ATG8ylation of vacuolar membrane protects plants against cell wall damage

Abstract

Vacuoles are essential for cellular metabolism and growth and the maintenance of internal turgor pressure. They sequester lytic enzymes, ions and secondary metabolites that, if leaked into the cytosol, could lead to cell death. Despite their pivotal roles, quality control pathways that safeguard vacuolar integrity have remained elusive in plants. Here we describe a conserved vacuolar quality control pathway that is activated upon cell wall damage in a turgor-pressure-dependent manner. Cell wall perturbations induce a distinct modification-ATG8ylation-on the vacuolar membrane (tonoplast) that is regulated by the V-ATPase and ATG8 conjugation machinery. Genetic disruption of tonoplast ATG8ylation impairs vacuolar integrity, leading to cell death. Together, our findings reveal a homeostatic pathway that preserves vacuolar integrity upon cell wall damage.

Fig. 1. Cell wall damage induces ATG8ylation of the tonoplast.

a, Confocal micrographs of root cells in the early elongation zone of A. thaliana, showing mCherry–ATG8A (mCh–ATG8A, magenta) to illustrate the relocalization of ATG8 to the tonoplast upon cell wall damage. A single optical slice and a maximum intensity projection (max. project.) of a whole cell (20 µm depth), alongside a merged image with VAMP711–YFP (tonoplast marker) and a corresponding bright field (BF) image, are shown. Pearson and Spearman colocalization values indicate the association between ATG8A and the tonoplast. The treatment conditions include mock, Torin (1.5 h, 9 µM), EGCG (30 mins, 50 µM), ES20-1 (8 h, 100 µM), isoxaben (3 days, 3 nM) and Driselase (1 h, 1%). Scale bars, 10 µm. b, Quantification of autophagosomes under the treatment conditions depicted in a. One-sided Wilcoxon tests compared the treatments (n = 10) to mock; significant differences (P < 0.01) are indicated with asterisks. In each box plot, the central line indicates the median, and the upper and lower bounds represent quartile 3 (75th percentile) and quartile 1 (25th percentile), respectively. The whiskers denote the minima and maxima of the data points. c, Electron microscopy images displaying APEX2–ATG8A localization after Torin (1.5 h, 9 µM) or ES20-1 treatments (8 h, 100 µM), with (mock) or without DAB staining (negative control). The Torin-treated samples show typical autophagosome structures, whereas ES20-1 treatments lead to the labelling of the tonoplast. The insets show densely labelled tonoplast invaginations. N, nucleus; V, vacuole; A, autophagosome (orange arrowheads). Representative images from three seedlings were analysed under each treatment. Scale bars, 1 µm. d, Confocal micrographs of A. thaliana root cells expressing the GFP–ATG8A-G117A mutant, which is incapable of conjugating to membranes. Images are shown after treatment with Torin (1.5 h, 9 µM) or ES20-1 (8 h, 100 µM). Representative images from ten seedlings were analysed under each treatment. Scale bars, 10 µm. e, Confocal micrographs of M. polymorpha, comparing GFP–ATG8A localization under mock or ES20-1 (8 h, 100 µM) treatment conditions. MDY-64 (1 h, 1 µM) staining is used to mark tonoplast localization. Representative images from ten gemmae were analysed under each treatment. Scale bars, 10 µm.

Fig. 2. Genetic basis of tonoplast ATG8ylation.

