AtATM is essential for meiosis and the somatic response to DNA damage in plants

Abstract

In contrast to yeast or mammalian cells, little is known about the signaling responses to DNA damage in plants. We previously characterized AtATM, an Arabidopsis homolog of the human ATM gene, which is mutated in ataxia telangiectasia, a chromosome instability disorder. The Atm protein is a protein kinase whose activity is induced by DNA damage, particularly DNA double-strand breaks. The phosphorylation targets of Atm include proteins involved in DNA repair, cell cycle control, and apoptosis. Here, we describe the isolation and functional characterization of two Arabidopsis mutants carrying a T-DNA insertion in AtATM. Arabidopsis atm mutants are hypersensitive to gamma-radiation and methylmethane sulfonate but not to UV-B light. In correlation with the radiation sensitivity, atm mutants failed to induce the transcription of genes involved in the repair and/or detection of DNA breaks upon irradiation. In addition, atm mutants are partially sterile, and we show that this effect is attributable to abundant chromosomal fragmentation during meiosis. Interestingly, the transcription of DNA recombination genes during meiosis was not dependent on AtATM, and meiotic recombination occurred at the same rate as in wild-type plants, raising questions about the function of AtAtm during meiosis in plants. Our results demonstrate that AtATM plays a central role in the response to both stress-induced and developmentally programmed DNA damage.

Figure 1.

AtATM T-DNA Insertion Mutants. (A) Genomic map showing the positions of the T-DNA insertions at the AtATM locus. The arrow indicates the transcriptional start site, and black rectangles represent exons. The triangles represent the T-DNAs inserted in intron 64 (atm-2) and exon 78 (atm-1). (B) Protein gel blot analysis of the AtAtm protein with an anti-AtAtm specific polyclonal antibody in wild-type (ATM) and atm-1 mutant calli (cal) and seedlings (se), both in the absence (C) and 1 h after γ-irradiation (γ; 100 Gy). The amounts of total protein loaded were 60 μg for seedlings and 250 μg for calli. M, molecular mass markers. (C) Sequences of the atm-1 and atm-2 insertion sites. The T-DNA inserts are boxed. LB and RB indicate the left border and right border, respectively. Numbers below the sequence indicate the positions on the sequence of BAC T24C20. The right border of the T-DNA in atm-2 could not be amplified with T-DNA primers. The junction was amplified by inverse PCR using primers on the genomic DNA. The atm-2 T-DNA is accompanied by the insertion of filler DNA (lowercase italics) from other regions of the Arabidopsis genome.

Figure 2.

atm-1 Mutants Are Partially Sterile. Flowering stems ([A] and [B]) and siliques ([C] and [D]) of the wild type ([A] and [C]) and atm-1 mutants ([B] and [D]) are shown. The siliques in the atm-1 mutants are shorter than those in the wild type. Wild-type seeds (E) are reduced in size compared with atm-1 seeds (F). Bars = 1 cm for (C) and (D) and 1 mm for (E) and (F).

Figure 3.

High Gametophytic Lethality in atm-1 Mutants. (A) to (D) Pistils were stained according to Christensen et al. (1996). Wild-type ovules show a normal development of embryo sacs (A), whereas in atm-1 mutants, the functional megaspore degenerates ([B] and [D]). Only a degenerate cell can be seen (arrow) in atm-1 ovules, and no embryo sac has developed. Bars = 30 μm for (A) and (B) and 10 μm for (C) and (D). (E) to (H) Staining of mature anthers according to Alexander (1969). The pollen cell wall is stained green and the cytoplasm is stained pink, indicating viability. In the wild type ([E] and [F]), most pollen grains appear viable. In atm-1 anthers ([G] and [H]), only a few pollen grains are viable, and the dead grains are green (arrow). Bars = 150 μm for (E) and (G) and 25 μm for (F) and (H).

Figure 4.

Meiosis in Wild-Type and atm-1 Mutant Plants. (A) to (G) Wild type. (H) to (T) atm-1. (A) and (H) Pachytene stage. (B) and (I) to (K) Diakinesis. (C) and (L) Metaphase I. (D), (M), and (N) Telophase I. (E), (O), and (P) Metaphase II. (F), (G), and (Q) to (S) Telophase II. (T) A “polyad” with more than four microspores. Anthers were stained with DAPI. Two juxtaposed nuclei are present in (B). During anaphase/telophase I, chromosomal fragments are scattered through the pollen mother cells in the atm-1 mutant. At the end of meiosis, extramicrosporial chromosomal fragments are visible outside of the four groups of chromosomes in the mutant tetrads. Arrows point to either bridges or abnormal chromosomal fragments. Bars = 10 μm.

Figure 5.

Hypersensitivity of the atm-1 Mutants to γ-Rays and MMS. (A) Wild-type (ATM/ATM) and mutant (atm-1/atm-1) 5-day-old seedlings were either left untreated (0 Gy) or subjected to 100 Gy of irradiation. The photographs were taken at 21 days after irradiation. The growth of wild-type plants has resumed (top right), whereas the growth of mutant seedlings is arrested (bottom right). (B) Daily root growth after 80 Gy of irradiation of wild-type (gray circles) and atm-1 (blue squares) 5-day-old seedlings. The inset shows a plot of the cumulative root growth in the absence of irradiation versus time (in days). atm-1 mutant roots are ∼30% longer than wild-type roots. (C) Wild-type and atm-1 5-day-old seedlings were grown in the presence of the indicated concentrations of MMS (in ppm), and the photographs were taken on day 21.

Figure 6.

Transcriptional Activation of Genes Involved in the Cellular Response to DSBs Is Abolished or Delayed in atm-1 Mutants. Total RNA was extracted from 5-day-old seedlings untreated (C) or 30 min, 1 h, 2 h, 4 h, and 6 h after treatment with 100 Gy of irradiation. RNA gel blots (20 μg of total RNA per lane) were hybridized with probes from the AtRAD51, AtPARP1, ATGR1, and AtLIG4 genes. Equal loading was checked by hybridization with a 25S ribosomal probe.