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MYB74 transcription factor guides de novo specification of epidermal cells in the abscission zone of Arabidopsis

Nature Plants volume 11, pages 849–860 (2025)Cite this article

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Abstract

The waxy cuticle layer is crucial for plant defence, growth and survival, and is produced by epidermal cells, which were thought to be specified only during embryogenesis. New surface cells are exposed during abscission, by which leaves, fruits, flowers and seeds are shed. Recent work has shown that nonepidermal residuum cells (RECs) can accumulate a protective cuticle layer after abscission, implying the potential de novo specification of epidermal cells by transdifferentiation. However, it remains unknown how this process occurs and what advantage this mechanism may offer over the other surface protection alternative, the wound healing pathways. Here we followed this transdifferentiation process with single-cell RNA sequencing analysis of RECs, showing that nonepidermal RECs transdifferentiate into epidermal cells through three distinct stages. During this vulnerable process, which involves a transient period when the protective layer is not yet formed, stress genes that protect the plant from environmental exposure are expressed before epidermis formation, ultimately facilitating cuticle development. We identify a central role for the transcription factor MYB74 in directing the transdifferentiation. In contrast to alternative protective mechanisms, our results suggest that de novo epidermal specification supports the subsequent growth of fruit at the abscission site. Altogether, we reveal a developmental programme by which plants use a transdifferentiation pathway to protect the plant while promoting growth.

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Fig. 1: Transdifferentiation of RECs into epidermal cells at single-cell resolution.
Fig. 2: Expression dynamics of the cell states during the transdifferentiation of RECs into epidermal cells.
Fig. 3: MYB74 plays a central role in the transdifferentiation of RECs into epidermal cells.
Fig. 4: De novo specification of epidermal cells in the AZ promotes fruit growth.

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Data availability

Data generated in this study are included in the article and Supplementary Information (Supplementary Tables 1–3). Single-cell RNA-seq data (accession number PRJNA1013602) and bulk RNA-seq data (accession number PRJNA1032145) generated in this study are available at the Sequence Read Archive database. Requests for materials and correspondence should be addressed to corresponding author J.M.K.

Code availability

The code used to analyse the scRNA-seq data in this study is available via GitHub at https://github.com/CB-postech/scRNA-seq_prEXL1_REC.

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Acknowledgements

We thank Y. Lee (Seoul National University) for her initial contribution and advice. We also thank J. Jeon, J. Choi and M. Kim for their technical support. We are grateful to W.T. Kim (Yonsei University), P.O. Lim (DGIST), Z.-M. Pei (Duke University) and Life Science Editors for their critical reading of the paper. This work was supported by grants from Samsung Science and Technology Foundation (SSTF-BA2101-10) and the National Research Foundation (grant number: 2022R1A2C3007309) and in part by a grant from DGIST (22-CoE-BT-04) to J.M.K.

Author information

Author notes

  1. Taek-Han Yoon

    Present address: Aptamer Sciences Inc., Seongnam, Republic of Korea

  2. Heejin Lim

    Present address: Center for Scientific Instrumentation, Korea Basic Science Institute, Cheongju, Republic of Korea

  3. These authors contributed equally: Xiaohong Wen, Chan Woong Lee, Seonghwan Kim.

Authors and Affiliations

  1. Department of New Biology, DGIST, Daegu, Republic of Korea

    Xiaohong Wen, Seonghwan Kim, Jae-Ung Hwang, Eunmin Lee, Taek-Han Yoon, Dong Gon Cha, Seulbee Lee, Heejeong Son, Jiwon Son, Su Hyun Jung, Jiyoun Lee, Heejin Lim, Huize Chen & June M. Kwak

  2. Department of Life Science, POSTECH, Pohang, Republic of Korea

    Chan Woong Lee, Yoon Ha Choi & Jong Kyoung Kim

  3. Department of Biological Science, Ajou University, Suwon, Republic of Korea

    Soon-Ki Han

  4. Shanxi Key Laboratory of Plant Macromolecules Stress Response, Taiyuan, People’s Republic of China

    Huize Chen

  5. School of Life Sciences, Shanxi Normal University, Taiyuan, People’s Republic of China

    Huize Chen

  6. Institute for Convergence Research and Education in Advanced Technology, Yonsei University, Seoul, Republic of Korea

    Jong Kyoung Kim

  7. New Biology Research Center, DGIST, Daegu, Republic of Korea

    June M. Kwak

  8. Center for Cell Fate Reprogramming and Control, DGIST, Daegu, Republic of Korea

    June M. Kwak

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Contributions

J.M.K. conceived the study, and J.M.K., J.K.K., J.-U.H., S.K. and X.W. designed the experiments. C.W.L., E.L., D.G.C. and J.K.K. analysed the scRNA-seq. S.K., J.-U.H., S.-K.H. and J.K.K. analysed the bulk RNA-seq data. X.W., S.K., J.-U.H., Y.H.C., S.-K.H., T.-H.Y., S.L., H.S., S.H.J., J.L., H.L. and H.C. performed the experiments. X.W., S.K., C.W.L., J.-U.H., J.K.K. and J.M.K. wrote the paper.

