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Analysis of hormonal regulation of promoter activities of Cannabis sativa prenyltransferase 1 and 4 and salicylic acid mediated regulation of cannabinoid biosynthesis

Cannabis sativa prenyltransferase 4 (CsPT4) and prenyltransferase 1 (CsPT1) have been shown to catalyze the step in the cannabinoid biosynthetic pathway that generates cannabigerolic acid (CBGA), the substrate for the end-point enzymes that generate cannabidiolic acid (CBDA) and tetrahydrocannabinolic acid (THCA). Prior studies from our lab suggest that CBGA production rate-limits the pathway. There is a lack of understanding concerning how important cannabinoid biosynthetic genes are regulated as cannabinoid synthesis increases during female flower development. Both CsPT genes were shown to be highly expressed in flowers. The genes were also found to be present in leaves and roots. GUS staining also detected the promoter activities in leaves of seedlings, and the promoter activities were drastically stronger in the section of the sugar leaves where glandular trichomes are formed. In silico analysis of the two CsPT genes revealed several hormone and transcription factor responsive elements. Dual luciferase assays were conducted to determine whether a hormone could alter the promoter activities of CsPT1 and CsPT4. The results showed that CsPT4 pro was activated following treatment from salicylic acid (SA), gibberellic acid (GA), ethylene, ABA, and cytokinin, while the CsPT1 promoter was activated following SA, ethylene, ABA, and auxin treatment. In parallel studies, a correlation was observed between multiple cannabinoid biosynthetic pathway genes and SA application to the cannabis growing medium, along with a correlation between MeSA floral application and an increase in cannabinoid content. The results from all aspects of this study demonstrated an interaction between certain hormones and cannabinoid synthesis.