a positive role for HBI1 in regulating rates of blue light. We further analyzed the hypocotyl pheno- hypocotyl elongation under blue light. type of HBI1-OX and HBI1-EAR-OX plants in darkness, red, Next, we analyzed the blue light fluence rate response of and far-red light. The results demonstrated that HBI1-OX HBI1-OX and HBI1-EAR-OX seedlings, and found that the seedlings developed slightly taller hypocotyls than the WT in hypocotyls of both lines were shortened as the fluence rate of these conditions, whereas HBI1-EAR-OX seedlings had dra- blue light increased, and that the hypocotyls of HBI1-OX seed- matically shorter hypocotyls than the WT (Supplementary lings were always taller than those of the WT at all blue light Fig.  S2). Furthermore, we analyzed the red and far-red light fluence rate examined, whereas those of HBI1-EAR-OX seed- fluence rate responses of HBI1-OX and HBI1-EAR-OX seed- lings were always shorter than those of the WT (Supplementary lings, and found that both lines basically displayed a similar Fig. S1). However, the highest hypocotyl length difference be- tendency as observed under different fluence rates of blue light tween HBI1-EAR-OX and the WT was observed at a fluence (Supplementary Fig. S3), and that the highest hypocotyl length –2 –1 rate <2 μmol m s , indicating that HBI1 might function to difference between HBI1-EAR-OX and the WT under red Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 Regulation of photomorphogenesis by CRY1–HBI1 interaction | 3875 Fig. 6. HBI1 acts genetically downstream of CRY genes to promote hypocotyl elongation. (A) Seedling phenotypes of the transgenic lines overexpressing –2 –1 HBI1-EAR in the cry1cry2 mutant background (HBI1-EAR-OX/cry1cry2) grown on half-strength MS medium under 2 μmol m s blue light for 5 d. Scale bar=5 mm. (B) Statistical analysis of hypocotyl length of seedlings shown in (A). Data are presented as means ±SD. Asterisks denote a significant difference between the indicated lines and cry1cry2 double mutant (t-test, P<0.001), n=30. (C) Western blot analysis showing Myc-HBI1–EAR fusion protein expression in corresponding lines shown in (A). Actin served as a loading control. HBI1-EAR/Actin indicates the relative band intensities of Myc- HBI1–EAR normalized to Actin and presented relative to that in HBI1-EAR-OX/cry1cry2 #6 set at unity. (D) Seedling phenotype of siblings segregated –2 –1 from the T heterozygous HBI1-EAR-OX/cry1cry2 #9 grown on half-strength MS medium under 2 μmol m s blue light for 5 d. Scale bar=5 mm. S, M, and L denote the segregated seedlings with short, medium, and long hypocotyls, respectively, and the numbers of these seedlings are shown under these letters. –2 and far-red light was observed at a fluence rate <30 μmol m under blue light. The expression of the Myc-HBI1–EAR –1 –2 –1 s and 1  μmol m s , respectively (Supplementary Fig.  S3). fusion protein in these lines was detected by western blot These results suggest that HBI1 might also promote hypocotyl (Fig. 6C). We selected a heterozygous HBI1-EAR-OX/cry1cry2 elongation preferentially under low fluence rates of red and line (HBI1-EAR-OX/cry1cry2 #9) to analyze the hypocotyl far-red light. Since the HBI1-EAR-OX lines that expressed phenotype in T . As shown in Fig. 6D, the siblings segregated high level of HBI1–EAR had extremely compact morph- from the T heterozygous line were divided into three different ology and failed to survive after transfer to soil, we selected classes of seedlings with short, medium, and very long hypoco- two heterozygous HBI1-EAR-OX lines (HBI1-EAR-OX #3 tyls at a ratio of ~1:2:1, further indicating that the severity and #9) to analyze the hypocotyl phenotype in T . As shown 2 of the shortened hypocotyl phenotype is positively correlated in Supplementary Fig. S4, the T siblings segregated from these 2 with the HBI1–EAR protein levels. These results suggest that heterozygous lines were divided into three different classes of HBI1 acts downstream of CRY genes to regulate hypocotyl seedlings with short, medium, and long hypocotyls at a ratio elongation under blue light. of ~1:2:1, further implying that the severity of the shortened We also examined the phenotypes of adult plants of hypocotyl phenotype is positively correlated with the HBI1– the WT, cry1cry2, HBI1-OX, HBI1-EAR-OX, and HBI1- EAR protein levels. Taken together, these results suggest that EAR-OX/cry1cry2, and observed that HBI1-OX and cry1cry2 HBI1 positively regulates hypocotyl elongation in darkness mutant plants developed longer rosette leaves and siliques and in blue, red, and far-red light. than the WT, whereas CRY1-OX plants had shorter rosette leaves and siliques than the WT, and that HBI1-EAR-OX HBI1 acts downstream of CRYs to promote hypocotyl plants hardly developed functional rosette leaves and siliques elongation (Supplementary Fig. S5). Moreover, HBI1-EAR-OX/cry1cry2 plants had much shorter rosette leaves and siliques than the With the demonstration that CRYs physically interact with HBI1, we next explored the genetic interaction of CRY genes cry1cry2 mutant. Taken together, these results suggest that with HBI1 by generating transgenic cry1cry2 mutant plants CRYs and HBI1 act antagonistically to regulate not only overexpressing Myc-HBI1–EAR (HBI1-EAR-OX/cry1cry2). hypocotyl elongation during early seedling photomorpho- As shown in Fig. 6A, B, HBI1-EAR-OX/cry1cry2 lines devel- genesis, but also leaf and silique elongation during vegetative oped dramatically shorter hypocotyls than the cry1cry2 mutant and reproductive development. Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 3876 | Wang et al. Fig. 7. CRYs and HBI1 antagonistically regulate the expression of genes promoting cell elongation. (A) Venn diagram showing the overlap of genes regulated by HBI1 and CRYs. (B) Hierarchical cluster analysis of 794 overlapping genes shown in (A). Red and green colors in the heatmap represent induced and repressed genes, respectively. The gradient bar denotes the log -fold change relative to the control sample. (C and D) qRT-PCR analyses showing the expression of direct target genes of HBI1 in multiple genotypes indicated. All the seedlings were grown in darkness for 5 d and then –2 –1 transferred to 30 μmol m s blue light for 0.5 h. Expression levels were normalized to an internal control PP2A, and the WT level was arbitrarily set to 1. Data are represented as means ±SD (n=3). The lower case letters ‘a’ to ‘d’ indicate statistically significant differences among means for gene expression levels of the indicated genotypes, as determined by Tukey’s LSD test (P≤0.01). CRYs and HBI1 antagonistically regulate the and HBI1 were largely involved in the regulation of the bio- logical processes including cell wall organization and cell wall expression of a set of genes related to cell elongation loosening, whereas those co-regulated by CRYs and HBI1 in To explore whether the regulation of hypocotyl elongation by the same direction were not (Supplementary Fig. S6). CRYs and HBI1 might proceed through the co-regulation of We performed qRT-PCR analysis of five representative genes related to cell elongation, we first compared the 8498 direct target genes of HBI1, which are positively regulated genes regulated by CRYs with the 1239 genes regulated by by HBI1 and known to promote hypocotyl elongation (Fan HBI1, which were obtained through RNA-Seq (Fan et al., 2014; et al., 2014), in WT , cry1cry2 mutant, CRY1-OX, and CRY2-OX He et al., 2015). We found that, of the 1239 HBI1-controlled seedlings that were exposed to blue light. As shown in Fig. 7C, genes, 794 genes (64.1%) were also affected by CRYs (Fig. 7A). the expression of these genes was enhanced to varying degrees A heatmap revealed that, among these co-regulated genes, 393 in the cry1cry2 mutant, but reduced to varying degrees in genes (50%) were affected in the opposite way (Fig. 7B). GO CRY1-OX and CRY2-OX plants. We also analyzed the expres- analysis suggests that the genes oppositely regulated by CRYs sion of these genes in HBI1-EAR-OX/cry1cry2 and HBI1-OX Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 Regulation of photomorphogenesis by CRY1–HBI1 interaction | 3877 seedlings exposed to blue light and found that these genes Discussion were expressed at significantly lower levels in HBI1-EAR-OX/ Previous reports have revealed that CRY1 and CRY2 inter- cry1cry2 than in the cry1cry2 mutant and HBI1-OX seedlings act with COP1 through their C-terminus to inhibit COP1 (Fig. 7D). Taken together, these results indicate that CRY in- activity and enhance the accumulation of HY5 to promote hibition of hypocotyl elongation is mediated, at least in part, photomorphogenesis (Yang et  al., 2000, 2001; Wang et  al., through repression of the expression of HBI1 target genes that 2001). Moreover, later studies introduced SPAs into CRY act to promote cell elongation. signaling pathway and demonstrated that CRYs interact with SPAs in a blue light-dependent manner, resulting in inhib- CRY1 inhibits the transcriptional activity of HBI1 ition of the function of the COP1–SPA complex and further through its N-terminus repression of COP1 activity (Lian et al., 2011; Liu et al., 2011; Zuo et al., 2011). These studies led to the establishment of the To explore how CRY1 might regulate HBI1 activity, we ana- CRY signaling cascade, which consists of CRYs, SPAs, COP1, lyzed whether HBI1 gene expression or HBI1 protein expres- and HY5. However, recent studies suggest that the CRY1 sion might be regulated by blue light using the dark-grown N-terminus and C-terminus probably regulate hypocotyl WT seedlings or HBI1-OX seedlings exposed to blue light elongation through different mechanisms (W.X. Wang et  al., for increasing lengths of time. The