Arabidopsis RAD51C gene is important for homologous recombination in meiosis and mitosis

Abstract

Rad51 is a homolog of the bacterial RecA recombinase, and a key factor in homologous recombination in eukaryotes. Rad51 paralogs have been identified from yeast to vertebrates. Rad51 paralogs are thought to play an important role in the assembly or stabilization of Rad51 that promotes homologous pairing and strand exchange reactions. We previously characterized two RAD51 paralogous genes in Arabidopsis (Arabidopsis thaliana) named AtRAD51C and AtXRCC3, which are homologs of human RAD51C and XRCC3, respectively, and described the interaction of their products in a yeast two-hybrid system. Recent studies showed the involvement of AtXrcc3 in DNA repair and functional role in meiosis. To determine the role of RAD51C in meiotic and mitotic recombination in higher plants, we characterized a T-DNA insertion mutant of AtRAD51C. Although the atrad51C mutant grew normally during vegetative developmental stage, the mutant produced aborted siliques, and their anthers did not contain mature pollen grains. Crossing of the mutant with wild-type plants showed defective male and female gametogeneses as evidenced by lack of seed production. Furthermore, meiosis was severely disturbed in the mutant. The atrad51C mutant also showed increased sensitivity to gamma-irradiation and cisplatin, which are known to induce double-strand DNA breaks. The efficiency of homologous recombination in somatic cells in the mutant was markedly reduced relative to that in wild-type plants.

Figure 1.

Molecular analysis of atrad51C T-DNA insertion. A, Genomic organization of the AtRAD51C locus, and the position of the T-DNA insertion at the AtRAD51C locus. Black boxes represent the exons, and white boxes represent 5′ and 3′ untranslated regions. Thick lines indicate probe regions for Southern- and northern-blotting analyses (probe A for Southern blotting and probe B for northern blotting). Arrows indicate PCR primer positions to determine the junction sequences of the T-DNA insertion. B, Southern-blotting analysis of the T-DNA insertion in the AtRAD51C locus. In these experiments, 5 μg of genomic DNA from AtRAD51C^+/+ (wild type), AtRAD51C^+/− (heterozygous), and atrad51C^−/− (homozygous) plants were digested with BamHI or XhoI. The digested DNAs were blotted and probed with the DIG-labeled probe A shown in section A. Asterisks show the nonspecifically hybridized bands because these bands were observed in all lanes regardless of genotypes (+/+, +/−, −/−). C, Northern-blotting analysis of AtRAD51C expression in flower buds of AtRAD51C^+/+, and atrad51C^−/− plants. In these experiments, 10 μg of total RNAs from flower buds of wild-type and mutant plants were blotted and probed with DIG-labeled probe B shown in section A. RNA M_r markers (0.5–5.0 kb) are shown on the left.

Figure 2.

atrad51C mutant plants are sterile. A, Fifteen-day-old AtRAD51C^+/+, AtRAD51C^+/−, and atrad51C^−/− plants on MS agar medium. +/+, +/−, and −/− indicate AtRAD51C^+/+, AtRAD51C^+/−, and atrad51C^−/− plants, respectively. B and C, Open flowers of wild type (B) and atrad51C mutant plants (C). D and E, Siliques of wild-type (D) and atrad51C mutant plants (E). F and G, Siliques resulting from cross-fertilization between wild-type female and wild-type male (F; as a control), and atrad51C mutant and wild-type male (G). H to K, Anthers of wild-type (H and J) and atrad51C mutant plants (I and K). Anthers from 2-mm buds were stained with Alexander's solution (1969; H and I) or stained with I₂-KI (J and K). L and M, Tetrad of wild-type (L) and atrad51C mutant plants (M) stained with aniline blue. N, AtRAD51C expression was detected in only PMCs by in situ hybridization. Black arrows indicate signals. O, No hybridization signal was detected when a sense probe was used.

Figure 3.

Mitosis in the petal of wild-type plants (A–E) and atrad51C^−/− mutant plants (F–J). A and F, Interphase; B and G, prophase; C and H, metaphase; D and I, anaphase; E and J, telophase. Calibration bar = 10 μm.

Figure 4.

