In addition to the well-known diterpenoid steviol glycosides, Stevia rebaudiana (Stevia) produces many labdane-type diterpenoids and a wide range of mono- and sesquiterpenoids. However, biosynthesis of mono- and sesquiterpenoids in Stevia remains unknown. Here we analyzed the extracts of Stevia leaves, flowers, stems, and roots by Gas Chromatography–Mass Spectrometry and putatively identified a total of 69 volatile organic compounds, most of which were terpenoids with considerably varied quantities among the four tissues of Stevia. Using Stevia transcriptomes, we identified and functionally characterized five terpene synthases (TPSs) that produced major mono- and sesquiterpenoids in Stevia. Transcript levels of these Stevia TPSs and levels of corresponding terpenoids correlated well in Stevia tissues. Particularly, the root-specific SrTPS4 and SrTPS5 catalyzed the formation of γ-curcumene/zingiberene/β-sesquiphellandrene and α-longipinene/β-himachalene/himachalol as multifunctional sesqui-TPSs, respectively. Most of the SrTPSs were highly responsive to various environmental stresses in a tissue-specific manner. Taken together, our results provide new insights into how Stevia produces diverse terpenoids to confer differential responses to various environmental factors in each tissue.

In recent years, the perspective towards the use of cannabis has slowly shifted from being an illicit drug to a medicinal plant. The pathway and enzymes involved in the production of cannabinoids are known; however, studies evaluating the production of cannabinoids in heterologous plants and cell cultures are still limited. In this study, we assessed the potential use of N. benthamiana (Nicotiana benthamiana) plants as a heterologous host for producing natural and novel cannabinoids. Transgenic N. benthamiana plants expressing genes encoding cannabis acyl-activating enzyme and olivetol synthase were generated, which were then used for transiently expressing other downstream pathway genes. Production of olivetolic acid and divarinic acid, the universal precursors for major and minor cannabinoids, respectively, was observed in transgenic N. benthamiana plants. To produce novel cannabinoid precursors with different side chains, various fatty acids were infiltrated into the transgenic N. benthamiana plants and the production of novel derivatives was observed. Although we were not able to derive the core intermediate, cannabigerolic acid, from our transgenic plants, possibly due to the low production levels of the precursors, our transgenics plants still serve as a high-potential platform for further development and exploring the N. benthamiana chemical space for generating novel cannabinoids.

Cannabis is one of the few plant genera capable of producing cannabinoids, the effects of which are synergized by terpene interactions. As the biosynthesis of both metabolite classes requires the same intracellular feedstocks, this work describes the coordinated regulation of global metabolic pathways that allows for their joint copious production in vivo. To this end, a transcriptomics-based approach to characterize the glandular trichomes of five Cannabis cultivars was pursued. Besides revealing metabolic traits that enhanced and proportionated the supply of critical carbon precursors, in-depth analysis showed significantly increased gene expression of two particular enzymes to meet the huge nicotinamide adenine dinucleotide phosphate (NADPH) demand of secondary metabolite production. Furthermore, it led to a hypothesis that the methyl-d-erythritol 4-phosphate pathway might be utilized more than the mevalonic acid pathway in Cannabis trichomes. While both pathways were found to be activated in a modular and calibrated way that reflected their broad participation in physiological processes, the genes for hexanoate, cannabinoid, and terpene biosynthesis were, in contrast, up-regulated in an en bloc and multi-loci manner due to their specific roles in secondary metabolite production. In addition, three new terpene synthases were characterized based on both in silico and experimental assays. Altogether, the study enhances the current understanding of secondary metabolite production in Cannabis cultivars, which may assist in their characterization and development.