Genome-wide Analysis Of The XTH Gene Family And Functional Analysis Of XTH23.5/25 During Early Somatic Embryogenesis In Dimocarpus Longan

Longan(Dimocarpus longan Lour.)belongs to the Sapindaceae family and the genus Dimocarpus.It is a tropical and subtropical evergreen fruit tree and is also an important economic crop.The developmental state of the longan embryo directly affects the yield and quality of the fruit.Due to the difficulty in obtaining early zygotic embryos,the molecular-level study of longan embryo development has been limited.However,the high synchronicity,frequency,and strong regeneration ability of longan somatic embryogenesis provide a good alternative material for studying longan embryo development.Xyloglucan endotransglucosylase/hydrolase(XTH)is a cell wall-modifying protein that affects cell expansion and wall relaxation and is involved in plant growth and development.Studies have shown that changes in cell wall physiology and biochemistry accompany somatic embryogenesis(SE)in plants.The mechanism by which XTH modifies the cell wall and participates in the longan early somatic embryogenesis requires further study.Therefore,this study conducted a genome-wide identification of the longan XTH gene family and analyzed its expression patterns during early somatic embryogenesis and under different treatments in embryogenic callus(EC)of longan.Additionally,DlXTH23.5/25 was screened and its role in cell wall modification during early longan somatic embryogenesis was explored by constructing and validating a transcription factor(TF)regulatory network.The main results of this study are as follows:1.Genome-wide identification and expression analysis of the XTH gene family during early longan somatic embryogenesisThe whole genome of longan XTH family members was identified based on three generations of longan ’HHZ’ genome.There are 25 members of longan XTH family,including two pairs of intraspecies collinear genes and 11 pairs of collinear genes with Arabidopsis thaliana,as well as five pairs with Oryza sativa.The promoter cis-elements analysis revealed that DIXTH family members contain a large number of stress response elements and stress-responsive cis-elements.Most DlXTH family members were highly expressed in early embryogenic callus(EC)(eight)and globular embryos(GE)(eight),suggesting that they may help to maintain the cell embryonic state and promote somatic embryogenesis.Among the 16 expressed DlXTH genes,13 were upregulated under high temperature stress,indicating that members of the DlXTH family are responsive to high temperature stress regulation.The combination of RNA-seq,ATAC-seq,and ChIP-seq(H3K4me1 modification)results revealed that most of the differentially expressed DlXTH genes during the early SE process were associated with changes in chromatin accessibility and high levels of H3K4me1 modification.Based on the early transcriptome database of longan somatic embryogenesis,differentially expressed genes DlXTH23.5/25 were selected.The qRT-PCR results showed that DlXTH23.5/25 was significantly upregulated during the early stages of somatic embryogenesis in longan at the GE stage,suggesting that the accumulation of its transcript level may be advantageous for promoting longan somatic embryogenesis.XET activity assay showed that during the early stages of somatic embryogenesis in longan,XET activity gradually accumulated and reached its highest point in the incompleteembryotic compact structure,then gradually decreased in the globular embryo stage.This suggests that the dynamic changes in XET content may have an impact on the early stages of somatic embryogenesis in longan.2.Subcellular localization of XTH23.5/25 and transient expression of longan embryonic callusBased on the previous study,the evolutionary tree of DlXTH23.5/25 were conducted among different species,protein sequence alignment,and secondary and tertiary structure prediction.The results showed that DlXTH23.5/25 is highly conserved and may be located in membranerelated structures and contain a signal peptide.By transiently transforming tobacco with DlXTH23.5/25 overexpression,it was confirmed that the DlXTH23.5/25 protein is located on the cell membrane.In addition,punctate fluorescence signals were observed in the cytoplasm,suggesting that the DlXTH23.5/25 protein is located on the newly secreted xyloglucan in the endoplasmic reticulum,and it catalyzes the transport and transglycosylation of xyloglucan.Transient overexpression of DIXTH23.5/25 in longan