a, Confocal micrographs of GFP–ATG8A expressed in the atg5, atg11 and atg16 mutant backgrounds of A. thaliana root cells treated with Torin (1.5 h, 9 µM) or ES20-1 (8 h, 100 µM). A single optical slice, a maximum-intensity projection (20 µm depth) and a bright field image are shown. Representative images from ten seedlings were analysed under each treatment. Scale bars, 10 µm. b, Same setup as in a but showing the atg16 mutant complemented with ΔCASM, which retains canonical autophagy but lacks non-canonical autophagy. Representative images from ten seedlings were analysed under each treatment. Scale bars, 10 µm. c, Schematic of ATG16 protein domains (coiled-coil domain (CCD) and WD40) showing the truncations used to complement atg16 mutants, with colours indicating the retained regions: 1–195 (green), 1–253 (blue), 1–295 (purple, ΔCASM), 1–379 (yellow) and full-length (FL) (orange). d, Carbon starvation assay across three replicates for wild-type (Col0), atg5, atg16 and complemented lines. ΔCASM and longer variants resist carbon starvation, unlike atg16 and other mutants. e, Western blot analysis of plant material expressing GFP–ATG8A in the Col0, ΔCASM/atg16 and atg16 backgrounds under mock, Torin (4 h, 9 µM) or ES20-1 (8 h, 100 µM) treatments. The blots were probed with anti-NBR1 and anti-GFP. Amido black staining (ABS) was used as the loading control. NBR1 intensity values are normalized to the loading control and presented as the average of three replicates. ATG8 intensity values are the ratio of GFP–ATG8A against free GFP and are the average of three replicates. f, Confocal micrographs showing NBR1–GFP localization (green) and mCh–ATG8F (magenta) in Arabidopsis root cells, under mock, Torin (1.5 h, 9 µM) and ES20-1 (8 h, 100 µM) treatments. The images include separate channels for NBR1–GFP and mCh–ATG8F, a merged image and a corresponding bright field image. Scale bars, 10 µm. g, Quantification of NBR1 puncta across treatments. One-sided Wilcoxon tests compared the treatments (n = 10) to mock; significant differences (P < 0.01) are indicated with asterisks. In each box plot, the central line indicates the median, and the upper and lower bounds represent quartile 3 (75th percentile) and quartile 1 (25th percentile), respectively. The whiskers denote the minima and maxima of the data points. Source data

Fig. 3. Tonoplast ATG8ylation maintains vacuolar integrity upon cell wall damage in a turgor-pressure-dependent manner.

a, Confocal micrographs of Arabidopsis root cells expressing mCh–ATG8A and VAMP711–YFP, highlighting ATG8A localization and tonoplast integrity under mock, ES20-1 (8 h, 100 µM), sorbitol (8 h, 50 mM) and combined sorbitol (8 h, 50 mM) + ES20-1 (8 h, 100 µM) treatments. A single slice of mCh–ATG8A, a maximum projection, a merged image with VAMP711–YFP and a corresponding bright field image are shown. Pearson and Spearman colocalization analyses are presented to quantify colocalization under each condition. Scale bars, 10 µm. b, Fluorescence lifetime imaging microscopy (FLIM) analysis of the Col0 and ΔCASM backgrounds treated with CarboTag-BDP, a fluorescent cell wall mechanoprobe, for 30 min at 10 µM concentration, following mock or ES20-1 (8 h, 100 µM) treatments. The fluorescence lifetime of the probe across three biological replicates is shown, with average lifetimes reported in nanoseconds. Four comparative graphs detail the lifetime variance for each treatment and genotype. Scale bars, 40 µm. c, FLIM analysis using Sulfo-BDP, a vacuolar mechanoprobe, to assess vacuolar crowding under the same conditions. Lifetime measurements in nanoseconds highlight differences in vacuolar crowding between treatments and genetic backgrounds. Average lifetime was measured for all values below 2 ns (peak 1) and above 2 ns (peak 2). Scale bars, 40 µm. d, Transmission electron micrographs demonstrating vacuolar morphology changes in the Col0 and ΔCASM backgrounds under mock and ES20-1 (8 h, 100 µM) treatments. The images reveal the significant fragmentation and invaginations upon cell wall damage in the ΔCASM line. Scale bars, 5 µm. e, Electron tomography analysis of a ΔCASM root cell treated with ES20-1 (8 h, 100 µM), providing a detailed 3D visualization of vacuolar morphology and the surrounding cellular environment. The tomogram is presented with a 180° rotation to enhance structural observation. Scale bar, 5 µm. f, PI staining of root cells from the Col0 and ΔCASM backgrounds under mock and ES20-1 (8 h, 100 µM) treatments, assessing cell viability and membrane integrity. Three replicates are shown for each genotype and treatment. Scale bars, 10 µm.

