| DNA methylation is an important epigenetic mark that regulates the expression of genes and transposable elements.RNA-directed DNA methylation（RdDM）is the main biochemical machinery for de novo DNA methylation in plants.The molecular mechanism of RdDM has been well illustrated in Arabidopsis,but largely unknown in other plant species.In this study,we used ethyl methyl sulfonate（EMS）to treat the seeds of transgenic silenced 35S::OsGA2ox1,and isolated a suppressor for gene silencing,five elements mountain 1（fem1）.Map-based cloning demonstrated that FEM1 encodes RNA-dependent RNA polymerase 2（OsRDR2）.The roles of OsRDR2-dependent methylation and 24-nt siRNAs on development of reproductive organs were revealed through technology related to molecular genetics,cell biology and bioinformatics.The main results of this study as follow:1.Mechanism of OsRDR2 regulating reproductive development in riceIn this study,the 35S promoter was used to drive the overexpression of OsGA2ox1 to generate the transgenic rice plants,OsGA2ox1 ectopic expression（GAE）lines exhibited dwarfism and dark green leaves.The offspring of GAE included partially recovered plants with semi-dwarfism and completely recovered plants with WT-like stature（OsGA2ox1silencing,GAS）.Whole genome-wide bisulfite sequencing and high-throughput small RNA sequencing revealed that non-canonical RdDM mediated gene silencing occurs in semi-dwarf GAE,and canonical RdDM mediated gene silencing occurs in GAS.In order to screen key factors for transgene silencing in rice,we performed EMS mutagenesis for GAS seeds and screened five elements mountain 1（fem1）which recovered the overexpression of35S::OsGA2ox1 transgene in the M2generation.Then we found CHH（H=A,T,or C）methylation levels of fem1 were decreased on the 35S promoter.Map-based cloning and genetic complementary demonstrated that FEM1 encodes RNA-dependent RNA polymerase 2（OsRDR2）.The fem1 mutants showed a severe gibberellin absent phenotype in the OsGA2ox1-containing transgenic background.In order to observe the real phenotype of fem1 mutants,we isolated homozygous fem1 without the transgene of 35S::OsGA2ox1by crossing heterozygous fem1/FEM1 with the wild-type（WT）.Compared with the WT,the fem1 mutants without the transgene of 35S::OsGA2ox1 exhibited shorter stature,smaller panicle and reducing seed setting rate.In addition,we obtained several osrdr2 allelic mutants by using CRISPR/Cas9（Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9）technology to knockout OsRDR2 in Nipponbare.Compared with the WT,the osrdr2 mutants had shorter stature,smaller panicles and exhibited almost completely sterility,suggesting that OsRDR2 plays a crucial role in reproductive development in rice.We performed whole genome-wide bisulfite sequencing and analysis for panicles and seedlings of WT and osrdr2,and found that the CHH methylation levels were substantially reduced in panicles and seedlings of osrdr2 than the counterparts of WT,suggesting that OsRDR2 is required for the maintenance of CHH methylation in rice.The s RNA-seq analysis revealed that the 24-nt s RNA biosynthesis is dependent on OsRDR2 in both panicles and seedlings.Further comparison found that due to the genome-wide CHH methylation level in panicles is significantly higher than that in WT seedlings,the number of CHH hypo-DMRs in osrdr2 panicles was larger than that in osrdr2 seedlings.CHH hyper-DMRs between panicles and seedlings were mostly overlapped with CHH hypo-DMRs between osrdr2 panicles and WT panicles,suggesting that the increase of CHH methylation level in WT panicles than in WT seedlings is dependent on OsRDR2.Genome-wide CHH methylation levels were increased in stamens and pistils than in seedlings and dependent on OsRDR2 as well.These results suggested that the obvious increase of genome-wide CHH methylation levels in reproductive organs than in seedlings is dependent on OsRDR2.The most striking phenotype of osrdr2 mutants is abnormalities in the reproductive organs,manifested in partial anther lobes exhibited abnormal differentiation;pollen viability was significantly decreased;pollen walls were wrinkled;some microspores undergone abnormal meiosis.Consistent with these phenotypic defects,transcription levels of several key genes involved in anther differentiation,meiosis of pollen mother cell,and development of pollen wall were downregulated.In addition,CHH methylation levels of these genes were significantly decreased in osrdr2 mutants,suggesting that DNA methylation in male organs is necessary to ensure the high expression of genes involved in reproductive development.Most of the genes encoding the components of RdDM were upregulated in panicles and pistils than in seedlings.The protein levels of OsRDR2 were significantly increased in reproductive organs such as panicles,anthers and pistils than in vegetative organs such as seedlings,leaves and sheaths.The upregulated RdDM encoding genes mostly were associated with OsRDR2-dependent DMRs in their promoters.Mutation in OsRDR2decreased CHH methylation levels in their promoters and thus changed their transcriptional levels.These results indicated that RdDM-mediated DNA methylation is partly responsible for enhancing the high expression of RdDM pathway genes.2.The dynamics and features of bill-siRNAs in different reproductive organs of riceAlthough genome-wide CHH methylation levels in rice panicle was significantly higher than in seedling,the 24-nt siRNAs abundance was significantly lower in panicle than in seedlings on the hyper-DMR between panicle and seedling.The inconsistency between CHH methylation levels and 24-nt siRNA abundance in panicles and seedlings prompted us to analyze s RNAs clusters.Among the identified 20,200 24-nt siRNA clusters in panicles,the top 0.81%highly expressed 163 clusters accounted for more than 68%of the total 24-nt siRNA reads.These highly expressed siRNAs were named as billionaire siRNAs（bill-siRNAs）,the lower expressed siRNAs as pauper siRNA（pau-siRNA）.We further analysis the s RNA-seq data of pistil,stamen,bract and branch in panicle,and found the bill-siRNAs in panicle were mainly from pistil（158 bill-siRNA clusters）.Both the egg cell and the ovary minus egg cell produced similar levels of bill-siRNAs as pistil.However,male organs generated fewer bill-siRNAs（65 clusters）than female organs.In embryos which developed from fertilized egg cell,seedlings and endosperm which developed from fertilized central cell,the gamete bill-siRNAs was disappeared.While highly expressed siRNAs were produced in different specific genomic regions in endosperm.Distincted with embryo and endosperm,the pistil bill-siRNAs were maintained in seed coat.In panicle,pistil,stamen,endosperm and seed coat,the length of bill-siRNA clusters were significantly longer than that of pau-siRNAs clusters.Similar to the pau-siRNAs,the first base of bill-siRNAs enriched at adenine,suggesting that bill-siRNAs are likely loaded into OsAGO4 to direct DNA methylation.These bill-siRNA clusters were enriched for more H3K9me2 modification than pau-siRNA clusters,suggesting that they might locate in heterochromatic regions.Moreover,these bill-siRNAs were highly conserved among TP309 and Nipponbare.In summary,mutation in FEM1/OsRDR2 abolished the accumulation of 24-nt siRNAs,consequently reduced CHH methylation levels.The genome-wide CHH methylation levels in reproductive organs were increased than in vegetative organ,which is resulted from higher RdDM activity in reproductive organs.The hypo-DMRs produced in the promoter regions of reproductive developmental genes caused disturbance of gene expression in osrdr2 mutants,thus led to osrdr2 abortion.There were more than 100 highly expressed bill-siRNA clusters,whose biosynthesis is dependent on FEM1/OsRDR2 in the panicle,pistil,and egg cell.The bill-siRNAs in pistil were maintained highly expressed in seed coat,but depleted in embryo and endosperm.In endosperm,bill-siRNAs were produced in different genomic regions. |
