Flowering stage- and cultivar-specific regulation of cannabinoid biosynthesis in hemp: An integrated transcriptomics and metabolomic analyses

Abstract

Cannabis sativa (hemp), produces cannabidiol (CBD), a major cannabinoid. Cultivars with stable cannabinoid levels enable efficient extraction and have substantial industrial value. However, cannabinoid content varies widely among cultivars, making it essential to understand the developmental and genotype-specific regulation for breeding. In this study, we investigated the molecular mechanisms underlying cannabigerolic acid (CBGA) and cannabidiolic acid (CBDA) biosynthesis by integrating transcriptome analysis with metabolite quantification of floral tissues. Using two hemp cultivars, CBG-Ambit and CBG-limonene, we compared cannabinoid accumulation and gene expression between early flowering stage, characterized by visible pistil emergence and early inflorescence development (approximately 2–3 weeks after flowering initiation), and the late flowering stage, defined by matured inflorescence with abundant glandular trichomes under non-pollinated female conditions (approximately 6–7 weeks after flowering initiation). Differential expression analysis identified over 2400 genes with stage- and cultivar-specific patterns. Key candidates included cytochrome P450 78A9 (CYP78A9), 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (HMGCR1), cannabidiolic acid synthase (CBDAS), tetrahydrocannabinolic acid synthase (THCAS), auxin transporter-like protein 2 (LAX2), myrcene synthase (MyrS), terpene synthase 29 (TS-29), geranylpyrophosphate (GPP), ABSCISIC ACID-INSENSITIVE 5-like protein 7 (ABI5), MYB transcription factor 1 (MYB1), histone-lysine N-methyltransferase (SUVH6), TIFY 10 A, tetraketide alpha-pyrone reductase 2 (TKPR2), ethylene-responsive transcription factor (ERF109), berberine bridge enzyme-like 8 (BBE8), and monoterpene synthase (MTS1). RT-qPCR validation confirmed stage- and cultivar-dependent gene expression. Functional enrichment analyses revealed that DEGs were primarily associated with secondary metabolism, oxidoreductase activity, and transcriptional regulation. Ultra-performance liquid chromatography (UPLC-MS) quantification revealed cannabinoid shifts; CBG-Ambit maintained elevated CBGA with limited conversion to CBDA, whereas CBG-limonene exhibited efficient CBGA-to-CBDA transition during late flowering. These findings indicate that developmental stage and genetic background jointly regulate cannabinoid biosynthesis, shaping cultivar-specific chemotypes. Overall, this study provides insight into CBD biosynthesis and facilitates genetic optimization for improved cannabinoid production in Cannabis sativa.