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.

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The plant-specific homeodomain zipper family (HD-ZIP) of transcription factors plays central roles in regulating plant development and environmental resistance. HD-ZIP transcription factors IV (HDZ IV) have been involved primarily in the regulation of epidermal structure development, such as stomata and trichomes. In our study, we identified nine HDZ IV-encoding genes in Cannabis sativa L. by conducting a computational analysis of cannabis genome resources. Our analysis suggests that these genes putatively encode proteins that have all the conserved domains of HDZ IV transcription factors. The phylogenetic analysis of HDZ IV gene family members of cannabis, rice (Oryza sativa), and Arabidopsis further implies that they might have followed distinct evolutionary paths after divergence from a common ancestor. All the identified cannabis HDZ IV gene promoter sequences have multiple regulation motifs, such as light- and hormone-responsive elements. Furthermore, experimental evidence shows that different HDZ IV genes have different expression patterns in root, stem, leaf, and flower tissues. Four genes were primarily expressed in flowers, and the expression of CsHDG5 (XP_030501222.1) was also correlated with flower maturity. Fifty-nine genes were predicted as targets of HDZ IV transcription factors. Some of these genes play central roles in pathogen response, flower development, and brassinosteroid signaling. A subcellular localization assay indicated that one gene of this family is localized in the Arabidopsis protoplast nucleus. Taken together, our work lays fundamental groundwork to illuminate the function of cannabis HDZ IV genes and their possible future uses in increasing cannabis trichome morphogenesis and secondary metabolite production.

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.