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First published online May 27, 2005; 10.1104/pp.104.058347 Plant Physiology 138:965-976 (2005) © 2005 American Society of Plant Biologists The AtRAD51C Gene Is Required for Normal Meiotic Chromosome Synapsis and Double-Stranded Break Repair in Arabidopsis1Department of Biology and the Huck Institutes of the Life Sciences (W.L., Z.L., L.T., R.X., H.M.) and Intercollege Graduate Program in Plant Physiology (W.L., R.X., H.M.), the Pennsylvania State University, University Park, Pennsylvania 16802; Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056 (X.Y., C.A.M.); and Department of Gene Technology, Tallinn University of Technology, Tallinn 12618, Estonia (L.T.)
Meiotic prophase I is a complex process involving homologous chromosome (homolog) pairing, synapsis, and recombination. The budding yeast (Saccharomyces cerevisiae) RAD51 gene is known to be important for recombination and DNA repair in the mitotic cell cycle. In addition, RAD51 is required for meiosis and its Arabidopsis (Arabidopsis thaliana) ortholog is important for normal meiotic homolog pairing, synapsis, and repair of double-stranded breaks. In vertebrate cell cultures, the RAD51 paralog RAD51C is also important for mitotic homologous recombination and maintenance of genome integrity. However, the function of RAD51C in meiosis is not well understood. Here we describe the identification and analysis of a mutation in the Arabidopsis RAD51C ortholog, AtRAD51C. Although the atrad51c-1 mutant has normal vegetative and flower development and has no detectable abnormality in mitosis, it is completely male and female sterile. During early meiosis, homologous chromosomes in atrad51c-1 fail to undergo synapsis and become severely fragmented. In addition, analysis of the atrad51c-1 atspo11-1 double mutant showed that fragmentation was nearly completely suppressed by the atspo11-1 mutation, indicating that the fragmentation largely represents a defect in processing double-stranded breaks generated by AtSPO11-1. Fluorescence in situ hybridization experiments suggest that homolog juxtaposition might also be abnormal in atrad51c-1 meiocytes. These results demonstrate that AtRAD51C is essential for normal meiosis and is probably required for homologous synapsis.
Meiosis is essential for eukaryotic sexual reproduction, allowing the production of haploid gametes. In addition, meiotic recombination during the early stages of meiosis allows the exchange of genetic information, serving as an important source of genetic diversity. The success of meiosis depends on a complex and prolonged prophase I that involves homologous chromosome (homolog) pairing, synapsis, and recombination (Zickler and Kleckner, 1999  ; Page and Hawley, 2003; Schwarzacher, 2003). After pairing, the homologs continue to associate and this interaction has been referred to as homolog juxtaposition (Zickler and Kleckner, 1999). Recombination results in crossover events that correspond to cytologically observed chiasmata, which, in combination with sister chromatid cohesion, maintain the association between homologs in the form of bivalents, ensuring proper segregation of homologs at anaphase I. Synapsis, the formation of synaptonemal complexes (SCs) between closely associated chromosomes, has also been implicated to play important roles in meiotic prophase I, although its relationship with recombination differs among organisms.
Cytological and molecular genetic studies support the idea that homolog pairing, synapsis, and recombination are closely coupled events in normal meiosis. In particular, recombination and synapsis are often interdependent. In fact, a number of meiotic genes are required for both normal synapsis and recombination in yeast (Saccharomyces cerevisiae; Zickler and Kleckner, 1999
In the widely accepted DSB repair model for recombination (Szostak et al., 1983
Five RAD51 paralogs have been identified in mammals and birds, including RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3 (Shinohara et al., 1993
A putative RAD51C ortholog, AtRAD51C, is present in the Arabidopsis genome (Osakabe et al., 2002
Identification of a T-DNA Insertion in the AtRAD51C Gene
Both Arabidopsis genomic and cDNA sequences reveal a putative homolog of the human RAD51C gene, At2g45280, which was named AtRAD51C (Osakabe et al., 2002
To understand the in vivo function of AtRAD51C, we obtained a T-DNA insertional line (SALK_021960) that contained an insertion in the second intron of the AtRAD51C gene (Fig. 1A). The position of the insertion, named atrad51c-1, was confirmed by PCR amplification with AtRAD51C-specific (oMC1451) and T-DNA-specific (oMC 645; LBb1) primers (Fig. 1A). Molecular characterization indicated that the atrad51c-1 insertion contains two copies of T-DNAs with their left border-AtRAD51C junctions. This insertion is also associated with the loss of a 141-bp sequence in the second AtRAD51C intron (Fig. 1A). RT-PCR with gene-specific primers did not detect AtRAD51C transcripts even after 43 cycles of amplification (Fig. 2M), indicating that atrad51c-1 is likely a null mutation. Segregation analyses indicated that atrad51c-1 was tightly linked with a sterility phenotype (see below): Of the 359 plants examined, every plant (92) that was homozygous for atrad51c-1 was found to be sterile. In addition, no sterile plants were found that were not homozygous for atrad51c-1.
