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Literature Sharing | MORE FLORET1 Interacts with C-type Replication Protein A Complex and Regulates Male Meiosis in Rice
Release time:
2025-06-12
This study investigates the role of MOF1 in male meiosis in rice. Two MOF1 mutant lines in the Nipponbare background showed reduced seed setting and pollen fertility due to impaired male meiotic bivalent formation. RNA-seq and RT-qPCR revealed that MOF1 does not affect the expression of known meiotic genes, suggesting it may not act as a transcription factor in meiosis. Protein interaction assays (Y2H and BiFC) demonstrated that MOF1 interacts with RPA1c, RPA2c, and FAR1, with RPA1c and RPA2c known to be involved in bivalent formation. Co-expression and nuclear localization of these proteins during anther development support a regulatory role for MOF1 in meiosis through protein-protein interactions. These findings offer new insights into the molecular mechanisms underlying male reproductive development in plants.
Homologs of MOF1 (LOC_Os04g47890) are present in various plant species, including Arabidopsis, maize, wheat, barley, and soybean. LOC_Os02g45080 is identified as a paralog of MOF1 in rice. However, the functions of these homologs and paralogs remain largely unknown. Haplotype analysis using the RFGB database revealed 8 coding sequence (CDS) haplotypes (Hap1–Hap8) of MOF1 across 2,975 cultivated rice germplasms, based on 4 SNPs. Hap1 predominates in Japonica varieties (98.94%), while Hap2 and Hap3 are more common in Indica varieties, appearing in 46.45% and 38.61% of accessions, respectively.
Fig. 1 Homologous genes and haplotype analysis of MOF1.
To further investigate the role of MOF1 in reproduction, two additional homozygous CRISPR/Cas9-induced mutant lines, Nip-mof-3 and Nip-mof-4, were generated in the Nipponbare background. Nip-mof-3 had “A” deletions, and Nip-mof-4 had “A” insertions at both target sites, resulting in frameshift mutations and premature stop codons in the MOF1 protein. Despite normal vegetative growth, both mutants showed dramatically reduced seed setting rates (3.81% and 5.21%) and pollen fertility (61.82% and 46.30%) compared to the wild type. Additionally, the mutants exhibited significantly reduced plant height and grain number, while traits such as flag leaf size and panicle length remained unaffected.
Fig. 2 Phenotypical analyses of Nip-mof1
Cytological analysis of pollen mother cells (PMCs) was conducted to assess the role of MOF1 in male meiosis. In wild-type plants, 98.66% of PMCs showed normal bivalent formation (12 bivalents) and proper chromosome alignment. In contrast, 50% of PMCs in the Nip-mof1-3 mutant displayed defective bivalent formation, with varying numbers of univalents. Additionally, 52.32% of mutant PMCs showed abnormal chromosome segregation at metaphase I. These results demonstrate that MOF1 is crucial for proper bivalent formation and chromosome behavior during male meiosis in rice.
Fig. 3 Meiosis process observation of Nip-mof1-3.
To explore the regulatory role of MOF1 in meiosis, expression analysis of 59 known meiotic genes revealed no significant changes in mof1 mutants, suggesting MOF1 likely does not act as a transcription factor. Yeast two-hybrid (Y2H) assays identified physical interactions between MOF1 and meiotic proteins RPA1c and RPA2c, both essential for crossover formation. These interactions were further confirmed by bimolecular fluorescence complementation (BiFC) in Nicotiana benthamiana, showing nuclear localization. Domain mapping through truncation experiments revealed that both N- and C-terminal regions of MOF1 interact with RPA2c. Additionally, FAR1, a transcription factor with DNA-binding and transposase domains, was identified as another MOF1 interactor and also interacted with RPA1c and RPA2c in Y2H and BiFC assays. These findings suggest that MOF1 may regulate male meiosis through a protein interaction network involving RPA1c, RPA2c, and FAR1 in rice.
Fig. 4 Interaction of among MOF1, RPA1c and RPA2c
Previous studies reported colocalization of RPA1c and RPA2c in meiotic PMCs, and high expression of MOF1 during meiosis. RNA-seq data confirmed co-expression of MOF1, RPA1c, RPA2c, and FAR1 in anthers. RT-qPCR showed FAR1 is highly expressed from stages S6 to S11 of anther development. Subcellular localization assays in tobacco cells revealed that MOF1-GFP and FAR1-GFP both localize to the nucleus. RT-qPCR of meiotic anthers from Nip and mof1 mutants showed no significant changes in RPA1c, RPA2c, or FAR1 expression, suggesting MOF1 does not regulate their transcription.
Fig. 5 Expression analyses among MOF1-interacted genes.
This study confirms that MOF1 mutation leads to pollen sterility and abnormal meiosis in diploid rice. MOF1 interacts with meiotic regulators RPA1c and RPA2c, and forms a protein complex with FAR1. These genes are co-expressed during anther development. The findings suggest that MOF1 functions as a protein interactor, not a transcriptional regulator, to modulate male meiosis and pollen development in rice, providing new insights into the molecular network underlying rice fertility.
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