The Arabidopsis homologue of Xrcc3 plays an essential role in meiosis

Abstract

The eukaryotic RecA homologue Rad51 is a key factor in homologous recombination and recombinational repair. Rad51-like proteins have been identified from yeast (Rad55, Rad57 and Dmc1) to vertebrates (Rad51B, Rad51C, Rad51D, Xrcc2, Xrcc3 and Dmc1). These Rad51-like proteins are all members of the genetic recombination and DNA damage repair pathways. The sequenced genome of Arabidopsis thaliana encodes putative homologues of all six vertebrate Rad51-like proteins. We have identified and characterized an Arabidopsis mutant defective for one of these, AtXRCC3, the homologue of XRCC3. atxrcc3 plants are sterile, while they have normal vegetative development. Cytological observation shows that the atxrcc3 mutation does not affect homologous chromosome synapsis, but leads to chromosome fragmentation after pachytene, thus disrupting both male and female gametogenesis. This study shows an essential role for AtXrcc3 in meiosis in plants and possibly in other higher eukaryotes. Furthermore, atxrcc3 cells and plants are hypersensitive to DNA-damaging treatments, supporting the involvement of this Arabidopsis Rad51-like protein in recombinational repair.

Figure 1

Molecular characterization of atxrcc3 T-DNA insertion. (A) Diagram showing the position of the T-DNA insertion at the AtXRCC3 locus. The black arrow indicates the AtXRCC3 transcriptional start site, the grey box represents the AtXRCC3 coding sequence and the white box the intron spliced in AtXRCC3 alpha mRNA. The triangle represents the T-DNA inserted in position +704 of the AtXRCC3 coding sequence. (B) Sequence of the atxrcc3 insertion site. The white box indicates the T-DNA insertion. LB1 and LB2 indicate the two left borders surrounding the T-DNA insertion, and their orientations are indicated in (A). The grey and white triangles represent base insertion and deletion, respectively. The in-frame TAA STOP codon resulting from LB1 integration is indicated in grey. The numbers represent positions relative to the start codon. (C) RT–PCR detection of AtXRCC3 transcript. WT and atxrcc3 indicate RT–PCR amplifications performed on wild-type and atxrcc3 total mRNAs extracted from cell suspensions. AtXRCC3 is the product amplified using primers o392 and o443 represented in (A). Amplification of the APT1 mRNA has been used as a control for reverse transcription.

Figure 2

atxrcc3 mutant plants are sterile. (A) Wild-type (left) and atxrcc3 (right) 3-week-old plants. Flowering stems (B) and siliques (C) of the wild-type and atxrcc3 plants.

Figure 3

Gametophytic lethality in atxrcc3 mutant plants. Anthers of wild-type (A) and atxrcc3 (B) plants have been stained according to Alexander (1969). Anthers from 2 mm buds (up) and mature flower (down) have been compared. The right of each panel presents enlargement of some pollen grains. The red-purple-stained cytoplasm indicates viability, while the pollen cell wall is counterstained in green. (C–J) Differential interference contrast (DIC) microscopy observations of embryo sac development in wild-type (C–F) and atxrcc3 (G–J) ovules, after clearing. The bottom of each panel presents an enlargement of the nuclei. MMC=megaspore mother cell, N=nucleus, DC=degenerative cell, bars=20 μm.

Figure 4

Meiosis in wild-type Arabidopsis: (A) zygotene, (B) pachytene, (C) diplotene, (D) diakinesis, (E) metaphase I, (F) anaphase I, (G) telophase I, (H) metaphase II, (I) anaphase II, (J) telophase II, (K) tetrad and (L) microspores. Bars=10 μm.

Figure 5

Meiosis is severely disrupted in atxrcc3 mutant plants: (A) zygotene, (B) pachytene, (C) diplotene, (D) diakinesis, (E) metaphase I, (F) anaphase I, (G) telophase I, (H) metaphase II, (I) anaphase II, (J) telophase II, (K) polyads and (L) microspores. Yellow arrows=bivalents, red arrows=chromosome fragments, b=bridge, mn=micronucleus, bars=10 μm.

Figure 6

Pachytene chromosomes of atxrcc3 hybridized with 5S rDNA (red) and DAPI counterstained: (A) DAPI, (B) 5S rDNA signals and (C) merged images. Arrows=5S rDNA signals.

Figure 7

atxrcc3 cells and plants are hypersensitive to DNA crosslinking agents, but not to DSB-inducing agents. (A, C) Five-day-old wild-type and atxrcc3 cell cultures were transferred and grown on plates containing increasing doses of mitomycin C (A) or bleomycin (C). After 3 weeks, callus growth was used to score sensitivity to DNA-damaging agent. (B) Seeds of wild-type and atxrcc3^+/− heterozygote plants were sown on plates containing increasing doses of mitomycin C. After 14 days, the percentage of hypersensitive plants (plants with three leaves or less) was used to produce a mitomycin C dose–response curve. Values represent three replicates, each replicate containing an average of 100 plants per dose. Error bars=standard deviation.