The FF of C. sativa have historically been the most utilized part of the plant due to their psychotropic and medicinal properties. In plant physiology, secondary metabolite production is commonly associated with defense responses against biotic and abiotic stresses. In cannabis, this defensive role is largely mediated by glandular trichomes—specialized epidermal structures on FF that synthesize and store a wide array of secondary metabolites, including cannabinoids and terpenoids. To gain deeper insight into the functional roles of trichome-specific genes identified in CannAtlas (Barbosa-Xavier et al., 2024), we performed enrichment analyses for Gene Ontology (GO), metabolic pathway (KEGG), and TF families. The enriched GO and KEGG terms strongly point to the biosynthesis, modification, and storage of secondary metabolites (Figure 4).

Among the enriched GO terms (Figure 4a), S-adenosylmethionine-dependent methyltransferase activity and methylation are particularly relevant, reflecting the role of methylation in enhancing the bioactivity, chemical stability, and defense potential of secondary metabolites such as cannabinoids and terpenoids (C. Zhang et al., 2021). Additional enrichment in ion binding, heme binding, and monooxygenase activity (Figure 4a) highlights the contribution of cytochrome P450 enzymes, which participate in the structural diversification of secondary metabolites and modulate their chemical properties (Layer et al., 2010). The enrichment of oxidoreductase activity (Figure 4a) underscores the oxidative processes necessary for synthesizing bioactive compounds. Terpene synthase activity (Figure 4a) was also significantly enriched, consistent with the role of volatile terpenes in repelling herbivores and inhibiting microbial pathogens (MacWilliams et al., 2023). GO terms related to the diterpenoid biosynthesis and lipid metabolism (Figure 4a) support the production of cannabinoids and their fatty acid precursors. KEGG enrichment analysis (Figure 4b) corroborated these patterns. Enrichment of unsaturated fatty acid biosynthesis and fatty acid metabolism point to the generation of precursors for signaling molecules like jasmonic acid, a key hormone in plant defense (Luo et al., 2019; Marks et al., 2009). The enrichment of sesquiterpenoid and triterpenoid biosynthesis highlights the trichome role in producing antimicrobial and insect-repellent compounds, while the broad enrichment of secondary metabolite biosynthesis emphasizes the specialized metabolic capacity of trichomes. TF enrichment (Figure 4c) revealed the SRS (SHI-related sequence) family as significantly overrepresented. This TF family regulates both trichome development and secondary metabolism (J. Zhang et al., 2020), suggesting that SRS TFs coordinate structural and chemical components of the cannabis defense system.Taken together, these results highlight cannabis glandular trichomes as hubs of defensive metabolism, integrating gene regulation, specialized metabolite biosynthesis, and adaptive responses to environmental challenges.

Cannabinoids and terpenes are the most extensively investigated compounds in C. sativa for their medicinal or recreational importance. A detailed understanding of their metabolic pathways is essential for strategies aimed at manipulating metabolite content in cannabis plants. To identify key genes involved in these pathways, we performed BLAST searches in CannAtlas using proteins previously characterized in cannabinoid and terpenoid biosynthesis (Kovalchuk et al., 2020), including those from upstream pathways such as polyketide, methylerythritol (MEP), and mevalonate (MEV) (Kovalchuk et al., 2020). This search identified 42 genes (Table S6). Given that these secondary metabolites are synthesized and stored in glandular trichomes of FF, we assessed their expression in flower and trichome samples (Figure 5). The polyketide pathway, which produces olivetolic acid, involves three core enzymes: Acyl-Activating Enzymes (AAEs), Olivetol Synthase (OLS), and Olivetolic Acid Cyclase (OAC), with OLS and OAC being directly implicated in cannabinoid biosynthesis (Thomas et al., 2020). We identified 13 polyketide-related genes (Table S6), including six AAEs classified as “expressed-in-all” and two as “mixed” in CannAtlas. Two OLS genes (F8388_010207, F8388_010208) and two OAC genes (F8388_012844, F8388_012845) showed trichome-specific expression, while another OLS gene (F8388_010206) was enriched in trichomes and IMF (Table S7). Of the eight MEP pathway genes identified (Table S6), four were expressed in all samples, two showed trichome-specific expression—including HDS (F8388_013291) and HDR (F8388_013980)—and one was leaf-specific (Table S7). All six MEV pathway genes were classified as “expressed-in-all”. Notably, isopentenyl-diphosphate delta-isomerase (IDI), which interconverts the MEP- and MEV-derived IPP and DMAPP to form the isoprenoid backbone (Krause et al., 2023), was represented by two genes, one of which (F8388_016754) was trichome-specific. Together, these results indicate that genes from the polyketide and MEP pathways are preferentially expressed in trichomes. Importantly, no consistent expression differences were observed between hemp and marijuana varieties, nor between MF and FF, suggesting conservation of upstream cannabinoid biosynthetic pathways across chemotypes and sexual morphs.