atrad51c-1 Is Normal in Vegetative Development, But It Is Female and Male Sterile Plants containing the atrad51c-1 mutation resembled wild-type plants during vegetative development under standard growth conditions (Fig. 2, A and B). Flowers from atrad51c-1 plants had normal morphology, including normal floral organ identity and number (Fig. 2, C and D). However, the atrad51c-1 plants were completely sterile, with small, empty seedpods (Fig. 2, G and H). Examination of the mutant flowers indicated that they were male sterile and mutant anthers failed to produce functional pollen grains (Fig. 2, D, F, and J). Some pollen grains were produced in the mutant, but they had abnormal sizes (Fig. 2F) and stained blue with Alexander's solution, indicating that they were not viable (Fig. 2J). At the tetrad stage, atrad51c-1 anthers contained polyads with five to 10 microspores (Fig. 2L) instead of four in the wild type (Fig. 2K). When the stigmas of atrad51c-1 flowers were pollinated with wild-type pollen (16 flowers crossed), they failed to set seed, indicating that the atrad51c-1 mutant is also female sterile.
Because mutations in human RAD51C affect the DNA repair in somatic cells, and our RT-PCR results as well as microarray data (Obayashi et al., 2004
Male and Female Meiosis Is Disrupted in atrad51c-1 Plants Our morphological and genetic analyses suggest that atrad51c-1 may be defective in both male and female meiosis. To test this hypothesis, we compared both male and female meioses in wild-type and atrad51c-1 plants grown under standard conditions, using chromosome spreads stained with 4',6-diamidino-2-phenylindole (DAPI). Meiotic abnormalities were identified in both male and female meiocytes (Fig. 4). In wild-type plants, male meiosis I starts with the condensation of chromatin, forming distinct thin chromosome threads at leptotene (Fig. 4A). After further condensation and pairing of homologous chromosomes during zygotene (Fig. 4B), juxtaposed homologs are observed at pachytene (Fig. 4C). Following further condensation (Fig. 4, D and E), the chromosomes then aligned at the division plane (Fig. 4F). No obvious alterations were observed in atrad51c-1 meiocytes at leptotene (Fig. 4G); however, dramatic abnormalities were observed from zygotene through telophase I. In contrast to the wild type (Fig. 4B), close association of homologs was rarely detected in mutant meiocytes (Fig. 4H). In addition, typical pachytene images could not be found in atrad51c-1 meiocytes (Fig. 4I), suggesting that fully juxtaposed chromosomes may not be present. Subsequent abnormalities were also observed during diplotene (Fig. 4J) and diakinesis (Fig. 4K). A striking feature of the atrad51c-1 mutant is the presence of numerous brightly stained spots. The abnormally large number of chromosomal entities was detected beginning at approximately diakinesis, suggesting that some DSBs were not repaired and subsequently were manifest as broken chromosomes. The chromosome fragments became more obvious at metaphase II, when they are highly condensed (atrad51c-1, an average of 28.7 ± 4.2 spots from 29 cells; for comparison, 31 atrad51-1 mutant cells have an average of 32.4 ± 4.9 spots; only well-spread cells were counted, although >1,000 were observed with fragmentation for each mutant). To test the possibility that the observed chromosome fragmentation might be resulting in part due to the force of chromosome spreading, we also examined intact atrad51c meiocytes (n = 324) and found similar chromosome fragments (data not shown), whereas wild-type cells had the expected five bivalents at late diakinesis. Other abnormalities observed in atrad51c-1 meiosis II were likely the consequence of defects in meiosis I (Fig. 4, S–X).