Meiosis is severely disrupted in atrad51C mutant plants. A to F, Wild type; G to P, atrad51C. A and G, Pachytene stage; B and H, diakinesis; C and I, metaphase I; D and J, anaphase I; E and K, metaphase II; F and L, anaphase II; M to P, telophase II. Arrows with b in sections I, J, and L indicate bridges. Arrows indicate abnormal chromosomal fragments. Calibration bar = 10 μm.

Figure 5.

Cisplatin sensitivity of wild-type, atrad51C, and atrad51B plants. A, Production of true leaves in wild-type, atrad51C, and atrad51B plants at 12 d after treatment with 15 to 75 μm cisplatin. Asterisks indicate true leaf production with small and slender leaves. These leaves were not fully opened. Data represent mean ± sd of 50 plants. B, Fifteen-day-old wild-type, atrad51C, and atrad51B plants treated or untreated with cisplatin. C, Three-week-old wild-type, atrad51C, and atrad51B plants treated or untreated with cisplatin. Fifteen-day-old plants untreated and treated with 30 μm and 50 μm cisplatin were transferred on MS agar medium without cisplatin, and cultured for additional 6 to 7 d. Top of each section, wild-type plants; center of each section, atrad51C plants; bottom of each section, atrad51B plants. D, Comparison of the root growth of AtRAD51C^+/+, AtRAD51C^+/−, and atrad51C^−/− plants after 3-d cisplatin treatment. Six-day-old seedlings were transferred to cisplatin-containing MS agar medium and incubated for another 3 d. Root length of each plant was measured using the public domain NIH Image program (developed at the United States National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image). Data represent mean ± sd of 30 to 40 measurements.

Figure 6.

A, Cisplatin sensitivity of wild-type and atrad51C-cultured cells. Calli were obtained from excised root segments of wild-type and atrad51C plants. These calli were maintained on CIM and subcultured every 3 weeks. Seven-day-old calli were transferred and grown on CIM containing 0 to 50 μm cisplatin. The sensitivity to cisplatin was scored visually 3 weeks later. B, Complementation of atrad51C. Calli from wild-type roots transformed with pZH1 (wild type [pZH1]), calli from the atrad51C roots transformed with pZH1 (atrad51C [pZH1]), and calli from the atrad51C roots transformed with pZHg51C (atrad51C [pZHg51C]) were maintained on CIM containing hygromycin. Sensitivity to cisplatin of transgenic callus was scored as mentioned above. The expression of AtRAD51C mRNA in calli from the atrad51C roots transformed with pZHg51C was similar to that in wild-type callus based on reverse transcription-PCR analysis (data not shown).

Figure 7.

Sensitivity of wild-type and atrad51C^−/− plants to γ-irradiation. In these experiments, 3-d-old seedlings of wild-type and mutant plants were either left untreated (as 0 Gy) or irradiated with a dose of 100 Gy or 200 Gy. The photographs were taken at 12 d after irradiation. Top of each section, wild-type plants; bottom of each section, atrad51C plants.

Figure 8.

Frequencies of intrachromosomal HR in atrad51C and wild-type plants. A to F, Frequency distribution histogram shows the proportions of plants with a given number of blue GUS spots in the direct repeat (A, without genotoxic stresses; C, with cisplatin treatment; E, with bleomycin treatment) and inverted repeat (B, without genotoxic stresses; D, with cisplatin treatment; F, with bleomycin treatment) populations. atrad51C mutant and wild-type plants are shown as black and white bars, respectively. Images, Visualization by histochemical staining of recombination events in the direct-repeat line 1406. Top left, Wild-type plant without genotoxic stress; top right, wild-type plant with cisplatin treatment; bottom left, atrad51C plant without genotoxic stress; bottom right D, atrad51C plant with cisplatin treatment.

Figure 8.

Frequencies of intrachromosomal HR in atrad51C and wild-type plants. A to F, Frequency distribution histogram shows the proportions of plants with a given number of blue GUS spots in the direct repeat (A, without genotoxic stresses; C, with cisplatin treatment; E, with bleomycin treatment) and inverted repeat (B, without genotoxic stresses; D, with cisplatin treatment; F, with bleomycin treatment) populations. atrad51C mutant and wild-type plants are shown as black and white bars, respectively. Images, Visualization by histochemical staining of recombination events in the direct-repeat line 1406. Top left, Wild-type plant without genotoxic stress; top right, wild-type plant with cisplatin treatment; bottom left, atrad51C plant without genotoxic stress; bottom right D, atrad51C plant with cisplatin treatment.