Figure 3.

AtPolλ protein accumulation in wild-type Arabidopsis seedlings after high salt and MMC treatment. A and B, Immunodetection of AtPolλ protein using anti-AtPolλ polyclonal antibody in total protein extracts (40 μg) from 7-d-old wild-type seedlings exposed to increasing concentrations of NaCl (0–200 mm) or MMC (0–10 μg mL⁻¹) for 8 h (top panels). C and D, Immunoblot analysis of protein extracts from 7-d-old wild-type seedlings transferred to one-half-strength MS liquid medium for recovery at various time points after 200 mm NaCl or 5 μg mL⁻¹ MMC treatment for 8 h (top panels). E and F, Accumulation of AtPolλ protein in the roots (E) and leaves (F) of 2-week-old wild-type Arabidopsis seedlings exposed to 200 mm NaCl for various time points analyzed by immunoblotting (top panels). The level of actin protein, detected using anti-actin antibody, is shown as a loading control (bottom panels in A–F). The 0-h time point served as the control. Representative gel images from at least three independent experiments are shown. G to I, Immunolocalization of AtPolλ in leaf cells of 7-d-old wild-type Arabidopsis seedlings from untreated control (G) and after 1 h (H) and 2 h (I) of recovery following 200 mm NaCl treatment for 6 h. Nuclei were stained with 4′,6-diamino-phenylindole (red fluorescence in panel I). AtPolλ was detected using anti-AtPolλ IgG coupled to fluorescein isothiocyanate (green fluorescence in panel II). Panel III shows differential interference contrast images, and nuclear localization of AtPolλ is shown in the merged images of panels I and II (panel IV; yellow fluorescence zones indicated by arrows). J, Relative accumulation levels of DSBs in 7-d-old wild-type (Wt) and atpolλ mutant seedlings exposed to various concentrations of NaCl for 6 h. The extent of DNA damage is represented by the fraction of DNA migrating in the comet tail. Bars represent mean values from three independent trials. K, Mean percentage of DNA in the comet tails of 7-d-old wild-type and atpolλ mutant seedlings at various recovery time points. Maximum damage is normalized as 100% at 0 h for all lines. Data shown are means ± sd of three independent replications. *P < 0.05, **P < 0.01 relative to respective controls (n = 3). [See online article for color version of this figure.]