We identified five genes directly involved in the cannabinoid biosynthetic pathway: three prenyltransferases (F8388_001433, F8388_008405, and F8388_013365), one Cannabidiolic Acid Synthase (CBDAS, F8388_004088), and one Tetrahydrocannabinolic Acid Synthase (THCAS, F8388_024901) (Table S6). Of these, THCAS and the prenyltransferase F8388_001433 were trichome-specific; CBDAS is group-enriched in FF and stems; the prenyltransferase F8388_008405 is mixed; and F8388_013365 is expressed-in-all tissues (Table S7). The classification of CBDAS as group-enriched in FF and stems, rather than trichome-specific, likely reflects both chemotype distribution and the methodology used by Barbosa-Xavier et al. (2024), which relies on median expression across samples. Approximately 70% of the trichome samples in the dataset originated from type I chemotypes, which typically show low CBDAS expression, potentially underestimating its trichome specificity. A similar bias could have occurred for THCAS had the dataset been skewed toward type III chemotypes. These observations highlight how sample composition and classification thresholds can affect the apparent tissue specificity of cannabinoid biosynthesis genes.

Cannabigerolic acid (CBGA), the central precursor of cannabinoids, is synthesized via alkylation of OAC and GPP, a reaction catalyzed by a CsPT1 aromatic prenyltransferase (CBGAS) (Innes & Vergara, 2023). The gene F8388_001433 shows high sequence identity to several prenyltransferases (Table S6), including CsPT7 (98.4%), CsPT1 (83.9%), and CsPT4 (78.7%). CsPT4 and CsPT7 are closely related to CsPT1, and CsPT4 has also been shown to catalyze CBGA formation in vivo (Luo et al., 2019; Rea et al., 2019). These similarities strongly suggest that F8388_001433 functions as an aromatic prenyltransferase involved in CBGA biosynthesis. Expression data support this functional assignment: THCAS, CBDAS, and CBGAS (F8388_001433) were more highly expressed in trichomes than in whole flowers (Figure 5b). Moreover, THCAS and CBGAS were preferentially expressed in type I and II chemotypes, while CBDAS was more highly expressed in type II and III chemotypes (Figure 5b), consistent with their roles in defining cannabinoid profiles.

Unexpected expression patterns were detected in certain type III hemp samples. Trichome samples SAMN09747685, SAMN09747688, and SAMN09747691, labeled as type III hemp (Finola strain) (Livingston et al., 2020), displayed type II-like expression profiles (Figure 5b). These samples are biological replicates from BioProject PRJNA483805, which otherwise shows consistent type III expression. We hypothesize that these samples may have been inadvertently contaminated with trichomes from type I varieties, such as Purple Kush or Hindu Kush, which were analyzed alongside Finola by Livingston et al. (2020) using two-photon microscopy and disc cell counting analysis. A second discrepancy was observed in the samples SAMN42101035, SAMN42101034, and SAMN42101033 from the MW6-15 cultivar (BioProject PRJNA1128734). Although described by Welling et al. (2023) as an industrial hemp line (type III), these samples displayed a type I expression profile, characterized by high THCAS expression and complete absence of CBDAS (Figure 5b). Such inconsistencies may reflect sample mislabeling, cross-contamination, or previously unrecognized chemotypic diversity, and warrant further validation. Regarding terpenoid biosynthesis, we identified eight candidate genes (Table S6): three with root-specific expression, three with trichome-specific, one group-enriched in trichomes and IMF, and one with low expression (Table S7). Trichome-specific genes (F8388_008713, F8388_024573, F8388_026422) and the group-enriched gene (F8388_017921) all exhibited markedly higher expression in trichomes compared to whole flower samples (Figure 5). Importantly, their expression showed no clear bias toward specific chemotypes. This pattern suggests that terpenoid biosynthesis is broadly active across both hemp and marijuana varieties, likely independent of cannabinoid chemotype-defining genes. Overall, despite chemotypic differences between hemp and marijuana, genes in the upstream cannabinoid and terpenoid pathways show similar expression profiles (Figure 5), highlighting conserved mechanisms of defense specialization in C. sativa.