Understanding the role of oxylipins in Cannabis to enhance cannabinoid production

Abstract

Phytocannabinoids are medically important specialized defense compounds that are sparsely distributed among plants, yet Cannabis sativa can synthesize unprecedented amounts of these compounds within highly specialized surface cell factories known as glandular trichomes. The control mechanisms that allow for this high level of productivity are poorly understood at the molecular level, although increasing evidence supports the role of oxylipin metabolism in phytocannabinoid production. Oxylipins are a large class of lipid-based oxygenated biological signaling molecules. Although some oxylipins are known to participate in plant defense, roles for the majority of the ca. 600 plant oxylipins are largely unknown. In this review, we examine oxylipin gene expression within glandular trichomes and identify key oxylipin genes that determine the fate of common lipid precursors. Mechanisms by which oxylipins may be interacting with phytocannabinoid metabolism, as well as specialized plant metabolism more broadly, are discussed and a model summarizing these contributions proposed.

Keywords: glandular trichomes; green leaf volatiles; jasmonates; lipoxygenase; oxylipins; specialized metabolites.

Figure 1

Role of oxylipins in plant growth, development, biotic and abiotic stress tolerance. Oxylipins play multifaceted roles in regulating plant growth, development, and responses to biotic and abiotic stresses. Jasmonates (JAs) mediate developmental processes, including lateral root formation, tuber formation, flowering, and trichome development. Several JAs are also known to attract pollinators and influence plant reproductive strategies. JAs activate defense genes in plants to protect against biotic and abiotic stress (nutrient deficiency, heavy metals [copper (Cu), cadmium (Cd)] and ozone (O₃)). Green leaf volatiles (GLVs) contribute to fruit ripening and act as direct defense molecules against pathogenic microbes. GLVs also induce other defense responses, such as attracting natural predators and promoting the production of specialized metabolites. Oxylipins like epoxy alcohols and divinyl ethers serve as direct defense molecules against various pests and pathogens. Image created in BioRender.com.

Figure 2

Spatial localization of the phytocannabinoid (PC) pathway in Cannabis. (A) Image of Cannabis female inflorescence. (B) Microscopic view of a capitate stalked glandular trichome on a perigonal bract. (C) Schematic representation of a capitate stalked glandular trichome, the major site of PC biosynthesis and storage. This consists of multicellular stalk (cream), stipe cells at the base of the trichome head (grey), secretory disc cells (light green) and large extracellular storage cavity (yellow). (D) The PC biosynthesis pathway in Cannabis. The methylerythritol phosphate (MEP) pathway takes place in non-photosynthetic chloroplasts of glandular trichome disc cells, while the polyketide pathway is localized in the cytosol. Geranyl pyrophosphate (GPP) is formed from the MEP pathway. The polyketide pathway requires an activated fatty acid starter unit (e.g., hexanoyl-CoA) and three molecules of malonyl-CoA and gives rise to olivetolic acid (OA). Prenylation with GPP by an aromatic prenyltransferase forms CBGA. This is exported to the extracellular cavity where cannabinoid synthases perform oxidative cyclization of the isoprenoid moiety of CBGA, forming the major acid PCs; CBDA and Δ⁹-THCA. In the final reaction, H₂O₂ is produced as a biproduct when CBGA is converted to PCs in the presence of O₂. Solid dark grey arrows represent known steps in pathways while the brown dashed arrows represent steps of the pathway that have not been resolved. OA, olevetolic acid; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl pyrophosphate; CBGA, cannabigerolic acid; CBDA, cannabidiolic acid; Δ⁹-THCA, Δ⁹-tetrahydrocannabinolic acid; AAE, acyl activating enzyme; TKS, tetraketide synthase; OA, olivetolic acid cyclase; GPPS, geranyl pyrophosphate synthase; aPT, aromatic prenyltransferase; CBGAS, cannabigerolic acid synthase, CBDAS, cannabidiolic acid synthase; D₉-THCAS, D9-tetrahydrocannabinolic acid synthase.

