ATR regulates a G2-phase cell-cycle checkpoint in Arabidopsis thaliana

Abstract

Ataxia telangiectasia-mutated and Rad3-related (ATR) plays a central role in cell-cycle regulation, transmitting DNA damage signals to downstream effectors of cell-cycle progression. In animals, ATR is an essential gene. Here, we find that Arabidopsis (Arabidopsis thaliana) atr-/- mutants were viable, fertile, and phenotypically wild-type in the absence of exogenous DNA damaging agents but exhibit altered expression of AtRNR1 (ribonucleotide reductase large subunit) and alteration of some damage-induced cell-cycle checkpoints. atr mutants were hypersensitive to hydroxyurea (HU), aphidicolin, and UV-B light but only mildly sensitive to gamma-radiation. G2 arrest was observed in response to gamma-irradiation in both wild-type and atr plants, albeit with slightly different kinetics, suggesting that ATR plays a secondary role in response to double-strand breaks. G2 arrest also was observed in wild-type plants in response to aphidicolin but was defective in atr mutants, resulting in compaction of nuclei and subsequent cell death. By contrast, HU-treated wild-type and atr plants arrested in G1 and showed no obvious signs of cell death. We propose that, in plants, HU invokes a novel checkpoint responsive to low levels of deoxynucleotide triphosphates. These results demonstrate the important role of cell-cycle checkpoints in the ability of plant cells to sense and cope with problems associated with DNA replication.

Figure 1.

Phylogenetic Analysis, Structure, and T-DNA Insertions of AtATR. (A) The neighbor-joining tree of Rad3-like protein sequences was performed as described (Culligan et al., 2000). Numbers above each branch represent the number of times the branch was found in 1000 bootstrap replicas. Arabidopsis sequences are in bold. No DNA-PKcs (DNA–protein kinase catalytic subunit)-like sequences were identified in the Arabidopsis genome. (B) Overview of the AtATR gene, positions of atr-1, atr-2, and atr-3 T-DNA insertions, and its corresponding cDNA structure. Numbered gray boxes indicate exons of AtATR; the positions of the T-DNA insertions are denoted as triangles. The cDNA structure (bottom) shows the corresponding highly conserved domains found in ATM, ATR, TOR, and FKBP12-rapamycin-associated protein sequences, including the kinase domain denoted as a black rectangle (PI3Kc). The cDNA sequence was confirmed by isolating and sequencing independent cDNAs of AtATR (ecotype Ws) using RT-PCR. The resulting sequence predicts 17 exons that encode a protein of 2702 amino acids. (C) Structure of the atr-3 T-DNA insertion. Capital letters denote T-DNA insertion sequence. Corresponding amino acids are shown above the DNA sequence. The left-border sequence encodes 10 new amino acids within the AtATR reading frame before a premature stop (boxed as TGA). The approximate size of the entire insertion is 6 kb. (D) Partial alignment of the kinase domain where atr-3 is inserted: 1 denotes a 30-bp deletion (equivalent to 10 amino acids) caused by the insertion, and 2 denotes the position of the catalytic Asp required for kinase function.

Figure 2.

Siliques Harvested from Mature Wild-Type, atr, and atm-1 (Ecotype Ws) Mutants. Wild-type, atr−/−, and atr−/− atm+/− on average produce ∼50 seeds per silique. atm plants, which are partially sterile, produce a range of silique sizes with much fewer (<10 on average) seeds. The siliques from the double atr atm line produced no seeds and were unable to outcross as males or females, suggesting complete sterility.

Figure 3.

RNA Gel Blot Analysis of the Wild Type and atr. RNA samples were prepared from 7-d-old wild-type and atr plants (whole seedlings) sown on MS agar plates plus 1 mM HU, in the presence of UV-B (chronic dose), or left untreated (n.t.). For acute doses of UV-B light, the wild type and atr were grown for 7 d on MS agar plates and irradiated with UV-B light (10 kJ/m²) in the dark and then harvested 2 h or 6 h after UV irradiation. Approximately 30 μg (lanes 1 to 6) or 25 μg (lanes 7 to 10) of total RNA from each sample was blotted and probed with a putative AtRNR1 (GenBank AF092841) mRNA sequence. The AtRNR1 mRNA sequence is 2.65 kb. The bands corresponding to 4.4 and 2.4 kb of the RNA ladder (Ambion) are shown at the left and denoted as arrows. The ethidium bromide–stained gel is shown (bottom), and the relative rates of expression (Rel. exp.) were determined with respect to wild-type no-treatment control. This experiment was repeated with independently isolated RNA samples with very similar results.

