Cannabinoids are predominantly produced in the glandular trichomes on cannabis female flowers. There is little known on how cannabinoid biosynthesis is regulated during female flower development. We aim to understand the rate-limiting step(s) in the cannabinoid biosynthetic pathway. We investigated the transcript levels of cannabinoid biosynthetic genes together with cannabinoid contents during 7 weeks of female flower development. We demonstrated that the enzymatic steps for producing cannabigerol (CBG), which involve genes GPPS, PT, TKS, and OAC, could rate limit cannabinoid biosynthesis. Our findings further suggest that upregulation of cannabinoid synthases, CBDAS and THCAS in a commercial hemp and medical marijuana variety, respectively, is not critical for cannabinoid biosynthesis. The cannabinoid biosynthetic genes are generally upregulated during flower maturation; increased expression occurs coincident with glandular trichome development and cannabinoid production in the maturing flower. The results also suggest that different cannabis varieties may experience discrete transcriptional regulation of cannabinoid biosynthetic genes. In addition, we showed that methyl jasmonate (MeJA) can potentially increase cannabinoid production. We propose that biweekly applications of 100 μM MeJA starting from flower initiation would be efficacious for promoting cannabinoid biosynthesis. Our findings provide important genetic information for cannabis breeding to generate new varieties with favorable traits.

SUMMARYCannabinoids are predominantly produced in the glandular trichomes on cannabis female flowers. There is little known on how cannabinoid biosynthesis is regulated during female flower development. We aim to understand the rate-limiting step(s) in the cannabinoid biosynthetic pathway.We investigated the transcript levels of cannabinoid biosynthetic genes as well as cannabinoid contents during 7 weeks of female flower development. We demonstrated that the enzymatic steps for producing CBG, which involve genes GPPS, PT and OAC, could be rate limit cannabinoid biosynthesis. Our findings further suggest that cannabinoid synthases, CBDAS and THCAS in a hemp and medical marijuana variety respectively, are not critical for cannabinoid biosynthesis. The cannabinoid biosynthetic genes are generally upregulated during flower maturation, which indicate glandular trichome development.MeJA can potentially increase cannabinoid production. We propose that biweekly application of 100 μM MeJA staring from flower initiation would be efficacious for promoting cannabinoid biosynthesis.Our findings suggest that the step of CBG production could rate limit the terminal cannabinoid biosynthesis. In addition, different cannabis varieties demonstrated discrete transcriptional regulation of cannabinoid biosynthetic genes.

Pathogen-associated molecular patterns, PAMPs, are a diverse group of molecules associated with pathogenic microbes and are known to activate immune response and in some cases enhance growth in plants. Two PAMPs, harpin and flg22, have shown these affects in various plant species. PAMPs are known to activate basal immunity, the ethylene signaling pathway, alter gene expression and change plant composition. Pretreatment with harpin enhanced hemp seedling resistance to Pythium aphanidermatum, while flg22 failed to induce the defense mechanism towards P. aphanidermatum. In the absence of the pathogen, both harpin and flg22 enhanced seedling growth when compared to the water control. Ethylene is a hormone involved in both plant defense signaling and growth. Both harpin and flg22 pretreatment induced certain ethylene responsive genes but not all the genes examined, indicating that harpin and flg22 act differently in ethylene and potentially defense signaling. In addition, both harpin and flg22 induced CsFRK1 and CsPR1, two marker genes for plant innate immunity. Both PAMPs can enhance growth but likely induce different defense signaling pathways.

Cannabissativa aromatic prenyltransferase 4 (CsPT4) and 1 (CsPT1) have been shown to catalyze cannabigerolic acid (CBGA) biosynthesis, a step that rate-limits the cannabinoid biosynthetic pathway; both genes are highly expressed in flowers. CsPT4 and CsPT1 promoter driven β-glucuronidase (GUS) activities were detected in leaves of cannabis seedlings, and strong CsPT4 promoter activities were associated with glandular trichomes. Hormonal regulation of cannabinoid biosynthetic genes is poorly understood. An in silico analysis of the promoters identified putative hormone responsive elements. Our work examines hormone-responsive elements in the promoters of CsPT4 and CsPT1 in the context of physiological responses of the pathway to the hormone in planta. Dual luciferase assays confirmed the regulation of promoter activities by the hormones. Further studies with salicylic acid (SA) demonstrated that SA pretreatment increased the expression of genes located downstream of the cannabinoid biosynthetic pathway. The results from all aspects of this study demonstrated an interaction between certain hormones and cannabinoid synthesis. The work provides information relevant to plant biology, as we present evidence demonstrating correlations between molecular mechanisms that regulate gene expression and influence plant chemotypes.

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.