Molecular Mechanism Of OsEDM2L Regulating Male Development In Rice(Oryza Sativa L.)

Plant male fertility and its molecular mechanism are critical theory basis of yield and heterosis utilization.Male sterility is controlled by cytoplasmic male sterility(CMS)and genic male sterility(GMS).GMS generated from mutated nuclear genes generally causes partial or complete abortion of pollen.Study of these male sterile lines and the molecular mechanisms of related genes will deepen our understanding on plant male development and provide the theory basis for hybrid breeding.This study identified a male sterility mutant and its regulated gene from a mutant library generated by CRISPR/Cas9-based knock out of the anther-specific genes in a japonica rice variety ZH11.We studied the mutant phenotype and molecular mechanism of targeted gene controlling male fertility by genetic,cytological and molecular biological methods.The main results are showed as follows:1.Genetic analysis verified that the target gene for this male sterile mutant was derived from the mutation of LOC?Os08g39250(OsEDM2L).The mutant osedm2l exhibited normal vegetative growth and female development,but abnormal anthers harboring mostly abortive pollen grains.The mutant producted about 26%seed setting rate by self-pollination.2.The tapetum in the mutant showed abnormal development,leading to aberrant tetrad in late meiosis,and microspores degraded at the early uninucleate microspore stage and middle uninucleate microspore stage.At the mature pollen stage,only few fertile pollen grains existed in the anther locules.The TUNEL assay exhibited delayed programmed cell degradation(PCD)of the tapetum in this mutant to the late uninucleate microspore stage in comparing with the wild-type(WT).3.OsEDM2L showed transcriptional expression in pistil and anthers before the bicellar pollen stage,but not in vegetative organs.GUS staining assay showed the GUS reporter gene mainly expressed in anthers before the early uninucleate microspore stage.4.OsEDM2L encodes a nucleus-localized protein of 694 amino acids,which contains a N~6-adenine methyltransferase-like domain.The prediction of three-dimensional structure indicates that OsEDM2L may have potential methyltransferase activity,which mediates m~6A epigenetic modification.Phylogenetic analysis showed that there are totally seven members of EDM2-like genes in rice.Knocking-out of the other six genes by CRISPR/Cas9 indicated that they are not related to male reproduction.5.Seven interactors were obtained by screening anthers'c DNA library.Of these interactor genes,MADS16 is known for floral organ regulation.Knockout of these genes by CRISPR/Cpf1 showed that ZF970 and Dp760 might be involved in male fertility due to reduced pollen fertility in the mutant lines,and the other interacted genes are not related with male fertility.The interactional analysis among these interactors and OsEDM2L suggested that these proteins may form a functional complex.6.RNA-seq showed that many genes were significantly changed between WT and mutant.These differential genes are distributed to many different pathways such as energy conversion,biosynthesis,phytohormone and signal transduction.In these pathways,RNA metabolism pathways may be directly correlated with the function of OsEDM2L.7.The q RT-PCR analysis of 15 anther functional maker genes in 7 stages of WT and osedm2l mutant showed delaying expression of most of these genes in later stages of anthers,which might be due to the delayed tapetum degradation.DTD(EAT1)and its downstream genes Os C4,Os C6,Os AP25 and Os AP37 exhibited the highest delayed expression,while RTS was obviously down-regulated.The genes b HLH142,TDR and Os CP1 were up-regulated in all stages of anthers.Os GAMYB,UDT1 and API5 were no significantly changed in earlier stages of anthers.8.OsEDM2L can interact with b HLH142 and TDR to regulate the expression of DTD and Os CP1,DTD further interact with OsEDM2L to regulate Os C4,Os C6,Os AP25 and Os AP37.ZF970 and Dp760 interacts with TDR,Dp760 also interacts with b HLH142.9.The transcriptome m~6A quantification analysis showed that the m~6A level in the mutant anthers at meiosis stage was significantly lower than that of WT.It is known that m~6A modification generally influences transcript alternative poly(A)and alternative splicing.Further analysis indicated that alternative poly(A)and alternative splicing of DTD/EAT1 and Os CP1 were obviously different between WT and the mutant.These results suggested that OsEDM2L encodes a N~6-adenine methyltransferase-likeprotein OsEDM2L.OsEDM2L,ZF970,Dp760,b HLH142 and TDR compose a five-factor complex to regulate the expression of downstream genes,further modulates the development of tapetum and pollen fertility.Otherwise,OsEDM2L involves in mediating m~6A modification,and regulates alternative poly(A)and alternative splicing of DTD and Os CP1transcripts.In osedm2l mutant,obviously delayed tapetal degredation results in aberrant microspores that do not develop into mature pollen grains.