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.

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.

Somatic embryogenesis (SE), like zygotic embryo development, is a progressive process. Early SE is the beginning of a switch from a somatic to an embryogenic state and is an important stage for initiating chromatin reprogramming of SE. Previous studies suggest that changes in chromatin accessibility occur during early SE, although information on the 3D structure of chromatin is not yet available. Here, we present a chromosome-level genome assembly of longan (Dimocarpus longan) using PacBio combined with Hi-C scaffolding, which resulted in a 446 Mb genome assembly anchored onto 15 scaffolds. During early SE, chromatin was concentrated and then decondensed, and a large number of long terminal repeat retrotransposons (LTR-RTs) were enriched in the local chromatin interaction region, suggesting LTR-RTs were involved in chromatin reorganization. Early SE was accompanied by the transformation from A to B compartments, and the interactions between B compartments were enhanced. Results from chromatin accessibility, monomethylation of histone H3 at lysine 4 (H3K4me1) modification, and transcription analyses further revealed a gene regulatory network for cell wall thickening during SE. Particularly, we found that the H3K4me1 differential peak binding motif showed abnormal activation of ethylene response factor (ERF) transcription factors and participation in SE. The chromosome-level genomic and multi-omics analyses revealed the 3D conformation of chromatin during early SE, providing insight into the molecular mechanisms underlying cell wall thickening and the potential regulatory networks of TFs during early SE in D. longan. These results provide additional clues for revealing the molecular mechanisms of plant SE.

Our aim in the experiment was to study the effects of methyl jasmonates (MeJA) on the active compounds of rosemary suspension cells, the metabolites’ change of contents under different concentrations of MeJA, including 0 (CK), 10 (M10), 50 (M50) and 100 μM MeJA (M100). The results demonstrated that MeJA treatments promoted the accumulation of rosmarinic acid (RA), carnosic acid (CA), flavonoids, jasmonate (JA), gibberellin (GA), and auxin (IAA); but reduced the accumulations of abscisic acid (ABA), salicylic acid (SA), and aspartate (Asp). In addition, 50 and 100 μM MeJA promoted the accumulation of alanine (Ala) and glutamate (Glu), and 50 μM MeJA promoted the accumulation of linoleic acid and alpha-linolenic acid in rosemary suspension cells. Comparative RNA-sequencing analysis of different concentrations of MeJA showed that a total of 30, 61, and 39 miRNAs were differentially expressed in the comparisons of CKvsM10, CKvsM50, CKvsM100, respectively. The analysis of the target genes of the differentially expressed miRNAs showed that plant hormone signal transduction, linoleic acid, and alpha-linolenic acid metabolism-related genes were significantly enriched. In addition, we found that miR160a-5p target ARF, miR171d_1 and miR171f_3 target DELLA, miR171b-3p target ETR, and miR156a target BRI1, which played a key role in rosemary suspension cells under MeJA treatments. qRT-PCR of 12 differentially expressed miRNAs and their target genes showed a high correlation between the RNA-seq and the qRT-PCR result. Amplification culture of rosemary suspension cells in a 5 L stirred bioreactor showed that cell biomass accumulation in the bioreactor was less than that in the shake flask under the same conditions, and the whole cultivation period was extended to 14 d. Taken together, MeJA promoted the synthesis of the active compounds in rosemary suspension cells in a wide concentration range via concentration-dependent differential expression patterns. This study provided an overall view of the miRNAs responding to MeJA in rosemary.