Three-dimensional Genome Construction In Longan And The Function Of Cell Wall Modification Genes BGAL9 And XTH22 During Early Somatic Embryogenesis

Longan(Dimocarpus longan Lour.)belongs to to the Sapindaceae family,and it is an important evergreen fruit tree in tropical/subtropical regions as well as an important economic fruit tree.The embryonic development of longan was closely related to its yield and fruit quality.Due to the difficulty in sampling early longan zygotic embryos,the application of longan embryos in genetic breeding is greatly limited.Longan somatic embryogenesis system is characterized by genetic stability and high regeneration frequency,which is an ideal material for studying the cell biology and regulation mechanism of embryogenesis.Somatic embryogenesis(SE)is a complex developmental process in which somatic cells are reprogrammed to have the ability to produce somatic embryos.The distribution of eukaryotic genome chromosomes in the nucleus is nonrandom.Chromatin is a complex three-dimensional conformation within the nucleus,and its spatial structure provides a mechanism for connecting distal enhancers to their target genes.Although there has been evidence that reprogramming the open state of somatic chromatin can activate the expression of cell totipotency factors to promote the formation of somatic embryos,the regulation of the fate of somatic cells by the threedimensional structure of chromatin during early SE is still in its infancy.Therefore,this study used the ‘Hong He Zi’ of longan as the experimental material to reveal chromatin interactions and structural changes during the early SE through longan genome assembly and Hi-C analysis.Combined multi-omics analysis showed that cell wall related pathways were involved in the early morphogenesis of longan somatic embryos.At the same time,cell wall modification genes BGAL9 and XTH22 were screened.Using the genetic transformation of longan,the dual luciferase system and yeast monohybrid technology,we constructed the transcription factor regulatory network and studied their function during early longan SE.The main research results are as follows:1.Genome assembly and Hi-C analysis of longan revealed chromatin interactions and structural changes during early somatic embryogenesisIn this study,a high-quality three-dimensional genome map of longan‘HHZ’ was constructed by combining Illumina Paired-End sequencing,Pac Bio SMRT sequencing and Hi-C sequencing.Combined with the changes in the three-dimensional chromatin structure of early SE,it was found that the three-dimensional chromatin structure had undergone large scale recombination.Chromatin concentration occurs in the process of EC to ICp EC,and deconcentration of chromatin from ICp EC to GE.Early SE was accompanied by a transition from A to B compartments,while the interaction between B compartments was enhanced.A large number of LTR-RTs were enriched in local chromatin interaction regions and participate in chromatin remodeling during somatic embryogenesis.The GO enrichment of LTR-RTs adjacent genes mainly plays a role in cell division,ribonucleotide binding,and histone modification.These results indicate that chromatin remodeling regulates somatic embryogenesis of longan.2.Multi-omics analysis showed that cell wall related pathways were involved in the process of early longan somatic embryogenesisThe above results indicate that chromatin status affects the cellular ability of longan somatic embryogenesis.Therefore,by combining chromatin accessibility,transcriptome,and H3K4me1 histone modifications,key pathways during early longan SE can be screened.Transcriptome sequencing was used to analyze EC,ICp EC and GE during early longan SE.A large number of differentially expressed genes were detected in ICp EC vs.GE,which was much larger than that in EC vs.ICp EC.The GO enrichment of differentially upregulated genes at the ICp EC stage showed that differentially upregulated genes at the ICp EC stage were mainly enriched in cell wall biosynthesis and metabolism related pathways.This indicates that the molecular mechanism of cell wall synthesis and metabolism has undergone a large-scale transformation at ICp EC stage.In KEGG enrichment analysis,genes differentially upregulated at ICp EC stage were mainly enriched in "ribosome" and "plant hormone signal transduction" pathways,indicating that the somatic embryogenesis of longan underwent extensive cell division at ICp EC stage.ATAC-seq analysis showed that chromatin opening durong early SE was correlated with gene expression.The chromatin opening signal in ICp EC was much lower than that in EC and GE,which further verified that chromatin concentration in ICp EC was higher than that in EC and GE.The differential peak associated genes of ATAC signal enhancement in ICp EC and GE were mainly concentrated in the “extracellular region”,“cell wall”,“cell wall modification” and “cell wall organization” pathways.In Ch IPseq analysis,it was found that more H3K4me1 modified peak signals were identified in EC and GE than in ICp EC stage.Combined with the enrichment results of different peak associated genes enrichment in the KEGG and GO analysis,it was found that cell wall synthesis and metabolism related pathways were enriched in the three stages of early SE.A total of 119 differentially expressed genes related to cell wall were identified during early longan SE and most of the genes that related to lignin and hemicellulose were highly expressed at ICp EC and GE.BGAL,SUS and Ces A were activated to participate in cell wall recombination of early SE.Combining chromatin accessibility and H3K4me1 histone modification,it was found that changes in chromatin accessibility of differentially expressed genes related to cell walls during early SE were associated with high levels of H3K4me1 modification.H3K4me1 differential peak binding motif showed an abnormal enrichment of ERF binding motifs,mainly concentrated in the ICp EC stage.In predicting the regulatory network of transcription factors and cell wall related genes,a large number of transcription factors have been found to target the promoter region of the XTH genes.At the same time,it was found that the chromatin accessibility of the somatic embryo marker gene Dlo004817＿AGL80 and Dlo019949＿ERF1 were positively correlated with gene expression.During early SE,Dlo004817＿AGL80 and Dlo019949＿ERF1 were differentially enriched in H3K4me1 modification.3.AGL61/80 activates the expression of cell wall