## 3 Discussion  
The biosynthesis of terpenoids primarily relies on the MEP pathway (plastids) and MVA pathway (cytosol). Darkness indirectly regulates terpenoid biosynthesis genes by suppressing light-signaling transduction pathways. However, the molecular mechanism of dark-affecting terpenoid synthesis in _L. cubeba_ is unknown. To address this knowledge gap, we subjected _L. cubeba_ fruits to both light and darkness treatments. Light treatment exhibited no significant impact on the total monoterenoid content in _L. cubeba_ fruits (Figure 1A). However, certain monoterenoids, including \(\alpha\)-pinene, camphene, and \(\beta\)-pinene, showed significant increases following 6 h of light exposure, demonstrating higher light responsiveness compared to other monoterenoids. (Figure 1B). These results align with findings  
Figure 8: qRT-PCR validation of six differentially expressed key genes across four stages (D1–D4) under darkness treatment. _ACOT_ (acyl-CoA thioeaterase), _HMGS_ (hydroxymethylglutaryl-CoA synthase), _DXS_ (1-deoxy-D-xylulose-5-phosphate synthase), _DXR_ (1-deoxy-D-xylulose 5-phosphate reducto isomerase), _MDS_ (2-C-methyl-D-erythtrol-2,4-cyclodiphosphate synthase), and _TPS_ (terpene synthase). _UBC_ was used as an internal reference gene for normalization. D2, D3, and D4 are compared with D1. Student’s \(t\)-test was used for significance analysis. All data are expressed as means \(\pm\) SD of three biological replicates (\({}^{\star}\)_p_ \(<\) 0.05, \({}^{\star}\)_p_ \(<\) 0.01). in mint (_Mentha_ spp.) and wild mint (_Mentha arensis_), indicating that light influences monoterenoid accumulation [27; 52; 53]. Given these findings, we next constructed transcriptome sequencing libraries under light and darkness treatments and investigated gene expression changes during terpenoid synthesis in _L. cubeba_. Transcriptome-based candidate gene screening has proven to be an effective, precise, and robust tool for use in molecular breeding in various plant species, including _Camelia vietnamensis_ [PERSON], _Tanactum cocineurin_, _Dendrobium catenatum_, _Pyrus communis_, _Taxus chinensis_, and _Carthamus titorious_ L. [54; 55; 56; 57; 58]. In this study, we established two RNA-seq libraries and profiled gene expression in _L. cubeba_ fruits following light and darkness treatments. Approximately 7 Gb of high-quality reads were generated, yielding 37,791 unigenes. Principal component analysis (PCA) identified strong inter-sample correlations (Figure 4C). Through reference genome alignment, we annotated gene structures (e.g., length, initiation sites) and predicted gene function. Comparative analysis of _L. cubeba_ fruit transcriptomes following light and darkness treatments identified 6227 upregulated and 6847 downregulated DEGs, with some DEGs lacking annotations in public databases. These unannotated DEGs may represent _L. cubeba_-specific candidate genes involved in terpenoid biosynthesis, aligning with prior findings [6]. Through KEGG pathway enrichment analysis, which enables functional prediction of genes and the elucidation of their roles within metabolic networks [59], we found that light-responsive DEGs were potentially involved in pathways such as metabolism of terpenoids and polyketides, biosynthesis of other secondary metabolites, and signal transduction (Figure S1), which facilitated the identification of candidate terpenoid biosynthesis-related genes. Plant terpenoid biosynthesis is predominantly regulated by environmental cues, such as light [60], and plant hormones, such as methyl jasmonate [27; 61]. Monoterpenes are usually biosynthesized in plastids, with photosynthesis providing precursors, ATP, and NADPH [62]. Transcriptome analysis revealed that both the total terpenoid content and the levels of key monoterpenoids were significantly reduced under darkness treatment (Figure 1). Notably, darkness treatment significantly suppressed the expression of genes in the MVA and MEP pathways, particularly _LcHMGS1_, _LcPMK_, _LcDXS3_, _LcDXS6_, _LcGGPPS2_, _LcGGPPS4_, and _LcTPS42_[63] (Figure 7).