Guangxi Key Laboratory of Superior Timber Trees Resource Cultivation, Guangxi Forestry Research Institute, Nanning 530002, China.
G3 (Bethesda). 2021 Apr 15;11(4). doi: 10.1093/g3journal/jkab070.
MicroRNAs (miRNAs) are small noncoding RNAs (18-24 nt) and function in many biological processes in plants. Although Eucalyptus trees are widely planted across the world, our understanding of the miRNA regulation in the somatic embryogenesis (SE) of Eucalyptus is still poor. Here we reported, for the first time, the miRNA profiles of differentiated and dedifferentiated tissues of two Eucalyptus species and identified miRNAs involved in SE of Eucalyptus. Stem and tissue culture-induced callus were obtained from the subculture seedlings of E. camaldulensis and E. grandis x urophylla and were used as differentiated and dedifferentiated samples, respectively. Small RNA sequencing generated 304.2 million clean reads for the Eucalyptus samples (n = 3) and identified 888 miRNA precursors (197 known and 691 novel) for Eucalyptus. These miRNAs were mainly distributed in chromosomes Chr03, Chr05, and Chr08 and can produce 46 miRNA clusters. Then, we identified 327 and 343 differentially expressed miRNAs (DEmiRs) in the dedifferentiation process of E. camaldulensis and E. grandis x urophylla, respectively. DEmiRs shared by the two Eucalyptus species might be involved in the development of embryonic callus, such as MIR156, MIR159, MIR160, MIR164, MIR166, MIR169, MIR171, MIR399, and MIR482. Notably, we identified 81 upregulated and 67 downregulated miRNAs specific to E. camaldulensis, which might be associated with the high embryogenic potential. Target prediction and functional analysis showed that they might be involved in longevity regulating and plant hormone signal transduction pathways. Further, using the gene expression profiles, we observed the negative regulation of miRNA-target pairs, such as MIR160ARF18, MIR396GRF6, MIR166ATHB15/HD-ZIP, and MIR156/MIR157SPL1. Interestingly, transcription factors such as WRKY, MYB, GAMYB, TCP4, and PIL1 were found to be regulated by the DEmiRs. The genes encoding PIL1 and RPS21C, regulated by upregulated miRNAs (e.g., egd-N-miR63-5p, egd-N-miR63-5p, and MIR169,) were downregulated exclusively in the dedifferentiation of E. camaldulensis. This is the first time to study the miRNA regulation in the dedifferentiation process of Eucalyptus and it will provide a valuable resource for future studies. More importantly, it will improve our understanding of miRNA regulation during the somatic embryogenesis of Eucalyptus and benefit the Eucalyptus breeding program.
微 RNA（miRNA）是一类小型非编码 RNA（18-24nt），在植物的许多生物学过程中发挥作用。尽管桉树在全球范围内广泛种植，但我们对桉树体细胞胚胎发生（SE）过程中的 miRNA 调控机制仍知之甚少。在这里，我们首次报道了两种桉树的分化和去分化组织的 miRNA 图谱，并鉴定了参与桉树 SE 的 miRNA。从赤桉和尾巨桉杂交无性系幼苗的继代培养中获得茎和组织培养诱导的愈伤组织，分别作为分化和去分化样本。对桉树样本进行小 RNA 测序，共获得 30420 万条清洁读数（n=3），鉴定出 197 个已知和 691 个新的桉树 miRNA 前体。这些 miRNA 主要分布在 Chr03、Chr05 和 Chr08 染色体上，并能产生 46 个 miRNA 簇。然后，我们分别鉴定出赤桉和尾巨桉去分化过程中 327 和 343 个差异表达的 miRNA（DEmiRs）。两种桉树共有的 DEmiRs 可能参与了胚胎愈伤组织的发育，如 MIR156、MIR159、MIR160、MIR164、MIR166、MIR169、MIR171、MIR399 和 MIR482。值得注意的是，我们鉴定出 81 个上调和 67 个下调的仅在赤桉中特异性表达的 miRNAs，这些 miRNAs 可能与赤桉高胚胎发生潜力有关。靶基因预测和功能分析表明，它们可能参与长寿调控和植物激素信号转导途径。此外，我们还观察到 miRNA 靶基因对的负调控，如 MIR160ARF18、MIR396GRF6、MIR166ATHB15/HD-ZIP 和 MIR156/MIR157SPL1。有趣的是，发现转录因子如 WRKY、MYB、GAMYB、TCP4 和 PIL1 受到 DEmiRs 的调控。基因编码 PIL1 和 RPS21C，受上调 miRNA（如 egd-N-miR63-5p、egd-N-miR63-5p 和 MIR169）调控，在赤桉去分化过程中特异性下调。这是首次研究桉树去分化过程中的 miRNA 调控，为今后的研究提供了有价值的资源。更重要的是，它将提高我们对桉树体细胞胚胎发生过程中 miRNA 调控的认识，有利于桉树的育种计划。