Corresponding authors

Correspondence to Jong Kyoung Kim or June M. Kwak.

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Nature Plants thanks Nobutoshi Yamaguchi 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 Single-cell RNA-seq analysis of residuum cells.

a, Workflow for single-cell RNA-sequencing (scRNA-seq) analysis of prEXL1:nls-GFP-expressing residuum cells (RECs). Receptacle tissues were collected, which are indicated by dotted yellow rectangles. Prior to protoplasting, tissues were observed under bright-field and GFP fluorescence to confirm GFP expression. After protoplasting, approximately 100,000 GFP-positive cells were isolated via fluorescence-activated cell sorting. These cells were then subjected to droplet-based scRNA-seq. Following raw data filtering, we obtained high-quality data for further analysis. b, A Uniform Manifold Approximation and Projection (UMAP) projection before the removal of non-RECs. After performing the dimensional reduction on 3,489 individual transcriptomes from AZ cells, we identified 23 cell clusters, each represented by a different color. Cell clusters corresponding to RECs are labeled with bold red numbers, whereas non-RECs are labeled with black numbers. c, UMAP plots showing the signature scores of non-REC cells, which are calculated based on the expression levels of cell type-specific marker genes. d, FACS gating strategy for GFP-positive REC protoplasts. Debris and doublets were excluded based on the forward scatter (FSC) and side scatter (SSC) properties. This is achieved by comparing FSC area (FSC-A) vs. SSC area (SSC-A), FSC width (FSC-W) vs. FSC height (FSC-H), and SSC width (SSC-W) vs. SSC height (SSC-H) sequentially. GFP-positive protoplasts were identified based on fluorescence intensity in the FITC channel, using FITC-A vs. FSC-A to differentiate cell populations. The final gated GFP-positive protoplasts were then isolated for downstream analysis.

Extended Data Fig. 2 scRNA-seq analysis identifies three distinct cell states during REC transdifferentiation.

a, UMAP projection yields five major clusters after removing non-RECs, showing the distribution of 2,823 RECs. Each cluster is color-coded. b–d, Violin plots displaying cell quality control statistics for the 2,823 RECs (n = 1): (b) the total number of unique molecular identifiers (UMIs), (c) the number of detected genes, and (d) the percentage of mitochondrial and chloroplast mRNA content per cell. The box plots display the median and interquartile range (25th-75th percentiles). The whiskers extend to 1.5 times the interquartile range. e, Pearson correlation analysis reveals three distinct developmental states among the five cell clusters. The colored bar indicates the scaled correlation. The color code for the cell clusters is consistent with that used in (a). f, UMAP projection shows the distribution of 2,823 cells across three developmental states. g, Heatmap illustrating gene expression correlations between cell states, calculated using Spearman rank correlation analysis. Cells are ordered along pseudotime. Within each pseudotime bin, cell expression profiles were averaged before calculating correlation coefficients. The color-coded bar at the top represents the relative proportion of developmental states within each pseudotime bin. h, Violin plots showing intercellular Spearman rank correlation coefficients for each developmental state from the 2,823 RECs (n = 1). Statistical significance was determined using unpaired two-sided Student’s t-tests (****p < 0.0001; state 1 vs 2: p < 2e-16, state 1 vs 3: p < 2e-16, state 2 vs 3: p < 2e-16). The box plots display the median and interquartile range (25th-75th percentiles) and the whiskers extend to 1.5 times the interquartile range. i, The heatmap shows the correlation between RECs in different developmental states and four inferred cell types after transfer analysis using Arabidopsis leaf scRNA-seq datasets27. Values were calculated using Pearson correlation coefficient analysis. The same color code was used for developmental states, as in panels e–i.

Extended Data Fig. 3 Expression profiles of cell state-specific genes during REC transdifferentiation.

a, Venn diagram illustrates the number of genes preferentially expressed in each developmental state. b, Heatmap displays the expression dynamics of cell state-specific genes over pseudotime. The mean gene expression values (that is, z-scores) are shown within each pseudotime bin. The color-coded bar at the top represents the relative proportion of developmental states within each pseudotime bin. The same cell state color code was used for all main and extended data figures.