Female meiosis in atrad51c-1 exhibited similar, but less severe, defects (Fig. 4EE–JJ) as those observed during male meiosis. In atrad51c-1 female meiocytes, association of homologs at zygotene was also altered (Fig. 4FF), and fully juxtaposed chromosomes as seen in normal pachytene could not be observed (Fig. 4GG). Mutant female meiocytes also exhibited abnormal chromosome alignment and the presence of more than 10 chromosomal entities. However, fewer fragments were detected at metaphase I (Fig. 4II) and anaphase I (Fig. 4JJ) in female meiocytes than male meiocytes (13–15 chromosomal entities in 23 cells), suggesting that female meiosis may be less affected by the mutation.
The absence of normal pachytene chromosomes suggested that the atrad51c-1 mutant may be defective in synapsis and/or in homolog juxtaposition. To test the latter, we examined both wild-type and atrad51c-1 meiocytes using fluorescence in situ hybridization (FISH). From interphase to early zygotene, eight to 10 centromere signals were observed in wild-type and atrad51c-1 meiocytes (Fig. 5, A, B, E, and F). By late zygotene, 72% of the wild-type male meiocytes had five or fewer centromere signals (>100 cells); at pachytene and diplotene/early diakinesis (Fig. 5, C and D), 100% of the cells had five or fewer centromere signals (>100 cells for each stage). In contrast, about one-half of the atrad51c-1 meiocytes had more than six centromere signals, with an average of 5.7, during mid-to-late stages of prophase I (approximately equivalent to the wild-type pachytene, diplotene, and early diakinesis stages, >100 cells for each stage; Fig. 5, G and H). This suggests that the atrad51c-1 mutation affects the juxtapositioning of the centromere regions of some chromosomes. Furthermore, most atrad51c-1 cells observed had one or two centromere signals that were larger than the rest and those in the wild type, whereas other centromere signals were often smaller and not as brightly stained as wild-type centromeres. These observations suggest that centromere regions in atrad51c-1 meiocytes may have abnormal structure.
Because paired centromeres could be nonhomologous, we next used a telomere-derived probe that labels, in addition to telomeres, an arm sequence adjacent to the centromere only on chromosome 1 (Armstrong et al., 2001 ), in order to further assess homolog interaction in atrad51c-1 meiocytes. Two strong signals corresponding to chromosome 1 were observed during interphase, leptotene, and early zygotene in both wild-type and atrad51c-1 meiocytes (Fig. 6). Beginning at late zygotene and extending through pachytene, diplotene, and diakinesis, one signal was observed in wild-type meiocytes (Fig. 6). In contrast, two sometimes adjacent signals were observed in the meiocytes of atrad51c-1 plants at the stages corresponding to the normal pachytene, diplotene, and diakinesis stages (Fig. 6). atrad51c-1 cells containing a single chromosome 1 signal were rarely (<20/300) observed. These results are consistent with our observations from DAPI staining and centromere FISH and confirm that, while there may be some association of homologs, the normal homolog juxtaposition is altered in the mutant. In addition to the chromosome 1 signal(s), similar numbers of telomere signals were observed in both wild type and atrad51c-1 (Fig. 6). However, the overall sizes of the telomere signals seemed to be smaller in atrad51c-1 meiocytes than those in wild-type cells (Fig. 6). In some instances, a telomere signal was not detected at some chromosome ends, possibly due to chromosome breakage.
To investigate possible atrad51c-1 defects in synapsis, SC formation was analyzed using TEM (Fig. 7). Wild-type meiotic chromosomes start to synapse at early zygotene (Fig. 7, A and B). Two lateral elements, aligned in parallel, become connected by transverse filaments to the central element forming the SC. Early recombination nodules associated with the SC structures can be observed in wild-type nuclei (Fig. 7B). In mutant meiocytes, at the stages corresponding to zygotene or early pachytene in wild-type meiocytes, SC structures can be seen as short stretches (less than 10% in total length of that in wild type), indicating that most chromosome regions remain unsynapsed (Fig. 7, D–F). Recombination nodules were not observed on the mutant SC central element among 73 cells that were examined with 25 to 30 sections for each cell. Furthermore, abnormal SCs consisting of more than two lateral elements (Fig. 7E) were sometimes observed (approximately one-third of the SCs in the mutant), suggesting that synapsis might have occurred between nonhomologous chromosomes. However, no SC or SC-like structures were observed in mutant nuclei at late pachytene, suggesting that partially synapsed chromosomes did not proceed to become fully synapsed and that the partial SCs precociously disassembled during the stage corresponding to wild-type pachytene.
