Homologous Recombination Defective Arabidopsis Mutants Exhibit Enhanced Sensitivity to Abscisic Acid

Abstract

Abscisic acid (ABA) acts as an important plant hormone in regulating various aspects of plant growth and developmental processes particularly under abiotic stress conditions. An increased ABA level in plant cells inhibits DNA replication and cell division, causing plant growth retardation. In this study, we have investigated the effects of ABA on the growth responses of some major loss-of-function mutants of DNA double-stand break (DSB) repair genes in Arabidopsis during seed germination and early stages of seedling growth for understanding the role of ABA in the induction of genome instability in plants. A comparative analysis of ABA sensitivity of wild-type Arabidopsis and the knockout mutant lines related to DSB sensors, including atatm, atatr, the non-homologous end joining (NHEJ) pathway genes, and mutants related to homologous recombination (HR) pathway genes showed relatively enhanced sensitivity of atatr and HR-related mutants to ABA treatment. The expression levels of HR-related genes were increased in wild-type Arabidopsis (Col-0) during seed germination and early stages of seedling growth. Immunoblotting experiments detected phosphorylation of histone H2AX in wild-type (Col-0) and DSB repair gene mutants after ABA treatment, indicating the activation of DNA damage response due to ABA treatment. Analyses of DSB repair kinetics using comet assay under neutral condition have revealed comparatively slower DSB repair activity in HR mutants. Overall, our results have provided comprehensive information on the possible effect of ABA on DNA repair machinery in plants and also indicated potential functional involvement of HR pathway in repairing ABA induced DNA damage in Arabidopsis.

Fig 1. Comparative analysis of ABA sensitivity of wild-type Arabidopsis and DNA double-strand break (DSB)-repair related mutants in presence of exogenously applied abscisic acid during seed germination.

Arabidopsis seeds from the wild type (Wt Col) and DSB-related mutants were germinated on MS-agar medium containing different concentrations of ABA. Plates were kept in the dark at 4°C for 3–5 days and then transferred to light chambers maintained at 22°C with 16-h-light/8-h-dark periods, respectively. One-week-old plants were tested for their sensitivity. (A) Satatistical analyses of germination rates in wild-type and DSB-related mutant plants grown in the absence or presence of increasing concentrations of ABA. (B) Similar experimental conditions were used as in (A) for analyzing the germination rates of wild-type and other DSB-related mutant lines. The bars represent mean values of from three independent observations. *P < 0.05, **P < 0.01 relative to respective controls (n = 3). In each case approximately 75 seeds were counted.

Fig 2. Growth responses of DSB-related mutants in presence of increasing concentrations of ABA during early seedling stage.

7-days-old pre-germinated wild-type (Wt-Col) and DSB-related mutant seedlings were transferred to MS medium containing different concentrations of ABA and maintained under 16 h light/8 h dark photoperiod for another 7-days and then growth responses were analysed. (A) and (B) Satatistical analyses of hypocotyl length of wild-type and mutant seedlings grown in absence or presence of increasing concentrations of ABA. Data shown are means ± SD of three independent replications. At least 75 seedlings were counted in each time. *P < 0.05, **P < 0.01 relative to respective controls (n = 3).

Fig 3. ABA sensitivity of DSB-related mutant seedlings.

(A) and (B) Measurement of primary root length in wild-type and DSB-related mutants grown in the absence or presence of increasing concentrations of ABA. 7-days-old pre-germinated wild-type (Wt-Col) and DSB-related mutant seedlings were transferred to MS medium containing different concentrations of ABA and maintained under 16 h light/8 h dark photoperiod for another 7-days before analyzing the relative ABA sensitivity of the seedlings. Bars represent mean values from three independent trials. At least 75 seedlings were counted in each time. *P < 0.05, **P < 0.01 relative to respective controls (n = 3).

Fig 4. ABA sensitivity and relative levels of lateral root formation in wild-type Arabidopsis and DSB-related mutant seedlings.

