Cannabinoids are synthesized in glandular stalked trichomes on the female flowers of Cannabis sativa (cannabis). The regulation of glandular trichome development has not been characterized in cannabis. We recently identified an R2R3-MYB transcription factor, CsMIXTA, which could be involved in trichome morphogenesis in cannabis. Some homologous genes of CsMIXTA are known to function in glandular trichome initiation in other plant species. CsMIXTA is highly expressed in flower tissue compared to vegetative tissues. Interestingly, CsMIXTA is also highly expressed in trichomes isolated from female flower tissue. In addition, CsMIXTA is upregulated during the peak stages of female flower maturation in correlation with some cannabinoid biosynthetic genes. Transient expression in Nicotiana benthamiana showed that CsMIXTA is localized in the nucleus. Furthermore, yeast transcriptional activation assay demonstrated that CsMIXTA has transactivation activity. Overexpression of CsMIXTA in Nicotiana tabacum resulted in higher trichome density, larger trichome size, and more branching on stalked glandular trichomes. The results indicate that CsMIXTA not only promotes glandular trichome initiation in epidermal cells, but also regulates trichome development in tobacco leaves. In this report, we characterized the novel function of the first cannabis transcription factor that may be critical for glandular trichome morphogenesis.

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.