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Identification of BBX proteins as rate-limiting cofactors of HY5

Abstract

As a source of both energy and environmental information, monitoring of incoming light is crucial for plants to optimize growth throughout development1. Concordantly, the light signalling pathways in plants are highly integrated with numerous other regulatory pathways2,3. One of these signal integrators is the basic leucine zipper domain (bZIP) transcription factor LONG HYPOCOTYL 5 (HY5), which has a key role as a positive regulator of light signalling in plants4,5. Although HY5 is thought to act as a DNA-binding transcriptional regulator6,7, the lack of any apparent transactivation domain8 makes it unclear how HY5 is able to accomplish its many functions. Here we describe the identification of three B-box containing proteins (BBX20, BBX21 and BBX22) as essential partners for HY5-dependent modulation of hypocotyl elongation, anthocyanin accumulation and transcriptional regulation. The bbx20 bbx21 bbx22 (bbx202122) triple mutant mimics the phenotypes of hy5 in the light and its ability to suppress the cop1 mutant phenotype in darkness. Furthermore, 84% of genes that exhibit differential expression in bbx202122 are also regulated by HY5, and we provide evidence that HY5 requires the B-box proteins for transcriptional regulation. Finally, expression of a truncated dark-stable version of HY5 (HY5(ΔN77)) together with BBX21 mutated in its VP motif strongly promoted de-etiolation in dark-grown seedlings, demonstrating the functional interdependence of these factors. In sum, this work clarifies long-standing questions regarding HY5 action and provides an example of how a master regulator might gain both specificity and dynamicity through the obligate dependence of cofactors.

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Fig. 1: BBX20–22 and HY5 interdependently promote photomorphogenesis.
Fig. 2: HY5 requires BBX proteins for transcriptional regulation.
Fig. 3: COP1 suppression of the HY5–BBX module inhibits de-etiolation in darkness.

Data Availability

The RNA-seq data has been deposited at the NCBI Gene Expression Omnibus under the accession number GSE137147. Source data are provided with this paper.

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Acknowledgements

We thank K. Halliday for proof-reading the manuscript. This project was supported by Deutsche Forschungsgemeinschaft (DFG grant JO 1409/1-1 to H.J. and JO 1409/2-1 to K.B.) and a Royal Society Grant (RG150711 to G.T.-O). Work in Geneva was supported by the Swiss National Science Foundation grant 31003A_175774 to R. Ulm.

Author information

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Authors

Contributions

H.J. conceived, designed and directed the project. G.T.-O. and M.P. performed ChIP–qPCR experiments and M.L created bbx20-1 and higher-order mutants. K.B. and C.B. performed the protoplast assays while H.J. and K.B. performed all other experiments. H.J. and K.B. analysed the data. H.J., K.B. and G.T.-O. wrote the manuscript and all authors revised the manuscript.

Corresponding author

Correspondence to Henrik Johansson.

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The authors declare no competing interests.

Additional information

Peer review information Nature Plants thanks Enamul Huq and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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

Extended data

Extended Data Fig. 1 Creation and validation of the bbx20-1 mutant.

a, Schematic representation of the BBX20 locus indicating two available T-DNA insertion lines and the sequence targeted by CRISPR/Cas9. Orange areas indicate 5’ and 3’ UTR while black areas indicate the two exons of BBX20. Blue and red text indicate the gRNA and PAM sequence, respectively, used for CRISPR/Cas9 induced mutagenesis of BBX20. The recovered bbx20-1 mutant harbored a 1-bp deletion 4-bp upstream of the PAM sequence, resulting in the loss of a HindIII recognition sequence available in the WT. b, Expected amino acid sequence of the bbx20-1 mutant caused by the 1-bp frameshift. Frameshifted amino acids are labelled in red and the asterisk indicates an early stop-codon. c, Hypocotyl measurements of 68 5-day-old seedlings from a bbx20-1 heterozygote parental plant grown in 100 µmol m−2 s−1 of red light. After measurements of the individual hypocotyls, PCR based genotyping revealed 14 WT, 38 heterozygote and 16 bbx20-1 homozygote seedlings allowing for grouping each measurement into the three genotypes. d, Hypocotyl measurements of Col-0, bbx20-1 and T1 bbx20-1 seedlings complemented with a genomic BBX20 construct, utilizing the pFAST vector system for identification of transgenic seeds, grown as in (c). e, Photo of representative seedlings from (d). Box plots represent medians and interquartile ranges with whiskers extending to the largest/smallest value and outliers are shown as dots. Different letters represent statistical significant differences (p < 0.05) as determined by one-way ANOVA followed by Tukey’s Post Hoc test. Source data

