Programmed induction of endoreduplication by DNA double-strand breaks in Arabidopsis

Abstract

Genome integrity is continuously threatened by external stresses and endogenous hazards such as DNA replication errors and reactive oxygen species. The DNA damage checkpoint in metazoans ensures genome integrity by delaying cell-cycle progression to repair damaged DNA or by inducing apoptosis. ATM and ATR (ataxia-telangiectasia-mutated and -Rad3-related) are sensor kinases that relay the damage signal to transducer kinases Chk1 and Chk2 and to downstream cell-cycle regulators. Plants also possess ATM and ATR orthologs but lack obvious counterparts of downstream regulators. Instead, the plant-specific transcription factor SOG1 (suppressor of gamma response 1) plays a central role in the transmission of signals from both ATM and ATR kinases. Here we show that in Arabidopsis, endoreduplication is induced by DNA double-strand breaks (DSBs), but not directly by DNA replication stress. When root or sepal cells, or undifferentiated suspension cells, were treated with DSB inducers, they displayed increased cell size and DNA ploidy. We found that the ATM-SOG1 and ATR-SOG1 pathways both transmit DSB-derived signals and that either one suffices for endocycle induction. These signaling pathways govern the expression of distinct sets of cell-cycle regulators, such as cyclin-dependent kinases and their suppressors. Our results demonstrate that Arabidopsis undergoes a programmed endoreduplicative response to DSBs, suggesting that plants have evolved a distinct strategy to sustain growth under genotoxic stress.

Fig. 1.

DSB-induced cell enlargement in root tips. (A) Propidium iodide-stained root tips. Five-day-old seedlings were treated with 0 or 10 μM zeocin for 24 h. (B) Measurement of root cell size. Five-day-old seedlings (n = 5) were treated with 0 or 10 μM zeocin, and epidermal cell area was measured after 24 h. Regression lines are included; R² = 0.61 (control) and 0.81 (+zeocin). The statistical significance of regression was estimated from the F test; F < 0.001 for both control and +zeocin. (C) DNA ploidy distribution in root tips. Seven-day-old seedlings were treated with 10 μM zeocin, and nuclear ploidy in 0.5-cm root tips was measured with a ploidy analyzer at the indicated time points. (D and E) Measurement of cell size and root growth upon genotoxic stress. Five-day-old seedlings (n ≥ 4 for D; n ≥ 11 for E) were irradiated with 150 Gy gamma or 1 kJ/m² UV rays or treated with 10 mM HU, 50 μM cisplatin (CP), or 100 ppm MMS. Epidermal cell area (D) and root growth (E) were measured after 24 h. Regression lines are included in D; R² = 0.21 (−gamma rays), 0.64 (+gamma rays), 0.62 (−UV), 0.70 (+UV), 0.26 (−HU), 0.56 (+HU), 0.38 (−CP and +CP), 0.37 (−MMS), and 0.52 (+MMS); F < 0.001 for all regression analyses. The error bars in E represent SD.

Fig. 2.

DSB-induced endoreduplication via the ATM/ATR–SOG1 pathway. (A and B) Five-day-old wild-type and mutant seedlings (n = 5) were treated with 0 or 10 μM zeocin, and epidermal cell area was measured after 24 h. Regression lines are included; R² = 0.61 (Col, control), 0.81 (Col, +zeocin), 0.62 (atm-2, control), 0.64 (atm-2, +zeocin), 0.49 (atr-2, control), 0.68 (atr-2, +zeocin), 0.60 (atm-2 atr-2, control), 0.63 (atm-2–atr-2, +zeocin), 0.49 (wee1-3, control), 0.67 (wee1-3, +zeocin), 0.42 (Col/Ler, control), 0.62 (Col/Ler, +zeocin), 0.51 (sog1-1, control), and 0.45 (sog1-1, +zeocin); F < 0.001 for all regression analyses. (C) Five-day-old seedlings of wild-type, wee1-3, and sog1-1 were treated with 0 (blue), 2 μM (yellow), or 10 μM (red) zeocin, and root growth was measured. The error bars represent SD (n ≥ 23). Hybrids of Columbia and Landsberg erecta (Col/Ler) were used as a control for sog1-1.

Fig. 3.

DSB-induced endoreduplication in Arabidopsis cultured cells. (A) DNA ploidy distribution in MM2d cells treated with or without 50 μM zeocin for 72 h. (B) DAPI-stained MM2d cells treated with or without 50 μM zeocin for 48 h. (C) Kinetochore number and nuclear area in MM2d cells treated with or without 50 μM zeocin for 72 h. (D) DNA ploidy analysis of partially synchronized MM2d cells. A 7-d-old culture was subcultured into medium with or without 50 μM zeocin and cultured for the indicated times. Positions of 6C (*), 12C (**), and 24C (Δ) peaks are shown.

Fig. 4.

Expression profiles of CDKs in response to DSBs. MM2d cells were treated with or without 50 μM zeocin as shown in Fig. 3D. (A) Transcript accumulation of CDKs. Total RNA was subjected to real-time RT-PCR. Expression levels were normalized to ACT8 and are indicated as relative values, with those for 0 h set to 1. The error bars represent the SD of three replicates. (B) Protein accumulation of CDKs. One hundred micrograms of protein extract were used for immunoblotting with specific antibodies against the indicated Arabidopsis CDKs. (C–E) Degradation of CDKB2 protein via the ATR–SOG1 pathway. Seven-day-old ProCDKB2;1:NT-GUS seedlings were treated with 10 μM zeocin or with 10 μM zeocin and 100 μM MG132 for 8 h (C). ProCDKB2;1:NT-GUS was introduced into xpf-2, xpf-2–sog1-1 (D), or atr-2 (E), and GUS expression was observed after treatment with or without 10 μM zeocin for 24 h.