Inefficient double-strand DNA break repair is associated with increased fasciation in Arabidopsis BRCA2 mutants

Abstract

BRCA2 is a breast tumour susceptibility factor with functions in maintaining genome stability through ensuring efficient double-strand DNA break (DSB) repair via homologous recombination. Although best known in vertebrates, fungi, and higher plants also possess BRCA2-like genes. To investigate the role of Arabidopsis BRCA2 genes in DNA repair in somatic cells, transposon insertion mutants of the AtBRCA2a and AtBRCA2b genes were identified and characterized. atbrca2a-1 and atbrca2b-1 mutant plants showed hypersensitivity to genotoxic stresses compared to wild-type plants. An atbrca2a-1/atbrca2b-1 double mutant showed an additive increase in sensitivity to genotoxic stresses compared to each single mutant. In addition, it was found that atbrca2 mutant plants displayed fasciation and abnormal phyllotaxy phenotypes with low incidence, and that the ratio of plants exhibiting these phenotypes is increased by gamma-irradiation. Interestingly, these phenotypes were also induced by gamma-irradiation in wild-type plants. Moreover, it was found that shoot apical meristems of the atbrca2a-1/atbrca2b-1 double mutant show altered cell cycle progression. These data suggest that inefficient DSB repair in the atbrca2a-1/atbrca2b-1 mutant leads to disorganization of the programmed cell cycle of apical meristems.

Fig. 1.

(A) Sensitivity to cisplatin of single mutants atbrca2a-1 and atbrca2b-1, and the double mutant atbrca2a-1/atbrca2b-1. Imbibed seeds were plated on MS agar medium containing 0–50 μM cisplatin. The numbers of true leaves in wild type (Nossen) and mutant plants were counted 14 d after plating. Data represent the mean ±SE of 50 plants in each group from three experiments. (B, C) Sensitivity of AtBRCA2 mutants to γ-irradiation. (B) Imbibed seeds (4 d at 4 °C) were irradiated with increasing doses of ⁶⁰Co γ-rays. After γ-irradiation, the seeds were immediately plated on MS agar medium. The number of true leaves was counted 10 d after irradiation, and the average number of leaves was calculated according to Harlow et al. (1994). Data represent the mean ±SE of 34 plants in each group from three experiments. (C) Fourteen-day-old plantlets [upper panels: wild type (Nossen), lower panels: atbrca2a-1/atbrca2b-1 double mutant plants] after γ-irradiation (0, 300, 600 Gy).

Fig. 2.

Morphological phenotypes of atbrca2 mutants. (A, B) Five-week-old wild-type plants (Nossen), (C, D, E) atbrca2a-1/atbrca2b-1 double mutant plants. (F, G) Cross-sections of stems from 5-week-old plants: wild type (Nossen) (F) and atbrca2a-1/atbrca2b-1 double mutant (G). (H, I) Longitudinal-sections of 2-week-old plants: wild type (Nossen) (H) and atbrca2a-1/atbrca2b-1 double mutant plants (I). (J, K, L) In situ hybridization analysis of the AtWUS gene in wild-type (Nossen) (J) and atbrca2a-1/atbrca2b-1 double mutant (K, L) plants.

Fig. 3.

γ-irradiation induces fasciation in wild-type (Nossen), atbrca2a-1 and atbrca2b-1 single mutants, and the atbrca2a-1/atbrca2b-1 double mutant.

Fig. 4.

Histochemical assay of the AtCYCB1;1::GUS reporter gene (Colon-Carmona et al., 1999) in 7-d-old wild-type and atbrca2a-1/atbrca2b-1 mutant seedlings with or without aphidicolin (A, B). GUS activity was detected after 3 d of treatment with aphidicolin. (A) GUS staining of SAMs and leaves. (B) GUS staining of root tips.

Fig. 5.

Histochemical assay of the AtCYCB1;1::GUS reporter gene (Colon-Carmona et al., 1999) in 7-d-old wild-type and atbrca2a-1/atbrca2b-1 mutant seedlings with or without cisplatin (A, B). GUS activity was detected after 3 d of treatment with cisplatin. (A) GUS staining of SAMs and leaves. (B) GUS staining of root tips.