Institute of Horticultural Biotechnology, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.
Institute of Biotechnology, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350003, China.
Plant Physiol. 2023 Jul 3;192(3):1799-1820. doi: 10.1093/plphys/kiad139.
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.
龙眼体细胞胚胎发生（SE）是一个体外生物学过程，其中双极结构被诱导形成体细胞并再生为完整的植物。microRNA（miRNA）是植物 SE 的重要参与者。然而，miR408 在 SE 中的机制仍然难以捉摸。在这里，我们使用龙眼胚胎发生愈伤组织中的稳定转基因技术来验证 miR408 如何促进龙眼早期 SE 中的细胞分裂和分化。dlo-miR408-3p 通过靶向 previously unidentified gene nudix hydrolase 23 (DlNUDT23) 来调节核黄素生物合成，该基因介导 N6-甲基腺苷（m6A）修饰，并影响龙眼早期 SE 中的 RNA 稳态和细胞周期基因表达。我们表明，DlMIR408 过表达（DlMIR408-OE）促进了 21-nt miRNA 的生物合成。在 DlMIR408-OE 细胞系中，dlo-miR408-3p 靶向并下调 DlNUDT23，促进核黄素生物合成，减少黄素单核苷酸（FMN）积累，促进 m6A 水平，并影响 miRNA 稳态。DNA 复制、糖基磷脂酰肌醇（GPI）-锚生物合成、戊糖磷酸途径以及牛磺酸和次牛磺酸代谢也与核黄素代谢密切相关。在核黄素喂养实验中，dlo-miR408-3p 和 pre-miR408 上调，DlNUDT23 下调，增加了龙眼球形胚状体中的 m6A 水平和细胞分裂分化。当核黄素生物合成受到抑制时，dlo-miR408-3p 下调，DlNUDT23 上调，m6A 修饰减少，细胞分裂受到抑制，但细胞分化不受抑制。FMN 人工去甲基化 m6A 修饰会影响前体 miRNA 和 miRNA 的稳态。我们的研究结果揭示了 dlo-miR408-3p 激活核黄素生物合成的机制，其中 DlNUDT23 是靶向的，m6A 修饰是动态介导的，细胞分裂受到影响，从而促进植物早期 SE。
