Data_Sheet_1_Integrated Four Comparative-Omics Reveals the Mechanism of the Terpenoid Biosynthesis in Two Different Overwintering Cryptomeria fortunei Phenotypes.docx

Chinese cedar (Cryptomeria fortunei) is a tree species with important ornamental, medicinal, and economic value. Terpenoids extracted from the essential oil of C. fortunei needles have been considered valuable ingredients in the pharmaceutical and cosmetic industries. However, the possible gene regulation mechanisms that limit terpenoid biosynthesis in this genus are poorly understood. Here, we adopted integrated metabolome analysis, transcriptome, small-RNA (sRNA), and degradome sequencing to analyze the differences in terpenoid regulatory mechanisms in two different overwintering C. fortunei phenotypes (wild-type and an evergreen mutant). A total of 1447/6219 differentially synthesized metabolites (DSMs)/unigenes (DEGs) were detected through metabolome/transcriptome analyses, and these DSMs/DEGs were significantly enriched in flavonoid and diterpenoid biosynthesis pathways. In C. fortunei needles, 587 microRNAs (miRNAs), including 67 differentially expressed miRNAs (DERs), were detected. Among them, 8346 targets of 571 miRNAs were predicted using degradome data, and a 72-miRNA-target regulatory network involved in the metabolism of terpenoids and polyketides was constructed. Forty-one targets were further confirmed to be involved in terpenoid backbone and diterpenoid biosynthesis, and target analyses revealed that two miRNAs (i.e., aly-miR168a-5p and aof-miR396a) may be related to the different phenotypes and to differential regulation of diterpenoid biosynthesis. Overall, these results reveal that C. fortunei plants with the evergreen mutation maintain high terpenoid levels in winter through miRNA-target regulation, which provides a valuable resource for essential oil-related bioengineering research.

柳杉（Chinese cedar, Cryptomeria fortunei）是一种兼具重要观赏、药用与经济价值的树种。从其针叶精油中提取的萜类化合物（terpenoids），被视为医药与化妆品工业中的珍贵原料。然而，该属植物中限制萜类生物合成的潜在基因调控机制，目前仍鲜为人知。本研究采用整合代谢组学分析、转录组学、小RNA（small-RNA, sRNA）及降解组测序（degradome sequencing）技术，对两种不同越冬表型的柳杉——野生型（wild-type）与常绿突变体（evergreen mutant）的萜类调控机制差异展开分析。通过代谢组/转录组分析，共检测到1447个差异合成代谢物（differentially synthesized metabolites, DSMs）与6219个差异表达单基因（unigenes, DEGs），上述DSMs与DEGs显著富集于黄酮类与二萜类生物合成通路。在柳杉针叶中，共检测到587个微小RNA（microRNAs, miRNAs），其中包括67个差异表达微小RNA（differentially expressed miRNAs, DERs）。基于降解组数据，共预测到571个miRNA的8346个靶基因（targets），并构建了涉及萜类与聚酮类（polyketides）代谢的72个miRNA-靶基因调控网络。进一步证实有41个靶基因参与萜类骨架（terpenoid backbone）与二萜类生物合成，靶基因分析显示，两个miRNA（即aly-miR168a-5p与aof-miR396a）可能与不同表型及二萜类生物合成的差异调控相关。综上，本研究结果表明，携带常绿突变的柳杉植株可通过miRNA-靶基因调控在冬季维持较高的萜类物质水平，这为精油相关的生物工程研究提供了宝贵的资源。