results showed that nei- 2016) and that the CRY1 N-terminus is able independently ther HBI1 gene expression nor HBI1 protein expression was to confer enhanced blue light inhibition of hypocotyl elong- affected by blue light (Fig. 8A, B). We then explored whether ation (He et al., 2015). Therefore, the CRY1 N-terminus may CRY1 might regulate the DNA binding activity of HBI1 function independently of the CRY1 C-terminus to medi- through CRY1–HBI1 interaction. To do this, we performed an ate CRY1 signaling by interacting with downstream com- EMSA to investigate whether the capacity of HBI1 to bind to ponents. Most recently, two reports have shown that CRYs the sequence of the promoter of one HBI1 target gene, DWF4 interact with PIF bHLH transcription factors to regulate (DWARF 4), whose expression is inhibited by CRYs (Fig. 7C), their transcriptional activity and mediate responses to canopy might be inhibited by CNT1. The control experiment showed shade, low blue light, or high temperature (Ma et  al., 2016; that HBI1 bound to the promoter sequence of the DWF4 Pedmale et al., 2016). gene that contains the E box sequence (CANNTG) in a pro- CRY1 is the primary blue light photoreceptor mediating tein concentration-dependent manner, and that the binding blue light inhibition of hypocotyl elongation. The loss-of- capacity was reduced by the competition from a cold com- function mutant of Arabidopsis CRY1 shows a dramatically petitor (Fig. 8C), whereas this binding was not detectable with enhanced hypocotyl elongation phenotype, whereas transgenic the control protein MBP. As anticipated, the DNA fragment plants overexpressing CRY1 display a considerably shortened bands shifted by HBI1 decreased as the amounts of CNT1 hypocotyl phenotype (Ahmad and Cashmore, 1993; Lin et al., increased (Fig. 8D), whereas they were not affected by increas- 1996). In this study, we further defined a role for a bHLH ing amounts of CCT1. These results indicated that CNT1 but transcription factor HBI1 in promoting hypocotyl growth in not CCT1 inhibited the DNA binding activity of HBI1 in blue, red, and far-red light. The results presented here demon- vitro. strate that Arabidopsis CRY1 interacts with HBI1 to inhibit its Next, we performed Dual-LUC assays to examine CRY1 DNA binding activity and hypocotyl elongation. Specifically, regulation of HBI1 transcriptional activity in tobacco cells. To we demonstrate by combined approaches of yeast two-hybrid do this, we isolated the 2  kb promoter region of the Exp16 assay, pull-down assay, protein co-localization, and co-IP that gene (Exp16 ), which is a HBI1 direct target gene (Fan et al. pro CRY1 interacts with HBI1 through its N-terminus in a blue- 2014), and made the reporter construct expressing LUC under light-dependent manner. The significance of the CRY1–HBI1 the control of Exp16 (Fig. 8E). The effector constructs were pro interaction is attested to by the following demonstrations. (i) transiently co-expressed in tobacco leaf cells with the reporter HBI1 acts downstream of CRYs to regulate hypocotyl elong- construct in different combinations. The results showed that, ation under blue light. (ii) CRYs act to co-regulate a set of consistent with HBI1 being a positive regulator of Exp16 (Fan genes that are involved in regulating cell elongation antag- et al., 2014), HBI1 alone strongly stimulated the Exp16 :LUC pro onistically with HBI1. (iii) The CRY1 N-terminus inhibits reporter activity (Fig.  8F). However, when CRY1 was co- HBI1–DNA binding activity in vitro. (iv) Both CRY1 and its expressed with HBI1, the activity of the Exp16 promoter was N-terminus are able to inhibit HBI1 transcriptional activity dramatically repressed (Fig.  8F). We then analyzed the effects in tobacco leaf cells. Based on our results, we propose a model of CNT1 and CCT1 on HBI1 regulation of the Exp16 :LUC pro describing CRY1 regulation of hypocotyl elongation via its reporter activity. As shown in Fig.  8G, HBI1 induction of interaction with HBI1 (Fig. 9). In darkness, CRY1 is not active Exp16 :LUC activity was considerably inhibited by CNT1, pro and cannot interact with HBI1 protein. Hence, HBI1 is fully but barely affected by CCT1. In addition, HBI1 activity was active, and able to bind to its target genes and regulate their inhibited by CRY1 and CNT1 in a dose-dependent manner expression, leading to active hypocotyl elongation. Upon blue (Supplementary Fig. S7). Taken together, these results suggest light irradiation, CRY1 is activated, and able to interact with that CNT1-mediated interaction of CRY1 with HBI1 leads HBI1 to inhibit its binding to target genes, leading to inhib- to inhibition of HBI1 DNA binding activity and its transcrip- ition of hypocotyl elongation. tional activity. Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 3878 | Wang et al. Fig. 8. CRY1 represses HBI1’s transcriptional activity through CNT1 by inhibiting the DNA binding activity of HBI1. (A) qRT-PCR analysis showing the –2 –1 expression of HBI1 in WT seedlings grown in darkness for 5 d with adaption in darkness (WT-DK) or an exposure to 30 μmol m s blue light (WT-BL) for 0.5 h. Expression levels were normalized to an internal control PP2A, and the WT-DK level was arbitrarily set to 1. Data are represented as means ±SD –2 –1 (n=3). (B) Immunoblot analysis of the HBI1-Flag fusion protein level in HBI1-OX seedlings grown in darkness for 5 d and then exposed to 30 μmol m s blue light for the durations indicated. An asterisk denotes a non-specific band serving as a loading control. HBI1/* indicates the relative band intensities of HBI1-Flag normalized to the non-specific band and presented relative to that in darkness set at unity. (C and D) EMSAs showing CNT1 inhibition of HBI1 DNA binding ability. EMSA was performed with MBP–HBI1 and MBP proteins using a labeled probe composed of a DNA fragment of the promoter of DWF4 and biotin (DWF4p-Bio). 1 × 2×, 3×, and 1×, 5×, 20× in (C) indicate the amounts of MBP–HBI1 and cold competitor DNA (DWF4p-Cold) relative to the initial concentration of MBP–HBI1 and DWF4p-Bio probes, respectively. 0.2×, 1×, and 5× in (D) indicate the amounts of His-TF–CNT1 and His-TF– CCT1 relative to one-tenth the amount of MBP–HBI1. Signals at the bottom indicate free probes. (E) Schematic diagram of the construct of the Dual-LUC assay reporter expressing REN under the control of 35S promoter and LUC under the control of the Exp16 promoter (Exp16 ). (F and G) Dual-LUC pro assays showing that CRY1 represses the transcriptional activity of HBI1 through CNT1. Tobacco leaves were transfected with Agrobacterium in different combinations of effectors and the Exp16 reporter, and then kept in dim light for 3 d. 0.05×, 3× in (F) and 0.1×, 3× in (G) indicate the culture volume of pro effectors relative to that of the Exp16 reporter. The ratio of LUC activity relative to REN activity of the expression control (CFP as effector) was arbitrarily pro set to 1, to which the ratios of other groups were normalized. Error bars represent ±SD (n=4). Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 Regulation of photomorphogenesis by CRY1–HBI1 interaction | 3879 It should be noted that we observed that CNT1 interacted with HBI1 in yeast cells and tobacco cells, it is possible that the with HBI1 regardless of blue light in yeast two-hybrid assays, signaling mechanism of CRY2 also involves interaction with whereas CNT2 interacted with HBI1 in blue light, but not in HBI1 under a low intensity of blue light. Moreover, our obser- darkness (Figs 2C, 4C). These differences might result from the vation that HBI1 acts to promote hypocotyl elongation in red amino acid sequence dissimilarity between CNT1 and CNT2. and far-red light (Supplementary Figs S2, S3) suggests a pos- Although CRY1 and CRY2 are homologous proteins, they sible role for HBI1 in mediating phytochrome signaling. Given have different functions and protein properties. For example, previous demonstrations that phytochromes interact with the CRY1 plays a role in inhibiting hypocotyl elongation under cryptochrome-interacting proteins such as COP1 and SPAs low, middle, and high fluence rates of blue light, whereas (Seo et al., 2004; Jang et al., 2010; Lu et al., 2015; Sheerin et al., CRY2 performs this role primarily under a low intensity of 2015), and that cryptochromes interact with the phytochrome- blue light (Lin et  al., 1998). Consistent with this, CRY2 is interacting factor PIF4 to mediate light signaling (Ma et  al., degraded under a high intensity of blue light, whereas CRY1 2016; Pedmale et  al., 2016), it is possible that phytochromes is stable (Shalitin et  al., 2002). The amino acid sequence dif- may interact directly with HBI1 to regulate hypocotyl elong- ferences between CRY1 and CRY2 might also lead to these ation. On the other hand, phytochromes might influence differences. In our Dual-LUC assays, we found that, similar to HBI1 function indirectly by regulating the biosynthesis of the a previous report (Ma et al., 2016), CRY1 or CNT1 alone is endogenous phytohormones BR and GA based on the fol- somehow able to activate the Exp16 reporter in tobacco cells lowing demonstrations: (i) PIF4 and PIF5 bind to the pro- pro (Fig. 8F, G). Given the demonstrations that CRY1 is not a tran- moter regions of the key BR biosynthetic genes DWF4 and scription factor and that soybean CRY2a interacts with CIB1 BR6ox2 to promote their expression directly (Wei et al. 2017); in a blue-light-dependent manner to inhibit CIB1 DNA bind- (ii) phytochromes regulate GA biosynthesis through PIF3- ing activity and repress leaf senescence (Meng et al., 2013), we LIKE5 (PIL5), which represses the expression of GA biosyn- postulate that CRY1 activation of Exp16 activity is not likely thetic genes GA3ox1 and GA3ox2 and activates the expression pro to be mediated through a direct mechanism, namely CRY1 of a GA catabolic gene GA2ox in Arabidopsis (Oh et al. 2006); binding to Exp16 . It is likely that CRY1–HBI1 interaction and (iii) HBI1 is involved in the signaling pathway of BR and pro might lead to a decrease in HBI1–DNA binding and transcrip- GA (Bai et al., 2012a). tional activity. The bHLH-type proteins constitute a large family of tran- In view of the fact that the signaling mechanism of both scription factors in eukaryotes, which is specifically classified CRY1 and CRY2 involves physical interactions with COP1, into dozens of subfamilies with a total of ~170 members in SPAs, and PIFs, and our demonstration that CRY2 also interacts Arabidopsis (Carretero-Paulet et  al., 2010). A  previous study Fig. 9. A model describing regulation of HBI1 transcription by CRY1. (A) In darkness, CRY1 is inactive and HBI1 is able to bind to the promoters of its direct target genes promoting cell elongation and activating their expression, leading to enhanced hypocotyl elongation. (B) Upon blue light irradiation, CRY1 is activated and able to interact with HBI1 through its N-terminus (CNT1) to inhibit HBI1 binding to its target genes, resulting in repression of expression of these genes and inhibited hypocotyl elongation. CCT1 denotes the CRY1 C-terminus. Thicker and thinner red arrows denote higher and lower gene expression, respectively. Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 3880 | Wang et al. reported that bHLH factors are capable of forming homodi- Acknowledgments mers or heterodimers through the HLH domains and bind- We thank Dr Lida Zhang for assistance in bioinformatic analysis of ing to specific DNA sequences through the basic domains RNA-Seq data. This work was supported by The National Key Research (Toledo-Ortiz et  al., 2003). HBI1 was identified as a typical and Development Program of China grant (2017YFA0503800) and The National Natural Science Foundation of China grants to HQY DNA-binding bHLH protein and closely related to many (31530085, 91217307, and 90917014) and to HLL (31570282 and bHLH factors belonging to different subfamilies. Among 31170266) them, CIBs (CIB4/5), CILs (CIL1/2), and BEEs (BEE1/2/3) are also shown to regulate hypocotyl elongation (Friedrichsen et  al., 2002; Ikeda et  al., 2012). Based on our results showing References that CRY1 may interact with these bHLH proteins through Ahmad M, Cashmore AR. 1993. HY4 gene of A.  thaliana encodes a its N-terminus, we postulate that these interactions may also protein with characteristics of a blue-light photoreceptor. Nature 366, result in inhibition of their DNA binding activity and reduced 162–166. expression of their target genes that are involved in positively Bai MY, Fan M, Oh E, Wang ZY. 2012a. A triple helix–loop–helix/basic helix–loop–helix cascade controls cell elongation downstream of multiple regulating cell elongation. hormonal and environmental signaling pathways in Arabidopsis. The Plant It has been demonstrated that the regulatory module Cell 24, 4917–4929. 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Cell 164, CRY2 with SPA1 regulates COP1 activity and floral initiation in Arabidopsis. 233–245. Current Biology 21, 841–847. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Experimental Botany Oxford University Press

CRY1 interacts directly with HBI1 to regulate its transcriptional activity and photomorphogenesis in Arabidopsis

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Abstract

Cryptochromes (CRYs) are blue light photoreceptors that mediate various light responses in plants and animals. In Arabidopsis, there are two homologous CRYs, CRY1 and CRY2, which mediate blue light inhibition of hypocotyl elongation. It is known that CRY2 interacts with CIB1, a basic helix–loop–helix (bHLH) transcriptional factor, to regu- late transcription and floral induction. In this study, we performed yeast two-hybrid screening and identified CIB1 as a CRY1-interacting protein. Moreover, we demonstrated that CRY1 physically interacted with the close homolog of CIB1, HBI1, which is known to act downstream of brassinosteroid (BR) and gibberellin acid (GA) signaling pathways to promote hypocotyl elongation, in a blue light-dependent manner. Transgenic and genetic interaction studies showed that overexpression of HBI1 resulted in enhanced hypocotyl elongation under blue light and that HBI1 acted down- stream of CRYs to promote hypocotyl elongation. Genome-wide gene expression analysis indicated that CRYs and HBI1 antagonistically regulated the expression of genes involved in regulating cell elongation. Moreover, we dem- onstrated that CRY1–HBI1 interaction led to inhibition of HBI1’s DNA binding activity and its target gene expression. Together, our results suggest that HBI1 acts as a new CRY1-interacting protein and that the signaling mechanism of CRY1 involves repression of HBI1 transcriptional activity by direct CRY1–HBI1 interaction. Keywords: Arabidopsis, blue light, cell elongation, cryptochrome, HBI1, protein interaction, transcriptional activity. Introduction Plants on the earth are influenced by many kinds of environ- source of energy for photosynthesis but also regulatory signals for mental factors during their life cycle from seed germination to growth and development. Plants have evolved multiple photo- flowering, among which light provides not only the essential receptors to perceive light quality, quantity, and direction, which Abbreviations: 3AT, 3-amino-1,2,4-triazole;bHLH, basic helix–loop–helix; BR, brassinosteroid; BEE2, BR enhanced expression 2;CCT1, cryptochrome 1 C-terminal domain; CCT2, cryptochrome 2 C-terminal domain;CIB1, cryptochrome interacting basic helix–loop–helix 1; CIL1, CIB1-like protein 1; CNT1, cryptochrome 1 N-terminal domain; CNT2, cryptochrome 2 N-terminal domain; CO, CONSTANS; COP1, constitutively photomorphogenic 1; CRY1, cryptochrome 1; CRY2, cryp- tochrome 2; DWF4, dwarf 4; GA, gibberellin; HBI1, homolog of BEE2 interacting with IBH 1; IBH1, ILI1-binding bHLH protein 1; NB, nuclear body; PRE1, paclob- trazol resistance 1. © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 3868 | Wang et al. include the UV-B light photoreceptor UVR8, the blue light interacting basic helix–loop–helix1). It has been shown that photoreceptors cryptochromes (CRYs) and phototropins, and CRY2 interacts with CIB1 through its N-terminus to regulate the red/far-red light photoreceptors phytochromes (Cashmore the transcriptional activity of CIB1 and floral initiation (H. Liu et  al., 1999; Briggs and Christie, 2002; Quail, 2002; Rizzini et al., 2008). HBI1 (HOMOLOG OF BEE2 INTERACTING et al., 2011). Among these photoreceptors, cryptochromes play WITH IBH 1) is also a basic helix–loop–helix (bHLH) tran- critical roles not only in plants, but also in Drosophila and mam- scriptional factor that shows high amino acid sequence simi- mals. In Arabidopsis, there are two well-studied homologous larity to CIB1 and its homologs, CIL1 (CIB1 like protein CRYs, CRY1 and CRY2, which play relatively major roles in 1) and BEE2 (BR enhanced expression 2) (Bai et al., 2012a). regulating photomorphogenesis under blue light and photo- Previous studies have demonstrated that HBI1, as well as CIBs periodic flowering, respectively (Ahmad and Cashmore, 1993; (CIB4/5), CILs (CIL1/2) ,and BEEs (BEE1/2/3), have simi- Guo et al., 1998; Lin et al., 1998). Photomorphogenesis is one lar functions and work to promote hypocotyl elongation in of the critical and best studied light-controlled processes in Arabidopsis, respectively (Friedrichsen et  al., 2002; Bai et  al., Arabidopsis, which is characterized by shortened hypocotyls, 2012a; Ikeda et  al., 2012). It has been demonstrated that expanded cotyledons, and accumulation of chlorophyll under ILI1 BINDING bHLH PROTEIN1 (IBH1) interacts with light, and cryptochromes and phytochromes are shown to play HBI1 to inhibit HBI1 transcriptional activity and hypocotyl critical roles in regulating this process (Pepper et  al., 1994; elongation by repressing the DNA binding ability of HBI1, McNellis and Deng, 1995; Cashmore et  al., 1999; Deng and and that the inhibitory effects of IBH1 on HBI1 transcrip- Quail, 1999; Quail, 2002; Lin and Shalitin, 2003). Furthermore, tional activity is counteracted by PRE1 (PACLOBTRAZOL both CRY1 and CRY2 have been shown to entrain the circa- RESISTANCE1) through its direct interaction with IBH1 dian clock and mediate blue light induction of stomatal open- (Bai et  al., 2012a). Whether CRY1 interacts with CIB1 and ing and development (Somers et  al., 1998; Mao et  al., 2005; its homologs through its N-terminus to regulate hypocotyl Kang et al., 2009). In Drosophila, cryptochrome acts in the input elongation remains unknown. pathway to entrain the circadian clock (Emery et al., 1998) and, In this study, we sought potential CRY1 N-terminus in mammals, cryptochromes serve as integral components of (CNT1)-interacting proteins through yeast two-hybrid the central oscillator of the circadian clock (Kume et al., 1999). screening, and identified CIB1 as a candidate. We confirmed In migratory birds, cryptochromes act as the magnetoreceptors by yeast two-hybrid and protein co-localization assays that to perceive magnetic fields and navigate during long-distance CNT1 interacts with CIB1 and its homologs BEE2 and CIL1. migration (Gegear et al., 2010). Furthermore, we demonstrated by yeast two-hybrid, pull- Cryptochomes are structurally divided into an N-terminal down, protein co-localization, and co-immunoprecipitation domain that shares high sequence similarity to photolyases, and (co-IP) assays that CRY1 interacted with HBI1 in a blue light- a distinguishing