MEIOTIC F-BOX Is Essential for Male Meiotic DNA Double-Strand Break Repair in Rice

Abstract

F-box proteins constitute a large superfamily in plants and play important roles in controlling many biological processes, but the roles of F-box proteins in male meiosis in plants remain unclear. Here, we identify the rice (Oryza sativa) F-box gene MEIOTIC F-BOX (MOF), which is essential for male meiotic progression. MOF belongs to the FBX subfamily and is predominantly active during leptotene to pachytene of prophase I. mof meiocytes display disrupted telomere bouquet formation, impaired pairing and synapsis of homologous chromosomes, and arrested meiocytes at late prophase I, followed by apoptosis. Although normal, programmed double-stranded DNA breaks (DSBs) form in mof mutants, foci of the phosphorylated histone variant γH2AX, a marker for DSBs, persist in the mutant, indicating that many of the DSBs remained unrepaired. The recruitment of Completion of meiosis I (COM1) and Radiation sensitive51C (RAD51C) to DSBs is severely compromised in mutant meiocytes, indicating that MOF is crucial for DSB end-processing and repair. Further analyses showed that MOF could physically interact with the rice SKP1-like Protein1 (OSK1), indicating that MOF functions as a component of the SCF E3 ligase to regulate meiotic progression in rice. Thus, this study reveals the essential role of an F-box protein in plant meiosis and provides helpful information for elucidating the roles of the ubiquitin proteasome system in plant meiotic progression.

Figure 1.

Phenotypic Analysis of mof. (A) Wild-type and mof plants after bolting. (B) Wild-type and mof inflorescences at the heading stage. (C) Wild-type and mof spikelets before anthesis. (D) Wild-type and mof spikelets after removing the lemma and palea. (E) A wild-type anther (left) and a mof mutant pale-yellow, smaller anther (right). (F) and (G) I₂-KI staining of the pollen grains within the anther of the wild type (F) and mof (G). (H) to (Q) Transverse section analysis of the anthers. The images are cross sections of a single locule. The wild-type anther is shown in (H), (J), (L), (N), and (P) and the mof mutant anther in (I), (K), (M), (O), and (Q). Stage 7 (meiosis prophase I) ([H] and [I]); stage 8a (dyad stage) ([J] and [K]); stage 8b (tetrad stage) ([L] and [M]); stage 9 (early microspore stage) ([N] and [O]); and stage 12 ([P] and [Q]). DMC, degenerated meiocyte cell; DMs, degenerated microspores; Dy, dyad; E, epidermis; En, endothecium; M, middle layer; MC, meiocyte cell; Mp, mature pollen; Ms, microspores; T, tapetal layer; Tds, tetrads; gl, glume; le, lemma; pa, palea; st, stamen. Bars = 20 cm in (A), 2 cm in (B), 2 mm in (C) to (E), 10 μm in (F) and (G), and 15 μm in (H) to (Q).

Figure 2.

Meiotic Chromosome Behaviors of Male Meiocytes in the Wild Type and mof. Chromosome behavior of male meiocytes of wild type ([A] to [I]) and mof ([J] to [O]) at various stages. Leptotene ([A] and [J]); zygotene ([B] and [K]); pachytene (C); diakinesis (D); metaphase I (E); anaphase I (F); metaphase II (G); anaphase II (H); telophase II (I); stages after zygotene ([L] to [O]). Bars = 5 μm.

Figure 3.

MOF Is Required for Bouquet Formation and Homologous Pairing. (A) and (E) Telomere bouquet formation analysis revealed by FISH using the telomere sequence as a probe (red) in wild-type (A) and mof (E) nuclei. Chromosomes (blue) are stained with DAPI. (B) and (F) Immunolocalization of OsCenH3 (red) showing paired and unpaired homologous chromosomes in wild-type (B) and mof (F) nuclei. (C) and (G) Homologous pairing analysis revealed by FISH using 5S rDNA probe (green; indicated by yellow triangle) in wild-type (C) and mof (G) nuclei. (D) and (H) Homologous pairing analysis revealed by FISH using probes prepared from BAC P0671B11 (red; indicated by orange triangle) in wild-type (D) and mof (H) nuclei. Bars = 5 μm.

