## 2 Results
### Functional Analysis of DEGs  
Gene Ontology (GO) enrichment analysis of the DEGs (L1-L2-L3-L4 vs. D1-D2-D3-D4) allows us to better understand the functions of genes. This analysis, based on all reference genes, categorizes functions into three categories (Figure S1). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the DEGs (L1-L2-L3-L4 vs. D1-D2-D3-D4) between light and darkness treatments showed that the DEGs were enriched in five aspects, namely, metabolism, genetic information processing, environmental information processing, cell processes, and organismal systems. The largest proportion of these DEGs was associated with metabolism, which included eleven sub-categories. In total, 136 genes were enriched in the metabolism of terpenoids and 185 were enriched in the biosynthesis of other secondary metabolites (Figure S1). Figure 4: Transcript length distribution, sample correlations, and differential gene expression in _L. cubeba_ under light and darkness treatments. (**A**) Transcripts were categorized into five groups of differing bp lengths based on alignment to the reference genome: <305 bp, 305–500 bp, 501–1500 bp, 1501–2000 bp, and >2000 bp. (**B**) Correlation analysis of 24 samples under light and darkness treatments. Higher correlation coefficients indicate greater inter-sample consistency and sequencing quality. (**C**) Comparison of the number of repetitive genes in the transcriptome. Overlapping regions represent shared genes. In total, 2290 and 3267 genes were uniquely expressed under darkness and light treatments, respectively. (**D**) The numbers of DEGs between different samples. The DEGs between the L1 (Light-0 h) and D1 (Dark-0 h) stages; the L2 (Light-6 h) and D2 (Dark-6 h) stages; the L3 (Light-12 h) and D3 (Dark-12 h) stages; and the L4 (Light-18 h) and D4 (Dark-18 h) stages. Up: significantly upregulated, down: significantly downregulated. Terpenoid content analysis revealed a significant decrease in _L. cubbeha_ monoterepenoid content during the D2 stage (Dark-6 h). To further identify key genes involved in terpenoid synthesis during the L2 and D2 stages, we performed GO and KEGG enrichment analyses on all DEGs from the L2 vs. D2 comparison. The GO analysis demonstrated that DEGs in L2 vs. D2 were significantly enriched in metabolic processes, comprising 843 upregulated genes and 696 downregulated genes. This was followed by enrichment in cellular processes, including 686 upregulated genes and 594 downregulated genes. Within the biological process category, only four pathways exhibited more downregulated than upregulated genes: localization, cellular component organization or biogenesis, immune system process, and positive regulation of biological process (Figure 6A). KEGG enrichment analysis revealed that 950 DEGs were collectively enriched in the global and overview maps pathway. The biosynthesis of carbohydrate metabolism pathway showed an enrichment of 327 DEGs. The amino acid metabolism pathway contained 190 enriched DEGs. These three pathways had the most abundant genes. Subsequent analysis identified 85 DEGs enriched in the metabolism of terpenoid and polyketides pathway. Among these, nine genes were closely associated with monoterepne synthesis, including six terpene syntheses (_Lcu09G_26017\(g\); _Lcu05G_15831\(g\); _Lcu01G_01644\(g\); _Lcu08G_22878\(g\); _novel.2952_; _Lcu01G_01640_g_), two short-chain dehydrogenases (_Lcu01G_00353_g and _Lcu01G_00332_g_), and one geraniol synthases (_Lcu06G_19648_g_) (Figure 6B). These DEGs are likely to be involved in terpenoid synthesis in _L. cubbeha_. Figure 5: Differential gene expression profiles. (**A**) Upregulationated (dark blue) and downregulated (sky blue) DEGs between light and darkness treatments (L1-L2-L3-L4 vs. D1-D2-D3-D4). The horizontal dashed line indicates the significance threshold (a _p_-value of <0.05). (**B**) Hierarchical clustering of the DEGs between light and darkness treatments (L1–L4 and D1–D4).