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Wounding induces multilayered barrier formation in mature leaves via phytohormone signalling and ATML1-mediated epidermal specification

Nature Plants volume 11, pages 1298–1315 (2025) Cite this article

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Abstract

The epidermis of plants forms a protective barrier against biotic and abiotic stress. Little is known about how breaches in the epidermis are repaired, especially in mature leaves. Here we investigated wound healing in the mature leaves of Arabidopsis. We discovered a wound protection mechanism comprising a multilayered ligno-suberized barrier covered with cuticular wax. This barrier is formed by mesophyll cells that adopt an epidermal fate. This cell fate transition is regulated in two steps by ATML1, a key transcription factor in epidermal specification. First, mesophyll cells of protective layer 1, just beneath the wound, transition into epidermal cells and seal the wound by depositing cuticle, a mechanism that involves signalling through ethylene and reactive oxygen species produced by RbohE. This signalling also promotes cell death in protective layer 1, ensuring wax accumulation on the surface. Second, the underlying protective layer 2 undergoes ligno-suberization, driven by jasmonic acid and RbohD, forming a cork-like layer on the leaf surface. ATML1 regulates this process in protective layer 2 as well. Wound healing in mature leaves thus involves the integration of ethylene and jasmonic acid signalling with ATML1-mediated epidermal cell specification to coordinate cell-layer-specific functions, including cuticular wax formation and ligno-suberization. This protective mechanism also occurs in the leaves of tobacco and Capsella, suggesting it is widespread.

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Fig. 1: Wounding induces multilayered reorganization of mesophyll cells in mature leaves.
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Fig. 2: Changes to the transcriptome during wound healing.
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Fig. 3: The wound surface is sealed by an ATML1-mediated cuticle at P1.
The alternative text for this image may have been generated using AI.
Fig. 4: Ethylene–RbohE signalling is crucial for P1 cell death and cuticular wax formation.
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Fig. 5: JA–RbohD signalling regulates the lignification of the P2 layer.
The alternative text for this image may have been generated using AI.
Fig. 6: The ATML1 module mediates P2 specification.
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Fig. 7: Wound-induced multilayered protective barrier formation in plants.
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Data availability

The raw data files for the RNA-seq analysis reported in this paper can be found at GenBank under the accession number GSE275743. Sequencing reads were aligned to the TAIR10.1 reference genome (GCF_000001735.4; publicly available). Source data are provided with this paper.

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Acknowledgements

We thank N. Geldner, M. C. Seo, J.-H. Jung and D. Gasperini for sharing plant materials. We also thank Life Science Editors for paper editing. This work was supported by grants from the Suh Kyungbae Foundation (no. SUHF-19010003) and the National Research Foundation of Korea (nos RS-2021-NR060084 and RS-2023-NR076388) to Y.L., the National Research Foundation of Korea (nos RS-2022-NR071952 and RS-2022-NR067491) to Y.Y., the National Research Foundation of Korea (no. RS-2023-00301976) to Y.J. and the National Research Foundation of Korea (no. RS-2022-NR071113) to K.K. J.-M.L. was supported by the Hyundai Motor Chung Mong-Koo Scholarship. J.-M.L. and M.H. were supported by the Stadelmann-Lee Scholarship Fund at Seoul National University, Republic of Korea.

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Authors and Affiliations

  1. School of Biological Sciences, Seoul National University, Seoul, Republic of Korea

    Jung-Min Lee, Woo-Taek Jeon, Minsoo Han, Min-Soo Choi, Myung Kwon, Geon Heo, Youngsung Joo & Yuree Lee

  2. Research Institute of Basic Sciences, Seoul National University, Seoul, Republic of Korea

    Kyungyoon Kim & Hoon Jung

  3. Department of Biotechnology, Catholic University of Korea, Bucheon, Republic of Korea

    Sujeong Je & Yasuyo Yamaoka

  4. Institute for Data Innovation in Science, Seoul National University, Seoul, Republic of Korea

    Youngsung Joo

  5. Research Center for Plant Plasticity, Seoul National University, Seoul, Republic of Korea

    Yuree Lee

  6. Plant Genomics and Breeding Institute, Seoul National University, Seoul, Republic of Korea

    Yuree Lee

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  1. Jung-Min Lee
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Contributions

Y.L. and J.-M.L. conceived of the study and designed the experiments. The experiments were performed by J.-M.L., W.-T.J., M.H., M.-S.C., M.K., K.K. and S.J. under the supervision of Y.J., Y.Y. and Y.L. J.-M.L., M.H. and G.H. generated the transgenic lines. W.-T.J. and H.J. performed the computational analyses and discussed the results with J.-M.L. and Y.L. Y.L. and J.-M.L. drafted the paper, and all authors contributed to its revision. All authors have read and approved the final version.