Figure 3

Key oxylipins in the oxylipin pathway. The oxylipin pathway starts with the hydroperoxidation of polyunsaturated fatty acids (PUFA) by lipoxygenase (LOX) enzymes. The position of oxygenation is either at the 9^th or 13^th carbon from the carbonyl end of the fatty acid. The resulting polyunsaturated hydroperoxides include 9-/13-hydroperoxy linoleic acid (9/13-HPOD) and 9-/13-hydroperoxy linolenic acid (9/13-HPOT). This reaction represents a key branchpoint for oxylipin biosynthesis and provides substrate for several enzymatic pathways, including allene oxide synthase (AOS), hydroperoxide lyase (HPL), divinyl ether synthase (DES), epoxy alcohol synthase (EAS) and peroxygenase (POX) pathways. The AOS and allene oxide cyclase (AOC) cascade leads to the formation of jasmonates (JAs), while the HPL branch leads to the production of green leaf volatiles (GLVs) and 12-oxo acids/traumatins. The other three pathways produce several groups of molecules that act in plant defense and signaling, including epoxy alcohols and divinyl ethers (e.g., colneleic acid). Abbreviations for compounds: α-LeA, α-linolenic acid; LA, Linoleic acid, 9-HPOD, (9S)-hydroperoxy-(10E,12Z)-octadecadienoic acid; 9-HPOT, (9S)-hydroperoxy-(10E,12Z,15Z)-octadecatrienoic acid; 13-HPOD, (13S)-hydroperoxy-(9Z,11E)-octadecadienoic acid; 13-HPOT, (13S)-hydroperoxy-(9Z,11E,15Z)- octadecatrienoic acid; epoxy alcohol, 13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoic acid; cis-(+)-OPDA, cis-(+)-12-oxo-phytodienoic acid; OPC-8, 3-oxo-2-(2-pentenyl)-cyclopentane-1-octanoic acid; (+)-7-iso-JA, jasmonic acid; (+)-7-iso-JA-Ile, jasmonic acid isoleucine conjugate;. (9Z)-traumatin, 12-oxo-(9Z)-dodecenoic acid; (10E)-traumatin, 12-oxo-(10E)-dodecenoic acid, (2E)-traumatic acid; (2E)-dodecanedioic acid; JAR1, jasmonoyl amino acid conjugate synthase; OPR, 12-oxo-phytodienoic acid reductase; AOC, allene oxide cyclase, HI, hexenal isomerase. 2-AR, 2-alkyl reductase.

Figure 4

Phylogenetic relationship of LOX proteins and tissue series gene expression in Cannabis. (A) Phylogenetic relationship of lipoxygenase (LOX) proteins in Cannabis to several plant species, including Arabidopsis thaliana (AtLOX), Camelia sinensis (CamLOX), Nicotiana attenuata (NaLOX), Oryza sativa (OsLOX) Solanum lycopersicum (SlLOX), Vitis vinifera (VvLOX) and Zea mays (ZmLOX). Branches with Bootstrap values above 80 shown by greater line thickness (black). Cannabis LOX proteins are shown in bold. Functionally validated genes for corresponding LOXs are shaded as indicated by the key. Absolute expression (Log2 (mean TPM) of LOX genes in different tissue types of (B) high Δ⁹-THC and (C) high CBD chemotypes. RNA-Seq data reported by Jost et al. (2025). Protein sequences of Arabidopsis thaliana (At), Cannabis sativa (Cs), Camelia sinensis (Cam), Nicotiana attenuata (Na), Glycine max wild type (Gm) Oryza sativa (Os), Solanum lycopersicum (Sl) Zea mays (Zm) and Vitis vinifera (Vv) were used for phylogenetic analysis and PLAT/LH2 and lipoxygenase (LOX) domains identified using InterPro (v.98.0). Non-Cannabis sequences without both domains were removed from the analysis. Ninety-one higher plant LOX proteins were used for phylogenetic analysis. The Marchantia polymorpha (common liverwort) LOX3 protein served as an outgroup. The alignment was trimmed of spurious sequences and only residues of the Interpro lipoxygenase super family (IPR000907) domain was compared. The multiple sequence alignment was generated using clustal omega (ver. 1.2.2) in Geneious prime (ver. 2023.0.4), and the phylogenetic tree was generated using the neighbor-joining method (Sievers and Higgins, 2021) and visualized using Interactive Tree Of Life (iTOL; v. 6.8.1) (Letunic and Bork, 2007).