Figure 4.

Immunolocalization of SC-Related Proteins in mof. Immunolocalization of PAIR2 (magenta) (A), PAIR3 (magenta) (B), and ZEP1 (magenta) (C) at zygotene in both the wild type and mof. Bars = 5 μm.

Figure 5.

MOF Is Not Required for DSB Formation but Is Essential for Meiotic Progression. Immunolocalization of γH2AX (magenta) at zygotene (A) and pachytene (B), and COM1 (magenta) (C) and RAD51C (magenta) (D) at zygotene in both the wild type and mof. REC8 signals (green) were used to indicate the meiotic chromosome axes. Bars = 5 μm.

Figure 6.

Molecular Characterization of MOF. (A) Fine mapping of MOF on chromosome 4. Names and positions of the markers are noted. cM, centimorgans. (B) A schematic representation of three exons and two introns of Os04g39080. The +1 indicates the putative starting nucleotide of translation, and the stop codon (TGA) is +1813. Blue boxes indicate exons, and intervening lines indicate introns. Numbers indicate the exon length (bp). The deletion site in mof is shown (arrow). (C) Phylogenetic analysis of MOF and its related homologs. A maximum likelihood analysis was performed using MEGA 4.0 using MOF-related sequences from Amborella trichopoda (Amt), Arabidopsis thaliana (At), Brachypodium distachyon (Bd), Gossypium raimondii (Gr), Linum usitatissimum (Lu), Medicago truncatula (Mt), Oryza sativa (Os), Sorghum bicolor (Sb), and Zea mays (Zm). The green shaded box indicates the dicotyledon cluster, and the turquoise shaded box indicates the monocotyledon cluster.

Figure 7.

Physical Interaction between MOF and OSK1. (A) Yeast two-hybrid assay for interaction of MOF with OSK1. A schematic diagram of MOF and the truncations used is shown. The interactions were verified by the growth of yeast strains on the -Leu-Trp-His-Ade+X-Gal selection medium. (B) BiFC assay for interaction between the MOF and OSK1 in rice protoplasts. (C) Coimmunoprecipitation of MOF-HA and OSK1-FLAG based on anti-FLAG immunoprecipitation from transfected Nicotiana benthamiana leaves.

Figure 8.

Expression Pattern of MOF. (A) Spatial and temporal expression analysis of MOF by RT-qPCR. RNAs were extracted from the root, stem, leaf, lemma/palea, and anthers of <stage 6, stage 7, stage 8a, stage 8b, stage 9, stage 10 (early), stage 10 (late), stage 11, stage 12, and stage 13/14. L/P, lemma/palea. Each reaction had three biological repeats. Error bars indicate sd. Actin served as a control. (B) to (I) In situ analyses of MOF in wild-type anthers. Anthers at stage 7, stage 8a, stage 8b, and stage 9 with MOF antisense probe ([B] to [E]) and sense probe ([F] to [I]) showing MOF expression in tapetal cells and meiocytes. MC, meiocyte cell; Ms, microspores; T, tapetal layer. Bars = 50 μm.

Figure 9.

Dual Immunolocalization of REC8 and MOF in Meiocyte Cells of the Complemented Transgenic Line. (A) Leptotene; (B) zygotene; (C) early pachytene; and (D) late pachytene. REC8, green; MOF, magenta. Bars = 5 μm.

Figure 10.

Proposed Working Model for MOF in the Regulation of Meiotic Progression. At early prophase I, MOF interacts with OSK1 to form an SCF complex E3 ligase complex that works together to promote early meiotic events, especially DSB processing and repair. Loss of function of MOF results in failure of homologous chromosome synapsis and recombination, which triggers meiotic arrest at late prophase I, and a prolonged arrest induces apoptosis and meiocyte elimination.