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A redundant transcription factor network steers spatiotemporal Arabidopsis triterpene synthesis

Nature Plants volume 9, pages 926–937 (2023)Cite this article

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Abstract

Plant specialized metabolites modulate developmental and ecological functions and comprise many therapeutic and other high-value compounds. However, the mechanisms determining their cell-specific expression remain unknown. Here we describe the transcriptional regulatory network that underlies cell-specific biosynthesis of triterpenes in Arabidopsis thaliana root tips. Expression of thalianol and marneral biosynthesis pathway genes depends on the phytohormone jasmonate and is limited to outer tissues. We show that this is promoted by the activity of redundant bHLH-type transcription factors from two distinct clades and coactivated by homeodomain factors. Conversely, the DOF-type transcription factor DAG1 and other regulators prevent expression of the triterpene pathway genes in inner tissues. We thus show how precise expression of triterpene biosynthesis genes is determined by a robust network of transactivators, coactivators and counteracting repressors.

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Fig. 1: Single-cell transcript patterns of genes in the triterpene BGCs in root meristem cells upon JA treatment.
Fig. 2: Triterpene BGC gene expression depends partially on MYC2.
Fig. 3: Cell-specific activation of genes in the triterpene BGCs is codetermined by MYC2 and bHLH clade IVa TFs.
Fig. 4: Expression of triterpene genes in the BGCs is coactivated by GL2 and repressed in inner root tissues by the DOF factor DAG1.

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Data availability

The scRNAseq data are accessible via an online browser tool (http://bioit3.irc.ugent.be/plant-sc-atlas/) and raw data are deposited at NCBI with GEO numbers GSE179820 and GSE212826 for the mock- and JA-treated root tips, respectively. All other data generated for this study are included either in the main paper, Extended Data, or the Supplementary Information. Material requests should be directed to the corresponding author. Published data for TF motif mapping were retrieved from CisBP 2.00 (downloaded in December 2019: http://cisbp.ccbr.utoronto.ca/) and JASPAR2020 (https://jaspar.genereg.net/). The regulatory regions used for motif mapping were downloaded from PLAZA Dicots 4.5 (https://bioinformatics.psb.ugent.be/plaza/versions/plaza_v4_5_dicots/). Source data are provided with this paper.

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Acknowledgements

This Article was written in loving memory of A. Van Moerkercke (1979–2021). The authors thank A. Bleys for critically reading the manuscript; D. Gasperini for kindly sharing the ProMYCs:NLS-VENUS reporter lines, and P. Vittorioso for the dag1 mutant, ProDAG1:GUS and DAG1 over-expressing lines; J. R. Wendrich and T. Eekhout for assistance in the launching and analysis of the scRNAseq experiment; and S. Desmet and G. Goeminne from the VIB Metabolomics Core – Ghent for the thalianol profiling. This work was supported by the European Community’s Seventh Framework Program (FP7/2007–2013) under grant agreement 613692-TriForC and H2020 Program under grant agreement 760331-Newcotiana to A.G.; the Special Research Fund from Ghent University to A.G. and A.R. (project BOF18/GOA/013), and M.M. (project BOF20/GOA/012); the Flemish Government (AI Research program) to Y.S.; the Research Foundation Flanders with research project grants to A.G. (G004515N and G008417N) and a postdoctoral fellowship to P.F.-C.; a Swiss National Science Foundation postdoctoral fellowship (P300PA_177831) to M.C.; and a China Scholarship Council PhD scholarship to Y.B. A.O. acknowledges funding support from the John Innes Foundation and the BBSRC Institute Strategic Program Grant ‘Molecules from Nature – Products and Pathways’ (BBS/E/J/000PR9790).

