The Role And Mechanism Of DNA Methylation And CircRNA During The Early Somatic Embryogenesis Of Dimocarpus Longan Lour.

Longan(Dimocarpus longan),which belongs to the genus longan of Sapindaceae family,is a characteristic subtropical fruit plant species in southern China.The embryonic developmental status of longan is closely related to seed size,fruit quality,fruit set and yield.Thus,longan embryological studies are essential for the longan industry.However,it is difficult to collect longan early-stage zygotic embryos;they are genetically highly heterozygous,exhibit poor material synchronization,have uncontrollable developmental processes,and are easily affected by the environment,all of which have become challenges in the embryological study of longan embryos.The physiological and morphological changes associated with longan SE(somatic embryogenesis)are similar to those associated with the zygotic embryo development process;as such,zygotic embryo development serves as an ideal model for studying the molecular mechanisms of longan embryos.Plant SE is an intricate developmental process that involves cell division and cellular reprogramming,and the spatial and temporal expression of genes is under the control of hormones,stress signals and genetic and epigenetic mechanisms.Increasing evidence demonstrates that changes in DNA methylation are importantly associated with plant SE development.However,very few studies have examined DNA methylation changes in genome-wide single-base resolution during the early stage development of SE.Moreover,as an important regulator in epigenetic modification,the function of circRNA(circularRNA),in plant SE is still unclear.In this study,to elucidate the molecular mechanisms underlying the DNA methylation and circRNA during early longan SE.The application of DNA methylation inhibitor 5-azacytidine(5-AzaC)in EC(embryogenic callus)confirmed the function of DNA methylation in longan SE development.Single-base resolution maps of DNA methylation at EC,ICp EC(incomplete compact pro-embryogenic cultures),and GE(globular embryos)stages were generated by whole genome bisulphite sequencing(WGBS),and the gene regulatory profiles were investigated by transcriptome sequencing.DNA methylation-related genes in the longan genome were identified,and the expression patterns of these genes during longan early SE were investigated.Additionally,we performed a genome-wide identification of circRNAs in longan early SE,characterized the global expression patterns of circRNAs,and discussed the underlying biological implications of circRNAs during longan early SE.We also constructed a ceRNA(competing endogenousRNA)interaction network of DNA methylation pathway-related genes,circRNA,long noncodingRNAs(lncRNAs)acting in concert with microRNAs(miRNAs),revealing potential regulatory mechanisms of circRNA involved in DNA methylation during longan early SE.Main results are as follows:1.Global DNA methylation levels during longan early SE and the effect of 5-AzaC on longan SEGlobal DNA methylation levels of EC,ICp EC and GE in the early stage of longan SE were measured by DNA methylation detection kit.The results showed that global DNA methylation levels decrease from EC to ICp EC and then a slight increase from ICp EC to GE.To explore the significance of DNA hypomethylation for longan somatic cell proliferation and SE development,as well as effect of the 5-AzaC treatment on global DNA methylation of longan somatic embryogenic cultures,the DNA methylation inhibitor 5-AzaC was used in EC.The results showed that 2.5 μM 5-AzaC treatment in EC promoted cell formation proliferation and GE morphogenesis.Additionally,DNA methylation levels were quantified in control and treated cultures of longan after 9 days 5-AzaC treatments.The results showed global DNA methylation decreased in longan cultures after the 5-AzaC treatments.Taken together,our results suggested that DNA demethylation has important roles in longan SE development.2.Genome-wide investigation of DNA methylation dynamics during longan early SETo further explore dynamic changes of DNA methylation during longan early SE and assess the relationship between DNA methylation and gene expression.In this study,we carried out WGBS and transcriptome sequencing analyses for EC,ICp EC and GE in an early SE system.The results showed that,at a global level,the DNA5-methylcytosine(5-m C)content in EC,ICp EC and GE was 24.59%,19.65% and 19.74%,respectively,suggesting a global decrease in DNA methylation from EC to ICp EC and then a slight increase from ICp EC to GE.Differentially methylated region(DMR)analysis showed that methylation changes mainly occurred in CHH contexts.Gene Ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)analysis of hypomethylated(hypo)-CHH-DMR-associated genes revealed that zein biosynthesis,fatty acid biosynthesis,circadian rhythm and mitophagy pathways were involved in longan early SE.The correlation between DNA hypomethylation and gene expression revealed that decreased DNA methylation did not cause extensive changes in gene expression during early longan SE and that gene expression may be affected by methylation changes in gene and downstream regions.3.Genome-wide identification and expression analysis of DNA methylation pathway-related genes in longanBased on the longan genome information,the genes related to the DNA methylation pathways in longan were