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Transcriptional regulation of strigolactone signalling in Arabidopsis

Abstract

Plant hormones known as strigolactones control plant development and interactions between host plants and symbiotic fungi or parasitic weeds1,2,3,4. In Arabidopsis thaliana and rice, the proteins DWARF14 (D14), MORE AXILLARY GROWTH 2 (MAX2), SUPPRESSOR OF MAX2-LIKE 6, 7 and 8 (SMXL6, SMXL7 and SMXL8) and their orthologues form a complex upon strigolactone perception and play a central part in strigolactone signalling5,6,7,8,9,10. However, whether and how strigolactones activate downstream transcription remains largely unknown. Here we use a synthetic strigolactone to identify 401 strigolactone-responsive genes in Arabidopsis, and show that these plant hormones regulate shoot branching, leaf shape and anthocyanin accumulation mainly through transcriptional activation of the BRANCHED 1, TCP DOMAIN PROTEIN 1 and PRODUCTION OF ANTHOCYANIN PIGMENT 1 genes. We find that SMXL6 targets 729 genes in the Arabidopsis genome and represses the transcription of SMXL6, SMXL7 and SMXL8 by binding directly to their promoters, showing that SMXL6 serves as an autoregulated transcription factor to maintain the homeostasis of strigolactone signalling. These findings reveal an unanticipated mechanism through which a transcriptional repressor of hormone signalling can directly recognize DNA and regulate transcription in higher plants.

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Fig. 1: GR244DO efficiently stimulates strigolactone signalling.
Fig. 2: BRC1 and TCP1 regulate shoot branching and leaf shape, respectively, in strigolactone signalling.
Fig. 3: Strigolactones promote anthocyanin accumulation by inducing transcription of PAP1 and anthocyanin-biosynthesis genes.
Fig. 4: SMXL6 binds directly to the SMXL7 promoter.
Fig. 5: Proposed model of transcriptional regulation by SMXL6.

Data availability

Uncropped gels and blots are provided in Supplementary Fig. 1. The RNA-seq and ChIP–seq data have been deposited in the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/) under the accession numbers GSE126331 and GSE140705. Materials and reagents are available from the corresponding authors on request. Source data are provided with this paper.

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Acknowledgements

We thank J. Qiu for providing the pap1-D mutant; Q. Xie for providing the CRISPR–CAS9 system driven by the YAO promoter; and Q. Chen for providing the CRISPR–CAS9 system driven by the egg-cell-specific promoter. This work was supported by grants from the National Natural Science Foundation of China (31788103, 31600221 and 31661143025) and the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2019099).

Author information

Authors and Affiliations

Authors

Contributions

J.L. and B.W. designed and supervised the overall research. L.W. and B.W. carried out chemical treatments, gene-expression analysis and phenotype observations. L.W. carried out protein-degradation analysis, ChIP analysis, EMSAs, anthocyanin measurements and assays of transcriptional activity. H.Y., T.L. and L.K. processed RNA-seq and ChIP–seq data. H.G., A.W., N.S. and H.M. contributed to genetic analyses and plasmid construction. X.L. and J.Y. synthesized the strigolactone analogues GR244DO, GR24ent-4DO and GR245DS. J.C. and B.W. measured ABA content. L.W., B.W. and G.X. performed statistical analyses. B.W., L.W. and J.L. analysed data and wrote the paper with input from all other authors.