C-terminal extension that is absent in pho- dependent manner. By making transgenic lines in which HBI1 tolyases (Sancar, 1994; Cashmore et al., 1999; Lin and Shalitin, function was either enhanced or repressed, we demonstrated 2003). It has been shown that the C-terminal domains of that HBI1 acted to regulate hypocotyl elongation positively Arabidopsis CRY1 and CRY2 mediate CRY1/2 signaling under blue, red, and far-red light, respectively. Moreover, by through direct interactions with COP1 (Yang et  al., 2000, creating transgenic lines in which HBI1 function was sup- 2001; Wang et  al., 2001), a RING-finger E3 ubiquitin ligase pressed in the cry1cry2 mutant background, we demonstrated (Deng et  al., 1992) that interacts with and targets the deg- that HBI1 acted downstream of CRY1 and CRY2 to regu- radation of a set of transcription factors, such as HY5/HYH late hypocotyl elongation under blue light. We demonstrated and CONSTANS (CO), to regulate photomorphogenesis and by genome-wide gene expression studies that CRY1/2 and flowering under long days, respectively (Putterill et  al., 1995; HBI1 acted antagonistically to regulate the expression of a set Oyama et  al., 1997; Osterlund et  al., 2000; Lee et  al., 2007; of genes related to cell elongation. We also showed by EMSA Jang et  al., 2008; L.J. Liu et  al., 2008). Moreover, CRY1 and and dual luciferase (Dual-LUC) assays that CRY1 inhibited CRY2 also interact with the COP1 enhancer, SPA1 (Lian the transcriptional activity of HBI1 through its N-terminus. et al., 2011; Liu et al., 2011; Zuo et al., 2011), which interacts This study therefore reveals a new mechanism of CRY1 sign- with COP1 to enhance its E3 ligase activity (Seo et al., 2003). aling, which involves direct blue light-dependent interaction The outcome of interactions of CRY1/CRY2 with COP1/ with the transcriptional factor HBI1 to regulate its transcrip- SPA1 is disruption of the COP1–SPA1 core complex, thus sta- tional activity and photomorphogenesis. bilizing HY5/CO proteins and promoting their accumulation. The N-terminal domain of CRY1 and CRY2 mediate CRY dimerization (Sang et al., 2005; Yu et al., 2007), and it is shown Materials and methods that CRY2 dimerization is inhibited by Blue-light Inhibitor of Plant materials and growth conditions Cryptochromes 1 (BIC1) (Q. Wang et al., 2016). Recently, the Arabidopsis thaliana ecotype Columbia (Col-0) was used as the wild-type CRY1 N-terminus has been shown to mediate CRY1 signal- (WT) control. cry1cry2, phyA-211, and phyB-9 mutants, and transgenic ing independent of the CRY1 C-terminus (He et  al., 2015). lines overexpressing Myc-CRY1 (CRYI-OX), Myc-CRY2 (CRY2-OX), However, the underlying mechnism is not well understood. and GUS-CCT1 (GUS-CCT1) were described previously (Yang et  al., The discovery showing direct cryptochrome regulation of 2000; Mao et al., 2005; Sang et al., 2005). HBI1-related transgenic plants transcription comes from screening for CRY2-interacting fac- were generated using corresponding constructs by an in planta method (Bechtold and Pelletier, 1998). For phenotype analyses of seedlings, seeds tors, which leads to the identification of CIB1 (cryptochrome Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 Regulation of photomorphogenesis by CRY1–HBI1 interaction | 3869 were sown on half-strength Murashige and Skoog (MS) medium plus (~0.2 μg) of prey proteins of His-TF, His-TF–CNT1, and His-TF–CCT1 1% sucrose with 0.8% agar and were cold treated for 4 d at 4 °C. After in a final volume of 1 ml at 4 °C for 0.5 h. Then the beads were washed exposure to white light for 12  h to promote germination, plates with with TBST three times, dissolved in 30 μl of 2× SDS loading buffer, and seeds were transferred to appropriate light conditions for 4 d at 22 °C. subjected to western blot or Coomassie Brilliant Blue staining. For adult plants, seeds were sown as above and grown under continuous For the semi-in vivo pull-down assay with Arabidopsis protein extracts, white light for 7 d, then transferred to soil to continue growing for the CRY1-OX seedlings grown in soil for ~2 weeks under white light were time indicated. kept in darkness for 2 d before they were transferred to red light (30 μmol –2 –1 –2 –1 –2 –1 m s ), blue light (50 μmol m s ), far-red light (10 μmol m s ), or continuing darkness for 3 h. These seedlings were quickly frozen by liquid Plasmid construction nitrogen and homogenized with TBST buffer containing 1 mM protein- ase inhibitor (Pefabloc and Cocktail, Roche). The supernatant adjusted The constructs expressing COP1 in yeast, and cLUC-Flag, CRY1–yel- to contain the same amount of total protein quantified by Bradford assay low fluorescent protein (YFP), CRY2–YFP, β-glucuronidase (GUS)– (Biorad) was infused to tubes containing MBP–HBI1-conjugated beads, nuclear localization signal (NLS)–YFP, and CNT1–NLS–YFP in incubated for 30 min at 4 °C, and washed three times with 1 ml of TBST plants were described previously (Lian et  al., 2011; He et  al., 2015; Xu buffer each time, then the precipitates were eluted into 30 μl of 2× SDS et  al., 2016). PCR-amplified fragments encoding HBI1, HBI1– ERF- loading buffer and subjected to western blot analysis with anti-Myc anti- associated amphiphilic repression (EAR), CNT1–NLS, NLS–CCT1, body (Millipore). CIB1, CNT2–NLS, NLS–CCT2, CRY2, BEE2, CIL1, CO, and NLS– GUS–CCT1 were cloned into the multiple cloning sites (MCS) of pHB- MCS-3×Flag, pHB-6×Myc-MCS, pGBKT7, pGADT7, pHB-MCS-YFP Protein co-localization assay [cyan fluorescent protein (CFP)], and pHB-YFP (CFP)-MCS, respect- ively. All of the constructs and primers used in this study are listed in Agrobacterium tumefaciens strain GV3101 harboring the constructs express- Supplementary Table S1 at JXB online. ing the studied proteins or p19 plasmid were diluted by MS liquid medium to OD =0.6 individually and treated with 200 μM acetosyrin- gone and 10 mM MES (pH 5.6) for 3 h at room temperature. Then the Analyses of RNA-Seq data mixture of Agrobacterium harboring the constructs expressing CFP and YFP fusion proteins, and p19 plasmid with a volume ratio of 1:1:1 were The RNA sequencing (RNA-Seq) data indicating the genes regu- introduced into tobacco (Nicotiana benthamiana) leaf epidermal cells by lated by HBI1 and CRYs have the accession numbers GSE53078 and infiltration. After incubation for 2 d or 3 d in dim light, leaves were col- GSE58552, respectively (Fan et al., 2014; He et al., 2015). A Venn diagram lected for confocal microscopic examination (Leica TCS SP5II confocal was generated in Venny (http://bioinfogp.cnb.csic.es/tools/venny/index. laser scanning microscope). html). A heatmap was generated with hierarchical clustering analysis by the MeV 4.7 software. Gene Ontology (GO) analysis was performed in agriGO v2.0 (http://systemsbiology.cau.edu.cn/agriGOv2/) (Tian et al., Co-immunoprecipitation assay 2017), with TAIR10 being as the reference and hypergeometric statistical test method and complete GO type. Tobacco leaves were infiltrated with a mixture of Agrobacterium culture (OD =0.6) harboring the constructs expressing p19, HBI1-Flag, and YFP fusion proteins or p19, CNT1–NLS–YFP, and Flag-tagged fusion Quantitative real-time PCR proteins, with a volume ratio of 1:1:1 and then kept in dim light for 3 d as for co-localization assays. Samples from tobacco leaves were collected We used WT, cry1cry2, CRY1-OX, CRY2-OX, HBI1-EAR-OX/cry1cry2, and homogenized in TBST containing proteinase inhibitors. After cen- and HBI1-OX seedlings for quantitative real-time PCR (qRT-PCR). trifugation, protein supernatant was incubated with 10 μl of protein G Seeds were sown as above and then subjected to white light treatment for magnetic beads (Invitrogen) [which had been incubated with 1  μl of 12 h to promote germination. The plates with seeds were kept in darkness anti-Flag antibody (Sigma) overnight at 4 °C] for 1 h at 4 °C. The immu- for 4 d before being exposed to blue light irradiation. Total RNAs were noprecipitate was washed three times with TBST and eluted with 0.5 μg isolated according to the manufacturer’s instructions (TIANGEN). The –1 μl 3×Flag peptide (Sigma) in 30 μl of TBST. A 24 μl aliquot of eluate detailed method of qRT-PCR was described previously (Zhang et  al. was resuspended with 6 μl of 5× SDS loading buffer, boiled for 5 min, 2014). PP2A was used as an internal control. The primers used are listed and then subjected to immunoblot analysis with anti-Flag (Sigma) and in Supplementary Table S1. anti-green fluorescent protein (GFP; Abmart) antibodies. Yeast two-hybrid assay Hypocotyl measurement Yeast two-hybrid assay was performed according to the user manual Seedlings were photographed, and then the digital photographs were for the Yeastmaker Yeast Transformation System (Clontech Laboratories, utilized to measure hypocotyl length through Image J software (http:// Mountain View, CA, USA). Combinations containing the fragments fused rsbweb.nih.gov/ij/). in pGBKT7 and pGADT7 vectors were co-transformed into the AH109 strain via the PEG/LiAc transformation procedure. At 3 d after trans- formation, yeast cells were spread on selective media with 3-amino-1,2,4- Western blotting triazole (3AT) in continuous blue light or darkness for 3 d. Arabidopsis seeds of different genotypes were grown on half-strength MS medium under continuous white light for 5 d. Seedlings with differ- Pull-down assay ent light treatments were collected and homogenized in TBST described above, and the protein supernatant, after quantification by Bradford For in vitro pull-down assay, the fragments encoding CNT1, CCT1, and assay (BioRad), was subjected to immunoblot analysis with the related HBI1 were each cloned into pCold-trigger factor (TF) or pMAL-c2X