Figure 4.

Root Growth and Cell-Cycle Arrest Phenotypes of γ-Irradiated Wild-Type and atr Plants. (A) Wild-type, atm, and atr seeds were γ-irradiated at a dose of 200 Gy. The seeds were immediately germinated on MS agar plates, and root growth was measured for 2 weeks. No-irradation controls grew as described (Garcia et al., 2003) and as in Figure 5D. (B) Five-day-old wild-type and atr seedlings containing the P_cyclinB1:GUS fusion construct (as described in the text and Figure 6) were γ-irradiated with 400 Gy, and individual wild-type and atr plants were harvested at 8 h, 1 d, and 2 d time points for GUS staining (n > 12). Two representative examples from each treatment are shown.

Figure 5.

Phenotypes of atr Challenged with HU, Aphidicolin, or UV-B Light. (A) Wild-type and atr seeds were germinated on MS plates or MS plus 1 mM HU and grown for 3 weeks. (B) Wild-type and atr seeds were germinated on MS plates and grown for 3 weeks in the presence or absence of UV-B light (see Methods). (C) Wild-type and atr seeds were germinated on MS plates and grown for 5 d and then transferred to plates containing 12 μg/mL of aphidicolin (Aph) for 5 d. (D) Five-day-old wild-type and atr seedlings were grown as above in the presence or absence (n.t., no treatment) of HU or aphidicolin (Aph). However, to make a more direct comparison of the effects of HU versus aphidicolin, we employed concentrations of HU and aphidicolin that inhibited wild-type root growth by ∼40% for both treatments, in this case, 1 mM HU and 12 μg/mL of aphidicolin. We measured root growth (n > 30) from each treatment for 8 d after transfer. Standard deviations are shown as error bars.

Figure 6.

GUS Staining of Root Tips. Five-day-old wild-type and atr-3 seedlings (carrying P_cyclinB1:GUS) were transferred to aphidicolin (Aph), or HU, or control (n.t.) plates, and root growth was measured every other day after transfer for 8 d (Figure 5D). Individual wild-type and atr plants were harvested at each time point for GUS staining of root tips. Wild-type and atr were grown for 5 d on MS plates and then transferred to control plates or to plates containing 1 mM HU or 12 μg/mL of aphidicolin. These experiments were repeated several times with very similar results; thus, representative pictures are shown. GUS stained root tips from 2, 4, and 6 d after transfer are shown. At least 12 individual plants were harvested and stained at each treatment and time point. Two representative examples are shown for each treatment.

Figure 7.

Semiquantitative RT-PCR Analysis of G2-Phase and S-Phase–Specific Genes. Wild-type and atr plants were grown as described in Figure 6. Root tips (n > 200) were excised (1 to 2 mm from the root cap) from wild-type and atr plants 4-d after transfer for RNA extraction, cDNA preparation, and RT-PCR analysis. Twenty-five microliters of each RT-PCR sample was loaded on an ethidium bromide–stained agarose gel, and representative gels are shown. All RT-PCR products were between 400 bp and 850 bp in size. The RT-PCR product of eIF4A (eukaryotic initiation factor) was employed as a standard for RT-PCR amplification. Numbers below each gel band denote its relative expression (first normalized to eIF4A) to the wild-type untreated (n.t.) sample. Standard deviations for two replicates are shown in parentheses. Aph, aphidicolin.

Figure 8.

DAPI-Stained Root Tips from Wild-Type and atr Grown as in Figure 6. (A) Shown are root tips day 4 and day 6 after transfer. (B) Close-up of 6 d after transfer to aphidicolin wild-type (left) and atr (right) DAPI-stained root tips.