Fig. 4. Molecular basis of tonoplast ATG8ylation.

a, Proximity-dependent biotin labelling of ATG8A-interacting proteins during cell-wall-damage-induced tonoplast ATG8ylation. TID, TurboID. b, Venn diagram summarizing the results of TurboID-based proximity-labelling proteomics of TurboID–ATG8A under Torin (4 hs, 9 µM) and ES20-1 (8 h, 100 µM) treatments, highlighting overlap and unique proteins. TurboID alone is used as a negative control. c, Volcano plot of TurboID–ATG8A ES20-1 treatment versus Torin (n = 3 replicates, 348 total proteins). The yellow dots indicate proteins enriched in ES20-1 (Student’s t-test, P < 0.05, n = 81); the blue dots indicate those enriched in Torin (P < 0.05, n = 34). d, Proteins in five categories shown as dot plots. Circle colour represents log fold change; edge colour indicates confidence (Student’s t-test with false discovery rate (FDR) correction, FDR < 0.05, grey; FDR < 0.01, black). e, Confocal micrographs showing the localization of TBC/RabGAP1–GFP and mCh–ATG8E in A. thaliana root cells, under mock or ES20-1 (8 h, 100 µM) treatments. A single optical slice, a maximum projection, a merged image and a bright field image are shown. Representative images from ten seedlings were analysed under each treatment. Scale bars, 10 µm. f, PS biosensor mCITRINE–2xPH–EVECTIN2 changes localization upon cell wall damage. Confocal micrographs of A. thaliana root cells under mock, Torin (1.5 h, 9 µM) and ES20-1 (8 h, 100 µM) treatments are shown. Maximum-intensity projections are shown in green and inverted greyscale. Representative images from ten seedlings were analysed under each treatment. Scale bars, 10 µm. g, Confocal micrographs depicting the localization of six different PIP sensors in A. thaliana root cells, under mock and ES20-1 (8 h, 100 µM) treatments. 1xFYVE^HRS and 1xPX^P40 target phosphatidylinositol 3-phosphate (PI3P), 1xPH^FAPP1 and 1xPH^OSBP target phosphatidylinositol 4-phosphate (PI4P) and 1xPH^PLδ1 and 1xTUBBY-C target phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). All images are represented as inverted greyscale. Representative images from ten seedlings were analysed under each treatment. Scale bars, 10 µm. h, Cell wall damage increases vacuolar pH. Confocal images of Col0 roots treated with LysoSensor Yellow/Blue DND-160 under mock or ES20-1 (8 h, 100 µM) conditions are presented, showing dual emission (Em) in yellow and blue on the basis of pH. Representative images from ten seedlings were analysed under each treatment. Scale bars, 10 µm.

Fig. 5. Vacuolar ionophore monensin changes vacuolar pH and triggers tonoplast ATG8ylation.

a, Confocal images of Col0, ΔCASM and atg16 roots treated with LysoSensor Yellow/Blue DND-160 under mock or ES20-1 (8 h, 100 µM) conditions, showing dual emission in yellow and blue on the basis of pH. Representative images from ten seedlings were analysed under each treatment. Scale bars, 10 µm. b, Confocal micrographs of root cells in the early elongation zone of A. thaliana, highlighting the localization of mCh–ATG8A (magenta) illustrating tonoplast ATG8ylation. The panel includes a single optical slice and a maximum-intensity projection of a whole cell (20 µm depth), alongside a merged image with VAMP711–YFP (tonoplast marker) and a corresponding bright field image. Scale bars, 10 µm. Pearson and Spearman colocalization values are presented, showing the association between ATG8A and the tonoplast. The treatment conditions include mock, Torin (1.5 h, 9 µM) and monensin (0.5 h, 200 µM) treatments. c, Quantification of autophagosomes under the treatment conditions depicted in b. One-sided Wilcoxon tests compared the treatments (n = 10) to mock; significant differences (P < 0.01) are indicated with asterisks. In each box plot, the central line indicates the median, and the upper and lower bounds represent quartile 3 (75th percentile) and quartile 1 (25th percentile), respectively. The whiskers denote the minima and maxima of the data points. d, Western blot analysis of NBR1 flux under mock, Torin (4 h, 9 µM) and monensin (0.5 h, 200 µM) treatments. NBR1 intensity values are normalized to the loading control and presented as the average of three replicates. e, Two replicates of the western blot in d. f, Confocal micrographs of GFP–MpATG8A and GFP–MpATG8B expressing M. polymorpha cells under mock or monensin (0.5 hs, 200 µM) treatments. MDY-64 (1 h, 1 µM) staining was used to mark tonoplast localization. Representative images from ten gemmae were analysed under each treatment. Scale bars, 10 µm. Source data

