The F-Box Protein ZYGO1 Mediates Bouquet Formation to Promote Homologous Pairing, Synapsis, and Recombination in Rice Meiosis

Abstract

Telomere bouquet formation, a highly conserved meiotic event, plays an important role in homologous pairing and therefore progression of meiosis; however, the underlying molecular mechanism remains largely unknown. Here, we identified ZYGOTENE1 (ZYGO1), a novel F-box protein in rice (Oryza sativa), and verified its essential role in bouquet formation during early meiosis. In zygo1 mutants, zygotene chromosome aggregation and telomere clustering failed to occur. The suppressed telomere clustering in homologous pairing aberration in rice meiosis1 (pair1) zygo1 and rice completion of meiotic recombination (Oscom1) zygo1 double mutants, together with the altered localization of OsSAD1 (a SUN protein associated with the nuclear envelope) in zygo1, showed that ZYGO1 has a significant function in bouquet formation. In addition, the interaction between ZYGO1 and rice SKP1-like protein 1 suggested that ZYGO1 might modulate bouquet formation as a component of the SKP1-Cullin1-F-box complex. Although double-strand break formation and early recombination element installation occurred normally, zygo1 mutants showed defects in full-length pairing and synaptonemal complex assembly. Furthermore, crossover (CO) formation was disturbed, and foci of Human enhancer of invasion 10 were restricted to the partially synapsed chromosome regions, indicating that CO reduction might be caused by the failure of full-length chromosome alignment in zygo1 Therefore, we propose that ZYGO1 mediates bouquet formation to efficiently promote homolog pairing, synapsis, and CO formation in rice meiosis.

Figure 1.

ZYGO1 Encodes a Novel F-Box Protein in Rice. (A) Gene structure of ZYGO1 and the mutation sites of zygo1 mutants. Black blocks and lines represent exons and introns, respectively. Untranslated regions are shown as gray boxes. (B) An unrooted tree based on the full-length protein sequences of ZYGO1 and its homologs from seven plant species. ZYGO1 is shown in red. Zm, Zea mays; Sb, Sorghum bicolor; Os, Oryza sativa; Bd, Brachypodium distachyon; Pp, Physcomitrella patens; At, Arabidopsis thaliana; Sl, Solanum lycopersicum; Vv, Vitis vinifera. (C) ZYGO1 contains a gamma-crystallin-like motif, an F-box domain, and a cysteine proteinase motif. (D) Multiple sequence alignment of the F-box domain of ZYGO1 with its homologs from five plant species. Amino acid sequence of the F-box domain is underlined and conserved amino acids are highlighted in red. (E) ZYGO1 interacts with OSK1 in yeast two-hybrid assays. Interactions were verified by the growth and the blue color of yeast cells on SD/QDO/X/A (SD-Ade-His-Leu-Trp + X-α-gal + Aureobasidin) medium. BD, bait vector; AD, prey vector; ZYGO1-N, the N-terminal residues (amino acids 1–137) of ZYGO1; ZYGO1-F, the F-box domain of ZYGO1 (amino acids 138–172); ZYGO1-C, the C-terminal residues (amino acids 173–417) of ZYGO1; ZYGO1-N+F, 1 to 172 amino acids of ZYGO1; ZYGO1-F+C, 138 to 417 amino acids of ZYGO1. (F) BiFC assays showing the interaction between OSK1 and the full-length protein sequence or the F-box domain of ZYGO1 in rice protoplasts. Bars = 5 µm.

Figure 2.

The Chromosome Behavior of Meiocytes in the Wild Type and zygo1. (A) to (H) The detailed chromosome behavior of meiocytes from leptotene to diakinesis in the wild type. Leptotene (A), late leptotene (B), leptotene to zygotene (C), early zygotene (D), zygotene (E), late zygotene (F), pachytene (G), and diakinesis (H). CM, chromosome mass. (I) to (L) The chromosome behavior of meiocytes from leptotene to diakinesis in zygo1. (I) and (J) Leptotene (I) and early post-leptotene (J). Chromosomes remained dispersed in the nucleus in zygo1. (K) Late post-leptotene. Some partially paired regions are indicated with arrows. (L) Diakinesis. Some univalents are indicated with arrows. Chromosomes were fixated with ethanol-acetic acid (3:1) solution and stained with DAPI. Bar = 5 µm.

Figure 3.