EC also showed that DIXTH23.5/25 can promote the accumulation of XET,suggesting that DIXTH23.5/25 may participate in the early process of longan somatic embryogenesis by regulating XET activity.3.Validation of the regulatory network involving WRKY31,ERF1/5,and XTH23.5/25 and their regulation of expression during the early stages of longan SE under high-temperature stressTo further validate the regulatory network of DIXTH family,DlWRKY31 and DlERF1/5 transcription factors were predicted and screened from the transcriptional database of early longan somatic embryogenesis.qRT-PCR results showed that the expression levels of the TFs were highest at the GE stage,consistent with the expression trend of DlXTH23.5/25 in the early stages of longan somatic embryogenesis,suggesting that the transcription factors DlWRKY31 and DlERF1/5 positively regulate the expression of DlXTH23.5/25.In vivo fluorescence imaging and dual-luciferase reporter gene assays showed that the transcription factors DlWRKY31 and DlERF1/5 can activate the expression of DlXTH23.5/25,suggesting that the TFs may bind to the promoter region of DlXTH23.5/25 and activate its transcription.Overexpression TFs in transiently transformed longan EC and protoplasts further confirmed that the transcription factors DlWRKY31 and DlERF1/5 promote the expression of DlXTH23.5/25.Transcriptomic data of longan EC under different temperature treatments showed that transcription factors DlWRKY31,DlERF1/5,and DlXTH23.5/25 can all respond to high temperature stress(35 ℃).The qRTPCR results further showed that the expression level of DlXTH23.5/25 was significantly up-regulated and XET activity increased under high temperature stress.The overexpression transcription factors DlWRKY31,DlERF1/5,and DlXTH23.5/25 can promote the accumulation of XET in tobacco,indicating that the regulatory network composed of TFs and DIXTH23.5/25 may participate in the regulation pathway of longan under high temperature stress by regulating the activity of XET.4.The Functional Study of longan XTH23.5/25To further investigate the function of XTH23.5/25 in longan,stable cell lines were obtained by overexpression DlXTH23.5 transforming longan EC.These cell lines were then differentiated into somatic embryos to explore the role of DlXTH23.5 in the early stages of longan somatic embryogenesis.The results showed that overexpression DlXTH23.5 can promote the early stages of somatic embryogenesis in longan.To further validate the involvement of DlXTH23.5/25 in cell wall modification during longan growth and development,a genetic transformation system mediated by Agrobacterium rhizogenes was used,and it was found that overexpression DlXTH23.5/25 can promote the growth of longan hairy roots and the accumulation of XET content.Histological sections and toluidine blue staining showed that the cell walls of DlXTH23.5/25 transgenic longan hairy roots were thickened,indicating that DlXTH23.5/25 may regulate cell wall thickening by promoting XET activity.The qRT-PCR results show that the expression level of DlXTH23.5/25 is significantly upregulated in the roots of longan treated with high temperature stress for six days.In addition,overexpression of DlXTH23.5/25 in longan hairy roots resulted in increased XET activity and decreased ROS levels under high temperature stress.Combining the expression patterns of genes related to ROS scavenging agents,it is speculated that DlXTH23.5/25 may reduce its own ROS levels by regulating the content of ROS scavenging agents,thereby reducing the damage caused by high temperature stress.In summary,25 DlXTH genes were identified from the longan genome,and the differentially expressed gene DlXTH23.5/25 was selected during longan early somatic embryogenesis.DlXTH23.5/25 was found to regulate the XET content,leading to thickening of the cell wall and participating in the growth and development of longan.Under high temperature stress(35℃),DlXTH23.5/25 may participate in the thickening of the cell wall by increasing the XET content,and thus respond to high temperature stress.DlXTH23.5/25 can also reduce its own ROS content,thereby reducing the damage caused by high temperature stress.In addition,transcription factors DlWRKY31 and DlERF1/5 bind to the DlXTH23.5/25 promoter and activate its transcription.Under high temperature stress,the regulatory network composed of TFs and DlXTH23.5/25 may participate in the early stages of longan embryogenesis under high temperature stress by regulating the activity of XET.