antibody. vector. The fusion proteins were expressed in Escherichia coli (Rosetta) and purified according to the manufacturer’s protocol (QIAGEN and NEB). Maltose-binding protein (MBP)–HBI1 was first incubated with EMSA amylose–magnetic beads (NEB) in TBST buffer (50 mM Tris–HCl, pH 7.5, 150  mM NaCl, 0.2% Triton X-100, and 10% glycerol) containing The proteins used in this experiment were expressed and purified as 1 mM proteinase inhibitor (Pefabloc, Roche) at 4 °C for 4 h, then the described above prior to being quantified by comparison with the BSA amylose–magnetic beads affiliated with MBP–HBI1 serving as bait was standard, then 1.5 μg of MBP and 0.5 μg of MBP–HBI1 were utilized. washed with TBST three times and incubated with the same amounts Biotin-labeled probes were commercially synthesized and annealed. The Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 3870 | Wang et al. binding reaction was carried out in 20 μl of reaction buffer [10× Binding prompted us to look for potential CNT1-interacting proteins. –1 buffer, 100 mM MgCl , 1 μg μl poly(dI dC), 1% NP-40] with 20 fM To this end, we carried out GAL4 yeast two-hybrid screening probe and purified proteins according to the manufacturer’s instructions against CNT1 and identified 15 clones encoding CIB1, a bHLH (Thermo Scientific). The reactions were resolved by 6% native PAGE at transcriptional factor with which CRY2 interacts through its 4 °C after 15 min incubation at room temperature, and then the DNA– N-terminus to regulate photoperiodic flowering (H. Liu et al., protein complexes were transferred to a nylon membrane. Biotin-labeled probes were detected by horseradish peroxidase (HRP)-conjugated 2008). To confirm the interaction of CNT1 with CIB1, we streptavidin and visualized with an ECL detection kit. performed GAL4 yeast two-hybrid assays by co-transforming yeast cells with the bait construct expressing CNT1 fused to the GAL4 DNA-binding domain (BD) tagged by an NLS sequence Dual-LUC assay (Fig. 1A), together with a prey construct expressing the GAL4 An ~2  kb fragment upstream of the start codon of Exp16 was ampli- fied and cloned into pGreen0800 vector, which expresses Renilla lucif- activation domain (AD) fused to CIB1 (Fig.  1B). The results erase (REN) driven by the 35S promoter serving as an internal reference showed that CNT1 interacted with CIB1 (Fig.  1C). We then and firefly luciferase (LUC) driven by the Exp16 promoter serving as a performed an in vivo protein co-localization assay by making reporter. Agrobacterium culture (OD =0.6) harboring the related con- constructs expressing CNT1 fused to the NLS followed by YFP, structs was mixed with the volume ratio indicated in Fig.  8F, G and CIB1, and its homologs BEE2 and CIL1 fused to cyan CFP Supplementary Fig. S7; the lack of any effector in all groups was supple- mented with liquid MS medium to the same volume. The mixture was (Fig.  1D). As shown in Fig.  1E, expression of CNT1–NLS– introduced into tobacco leaves by infiltration and kept in dim white light YFP produced nuclear bodies (NBs) in tobacco leaf epidermal for 3 d, and then the samples were collected for measurements of lucif- cells, whereas expression of CFP–CIB1, CFP–BEE2, or CIL1– erase activity using commercial reagents according to the manufacturer’s CFP did not. However, when co-expressed with CNT1–NLS– instructions (Promega). YFP, CFP–CIB1, CFP–BEE2, and CIL1–CFP proteins were co-localized to the same NBs as CNT1–NLS–YFP, indicating interactions of CNT1 with these proteins. Results The CRY1 N-terminus interacts with CIB1 and its CNT1 physically interacts with HBI1 in yeast cells and homologs BEE2 and CIL1 in yeast and tobacco cells in vitro Our previous demonstration that CNT1 is able to mediate Among the CIBs and CIB-like proteins, HBI1 is preferen- CRY1 signaling independent of its C-terminus (He et al., 2015) tially expressed in hypocotyl and cotyledon, and acts as a Fig. 1. CNT1 physically interacts with CIB1, BEE2, and CIL1 in yeast and tobacco cells. (A) Bait protein in yeast two-hybrid assay. CNT1 is fused with the GAL4-binding domain (BD) and nuclear localization signal (NLS) sequence. (B) Prey protein in yeast two-hybrid assay. CIB1 is fused with the GAL4 activation domain (AD). (C) Analysis of CNT1–CIB1 interaction in yeast cells. In this and other figures, all vector combinations are given as bait/prey. Yeast cells co-expressing CNT1/CIB1 were grown on basic (SD-W-L) or selective media with 10 mM 3AT (SD-W-L-H-A+3AT) in darkness for 3 d. (D) Schematic diagram of constructs expressing CNT1 fused to the NLS sequence and yellow fluorescent protein (YFP) and constructs expressing CIB1, BEE2, and CIL1 fused to cyan fluorescent protein (CFP). (E) Co-localization assay showing that CNT1 and these homologous proteins, CIB1, BEE2, and CIL1, localize together to the same nuclear bodies (NBs) in tobacco cells. Dic, differential interference contrast. Scale bars=5 μm. Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 Regulation of photomorphogenesis by CRY1–HBI1 interaction | 3871 positive regulator of cell elongation downstream of the BR CRY1 interacts with HBI1 through its N-terminus in (brassinosteroid) and GA (gibberellin acid) signaling pathways plant cells through the PREs (PACLOBTRAZOL RESISTANCE1)– With the demonstration that CNT1 interacts with HBI1 in IBH1 (ILI1 BINDING bHLH PROTEIN1)–HBI1 module yeast cells and in an in vitro pull-down assay, we asked whether (Bai et al. 2012a). We therefore focused on exploring whether CNT1 might interact with HBI1 in vivo. To test this possibility, CRY1 might interact directly with HBI1 through CNT1 we first performed a protein co-localization assay in tobacco to regulate hypocotyl elongation. To test this possibility, we cells using a construct expressing HBI1 tagged by CFP (Fig. 3A) first performed a yeast two-hybrid assay with bait constructs and a construct expressing CNT1–NLS–YFP (Fig.  1D). The expressing CNT1–NLS (Fig. 1A) and the CRY1 C-terminus results showed that, CO–CFP (Fig.  3A) and CNT1–NLS– fused to an NLS (NLS–CCT1) (Fig. 2A), and prey constructs YFP expressed in the same tobacco cells were not merged expressing the full-length HBI1 (Fig.  2B) and COP1 (Lian together and localized to different NBs, while CNT1–NLS– et al., 2011), respectively. As shown in Fig. 2C, CNT1 inter- YFP and HBI1–CFP proteins were co-localized to the same acted with HBI1 in both darkness and blue light, whereas NBs (Fig. 3B), indicating a possible interaction of CNT1 with CCT1 did not interact with HBI1 in either darkness or blue HBI1. Next, we performed a co-IP assay with tobacco leaves light but interacted with the positive control, COP1 (Yang transiently co-expressing CNT1–NLS–YFP, YFP–NLS–GUS– et  al., 2001). These results indicated that CNT1 interacted CCT1, or GUS–NLS–YFP with Flag-tagged HBI1 proteins with HBI1 in yeast cells. We then performed an in vitro pull- to confirm the interaction of CNT1 with HBI1. The results down assay to confirm the interaction of CNT1 with HBI1 showed that CNT1 was immunoprecipitated by HBI1, but not using CNT1 and CCT1 polypeptides tagged by His-tagged by CCT1 or the control protein GUS (Fig. 3C), further indicat- TF, and HBI1 protein tagged by MBP expressed in E. coli. The ing an interaction of CNT1 with HBI1. Moreover, co-IP assay results showed that CNT1 was pulled-down by HBI1, but with tobacco leaves transiently co-expressing Flag-tagged HBI1 CCT1 was not (Fig.  2D). These results indicate that CNT1 or cLUC (the C-terminus of firefly luciferase) with CNT1– rather than CCT1 might mediate the interaction of CRY1 NLS–YFP confirmed that the binding of CNT1 to HBI1 is with HBI1. Fig. 2. CNT1 physically interacts with HBI1 in yeast cells and in vitro. (A) Bait protein. CCT1 is fused with the GAL4-binding domain (BD) and NLS sequence. (B) Prey protein. HBI1 is fused with the GAL4 activation domain (AD). (C) HBI1 interacts with CNT1 rather than CCT1 in yeast cells. Yeast cells –2 –1 co-expressing CNT1/HBI1 were grown on basic (SD-W-L) or selective media with 10 mM 3AT (SD-W-L-H-A+3AT) in continuous 30 μmol m s blue light (BL) or darkness (DK) for 3 d at the top or cells co-expressing CCT1/HBI1 were grown on basic (SD-W-L) or selective media with 2 mM 3AT (SD-W- L-H-A+3AT) in darkness for 3 d at the bottom. (D) MBP pull-down assay showing the interaction of HBI1 with CNT1 rather than CCT1. CBB Staining denotes Coomassie Brilliant Blue staining. His-TF was used as negative control. All these experiments were independently repeated three times. Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 3872 | Wang et al. Fig. 3. CRY1 physically interacts with HBI1 through its N-terminus in a blue light-dependent manner. (A) Schematic diagram of constructs expressing HBI1 and the negative control CO fused to CFP. (B) Co-localization assay of studied proteins in tobacco cells. CNT1 and HBI1 localize together to the same NBs, whereas CNT1 and CO localize to different NBs. Dic, differential interference contrast. Scale bars=5 μm. (C) Co-IP assay showing interaction of CNT1 with HBI1. YFP-tagged CNT1, CCT1, and the negative control, GUS protein, were co-expressed with Flag-tagged HBI1 in tobacco leaves. The immunoprecipitates were detected by anti-GFP and anti-Flag antibodies. A single asterisk and double asterisks denote a non-specific band and the heavy chain of IgG, respectively. (D) Co-IP assay showing that CNT1 interacts specifically with HBI1. Flag-tagged HBI1 and the negative control, cLUC protein, were co-expressed with YFP-tagged CNT1 in tobacco leaves. The immunoprecipitates were detected by anti-Flag and anti-GFP antibodies. A single asterisk and double asterisks denote a non-specific band and the heavy chain of IgG, respectively. (E) Semi-in vivo pull-down assay showing the blue light-specific interaction of CRY1 with HBI1. MBP–HBI1 served as bait. Myc-CRY1-containing protein extracts from CRY1-OX seedlings that were adapted to darkness –2 –1 –2 –1 –2 –1 (DK) or exposed to 50 μmol m s blue (BL), 30 μmol m s red (RL), or 10 μmol m s far-red (FRL) light for 3 h served as preys. CBB Staining denotes Coomassie Brilliant Blue staining. All experiments above were independently repeated at least three times. specific (Fig.  3D). Furthermore, we performed a semi-in vivo whereas CRY2 regulates this process mainly under low inten- pull-down assay using MBP–HBI1 fusion protein as a bait, and sity of blue light (Lin et  al., 1998). Given the previous dem- Myc-CRY1-containing protein extracts as preys, which were onstration that the signaling mechanism of both CRY1 and prepared from Arabidopsis seedlings overexpressing Myc-CRY1 CRY2 involves direct interactions with COP1, SPAs, and PIFs (CRY1-OX) adapted in the dark and exposed to blue, red, and (Wang et al., 2001; Yang et al., 2001; Lian et al., 2011; Liu et al., far-red light. The results showed that MBP–HBI1 pulled-down 2011; Ma et  al., 2016; Pedmale et  al., 2016), we first exam- Myc-CRY1 from CRY1-OX seedlings exposed to blue light, ined whether CRY2 might also interact with HBI1 through but not from those either adapted in darkness or exposed to red yeast two-hybrid assay using the bait construct expressing the or far-red light (Fig. 3E). These results indicate that CRY1 inter- full-length CRY2 (Fig. 4A) and the prey construct expressing acts with HBI1 in a blue light-dependent manner. HBI1 (Fig. 2B). The results showed that the full-length CRY2 interacted with HBI1 in the dark and in blue light (Fig. 4B). To determine further whether the CRY2 N-terminus or CRY2 interacts with HBI1 through its N-terminus C-terminus (designated as CNT2 and CCT2) might medi- It has been demonstrated that CRY1 regulates hypocotyl ate the interaction of CRY2 with HBI1, we made bait con- elongation under both low and high intensity of blue light, structs expressing CNT2–NLS and NLS–CCT2, respectively Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 Regulation of photomorphogenesis by CRY1–HBI1 interaction | 3873 Fig. 4. CRY2 interacts with HBI1 through its N-terminus in yeast and tobacco cells. (A) Bait proteins. All proteins, CNT2, CCT2, and CRY2, are fused with the GAL4 binding domain (BD), among which CNT2 and CCT2 are also tagged with the NLS sequence. (B) The physical interaction of CRY2 and HBI1 analyzed in yeast cells. Yeast cells co-expressing CRY2/HBI1 were grown on basic (SD-W-L) or selective media with 5 mM 3AT (SD-W-L-H+3AT) –2 –1 in continuous 30 μmol m s blue light (BL) or darkness (DK) for 3 d. (C) The physical interaction of CNT2 and HBI1 analyzed in yeast cells. Yeast cells –2 –1 co-expressing CNT2/HBI1 were grown on basic (SD-W-L) or selective media with 5 mM 3AT (SD-W-L-H+3AT) in continuous 30 μmol m s blue light (BL) or darkness (DK) for 3 d at the top or cells co-expressing CCT2/HBI1 were grown on basic (SD-W-L) or selective media with 2 mM 3AT (SD-W-L- H+3AT) in darkness for 3 d at the bottom. (D) Schematic diagram of constructs expressing CNT2 tagged by the NLS sequence and CRY2 fused to YFP. (E) Co-localization assay showing that HBI1 co-localizes in the same NBs with CNT2 and CRY2 in tobacco cells. Dic, differential interference contrast. Scale bars=5μm. (Fig. 4A), and performed a yeast two-hybrid assay again with and light signaling pathways to promote hypocotyl elongation these bait constructs and the prey constructs expressing HBI1 (Bai et  al., 2012a). Our demonstration that CRY1 physically and COP1. As shown in Fig. 4C, CNT2 interacted with HBI1 interacts with HBI1 suggests that CRY1-mediated blue light in blue light, but not in darkness, whereas CCT2 did not, but inhibition of hypocotyl elongation might proceed, at least in interacted with the positive control COP1 (Wang et al., 2001). part, through HBI1. To define a role for HBI1 in regulating Moreover, we performed a protein co-localization study to hypocotyl elongation under blue light, we first generated trans- confirm the interactions of CRY2 and CNT2 with HBI1 genic lines overexpressing HBI1 tagged with Flag (HBI1-OX) in tobacco cells using the constructs expressing CRY2–YFP (Fig.  5A, B), in which the expression of HBI1-Flag fusion and CNT2–NLS–YFP (Fig.  4D), and the construct express- protein was detected by western blot (Fig. 5C), and analyzed ing HBI1–CFP (Fig. 3A), respectively. The results showed that the hypocotyl elongation phenotype in blue light. The results HBI1 was localized to the same NBs of CRY2 and CNT2 showed that these lines exhibited a significantly taller hypoco- (Fig. 4E), indicating interactions of CRY2/CNT2 with HBI1. tyl phenotype than the WT under blue light (Fig. 5B, D). We Taken together, these results suggest that, like CRY1, CRY2 then made a construct expressing Myc-HBI1 fused to the EAR interacts with HBI1 through its N-terminus. motif (HBI1-EAR) (Fig. 5E), which is shown to inactivate the function of the native HBI1 and its close homologs (Bai et al., 2012a; Liu et al., 2013), and generated transgenic lines overex- HBI1 positively regulates hypocotyl elongation under pressing Myc-HBI1–EAR (HBI1-EAR-OX). Analysis of the blue, red, and far-red light hypocotyl phenotype demonstrated that these lines had sig- It has been demonstrated that HBI1 functions in a triantag- nificantly shorter hypocotyls than the WT under blue light, onistic cascade system downstream of BR, GA, temperature and that the severity of this shortened hypocotyl phenotype Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 3874 | Wang et al. Fig. 5. HBI1 acts as a positive regulator of hypocotyl elongation under blue light. (A and E) Schematic diagram depicting the constructs expressing fusion proteins of HBI1-Flag and Myc-HBI1–EAR used for transformation of WT plants. (B and F) Seedling phenotypes of HBI1-OX and HBI1-EAR-OX –2 –1 transgenic plants grown on half-strength MS medium under 2 μmol m s blue light for 5 d. Scale bars=5 mm. (C) Western blot analysis showing HBI1-Flag fusion protein expression in the corresponding lines shown in (B). An asterisk denotes a non-specific band recognized by the antibody, which served as a loading control. HBI1/* denotes the relative band intensities of HBI1-Flag normalized to the non-specific band and presented relative to that in HBI1-OX #2 set at unity. (D) Statistical analysis of hypocotyl length of seedlings shown in (B). Data are presented as means ±SD. Asterisks denote a significant difference between the indicated lines and the WT (t-test, P<0.001), n=30. (G) Western blot analysis showing Myc-HBI1–EAR fusion protein expression in the corresponding lines shown in (F). Actin served as a loading control. HBI1-EAR/Actin indicates the relative band intensities of Myc-HBI1– EAR normalized to Actin and presented relative to that in HBI1-EAR-OX #3 set at unity. (H) Statistical analysis of hypocotyl length of seedlings shown in (F). Data are presented as means ±SD. Asterisks denote a significant difference between the indicated lines and the WT (t-test, P<0.001), n=30. was positively correlated with the HBI1–EAR protein levels promote hypocotyl elongation preferentially under low fluence (Fig. 5F–H), confirming a positive role for HBI1 in regulating rates of blue light. We further analyzed the hypocotyl pheno- hypocotyl elongation under blue light. type of HBI1-OX and HBI1-EAR-OX plants in darkness, red, Next, we analyzed the blue light fluence rate response of and far-red light. The results demonstrated that HBI1-OX HBI1-OX and HBI1-EAR-OX seedlings, and found that the seedlings developed slightly taller hypocotyls than the WT in hypocotyls of both lines were shortened as the fluence rate of these conditions, whereas HBI1-EAR-OX seedlings had dra- blue light increased, and that the hypocotyls of HBI1-OX seed- matically shorter hypocotyls than the WT (Supplementary lings were always taller than those of the WT at all blue light Fig.  S2). Furthermore, we analyzed the red and far-red light fluence rate examined, whereas those of HBI1-EAR-OX seed- fluence rate responses of HBI1-OX and HBI1-EAR-OX seed- lings were always shorter than those of the WT (Supplementary lings, and found that both lines basically displayed a similar Fig. S1). However, the highest hypocotyl length difference be- tendency as observed under different fluence rates of blue light tween HBI1-EAR-OX and the WT was observed at a fluence (Supplementary Fig. S3), and that the highest hypocotyl length –2 –1 rate <2 μmol m s , indicating that HBI1 might function to difference between HBI1-EAR-OX and the WT under red Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 Regulation of photomorphogenesis by CRY1–HBI1 interaction | 3875 Fig. 6. HBI1 acts genetically downstream of CRY genes to promote hypocotyl elongation. (A) Seedling phenotypes of the transgenic lines overexpressing –2 –1 HBI1-EAR in the cry1cry2 mutant background (HBI1-EAR-OX/cry1cry2) grown on half-strength MS medium under 2 μmol m s blue light for 5 d. Scale bar=5 mm. (B) Statistical analysis of hypocotyl length of seedlings shown in (A). Data are presented as means ±SD. Asterisks denote a significant difference between the indicated lines and cry1cry2 double mutant (t-test, P<0.001), n=30. (C) Western blot analysis showing Myc-HBI1–EAR fusion protein expression in corresponding lines shown in (A). Actin served as a loading control. HBI1-EAR/Actin indicates the relative band intensities of Myc- HBI1–EAR normalized to Actin and presented relative to that in HBI1-EAR-OX/cry1cry2 #6 set at unity. (D) Seedling phenotype of siblings segregated –2 –1 from the T heterozygous HBI1-EAR-OX/cry1cry2 #9 grown on half-strength MS medium under 2 μmol m s blue light for 5 d. Scale bar=5 mm. S, M, and L denote the segregated seedlings with short, medium, and long hypocotyls, respectively, and the numbers of these seedlings are shown under these letters. –2 and far-red light was observed at a fluence rate <30 μmol m under blue light. The expression of the Myc-HBI1–EAR –1 –2 –1 s and 1  μmol m s , respectively (Supplementary Fig.  