| longan(i.e.,Dimocarpus longan Lour.),which originated in Fujian,Guangdong and Southeast Asia,is an important sapindaceae tropical/subtropical evergreen woody fruit tree.Longan fruits are rich in active substances such as polysaccharides,alkaloids and flavonoids,with high nutritional values.Under natural conditions,the quality of longan embryo development directly affects the development of longan fruit,poor embryo developmental quality will lead to low fruit setting rate,serious fruit dropping and form small size fruit.Thus,the study on the molecular mechanism of longan embryogenesis can provide theoretical basis for improving fruit yield and quality.However,collecting the longan zygotic embryo in the early developmental stage is a major challenge that hinders the elucidation of the molecular mechanism underlying zygotic embryo development.Thus,the longan somatic embryogenesis(SE)system has been used to study in vitro longan embryo development.Earlier research revealed that longan embryonic callus(ECs)comprise embryonic and non-embryonic cells,but only the embryonic cells with vigorous division ability have the potential of somatic embryogenesis.To data,the molecular mechanism of longan SE process has been studied based on genome sequencing,transcriptome sequencing and proteome sequencing technologies.However,these findings were theresult of analyses of the gene expression profiles at specific developmental stages in limited pooled cell populations.Unfortunately,the differences in the molecular regulation in embryonic and non-embryonic cells remain unknown.Moreover,the specific cell types in somatic embryos are unclear.In this study,based on the recently developed single-cell RNA sequencing(scRNA-seq)technology,this study conducted scRNA-seq for longan EC.The cell types of longan EC were classified and identified based on the gene expression patterns and functional annotation results of different cells obtained from the scRNA-seq results.Meanwhile,key regulatory factors were screened to verify their functions during the SE of longan.The main results are as follows:1 Single cell RNA sequencing analysis of the embryogenic callus in longanA total of 28,724 EC cells from two biological replicates were isolated and profiled via droplet-based scRNA-seq.Based on an unsupervised clustering analysis of gene expression patterns performed using the Seurat software,twelve distinct cell clusters were identified.To determine the potential cell types in these clusters,the top 20 most differentially expressed up-regulated genes were selected as cell-type-enriched marker genes,and their functions were annotated.According to the gene function annotation results and combined withmarker genes of different cell types,5 clusters of 4 cell types were identified in longan EC cell populations: proliferating cells(proliferating cell,PC,clusters 6 and 8),meristematic cells(meristematic cell,MC,cluster 9),vascular cells(vascular cell,VC,cluster 7),and epidermal cells(vascular cell,EPC,cluster 11).Combined with bulk RNA-seq results,we analyzed the expression patterns of the top 20 up-regulated expression genes using the longan early SE transcriptome dataset.Most of the top 20 up-regulated expression genes in clusters 8 and 9 tended to have decreasing expression levels from the EC stage to the globular embryo(GE)stage,speculating that these two clusters may contain the early SE-related cell populations.In contrast,most of the top 20up-regulated expression genes in clusters 2,5,7,and 11 tended to have increasing expression levels from the EC stage to the GE stage.On the basis of their increased activity in the GE stage,we predicted that these four clusters may contain the SE-related cell populations that differentiated relatively late.The cluster 8,9,7,and 11 EC cell populations were divided and differentiated into distinct cell types during SE.Thus,we selected clusters 8,9,7,and 11 to construct the cell differentiation trajectory.Cells from clusters 8 and 9 were assembled at the beginning of the pseudo-timeline.Cluster 7 was associated with the middle part of the pseudo-timeline,and cluster 11 was associated with a relatively late stage in the pseudo-timeline.The pseudo-timeline analysisdefined the continuous cell differentiation trajectories from early embryogenic cell division to vascular and epidermal cell differentiation.Meanwhile,key transcriptional regulators associated with cell fate decisions were revealed.Major,highly connected TF genes were detected throughout the cell differentiation process,including ETHYLENE RESPONSIVE FACTOR(ERF6),ERF20,ERF114,MYB9,MYB4,NAC58,and WRKY75.We identified the NAC83–XTH23 and NAC58–MYB9transcription factor–target gene regulatory networks and demonstrated that these two transcription factors activate the expression of their target genes.Furthermore,the up-regulated expression genes assigned to the‘autophagy’ metabolic pathway may be important for maintaining embryogenic cellular homeostasis and for mediating non-embryonic cellular apoptosis.2 Genome-wide identification and expression pattern analysis of the AP2/ERF transcription factor superfamily in longanBased on the scRNA-seq result,it was found that several AP2/ERF transcription factors may be involved in regulating the cell differentiation process of longan EC,such as