Fig. 6. Vacuolar ionophore monensin triggers tonoplast ATG8ylation.

a, Confocal micrographs displaying the localization of all nine GFP-tagged ATG8 isoforms (ATG8A to ATG8I) of A. thaliana under monensin (0.5 h, 200 µM) treatment. Representative images from ten seedlings were analysed under each treatment. Scale bars, 10 µm. b, Confocal micrographs of the GFP–ATG8A-G117A mutant, highlighting its localization in response to monensin (0.5 h, 200 µM) treatment. The images include single optical slices and maximum intensity projections. Representative images from ten seedlings were analysed under each treatment. Scale bars, 10 µm. c, Confocal micrographs of mCh–ATG8A (magenta) colocalized with the tonoplast marker VAMP711–YFP illustrating the recruitment of ATG8 to the tonoplast upon monensin treatment. The panel includes a single optical slice alongside a merged image with VAMP711–YFP and a corresponding bright field image. The images follow a time course treatment (0, 10, 20, 30 and 60 min) with monensin (200 µM). Representative images from ten seedlings were analysed under each treatment. Scale bars, 10 µm. d, Venn diagram summarizing the results of a TurboID-based proximity-labelling proteomics experiment with TurboID–ATG8A under Torin (1.5 h, 9 µM), ES20-1 (8 h, 100 µM) and monensin (2 h, 200 µM) treatments, highlighting the overlap and unique proteins identified across conditions. TurboID alone is used as a negative control.

Fig. 7. SidK expression induces tonoplast ATG8ylation in A. thaliana.

a, Western blot analysis from immunoprecipitation (IP) experiments using Flag–GFP, Flag–SidK or Flag–SidK-F62A to pull down the VHA-A subunit of V-ATPase. The blots were probed with anti-Flag and anti-VHA-A antibodies to detect the presence of VHA-A in the pull-down from each bait protein. b, IP followed by mass spectrometry (IP–MS) results, presenting the identification of V-ATPase subunits co-immunoprecipitated with Flag–SidK and Flag–SidK-F62A. A schematic representation of the V-ATPase complex is also shown, detailing all its subunits. The ones marked with bold letters were detected in the SidK IP–MS experiment. c, Inducible expression of SidK as a tool to probe V-ATPase function in Arabidopsis cells. Western blot analysis of GFP–ATG8A lines expressing either mCh–SidK or mCh–SidK-F62A under a DEX-inducible promoter is presented, showing protein levels with and without DEX induction. The blots were probed for anti-GFP and mCh to detect the fusion proteins. d, SidK expression changes vacuolar pH. Confocal micrographs of mCh–SidK or mCh–SidK-F62A expressing lines treated with LysoSensor Yellow/Blue DND-160 are shown, displaying blue, yellow and red emissions, alongside bright field images, with and without DEX induction. Representative images from ten seedlings were analysed under each treatment. Scale bars, 10 µm. e, SidK expression induces tonoplast ATG8ylation. Confocal micrographs of GFP–ATG8A co-expressing mCh–SidK or mCh–SidK-F62A, treated with the tonoplast marker MDY64 displayed in magenta, are shown. Images show the GFP channel, a merge of the magenta and green channels, red emissions and bright field, with and without DEX induction. Representative images from ten seedlings were analysed under each treatment. Scale bars, 10 µm. Source data

Fig. 8. Model of the plant response to vacuolar damage upon cell wall damage through ATG8ylation of the tonoplast.

