The Arabidopsis MTOPVIB Interacts With AtPRD1 And Is Involved In Meiotic DNA Double-strand Break Formation

In sexual reproduction of flowering plants,the meiocytes(2n)in ovules and anthers undergo meiosis to generate megaspores(n)and microspores(n),respectively.The megaspore(one out of four in each ovule)and microspores in anther further develop into female gametophyte(embryo sac)and male gametophytes(pollen grains),respectively.Meiosis is a key step of female and male gametophyte formation,which undergoes several important biological events,such as DNA replication,sister chromatids synechia,homologous chromosome pairing,recombination and separation.Disruption of meiosis at any point of the process may dramatically affect formation of female and male gametophytes,leading to sterility of plant.The homologous chromosome recombination is an important way for exchange of genetic informations.It is initiated by formation of DNA double-strand break(DSB).AtSPO11-1 and AtSPO11-2 are homologous to SPO11 and involved in DSB formation.AtPRDl and AtPRD2 are homologous to mice MEI1 and MEI4,respectively,also invovled in DSB formation.AtPRD3 and AtDFO are plant-specific DSB formaiotn proteins.Although these Arabidopsis proteins have been identified as being involved in DSB,very little has been kown about the genetic rmechanisms of DSB formation.Therefore,studying formation of female and male gametophytes as well as the homologous chromosome recombination not only enable us to understand molecular genetic mechanisms of sexual plant reproduction,but also may provide genetic theoretical references for crop breeding.This thesis was to study molecular genetic mechanism of female and male gametophyte formation by identification and characterization of reproductive development-defective mutant mt187 and the corresponding genes.mt187 was identified in screening for female and male gametophyte-defective mutants from the gene-trap and enhancer-trap Ds-insertion lines.It exhibited a phenotype of shorter silliques and reduction in fertility.Genetic analysis showed that the mutant phenotype was not linked to the insertion of the Ds element.Mapping cloning idendified the corresponding mutation point was the last base pair(G1890)of the eighth exon in Atlg60460 gene.The mutation of G1890A caused alternated splicing of the related introns and disrupted the function of the gene,resulting in disruption of female and male gametophyte formation and leading to significant reduction in fertility of the plants.Meanwhile,another T-DNA insertion mutant allele was obtained from ABRC,which exhibited a similar phenotype.These results indicate Atlg60460 plays an important role in female and male gametophyte formation.Atlg60460 encodes a protein that was recently renamed as MTOPVIB.Based on the fact that two mutant alleles for the gene had been identified previously,the mt187 was then renamed as mtopVIB-3.The T-DNA insertion allele mentioned above was identical to the mtopVIB-2 in science paper.Compared with mtopVIB-3,the mtopVIB-2 had a heavier phenotype with a lower seedset rate of 2%.The mtopVIB mutants were defective not only in male gametophyte development,but also in female gametophyte development.In particular,defects in the male tetrads resulted in abortion of pollen grains,while formation of female gametophyte was disrupted at the megasporocytic stage(FG1).These results indicated that the mutations might affect meiosis.Cell biological observation showed that the mtopVIB mutants had irregular tetrads and abnormal pollen grains.Cytological observation showed that the chromosomes in the mutants were not as thick as that in wild-type at the pachytene stage.Furthermore,more than five univalents appeared at the diakinesis stage in the mutants,unlike the typical five bivalents in wild-type.The mtopVIB mutants also exhibited abnormal centromere pairing and synapsis.The frequency of homologous recombination was obviously reduced compared to that in wild-type.The double mutants,which were generated by crosses of the mtopVIB-2 with the different DSB repair-defective muants(Atcoml-1,Atrad50-1,Atrad51-1 and Atmre11-1),all had only 10 univalents and did not have bivalents at metaphase I,like that in the mtopVIB-2,indicating that the mtopVIB-2 was defective in DSB formation.These results suggested that MTOPVIB palyed important roles in DSB formation.Real-time PCR assays showed that MTOPVIB is constitutively expressed in many tissues,with a higher expression level in inflorescences.The MTOPVIB belongs to a kind of highly-conservative proteins,and structurally similar to the B subunit of the Topo VI complex.It has an ATP-binding Bergerat domain at its N-terminal and a transducer domain which can induce conformational change at C-terminal.Y2H and BiFC assays showed that MTOPVIB could interact with AtSPO11-1 and AtSP011-2,respectively.Furthermore,Y3H and BiFC assays showed that MTOPVIB could interact with AtSPO11-1 and AtSPO11-2 at the same time,implying that the three proteins could form a Topo ?-like complex.Moreover,Y2H and BiFC assay showed that AtPRDl could interact with MTOPVIB and AtSPO11-2,respectively,indicating that AtPRD1 could interact with the components of the Topo?-like complex.The assay using the same method showed that AtPRD1 could interact with AtPRD3 and AtDFO,respectively.In addition,Y3H and BiFC assays showed that AtPRDl could interact with AtPRD3 and AtDFO at the same time,indicating that AtPRDl may be an important protein for DSB formation.In summary,in Arabidopsis,the MTOPVIB protein interacts with AtPRD1 and is involved in meiotic DNA double-strand break formation,by coordinately working with other proteins,such as AtSPO11-1,AtSPO11-2,AtPRD3,AtDFO.The results provide a new experimental evidence for the knowledge about the the mechanism of meiotic DSB formation in Arabidopsis.