The Dynamics Of DNA Methylation And The Biogenesis Of Circular RNA In Maize Roots Under Drought Stress

Maize(Zea mays L.)is not only highest-yielding crop in the world but also widely used as industrial raw-material and biofuel,the production of maize play an important role in food security and economic development.However,various abiotic stresses damaged corn yield and threaten global food security seriously,especially under drought stress.Plant resistance to drought is a very complex trait.In the past few decades,studies of molecular mechanism of plant drought-tolerance benefited from the development of molecular biology,several key genes and regulatory modules have been identified in maize.Epigenetic changes,such as DNA methylation and histone modification,play an important role in stress-induced gene expression and transcriptional regulation under drought stress.Analysis of epigenetic modifications can provide new insights into improving regulatory network and understanding plant drought tolerance.To study response and function of DNA methylation under drought stress in maize roots,the study selected extreme drought-tolerance and drought-sensitive RIL offsprings in recombinant inbred lines derived from maize droughttolerant inbred line AC7643 and drought-sensitive inbred line AC7729/TZSRW.The profile of whole-genome DNA methylation was scanned on the maize root under Well Water(WW)and Water Stress(WS)by Me DIP(Methylated DNA Immunoprecipitation)sequencing.We explored the response pattern of DNA methylation to drought stress and the roles of DNA methylation in the epigenetic regulation of genes expression.Evidence had shown that DNA methylation has a significant impact on exon splicing.As a special type of splicing product,the relationship between circular RNA(circ RNA)in maize root and drought stress is unclear.circ RNA sequencing of maize root under WW and WS was carried out to identify the expression and response of circ RNA.On the other hand,the multi-omics analysis of the whole transcriptome and histone epigenetic modification was used to explore the biogenesis and possible roles of stress-induced circ RNA under drought stress.The main findings are as follows:1.The distribution pattern of DNA methylation in different genomic elements was notably inconsistent.DNA methylation modification levels in transposons was significantly higher than that of genes,while various classes of transposon exhibited distinct characteristics of DNA methylation distribution.Except for promoter,exon also enriched DNA methylation.Analysis of the conservation of DNA methylation found about 80% of DNA methylation were shared among under different water conditions or different droughttolerance inbred lines.The proportion of DNA methylation shared between the parental inbred lines and RIL offsprings was significantly lower,but overall DNA methylation was conserved.Further analysis revealed that the consistent DNA methylation modification between parental lines and RILs was increased significantly under WS.Besides,the proportion of heritable DNA methylation in recombination hotspots and copia transposon was significantly lower than that of random DNA segments or other transposons.2.Under drought stress,The number of genomic regions where DNA methylation level increased(hyper-DMRs)under WS was significantly higher than that of regions with decreased DNA methylation(hypo-DMRs).DNA methylation of drought-sensitive materials was more unstable under WS,the number and proportion of hyper-DMR was both higher than that of drought-tolerant materials.In addition,there were significantly different among gene with DNA methylation change in different drought-tolerance inbred lines: production of sm RNA involved in gene silencing and RNA interference only enriched in genes with increased DNA methylation modification level in drought-tolerant lines,while methyltransferase activity only enriched in genes which DNA methylation modification level was increased in drought-sensitive inbred lines.Furthermore,differentially expressed genes in drought-sensitive inbred lines were significantly enriched DNA assembly and protein-DNA complex,where genes involved in si RNA production were up-regulated in drought-tolerant materials.This consistency implied that distinct differentially methylated genes in different inbred lines may be involved in the regulation of drought-tolerance in different inbred lines through adjusting the expression of stress-responsive genes.3.DNA methylation modification in gene body was significantly negatively correlated with gene expression level(r =-0.31),while DNA methylation level of exon was significantly positively correlated with exon expression(r = 0.18).Also,DNA methylation was closely related to gene alternative splicing(AS).Genes with more AS events had higher DNA methylation modification level,and retained intron modified by DNA methylation had higher percent spliced-In.However,DNA methylation variation and gene expression and splicing changes under WS weren’t always consistent.Among genes with significantly increased DNA methylation levels in the promoter of genes under WS,the proportion of upregulated genes was significantly higher than that of down-regulated genes.However,whether DNA methylation increased or decreased in gene body,there was significantly more down-regulated genes.Besides,various classes of transposons had distinct fundamental roles in the regulation of nearby gene expression.Lower DNA methylated proportion of TIR transposons in expressed genes was observed,while other transposons exhibited the opposite trend.The response of si RNA in maize roots under drought stress was similar to that of DNA methylation.There was a significant positive correlation between si RNA abundance and DNA methylation modification level of gene body(r = 0.38).This correlation was the highest in the gene body and gradually weakened toward the flanking region of genes.Among various classes of transposons,the si RNA expression level of copia was highest positively correlated with DNA methylation level of copia,while si RNA abundance of gypsy shown weakest correlated with DNA methylation.4.The analysis of circ RNA-seq data found that circ RNA had shown stronger expression-specificity than linear gene,and circ RNA-producing genes expressed constitutively.The number and expression level of circ RNA increased under WS.The number of circ RNAs expressed in the inbred lines categorized in the drought-sensitive group was significantly higher than that in the drought-tolerant group,which is quite different from the results obtained for linear transcripts.5.The factors that affect circ RNA expression under WS mainly included the characteristics of flanking sequences,epigenetic modification and the abundance of splicingrelated protein.Longer flanking introns and enrichments of LINE and Copia contributed to the enhancement of circ RNA expression.Epigenetic modifications were involved in the synthesis of circ RNA: The epigenetic marks placed on circ RNA-producing genes were in less active states,with totally different signals at the back-splicing junctions although circ RNAs spliced in a similar pattern to linear transcripts.Among all tested epigenetic marks involved in the regulation of circ RNA expression,H3K9 AC had the greatest impact.In addition,the expression variation of RNA-binding proteins and ABA pathway genes under WS also contributed to the increase of circ RNA expression.6.Most circ RNA only harboured fewer mi RNA binding sites,which are not enough to serve as mi RNA "sponges".However,the abundance of si RNA on circ RNA were significantly higher,the enrichment of si RNA may be originated from sequences with reverse complementary to linear transcripts and cleavage by mi RNAs.The screening of predicted IRES sites and coding potential of circ RNA in our research revealed a higher proportion(59.23%)of circ RNAs with protein-coding potential than expected,providing further evidence for the assumption that more circ RNAs functions as proteins and these circ RNAs with coding potential tend to form amino acid that differs from that of their host genes.By using Genome-Wide Association Analysis of drought-tolerance traits,circ RNAproducing genes was found that significantly enriched for SNPs associated with plant survival rate under drought stress in 368 maize inbred lines and QTLs associated traits under WS in bi-parental populations.