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Slow release of a synthetic auxin induces formation of adventitious roots in recalcitrant woody plants

Nature Biotechnology (2024)Cite this article

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Abstract

Clonal propagation of plants by induction of adventitious roots (ARs) from stem cuttings is a requisite step in breeding programs. A major barrier exists for propagating valuable plants that naturally have low capacity to form ARs. Due to the central role of auxin in organogenesis, indole-3-butyric acid is often used as part of commercial rooting mixtures, yet many recalcitrant plants do not form ARs in response to this treatment. Here we describe the synthesis and screening of a focused library of synthetic auxin conjugates in Eucalyptus grandis cuttings and identify 4-chlorophenoxyacetic acid–l-tryptophan-OMe as a competent enhancer of adventitious rooting in a number of recalcitrant woody plants, including apple and argan. Comprehensive metabolic and functional analyses reveal that this activity is engendered by prolonged auxin signaling due to initial fast uptake and slow release and clearance of the free auxin 4-chlorophenoxyacetic acid. This work highlights the utility of a slow-release strategy for bioactive compounds for more effective plant growth regulation.

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Fig. 1: A chemical screen for rooting enhancers of difficult-to-root cuttings highlighted 4-CPA-Trp-OMe (1q).
Fig. 2: The 1q combines high penetration with slow 4-CPA release to facilitate prolonged auxin signaling.
Fig. 3: Bypassing key auxin homeostasis regulators supports 4-CPA long-term signaling.
Fig. 4: Hydrolysis of 1r to 4-CPA is enzymatically regulated in E. grandis and Arabidopsis.
Fig. 5: Structural conservation of ILR1 ligand-binding pocket contributes to 4-CPA release.
Fig. 6: 1q is a robust rooting enhancer for woody cuttings.

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Data availability

All data supporting the findings of this study are available within the paper and its Supplementary Information. RNA sequencing data associated with this work is available at BioProject accession PRJNA1029024 (manuscript refs. 90,130). Source data are provided with this paper.

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Acknowledgements

We thank the undergraduate students A. Verblun and G. Yehezkely in the laboratory of R.W. for supporting experiments. We also thank E. Shani for providing DR5:Luciferase, DR5:Venus and aux1–7 Arabidopsis lines; M. Bennett and R. Swarup for providing the aux/lax quadruple mutant; M. Estelle for providing Y2H vectors; K. Ljung for providing the gh3 octuple mutant and B. Bartel for providing the ilr1-1 ill2-1 iar3-2 triple mutant and vectors expressing GST-recombinant version of ILR1, ILL2 and IAR3. We also thank I. Mayrose and K. Halabi for their assistance in constructing ILR1/ILLs phylogenetics. This work was supported by funding from the Israel Science Foundation (grant number 1057/21 to R.W.), the Chief Scientist of the Ministry of Agriculture and Rural Development, Israel (grant numbers 20-10-0067, 13-37-0005 and 20-01-0270 to R.W. and E.S.), the United States–Israel Binational Agricultural Research and Development Fund (BARD, grant number IS-5195-19R to R.W, E.S and C.J. Staiger (Purdue University, IN)), the Yuri Milner 70@70 Fellowship (to O.R.), the Deutsche Forschungsgemeinschaft (SFB024 TPB12 to C.D and SFB924 TPA08 and HA3468/6-3 to U.Z.H) and the Tel Aviv University Center for AI and Data Science (to N.B.-T).

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Authors and Affiliations

  1. School of Plant Sciences and Food Security, Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel

    Ohad Roth, Inna Vints & Roy Weinstain

  2. The Institute of Plant Sciences, The Volcani Center, Ministry of Agriculture and Rural Development, Rishon LeZion, Israel

    Sela Yechezkel, Ori Serero, Avi Eliyahu, Pan Tzeela, Vikas Dwivedi, Mira Carmeli-Weissberg, Felix Shaya, Adi Faigenboim-Doron & Einat Sadot

  3. The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel

    Ori Serero, Avi Eliyahu, Pan Tzeela & Joseph Riov

  4. Department of Electrical and Computer Engineering, University of Washington, Seattle, WA, USA

    Alberto Carignano & Eric Klavins

  5. Chair of Plant Systems Biology, Technical University of Munich, Freising, Germany

    Dorina P. Janacek & Ulrich Z. Hammes

  6. Chair of Food Chemistry and Molecular and Sensory Science, Technical University of Munich, Freising, Germany

    Verena Peters & Corinna Dawid

  7. Department of Biochemistry and Molecular BiologySchool of Neurobiology, Biochemistry & Biophysics, Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel

    Amit Kessel & Nir Ben-Tal

  8. Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    Kien Lam Ung & Bjørn Panyella Pedersen

  9. School of Life Sciences, University of Warwick, Coventry, UK

    Richard Napier

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Contributions

J.R., E.S. and R.W. conceived of the project and E.S. and R.W. supervised the project. O.R. designed and ran experiments and analyzed data. S.Y., O.S., A.E. and P.T. ran rooting experiments of cuttings from the different trees. I.V. synthesized and characterized compounds. A.E. and V.D. performed RNA sequencing and qPCR experiments. F.S. ran mass-spectrometry analyses of auxins and conjugates and analyzed data under the supervision of M.C.-W. A.K. designed and ran molecular simulation experiments with input from O.R. under the supervision of N.B.-T. A.F.-D. performed bioinformatics analysis of the RNA sequencing data. R.N. designed and performed SPR measurements. D.P.J. performed oocyte uptake assay under the supervision of U.Z.H. U.Z.H. performed SSM assays. V.P. performed mass spectrometry analyses on oocyte extracts and membranes under supervision of C.D. K.L.U. performed PIN purification under supervision of B.P.P. A.C. performed and analyzed binding assays of auxin receptors under the supervision of E.K. E.S., R.W. and O.R. wrote the manuscript with inputs from all co-authors.

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Correspondence to Einat Sadot or Roy Weinstain.

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Supplementary Figs. 1–26, Tables 1–3 and 1H- and 13C-NMR and mass-spectrometry data.

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Roth, O., Yechezkel, S., Serero, O. et al. Slow release of a synthetic auxin induces formation of adventitious roots in recalcitrant woody plants. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-023-02065-3

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