The cyclin-A CYCA1;2/TAM is required for the meiosis I to meiosis II transition and cooperates with OSD1 for the prophase to first meiotic division transition

Abstract

Meiosis halves the chromosome number because its two divisions follow a single round of DNA replication. This process involves two cell transitions, the transition from prophase to the first meiotic division (meiosis I) and the unique meiosis I to meiosis II transition. We show here that the A-type cyclin CYCA1;2/TAM plays a major role in both transitions in Arabidopsis. A series of tam mutants failed to enter meiosis II and thus produced diploid spores and functional diploid gametes. These diploid gametes had a recombined genotype produced through the single meiosis I division. In addition, by combining the tam-2 mutation with AtSpo11-1 and Atrec8, we obtained plants producing diploid gametes through a mitotic-like division that were genetically identical to their parents. Thus tam alleles displayed phenotypes very similar to that of the previously described osd1 mutant. Combining tam and osd1 mutations leads to a failure in the prophase to meiosis I transition during male meiosis and to the production of tetraploid spores and gametes. This suggests that TAM and OSD1 are involved in the control of both meiotic transitions.

Figure 1. The tam mutations.

(A) The CYCA1;2/TAM gene contains nine exons. The positions of the three insertions in the tam-2, tam-3 and tam-4 mutants are indicated as are the three point mutations in tam-5, tam-6 and tam-7. The tam-2 insertion is accompanied by a deletion. (B) Male meiotic products stained with toluidine blue: a tetrad from the wild-type and dyads from the tam-2 and tam-4 mutants.

Figure 2. Male meiosis in wild type.

Micrographs of Arabidopsis chromosome spreads stained with 4',6-diamidino-2-phenylindole (DAPI) at (A) Pachytene; (B) Diakinesis; (C) Metaphase I; (D) Anaphase I; (E) Telophase I; (F) Metaphase II; (G) Anaphase II; and (H) Telophase II. Scale bar = 10 µm.

Figure 3. Male meiosis in the tam-2 mutant.

Micrographs of DAPI stained Arabidopsis chromosomes show that tam-2 Meiosis I including (A) Pachytene; (B) Diakinesis; (C) Metaphase I; (D, E) Anaphase I; and (F) Telophase is indistinguishable from the wild type but no figures characteristic of a second division were observed. I. Scale bar = 10 µm.

Figure 4. Female meiosis in wild type.

Micrographs of DAPI stained Arabidopsis chromosomes during (A) Pachytene; (B) Diakinesis; (C) Metaphase I; and (D) Anaphase I. Scale bar = 10 µm.

Figure 5. Female meiosis in the tam-2 mutant.

Micrographs of DAPI stained Arabidopsis chromosomes in tam-2 show that both meiosis I (A) Pachytene; (B) Diakinesis with five bivalents; (C) Metaphase I; (D) Anaphase I; (E) Telophase I and meiosis II (F) Metaphase II and anaphase II were indistinguishable from wild type. Scale bar = 10 µm.

Figure 6. Single-locus (FTL3253) segregation patterns in pollen.

A tetrad of wild-type (qrt1-2 background) pollen (A) from a plant that was hemizygous for a transgene encoding a cyan fluorescent protein has two fluorescent and two non-fluorescent pollen grains. Dyads of pollen (B and C) from the osd1-1 and tam-2 mutants, respectively, having one fluorescent and one non-fluorescent pollen grain in each case. Dyads (D and E) from the osd1-1 and tam-2 mutants, respectively, each showing two fluorescent pollen grains (osd1 n = 173, tam n = 214). Interpretations, of chromosome segregation and crossover position, are shown on the left. Scale bar = 20 µm.

Figure 7. Two-locus (FTL1273 red, FTL993 blue) segregation pattern in pollen.

(A) Tetrad of wild type pollen with two blue fluorescent and two red fluorescent pollen grains, indicative of an absence of recombination between the markers. A crossover between the centromere and the upper marker may or may not have occurred. (B) Tetrad of wild-type pollen with one blue fluorescent, one red fluorescent pollen, one blue and red fluorescent and one non-fluorescent pollen grain, indicative of a crossover between the two markers. (C, D) Dyads of pollen from the osd1-1 and tam-2 mutants, respectively, each with one blue fluorescent and one red fluorescent pollen grain. (E, F) Dyads from osd1-1 and tam-2 mutants, respectively, each with one blue and red fluorescent pollen grain and one blue fluorescent pollen grains. (G, H) Dyads from osd1-1 and tam-2 mutants, respectively, each with two blue and red fluorescent pollen grain. (I, J) Dyad in osd1-1 and tam-2, respectively, with one blue and red fluorescent pollen grain and one red fluorescent pollen grain. (K, L) Dyads from MiMe-1 and MiMe-2 plants, respectively, each with two blue and red fluorescent pollen grains. Interpretations of chromosome segregation and crossover position, are shown on the left. (wild type n = 266, osd1 n = 263, tam n = 369). Rare cases indicative of multiple crossovers are not shown. Scale bar = 20 µm.

Figure 8. Genetic make-up of the osd1, tam, tam/osd1, MiMe, and MiMe-2 male and female diploid gametes.

Genotype of pollen grains, for the FTL loci (Figure 6 and Figure 7). In addition triploid offspring from mutant x wild-type crosses were genotyped for several molecular markers. The percentage of heterozygous gametes was determined (n is indicated into brackets). The frequency of heterozity is indicated by a color code, from 0% (green) to 100% (red). The positions of each marker (red) and the centromeres (blue) are indicated on the chromosomes. ^adata from .

Figure 9. Mitosis-like division instead of meiosis, in MiMe-2 plants.

(A–D) Male Meiosis. (A) Metaphase I. (B) Anaphase I. (C, D) Telophase I. (E–F) Female meiosis. (E) Metaphase I. (F) Anaphase I. Scale bar = 10 µm.

Figure 10. Female meiosis in the tam-2/osd1-1 double mutant.

Meiosis I is indistinguishable from the wild type but no figures characteristic of a second division were observed. (A) Pachytene. (B) Metaphase I. (C) Anaphase I. (D) Telophase I. Scale bar = 10 µm.

Figure 11. Pollen grains in wild type, osd1, tam and tam/osd1.

Anthers were stained as described by Alexander . (A) Wild type. (B) tam-2 (C) osd1-1. (D–F) tam-2/osd1. Scale bar = 100 µm.

Figure 12. Male meiotic products stained with toluidine blue.

(A) Wild type. (B) tam-2. (C) osd1-2. (D) tam-2/osd1-1. Scale bar = 10 µm.

Figure 13. Male meiosis in tam-2/osd1-1.

Prophase was indistinguishable from wild type. (A) Leptotene. (B) Pachytene. (C) Diplotene. (D) Diakinesis. (E) Spore formation. (F) Spore. Scale bar = 10 µm.