Fig. 4: ATG8 facilitates the intralumenal vesicles formation in vacuoles.

a 3D projection of GFP-ATG8a and tonoplast marker Vamp711-mCherry after monensin treatment. Arrowheads indicated representative intralumenal vesicles. A plot profile analysis of the overlapping signals between GFP-ATG8a and Vamp711-mCherry was shown on the right. Scale bar, 20 μm. b Representative electron microscopic images illustrated the morphological changes in the vacuole following a 1 h treatment with monensin. Invaginating vesicles were denoted by magenta triangles. Similar TEM results were obtained in six individual blocks with three replicates. Scale bars, 1 μm. c Electron tomography analysis of the vacuolar morphology after monensin treatment for 1 h. The 3D model reconstructed from the tomography was presented on the right. Magenta triangles in individual tomogram slices indicate invaginating vesicles. In the 3D model, red color indicates mitochondria, yellow color indicates ER, and blue color indicates small vesicles. The 3D tomography was repeated three times independently, with similar results. Scale bars, 1 μm. d Time-series analysis of the ATG8-positive vesicles invaginated from the vacuolar membrane. A representative invaginating vesicle was indicated by the arrowhead. The magenta arrow pointed to a ruptured membrane structure that curved back to form a vesicle. Scale bar, 10 μm. e Time-series analysis of the effect of ConcA on ATG8-positive vesicles invaginated from the vacuolar membrane. Scale bar, 10 μm. f Time-series analysis of the vacuolar membrane invagination in atg5-1 mutant upon monensin treatment. Scale bar, 10 μm. Similar confocal imaging results in (a, d–f) were obtained in at least six individual roots with three replicates. Source data are provided as a Source Data file.