Tonoplast ATG8ylation is a vacuolar quality control mechanism. After cell wall damage (1), the vacuole gets damaged (2). This induces the assembly of the V-ATPase (3) and subsequent tonoplast ATG8ylation (4). This facilitates tonoplast repair (5). Figure created with BioRender.com.

Extended Data Fig. 1. Cell wall damage triggers ATG8ylation of the tonoplast.

(a) Confocal micrographs of the early elongation zone root cells of Arabidopsis thaliana, depicting the co-localization of mCherry-ATG8A (magenta) with the tonoplast marker γ-tip-GFP. The set includes a single optical slice and a maximum intensity projection of an entire cell (20 µm depth), along with a merged image incorporating γ-tip-GFP and a corresponding bright field image. Scale bar, 10 µm. Pearson and Spearman co-localization scores are provided to quantify the association between ATG8A and the tonoplast, under treatment conditions including mock, Torin (1.5 hours, 9 µM), EGCG (30 minutes, 50 µM), ES20 (8 hours, 100 µM), ES20-1 (8 hours, 100 µM), Isoxaben (3 days, 3 nM), and Driselase (1 hour, 1%). (b) Quantitative analysis of autophagosome numbers under the treatment conditions shown in Supplementary Fig. 1a. One-sided Wilcoxon test compared treatments (n = 10) to mock; significant differences (p < 0.01) are indicated as asterisks. The central line indicates the median, and the upper and lower bounds represent quartile 3 (75th percentile) and quartile 1 (25th percentile), respectively. The whiskers denote the minima and maxima of the data points.

Extended Data Fig. 2. All Arabidopsis and Marchantia ATG8 isoforms are recruited to the tonoplast upon cell wall damage.

(a) Confocal micrographs displaying the localization of all nine GFP-tagged ATG8 isoforms (GFP-ATG8A to GFP-ATG8I) in Arabidopsis thaliana root cells. For each isoform, images under mock conditions include a single optical slice, a maximum intensity projection of an entire cell (20 µm depth), and a corresponding bright field image. The same set of images is presented for cells treated with ES20-1 (8 hours, 100 µM). Representative image from 10 seedlings analyzed under each treatment. Scale bar, 10 µm. (b) Electron microscopy (EM) images displaying APEX2-ATG8A localization post DAB staining to highlight unspecific labeling of the Golgi stacks in APEX2-ATG8A lines. N: Nucleus, G and orange arrowheads: Golgi apparatus. Representative image from 3 seedlings analyzed under each treatment. Scale bar, 1 µm. (c) Confocal micrographs of Marchantia polymorpha, comparing GFP-ATG8B localization under mock or ES20-1 (8 hours, 100 µM) treatment conditions. MDY-64 (1 hour, 1 µM) staining is used to mark tonoplast localization. Representative image from 10 gemmae analyzed under each treatment. Scale bar, 10 µm. (d) Alkaline pH treatment induces ATG8ylation of the tonoplast. Confocal micrographs of root cells in the early elongation zone of Arabidopsis thaliana, highlighting the localization of mCherry-ATG8A (magenta) to illustrate re-localization of ATG8 to the tonoplast upon alkaline stress. The panel includes a single optical slice and a maximum intensity projection of a whole cell (20 µm depth), alongside a merged image with VAMP711-YFP (tonoplast marker) and a corresponding bright field image. Treatment conditions include pH 5, 6, 7, 7,5 and 8 for 3 hours. Representative image from 10 seedlings analyzed under each treatment. Scale bar, 10 µm.

Extended Data Fig. 3. Genetic basis of tonoplast ATG8ylation.

