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.

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