Background: Somatic embryogenesis (SE) is a process of somatic cells that dedifferentiate to totipotent embryonic stem cells and generate embryos in vitro. Longan SE has been established and wildly used as model system for studying embryogenesis in woody plants, SE-related genes had been characterized. In spite of that, a comprehensive overview of SE at a molecular level is still absent. To understand the molecular mechanisms during longan SE, we examined the transcriptome changes by using Illumina HiSeq from the four distinct developmental stages, including non-embryogenic callus (NEC), embryogenic callus (EC), incomplete compact pro-embryogenic cultures (ICpEC), globular embryos (GE).Results: RNA-seq of the four samples generated a total of 243.78 million high quality reads, approximately 81.5% of the data were mapped to longan genome. The cDNA libraries of NEC, EC, ICpEC and GE, generated 22,743, 19,745, 21,144, 21,102 expressed transcripts, 1935, 1710, 1816, 1732 novel transcripts, 2645, 366, 505, 588 unique genes, respectively. Comparative transcriptome analysis showed that a total of 10,642, 4180, 5846 and 1785 genes were differentially expressed in the pairwise comparisons of NEC_vs_EC, EC_vs_ICpEC, EC_vs_GE, ICpEC_vs_GE, respectively. Among them, plant hormones signalling related genes were significantly enriched, especially the auxin and cytokinin signalling components. The transcripts of flavonoid biosynthesis related genes were mainly expressed in NEC, while fatty acid biosynthesis related genes mainly accumulated in early SE. In addition, the extracelluar protein encoding genesLTP,CHI,GLP,AGP,EP1were related to longan SE. Combined with the FPKM value of longan nine tissues transcription, 27 SE specific or preferential genes (LEC1,LEC1-like,PDF1.3,GH3.6,AGL80,PIN1,BBM,WOX9,WOX2,ABI3, et al.) and 28 NEC preferential genes (LEA5,CNOT3,DC2.15,PR1–1,NsLTP2,DIR1,PIP1,PIP2.1,TIP2–1,POD-P7andPOD5et al.) were characterized as molecular markers for longan early SE. qRT-PCR validation of SE-related genes showed a high correlation between RNA-seq and qRT-PCR data.Conclusion: This study provides new insights into the role of the transcriptome during early SE in longan. Differentially expressed genes reveal that plant hormones signalling, flavonoid and fatty acid biosynthesis, and extracelluar protein related genes were involved in longan early SE. It could serve as a valuable platform resource for further functional studies addressing embryogenesis in woody plants.

Mother of FT and TFL1 (MFT) belongs to phosphatidylethanolamine-binding protein (PEBP) family, which plays an important role in flowering time regulation, seed development, and germination. To gain insight into the molecular function of DlMFT in Dimocarpus longan Lour., we isolated DlMFT and its promoter sequence from longan embryogenic callus (EC). Bioinformatic analysis indicated that the promoter contained multiphytohormones and light responsive regulatory elements. Subcellular localization showed that the given the DlMFT signal localized in the nucleus, expression profiling implied that DlMFT showed significant upregulation during somatic embryogenesis (SE) and zygotic embryogenesis (ZE), and particular highly expressed in late or maturation stages. The accumulation of DlMFT was mainly detected in mature fruit and seed, while it was undetected in abortive seeds, and notably decreased during seed germination. DlMFT responded differentially to exogenous hormones in longan EC. Auxins, salicylic acid (SA) and methyl jasmonate (MeJa) suppressed its expression, however, abscisic acid (ABA), brassinosteroids (BR) showed the opposite function. Meanwhile, DlMFT differentially responded to various abiotic stresses. Our study revealed that DlMFT might be a key regulator of longan somatic and zygotic embryo development, and in seed germination, it is involved in complex plant hormones and abiotic stress signaling pathways.

