Extranuclear protection of chromosomal DNA from oxidative stress

Abstract

Eukaryotic organisms evolved under aerobic conditions subjecting nuclear DNA to damage provoked by reactive oxygen species (ROS). Although ROS are thought to be a major cause of DNA damage, little is known about the molecular mechanisms protecting nuclear DNA from oxidative stress. Here we show that protection of nuclear DNA in plants requires a coordinated function of ROS-scavenging pathways residing in the cytosol and peroxisomes, demonstrating that nuclear ROS scavengers such as peroxiredoxin and glutathione are insufficient to safeguard DNA integrity. Both catalase (CAT2) and cytosolic ascorbate peroxidase (APX1) play a key role in protecting the plant genome against photorespiratory-dependent H(2)O(2)-induced DNA damage. In apx1/cat2 double-mutant plants, a DNA damage response is activated, suppressing growth via a WEE1 kinase-dependent cell-cycle checkpoint. This response is correlated with enhanced tolerance to oxidative stress, DNA stress-causing agents, and inhibited programmed cell death.

Fig. 1.

Tolerance of apx1/cat2 to oxidative stress. (A and B) Photographs (A) and survival rates (B) of WT (black bars), apx1 (medium gray bars), cat2 (dark gray bars), and apx1/cat2 (light gray bars) seedlings grown under LL or HL conditions. (C) DAB staining (brown) indicating accumulation of H₂O₂ in seedlings grown at LL or subjected to HL stress in the absence or presence of 10 μM DPI (an inhibitor of NADPH oxidase). (D) Protein blot analysis of catalase (CAT) and ascorbate peroxidase (APX) in WT, apx1, cat2, and apx1/cat2 plants and detection of ribulose-1,5-bisphosphate carboxylase (RbcL) protein oxidation in leaf extracts obtained from HL-treated plants (900 μmol·m⁻²·s⁻¹, 1 h). (E) Survival rates in response to heat stress, showing enhanced basal and acquired thermotolerance of apx1/cat2 plants compared with cat2 plants. (F) Root growth in the presence of increasing concentrations of paraquat. Root growth of WT, apx1, and cat2 seedlings was severely reduced, but apx1/cat2 plants displayed high levels of tolerance to oxidative stress. (G) Photograph of WT, apx1, cat2, and apx1/cat2 plants grown under ambient air and exposed to HL stress (1,000 μmol·m⁻²·s⁻¹, 24 h). Lesions are apparent only on the leaves of cat2 plants. (H) Photograph of 4-wk-old plants grown at high CO₂ (3,000 ppm). High CO₂ abolished growth retardation in cat2 and apx1/cat2 plants. (I) Photograph of plants grown in high CO₂ and transferred to ambient air and subjected to 24 h of HL stress. Lesions also appeared on leaves of apx1/cat2 plants and were as prominent as in cat2 plants. Error bars in B, E, and F show SEM (n = 60); **P < 0.01 (Student's t test).

Fig. 2.

Functionality of the H₂O₂-dependent DDR in apx1/cat2 plants. (A) Venn diagram showing the overlap between constitutive apx1/cat2-specific transcripts and transcripts accumulating in cells in response to three different DNA stress-generating conditions: γ irradiation (γ-rad), hydroxyurea (HU), and a combination of bleomycin and mitomycin C (BM+MMC). In total, 75 apx1/cat2-specific transcripts were positively regulated in at least one DNA stress experiment, and 22 transcripts accumulated in all DNA stress experiments. (B and C) Accumulation of DNA stress marker transcripts (CYCB1;1, PARP2, BRCA1, and RAD51) in WT, apx1, cat2, and apx1/cat2 plants grown in ambient air (−CO₂) or high CO₂ (+CO₂; 3,000 ppm) (B) and in apx1/cat2 plants released from a high-CO₂ environment to ambient air (AA) at LL (C). (D–F) Photographs showing a time course in which different groups of WT, apx1, cat2, and apx1/cat2 plants grown under high CO₂ were transferred to ambient air for 0 (D), 1 (E), or 2 d (F) at LL and subsequently treated with HL. (G) Root growth kinetics of WT, apx1, cat2, and apx1/cat2 seedlings grown in the presence of aphidicolin (12 μg/mL). Error bars show SEM (n = 60). **P < 0.01 (Student's t test).

Fig. 3.

Cell-cycle checkpoints and the ER–PCD pathway in apx1/cat2 mutants and abiotic stress. (A) DNA ploidy-level distribution in the first leaves of 9-d-old seedlings. (B) Cell size and number in first leaves of 21-d-old seedlings. Average leaf blade area (±SEM) is shown at the top of the frame. (C) Photographs of apx1/cat2 and apx1/cat2/wee1 plants grown under ambient air conditions. (D) Accumulation of DNA stress marker transcripts (CYCB1;1, BRCA1, PARP2, and RAD51) in WT, apx1/cat2, and apx1/cat2/wee1 plants. (E) Root growth of WT and DNA stress checkpoint mutants (wee1-1, etg1-1, and atr-2) in the presence of increasing concentrations of tertiary butyl hydroperoxide (tButyl). (F) Survival of WT, wee1-1, etg1-1, and atr-2 plants grown under LL or HL conditions. (G) Survival rates of WT (black bar), apx1 (medium gray bar), cat2 (dark gray bar), and apx1/cat2 (light gray bar) plants following HL stress in the absence (control) or presence of ER–PCD pathway–blocking agents PBA (0.1 mM) and TUDCA (1 mM). Application of PBA and TUDCA rescued cat2 plants. (H) Survival rates of WT and bi1 seedlings grown under LL or HL conditions. Error bars in A, B, and E–H show SEM (n = 3–5). *P < 0.05; **P < 0.01 (Student's t test).

Fig. 4.

Model for extranuclear protection of chromosomal DNA from oxidative stress. (A) ROS are maintained in cells by a network of scavenging enzymes such as cytosolic ascorbate peroxidase 1 (cApx1) and peroxisomal catalases (perCat) that protect cells against oxidative DNA damage. (B) Under HL conditions, a lack of perCat leads to the accumulation of ROS that overcomes the scavenging ability of cApx1 and triggers PCD that probably is mediated by the ER pathway. (C) The absence of both cApx1 and perCat in plants grown under LL triggers a network of DNA repair, cell-cycle control, and the ER–PCD pathway–suppressing BI-1 that renders cells highly tolerant of HL and oxidative stress conditions.