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Correspondence to Yuree Lee.

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Nature Plants thanks Idan Efroni, Shinobu Takada and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Three-dimensional cellular architecture of the wounded leaf.

Maximum projection images of the wounded leaf were obtained using micro-CT at 0, 1, and 5 DPW. Scale bars, 100 μm. All experiments were independently repeated at least three times.

Extended Data Fig. 2 Overview of weighted gene co-expression network (WGCNA) analysis.

a, Principal Component Analysis (PCA) of all 36 time series samples. b, WGCNA cluster dendrogram of all 36 time series samples, grouping genes into 23 distinct modules. c-d, Scale independence and mean connectivity for calculating soft-thresholding powers.

Extended Data Fig. 3 The wound surface is sealed by an ATML1-mediated cuticle at P1.

a, Confocal micrographs show suberin deposition in wounded leaves stained with Fluorol Yellow. The left two panels show palisade mesophyll cells from a top view, while the right panel presents a vertical cross-section of the leaf obtained with a vibratome. b, Promoter-GUS analyses of GPAT4 and GPAT5. c, Toluidine blue (TB) staining shows the permeability of wounded site at 5 DPW. d, Cutin composition in WT and gpat4 gpat8 wounded leaves (n = biological replicates; same as in Fig. 3d). e, A heatmap showing expression patterns of genes associated with epidermal specification factors. f-g, Promoter-GUS histochemical analysis of ATML1 and PDF2 (f) and sectioned images of ATML1 expressed wounded leaf with Safranin staining (g). h, Confirmation of ATML1–SRDX induction assessed by western blot. Protein samples were extracted from the leaves of 4-week-old proUBQ10::XVE»ATML1–SRDX plants treated with 10 μM estradiol or mock (DMSO) after 1-day treatment. i, Confirmation of ATML1–SRDX induction in proUBQ10::XVE»ATML1–SRDX plants using RT-qPCR. RNA was extracted at 4 HPW. Expression was normalized to PP2A and shown relative to mock control (N = 3 biological replicates). ATML1-SRDX, reverse primer detects the SRDX sequence; ATML1-endo, reverse primer detects the 3′-untranslated region of ATML1 to examine ATML1 endogenous expression. j, Representative images showing deficient epidermal differentiation of 7-day-old proUBQ10::XVE»ATML1–SRDX #5 plants grown on plates containing 1 μM estradiol or mock (DMSO). k, Representative images of toluidine blue stained proUBQ10::XVE»ATML1–SRDX #5 plants in the absence (mock, DMSO) or presence of 1 μM estradiol for 5-day treatment without wound. l-m, Permeability to toluidine blue staining (l), and transmission electron micrographs of cuticle structure (m) of proATML1::XVE»ATML1-SRDX plants at 5 DPW. Three individual T1 transgenic plants (#3, #10, #13) were analyzed. In (c,i,l,m), 1 μM (ATML1-SRDX) and 10 μM (CDEF1) estradiol or mock (DMSO) was treated immediately before wounding. All experiments were independently repeated at least three times. Data were analyzed using a Kruskal–Wallis test with Holm correction, followed by Dunn’s post-hoc test (d) or two-tailed Student’s t-test (i). Different letters above the bars indicate significant differences (p < 0.05). Error bars (d,i) indicate mean ± SD. The raw data and exact P values are provided in Source Data Extended Data Fig. 3. ***p < 0.001, ****p < 0.0001; Scale bars, 50 μm (a,g), 100 μm (b,c,f,l), 500 μm (j), 1 mm (k), 50 nm (m).

Source data

Extended Data Fig. 4 Ethylene–RbohE signaling is critical for PCD-mediated P1 maturation.