Author information

Author notes

  1. Yuechen Bai

    Present address: School of Life Sciences, Fudan University, Shanghai, P.R. China

  2. Patricia Fernández-Calvo

    Present address: Misión Biolóxica de Galicia, CSIC, Pontevedra, Spain

  3. Patricia Fernández-Calvo

    Present address: Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM)-Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM, Madrid, Spain

  4. Maite Colinas

    Present address: Department of Natural Product Biosynthesis, Max Planck Institute for Chemical Ecology, Jena, Germany

  5. These authors jointly supervised this work: Andrés Ritter, Alain Goossens.

Authors and Affiliations

  1. Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium

    Trang Hieu Nguyen, Louis Thiers, Alex Van Moerkercke, Yuechen Bai, Patricia Fernández-Calvo, Max Minne, Thomas Depuydt, Maite Colinas, Bert De Rybel, Klaas Vandepoele, Andrés Ritter & Alain Goossens

  2. VIB Center for Plant Systems Biology, Ghent, Belgium

    Trang Hieu Nguyen, Louis Thiers, Alex Van Moerkercke, Yuechen Bai, Patricia Fernández-Calvo, Max Minne, Thomas Depuydt, Maite Colinas, Bert De Rybel, Klaas Vandepoele, Andrés Ritter & Alain Goossens

  3. Plant Sciences Unit, Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Melle, Belgium

    Louis Thiers

  4. VIB Single Cell Core, Ghent-Leuven, Belgium

    Kevin Verstaen

  5. VIB Center for Inflammation Research, Data Mining and Modelling for Biomedicine, Ghent, Belgium

    Kevin Verstaen & Yvan Saeys

  6. VIB Flow Core, VIB Center for Inflammation Research, Ghent, Belgium

    Gert Van Isterdael

  7. Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium

    Gert Van Isterdael

  8. Department of Biochemistry and Metabolism, John Innes Centre, Norwich, UK

    Hans-Wilhelm Nützmann & Anne Osbourn

  9. Department of Biology and Biochemistry, The Milner Centre for Evolution, University of Bath, Bath, UK

    Hans-Wilhelm Nützmann

  10. Department of Applied Mathematics, Computer Science and Statistics, Ghent University, Ghent, Belgium

    Yvan Saeys

  11. Bioinformatics Institute Ghent, Ghent University, Ghent, Belgium

    Klaas Vandepoele

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  1. Trang Hieu Nguyen
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Contributions

A.G., T.H.N., A.V.M., P.F.-C. and A.R. conceptualized the project. T.H.N., L.T., M.M., T.D., M.C., K. Verstaen, G.V.I., Y.S., B.D.R., K. Vandepoele, H.-W.N., A.O., A.R. and A.G. developed the methodology. T.H.N., Y.B., A.V.M., P.F.-C., L.T., T.D., M.C., G.V.I., A.R. and A.G. conducted the investigations. T.H.N., L.T., A.R., K. Verstaen and T.D. performed visualization. A.G, Y.S. and A.O. acquired funding. A.G. and A.R. administered the project. A.G., A.R., B.D.R., K. Vandepoele and Y.S. supervised the project. A.V.M., A.R. and A.G. wrote the original draft. T.H.N. and B.D.R. reviewed and edited the manuscript.

Corresponding author

Correspondence to Alain Goossens.

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Extended data

Extended Data Fig. 1

Violin plots showing cell-specific expression of the thalianol biosynthesis genes under mock and JA treatments.

Extended Data Fig. 2

Violin plots showing cell-specific expression of the marneral biosynthesis genes under mock and JA treatments.

Extended Data Fig. 3 MYC2 expression is induced by protoplasting in root tips.

a, Violin plots showing cell-specific expression of MYC2, MYC3 and MYC4 upon mock and JA treatments. b, RT-qPCR expression analysis of intact roots tips and root tip protoplasts upon mock and JA treatments. Values on the Y-axis represent fold-induction compared to mock-treated intact roots (set to 1). The error bars designate the SE of the mean (n = 3 biologically independent samples). Statistical significance was determined using the Student’s t-test (*P < 0.05, **P < 0.005, ***P < 0.0005).

Source data

Extended Data Fig. 4 Heatmap of THAS single-cell co-expression analysis.

Top 100 genes co-expressed with THAS under JA treatment across all root tip tissue types. Expression values as log fold change of JA compared to mock conditions are shown for each of the separate tissue types.

