Terpene Synthases and Terpene Variation in Cannabis sativa

Abstract

Cannabis (Cannabis sativa) resin is the foundation of a multibillion dollar medicinal and recreational plant bioproducts industry. Major components of the cannabis resin are the cannabinoids and terpenes. Variations of cannabis terpene profiles contribute much to the different flavor and fragrance phenotypes that affect consumer preferences. A major problem in the cannabis industry is the lack of proper metabolic characterization of many of the existing cultivars, combined with sometimes incorrect cultivar labeling. We characterized foliar terpene profiles of plants grown from 32 seed sources and found large variation both within and between sets of plants labeled as the same cultivar. We selected five plants representing different cultivars with contrasting terpene profiles for clonal propagation, floral metabolite profiling, and trichome-specific transcriptome sequencing. Sequence analysis of these five cultivars and the reference genome of cv Purple Kush revealed a total of 33 different cannabis terpene synthase (CsTPS) genes, as well as variations of the CsTPS gene family and differential expression of terpenoid and cannabinoid pathway genes between cultivars. Our annotation of the cv Purple Kush reference genome identified 19 complete CsTPS gene models, and tandem arrays of isoprenoid and cannabinoid biosynthetic genes. An updated phylogeny of the CsTPS gene family showed three cannabis-specific clades, including a clade of sesquiterpene synthases within the TPS-b subfamily that typically contains mostly monoterpene synthases. The CsTPSs described and functionally characterized here include 13 that had not been previously characterized and that collectively explain a diverse range of cannabis terpenes.

Recent advances in Cannabis sativa genomics research

“…Cannabis genome assemblies other than cs10 also have much to offer. For instance, the PK, FN and the contig‐level Jamaican Lion trio assemblies have been key to confirming findings from earlier, lower‐resolution studies as well as uncovering important biology of the Cannabis genus (Van Bakel et al ., 2011; Laverty et al ., 2019; Prentout et al ., 2019; Vergara et al ., 2019; Booth et al ., 2020; McKernan et al ., 2020). Further improvement of these assemblies will assist in fully realising their potential.…”

“…Gene co‐expression network analysis identified functionally relevant cannabis terpene synthases ( CsTPS ) involved in mono‐ and sesquiterpene accumulation (Zager et al ., 2019). The differential expression of CsTPS genes among cultivars also has been linked to variation in terpene profiles of these cultivars (Booth et al ., 2020). However, the genes underlying the synthesis of many minor cannabinoids and associated expression patterns are less well‐defined (Pollastro et al ., 2011; Citti et al ., 2019; Welling et al ., 2019; Basas‐Jaumandreu & de las Heras, 2020).…”

“…Despite their therapeutic potential and similar biosynthetic origin, genetic prediction of terpene composition is challenging. Genomic analysis of 55 CsTPS genes suggests a complex genetic background characterized by CsTPS nonhomologous gene clusters and tandem duplication events (Allen et al ., 2019; Booth et al ., 2020; McKernan et al ., 2020). Further, the presence of extensive CNVs within the CsTPS‐a (sesquiterpene synthase) and CsTPS‐b (monoterpene synthase) gene subfamilies point to their highly diverse nature (Booth et al ., 2020).…”

“…The diversity of the CsTPS genes complicates studies of gene regulation at various levels. Terpene composition can vary between cultivars, tissue types, trichome morphotypes and across development, whilst nonenzymatic modifications such as the oxidation of β ‐myrcene cause variation independent of genomic and transcriptomic regulation (Marchini et al ., 2014; Aizpurua‐Olaizola et al ., 2016; Allen et al ., 2019; Livingston et al ., 2019; Booth et al ., 2020). Consequently, future transcriptional studies would need to consider gene‐environment interactions, as well as organ, tissue and cell‐type specificity.…”

The Cream of the Crop: Biology, Breeding and Applications of Cannabis sativa

“…Other studies have characterised the transcriptome of hemp lines grown under salinity and drought stress (Gao et al, 2018;Liu et al, 2016), as well as during bast fibre development (Behr et al, 2016;Guerriero et al, 2017). Three further studies have focused on sequencing the transcriptome of glandular trichomes, with the aim of profiling the expression of genes involved in terpene and phytocannabinoid biosynthesis (Booth et al, 2020;Livingston et al, 2020;Zager et al, 2019). Furthermore, two recent studies have focused on identifying the sex chromosomes based on characterising the expression of sex-linked genes in male and female plants (McKernan et al, 2020;Prentout et al, 2020).…”