Extended Data Fig. 2 BBX20 acts upstream of HY5 and downstream of COP1.

a, BBX20 transcript levels relative to the reference genes ACT2 and EF1A in 4-day-old WT and 35S::GFP-BBX20 transgenic seedlings grown in 75 µmol m−2 s−1 of constant white light. n = 4 biological replicates indicated by open circles. Data represents means ± SE. b-c, Hypocotyl measurements of 5-day-old seedlings grown in darkness, monochromatic red (80 µmol m−2 s−1), blue (14 µmol m−2 s−1) and far-red (1 µmol m−2 s−1) light. d, Hypocotyl measurements of 5-day-old seedlings grown in darkness. Box plots represent medians and interquartile ranges with whiskers extending to the largest/smallest value and outliers are shown as dots. Different letters represent statistical significant differences (p < 0.05) as determined by one-way ANOVA followed by Tukey’s Post Hoc test. Source data

Extended Data Fig. 3 Transcript analysis of genes inhibited by BBX20-22 and HY5.

Analysis of XTH18, PRX53 and IAA6 transcript abundance relative to the GADPH and TFIID reference genes in 4-day-old seedlings grown in 80 µmol m−2 s−1 of red light. Data represents means ± SE. n = 4 independent biological replicates. Different letters denote statistical significant differences (p < 0.05) as determined by one-way ANOVA followed by Tukey’s Post Hoc test. Open circles indicate single biological measurements. Source data

Extended Data Fig. 4 The bbx202122 phenotype is not due to reduced HY5 transcript abundance.

a, Transcript levels of HY5 relative to GADPH and TFIID in 4-day-old seedlings grown in 80 µmol m−2 s−1 of red light. n = 4 biological replicates indicated by open circles. Data represents means ± SE. Different letters represent statistical significant differences (p < 0.05) as determined by one-way ANOVA followed by Tukey’s Post Hoc test. b, Hypocotyl measurements of 5-day old seedlings grown as in (a) or constant darkness. Different letters represent statistical significant differences (p < 0.05) as determined by two-way ANOVA followed by Tukey’s Post Hoc test. c, Quantification of fluorescence intensity of YFP in the nuclei of bbx202122hy5hyh protoplasts transiently expressing HY5-YFP with or without CFP-BBX21. Different letters represent statistical significant differences (p < 0.05) as determined by Student’s t-test. Box plots represent medians and interquartile ranges with whiskers extending to the largest/smallest value and outliers are shown as dots. Source data

Extended Data Fig. 5 A predicted 9aaTAD of BBX21 promotes transcription in yeast.

a, Liquid yeast two-hybrid β-galactosidase assay using DBD-HY5 as bait and BBX20, BBX21 or BBX22 as prey not fused to an additional activation domain. Box plots represent medians and interquartile ranges with whiskers extending to the largest/smallest value and outliers are shown as dots. n = 6. b, Alignment of predicted TAD region of BBX20 and BBX21 using Clustal Omega (1.2.4). BBX21(mTAD) shows the sequence after the introduction of 5 alanine residues c, Graphical representation of four truncated BBX21 constructs, 21A-21D, all containing the predicted 9aaTAD region. B and DBD represent B-box domain and DNA-binding domain, respectively. d, Measurements of auto activation of 21A, 21B, 21C and 21D fragments in yeast. n = 6. e, Yeast two-hybrid assay using HY5 as bait and BBX21 or BBX21(mTAD) as prey. –LW and –LWUH indicate media lacking either Leu, Trp or Leu, Trp, Ura, His, respectively. 3-AT represents the addition of 3-amino-1, 2,4-triazol to the growth medium. The experiment was repeated with similar results using two independent sets of primary transformants. Single measurements are shown as open circles and statistical groups are indicated by letters as determined by one-way ANOVA followed by Tukey’s Post Hoc test. Source data

Extended Data Fig. 6 BBX20 and BBX21 associates with DNA dependent on HY5 in Arabidopsis.

a-d, Chromatin immunoprecipitation using no antibody (-Ab) or an anti-GFP antibody (+Ab) on samples harvested from 4-day-old 35S::GFP, 35S::GFP-BBX20 #2 and hy5 35S::GFP-BBX20 #2 (a, b) or 35S::GFP, 35S::GFP-BBX21 #2 and hy5 35S::GFP-BBX21 #2 (c, d) transgenic seedlings grown in 80 µmol m−2 s−1 of red light. p1 and p2 denotes primer pairs amplifying a non-binding control region and HY5 binding region, respectively. n = 3 biological replicates for +Ab samples and a single sample for -Ab). Data represents means ± SE. Single measurements are shown as open circles and statistical groups are indicated by letters as determined by one-way ANOVA followed by Tukey’s Post Hoc test. Source data