Corresponding authors

Correspondence to Bing Wang or Jiayang Li.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Comparison of rac-GR24 and GR24 stereoisomers in stimulating strigolactone signalling.

a, Chemical structures of GR24 stereoisomers with different stereochemical features. b, Stability of SMXL6–GFP in 35S:SMXL6-GFP transgenic seedlings treated with 2 μM rac-GR24, GR245DS, GR244DO or GR24ent-4DO. Proteins were detected by immunoblotting with an anti-GFP monoclonal antibody. Data represent three independent experiments. c, Expression of SMXL6 and SMXL8 in 10-day-old wild-type seedlings pretreated in half-strength MS liquid medium for 2 h, and then treated with 5 μM rac-GR24, GR245DS or GR244DO for 0 h, 2 h or 4 h. Data were normalized to mock treatment at specific time points and are means ± s.e.m.; n = 3 biologically independent samples. d, Induction of SMXL6 and SMXL8 upon GR244DO treatment is blocked by D14 mutation. Data were normalized to mock treatment in wild-type seedlings at specific time points and are means ± s.e.m.; n = 3 biologically independent samples. c, d, P values are shown; two-sided Student’s t-test.

Source data

Extended Data Fig. 2 Verification of strigolactone-responsive genes by qRT–PCR analyses.

ae, gi, Expression of strigolactone-responsive genes in 10-day-old wild-type (Col-0) seedlings pretreated in half-strength MS liquid medium for 2 h, and then treated with 5 μM GR244DO in half-strength MS liquid medium for 2 h or 4 h. The qRT–PCR data in ae, gi are means ± s.e.m.; n = 3 biologically independent samples (for SAUR22, SAUR29, SAUR61, AFL1, ELIP1, DLK2, TT7, ANS, HB52, WRKY38, WRKY49, MYB27, MAX4) or 4 biologically independent samples (for SAUR3, SAUR65, IAA19, IAA29, HB40, YUC3, KUF1, STH7/BZS1, AT5G56840, REM36, bHLH29, HSFA6B, AT1G64380, AT1G71520, HEC1, NRT2.6, BGLU47, DVL15, RR16, ABCG37, BEE3, EXP11, EXP12). P values are shown; two-sided Student’s t-test. f, Fold change in transcription-factor-encoding genes in response to GR244DO treatment based on RNA-seq data. ABCG37, ATP-BINDING CASSETTE G37; AFL1, At14a-LIKE 1; ANS, ANTHOCYANIDIN SYNTHASE; BEE3, BR-ENHANCED EXPRESSION 3; BGLU47, BETA-GLUCOSIDASE 47; bHLH29, BASIC HELIX-LOOP-HELIX PROTEIN 29; BRC1, BRANCHED 1; DLK2, D14-LIKE 2; DVL15, DEVIL 15; ELIP1, EARLY LIGHT-INDUCIBLE PROTEIN 1; EXP11, EXP12, EXPANSIN 11, 12; HB40, HB52, HOMEOBOX PROTEIN 40, 52; HEC1, HECATE 1; HSFA6B, HEAT SHOCK TRANSCRIPTION FACTOR A6B; IAA19, IAA29, INDOLE-3-ACETIC ACID INDUCIBLE 19, 29; KAN1, KANADI 1; KUF1, KAR-UP F-BOX 1; MAX4, MORE AXILLARY GROWTH 4; MYB27, MYB DOMAIN PROTEIN 27; NRT2.6, NITRATE TRANSPORTER 2.6; PAP1, PRODUCTION OF ANTHOCYANIN PIGMENT 1; PAR1, PHY RAPIDLY REGULATED 1; REM36, REPRODUCTIVE MERISTEM 36; RR16, RESPONSE REGULATOR 16; SAUR3, SAUR22, SAUR29, SAUR61, SAUR65, SMALL AUXIN UPREGULATED RNA 3, 22, 29, 61, 65; STH7/BZS1, SALT TOLERANCE HOMOLOG 7/BZR1-1D SUPPRESSOR 1; TCP1, TCP DOMAIN PROTEIN 1; TGA7, TGACG SEQUENCE-SPECIFIC BINDING PROTEIN 7; TT7, TRANSPARENT TESTA 7; WRKY38, WRKY49, WRKY DNA-BINDING PROTEIN 38, 49; YUC3, YUCCA 3.