Light is an important factor that affects the synthesis of functional metabolites in longan embryogenic calli (ECs). However, analysis of the effect of light on functional metabolites in longan ECs via RNA sequencing has rarely been reported and their light regulation network is unclear. The contents of various functional metabolites as well as the enzymatic activities of superoxide dismutase and peroxidase and the level of H₂O₂ in longan ECs were significantly higher under blue light treatment than under the other treatments (dark, white). In this study, we sequenced three mRNA libraries constructed from longan ECs subjected to different treatments. A total of 4463, 1639 and 1806 genes were differentially expressed in the dark versus blue (DB), dark versus white (DW) and white versus blue (WB) combinations, respectively. According to GO and KEGG analyses, most of the differentially expressed genes (DEGs) identified were involved in transmembrane transport, taurine and hypotaurine metabolism, calcium transport and so forth. Mapman analysis revealed that more DEGs were identified in each DB combination pathway than in DW combination pathways, indicating that blue light exerts a significantly stronger regulatory effect on longan EC metabolism than the other treatments. Based on previous research and transcriptome data mining, a blue light signaling network of genes that affect longan functional metabolites was constructed and HY5, PIF4 and MYC2 were shown to be the key regulatory genes in the network. The results of this study demonstrate that the expression levels of phase-specific genes vary with changes in longan EC functional metabolites.

Plant somatic embryogenesis (SE) is an in vitro biological process wherein bipolar structures are induced to form somatic cells and regenerate into whole plants. MicroRNA (miRNA) is an essential player in plant SE. However, the mechanism of microRNA408 (miR408) in SE remains elusive. Here, we used stable transgenic technology in longan (Dimocarpus longan) embryogenic calli to verify the mechanism by which miR408 promotes cell division and differentiation of longan early SE. dlo-miR408-3p regulated riboflavin biosynthesis by targeting nudix hydrolase 23 (DlNUDT23), a previously unidentified gene mediating N6-methyladenosine (m⁶A) modification and influencing RNA homeostasis and cell cycle gene expression during longan early SE. We showed that DlMIR408 overexpression (DlMIR408-OE) promoted 21-nt miRNA biosynthesis. In DlMIR408-OE cell lines, dlo-miR408-3p targeted and downregulated DlNUDT23, promoted riboflavin biosynthesis, decreased flavin mononucleotide (FMN) accumulation, promoted m⁶A level, and influenced miRNA homeostasis. DNA replication, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, the pentose phosphate pathway, and taurine and hypotaurine metabolism were also closely associated with riboflavin metabolism. In a riboflavin feeding assay, dlo-miR408-3p and pre-miR408 were upregulated and DlNUDT23 was downregulated, increasing the m⁶A level and cell division and differentiation in longan globular embryos. When riboflavin biosynthesis was inhibited, dlo-miR408-3p was downregulated and DlNUDT23 was upregulated, which decreased m⁶A modification and inhibited cell division but did not inhibit cell differentiation. FMN artificial demethylated m⁶A modification affected the homeostasis of precursor miRNA and miRNA. Our results revealed a mechanism underlying dlo-miR408-3p-activated riboflavin biosynthesis in which DlNUDT23 is targeted, m⁶A modification is dynamically mediated, and cell division is affected, promoting early SE in plants.

Polygonatum rhizomes are rich in various compounds with many biological activities and are widely used in functional foods and pharmaceutical products. In order to screen for superior Polygonatum cyrtonema Hua (P. cyrtonema) germplasm and also to elucidate the nutritional and medicinal values of rhizomes, the metabolic composition and quality traits of rhizomes from different germplasms and age sections of P. cyrtonema were analysed by widely targeted metabolomics, and the molecular mechanism of triacylglycerol synthesis was explored. The results showed that the different germplasms and age sections of P. cyrtonema were rich in different nutritional and medicinal components. Of these, the broad-leaved green stem (GK) germplasm is rich in polysaccharides, alkaloids, and lipids; the pointed-leaved green stem (JL) germplasm is rich in flavonoids, steroids, and amino acids, while the pointed-leaved purple stem (JZ) germplasm contains more phenolic acids. The one-year (AT) age section is rich in polysaccharides, steroids, organic acids, and lipids; the three years (CT) age section contains more flavonoids, alkaloids, and amino acid metabolites. Lipids were significantly enriched in the broad-leaved green stem germplasm and the one-year age section. Interestingly, the highest accumulation of triacylglycerols, an important component of lipids, was also found in the GK germplasm and the AT age section. Nineteen, 14, and 13 members of the glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAT), and diacylglycerol acyltransferase (DGAT) gene families, respectively, involved in triacylglycerol synthesis were also identified. The quantitative real-time PCR (qRT-PCR) results further suggested that the differentially expressed PcDGAT1, PcDGAT2.4, PcGPAT9.1, PcLPAT2.9, and PcLPAT4.3 genes may play important roles in triacylglycerol synthesis in P. cyrtonema. Therefore, this study provides a new theoretical reference for product development and the breeding of new varieties of Polygonatum species.