The promoter regions of CBDAS and PT genes of cannabinoid biosynthesis in Cannabis sativa respond to phytohormones and stress-related signals.

Main conclusion: The functional characterization of promoter regions of CBDAS and PT genes of cannabinoids biosynthesis suggests that multiple factors including tissue-specific, phytohormones, and stress-related signals modulate their activity. Cannabis sativa L. has tremendous potential as a future crop for producing clinically important cannabinoid metabolites. While the cannabinoid biosynthetic pathway is largely known, the mechanistic details about its regulation are less understood. Decrypting the environmental and developmental factors regulating cannabinoid biosynthesis pathway may prove beneficial in pathway engineering and molecular breeding programs. Functional characterization of the promoter regions of key cannabinoid biosynthesis genes can provide useful insights into their transcriptional regulation. This study, therefore, is focused to uncover the role of different phytohormones and abiotic factors in influencing the activity of CsCBDAS and CsPT1 promoters through the development of promoter-GUS fusion expressing transgenic lines of Nicotiana tabacum. Spatial analysis across different tissues revealed that CsCBDAS and CsPT1 promoters drive a high level of GUS staining in leaf and flowers of the transgenic lines. A strong GUS staining was detected in the glandular trichomes of both tobacco transgenic lines. The results showed that out of the five hormones, three (IAA, GA₃, and SA) and four (IAA, GA₃, SA, and ABA) caused significant activation of CsCBDAS and CsPT1 promoters, respectively. While the light, heat, cold, salt, and wound stress induced promoter activity of both CsCBDAS and CsPT1, the drought stress was found to induce the activity of CsCBDAS promoter only. Validation of the expression patterns of these genes under different conditions in C. sativa through qRT-PCR suggested that phytohormones and abiotic factors may influence the cannabinoid biosynthesis in C. sativa by modulating their promoter activity.