modification gene BGAL9 and participates in somatic embryogenesis and high temperature stress responseBased on multi-omics analysis,it was screened that cell wall-related pathways were involved in the morphogenesis of longan somatic embryos.Therefore,combining with the transcriptional regulation network of cell wall-related genes,the BGAL family of cell wall-modifying genes was identified for further study.There are 20 BGAL members in longan,including 6 pairs of intraspecial collinearity genes,16 pairs(Arabidopsis thaliana)and 3 pairs(rice)of interspecies collinearity genes.It was found that most of the BGALs promoter regions contain light signaling,hormone,stress,and developmental elements.Six of the 11 BGALs were highly expressed in GE,which may be beneficial for promoting early SE.DlBGAL could responded to temperature treatment,and most of the DlBGAL expressed were detected to be upregulated at high temperature.Combined with transcription,chromatin accessibility and H3K4me1 modification,it was found that most DlBGAL were differentially expressed during early SE could be identified the peak in ATAC-seq and H3K4me1 modification.The DlBGAL9 transcription factor regulatory network was constructed,and the transcription factors DlAGL61 and DlAGL80 were screened for transient transformation of longan EC.The regulatory effects of DlAGL61 and DlAGL80 on DlBGAL9 were preliminarily verified by q RT-PCR.Dual luciferase reporter gene assay showed that DlAGL61 and DlAGL80 could bind to the promoter region of DlBGAL9 and activate its transcription.The expression patterns of DlAGL61/80 and DlBGAL9 under different treatments of longan EC indicated that the expression patterns of DlAGL61/80 and DLBGAL9 responded to 2,4-D treatment and high temperature stress.At the same time,the β-GAL activity of early SE was significantly up-regulated at GE,while the pectin content was decreased.Under the high temperature,β-GAL activity was significantly downregulated at 6 d and 12 d,but significantly increased at 9 d.0.1 μM BR treatment could promote somatic embryogenesis,and according to expression pattern analysis,the DlAGL61/80-DlBGAL9 regulatory network may play an important role in longan response to BR treatment.The mechanism of cell wall regulation by DlAGL80 and DlBGAL9 was studied using the hairy root transformation system.Transgenic DlAGL80 and DlBGAL9 promote pectin degradation and thicken cell walls,indicating that DlAGL80 and DlBGAL9 may participate in cell wall modification during root growth by regulating pectin content.q RT-PCR was used to verify the expression patterns of DlAGL80 and DlBGAL9 under high temperature stress.The expression levels of DlAGL80 and DlBGAL9 were significantly upregulated under high temperature stress,and the activity of β-GAL decreased.Combined with the expression patterns of ROS scavenger related genes,it is speculated that DlAGL80 and DlBGAL9 may reduce their ROS content by regulating ROS scavenger content,thereby reducing the damage caused by high temperature stress.4.ERF1/5 activates expression of cell wall modification gene XTH22 and participates in somatic embryogenesis and high temperature responseBased on the transcriptional regulation network of cell wall related genes,the cell wall modification gene DlXTH22 was identified for further research.Using the longan ‘HHZ’ transcriptome database,the expression patterns of DlXTH22 in different transcriptome were analyzed.By constructing phylogenetic evolution trees of DlXTH22 in different species,and selecting the closest members for sequence alignment.The results show that DlXTH22 and other species related members contain XET conservative domains.Transient transformation of overexpressed DlXTH22 into tobacco showed that DlXTH22 was localized in the cell membrane and promoted the accumulation of XET activity.The overexpression of DlXTH22 into longan EC showed that the DlXTH22 longan EC cell line could promote the accumulation of XET activity during normal SE.Agrobacterium rhizogenes mediated genetic transformation system,it was found that DlXTH22 overexpression promoted the growth of longan lines and increased XET activity and hemicellulose content.The results of paraffin section showed that DlXTH22 transgenic longan hairy root cell wall thickened,suggesting that DlXTH22 may promote hemicellulose accumulation by regulating the synthesis of xyloglucan,thus thickening the cell wall.q RT-PCR was used to verify the expression pattern of DlXTH22 under high temperature stress.The expression level of DlXTH22 under high temperature stress was significantly up-regulated,and the activity of XET was increased,while the content of ROS was decreased.Combined with the expression patterns of ROS scavenger related genes,it was speculated that DlXTH22 might reduce its ROS content by regulating ROS scavenger content,thus reducing the damage caused by high temperature stress.The DlXTH22 transcription factor regulatory network was constructed,and the transcription factors DlERF1 and DlERF5 were screened for transient transformation of longan EC and protoplasts.The regulatory effects of DlERF1 and DlERF5 on DlXTH22 were preliminarily verified by q RT-PCR.Dual luciferase reporter gene experiment and Y1 H results showed that DlERF1 and DlERF5 could directly bind the promoter region of DlXTH22 and activate its transcription.Similarly,overexpression of DlERF1 promoted the accumulation of XET activity and the thickening of cell wall,suggesting that DlERF1 may regulate the content of XET by regulating the expression of DlXTH22,and participate in cell wall modification during root growth.In summary,this study constructed a high-quality three-dimensional genome map of longan ‘HHZ’.Combining the three-dimensional chromatin structure of longan early somatic embryogenesis,the interaction and structural changes of chromatin during the early somatic embryogenesis were revealed.Based on the results of multi omics analysis of RNA-seq,ATAC-seq,and Ch IP-seq(H3K4me1 modified)during early longan SE,it is revealed that cell wall related pathways were involved in the early morphogenesis of longan somatic embryos.By analyzing the differentially expressed genes related to the early cell wall of longan SE and constructing a transcriptional regulatory network,the potential function of DlAGL61/80-DlBGAL9 and DlERF1/5-DlXTH22 in regulating cell wall modification was revealed,which laid a foundation for elucidating the potential mechanism of cell wall modification during early longan SE.