ERF6,ERF20 and ERF114,which were significantly up-regulated expression in different cell types of single-cell transcriptomes.A genome-wide survey of the AP2/ERF superfamily was carried out to discover its evolution and function in longan.A total of 125 longan AP2/ERF genes were identified and classified them into the ERF,AP2,RAV families,and one Soloist.Based on the bulk RNA-seq result,the expression patterns of AP2/ERF transcription factor superfamily were analyzed.The results revealed that some AP2/ERF members regulated early SE and developmental processes in longan,and responded to light qualities,exogenous hormones,and temperature stresses.Combined with the single-cell transcriptome results,ERF6 and ERF20,which were up-regulated expression in single-cell transcriptome,were up-regulated expression in the GE stage and under high temperature stress.3 Genome-wide identification and expression analysis of Longan GPAT gene familyBased on the scRNA-seq results,two GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE(GPAT)family members,GPAT6.2 and GPAT8,were found to be specifically expression in EPC,revealing that they may be involved in the biological processes of longan growth and development and resistance to stress.A genome-wide survey of the GPAT family was carried out to discover its evolution and function in longan.A total of nine GPAT family genes were ultimately ascertained.Based on the bulk RNA-seq result,the expression patterns of GPAT family genes were analyzed.The results revealed that GPAT family members regulated early SE and developmental processes,and responded to light qulities and temperature stresses.The triacylglycerol(TAG)content during the early SE of longan was determined,and found that the TAG content wasincreased during the early SE of longan.Widely targeted metabolomics determination also showed similar results.Combined with single-cell transcriptome result,GPAT6.2 and GPAT8,which were up-regulated expression in single-cell transcriptome,were up-regulated in the GE stage and under high temperature stress of longan EC,and were highly correlated with the TAG content,suggesting that they may play an important role in longan.4 Verification of AP2/ERF-GPAT regulatory networks and analysis of the expression pattern during the early somatic embryos of longan under high temperature stressThe AP2/ERF-GPAT transcription factor-target gene regulatory network of longan was predicted,a total of 73 AP2/ERF transcription factors may regulate nine GPAT genes.Combined with single-cell transcriptome result,ERF6,which was up-regulated expression in single-cell transcriptome,may regulated all of the GPAT family members.The overexpression transient transformation protoplasts results showed that overexpression of ERF6 leaded to the up-regulated expression of almost all GPAT family genes.Dual-luciferase assay showed that ERF6 could activate the promoter activity of GPAT6.2 and GPAT8.To further study the response mechanism of ERF6 and GPATs during the early SE of longan under high temperature stress,the early somatic embryos of longan were treated with high temperature(35℃)stress.Theresults showed that high temperature stress inhibited the differentiation of the early somatic embryos in longan,and the protoembryos couldn’t differentiate into GEs under high temperature stress.After 9 days of high temperature stress treatment,the expression of of ERF6 and GPAT family genes were significantly up-regulated.The TAG content was significantly up-regulated after 6 and 9 days of high temperature stress,indicating that TAG biosynthesis pathway was also involved in the high temperature stress of longan.The results suggestted that ERF6 may be involved in the response of longan somatic embryos to high temperature stress by activating the expression of GPAT family genes and resulting in the biosynthesis of somatic embryos TAG.5 Functional study of the ERF6 through stable transformation of longan embryogenic callusThe function of ERF6 was verified by stable transformation of longan ECs.The results showed that the expression levels of GPAT family genes were significantly up-regulated in ERF6 overexpression lines,furthermore,the TAG content was increased significantly in ERF6 overexpression lines,which indicated that ERF6 was involved in the TAG biosynthesis in the longan ECs by activating the expression of GPATs.The screened ERF6 positive ECs were transferred to MS medium for SE observation,the results showed that overexpression of ERF6 could induced SE normally in longan.In addition,overexpression of ERF6inhibited the differentiation process of longan somatic embryos under high temperature stress,the TAG content of longan somatic embryos was increased significantly after overexpression of ERF6 under high temperature stress,and ERF6 can also activated the expression of GPAT family genes under high temperature stress.After exogenously adding melatonin(MT),the TAG content of longan somatic embryos was decreased after overexpression of ERF6 under high temperature stress,and the cell disintegration disappeared in the ERF6-overexpression somatic embryos.The results showed that ERF6 promoted the TAG biosynthesis by activating the expression of GPAT family genes,thus inhibited the differentiation of somatic embryos in longan under high temperature stress. |