AtSPO11-1 Is Important for Chromosome Fragmentation in atrad51c-1 Meiocytes It is known that, in yeast, SPO11-generated DSBs are required for normal recombination and synapsis and that the Arabidopsis AtSPO11-1 gene is required for pairing and recombination. The chromosome fragmentation observed in atrad51c-1 meiocytes suggests that they may represent a defect to process DSBs generated by the AtSPO11-1 protein. To test the relationship between AtSPO11-1-generated DSBs and chromosome fragmentation in atrad51c-1 plants, we generated the atspo11-1 atrad51c-1 double mutant and analyzed its meiosis (Fig. 8). The meiotic chromosome behavior in the double mutant was similar to that of the atspo11-1 single mutant, including the presence of many univalents and occasional bivalents (Fig. 8, D and J). This was very different from that in the atrad51c-1 mutant (Fig. 4). In the vast majority of cells (>110), no more than 10 chromosomes were observed in male meiocytes of the double mutant from metaphase I through metaphase II, and 20 newly separated sister chromatids were observed at anaphase II (Fig. 4V), although, in a few cells (3 out of 110 examined), there was an extra DAPI-staining spot in the double mutant. Therefore, chromosome fragmentation was nearly absent in the double mutant, indicating that AtRAD51C acts downstream of AtSPO11-1. We conclude that AtRAD51C likely plays an important role in processing AtSPO11-1-generated DSBs.
Our results clearly show that AtRAD51C is required to repair the meiotic DSBs generated by the AtSPO11-1 during meiosis I, consistent with the function of vertebrate RAD51C in mitosis. The atrad51c-1 mutant plants can undergo normal vegetative and flower development with apparently normal mitosis, although mutations in AtRAD51C and other RAD51 paralogs have recently been shown to cause increased sensitivity to the DNA cross-linking agent mitomycin C (Bleuyard et al., 2005 ), suggesting that AtRAD51C plays a role in DNA repair in the mitotic cell cycle. In vitro studies showed that mammalian RAD51C promotes branch migration and the processing of double Holliday junctions in homologous recombination (Liu et al., 2004). Our results also show that AtRAD51C is required for normal synapsis and may be involved in homolog juxtaposition, suggesting that AtRAD51C may be required for early meiosis before the formation of double Holliday junctions. Therefore, analyses reported here and from others indicate that RAD51C is likely involved in multiple processes during the meiotic prophase I and mitotic cell cycle in animals and plants.
Although RAD51C orthologs have not been identified in yeast and other fungal organisms, putative orthologs are found in plants and animals. In Drosophila, the spnD gene encodes a putative RAD51C homolog (Abdu et al., 2003
In contrast to mammalian and chicken RAD51C, AtRAD51C and spnD are not required for mitotic growth under normal conditions. A similar situation exists for RAD51 homologs. The mammalian and chicken RAD51 gene is required for the mitotic cell cycle and a mouse rad51 knockout is embryonic lethal (Lim and Hasty, 1996
Analyses in yeast, C. elegans, and Drosophila indicate that the relationship between synapsis and recombination is not conserved between yeast and invertebrate animals (Keeney et al., 1997
The relationship between pairing and recombination is much less clear than that for synapsis and recombination (Page and Hawley, 2003
Recent reports (Bleuyard and White, 2004
It is known that RAD51 can bind to ssDNA and catalyze strand invasion by the ssDNA into a double-stranded DNA, forming a D-loop (Petukhova et al., 2000
Mutant analysis has also been reported for the Arabidopsis DMC1 homolog, AtDMC1 (Couteau et al., 1999
The model proposed above for AtRAD51, AtRAD51C, and AtXRCC3 function may also explain why these proteins are dispensable for the mitotic cell cycle. It is likely that, during normal mitotic growth, only a small number of DSBs are generated, far fewer than the number of DSBs generated by SPO11. If so, the number of RAD51 foci needed during the mitotic cell cycle would be far fewer than during meiosis. Indeed, the RAD51 mRNA levels are higher in meiotic cells than other cells (Li et al., 2004
Plant Materials The atrad51c-1 T-DNA insertional line was obtained from the SALK stock of Arabidopsis (Arabidopsis thaliana) T-DNA insertion lines. Both the wild-type and the atrad51c-1 mutant plants are of the Columbia ecotype. Unless otherwise indicated, the plants were grown under long-day conditions (16-h day/8-h night) at 22°C ± 2°C.