Extended Data Fig. 7 HY5 binding to the MYB12 promoter in bbx202122 and 35S::GFP-BBX20 #1 correlate with HY5 transcript levels.

a, Chromatin immunoprecipitation using no antibody (-Ab) or an anti-HY5 antibody (+Ab) on samples harvested from 4-day-old WT, hy5, bbx202122 and 35S::GFP-BBX20 #1 seedlings grown in 80 µmol m−2 s−1 of red light. p2 denotes primer pairs amplifying a HY5 binding region of the MYB12 promoter as shown in Fig. 2a and ACT is used as negative control. n = 3 independent biological replicates and each replicate was normalized to WT pMYB12 p2 +AB. Data represents means ± SE. Single measurements from each biological repeat is indicated by an open circle, cross and plus sign, respectively. b, Measurements of HY5 transcript levels relative to PP2A in 4-day-old seedlings grown in 80 µmol m−2 s−1 of red light. Biological replicates indicated by open circles. Data represents means ± SE. Different letters represent statistical significant differences (p < 0.05) as determined by one-way ANOVA followed by Tukey’s Post Hoc test. Source data

Extended Data Fig. 8 Expression of BBX21(VP-AA) or a VP16 fusion is sufficient for HY5(ΔN77) to promote photomorphogenesis.

a, Photo of representative seeds, dissected embryos and seed coats of indicated genetic background. Similar observations were made over multiple generations. b, Alignment of VP-domain containing amino acids 35-47 of HY5, 236-248 of BBX24, 226-238 of BBX25 and 305-317 of BBX21, respectively, using Clustal Omega (1.2.4). The Val-Pro pair labelled red in BBX21 was modified to Ala-Ala to generate BBX21(VP-AA). c, Immunoblot analysis of total protein samples collected from transgenic seedlings expressing GFP-BBX21 and GFP-BBX21VP-AA driven by the 35S promoter, grown for 4 days in darkness. Anti-GFP and anti-ACT antibodies were used to detect the BBX proteins and the ACT loading control, respectively. 3 independent biological replicates are shown. d, BBX21 transcript levels relative to the GADPH and TFIID reference genes in WT, 35S::GFP-BBX21 and 35S::GFP-BBX21VP-AA seedlings grown in darkness for 4 days. n = 4. e, BBX21 transcript levels relative to the GADPH and TFIID reference genes in WT and XVE::BBX21VP-AA seedlings grown in darkness for 4 days with 20 µM of 17‐β‐oestradiol (+E2) or 0.1% ethanol (v/v) (Control). n = 4 biological replicates. f, Transcript levels of HY5 and BBX21 shown as relative to the GADPH and TFIID reference genes in the indicated crosses between WT, hy5, XVE::BBX21VP-AA and hy5 35S::HY5ΔN77 grown for 4 days in darkness on 20 µM of 17‐β‐oestradiol. Black and red letters indicate significance for HY5 and BBX21 levels, respectively. n = 4 biological replicates indicated by open circles. g-i, Analysis of XTH12, XTH13, XTH26 (g) PRX7, PRX26, PRX44 (h) MYB12, F3H and FLS1 (i) transcript abundance relative to GADPH and TFIID in 4 day old seedlings grown as in (f). n = 4 biological replicates indicated by open circles. Data represents means ± SE. Different letters represent statistical significant differences (p < 0.05) as determined by one-way (d, f-h) or two-way (e) ANOVA followed by Tukey’s Post Hoc test. j, Photo of representative 5-day-old dark grown hy5 mutant seedlings or T1 hy5 mutant seedlings transformed with 35S::VP16HY5ΔN77. Source data

Supplementary information

Supplementary Information

Supplementary Methods and Supplementary Table 1.

Reporting Summary

Supplementary Data 1

Lists of bbx202122 and hy5 DEGs from RNA sequencing, including a list of DEGs overlapping in the two mutants. n = 3 biological replicates. See Methods for details on statistical analysis.

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Bursch, K., Toledo-Ortiz, G., Pireyre, M. et al. Identification of BBX proteins as rate-limiting cofactors of HY5. Nat. Plants 6, 921–928 (2020). https://doi.org/10.1038/s41477-020-0725-0

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