Severe developmental defects, hypersensitivity to DNA-damaging agents, and lengthened telomeres in Arabidopsis MRE11 mutants

Abstract

The Mre11 protein is essential for the long-term genetic stability of the cell and acts to ensure the efficient repair of DNA damage. Vertebrate cells lacking Mre11 function are not viable. However, we report here that this is not the case in the model plant Arabidopsis. We have isolated two different Arabidopsis lines containing a T-DNA copy integrated at a different point in the MRE11 gene (AtMRE11). Both mutant plant lines were hypersensitive to DNA-damaging treatments but exhibited strikingly different developmental phenotypes. Furthermore, we also observed lengthened telomeres in these plant lines, showing that AtMre11 is involved in telomere maintenance. Thus, the lines we have isolated are unique tools with which to study in detail the role of AtMre11 in the mature plant.

Figure 1.

Alignment of Mre11 Proteins from Arabidopsis (Arab), Schizosaccharomyces pombe (Spom), S. cerevisiae (Scer), and Humans (Hsap). The phosphoesterase motifs (I to IV) conserved between the Mre11 proteins and the E. coli SbcC nuclease are indicated with black boxes (Sharples and Leach, 1995). The positions at which the Arabidopsis Mre11 protein is truncated by the T-DNA insertions in the AtMRE11-1 and AtMRE11-2 lines are indicated with arrows.

Figure 2.

Molecular Analysis of the T-DNA Insertions. (A) Scheme of the T-DNA used for mutagenesis. The orientations of the NPTII and β-glucuronidase (GUS) transcription cassettes are shown. The primers JL-202 and XR2 also are shown. LB, left border; RB, right border. (B) The genomic organization of AtMRE11 is shown (22 exons) with the positions of the integrated T-DNAs. The shaded rectangle represents the probe used for the DNA gel blot. The primers used for reverse transcriptase–mediated (RT) PCR are also shown (1, RT-MRE1; 2, RT-MRE2; 3, PRE3). In the AtMRE11-1 line, the left border of the T-DNA was linked to the AtMRE11 sequence at position 953. The right border end of the T-DNA was inserted at position 979. This results in a 26-bp deletion of genomic DNA in exon 9 of the gene. Two base pairs were lost from the left border T-DNA, whereas the right border end remained intact. In the AtMRE11-2 line, filler DNA was found on both sides of the inverted repeat. This filler DNA (65 and 59 bp) was linked to positions 1757 and 1759, respectively, in the AtMRE11 sequence. Therefore, in the AtMRE11-2 line, the integration of the T-DNA inverted repeat resulted in the deletion of a single nucleotide from the AtMRE11 locus (G at position 1758). E, EcoRI restriction site. (C) DNA gel blot of the AtMRE11-1 and AtMRE11-2 lines. Lane 1, wild- type seedlings (Wassilewskija); lane 2, dwarf kanamycin-resistant AtMRE11-1 seedlings; lane 3, normal kanamycin-resistant AtMRE11-1 seedlings; lane 4, normal kanamycin-resistant AtMRE11-2 seedlings. All DNA samples were digested with EcoRI. (D) RT-PCR performed on total RNA isolated from the two mutant lines. RT-PCR was performed using the primers RT-MRE1 and RT-MRE2 (top) to amplify the AtMRE11 cDNA. Amplification of the cyclophilin AtROC5 cDNA (bottom) was performed using primers RT-ROC3.1 and RT-ROC5.1. Lane 1, wild type (Wassilewskija); lane 2, AtMRE11-1^−/−; lane 3, AtMRE11-2^−/−. (E) Protein gel blot analysis using a polyclonal antibody raised against the N terminus of the AtMre11 protein. Lane 1, wild-type seedlings (Wassilewskija); lane 2, AtMRE11-1^−/− plants; lane 3, AtMRE11-2^−/− plants. (F) Sequence analysis of the T-DNA insertions in the AtMRE11-1 and AtMRE11-2 lines. The top lines show the genomic sequence of the AtMRE11 locus. Intron sequences are shown in lowercase italic letters. Exon sequences are shown in uppercase letters. The nucleotides derived from the T-DNA insertion are shown in uppercase boldface letters. The bottom lines show the predicted amino acid sequence as a result of the T-DNA insertions. Ten amino acids (NTQLKNVNMI) are derived from the filler DNA and form the C terminus of the predicted protein in the AtMRE11-2 line.

Figure 3.

Phenotypes of the AtMRE11-1 and AtMRE11-2 Lines. (A) At left, AtMRE11-1^−/− seedlings; at right, AtMRE11-1^+/− seedlings. (B) Mature plants. From left to right, AtMRE11-1^−/−, AtMRE11-1^+/−, AtMRE11-2^−/−, and the wild type. (C) Fasciation in mature AtMRE11-1^−/− plants. (D) to (G) AtMRE11-1^−/− seedlings. (H) Main root of an AtMRE11-1^+/− seedling. (I) Main root of an AtMRE11-1^−/− seedling. (J) Lateral root of an AtMRE11-1^−/− seedling. (K) Cotyledon epidermis of an AtMRE11-1^−/− seedling. (L) Dark-field view of a leaf from an AtMRE11-1^−/− seedling.

Figure 4.

Response of the AtMRE11-1 and AtMRE11-2 Lines to DNA-Damaging Treatments. (A) Hypersensitivity of the AtMRE11-1 and AtMRE11-2 lines to MMS. The MMS concentrations tested are given above the top row. Ws, Wassilewskija. (B) Hypersensitivity of AtMRE11-2^−/− plants to x-rays. The x-ray dosage (Gray) is shown above the top row.

Figure 5.

Telomere Length in the AtMRE11-1 and AtMRE11-2 Lines. Lane 1, wild type (Wassilewskija); lanes 2 to 4, three successive generations (T2 to T4) of AtMRE11-2^−/− plants; lane 5, AtMRE11-1^+/− plants (T3 generation); lane 6, AtMRE11-1^−/− plants.