(A) and (B) Comparative analysis of number of lateral root formation in wild-type (Wt-Col) and DSB mutant seedlings in absence and presence of different concentrations of ABA. 7-days-old pre-germinated seedlings were transferred to MS medium supplemented with increasing concentrations of ABA as described under ‘Material and Methods’. Data shown are means ± SD of three independent replications. *P <0.05, *P <0.01 relative to respective controls (n = 3).

Fig 5. Comparative analysis of fresh-weights of wild-type and DSB-related mutant seedlings under ABA stress.

(A) and (B) Determination of relative fresh weights of wild-type and DSB-related mutants grown in the absence or presence of increasing concentrations of ABA. 7-days-old pre-germinated wild-type (Wt-Col) and DSB-related mutant seedlings were transferred to MS medium containing different concentrations of ABA and maintained under 16 h light/8 h dark photoperiod for another 7-days before analyzing the relative ABA sensitivity of the seedlings. Bars represent mean values from three independent trials. At least 75 seedlings were counted in each time. *P < 0.05, **P < 0.01 relative to respective controls (n = 3).

Fig 6. ABA-induced DNA breaks and repair activity in wild-type and DSB-related mutants.

(A) Two-week-old wild-type and DSB-related mutant seedlings were treated with 30 μM ABA for 24 h at room temperature and the extent of DSB induction was analysed by the neutral (N/N) comet assays. The extent of DNA damage is represented by the fraction of DNA migrating in the comet tail. (B) DSB repair during various post-treatment recovery periods, including 0 h, 4 h, 8 h and 12 h, respectively. Additional data sets for 2 h and 6 h of post-treatment recovery periods are available on request. Mean percentage of DNA in the comet tails of wild-type and other mutant lines at various recovery time points under standard growth conditions. Maximum damage is normalized as 100% at t = 0 for all lines. Data shown are means (±SD) of three independent replications.

Fig 7. Detection of histone H2AX phosphorylation in wild-type and DSB-related mutants following ABA treatment.

(A) and (B) Two-week-old wild-type (Wt-Col), atatm-2, atatr, the ‘classic’ NHEJ-related mutants and HR mutant seedlings were exposed to 30 μM for 24 h following which histone protein extracts were prepared. Immunodetection of γ-H2AX was carried out using rabbit antiplant γ-H2AX polyclonal antibody in total protein extracts (60 μg). The upper panels in each figure represent Western bolts from untreated control seedlings while the lower panels indicate expression levels of actin protein, detected using anti-actin antibody, as a loading control. Figures are representative blots from two experiments.

Fig 8. ABA treatment induces expression of DSB-related HR genes during seed germination.

Relative expression levels of the major DSB-related marker genes during seed germination in wild-type Arabidopsis in the absence or presence of 0.5 and 5 μM ABA. The transcripts of each gene under normal condition were used a comparison standard. The expression levels were determined by real-time qPCR and normalized to ACTIN2 (ACT2). The data are representative from one of three independent trials with similar results. Error bars indicate ±SE (n = 3).

Fig 9. ABA treatment increases expression of HR genes during post-germination periods.

Two-week-old wild-type Arabidopsis seedlings were treated with 30 μM ABA for the indicated time points. The expression levels of the major DSB-related marker genes were determined by real-time qPCR and normalized to ACTIN2 (ACT2). The data are from one experiment with triple technical repeats. Error bars indicate ±SE, n = 3.

Fig 10. Proposed hypothetical model illustrating the interaction between ABA mediated genome instability and possible involvement of HR pathway.

ABA mediated reactive oxygen species (ROS) generation and oxidative damage inhibits DNA replication. Prolong replication block results in the formation of DNA single and double stand breaks, which activate ATR kinases and cell-cycle arrest. Activation of DNA double-strand break repair pathway for repairing damage during G1-S and S-G2 phases of cell-cycle. Possibility of HR repair during G1-S has been indicated with sign of interrogation.