Integrated Metabolomic and Transcriptomic Analyses Reveal the Potential Regulation of Flavonoids in the Production of Embryogenic Cultures during Early Somatic Embryogenesis of Longan (Dimocarpus longan Lour.).

Embryogenic cultures of longan (Dimocarpus longan Lour.) contain various metabolites with pharmacological properties that may function in the regulation of somatic embryogenesis (SE). In this study, based on widely targeted metabolomics, 501 metabolites were obtained from the embryogenic calli, incomplete compact proembryogenic cultures, and globular embryos during early SE of longan, among which 41 flavonoids were differentially accumulated during the SE. Using RNA sequencing, 36 flavonoid-biosynthesis-related genes and 43 MYB and 52 bHLH transcription factors were identified as differentially expressed genes. Furthermore, Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that the flavonoid metabolism-related pathways were significantly enriched during the early SE. These results suggested that the changes in flavonoid levels in the embryogenic cultures of longan were mediated by MYBs and bHLHs via regulating flavonoid-biosynthesis-related genes, thus potentially regulating early SE. The identified metabolites in the embryogenic cultures of longan can be used to develop pharmaceutical ingredients.

Riboflavin mediates m6A modification targeted by miR408, promoting early somatic embryogenesis in longan.

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.

A quantitative super-resolution imaging toolbox for diagnosis of motile ciliopathies.

Airway clearance of pathogens and particulates relies on motile cilia. Impaired cilia motility can lead to reduction in lung function, lung transplant, or death in some cases. More than 50 proteins regulating cilia motility are linked to primary ciliary dyskinesia (PCD), a heterogeneous, mainly recessive genetic lung disease. Accurate PCD molecular diagnosis is essential for identifying therapeutic targets and for initiating therapies that can stabilize lung function, thereby reducing socioeconomic impact of the disease. To date, PCD diagnosis has mainly relied on nonquantitative methods that have limited sensitivity or require a priori knowledge of the genes involved. Here, we developed a quantitative super-resolution microscopy workflow: (i) to increase sensitivity and throughput, (ii) to detect structural defects in PCD patients' cells, and (iii) to quantify motility defects caused by yet to be found PCD genes. Toward these goals, we built a localization map of PCD proteins by three-dimensional structured illumination microscopy and implemented quantitative image analysis and machine learning to detect protein mislocalization, we analyzed axonemal structure by stochastic optical reconstruction microscopy, and we developed a high-throughput method for detecting motile cilia uncoordination by rotational polarity. Together, our data show that super-resolution methods are powerful tools for improving diagnosis of motile ciliopathies.