Extended Data Fig. 4 Expression pattern of marker genes for cell states 2 and 3.

a, Expression of prABI5:nls-GFP (top) and prERF1A:nls-GFP (bottom) in receptacle AZs before and after abscission. Z-projected surface scan images show the spatiotemporal expression patterns of ABI5 and ERF1A in AZs over successive stages, thereby confirming the results of our scRNA-seq analysis. Scale bars, 100 µm. b, Expression patterns of state 3-specific genes (that is, BDG1, GPAT4, KCS20, LACS2, LTPG2, and WRI4) in AZs from S15 to S17m, as examined using promoter:nls-GFP reporters. Scale bars, 100 µm. c, Section images show the expression of KCS20, WRI4, LACS2, and LTPG2 only in the outermost layer of RECs after abscission (S17m), indicating that only one layer of RECs differentiates into epidermal cells. Scale bar, 50 µm.

Extended Data Fig. 5 Schematic diagram illustrating the progression of REC transdifferentiation and expression profiles of the cell state-specific transcription factors.

a, The schematic drawing illustrates which stages of fruit development correspond to the cellular states 1, 2, and 3. At flower development stage 15 (S15), sepals, petals, and stamens wilt and undergo abscission. Stages S15e (early) and S15l (late) indicate the flower stages immediately following S14 and preceding S16, respectively. At S16, the fruit is approximately twice the length of the petals. S17 includes stages beyond S16, with specific substages defined as follows: S17e (early) corresponds to fruit elongation, S17m (middle) to fruit lateral growth, and S17l (late) to the beginning of yellowing at the fruit tip. These stages also align with phases of embryo development: S17e relates to the globular to early heart stages, S17m corresponds to the late heart to cotyledon stages, and S17l aligns with the maturation stage. During S15, RECs exhibit mesophyll-like characteristics (cell state 1), lose their photosynthetic properties, and move to transition cell state 2. After completing floral organ abscission at S16 and the onset of longitudinal elongation of the silique at S17e, state 2 RECs differentiate into epidermal cells. Eventually, the floral abscission zones become covered by cuticle layers (cell state 3, S17m). RECs are depicted in pink, while SECs are in black. The flower development stages and their durations (h) are referenced according to Alvarez-Buylla et al.32. b, Heatmap showing the dynamic expression of transcription factors specific to each state over pseudotime. The color scale represents relative gene expression levels. The same cell state color code is used for all main and extended data figures.

Extended Data Fig. 6 Expression pattern of MYB74 and abscission-related genes in the AZ.

a-b, Spatiotemporal expression of prMYB74:nlsGFP-GUS in AZs: (a) the Z-projected surface scan view from S15 to S17l and (b) the transverse section view from S14 to S17m. In b, the top panel shows GUS expression in RECs, and the bottom panel shows GFP expression in the receptacle from which images were obtained using light-sheet microscopy. Scale bars, 50 µm (top) and 100 µm (bottom). c, Expression of abscission-related genes (BOP2, HSL2, and QRT2) in multiple cell layers of AZs before and after abscission. Scale bar, 50 µm. d, Expression of MYB74 in the transverse and longitudinal sections from the abscission-defective mutants hae hsl2 and ida, respectively. Scale bar, 50 µm.

Extended Data Fig. 7 RNA-seq analysis of MYB74-induced transcriptomic changes in AZ and receptacles shows that MYB74 controls the expression of genes associated with REC transdifferentiation.

a, RT-qPCR results show that MYB74 is induced following oestradiol treatment compared to mock treatment (Mock) and that the induction of MYB74 upregulates genes involved in the synthesis of cutin precursors. For all analyses, Actin2 was used as an internal control. The fold change was calculated relative to the mock-treated samples at 2 hours. The data shown were mean ± SD from three biological repeats. b, Heatmap displaying differentially expressed genes (DEGs) between oestradiol- and mock-treated samples (three biological replicates each). Statistical significance was evaluated using a two-sided Student’s t-test and graph showing all DEGs that met specific cut-off values (that is, an adjusted p < 0.05 and log2 fold change > 2). c, Heatmap showing the differential expression of genes related to photosynthesis and cuticle upon the induction of MYB74. d, Venn diagram showing that many REC-expressed MYB74 target genes (that is, 687 of 2176) show responses to MYB74 induction, with 272 and 415 being significantly up- and down-regulated, respectively. e, Gene ontology (GO) enrichment analysis for REC state-specific MYB74 target genes (2,176). Dot size represents the number of cells found to express a given gene, and its color represents its adjusted p-value (corrected for false discovery rate).