Plants were photographed using a Sony (Tokyo) digital camera DSC-F707, and photographs of other fresh plant samples (flowers and siliques) were obtained using a Nikon (Tokyo) dissecting microscope with an Optronics (Goleta, CA) digital camera. Developing microspores from fresh floral buds and anthers prior to dehiscence were processed as reported for examining gametophyte development (Azumi et al., 2002
RNA samples were extracted from young inflorescence tissues of wild-type and atrad51c-1 mutant plants. One microgram of total RNA from each sample was treated with RNase-free DNase I (Invitrogen, Carlsbad, CA) followed by inactivation of the DNase I. Reverse transcription was performed using Supertranscriptase II (Invitrogen) and synthesized cDNA was used as the template for PCRs. The primers used for the PCRs were oMC 1594 (5'-ATGATTTCATTTGGGCGGCGTA-3') and oMC 1593 (5'-CCTCCAAGACCACCACACTCA-3'). As a control, the APT1 cDNA in the same samples was amplified using primer pair oMC 571 (5'-TCCCAGAATCGCTAAGATTGCC-3') and oMC 572 (5'-CCTTTCCCTTAAGCTCTG-3'), based on a report that this gene is expressed constitutively in different Arabidopsis organs (Moffatt et al., 1994
Crosses were made between AtSPO11-1/atspo11-1 and AtRAD51C/atrad51c-1 plants and resulting double-heterozygous F1 plants (AtRAD51C/atrad51c-1; AtSPO11-1/atspo11-1) were identified with gene-specific primers. The progeny of the double-heterozygous plants were genotyped with gene-specific primers and chromosome behavior in male meiosis of double-mutant plants was analyzed following DAPI staining. The primers used for genotyping the plants include oMC 1662 (5'-TGAACTCCTCCTTGGAGCTATGTTGG-3'); oMC 1451 (5'-CCAATGCGATTTGCTCGGTGTAACT-3'); oMC 645 (5'-GCGTGGACCGCTTGCTGCAACT-3'); oMC 703 (5'-ACGTATCGGGCCTAAATTCC-3'); oMC 704 (5'-TTTGGAGATCTTCCTTCAGCC-3'); and oMC 705 (5'-ACTGGGATTCGTCTTGGACA-3').
Inflorescences were fixed in acetic alcohol (ethanol:glacial acetic acid, 3:1) for 2 h at room temperature and stored at –20°C after replenishing the fixative. Staged anthers were spread onto slides and subjected to FISH using previously published procedures (Fransz et al., 1996
For TEM, the fixation and infiltration procedures were as described previously (Owen and Makaroff, 1995
We thank J. Ecker and the Arabidopsis Biological Resource Center (ABRC) for providing the T-DNA insertional line, M. Grelon for the atspo11-1 seeds, Y. Hu for RNA in situ experiments, R. Cyr, G. Ning, M. Hazen, R. Haldeman, W. Hu, and A. Wijeratne for advice and technical assistance, A. Omeis and J. Wang for plant care, and C. Chen, O. Hamant, C. Hendrix, S.J. Hord, A. Wijeratne, and the reviewers for helpful comments on this manuscript. Received December 14, 2004; returned for revision March 27, 2005; accepted March 27, 2005.
1 This work was supported by the National Institutes of Health (grant no. R01 GM63871–01 to H.M.) and the National Science Foundation (grant nos. MCB–0092075 to H.M. and MCB–0322171 to C.A.M.). W.L. was partially supported by the Intercollege Graduate Program in Plant Physiology at the Pennsylvania State University. R.X. is supported by a University Graduate Fellowship from the Pennsylvania State University. H.M. gratefully acknowledges the support of the John Simon Guggenheim Memorial Foundation, the K.C. Wong Educational Foundation, and the Foreign Distinguished Young Scholar Award from the Chinese National Science Foundation.  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.058347. * Corresponding author; e-mail hxm16{at}psu.edu; fax 814–863–1357.
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ABSTRACT
RESULTS
-tubulin, respectively. Cells at different stages during the mitotic cell cycle were analyzed (preprophase, A, E, I, and M; metaphase, B, F, J, and N; anaphase, C, G, K, and O; and telophase, D, H, L, and P).