(a) Confocal micrographs of GFP-ATG8A expressed in atg2 mutant background of Arabidopsis thaliana root cells treated with Torin (1.5 hours, 9 µM) or ES20-1 (8 hours, 100 µM). Each panel includes a single optical slice and a corresponding bright field image to illustrate ATG8A behavior. Representative image from 10 seedlings analyzed under each treatment. Scale bar, 10 µm. (b) Confocal micrographs of GFP-ATG8F or GFP-ATG8I expressed in wild-type or atg4 mutant background of Arabidopsis thaliana root cells treated with Torin (1.5 hours, 9 µM) or ES20-1 (8 hours, 100 µM). Each panel includes a single optical slice and a corresponding bright field image to illustrate ATG8A behavior. Representative image from 10 seedlings analyzed under each treatment. Scale bar, 10 µm.

Extended Data Fig. 4. Generation of the atg16 mutant and the complementation lines.

(a) Diagram showing a representation of the ATG16 gene, the atg16 mutant generated by CRISPR and the different truncations used to complement the atg16 phenotype. The position of the gRNAs for CRISPR (orange) and the primers for two qPCRs (blue – qPCR1- and green -qPCR2-) are indicated. (b, c) Bar graph showing the relative gene expression levels of ATG16 on different genotypes: Col0, atg16 ATG16 FL, atg16 ATG16^1-379, atg16 ATG16^1-295, atg16 ATG16^1-253, atg16 ATG16^1-195 and atg16. Two different qPCR were performed, targeting the end of Exon 3 and beginning of Exon 4 (b) and targeting the middle of Exon 5 (c). Gene expression is presented as fold changes normalized to the reference gene (ACT2) and calculated using the ΔΔCt method. Data are presented as mean values from three independent biological replicates, each with three technical replicates, plus SEM. Statistical significance was determined by a two-tailed Student’s t-test (p < 0.05) and represented with an asterisk. (d) Replicates of the western blot analysis of plant material expressing GFP-ATG8A in Col0, ΔCASM/atg16, and atg16 backgrounds of Fig. 2e. Source data

Extended Data Fig. 5. Autophagy adaptor CFS1 is not recruited to the tonoplast upon cell wall damage.

(a) Confocal micrographs obtained from GFP-ATG8A mCh-CFS1 stably co-expressing Arabidopsis thaliana root cells under mock, Torin (1.5 hours, 9 µM), and ES20-1 (8 hours, 100 µM) treatments. The panel sequence includes single optical slices for GFP-ATG8A and mCh-CFS1, an additional panel for mCh-CFS1 with oversaturation to enhance visualization, a merge of both channels, corresponding bright field images and insets to enhance visualization. Scale bar, 10 µm. (b) Quantification of CFS1 puncta across the different treatment conditions used in (A). One-sided Wilcoxon test compared treatments (n = 10) to mock; significant differences (p < 0.01) are indicated as asterisks. The central line indicates the median, and the upper and lower bounds represent quartile 3 (75th percentile) and quartile 1 (25th percentile), respectively. The whiskers denote the minima and maxima of the data points.

Extended Data Fig. 6. Tonoplast ATG8ylation does not require FERONIA.

(a) Confocal micrographs of GFP-ATG8A expressed in Arabidopsis thaliana lrx3/4/5 triple mutant background (a) or fer4 mutant background (b), treated with mock, Torin (1.5 hours, 9 µM), and ES20-1 (8 hours, 100 µM). The set includes a single optical slice, a maximum intensity projection, and a bright field image for each treatment condition. Representative image from 10 seedlings analyzed under each treatment. Scale bar, 10 µm. (c) Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells of GFP-ATG8A, GFP-ATG8A fer-4 and GFP-ATG8A lrx3/4/5. MDY‐64 (yellow) staining depicts vacuolar membrane. One-sided Wilcoxon test compared treatments (n = 10) to GFP-ATG8A line; significant differences (p < 0.01) are indicated as asterisks. Scale bar, 10 µm. The central line indicates the median, and the upper and lower bounds represent quartile 3 (75th percentile) and quartile 1 (25th percentile), respectively. The whiskers denote the minima and maxima of the data points. Mean values are represented with ‘x’.