Source data

Extended Data Fig. 3 SMXL6, 7, 8 negatively regulate AFL1 expression and drought tolerance.

a, Induction of AFL1 upon GR244DO treatment is blocked in the d14 mutant. Data were normalized to mock treatment in wild-type seedlings at specific time points and are means ± s.e.m.; n = 3 biologically independent samples. b, Expression of AFL1 in 10-day-old seedlings of the wild type (Col-0) and s678 triple mutant. Data were normalized to the wild type and are means ± s.e.m.; n = 3 biologically independent samples. c, Phenotype and survival rate of wild-type and s678 plants. We exposed 2-week-old plants to drought stress by withholding of water for 2 weeks and then rewatered for 3 days. Data are means ± s.e.m.; n = 3 pools (24 plants each pool). ac, P values are shown; two-sided Student’s t-test.

Source data

Extended Data Fig. 4 The EAR motif of SMXL6 is essential for regulation of shoot and leaf development.

a, Phenotypes of wild-type (Col-0), s678, S6:S6-HA s678 and S6:S6ΔEAR-HA s678 plants. S6:S6-HA 1 s678 and S6:S6-HA 2 s678 represent independent transgenic lines with the S6:S6-HA transgene in the s678 background; S6:S6ΔEAR-HA 1 s678 and S6:S6ΔEAR-HA 2 s678 represent independent transgenic lines containing the S6:S6ΔEAR-HA transgene in the s678 background. Scale bar, 5 cm. b, Quantitative analysis of shoot branching in the adult plants shown in a. We counted the number of primary branches grown from the rosette leaf axil (left) and secondary branches grown from the cauline leaf axil (right) of at least 0.5 cm. Data are means ± s.e.m.; n = 20 plants. c, Ratio of leaf length to width for the fifth leaves of the wild-type (Col-0), s678, S6:S6-HA s678 and S6:S6ΔEAR-HA s678 plants after growth for 3 weeks. Data are means ± s.e.m.; n = 15 leaves. d, Leaf morphology of 3-week-old plants. The fifth leaves are marked by white arrows. Scale bar, 1 cm. b, c, P values are shown; Tukey’s HSD test.

Source data

Extended Data Fig. 5 BRC1 regulates strigolactone-mediated shoot branching.

a, Expression of BRC1 in buds of primary rosette (RI) and secondary cauline (CII) branches. Data were normalized to the wild type (Col-0) and are means ± s.e.m.; n = 3 biologically independent samples. b, Schematic representation of the T-DNA insertion mutant brc1-6. The T-DNA insertion site and positions of RT–qPCR primers are indicated by white and black triangles, respectively. c, Expression of BRC1 in 10-day-old seedlings using B1-1 and B1-2 primer pairs. Data were normalized to the wild type and are means ± s.e.m.; n = 3 biologically independent samples. d, Shoot-branching phenotypes of plants at the adult stage. Scale bar, 5 cm. Data represent 15 independent experiments. We counted the number of primary rosette branches and secondary cauline branches of at least 0.5 cm. Data are means ± s.e.m.; n = 15 plants. e, f, Rosette leaf number (e) and the ratio of primary rosette branch to rosette leaf number (f) in adult Col-0, d14-1, s678, brc1-6, and s678 brc1-6 plants. Data are means ± s.e.m.; n = 14 plants. g, Induction of HB40 upon GR244DO treatment is blocked in the d14 mutant. Data were normalized to mock treatment in wild-type seedlings at specific time points and are means ± s.e.m.; n = 3 biologically independent samples. h, ABA content in unelongated axillary buds of rosette leaves in Col-0, max3-9 and s678 plants. Data are means ± s.e.m.; n = 4 biologically independent samples. a, ch, P values are shown; two-sided Student’s t-test.

