Fig. 5: COP1 mutation-caused ADA2b accumulation leads to increased DNA damage resistance.
From: The COP1-ADA2b module mediates light regulation of DNA double-strand break repair in Arabidopsis
a–c Fluorescence imaging and immunoblot analysis showing that COP1 mediates dark-induced degradation of ADA2b. YFP-ADA2b-OX and YFP-ADA2b-OX/cop1-4 seedlings were grown for 5 d in constant dark (DK) or high-intensity white light (WL, 80 μmol/m2/s). Bar, 10 μm. b Quantification of YFP-ADA2b fluorescence intensity from (a). c Immunoblot analysis of protein extracts from seedlings treated as in (a), using anti-GFP and -HSP82 antibodies. The numbers below the bands indicate the relative abundance of YFP-ADA2b normalized to HSP82. d, e YFP-ADA2b-OX/cop1-4 seedlings showing longer root and increased root apical meristem region than YFP-ADA2b-OX seedlings in the dark. Six-day-old etiolated seedlings were photographed, and their root lengths were measured (d). Bar, 5 mm. Seedlings were stained with PI (e). The root apical meristem regions of the seedlings were indicated between the white arrowheads. Bar, 25 μm. f Immunofluorescence staining assays showing that YFP-ADA2b-OX/cop1-4 seedlings accumulate significantly less MMS-induced DNA damage than YFP-ADA2b-OX seedlings. Five-day-old dim white light-grown seedlings were transferred to 1/2 MS liquid medium containing 100 ppm MMS for 48 h. The nuclei were immunostained with the anti-γH2AX antibody and stained with DAPI. Bars, 10 μm. g Comet assay showing that the YFP-ADA2b-OX/cop1-4 seedlings accumulate significantly less MMS-induced DNA damage than YFP-ADA2b-OX seedlings. Six-day-old dim white light-grown seedlings were transferred to 1/2 MS liquid medium containing 100 ppm MMS for 2 h. Bar, 20 μm. Data are presented as mean ± SD (b, n = 10 seedlings, adjusted P values were analyzed by one-way ANOVA, Tukey’s test, different letters indicate significant differences; d n > 30 seedlings, e n = 15 seedlings, f n = 100 nuclei, g n > 100 nuclei, unpaired two-sided t-test, ****P < 0.0001). These experiments were repeated more than twice with similar results. Source data are provided as a Source Data file.