S3). fusion protein in these lines was detected by western blot These results suggest that HBI1 might also promote hypocotyl (Fig. 6C). We selected a heterozygous HBI1-EAR-OX/cry1cry2 elongation preferentially under low fluence rates of red and line (HBI1-EAR-OX/cry1cry2 #9) to analyze the hypocotyl far-red light. Since the HBI1-EAR-OX lines that expressed phenotype in T . As shown in Fig. 6D, the siblings segregated high level of HBI1–EAR had extremely compact morph- from the T heterozygous line were divided into three different ology and failed to survive after transfer to soil, we selected classes of seedlings with short, medium, and very long hypoco- two heterozygous HBI1-EAR-OX lines (HBI1-EAR-OX #3 tyls at a ratio of ~1:2:1, further indicating that the severity and #9) to analyze the hypocotyl phenotype in T . As shown 2 of the shortened hypocotyl phenotype is positively correlated in Supplementary Fig. S4, the T siblings segregated from these 2 with the HBI1–EAR protein levels. These results suggest that heterozygous lines were divided into three different classes of HBI1 acts downstream of CRY genes to regulate hypocotyl seedlings with short, medium, and long hypocotyls at a ratio elongation under blue light. of ~1:2:1, further implying that the severity of the shortened We also examined the phenotypes of adult plants of hypocotyl phenotype is positively correlated with the HBI1– the WT, cry1cry2, HBI1-OX, HBI1-EAR-OX, and HBI1- EAR protein levels. Taken together, these results suggest that EAR-OX/cry1cry2, and observed that HBI1-OX and cry1cry2 HBI1 positively regulates hypocotyl elongation in darkness mutant plants developed longer rosette leaves and siliques and in blue, red, and far-red light. than the WT, whereas CRY1-OX plants had shorter rosette leaves and siliques than the WT, and that HBI1-EAR-OX HBI1 acts downstream of CRYs to promote hypocotyl plants hardly developed functional rosette leaves and siliques elongation (Supplementary Fig. S5). Moreover, HBI1-EAR-OX/cry1cry2 plants had much shorter rosette leaves and siliques than the With the demonstration that CRYs physically interact with HBI1, we next explored the genetic interaction of CRY genes cry1cry2 mutant. Taken together, these results suggest that with HBI1 by generating transgenic cry1cry2 mutant plants CRYs and HBI1 act antagonistically to regulate not only overexpressing Myc-HBI1–EAR (HBI1-EAR-OX/cry1cry2). hypocotyl elongation during early seedling photomorpho- As shown in Fig. 6A, B, HBI1-EAR-OX/cry1cry2 lines devel- genesis, but also leaf and silique elongation during vegetative oped dramatically shorter hypocotyls than the cry1cry2 mutant and reproductive development. Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 3876 | Wang et al. Fig. 7. CRYs and HBI1 antagonistically regulate the expression of genes promoting cell elongation. (A) Venn diagram showing the overlap of genes regulated by HBI1 and CRYs. (B) Hierarchical cluster analysis of 794 overlapping genes shown in (A). Red and green colors in the heatmap represent induced and repressed genes, respectively. The gradient bar denotes the log -fold change relative to the control sample. (C and D) qRT-PCR analyses showing the expression of direct target genes of HBI1 in multiple genotypes indicated. All the seedlings were grown in darkness for 5 d and then –2 –1 transferred to 30 μmol m s blue light for 0.5 h. Expression levels were normalized to an internal control PP2A, and the WT level was arbitrarily set to 1. Data are represented as means ±SD (n=3). The lower case letters ‘a’ to ‘d’ indicate statistically significant differences among means for gene expression levels of the indicated genotypes, as determined by Tukey’s LSD test (P≤0.01). CRYs and HBI1 antagonistically regulate the and HBI1 were largely involved in the regulation of the bio- logical processes including cell wall organization and cell wall expression of a set of genes related to cell elongation loosening, whereas those co-regulated by CRYs and HBI1 in To explore whether the regulation of hypocotyl elongation by the same direction were not (Supplementary Fig. S6). CRYs and HBI1 might proceed through the co-regulation of We performed qRT-PCR analysis of five representative genes related to cell elongation, we first compared the 8498 direct target genes of HBI1, which are positively regulated genes regulated by CRYs with the 1239 genes regulated by by HBI1 and known to promote hypocotyl elongation (Fan HBI1, which were obtained through RNA-Seq (Fan et al., 2014; et al., 2014), in WT , cry1cry2 mutant, CRY1-OX, and CRY2-OX He et al., 2015). We found that, of the 1239 HBI1-controlled seedlings that were exposed to blue light. As shown in Fig. 7C, genes, 794 genes (64.1%) were also affected by CRYs (Fig. 7A). the expression of these genes was enhanced to varying degrees A heatmap revealed that, among these co-regulated genes, 393 in the cry1cry2 mutant, but reduced to varying degrees in genes (50%) were affected in the opposite way (Fig. 7B). GO CRY1-OX and CRY2-OX plants. We also analyzed the expres- analysis suggests that the genes oppositely regulated by CRYs sion of these genes in HBI1-EAR-OX/cry1cry2 and HBI1-OX Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 Regulation of photomorphogenesis by CRY1–HBI1 interaction | 3877 seedlings exposed to blue light and found that these genes Discussion were expressed at significantly lower levels in HBI1-EAR-OX/ Previous reports have revealed that CRY1 and CRY2 inter- cry1cry2 than in the cry1cry2 mutant and HBI1-OX seedlings act with COP1 through their C-terminus to inhibit COP1 (Fig. 7D). Taken together, these results indicate that CRY in- activity and enhance the accumulation of HY5 to promote hibition of hypocotyl elongation is mediated, at least in part, photomorphogenesis (Yang et  al., 2000, 2001; Wang et  al., through repression of the expression of HBI1 target genes that 2001). Moreover, later studies introduced SPAs into CRY act to promote cell elongation. signaling pathway and demonstrated that CRYs interact with SPAs in a blue light-dependent manner, resulting in inhib- CRY1 inhibits the transcriptional activity of HBI1 ition of the function of the COP1–SPA complex and further through its N-terminus repression of COP1 activity (Lian et al., 2011; Liu et al., 2011; Zuo et al., 2011). These studies led to the establishment of the To explore how CRY1 might regulate HBI1 activity, we ana- CRY signaling cascade, which consists of CRYs, SPAs, COP1, lyzed whether HBI1 gene expression or HBI1 protein expres- and HY5. However, recent studies suggest that the CRY1 sion might be regulated by blue light using the dark-grown N-terminus and C-terminus probably regulate hypocotyl WT seedlings or HBI1-OX seedlings exposed to blue light elongation through different mechanisms (W.X. Wang et  al., for increasing lengths of time. The results showed that nei- 2016) and that the CRY1 N-terminus is able independently ther HBI1 gene expression nor HBI1 protein expression was to confer enhanced blue light inhibition of hypocotyl elong- affected by blue light (Fig. 8A, B). We then explored whether ation (He et al., 2015). Therefore, the CRY1 N-terminus may CRY1 might regulate the DNA binding activity of HBI1 function independently of the CRY1 C-terminus to medi- through CRY1–HBI1 interaction. To do this, we performed an ate CRY1 signaling by interacting with downstream com- EMSA to investigate whether the capacity of HBI1 to bind to ponents. Most recently, two reports have shown that CRYs the sequence of the promoter of one HBI1 target gene, DWF4 interact with PIF bHLH transcription factors to regulate (DWARF 4), whose expression is inhibited by CRYs (Fig. 7C), their transcriptional activity and mediate responses to canopy might be inhibited by CNT1. The control experiment showed shade, low blue light, or high temperature (Ma et  al., 2016; that HBI1 bound to the promoter sequence of the DWF4 Pedmale et al., 2016). gene that contains the E box sequence (CANNTG) in a pro- CRY1 is the primary blue light photoreceptor mediating tein concentration-dependent manner, and that the binding blue light inhibition of hypocotyl elongation. The loss-of- capacity was reduced by the competition from a cold com- function mutant of Arabidopsis CRY1 shows a dramatically petitor (Fig. 8C), whereas this binding was not detectable with enhanced hypocotyl elongation phenotype, whereas transgenic the control protein MBP. As anticipated, the DNA fragment plants overexpressing CRY1 display a considerably shortened bands shifted by HBI1 decreased as the amounts of CNT1 hypocotyl phenotype (Ahmad and Cashmore, 1993; Lin et al., increased (Fig. 8D), whereas they were not affected by increas- 1996). In this study, we further defined a role for a bHLH ing amounts of CCT1. These results indicated that CNT1 but transcription factor HBI1 in promoting hypocotyl growth in not CCT1 inhibited the DNA binding activity of HBI1 in blue, red, and far-red light. The results presented here demon- vitro. strate that Arabidopsis CRY1 interacts with HBI1 to inhibit its Next, we performed Dual-LUC assays to examine CRY1 DNA binding activity and hypocotyl elongation. Specifically, regulation of HBI1 transcriptional activity in tobacco cells. To we demonstrate by combined