Functional And Applied Characterization Of OsATM On The Double-Strand Breaks Repair In Rice Meiosis

In sexual reproductive organism,the recombination of genetic material mainly depends on the random combination of non-homologous chromosomes from both parents and the exchange of non-sister chromatids between homologous chromosomes during meiosis.It is important to reveal the molecular mechanism of meiosis for fully understanding of the role of genetic recombination mechanism.In meiosis,homologous recombination is a very complex and critical biological process.It begins with the double-strand breaks(DSB)mediated by SPO11.Homologous recombination in DSB repair not only ensures the accurate segregation of chromosomes in meiosis,but also increase the genetic diversity of hybrid progenies.In human beings,ATM is the earliest identified protein involved in the repair of DNA damage in somatic cells.However,in mammals,due to the stagnation and apoptosis of pollen mother cells caused during meiosis by ATM gene mutation,its biological function is still unclear in the process of meiosis.To understand the function of OsATM in rice meiosis,Osatm allelic mutants,and two double mutants,Osatm Osspoll-1,Osatm Osdmcl were developed and well ivvestigated.The main results are as follow:1.Osatm-1 PMCs could normally complete homologous chromosome pairing and synapsis,and chromosome behaviors showed no obvious differences up to pachytene between wild type and Osatm-1.However,chromosomes entangled to form multivalent at diakinesis.These aberrant chromosome associations became more evident at metaphase I.At anaphase I,extensive chromosome bridges and fragmentations were observed.Chromosome bridges and fragmentations were also detected at anaphase Ⅱ and tetrads,and eventually formed micronucleus in the separated microspores.The chromosome behaviors of Osatm-2 and Osatm-3 were similar to that of Osatm-1,with aberrant chromosome associations and fragmentations.2.To determine the causes of chromosomal abnormalities in Osatm during meiotic DSB repair,we constructed the OsATM and OsSPO11-1 double mutants and performed genetic analysis.OsSPO11-1 mediates the meiotic DSBs formation.Due to the absence of meiotic DSBs,homologous chromosomes in Osspoll-1 PMCs failed to be paired at pachytene.The Osspo11-1 Osatm-1 double mutant exhibited the same chromosome phenotype as Osspo11-1 and no aberrant chromosome associations and fragmentations were observed,suggesting that OsATM functions downstream of OsSPO11-1-catalyzed DSB formation.3.In rice meiosis,OsDMC1 plays an important role in HR-mediated DSB repair,and the distruption of OsDMCl resulted in a typical synapsis defect at pachytene.When combining the mutation of OsDMCl and OsATM,we observed more severe associations and fragmentations in the Osdmcl Osatm-1 double mutant than either single mutant.At pachytene,Osdmcl Osatm-1 displayed destroyed synapsis,the same phenotype as Osdmcl.However,more severe chromosome entangles and fragmentations than that of Osatm-1 were observed at both metaphase Ⅰ and anaphase Ⅰ in Osdmcl Osatm-1,indicating that OsDMC1 may still have function in the Osatm-1 mutant.These suggested that OsDMC1 and OsATM play roles in two independent pathways.4.The immunofluorescence staining results of y-H2AX showed that extensive y-H2AX foci could be observed in Osatm-1 PMCs at zygotene,which was equivalent to that of the wild type.This result indicated that OsATM is not required for phosphorylation of H2AX during rice meiosis,which is different from the situation in mitosis of somatic cells.5.HEI10 is the member of ZMM protein family and can label the interference-sensitive CO(Crossover).The removal of OsDMCl from chromosomes in Osatm PMCs was delayed and the number of HEI10 foci decreased.Therefore,the loss of OsATM may partially reduce the number of interference-sensitive COs.Together,OsATM plays an essential role in the repair of meiotic DSBs in rice.These results are important for understanding of the molecular mechanism of meiosis control in rice.