For millennia, the diploid dioecious shrub Cannabis sativa, has been cultivated for its fiber, grain, and pharmacological properties [1]. Originating in Asia and now grown globally [2], C. sativa is a multibillion-dollar cash crop [3, 4] with many uses [5] including feed [6], textile [7], biofuel [8], medicine [9], and recreation [10, 11]. The latter two of which are due to the production of biologically active metabolites, known as cannabinoids, within the cannabis trichomes [12]. Cannabinoids are a large chemical family with 130 currently known distinct chemical species [13] grouped into 10 structural types [14]. Undeniably, the best understood are the cannabidiol (CBD) [15] and psychoactive Δ⁹-tetrahydrocannabinol (THC) [16]. Given their growing therapeutic uses and social acceptance, efforts are underway to produce chemotypes with explicit cannabinoid content [17, 18], platforms for heterologous production [19], and novel cannabinoid analogues [20].

The understanding of cannabinoid biosynthesis has made strides in recent years [12, 19, 21]. Briefly, cannabinoids are synthesized from the convergence of a fatty acid and terpene pathway. Hexanoic acid is activated into hexanoyl-CoA by acyl-activating enzyme 1 and subsequently elongated with three molecules of malonyl-CoA by TETRAKETIDE SYNTHASE (TSK, also known as OLIVETOL SYNTHASE). TKS functions in concert with OLIVETOLIC ACID CYCLASE to generate olivetolic acid. Geranyl pyrophosphate produced from the non-mevalonate-dependent isoprenoid pathway (MEP) is used by CANNABIGEROLIC ACID SYNTHASE to prenylate olivetolic acid into cannabigerolic acid (CBGA), the first bona fide cannabinoid. CBGA can then be shunted into one of at least three oxidocyclization subbranches to produce tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), and cannabichromenic acid (CBCA) by THCA SYNTHASE, CBDA-SYNTHASE, and CBCA SYNTHASE, respectively. Later, THCA, CBDA, CBCA may undergo nonenzymatic decarboxylation into the better-known THC, CBD, or cannabichromene (CBC). Lastly, cannabinoids may also undergo spontaneous rearrangements that are responsible for the structural variation found within this chemical family [12, 13, 22].

Despite the recent advances in the understanding of the cannabinoid biosynthetic pathway, the origin of hexanoic acid (also known as caproic acid) remains unclear. It is often the rate-limiting step in heterogenous systems [20, 23] requiring hexanoic acid as a feedstock [19]. In planta, trichome-specific transcriptomic analysis observed co-expression of genes involved in oxylipin biosynthesis, namely LIPOXYGENASE (LOX) and HYDROPEROXIDE LYASE (HPL), with genes previously established in cannabinoid biosynthesis [24, 25]. This has prompted the hypothesis that the oxylipin pathway provides the substrate required for cannabinoid biosynthesis. Further, this putative oxylipin-cannabinoid interaction invites exploration to understand the role of the oxylipins in cannabinoid biosynthesis and Cannabis biology.

Oxylipins are a large group of oxidized fatty acid-derivatives possessing potent signaling activities that govern a multitude of physiological processes including, growth, development, and defense [26]. In plants, the majority of oxylipin biosynthesis occurs via the LOX pathway. It begins with the regio- and stereo-specific incorporation of molecular oxygen at either the 9^th—or 13^th -carbon of linoleic (C18:2) or linolenic (C18:3) acid by 9- or 13-LOX isoforms [27]. The resulting 9- or 13-oxylipins can be fluxed into at least one of seven sub-branches to produce chemically diverse groups of distinct chemical species, including alcohols, aldehydes, divinyl ethers, esters, epoxides, hydroxides, hydroperoxides, ketols, ketones, and triols. Though the biological role for the vast majority of plant oxylipins remains to be deciphered, investigations of selected members of 13-oxylipins, namely jasmonates and green leaf volatiles (GLVs) have spearheaded a framework towards understanding their physiological and ecological roles.

Jasmonates are best known for their roles in providing defense against insects and necrotrophic pathogens [28]. Jasmonates are cyclopentones produced through the ALLENE OXIDE SYNTHASE (AOS) subbranch [29, 30]. Following the oxygenation of C18:3 to 13[S]-hydroperoxyoctadecatrienoic acid (13-HPOT) by 13-LOX activity, AOS [29, 31, 32] converts 13-HPOT to 12,13(S)-epoxy-octadecatrienoic acid (12,13-EOT) and ALLENE OXIDE CYCLASE (AOC) [33] converts 12,13-EOT to (+)-cis-12-oxo-phytodienoic acid (12-OPDA), the first jasmonate in the pathway. LOX, AOS, and AOC are closely associated with each other and participate in substrate channeling during jasmonate biosynthesis [34]. Aside from serving as a substrate for downstream reactions, 12-OPDA possesses its own distinct activity [35–37]. 12-OPDA is reduced via 12-OXO-PHYTODIENOIC ACID REDUCTASE (OPR) [38, 39] to 9S,13S-OPDA to 3-oxo-2-(2′[Z]-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0) and then undergoes three rounds of β-oxidations to produce (+)-7-iso-jasmonic acid (JA) [40]. Hexadecatrienoic acid (C16:3) may also serve as a substrate for jasmonate biosynthesis, eliminating the need for a round of β-oxidation [41–43]. While a multitude of JA derivates have been identified [29], (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile) is the best studied, owed to its service as a ligand in receptor-mediated perception and subsequent signaling [44].

GLVs are best known as the scent of cut grass and are involved in defense signaling, plant-to-plant and plant-to-insect communication [45]. They are C₆ volatile aldehydes, alcohols, and their acetyl esters produced through the HPL subbranch [46, 47]. Here, the 13-hydroperoxy fatty acids produced from 13-LOX activity from either C18:2 or C18:3 are cleaved, respectively, by 13-HPL [48–50] into the hexanal or (3Z)-hexenal and 12-oxo-(9Z)-dodecenoic acid. The latter of which is the progenitor of the traumatin sub-group, of which some members display signaling activity independent of GLVs [51]. Additionally, C16:3 may also serve as a fatty acid substrate for GLV biosynthesis, yielding (7Z)-10-oxo-decenoic acid in place of traumatin [52]. The GLV aldehydes are reduced to alcohols through reductase [53] and acetylated through acetyltransferase [54]. Finally, (3Z)-hexenal can also be isomerized through (3Z):(2E)-ENAL ISOMERASE [55, 56].

Thus, this study sought to elucidate the Cannabis oxylipin biosynthetic pathway as a source for cannabinoid substrate and molecular signals important in defense and development. Here, a census was conducted on the C. sativa genome to catalog the major oxylipin biosynthetic genes from the LOX, AOS, AOC, and OPR gene families. Respectively, their chromosomal locations, conserved protein domains, genetic structures, and phylogenetic relationships were determined. Collinearity analysis identified several genes in syntenic regions with Arabidopsis and tomato. The promoter analysis revealed evidence for tissue- and stimulus-specific transcriptional regulation, which stands in agreement with expression observed from a Cannabis transcriptome atlas and publicly-available transcriptomes. Finally, gene co-expressional network and functional enrichment analysis identified genetic modules that implicate oxylipin biosynthetic genes in distinct Cannabis physiological processes.