Fig. 7: ATG8ylation contributes to the restoration of vacuolar function after removal of monensin.

a Monensin-induced ATG8 binding to the vacuolar membrane is reversible. Scale bar, 20 μm. b Monensin induces ion exchange across the vacuolar membrane. The fluorescent dye CoroNa Green AM (1 μM) was used for staining Na⁺. Scale bar, 20 μm. c Analysis of vacuolar acidification dynamics in Col-0 and atg5-1 roots during the recovery phase after monensin treatment. Vacuolar acidification was measured by detecting the ratio of BCECF fluorescence. Scale bar, 20 μm. d Quantitative statistical analysis of the ratio (488/458) of BCECF fluorescence intensities. The ratio of BCECF fluorescence intensities from ten individual roots (n = 10) was quantified with ImageJ. Data are mean ± SD. Significance analysis using an unpaired two-sided Student’s t-test. e–h Analysis of the effect of monensin treatment on the root elongation of Col-0, atg11-1, and atg5-1 mutants. Five-day-old seedlings were treated with 20 μM monensin for 2 h and then transferred to fresh 1/2MS agar plates. Images (e and g) were captured at 0 and 2 days after transfer to the plate. Statistical analysis of root elongation (f and h) was performed by quantifying six roots per genotype (n = 6) using ImageJ. Scale bar, 10 mm. i Analysis the subcellular localization of ATG8 in response to alkaline stress induced by (NH₄)₂CO₃. Five-day-old seedlings were immersed in 10 mM (NH₄)₂CO₃ solution (pH 8.5) for 1 h, followed by confocal imaging. Magenta arrows indicate the rupture of an invaginated vesicle, white arrows mark the complete vesicle formation from a detached membrane. Scale bar, 10 μm. j Comparative analysis of alkaline stress sensitivity in Col-0, atg11-1, and atg5-1 mutants. Five-day-old seedlings were treated with 10 mM (NH₄)₂CO₃ solution (pH 8.5) for 12 h, then transferred to 1/2 MS liquid medium for 5-day recovery. Scale bar, 10 mm. k Statistical analysis of chlorophyll content in (j). Three biological replicates (n = 3) were used for quantification. Data are mean ± SD. Significance analysis using an unpaired two-sided Student’s ttest. Source data are provided as a Source Data file.