Comparative transcriptome analysis reveals putative transcription factors components of the regulatory network for cannabidiol biosynthesis in Cannabis sativa L.

Li Y.; Tang H.; Zhang T.; Li X.; Zheng B.; Cheng M.; Yang G.; Pan G. · Frontiers in plant science · 2026

**Background & Methods** This was a comparative transcriptome analysis study examining four cannabis tissues with distinct cannabidiol (CBD) content to identify transcriptional regulatory mechanisms controlling CBD biosynthesis. Researchers used Short Time-Series Expression Miner (STEM) analysis combined with strain-specific expression correlation analysis to identify differentially expressed genes and core transcription factors (TFs), followed by yeast one-hybrid and yeast two-hybrid assays to verify protein-DNA binding relationships and protein-protein interactions. **Key Findings** • Transcriptome analysis identified 1,314 genes specifically upregulated in female flowers, with 19 expression profiles identified via STEM clustering; Profile 8 contained 16 transcription factors and 6 CBD biosynthetic enzyme genes showing consistent expression patterns correlating with CBD content across tissues. • Two key transcription factors, CsMYB6 and CsMYB29, demonstrated strain-specific expression profiles positively correlated with CBD biosynthetic enzyme expression; CsMYB6 bound promoters of four CBD biosynthetic enzyme genes while CsMYB29 bound two promoter regions. • Protein interaction studies revealed CsMYB6 interacts with two regulatory proteins (CsMYB1 and CsWRKY1), while CsMYB29 interacts with CsWRKY1; both transcription factors localized to cell nuclei, consistent with their regulatory function. **Dosage & Administration** Not reported. **Safety & Adverse Effects** Not reported. **Evidence Quality** This is a molecular biology research review providing foundational mechanistic insights into CBD biosynthesis regulation. The study demonstrates moderate-to-good quality evidence through multiple complementary methodologies (transcriptomics, yeast assays, subcellular localization) validating identified transcription factors. However, significant limitations include: lack of functional validation through genetic manipulation studies; no in vivo confirmation of transcription factor effects on CBD production; examination limited to four tissue types; and inability to establish complete regulatory network components. The findings represent an important step toward understanding CBD biosynthesis mechanisms but require downstream functional studies to validate causal relationships and clinical translation potential for breeding applications.

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