N~6-methyladenine (m~6A) Regulates Root-developed Genes In Phyllostachys Edulis

N~6-methyladenine(m~6A)is the most important RNA modification in eukaryotes and plays critical roles in regulating plant growth and development.Moso bamboo(Phyllostachys edulis)is an important forest resource in our country,known for its significant economic value due to its rapid growth characteristics.The nutrients needed for the rapid growth of Moso bamboo depend on the expansion of the underground root system.However,our understanding of roots growth in bamboo at the molecular level is limited,particularly with respect to epigenetics of m~6A.Previous experiments have shown that m~6A hypomethylation caused growth retardation of roots in bamboo.In order to explore the role of m~6A and its impact on post-transcriptional regulation,we analyzed the epigenetic modification and post-transcriptional regulation of the roots in bamboo through Nanopore direct RNA sequencing(DRS)and RNA-seq.We identified the global m~6A hypomethylation after the treatment of m~6A inhibitor DZnep A(Deazaneplanocin A).The m~6A hypomethylation sites were enriched in the 3’untranslated region(UTR)with the AAACA and AAACT motifs.In bamboo,we observed differential distribution of m~6A on exons of different lengths,with enrichment at the end of exons with the length more than 1,000 nt,where the trend strengthens with the increase of exon length.Analysis of the full-length ratio of the reads revealed that global genes exhibited increasing full-length ratio accompanied by hypomethylated m~6A.Among them,2,614 genes including the 4 m~6A readers significantly increased in the full-length ratio,while only 14 genes decreased.Additionally,we also found that full-length reads had a higher ratio of m~6A modification than non-full-length reads.Detection of alternative splicing(AS)events based on transcripts assembly by DRS and RNA-seq revealed that intron retention was the most abundant AS event in roots,and most transcripts showed only one AS event.Additionally,we identified 844transcripts with differential AS events.Correlation m~6A analysis shows that about 60%reads with AS events had m~6A modification,and 72.37%in reads with mutually exclusive exons event.By identifying the 3’end sites of the DRS reads,we constructed the alternative polyadenylation(APA)landscape of the whole transcriptome in bamboo.We found that 18,227 coding genes(67%of expressed gene in this research)were regulated by APA,which mainly involved in mRNA transcription,protein translation and degradation.Furthermore,m~6A hypomethylation resulted in increased usage of proximal poly(A)sites in genes,including four m~6A readers,two m~6A erasers and root development genes.Dynamic analysis of poly(A)tail length(PAL)showed that the overall PAL of genes was shortened after DZnep A treatment,including many genes related to root development.Correlation analysis showed that PAL was positively correlated with gene expression and the proportion of full-length reads,but negatively correlated with m~6A modification.GO enrichment showed that genes with short poly(A)tails were related to protease,proteolysis and electron transport chain,while genes with long poly(A)tails were more involved in signal transduction of signal pathways.Differentially expressed genes analysis revealed that genes involving cell wall synthesis,cell oxidoreduction,and photosynthesis exhibited significant reduction,while expressions of genes related to secondary metabolite synthesis,glutathione metabolism,and cell differentiation were significantly increased.After correlating with m~6A modification,we found that gene expression was negatively correlated with m~6A modification.Genes related to glutathione metabolism and synthesis showed decreased m~6A modification and increased expression,while genes involved cell wall and cellulose synthesis had increased m~6A modification and decreased expression.The hypomethylation of m~6A in roots of bamboo is accompanied by the dynamic change of m~6A regulatory protein expression.The expression of two m~6A writers(Phe MTA and Phe FIONA1)was significantly up-regulated in DZnep A,the expression of two m~6A erasers was significantly down-regulated,and the expression of eight m~6A readers including Phe CPSF30 was up-regulated.By filtering coding RNA in transcripts assembled by DRS data,we identified5,500 high-confidence lncRNAs.The full-length ratio of lncRNA is higher than that of mRNA,and the distribution of m~6A on lncRNA transcript region has no preference.Compared with the control,1,175 lncRNA transcripts were differentially expressed,and the lncRNAs with increased differential expression showed a significant suppression in m~6A.In summary,this study comprehensively analyzed the epigenetic modification and post-transcriptional regulation mechanism of roots in bamboo through Nanopore direct RNA technology combined with Illumina RNA-seq,and initially explored the epigenetic regulatory network composed of m~6A modification and post-transcriptional regulation.The effect on the growth and development of roots will provide a molecular theoretical base for a comprehensive understanding of roots growth in Phyllostachys edulis.