Source data

Extended Data Fig. 6 TCP1 contributes to strigolactone-mediated leaf-shape regulation.

a, Ratio of leaf length to width for the fifth leaves in wild-type (Col-0), s678, brc1-6 and s678 brc1-6 plants grown for 3 weeks. Data are means ± s.e.m.; n = 12 leaves. b, The EAR motif is required for SMXL6-mediated regulation of the expression of TCP1 in seedlings. Data were normalized to wild type and are means ± s.e.m.; n = 3 biologically independent samples. c, Left, morphology of the fifth leaves of 3-week-old plants. 35S:TCP1-SRDX 1 and 35S:TCP1-SRDX 2 represent independent transgenic lines with the 35S:TCP1-SRDX transgene in the Col-0 background; 35S:TCP1-SRDX 1 s678 and 35S:TCP1-SRDX 2 s678 represent independent transgenic lines with the 35S:TCP1-SRDX transgene in the s678 background. Scale bar, 1 cm. Right, ratio of leaf length to width for the fifth leaves. Data are means ± s.e.m.; n = 12 leaves. d, Morphology of the fifth leaves of wild-type, s678, tcp1-1 and s678 tcp1-1 plants grown for 3 weeks. Scale bar, 1 cm. e, Mutation site of the tcp1-1 mutant. ac, P values are shown; Tukey’s HSD test (a, c) or two-sided Student’s t-test (b).

Source data

Extended Data Fig. 7 Strigolactones promote expression of anthocyanin-biosynthesis genes.

a, Induction of PAP2, MYB113 and MYB114 upon GR244DO treatment is blocked in the d14 mutant. Data were normalized to mock treatment in wild-type seedlings at specific time points and are means ± s.e.m.; n = 3 biologically independent samples. b, c, Anthocyanin content in Col-0 plants, strigolactone mutants and transgenic lines after growth for 3 weeks. Data are means ± s.e.m.; n = 3 pools (6 seedlings per pool). d, Expression of PAP1, PAP2, MYB113, MYB114 and DFR in 3-week-old wild-type (Col-0), max3-9, s678 and max3-9 s678 plants. Data were normalized to wild type and are means ± s.e.m.; n = 3 biologically independent samples. e, Expression of PAP1, PAP2, MYB113, MYB114 and DFR in 3-week-old wild-type, s678, S6:S6-HA s678 and S6:S6ΔEAR-HA s678 plants. Data were normalized to wild type and are means ± s.e.m.; n = 3 biologically independent samples. f, The pap1-D mutant rescued the anthocyanin-biosynthesis deficiency of the max3-9 mutant. Scale bar, 1 mm. Data represent 18 independent experiments. g, Mutation sites in the pap1-2 and pap2-1 mutants. ae, P values are shown; two-sided Student’s t-test (a, b, d, e) or Tukey’s HSD test (c).

Source data

Extended Data Fig. 8 Feedback inhibition of SMXL transcription in Arabidopsis.

a, SMXL6 binds to the SMXL6 and SMXL8 promoters in ChIP–seq (left) and ChIP–qPCR assays (right). Red lines below the peak regions show the locations of probes used in ChIP–qPCR assays and EMSAs. Chromatin from S6:S6-HA s678 and s678 seedlings was immunoprecipitated with anti-HA polyclonal antibodies. In ChIP–qPCR assays, the enrichment of target gene promoters is displayed as a percentage of input DNA. Values are means ± s.e.m.; n = 4 biologically independent samples. The TUA2 promoter was used as a nonspecific target. b, Diagrams showing the constructs used in transient expression assays in protoplasts. We included the −1598 to +402 region of SMXL6, −1607 to +393 region of SMXL7, −1890 to +110 region of SMXL8 and −2520 to +480 region of BRC1 in pSMXL6-LUC, pSMXL7-LUC, pSMXL8-LUC and pBRC1-LUC constructs, respectively. The small black rectangle represents the EAR motif in SMXL6. Firefly LUC, firefly luciferase reporter gene; Renilla LUC, Renilla luciferase reporter gene. Firefly LUC activity was normalized against that of Renilla LUC. c, Transcriptional regulation by SMXL6 and SMXL6ΔEAR of SMXL6 and SMXL8 promoters in transient expression assays. Data were normalized to samples expressing GFP and are means ± s.e.m.; n = 3 biologically independent samples. d, e, Expression of SMXL6, SMXL7 and SMXL8 in nonelongated axillary buds of primary rosette (RI) branches and secondary cauline (CII) branches of Col-0 and strigolactone mutants. Data were normalized to wild type and are means ± s.e.m.; n = 3 biologically independent samples. a, ce, P values are shown; two-sided Student’s t-test.

