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.

Long non-coding RNAs (lncRNAs) are involved in variable cleavage, transcriptional interference, regulation of DNA methylation and protein modification. However, the regulation of lncRNAs in plant somatic embryos remains unclear. The longan (Dimocarpus longan) somatic embryogenesis (SE) system is a good system for research on longan embryo development. In this study, 7643 lncRNAs obtained during early SE in D. longan were identified by high-throughput sequencing, among which 6005 lncRNAs were expressed. Of the expressed lncRNAs, 4790 were found in all samples and 160 were specifically expressed in embryogenic callus (EC), 154 in incomplete embryogenic compact structures (ICpECs), and 376 in globular embryos (GEs). We annotated the 6005 expressed lncRNAs, and 1404 lncRNAs belonged to 506 noncoding RNA (ncRNA) families and 4682 lncRNAs were predicted to target protein-coding genes. The target genes included 5051 cis-regulated target genes (5712 pairs) and 1605 trans-regulated target genes (3618 pairs). KEGG analysis revealed that most of the differentially expressed target genes (mRNAs) of the lncRNAs were enriched in the “plant-pathogen interaction” and “plant hormone signaling” pathways during early longan SE. Real-time quantitative PCR confirmed that 20 selected lncRNAs showed significant differences in expression and that five lncRNAs were related to auxin response factors. Compared with the FPKM expression trends, 16 lncRNA expression trends were the same in qPCR. In lncRNA-miRNA-mRNA relationship prediction, 40 lncRNAs were predicted to function as eTMs for 15 miRNAs and 7 lncRNAs were identified as potential miRNA precursors. In addition, we verified the lncRNA-miRNA-mRNA regulatory relationships by transient expression of miRNAs (miR172a, miR159a.1 and miR398a). Analyses of lncRNAs during early longan SE showed that differentially expressed lncRNAs were involved in expression regulation at each SE stage, and may form a regulatory network with miRNAs and mRNAs. These findings provide new insights into lncRNAs and lay a foundation for future functional analysis of lncRNAs during early longan SE.

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.

Amaranth plants contain large amounts of betalains, including betaxanthins and betacyanins. Amaranthin is a betacyanin, and its molecular structure and associated metabolic pathway differ from those of betanin in beet plants. The chlorophyll, carotenoid, betalain, and flavonoid contents in amaranth leaves were analyzed. The abundance of betalain, betacyanin, and betaxanthin was 2–5-fold higher in the red leaf sectors than in the green leaf sectors. Moreover, a transcriptome database was constructed for the red and green sectors of amaranth leaves harvested from 30-day-old seedlings. 22 unigenes were selected to analyze the expression profiles in the two leaf sectors. The RNA-sequencing data indicated that many unigenes are involved in betalain metabolic pathways. The potential relationships between diverse metabolic pathways and betalain metabolism were analyzed. The validation of the expression of 22 selected unigenes in a qRT-PCR assay revealed the genes that were differentially expressed in the two leaf sectors. Betalains were biosynthesized in specific tissues of the red sectors of amaranth leaves. Almost all of the genes related to betalain metabolism were identified in the transcriptome database, and the expression profiles were different between the red sectors and green sectors in the leaf. Amaranth plants consist of diverse metabolic pathways, and the betalain metabolic pathway is linked to a group of other metabolic pathways.

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.