Extended Data Fig. 7. Cell wall damage affects vacuolar and cytoplasmic crowding.

(a) FLIM analysis of Sulfo-BDP mechanoprobe to assess vacuolar crowding in single cells. Analysis of Col0 and ΔCASM backgrounds treated with Sulfo-BDP, a fluorescent vacuolar mechanoprobe, for 90 minutes at 10 µM concentration, following mock or ES20-1 (8 hours, 100 µM) treatments. The fluorescence lifetime of the probe across three biological replicates, with average lifetimes reported in nanoseconds. Average lifetime, minimum and maximum lifetime with over 2·10⁹ counts, and the subtraction of this maximum and minimum are provided in a supplemental table. Scale bar, 20 µm. (b) FLIM analysis of PEG-BDP mechanoprobe to assess cytoplasmic crowding. Analysis of Col0 and ΔCASM backgrounds treated with PEG-BDP, a fluorescent cytoplasmic mechanoprobe, for 30 minutes at 10 µM concentration, following mock or ES20-1 (8 hours, 100 µM) treatments. The fluorescence lifetime of the probe across three biological replicates, with average lifetimes reported in nanoseconds. Four comparative graphs detail the lifetime variance per treatment and genotype. Scale bar, 40 µm.

Extended Data Fig. 8. Tonoplast ATG8ylation contributes to vacuolar and cellular homeostasis upon cell wall damage.

(a) Vacuolar morphology analyses upon cell wall damage. Representative images and quantification of vacuolar morphology of Col0, ΔCASM and atg16 root cells from the early elongation zone. MDY‐64 (green) staining depicts vacuolar membrane. One-sided Wilcoxon test compared treatments (n = 10) to Col0 wild-type; significant differences (p < 0.01) are indicated as asterisks. The central line indicates the median, and the upper and lower bounds represent quartile 3 (75th percentile) and quartile 1 (25th percentile), respectively. The whiskers denote the minima and maxima of the data points. Scale bar, 10. (b) Transmission Electron Microscopy (EM) images visualizing the vacuole in Col0 and ΔCASM backgrounds under mock and ES20-1 (8 hours, 100 µM) treatments. The images reveal vacuolar fusion between cells in response to cell wall damage. Insets of cell wall damage upon ES20-1 treatment are also included. Representative image from 3 seedlings analyzed under each treatment. Scale bar, 5 µm for the zoom out images and 0,5 µm for the insets of the cell wall. (c) ΔCASM root cells are more sensitive to cell wall damage. Propidium Iodide (PI) staining of root cells from Col0 and ΔCASM backgrounds under mock and ES20-1 (8 hours, 100 µM) treatments, assessing cell viability and membrane integrity. Seven more replicates of Fig. 3f are shown for each genotype and treatment. Scale bar, 10 µm.

Extended Data Fig. 9. Key ESCRT proteins are not recruited to the tonoplast upon cell wall damage.

Confocal micrographs depicting the localization of ESCRT (Endosomal Sorting Complex Required for Transport) machinery components GFP-FREE1, GFP-ALIX, and VPS23-RFP in Arabidopsis thaliana root cells, under mock, Torin (1.5 hours, 9 µM), and ES20-1 (8 hours, 100 µM) treatments. Each set comprises a single optical slice, a maximum intensity projection, and a corresponding bright field image, facilitating the comparative analysis of ESCRT component dynamics under torin and ES20-1 treatments. Representative image from 10 seedlings analyzed under each treatment. Scale bar, 10 µm.

Extended Data Fig. 10. TBC/RabGAP1 is conserved in plants.

A plant phylogeny displaying the presence of TBC/RabGAP1 across diverse species. Major plant taxonomic groups are denoted with a colored ribbon. Bootstrap confidence above 70 is shown at the bottom of each new leaf.