Characteristics And Regularities Of Methylation Pattern Changes Induced By Space HZE Ions Of Different LET In Rice At Different Developmental Stages

Space is a complex environment in which biological samples are exposed to radiation from galactic cosmic rays,solar particle event and trapped belt radiation during spaceflight.Space radiation is a low dose,but high energy composite environment.Among these radiations,high charged and energy(HZE)particles from galactic cosmic rays can cause more damages in organisms because of their high LET,therefore,it has become one of the key issues in the study of space radiation biology.High LET values usually refer to heavy ions with LET which is greater than 10KeV/?m.Ground studies have shown that in the range of 10 to 100 KeV/?m,the relative biological effects increased with increasing LET value,but decreased when LET values exceed 100KeV/?m.This pattern was also found in the genomic expression analysis in rice obtained from space with different LET' values.Since DNA methylation can control gene expression,whether DNA methylation involved in the regulation of functional gene expression:induced by HZE of different LET values in the spatial environment required further analysis.CR-39 heavy ion detection membrane can detect heavy ions with a LET value greater than 5 KeV/?m.According to the CR-39 particle track analysis,it can be determined whether the HZE particle hits the embryo of the rice seed,and obtain the LET value of the HZE that hit the seed.Through CR-39,7 rice seeds which were hit by HZE particle with different LET values(60,100,150,200 KeV/?m)carried by the SJ-10 satellite and 1 rice seed which was not hit by HZE particle(RN)were selected for the analysis.After returning to earth,these seeds were kept in cold storage for 606 days,after that,these seeds were planted in an artificial climate chamber with ground:control group(CK),when growing to tillering and heading stages,rice leaves were harvested and analysed by whole genome bisulfite and RNA sequencing to get the following results:First,the landscape of methylation cytosine ratios in the whole genome of rice at tillering and heading stage were analyzed.It was found that from the tillering stage to the heading stage,the proportion of methylated cytosine at the CG and CHG sites in the RN and R60 treatment groups increased,while in the 100,150,and 200KeV/?m treatment groups,CG and CHG sites' methylation ratio remained low state.However,the CHH sites were different.Two 100KeV/?n treatment plants's methylation.ratio at CHH sites remained low and the other treatment groups have changed from low level to high from tillering to heading stage.Correlation analysis of the corresponding methyltransferases showed that CHH sites in different LET treatment groups at the tillering stage were significantly positively correlated with methyltransferase AGO1a(P<0.05).Although the proportion of methylated cytosine in other treatment groups was related to methyltransferase,it was not significant(P>.0.05).The number of differentially methylated regions(DMRs)in treatment groups with different LET values varies from 25,000 to 30,000 per plant.The proporti ons of DMR in the promoter,gene coding region and repetitive sequence region were 23%-26%,19%-27%and 50%-56%,respectively.The number of DMR and methylation patterns at CG,CHG and CHH sites in the promoter and gene coding region were analyzed,and it was found that except R100T2,the number of hypo DMR:at CG and CHG sites increased from tillering stage to heading stage,indicating an enhanced demethylation,At CHH sites,except R100-2 and R100-3,hypo DMR of other treatment groups decreased from tillering stage to heading stage,indicating that the CHH sites' methylation increased.Analysis of its correlation with the corresponding methyltransferase showed that in the promoter region,the correlation coefficient between the methylation level of the CG site and AGOla was 0.85(P<0.01)at tillering stage,and the correlation coefficients of methylation level at CG and CHG sites with AGO1a were 0.86 and 0.76,respectively(P<0.01,P<0.05).at heading stage.In the.gene coding region,the correlation coefficients between the methylation of CG and CHG sites and AGO1a reached 0.9(P<0.01)and 0.66,respectively at heading stage,indicating that the methylation of CG and CHG sites in the promoter and gene coding region was regulated by methyltransferase AGO1a.GO enrichment analysis showed that at tillering and heading stages,DMR related genes were enriched in protein phosphorylation,nucleic acid metabolism and polysaccharide binding function.And as the LET value increased,the number of significantly enriched GO terms also increased.Through KEGG pathway enrichment analysis,regardless of the high or low LET value,space radiation had a significant effect on the methylation patterns of genes related to diterpenoid and steroid biosynthesis which were related to plant growth and stress response.The methylation patterns and expression levels of some related genes in these two pathways were consistent,accounting for 63%and 83%,respectively,and it was found that the same methylation pattern changes occurred in different treatment groups,indicating space hotspots.This research showed that the space-induced methylation pattern changes of rice genome were mainly achieved by expression changes of methyltransferase AGO1,and caused 20,000 to 30,000 DMRs.Methylation patterns of promoter and genebody region have different CG,CHG and CHH specificities at the tillering and heading stages,and the overall methylation and demethylation levels were a dynamic regulatory process.In particular,the methylation modification at CHH sites in the promoter and gene coding region may be related to the different LET value.This study provided an important basis for the analysis of epigenetic mechanism and biological effects of HZE ions in the space environment.