The Arabidopsis AtRAD51 gene is dispensable for vegetative development but required for meiosis

Abstract

The maintenance of genome integrity and the generation of biological diversity are important biological processes, and both involve homologous recombination. In yeast and animals, homologous recombination requires the function of the RAD51 recombinase. In vertebrates, RAD51 seems to have acquired additional functions in the maintenance of genome integrity, and rad51 mutations cause lethality, but it is not clear how widely these functions are conserved among eukaryotes. We report here a loss-of-function mutant in the Arabidopsis homolog of RAD51, AtRAD51. The atrad51-1 mutant exhibits normal vegetative and flower development and has no detectable abnormality in mitosis. Therefore, AtRAD51 is not necessary under normal conditions for genome integrity. In contrast, atrad51-1 is completely sterile and defective in male and female meioses. During mutant prophase I, chromosomes fail to synapse and become extensively fragmented. Chromosome fragmentation is suppressed by atspo11-1, indicating that AtRAD51 functions downstream of AtSPO11-1. Therefore, AtRAD51 likely plays a crucial role in the repair of DNA double-stranded breaks generated by AtSPO11-1. These results suggest that RAD51 function is essential for chromosome pairing and synapsis at early stages in meiosis in Arabidopsis. Furthermore, major aspects of meiotic recombination seem to be conserved between yeast and plants, especially the fact that chromosome pairing and synapsis depend on the function of SPO11 and RAD51.

Fig. 1.

Mitosis in root tips of wild-type (A, B, E, F, I, J, M, and N) and atrad51-1 (C, D, G, H, K, L, O, and P) seedlings. Chromosomes (Left) and microtubule structures (Right) in the same cells were visualized with 4′,6-diamidino-2-phenylindole, and antibodies against β-tubulin were visualized at different stages during the mitotic cell cycle (preprophase, A and C; metaphase, E and G; anaphase, I and K; and telophase, M and O).

Fig. 2.

Phenotypes of wild-type (A, C, E, G, I, and L), atrad51-1 (B, D, F, H, J, K, and N), and atrad51-1/atrad51-1 35S::MYC::AtRAD51 transgenic plants (M). Shown are opened flowers (A and B), stamens and carpels dissected from unopened flowers (E and F), tetrad (C and D) with numbered microspores, pollen grain (G and H), ovule (I–K; arrows point at aborted ovules), and young siliques (L and N). Two independent 35S::MYC::AtRAD51 transgenic lines (plants 16 and 37) produced siliques (M) that were similar in size to the wild-type ones (L). RT-PCR analysis of these two lines and another sterile transgenic line was performed and shown in O. Lanes: 1, wild type; 2, T1 transgenic plant 16; 3, T1 transgenic plant 37; 4, T1 transgenic plant 22. APT1 expression was determined as a positive control.

Fig. 3.

AtRAD51 gene expression pattern in floral organs. AtRAD51 expression was detected in floral primordia (A) and at moderate levels in very young flower buds (B). In later anthers, the expression is strong in meiocytes (C) and restricted in the anther locules (D). Expression of AtRAD51 in carpels is restricted to ovules (E). There was no detectable expression in pollen grains (F) or gynoecium after meiosis (G) or when a sense probe was used (H).

Fig. 4.

Male and female meiosis in wild-type (male, A–F; female, M–R) and atrad51-1 (male, G–L; female, S–X) plants. For male meiosis, stages included for wild-type (A–F) and atrad51 (G–L) meiosis are leptotene (A and G), zygotene (B and H), pachytene (C and I), diplotene (D and J), diakinesis (E and K), and metaphase I (F and L). For female meiosis, stages included for wild-type (M–R) and atrad51-1 (S–X) meiosis are leptotene (M and S), zygotene (N and T), pachytene (O and U), diakinesis (P and V), metaphase I (Q and W), and anaphase I (R and X). The arrow in B indicates a pairing fork.

Fig. 5.

Transmission electron micrographs of male meiocyte nuclei in wild-type (A and B) and atrad51-1 (C–E) plants. SCs (arrows) were observed at zygotene (A) and pachytene (B) stages in wild type. In atrad51-1, the axial elements (arrowheads) remained unpaired in nuclei corresponding to zygotene (C) or pachytene (D) stages; an occasional SC was observed (E). Nu, nucleolus; RN, recombination nodule. (Scale bar, 100 nm.)

Fig. 6.

Male meiosis in atspo11-1 (A–F) and atspo11-1 atrad51-1 (G–L). Stages included are zygotene (A and G), diakinesis (B and H), metaphase I (C and I), anaphase I (D and J), metaphase II (E and K), and anaphase II (F and L). The arrows in D and I indicate the presence of bivalents. See Fig. 4 for a comparison of the wild-type and atrad51-1 meioses.