ATR cooperates with CTC1 and STN1 to maintain telomeres and genome integrity in Arabidopsis

Abstract

The CTC1/STN1/TEN1 (CST) complex is an essential constituent of plant and vertebrate telomeres. Here we show that CST and ATR (ataxia telangiectasia mutated [ATM] and Rad3-related) act synergistically to maintain telomere length and genome stability in Arabidopsis. Inactivation of ATR, but not ATM, temporarily rescued severe morphological phenotypes associated with ctc1 or stn1. Unexpectedly, telomere shortening accelerated in plants lacking CST and ATR. In first-generation (G1) ctc1 atr mutants, enhanced telomere attrition was modest, but in G2 ctc1 atr, telomeres shortened precipitously, and this loss coincided with a dramatic decrease in telomerase activity in G2 atr mutants. Zeocin treatment also triggered a reduction in telomerase activity, suggesting that the prolonged absence of ATR leads to a hitherto-unrecognized DNA damage response (DDR). Finally, our data indicate that ATR modulates DDR in CST mutants by limiting chromosome fusions and transcription of DNA repair genes and also by promoting programmed cell death in stem cells. We conclude that the absence of CST in Arabidopsis triggers a multifaceted ATR-dependent response to facilitate maintenance of critically shortened telomeres and eliminate cells with severe telomere dysfunction.

FIGURE 1:

Loss of ATR rescues the morphological defects of ctc1 mutants. The morphology of ctc1 mutants in the presence or absence of ATM or ATR is shown. (A) The phenotype of a ctc1 atm double mutant (right) resembles that of the ctc1 single mutant. (B, C) Morphological defects of ctc1 mutants are largely rescued when ATR is lost. Arrowheads indicate fasciated stems and flowers; arrows indicate irregular phyllotaxy. Images of second-generation (G2) ctc1 atr mutants are presented showing an intact plant (D) with curved, small leaves or malformed flowers (E) bearing a curved pistil and stamen and petal deficiency.

FIGURE 2:

ATR, but not ATM, contributes to telomere length maintenance in ctc1 and stn1 mutants. (A) TRF analysis of ctc1 crosses to atr and atm (lanes 1–9) and stn1 crosses to atm (lanes 10–16) and atr (lanes 17–24). (B) PETRA results for the 2R telomere in ctc1 atr mutants and the 3L telomere in stn1 atr mutants. (C) Quantification of telomere lengths from ctc1 atr PETRA analysis shown in B. Telomere length was calculated by subtracting the distance of the subtelomeric primer binding site relative to start of the telomere repeat array from the PETRA value. For all genotypes, n = 4. (D) Parent–progeny PETRA analysis of telomeres in G1 and G2 ctc1 atr mutants. Asterisk indicates interstitial telomeric repeats used as a loading control.

FIGURE 3:

ATR stimulates telomerase activity. Quantitative TRAP results for first-generation (G1), second-generation (G2), and fourth-generation (G4) mutants of different genotypes are shown. Q-TRAP was also performed on wild-type seedlings treated with 20 μM zeocin for 3 d. All samples were from flowers except G2 atr, G2 ctc1, and G2 ctc1 atr, which were from seedlings. Telomerase activity is plotted relative to wild type. For zeocin-treated seedlings, telomerase activity is relative to untreated-wild type seedlings. Error bars represent SD. n = 2 for all genotypes except G1 WT, n = 5; zeocin-treated WT, n = 6; G1 ctc1, n = 4; G2 atr, n = 3; and G4 atr, n = 4.

FIGURE 4:

End-to-end chromosome fusions increase in plants lacking CST and ATR. (A) Cytology of anaphases from pistils from G1 plants of the genotypes indicated. Spreads are stained with DAPI. (B) Quantification of anaphase bridges from cytology in A.

FIGURE 5:

Loss of CTC1 activates a transcriptional response, which is alleviated by ATR. Quantitative RT-PCR results are shown for the DDR transcripts PARP1, BRCA1, and RAD51 in floral organs. Expression levels are relative to wild type, and data for first-generation (G1) mutants are shown. For each genotype, n = 3, except for ctc1 atm, n = 2. *p < 0.05 relative to wild type; **p < 0.005 relative to wild type (Student's t test). Error bars represent SEM.

FIGURE 6:

ATR activates programmed cell death of the root apical meristem (RAM) of ctc1 mutants. (A) Representative images of G2 seedling root tips stained with propidium iodide (PI). (i) Diagram of a root tip. Stem cells and adjacent daughter cells are shaded gray. White cells in the RAM center are quiescent center cells. WT (ii) and atr (iii) roots are PI negative, but the RAMs of ctc1 (iv) and stn1 (v) mutants have numerous PI-positive (dead) cells. (vi) Fewer PI-positive cells are present in ctc1 atr mutants. (vii and viii) A subset of ctc1 or stn1 roots were PI negative but displayed severe morphological defects. (B) Quantification of PI-positive cells in different genetic backgrounds. The average number of PI-positive cells per root tip is shown. stn1 (n = 12), ctc1 (n = 17), ctc1 atr (n = 12). *p < 0.05 (Student's t test). Error bars represent SEM.

FIGURE 7:

Model depicting CST and ATR cooperation in maintaining telomeric DNA and genome integrity in Arabidopsis. (A) In wild-type plants, CST interacts with the 3′ overhang to protect the chromosome terminus from telomere shortening, end-to-end chromosome fusions (Song et al., 2008; Surovtseva et al., 2009), and activation of ATR-dependent DDR (this study). ATR facilitates replication fork progression. Similarly, CST is believed to stimulate replication fork restart within the telomeric duplex via interaction with DNA polymerase alpha (Price et al., 2010; Nakaoka et al., 2011). Telomeric DNA is represented by blue lines. (B) Plants lacking CST activate ATR-dependent DDR, initiating programmed cell death in stem cell niches. Replication fork progression is perturbed in the telomeric duplex, contributing to the loss of telomeric DNA. Telomerase action delays the onset of complete telomere failure. (C) Accumulating replicative stress in atr mutants triggers an ATR-independent DDR that results in telomerase inhibition. Telomeres in the wild-type size range can be maintained. (D) Catastrophic telomere shortening occurs in plants lacking both CST and ATR due incomplete replication of the duplex and failure of telomerase to act on critically shortened telomeres. See the text for details.