ZYGO1 Is Essential for Bouquet Formation. (A) Telomere behavior in the wild type and the zygo1 mutant during early prophase I. Immuno-FISH analysis was performed using antibodies against PAIR2 (red) and PAIR3 (blue) as well as the telomere probe pAtT4 (green). The loading pattern of PAIR2 and PAIR3 indicates the stage of meiocytes in the wild type and zygo1. Telomere bouquet was observed in wild-type meiocytes at early zygotene, whereas it was not observed in zygo1 throughout the post-leptotene stage. (B) Distribution of telomere foci in pair1, Oscom1, pair1 zygo1, and Oscom1 zygo1 at early zygotene or post-leptotene stage. Telomere clustering in pair1 and Oscom1 was suppressed in pair1 zygo1 and Oscom1 zygo1 double mutants. (C) Immunofluorescent investigation of OsSAD1 signal (red) in the wild type and zygo1. Chromosomes were stained with DAPI (blue). The polarized enrichment of OsSAD1 foci was not observed in zygo1 throughout the post-leptotene stage. Bars = 5 µm.

Figure 4.

ZYGO1 Is Necessary for Full-Length Homologous Pairing and Synapsis. (A) to (C) The chromosome pairing status revealed by FISH assays in the wild type and zygo1. Bulked oligonucleotide probes, 11S (green) and 11L (red), were used to track the short and long arms of chromosome 11, respectively. Chromosomes were stained with DAPI (blue). (A) Wild-type meiocyte with well-paired probe signals at pachytene. (B) zygo1 meiocyte with separated signals of these two probes. (C) zygo1 meiocyte with partially paired signals of these two probes. (D) to (G) Super-resolution images of the triple immunolocalization of PAIR2 (red), PAIR3 (green), and ZEP1 (blue) in the wild type and zygo1 during early prophase I. (D) Wild type. Zygotene with partial synapsis. (E) Wild type. Pachytene with full synapsis. (F) zygo1. Early post-leptotene with short fragments of ZEP1 signals. (G) zygo1. Late post-leptotene with more extensive but incomplete ZEP1 signals. (D1), (E1), (F1), and (G1) show magnified images of the framed parts in (D), (E), (F), and (G), respectively. Bars = 5 µm.

Figure 5.

ZYGO1 Is Not Required for the Localization of γH2AX, OsCOM1, OsDMC1, and OsZIP4 onto Chromosomes. (A) Immunolocalization of γH2AX (red), OsCOM1 (green), OsDMC1 (green), and OsZIP4 (green) at zygotene or post-leptotene stage in the wild type and zygo1. Bars = 5 µm. (B) to (E) Quantification of γH2AX (B), OsCOM1 (C), OsDMC1 (D), and OsZIP4 (E) foci per meiocyte in the wild type and zygo1, respectively. Values are means ± se. Statistical analyses revealed no significant differences (NS) between the wild type (n values are 20, 18, 20, and 19, respectively) and zygo1 (n values are 20, 20, 13, and 20, respectively). The P values are 0.1746, 0.4010, 0.6400, and 0.2404, respectively, from two-tailed Student’s t tests.

Figure 6.

CO Formation Is Affected in zygo1. (A) The distribution of 5S rDNA foci (red) revealed by FISH assays in the wild type and zygo1. Chromosomes at metaphase I were stained with DAPI (blue). Frequencies of associated or nonassociated 5S rDNA signals in the wild type and zygo1 are displayed. Bars = 5 µm. (B) Chiasmata per cell in the wild type, hei10, Oszip4, zygo1, hei10 zygo1, and Oszip4 zygo1. Values are means ± se. ****P < 0.0001. The n values are 58, 54, 53, 102, 65, and 67, respectively, from two-tailed Student’s t tests.

Figure 7.

ZYGO1 Promotes Homologous Pairing Independent of HEI10 and OsZIP4, but Affects HEI10 Distribution. (A) Chromosome behavior of meiocytes in hei10, Oszip4, hei10 zygo1, and Oszip4 zygo1 at pachytene or late post-leptotene stage. Full-length chromosome pairing in hei10 and Oszip4 is suppressed in hei10 zygo1 and Oszip4 zygo1 double mutants. Chromosomes were stained with DAPI. (B) Triple immunolocalization of OsREC8 (red), HEI10 (green), and ZEP1 (blue) in the wild type and zygo1 at late pachytene or late post-leptotene stage. The bright punctuated signals of HEI10 are restricted to the short ZEP1 stretches in zygo1. Bars = 5 µm.

Figure 8.

Proposed Model for ZYGO1 Function during Early Prophase I. In the wild type, accompanying homologous pairing, telomeres gradually aggregate from late leptotene to zygotene and finally are clustered into a typical bouquet at zygotene. Meanwhile, linear chromosomes gradually aggregate into a fused mass. The telomere bouquet and chromosome mass completely dissolve and homologous chromosomes are fully synapsed at pachytene. In zygo1, however, both the telomere bouquet and chromosome mass do not form. Although some chromosomes initiate pairing and synapsis, full-length chromosome pairing and synapsis are not completed. Finally, crossover formation is restricted to the partially synapsed region of chromosomes in zygo1.