identified and analyzed.The results showed that 93 DNA methylation pathway-related genes in longan were identified,including 33 de novo DNA methylation pathway-related genes,15 maintenance methylation pathway-related genes and 45 active demethylation pathway-related genes.Conserved domains and phylogenetic tree analysis of longan and Arabidopsis thaliana showed that homologous DNA methylation pathway-related genes had similar conserved domains,and the phylogenetic trees of these genes clustered closely,suggesting highly conserved DNA methylation pathway-related genes across the different plant species.In addition,multiple cis-acting elements were observed in DNA methylation-related genes of longan,including plant developmental response,stress response and hormone response-related elements.Expression patterns of DNA methyltransferase and demethylase genes during longan early SE suggested that the decrease in DNA methylation was probably regulated by DNA methyltransferase genes and the DNA demethylase gene REPRESSOR OF SILENCING 1(ROS1).The expression patterns of longan DNA methyltransferase and DNA demethylase gene under different hormones(auxin,gibberellin,methyl jasmonate,salicylic acid,ethylene and abscisic acid)indicated that DNA methylation plays important role in hormonal responses during longan early SE.4.Genome-wide circularRNA profiling and competing endogenousRNA regulatory network analysis during longan early SEHigh-throughput sequencing was used to identify circRNAs in longan EC,ICp EC and GE.A total of 5029 circRNAs were identified in the three stages of longan early SE.KEGG analysis revealed that differentially expressed(DE)circRNA host genes were enriched in the“non-homologous end-joining”(NHEJ)and “butanoate metabolism”pathways.In addition,the reactive oxygen species(ROS)content during longan early SE was determined.The results indicated that ROS-induced DNA double-strand breaks(DSBs)may not fully depend on the NHEJ repair pathway.Correlation analyses of the levels of related metabolites(glutamate,γ-aminobutyrate and pyruvate)and the expression levels of circRNAs and their host genes involved in butanoate metabolism were performed.The results suggested that circRNAs may act as regulators of the expression of cognate mRNAs,thereby affecting the accumulation of related compounds.A competing endogenousRNA(ceRNA)network of DE circRNAs,DE mRNAs,DE lncRNAs and DE miRNAs was constructed.The results showed that the putative targets of the noncodingRNAs(ncRNAs)were significantly enriched in the KEGG pathways“mitogen-activated protein kinase signaling” and “nitrogen metabolism”.Furthermore,the expression patterns of the candidate circRNAs,lncRNAs,miRNAs and mRNAs confirmed the negative correlation between miRNA and ceRNA.And two circRNA overexpression vectors were constructed based on chemical oligonucleotide synthesis and then transiently into longan EC protoplasts,which further verified the ceRNA network correlations in longan early SE.5.Identification and functional study of ncRNAs that regulate genes related to DNA methylation pathwaysThrough integrated multi-omics data,the potential role of ncRNA in regulating DNA methylation during longan early SE was investigated.Firstly,we identified potential miRNAs,lncRNAs,and circRNAs that regulate genes related to DNA methylation pathways,and analyzed the correlations between miRNAs,lncRNAs,and circRNAs and DNA methylation pathway-related genes.The results showed that 62 miRNAs,10 lncRNAs and 53 circRNAs may be involved in the regulation of genes related to DNA methylation pathways.Moreover,we overexpressed circ ROS1 to explore its role in regulating the parental gene Dl ROS1.The results showed that circ ROS1 promoted the expression of Dl ROS1.In addition,the ceRNA network of DNA methylation pathway-related genes,miRNAs,lncRNAs and circRNAs was constructed,and the targeting relationship between circ CTPA2,Dl CMT3 and mi R5813 was verified by dual luciferase reporter experiments.The results indicated that circ CTPA2 may act as a sponge of dlo-mi R5813,indirectly regulate of the gene expression DNA methylase Dl CMT3,thereby participating in longan early SE.Overall,in this study,we observed a positive effect on SE with application of a DNA methylation inhibitor,suggesting that a decrease in DNA methylation is crucial for longan SE development.Single-base resolution maps of DNA methylation at the EC,ICp EC and GE,revealing the dynamics and regulation mechanism of DNA methylation during longan early SE.Expression patterns of DNA methylation pathway-related genes during longan early SE suggested that DNA hypomethylation is mainly due to the decreased expression of DNA methyltransferase genes and increased expression of DNA demethylase gene ROS1.Moreover,our study involved the first genome-wide identification of circRNAs in longan early SE.The functions of DE circRNAs in regulating hosting genes and acting as ceRNAs were analyzed,which revealed their possible biological roles in longan early SE.Additionally,we identified ncRNAs that potentially regulate genes related to DNA methylation pathways,and mined two circRNAs(circ ROS1 and circ CTPA2)that might regulate DNA methylation during longan early SE.Our study revealed the potential role of DNA methylation and circRNAs in longan early SE,which provided new insights into the intricate regulatory mechanism underlying plant SE.