Fig. 5: Unequal chloroplast import machinery between two chloroplasts drives selective cpDNA degradation in zygotes.

a, b Proteasome-inhibitor treatment (MG132, 10 μM, 4 h) reversed TOC degradation in wild-type minus (a) and otu2p-ko (b) gametes. Gametes were incubated with MG132 at the indicated concentrations for 4 h and washed three times before mating. The TOC159, TOC75, and TOC34 proteins were quantified relative to the PsbD by western blot analysis. Values below the blot show the average % TOC contents relative to the samples treated with 10 μM MG132 (mean ± s.d.) Biological triplicates were loaded together. c Proteasome-inhibitor (MG132 or bortezomib) treatment protected minus cpDNA from degradation in wild-type zygotes. Differential interference contrast and overlaid fluorescent images of Hoechst 33342 (cyan)-stained zygotes with chlorophyll autofluorescence (magenta) were taken at 90 min after mating. Minus gametes were stained with MitoTracker Green to distinguish plus- from minus-derived chloroplasts. The percentage of the dominant cpDNA degradation pattern for each mating combination was calculated from three biological replicates (mean ± s.d.) Bar = 5 μm. “n” indicates the total number of zygotes examined. d Proteasome-inhibitor treatment established selective cpDNA degradation in otu2-ko zygotes. The average percentages of uniparental for plus (UPp), uniparental for minus (UPm), biparental (BP), and double degradation without cpDNA staining (DD) were calculated from three biological replicates. “n” indicates the total number of zygotes examined. Stacked bar graphs show four cpDNA degradation patterns, whose detailed values are provided in Supplementary Table 3.