Source data

Extended Data Fig. 9 Domain mapping of the SMXL7 promoter bound by SMXL6 in EMSAs.

a, Recombinant GST, GST–SMXL6, GST–SMXL7 and GST–SMXL8 proteins used in EMSA assays. Bands of target proteins are indicated by red arrows. Data represent four independent experiments. b, Direct binding of GST–SMXL6, GST–SMXL7 and GST–SMXL8 to the promoters of SMXL67, 8 in EMSAs. Data represent three independent experiments. c, d, Direct binding of GST–SMXL6 to the P7-3 and P7-3-3 regions of the SMXL7 promoter in EMSAs. Data represent four independent experiments.

Extended Data Fig. 10 Relationship between SMXL6, 7, 8 and SPL9, 15 in shoot branching.

a, b, Phenotypes and quantitative analysis of wild-type (Col-0), d14-1, s678, spl9-4 spl15-1 and s678 spl9-4 spl15-1 plants grown for 6 weeks. Scale bar, 3 cm. Data are means ± s.e.m.; n = 14 plants. c, Phenotypes of Col-0, s678, spl9-4 spl15-1 and s678 spl9-4 spl15-1 plants grown for 3 weeks. Scale bar, 3 cm. d, Rosette leaf number and average primary rosette branch number per rosette leaf in Col-0 plants and the indicated mutants. Data are means ± s.e.m.; n = 14 plants. e, Expression of BRC1 in primary rosette buds of Col-0 plants and the indicated mutants. Data were normalized to wild type and are means ± s.e.m.; n = 4 biologically independent samples. b, d, e, P values are shown; two-sided Student’s t-test.

Source data

Supplementary information

Supplementary Figure 1

This file contains the uncropped gel source data for Figure 4e, f, and Extended Data Figure 1b, Extended Data Figure 9a-d. Red boxes indicate gel region used in figures.

Reporting Summary

Supplementary Methods

This file contains the synthesis method of GR24 stereoisomers.

Supplementary Table 1

Differentially Expressed Genes (DEGs) upon GR244DO treatment. P values were determined by R package (version 3.3.1) DESeq (version 1.26.0); n = 3 biologically independent samples.

Supplementary Table 2

Enriched GO terms and KEGG pathways of DEGs. P values and FDR were determined by PlantGSEA program.

Supplementary Table 3

Transcription factor-encoding genes responsive to GR244DO treatment. P values were determined by R package (version 3.3.1) DESeq (version 1.26.0); n = 3 biologically independent samples.

Supplementary Table 4

Chromosomal regions of SMXL6 binding peaks. Peaks, P values, FDR, and fold enrichment were determined by MACS (version 1.4.2).

Supplementary Table 5

Overlapped chromosomal regions of SMXL6 binding peaks in ChIP-seq.

Supplementary Table 6

Overlapped genes targeted by SMXL6 in ChIP-Seq.

Supplementary Table 7

The GR244DO responsive genes associated with ChIP-seq targeted genes.

Supplementary Table 8

Primers and probes used in this study.

Supplementary Table 9

GenBank accession number of plasmids in this study.

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Wang, L., Wang, B., Yu, H. et al. Transcriptional regulation of strigolactone signalling in Arabidopsis. Nature 583, 277–281 (2020). https://doi.org/10.1038/s41586-020-2382-x

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