approaches of yeast two-hybrid do this, we isolated the 2  kb promoter region of the Exp16 assay, pull-down assay, protein co-localization, and co-IP that gene (Exp16 ), which is a HBI1 direct target gene (Fan et al. pro CRY1 interacts with HBI1 through its N-terminus in a blue- 2014), and made the reporter construct expressing LUC under light-dependent manner. The significance of the CRY1–HBI1 the control of Exp16 (Fig. 8E). The effector constructs were pro interaction is attested to by the following demonstrations. (i) transiently co-expressed in tobacco leaf cells with the reporter HBI1 acts downstream of CRYs to regulate hypocotyl elong- construct in different combinations. The results showed that, ation under blue light. (ii) CRYs act to co-regulate a set of consistent with HBI1 being a positive regulator of Exp16 (Fan genes that are involved in regulating cell elongation antag- et al., 2014), HBI1 alone strongly stimulated the Exp16 :LUC pro onistically with HBI1. (iii) The CRY1 N-terminus inhibits reporter activity (Fig.  8F). However, when CRY1 was co- HBI1–DNA binding activity in vitro. (iv) Both CRY1 and its expressed with HBI1, the activity of the Exp16 promoter was N-terminus are able to inhibit HBI1 transcriptional activity dramatically repressed (Fig.  8F). We then analyzed the effects in tobacco leaf cells. Based on our results, we propose a model of CNT1 and CCT1 on HBI1 regulation of the Exp16 :LUC pro describing CRY1 regulation of hypocotyl elongation via its reporter activity. As shown in Fig.  8G, HBI1 induction of interaction with HBI1 (Fig. 9). In darkness, CRY1 is not active Exp16 :LUC activity was considerably inhibited by CNT1, pro and cannot interact with HBI1 protein. Hence, HBI1 is fully but barely affected by CCT1. In addition, HBI1 activity was active, and able to bind to its target genes and regulate their inhibited by CRY1 and CNT1 in a dose-dependent manner expression, leading to active hypocotyl elongation. Upon blue (Supplementary Fig. S7). Taken together, these results suggest light irradiation, CRY1 is activated, and able to interact with that CNT1-mediated interaction of CRY1 with HBI1 leads HBI1 to inhibit its binding to target genes, leading to inhib- to inhibition of HBI1 DNA binding activity and its transcrip- ition of hypocotyl elongation. tional activity. Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 3878 | Wang et al. Fig. 8. CRY1 represses HBI1’s transcriptional activity through CNT1 by inhibiting the DNA binding activity of HBI1. (A) qRT-PCR analysis showing the –2 –1 expression of HBI1 in WT seedlings grown in darkness for 5 d with adaption in darkness (WT-DK) or an exposure to 30 μmol m s blue light (WT-BL) for 0.5 h. Expression levels were normalized to an internal control PP2A, and the WT-DK level was arbitrarily set to 1. Data are represented as means ±SD –2 –1 (n=3). (B) Immunoblot analysis of the HBI1-Flag fusion protein level in HBI1-OX seedlings grown in darkness for 5 d and then exposed to 30 μmol m s blue light for the durations indicated. An asterisk denotes a non-specific band serving as a loading control. HBI1/* indicates the relative band intensities of HBI1-Flag normalized to the non-specific band and presented relative to that in darkness set at unity. (C and D) EMSAs showing CNT1 inhibition of HBI1 DNA binding ability. EMSA was performed with MBP–HBI1 and MBP proteins using a labeled probe composed of a DNA fragment of the promoter of DWF4 and biotin (DWF4p-Bio). 1 × 2×, 3×, and 1×, 5×, 20× in (C) indicate the amounts of MBP–HBI1 and cold competitor DNA (DWF4p-Cold) relative to the initial concentration of MBP–HBI1 and DWF4p-Bio probes, respectively. 0.2×, 1×, and 5× in (D) indicate the amounts of His-TF–CNT1 and His-TF– CCT1 relative to one-tenth the amount of MBP–HBI1. Signals at the bottom indicate free probes. (E) Schematic diagram of the construct of the Dual-LUC assay reporter expressing REN under the control of 35S promoter and LUC under the control of the Exp16 promoter (Exp16 ). (F and G) Dual-LUC pro assays showing that CRY1 represses the transcriptional activity of HBI1 through CNT1. Tobacco leaves were transfected with Agrobacterium in different combinations of effectors and the Exp16 reporter, and then kept in dim light for 3 d. 0.05×, 3× in (F) and 0.1×, 3× in (G) indicate the culture volume of pro effectors relative to that of the Exp16 reporter. The ratio of LUC activity relative to REN activity of the expression control (CFP as effector) was arbitrarily pro set to 1, to which the ratios of other groups were normalized. Error bars represent ±SD (n=4). Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 Regulation of photomorphogenesis by CRY1–HBI1 interaction | 3879 It should be noted that we observed that CNT1 interacted with HBI1 in yeast cells and tobacco cells, it is possible that the with HBI1 regardless of blue light in yeast two-hybrid assays, signaling mechanism of CRY2 also involves interaction with whereas CNT2 interacted with HBI1 in blue light, but not in HBI1 under a low intensity of blue light. Moreover, our obser- darkness (Figs 2C, 4C). These differences might result from the vation that HBI1 acts to promote hypocotyl elongation in red amino acid sequence dissimilarity between CNT1 and CNT2. and far-red light (Supplementary Figs S2, S3) suggests a pos- Although CRY1 and CRY2 are homologous proteins, they sible role for HBI1 in mediating phytochrome signaling. Given have different functions and protein properties. For example, previous demonstrations that phytochromes interact with the CRY1 plays a role in inhibiting hypocotyl elongation under cryptochrome-interacting proteins such as COP1 and SPAs low, middle, and high fluence rates of blue light, whereas (Seo et al., 2004; Jang et al., 2010; Lu et al., 2015; Sheerin et al., CRY2 performs this role primarily under a low intensity of 2015), and that cryptochromes interact with the phytochrome- blue light (Lin et  al., 1998). Consistent with this, CRY2 is interacting factor PIF4 to mediate light signaling (Ma et  al., degraded under a high intensity of blue light, whereas CRY1 2016; Pedmale et  al., 2016), it is possible that phytochromes is stable (Shalitin et  al., 2002). The amino acid sequence dif- may interact directly with HBI1 to regulate hypocotyl elong- ferences between CRY1 and CRY2 might also lead to these ation. On the other hand, phytochromes might influence differences. In our Dual-LUC assays, we found that, similar to HBI1 function indirectly by regulating the biosynthesis of the a previous report (Ma et al., 2016), CRY1 or CNT1 alone is endogenous phytohormones BR and GA based on the fol- somehow able to activate the Exp16 reporter in tobacco cells lowing demonstrations: (i) PIF4 and PIF5 bind to the pro- pro (Fig. 8F, G). Given the demonstrations that CRY1 is not a tran- moter regions of the key BR biosynthetic genes DWF4 and scription factor and that soybean CRY2a interacts with CIB1 BR6ox2 to promote their expression directly (Wei et al. 2017); in a blue-light-dependent manner to inhibit CIB1 DNA bind- (ii) phytochromes regulate GA biosynthesis through PIF3- ing activity and repress leaf senescence (Meng et al., 2013), we LIKE5 (PIL5), which represses the expression of GA biosyn- postulate that CRY1 activation of Exp16 activity is not likely thetic genes GA3ox1 and GA3ox2 and activates the expression pro to be mediated through a direct mechanism, namely CRY1 of a GA catabolic gene GA2ox in Arabidopsis (Oh et al. 2006); binding to Exp16 . It is likely that CRY1–HBI1 interaction and (iii) HBI1 is involved in the signaling pathway of BR and pro might lead to a decrease in HBI1–DNA binding and transcrip- GA (Bai et al., 2012a). tional activity. The bHLH-type proteins constitute a large family of tran- In view of the fact that the signaling mechanism of both scription factors in eukaryotes, which is specifically classified CRY1 and CRY2 involves physical interactions with COP1, into dozens of subfamilies with a total of ~170 members in SPAs, and PIFs, and our demonstration that CRY2 also interacts Arabidopsis (Carretero-Paulet et  al., 2010). A  previous study Fig. 9. A model describing regulation of HBI1 transcription by CRY1. (A) In darkness, CRY1 is inactive and HBI1 is able to bind to the promoters of its direct target genes promoting cell elongation and activating their expression, leading to enhanced hypocotyl elongation. (B) Upon blue light irradiation, CRY1 is activated and able to interact with HBI1 through its N-terminus (CNT1) to inhibit HBI1 binding to its target genes, resulting in repression of expression of these genes and inhibited hypocotyl elongation. CCT1 denotes the CRY1 C-terminus. Thicker and thinner red arrows denote higher and lower gene expression, respectively. Downloaded from https://academic.oup.com/jxb/article/69/16/3867/5026618 by DeepDyve user on 18 July 2022 3880 | Wang et al. reported that bHLH factors are capable of forming homodi- Acknowledgments mers or heterodimers through the HLH domains and bind- We thank Dr Lida Zhang for assistance in bioinformatic analysis of ing to specific DNA sequences through the basic domains RNA-Seq data. This work was supported by The National Key Research (Toledo-Ortiz et  al., 2003). HBI1 was identified as a typical and Development Program of China grant (2017YFA0503800) and The National Natural Science Foundation of China grants to HQY DNA-binding bHLH protein and closely related to many (31530085, 91217307, and 90917014) and to HLL (31570282 and bHLH factors belonging to different subfamilies. 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Journal of Experimental Botany – Oxford University Press

Published: Jul 18, 2018

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