Extended Data Fig. 5 The expression of bHLH clade IVa genes is induced by JA in a COI1-dependent manner.

a, Violin plots showing cell-specific expression of bHLH18 and bHLH25 upon mock and JA treatments. b, Expression profile of ProbHLH19:GFP-GUS in wt root tips grown on mock or 50 µM JA for 24 h. Scale bars = 20 µm. At least 3 seedling roots were observed with similar results. c, RT-qPCR expression analysis of bHLH clade IV genes upon mock and JA treatments in the Col-0 wt and coi1-1 backgrounds. The error bars designate the SE of the mean (n = 3 biologically independent samples). Values on the Y-axis represent fold-induction compared to mock-treated wt (set to 1). Statistical significance was determined using the Student’s t-test (*P < 0.05, **P < 0.005, ***P < 0.0005).

Source data

Extended Data Fig. 6 Expression of bHLH clade IVa transcription factors is induced by MYCs.

a, RT-qPCR expression analysis of bHLH clade IV genes upon mock and JA treatments in the Col-0 wt and mycT backgrounds. The error bars designate the SE of the mean (n = 3 biologically independent samples). Values on the Y-axis represent fold-induction compared to mock-treated wt (set to 1). Statistical significance was determined using the Student’s t-test (*P < 0.05, **P < 0.005, ***P < 0.0005). b, Transactivation in N. tabacum protoplasts of the ProbHLH19 and ProbHLH20 fused to the fLUC reporter and cotransfected with either GUS, MYC2, MYC2D105N, bHLH19 or bHLH25. Values on the Y-axis are normalized fold-changes relative to protoplasts cotransfected with the reporter constructs and a pCaMV35S:GUS (GUS) control plasmid (set to 1). The error bars designate the SE of the mean (n = 8 biologically independent samples). Statistical significance was determined using the Student’s t-test (*P < 0.05, **P < 0.005, ***P < 0.0005).

Source data

Extended Data Fig. 7 Violin plots showing cell-specific expression of candidate root triterpene transcriptional regulators under mock- and JA-treated conditions.

Only genes from the HDG- and DOF-type families that show expression in our scRNAseq dataset are represented.

Extended Data Fig. 8 Homeodomain glabrous proteins coactivate the transcription of the THAS promoter.

Transactivation in N. tabacum protoplasts transfected with ProTHAS fused to the fLUC reporter, and cotransfected with combinations of GL2, HDG2, HDG5, MYC2D105N, bHLH19 or/and bHLH20. Values on the Y-axis are normalized fold-changes relative to protoplasts cotransfected with the reporter constructs and a pCaMV35S:GUS (GUS) control plasmid (set to 1). The error bars designate the SE of the mean (n = 8 biologically independent samples). Statistical significance was determined using the Student’s t-test (*P < 0.05, **P < 0.005, ***P < 0.0005).

Source data

Extended Data Fig. 9 vDOF1 proteins do not modulate transcription of the THAS promoter.

Transactivation in transfected N. tabacum protoplasts of the ProTHAS fused to the fLUC reporter, and cotransfected with combinations of vDOF1, MYC2D105N, bHLH19 or/and bHLH20. Values on the Y-axis are normalized fold-changes relative to protoplasts co-transfected with the reporter constructs and a pCaMV35S:GUS (GUS) control plasmid (set to 1). The error bars designate the SE of the mean (n = 8 biologically independent samples). Statistical significance was determined using the Student’s t-test (NS, Non-significant; ***P < 0.0005).

Source data

Extended Data Fig. 10

Model for the regulatory network that drives spatiotemporal expression of thalianol and marneral biosynthesis genes in Arabidopsis root tips.

Supplementary information

Supplementary Information

Supplementary Figs. 1–8 and Tables 1–5.

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Supplementary Data

Source data for supplementary figures.

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Nguyen, T.H., Thiers, L., Van Moerkercke, A. et al. A redundant transcription factor network steers spatiotemporal Arabidopsis triterpene synthesis. Nat. Plants 9, 926–937 (2023). https://doi.org/10.1038/s41477-023-01419-8

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  • DOI: https://doi.org/10.1038/s41477-023-01419-8

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