Extended Data Fig. 8 MYB transcription factors are expressed in RECs, rendering functional redundancy.

a, FA plots showing the expression of MYB family members enriched at the transition stage. The color scale represents the relative expression level. b-c, Surface-scanned fluorescent images of AZs (b) and longitudinally-sectioned images (c) reveal that MYB102 and MYB2 exhibit spatiotemporal expression similar to MYB74. Scale bar, 100 µm (b) and 50 µm (c). d, Loss-of-function mutations of myb74, myb74 myb102, and myb74 myb2 myb102 do not significantly affect cuticle layer formation. Cuticle formation and integrity were assessed by toluidine blue (TB) staining. Stage-17 siliques of the primary inflorescence at each flower position were analyzed. A representative result from three independent tests ( > 5 primary inflorescences for each test) is presented. Scale bars,100 µm. e, The first silique position that had a significant reduction in TB staining. TB-stained areas were quantified using Image J from acquired images in (d), and the position of the first silique where the stained area dropped to below 25% of the total AZ area was identified. The representative result is presented as the mean ± SD (n = 10). Statistical significance was determined using one-way ANOVA followed by Fisher’s test. The letters indicate no significant difference between genotypes (p = 0.8573).

Extended Data Fig. 9 MYB74 plays a role in REC transdifferentiation and cuticle layer formation.

a, Schematic representation of the prMYB74:MYB74-SRDX construct used to express the chimeric repressor MYB74-SRDX. The dark box indicates the SRDX fragment, fused to the MYB74 coding sequence (CDS). Blue arrows indicate the locations of primers used for RT-qPCR analysis of MYB74-SRDX expression. b, Compared to wild-type plants, the relative expression levels of MYB74-SRDX in prMYB74:MYB74-SRDX plants were examined using RT-qPCR assays. Total RNA was extracted from the receptacles of T2 plants from two independent transgenic lines (that is, #5 and #35). UBQ10 was used as an internal control. The error bars indicated mean ± SD from three biological replicates. c, Scanning electron microscope (SEM) images show the AZs and RECs’ surfaces of S17m siliques. In WT, an intact cuticle layer has formed, effectively filling the gaps between RECs. However, epidermis-specific expression of the cutinase gene CUTICLE DESTRUCTING FACTOR1 (CDEF1) impairs cuticle formation, resulting in unfilled gaps (indicated by an arrowhead). This plant line was able to flower but did not produce normal flowers. Scale bars, 50 µm (top) and 5 µm (bottom). d, Dual luciferase assay shows that MYB74 directly regulates GPAT4 promoter activity. A vector containing the multicloning site instead of the MYB74 coding sequence was a control. The error bars showed mean ± SD from three biological replicates. e, Co-expression of MYB74 and GPAT4 in the outermost layer of RECs, which differentiate into epidermal cells. Note the presence of inner RECs expressing only MYB74. Scale bars, 25 µm. f, Altered expression of the epidermal marker gene ATML1 in the RECs of plants carrying prUBQ10 > >MYB74-SRDX and prATML1:nls-GFP following MYB74-SRDX induction via one-day treatment with ß-estradiol. Scale bar, 50 µm.

Extended Data Fig. 10 The repressed function of MYB74 affects both receptacle and fruit growth.

a, Representative transverse section images of WT, MYB74-SRDX #35, and MYB74-SRDX #5 receptacles using resin embedding. These show the anatomical changes in transgenic plants, including reduced diameters, enlarged epidermal RECs, and cortical abnormalities. Scale bar, 100 µm. b, The distance from the pith center to the AZ vasculature was measured from section images in (a): Three consecutive 2 μm-thick transverse sections were taken from each receptacle sample. In each section, 1 to 4 sepal abscission zones were identified, each displaying lateral vasculatures. The radius was measured as the distance from the center of the pith to the sepal AZ vasculature (black lines). The mean radius from all measurements for each receptacle was then calculated. Five receptacles, each containing four sepal abscission zones, were examined for each genotype. From the 20 sepal abscission zones, 114 measurements were made from the section images for WT, #35, and #5 each. Box limits represent the 25th to 75th percentiles, whiskers show the entire data range, and the center line indicates the median. Statistical significance was determined via one-way ANOVA with Fisher’s test. c, Relative expression analysis showed that the induction of MYB74 by oestradiol upregulates five target MYB genes that negatively modulate lignification. Expression levels, based on TPM (Transcripts Per Million) were presented as mean ± SD from biological triplicates. d, Comparison of primary inflorescence stems between WT plants and transgenic plants carrying prMYB74:MYB74-SRDX. Arrowheads indicate siliques that are smaller than WT siliques. Scale bar, 1 cm.

Supplementary information

Reporting Summary

Supplementary Table 1

Supplementary Data S1: List of MYB74 target genes that are expressed in RECs and downregulated or upregulated in response to β-oestradiol induction.

Supplementary Table 2

Supplementary Data S2: List of putative target genes of MYB74 that are expressed in RECs.

Supplementary Table 3

Supplementary Data S3: List of primers used for this research.

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Wen, X., Lee, C.W., Kim, S. et al. MYB74 transcription factor guides de novo specification of epidermal cells in the abscission zone of Arabidopsis. Nat. Plants 11, 849–860 (2025). https://doi.org/10.1038/s41477-025-01976-0

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