Dynamic Study Of Epigenomic And Post-transcriptional Regulation During Vascular Cambium Periodicity In Populus Tomentosa

The division and differentiation of vascular cambium in trees contribute to the generation of secondary xylem and secondary phloem,which determines the thickening growth of perennial woody plants and is the basis of wood formation.The activity of vascular cambium of trees in temperate regions exhibits obvious periodicity along with seasonal change.Cambial stem cells are dormant in winter and reactivating in spring.The regulation of this periodicity process is of great significance in forestry production.However,the molecular mechanisms underlying dormant-active transition are still unclear.miRNA,DNA methylation,N~6-methyladenosine and alternative splicing play important roles in the regulation of genes at transcriptional and post-transcriptional levels.These results will provide valuable information for further studies on the annual cycle development of cambium and further improvement of wood quality.In the present study,we collected dormant,reactivating,and active cambium from Populus tomentosa,the important trees used for afforestation in China,for morphology observation and hormone determination by using frozen sectioning,semi-thin sectioning and LC-MS/MS.Furthermore,gene expression,miRNA profiling,DNA methylome,m~6A profiling and alternative splicing during vascular cambium periodicity were performed with high-throughput sequencing and bioinformatics methods.These results revealed the role of epigenetic and post-transcriptional regulation in the annual development of vascular cambium in woody plants.The main results are as follows:(1)The results of morphological observation showed obvious changes of cambium zones from dormant stage to active stage.The thickness and the number of cell layers in cambium zones increased obviously in the dormant-active cycle,with compactly arranged cells in dormant stage and loosely arranged cell in active stage.The results of hormone determination by LC-MS/MS showed the increased content of IAA and decrease content of ABA from dormant to active stage.The contents of ACC,TZR and IPR were the lowest in the dormant stage,but significantly increased in reactivating and active stage.(2)The results of transcriptome profiling showed that differentially expressed genes were associated with various biological processes involved in cambium activity periodicity,such as regulation of meristem growth,phloem and xylem growth,cell division and expansion,cell wall biosynthesis,and plant hormone signaling.The expression patterns of genes involved in plant hormone signaling,cell division and cell wall biosynthesis were validated with q RT-PCR.(3)The results of small RNA sequencing results showed that the length of small RNAs in the cambium of poplar was mainly ranged from 20 nt to 24 nt.A total of 360 miRNAs were identified after bioinformatics analysis,235 of which are known and 125 were newly predicted.Differentially expressed miRNAs and target genes showed stronger negative correlations than other miRNA/target pairs.The target genes of differentially expressed miRNAs are mainly involved in certain pathways,such as plant hormone signal transduction,cell wall-associated pathways,plant-pathogen interaction.Furthermore,the expression patterns of differentially expressed miRNAs were validated by q RT-PCR.(4)The results of global DNA methylation analysis showed that the CG methylation level in the cambium was 19.5–21.2%,that of CHG was 12.0–12.8%,and that of CHH was 5.7–6.5%.Moreover,CG methylation levels exhibited a positive relationship with gene expression in the gene body,but a negatively relationship in promoter and downstream.In contrast,CHG and CHH methylation were negatively correlated with gene expression mostly in the downstream region.In addition,we observed that the number of CHH-DMR changes was the greatest during vascular cambium development,and showed a decreasing trend in the transition from dormant phase to active phase.Most importantly,we found that the expression of genes with CHH DMRs in the promoter displayed higher correlations with the DNA methylation levels than that of non-CHH-DMR genes.Strikingly,these genes were involved in plant hormone signal transduction,starch and sucrose metabolism,phenylpropanoid biosynthesis,and plant-pathogen interactions.(5)The results of m~6A-seq showed m~6A/A ratio was the lowest at dormant stage,and increased at reactivating and active stage.Reactivating cambium harbored the greatest number of m~6A peaks and m~6A-containing genes,followed by the dormant stage and active stage.m~6A peaks were highly enriched around the stop codon and in the 3’UTR region,and the UGUA motif was a common conserved m~6A sequence in different samples.Differential m~6A genes were mainly involved in plant hormone,phenylpropane synthesis and carbon fixation.The genes with differential m~6A enrichment were verified by m~6A-IP-q PCR.Interestingly,we found that the enrichment of m~6A were different at different stages,such as 14-3-3protein,HSP18.2 and transcription factor YY1.Moreover,m~6A enrichment was positively correlated with the gene expression,which indicated that m~6A modification plays a role in stabilizing gene expression in vascular cambium of poplar.(6)The results of AS analysis showed the widespread dynamic difference of alternative splicing during cambium development in poplar,with the least proportion of genes underlying alternative splicing at active stage.Exon skipping,intron retention and alternative 3’splice site were the main types of differential alternative splicing events in the vascular cambium of different stages.DAS genes were mainly involved in biological processes such as spliceosome,carbon metabolism,protein processing and RNA transport.Differential AS events among different stages were verified by RT-PCR,and we found that the important genes involved in cambium development,such as ARF,WOX4 and CESA3,undergoing different alternative splicing events at different stages of vascular cambium,indicating the important role of alternative splicing in the annual development of vascular cambium in poplar.Taken together,this study intends to reveal the dynamic changes of epigenetic and post-transcriptional regulation during cambium periodicity in poplar,which will facilitate further understanding of the regulatory mechanisms during vascular activity periodicity in trees and provide new clues for revealing the molecular mechanisms underlying wood formation and tree breeding by molecular design.