a, Promoter-GUS analyses of RbohE and ETR1 at the wounded site. b, Superoxide accumulation detected using nitroblue tetrazolium (NBT) staining in wounded leaves of WT, rbohD, and rbohE at 1 HPW and 3 DPW, along with quantification of the NBT-stained area (n = 10 leaves). c-d, Confocal microscopy images of WT and cell death mutants at 1 to 3 DPW (c), along with quantification of cell length of P1 (d, n = 50 cells). e-f, Confocal microscopy images of mc1 mc2 lsd1 at 5 DPW (e), along with quantification of cell length of P1 (f, n = 50 cells). In (c,e), calcofluor-white was used to visualize the cell wall and white dotted lines indicate P1 cells. g, A heatmap illustrates the expression patterns of cutin and wax synthesis genes upregulated at 2-4 HPW in WT and rbohE, presented with a log2FC scale relative to the 0-hour control for each background. h, RT-qPCR analysis of expression of GPAT4, ABCG12, and BDG1 in WT and rbohE at 4 HPW, normalized to PP2A and shown relative to the 0-hour control for each background (N = 3 biological replicates). i-j, Transmission electron micrographs showing cuticle layer formation in WT and rbohE at 12 HPW (i), along with quantification of the thickness of the cuticular wax layer (j, n = 15 cells). k, Cuticular wax composition in wounded leaves of WT and various mutants at 5 DPW (n = biological replicates; same as in Fig. 4l). All experiments were independently repeated at least three times. Data were analyzed using one-way ANOVA, followed by Tukey’s post-hoc test (b,d), two-tailed Student’s t-test (f,h,j), or a Kruskal–Wallis test with Holm correction, followed by Dunn’s post-hoc test (k). Different letters above the bars indicate significant differences (p < 0.05). Error bars (b,d,f,h,j,k) indicate mean ± SD. The raw data and exact P values are provided in Source Data Extended Data Fig. 4. n.s., no significance; Scale bars, 100 μm (a), 250 μm (b), 50 μm (c,e), 100 nm (i).

Source data

Extended Data Fig. 5 Spatiotemporal pattern of wound-induced lignification.

a, Confocal micrographs show lignin deposition of wounded leaves at 2 and 5 DPW. Cell walls were visualized with calcofluor-white (cyan), and lignin with basic fuchsin (white). The left two panels show palisade mesophyll cells from a top view, while the right panel presents a vertical cross-section of the leaf obtained with a vibratome. b-d, Confocal micrographs (right panel) show lignin deposition in wounded leaves at diverse time points with the representative images of each leaf (left panel) (b). Either 10 µM piperonylic acid (PA) or mock was infiltrated before mechanical wounding. Lignin accumulation was quantified based on basic fuchsin fluorescence intensity in P1 (c, n = 100 cells) and P2 (d, n = 100 cells) relative to WT at 0 HPW for each control. Box-and-whisker plots (c,d) show the 10th–90th percentiles; boxes represent the interquartile range, lines within indicate the median. All experiments were independently repeated at least three times. Data were analyzed using one-way ANOVA followed by Tukey’s post-hoc test (c,d). The raw data and exact P values are provided in Source Data Extended Data Fig. 5. Scale bars, 50 μm (a), 100 μm (b-right), 0.5 cm (b-left).

Source data

Extended Data Fig. 6 JA–RbohD signaling regulates the lignification of P2.

a, Maximum projection images of confocal microscopy illustrate the broad expression patterns of lignin synthesis-related genes and the P2-specific expression pattern of laccases. GFP signals (Green) of RbohD, CAD5, LAC3, LAC5, and LAC13 were visualized by promoter::nls-GFP and JAZ10 by promoter::nls-3xVenus. b, Promoter-GUS analysis of JAZ10 and LAC3 in the wounded site at 1 HPW and 2 DPW, respectively. c, RT-qPCR analysis of expression of monolignol biosynthesis-related genes in proUBQ10::XVE»ATML1–SRDX #5, normalized to PP2A and shown relative to mock (N = 3 biological replicates). Estradiol was applied at 0 HPW, followed by RNA extraction at 4 HPW. d, RT-qPCR analysis of expression of monolignol biosynthesis-related genes in WT and various mutants at 4 HPW, normalized to the PP2A expression, and presented relative to WT (N = 3 biological replicates). e, Superoxide accumulation detected with nitroblue tetrazolium (NBT) staining in wounded leaves of WT and aos at 1 HPW, along with quantification of the NBT-stained area (n = 10 leaves). f, RT-qPCR analysis of expression levels of ATML1, GPAT4, ABCG11, and RbohE in aos and rbohD, normalized to the PP2A, and presented relative to each WT control (N = 3 biological replicates). All experiments were independently repeated at least three times. Data were analyzed using two-tailed Student’s t-test (c,e), and a Kruskal–Wallis test with Holm correction, followed by Dunn’s post-hoc test (d,f). Different letters above the bars indicate significant differences (p < 0.05). Error bars (c,d,e,f) indicate mean ± SD. The raw data and exact P values are provided in Source Data Extended Data Fig. 6. n.s., no significance, ****p < 0.0001; Scale bars, 50 μm (a), 250 μm (b,e).

