Fig. 2: Mutation in ATGs blocks autophagosome formation and leads to seed abortion.

a atg5 and atg7 Mutants were generated by CRISPR/Cas9 system. Schematic diagrams of ATG5 and ATG7 genomic sequences. Blue box, exon; gray line, intron; and pink box, untranslated region. The inserted or deleted nucleotides are indicated under the diagram. b Schematic diagrams showing the domains within the ATG5 and ATG7 proteins, as well as the mutant versions created by CRISPR/Cas9-edited mutagenesis. Missense sequences resulting from frameshift or fragment deletion are depicted as green boxes. c Protein levels of ATG5 and ATG7 in the wild type (WT) and their corresponding atg mutants were evaluated. β-Actin was used as the loading control. The immunoblot assay was repeated three times with similar results. d, f Representative images showing ATG8 immunofluorescence-labeled autophagosomes (d) and GFP-ATG8-labeled autophagosomes (f) in the integumentary tapetum from the WT and atg5-2 mutant. The lower rows show a magnified integumentary tapetal cell, which is outlined with the dot lines. PI propidium iodide, SR2200 SCRI Renaissance 2200. Arrows indicate autophagosomes. Scale bars: 20 µm (upper row) and 2.5 µm (lower row) in (d); 20 µm (upper row) and 2.5 µm (lower row) in (f). e, g The number of ATG8 immunofluorescence-labeled autophagosomes (e) and GFP-ATG8-labeled autophagosomes (g) in the integumentary tapetum are presented in box and whisker plots. Centerline, median values; top and bottom of boxes, 75th and 25th percentiles; top and bottom lines, maximum and minimum values. Data are from three independent experiments, each experiment with ten samples (n = 30). h The level of ATG8–PE adduct decreased in atg5-2 and atg7-1 mutants. β-actin serves as the loading control. ATG8–PE assay was repeated three times with similar results. i, j Mutation in ATG5 and ATG7 results in seed abortion. i Representative images showing seed set from the WT, atg5-2 and atg7-1 mutants, and proATG5:ATG5-GFP complementation lines. Arrows indicate aborted seeds. Scale bars: 1 mm. j The number of normal and aborted seeds in the WT, atg5 and atg7 mutants, and the complementation lines. Values are means ± SD (n = 4 independent ovaries), (two-tailed Student’s t test, ns no significant difference, P > 0.05; ***P < 0.001; ****P < 0.0001).