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Fig. 7. Analysis of the T-DNA (portion of the tumor-inducing plasmid that is transferred to plant cells) insertion in AtRAD51. (A) An illustration of the AtRAD51 locus and the T-DNA insertion. The original line 134A01 was found to have two T-DNA inserts that segregated independently. One of the two inserts is located between nucleotides 2830 and 2831 in the AtRAD51 gene, is a highly truncated copy of the pAC161 tagging vector, and carries neither a complete resistance marker nor an intact right border. The other T-DNA was unlinked to the AtRAD51 gene, consisted of at least two copies of the tagging vector, and carries a functional sulfadiazine-resistance gene (data not shown). The two-insert loci were separated by outcrossing, and plants that contained only the T-DNA tag in AtRAD51 were obtained as shown in A. Protein-coding regions of the AtRAD51 gene are shown as solid boxes, introns and UTRs are shown as gray lines, and the transcript is shown as solid boxes below the gene with an arrow at the end of the transcript, indicating the direction of transcription. The T-DNA insertion in the AtRAD51 gene was further confirmed by PCR using an AtRAD51 gene-specific primer and a T-DNA tag-specific primer suggested by GABI-KAT (data not shown). (B) Quantitative RT-PCR with AtRAD51 gene-specific primers. The results indicated that transcripts of the AtRAD51 gene were not present in plants homozygous for the T-DNA insertion. Lanes 1 and 2, AtRAD51/atrad51-1 plants; lanes 3 and 4, atrad51-1/atrad51-1 plants; lanes 5 and 6, wild-type plants. Lanes 1, 3, and 5 are from flower samples; lanes 2, 4, and 6 are from leaf samples. Expression of 18S rRNA was determined as a control.

Fig. 8. The atrad51-1 mutant exhibited normal vegetative development and defective reproduction. Wild-type (A and C) and atrad51-1 mutant (B and D) plants were similar in growth rate, height, and biomass. The growth was monitored in 14-day intervals from sawing to seed set. Under these conditions, homozygous mutants were indistinguishable from heterozygous plants and wild type until plants started to set seed. The same held true for plants grown under long-day conditions. Therefore, the loss of AtRAD51 function had no effect on vegetative development. However, mutant plants were completely sterile, had siliques that were remarkably reduced in size, and lacked seeds.

Fig. 9. Additional observations of male meiosis in wild-type (A–C and G–I) and atrad51-1 (D–F and J–L) plants. For wild-type male meiosis, after alignment at the division plane at metaphase I, the homologous chromosomes are segregated at anaphase I (A) and form two clusters of univalents at telophase I (B). Meiosis II starts with prophase II (C), followed by the alignment of chromosomes at metaphase II (G) and sister chromatid segregation at anaphase II (H), resulting in tetrads with four nuclei (I). In atrad51-1, multiple chromosome fragments were present at anaphase I (D), and these fragments were segregated abnormally at telophase I (E). The abnormal meiosis II (F; J–L) seems to be the consequence of abnormal meiosis I.