Source data

Extended Data Fig. 7 ATML1 module regulates P2 specification.

a, RT-qPCR analysis of time-serial ATML1 expression in WT, normalized to PP2A and shown relative to 0-hour control (N = 3 biological replicates). b-d, Wound-induced P2 specification at 5 DPW in proUBQ10::XVE»ATML1–SRDX #5 plants was shown by the quantification of lignin intensity (b, n = 100 cells), expansion of P2 cells (c, n = 50 cells), and P2 plastid size (d, n = 50). 1 μM Estradiol or mock (DMSO) treatment was applied to leaves at 0 or 12 HPW. e, Promoter-GUS histochemical analyses of PDF2. f-i, Wound-induced P2 specification at 5 DPW in atml1-3 and pdf2-4 mutants. Lignification and chloroplast images were obtained from confocal microscopy (f), along with the quantification of lignin intensity (g, n = 100 cells), P2 plastid size (h, n = 50) and expansion of P2 cells (i, n = 50 cells). j, Confocal micrographs show P2 lignification in proUBQ10::XVE»CDEF1 plants at 5 DPW. 10 μM Estradiol or mock (DMSO) treatment was applied to leaves before wounding. k-l, Confocal micrographs show the cell morphology of gpat4 gpat8 mutant at 5 DPW (k) and RT-qPCR analysis of expression of ethylene-related genes under lanolin oil treatment at 1 DPW, normalized to PP2A and shown relative to WT control (l, N = 3 biological replicates). Lanolin oil was applied directly to the wound site immediately after wounding. In (f,j,k), cell morphology, lignin, and chloroplasts were visualized using calcofluor-white, basic fuchsin, and autofluorescence, respectively. White and yellow dotted lines indicate P1 and P2 cells. In (b,g), lignin intensity was quantified relative to the mock or WT control. All experiments were independently repeated at least three times. Box-and-whisker plots (b,c,d,g,h,i) show the 10th–90th percentiles; boxes represent the interquartile range, lines within indicate the median. Data were analyzed using a Kruskal–Wallis test with Holm correction, followed by Dunn’s post-hoc test (a,l), one-way ANOVA, followed by Tukey’s post-hoc test (b,d,g,h), or two-tailed Student’s t-test (c,i). Different letters above the bars indicate significant differences (p < 0.05). Error bars (a,l) indicate mean ± SD. The raw data and exact P values are provided in Source Data Extended Data Fig. 7. n.s., no significance, ****p < 0.0001; Auto F., Auto Fluorescence; Scale bars, 100 μm (e), 50 μm (f, j, k).

Source data

Extended Data Fig. 8 Consistent wound healing processes in actively expanding younger leaves.

a-b, Time-series analysis of 8th leaf size of Arabidopsis from 17 DAS (Days After Sowing) to 30 DAS. Representative images of 8th leaf at each DAS (a), along with measurements of 8th leaf size at each DAS (b, n = 30 leaves from individual plants). Box-and-whisker plots show the 10th–90th percentiles; boxes represent the interquartile range, lines within indicate the median. c-e, The wound healing process in the 8th leaf at 21 DAS (an actively expanding younger leaf) was assessed through ATML1 expression at 2 HPW and 2 DPW (c), toluidine blue staining showing wound barrier formation at 5 DPW (d), and confocal micrograpy illustrating P2 lignification at 5 DPW (e). Cell walls were visualized with calcofluor-white (cyan), and lignin with basic fuchsin (white). f-g, The wound healing process in the 1st true leaf at 10 DAS was assessed through toluidine blue staining showing wound barrier formation at 5 DPW (f), and confocal micrographs illustrating wound-induced lignification at 5 DPW (g). In (f,g), the adaxial epidermis was peeled off with a sharp knife tip indicated by white dotted lines. In (f), toluidine blue was applied for 2 min. All experiments were independently repeated at least three times. Data were analyzed using two-tailed Student’s t-test (b). The raw data and exact P values are provided in Source Data Extended Data Fig. 8. n.s., no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; Scale bars, 1 cm (a), 100 μm (c,d,f), 50 μm (e,g).

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Lee, JM., Jeon, WT., Han, M. et al. Wounding induces multilayered barrier formation in mature leaves via phytohormone signalling and ATML1-mediated epidermal specification. Nat. Plants 11, 1298–1315 (2025). https://doi.org/10.1038/s41477-025-02028-3

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