Arabidopsis ATM and ATR kinases prevent propagation of genome damage caused by telomere dysfunction

Abstract

The ends of linear eukaryotic chromosomes are hidden in nucleoprotein structures called telomeres, and loss of the telomere structure causes inappropriate repair, leading to severe karyotypic and genomic instability. Although it has been shown that DNA damaging agents activate a DNA damage response (DDR), little is known about the signaling of dysfunctional plant telomeres. We show that absence of telomerase in Arabidopsis thaliana elicits an ATAXIA-TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR)-dependent DDR at telomeres, principally through ATM. By contrast, telomere dysfunction induces an ATR-dependent response in telomeric Conserved telomere maintenance component1 (Ctc1)-Suppressor of cdc thirteen (Stn1)-Telomeric pathways in association with Stn1 (CST)-complex mutants. These results uncover a new role for the CST complex in repressing the ATR-dependent DDR pathway in plant cells and show that plant cells use two different DNA damage surveillance pathways to signal telomere dysfunction. The absence of either ATM or ATR in ctc1 and stn1 mutants significantly enhances developmental and genome instability while reducing stem cell death. These data thus give a clear illustration of the action of ATM/ATR-dependent programmed cell death in maintaining genomic integrity through elimination of genetically unstable cells.

Figure 1.

Dysfunctional Telomeres in Plant Cells Activate a DDR. (A) Immunostaining and subtelomeric FISH labeling of root tip nuclei of the wild type (WT), tert, and ctc1 mutants. Nuclei were stained with DAPI (blue), γ-H2AX foci are colored in green, and FISH signals are colored in magenta. Images are collapsed Z-stack projections of a deconvolved three-dimensional image stack. Colocalized foci are indicated with white arrows. (B) Percentage of nuclei containing at least one γ-H2AX focus (% of nuclei+), the mean number of foci per nucleus (with se), the percentage of colocalization of the foci with the subtelomeric probes, the percentage of nuclei containing at least one TIF, and the mean number of TIFs per nucleus. The number of telomeric or nontelomeric foci and the percentage of nuclei were counted in 75 nuclei (15 nuclei from five different root tips) in each case. Bar in (A) = 2 μm.

Figure 2.

Short Telomeres in tert Mutant Plants Activate Both ATM and ATR. (A) The percentage of nuclei containing at least one TIF and the mean number of TIFs per nucleus (with se) after immunostaining and subtelomeric FISH labeling of root tip nuclei of atm, atr, tert, tert atm, and tert atr (± IATM) mutants. The number of telomeric foci and the percentage of nuclei were counted in 75 nuclei (15 nuclei of five different root tips) in each case. (B) Mean numbers of telomeric foci. Error bars indicate se (n = 75), and an asterisk indicates significant differences (Student’s t test; P < 0.05) between the indicated mutants.

Figure 3.

Increased H2AX Phosphorylation in S-Phase and Early G2-Phase Nuclei of tert atr Mutant Plants. (A) Immunofluorescence of root tip nuclei labeled with EdU and γ-H2AX in the tert mutant. DNA is stained with DAPI (blue), EdU incorporation is detected in green, and γ-H2AX foci are colored in magenta. (B) γ-H2AX foci in total interphase nuclei or in replicating (EdU+) nuclei (with se) in tert, tert atm, and tert atr mutants. Error bars indicate se (n = 75), and the asterisk indicates significant differences (Student’s t test; P < 0.05) between the tert atr mutant with or without EdU labeling. The number of foci was counted in 75 nuclei (15 nuclei of five different apices) in each case. Bar in (A) = 5 μm.

Figure 4.

CST Complex Mutants Activate an ATR-Dependent DDR. The percentage of nuclei containing at least one TIF and the mean number of TIFs per nucleus (with se) after immunostaining and subtelomeric FISH labeling of root tip nuclei of (A) ctc1, ctc1 atm, ctc1 atr, and ctc1 atr +IATM and (B) stn1, stn1 atm, stn1 atr, and stn1 atr +IATM mutant plants. The number of telomeric foci and the percentage of nuclei were counted in 75 nuclei (15 nuclei of five different apices) in each case. Error bars indicate se (n = 75), and an asterisk indicates significant differences (Student’s t test; P < 0.05).

Figure 5.

Increased Numbers of RPA Foci in the ctc1 Mutant. (A) Images illustrating the presence of RPA foci in ctc1 mutants after immunostaining and subtelomeric BAC-FISH of root tip nuclei. Nuclei were stained with DAPI (blue), RPA foci are colored in green, and FISH signals are colored in magenta. Images are collapsed Z-stack projections of deconvolved three-dimensional image stacks. Colocalized foci are indicated with white arrows. (B) Mean number of RPA foci per nucleus (with se), percentage of nuclei with at least one RPA focus (% of nuclei+), and the percentage of colocalization of RPA foci with the subtelomeric BAC FISH probes in the wild type (WT) and ctc1 mutants. The number of foci and the percentage of nuclei were counted in 100 nuclei (20 nuclei from five different slides) in each case. n.a., not analyzed; nb, the actual numbers. Bar in (A) = 2 μm.

Figure 6.

Increased Chromosomal Instability in ctc1 and stn1 Mutant Plants in the Absence of ATR and/or ATM. (A) The percentage of chromosomal bridges, the percentage of multiple bridges/aberrant, and the percentage of anaphases with subtelomeric signal in bridges observed after cytogenetic analysis of flower bud nuclei (from three different plants in each case) of atm, atr, ctc1, ctc1 atm, ctc1 atr, stn1, stn1 atm, stn1 atr, and ctc1 atm atr mutants. n.a., not analyzed; nb, actual numbers. (B) Examples of multiple bridges/aberrant anaphases observed in ctc1 atr. (C) Examples of bridges containing a subtelomeric signal in ctc1 atr analyzed by FISH with the nine subtelomeric BAC fluorescent probes (magenta). Bars in (B) and (C) = 2 μm.

Figure 7.

ATM/ATR-Dependent PCD of Stem Cells. (A) ATM/ATR-dependent cell death in root tips. The values represent the mean of the cell death area calculated with eight different root tips. Error bars are ±se (n = 8). An asterisk indicates significant differences (Student’s t test; P < 0.05) between the ctc1 and the ctc1 atm or the ctc1 atr mutants. (B) Representative images of root tips stained with propidium iodide, which stains dead cells (images are representative of eight root tips). No cell death is observed in wild-type (wt) plants, abundant cell death is observed in the region around the quiescent center in ctc1 or ctc1 atm mutants, and limited cell death is observed ctc1 atr mutants. (C) Percentages of mitoses showing at least one chromosome bridge in root tips of young plantlets and floral tissue of mature plants and the ratios of values found in roots versus buds. Numbers in parentheses are the actual counts. Bar in (B) = 50 μm.