Figure 5

Structural features of GmLOX1 and CsLOX15. (A) Lipoxygenase (LOX) reaction mechanism, 13-LOX convert polyunsaturated fatty acids i.e. linoleic acid (LA) into (13S)-hydroperoxy-(9Z,11E)-octadecadienoic acid ((13S)-HPOD) by a two-step reaction involving the reduction of Fe³⁺ to Fe²⁺ by proton coupled electron transfer and then insertion of oxygen. (B) In silico 3D structure of CsLOX15 showing β- sheet rich N-terminal domain and α- helix rich C-terminal domain. (C) Coordination geometry of the catalytic site and Fe ligand of WT GmLOX1, showing important residues involved in iron coordination (H499, H504, H690, N694, I839, OH/H₂O, Q495, Q697, N694) and substrate binding (I553, L546, L754). (D) In silico coordination geometry of CsLOX15, around the Fe ligand (superimposed to (5 A⁰)) showing iron coordination, substrate binding residues, and an additional side chain residue W585. (E) Amino acid residues in substrate entry of GmLOX1 facilitating H binding network. (F) Probable amino acid residues in CsLOX15 substrate entry. (G) Key amino acid residues within GmLOX1 substrate cavity. (H) Key amino acid residues within CsLOX15 substrate cavity. Orange sphere, LOX Fe ligand; blue rod, water molecule; yellow dashed lines, polar contacts. The secondary structure was predicted using Phyre2 (V 2.0) (normal mode) with 100% confidence (Kelley et al., 2015) and the validation of the model was carried out using PROCHECK program in SAVES v6.1 ( Supplementary Figure 2 ). The 3D-structure was modelled using PYMOL (Ver. 3.3) by superimposing with GmLOX1 (PDB entry 1F8N).

Figure 6

Proposed model for the interactions between oxylipin and phytocannabinoid metabolism in Cannabis. LOX-AOS and LOX-HPL branches of the oxylipin pathway may be interacting with phytocannabinoid biosynthesis. Potential mechanisms could include JA-mediated transcriptional regulation of phytocannabinoid biosynthesis genes or by providing a carbon source via the breakdown of PUFA. Solid orange lines represent known interactions; orange dashed lines indicate other possible interactions. Trichome-specific Cannabis LOX candidates for GLV and JA synthesis are shown ( Table 1 ); Black dashed lines represent enzymatic reaction not yet fully explored in Cannabis. CsLOX15 (dark grey) has been functionally characterized, while other CsLOXs (turquoise) remain uncharacterized. Image created in BioRender.com PUFA, poly unsaturated fatty acids; 13-HPOD, (13S)-hydroperoxy-(9Z,11E)-octadecadienoic acid; 13-HPOT, (13S)-hydroperoxy-(9Z,11E,15Z)- octadecatrienoic acid; cis-(+)-OPDA, cis-(+)-12-oxo-phytodienoic acid; (+)-7-iso-JA, jasmonic acid; (+)-7-iso-JA-Ile, jasmonic acid isoleucine conjugate; (9Z)-traumatin, 12-oxo-(9Z)-dodecenoic acid; (10E)-traumatin, 12-oxo-(10E)-dodecenoic acid, (2E)-traumatic acid; (2E)-dodecanedioic acid; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; OA, olivetolic acid; GPP, geranyl pyrophosphate; DAD, defective in anther dehiscence protein, a phospholipase (PLA1); FAD2/3, fatty acid desaturase 2/3; CsLOX, Cannabis lipoxygenase; HPL, hydroperoxide lyase; AOS, allene oxide synthase; AOC, allene oxide cyclase; OPR, 12-oxo-phytodienoic acid reductase; JAR1, jasmonoyl amino acid conjugate synthase; HI, hexenal isomerase; 2-AR, 2-alkynal reductase; ALDH, aldehyde dehydrogenase; AAE, acyl activating enzyme; TKS, tetraketide synthase; OAC, olivetolic acid cyclase; GPPS, geranyl pyrophosphate synthase; aPT, aromatic prenyltransferase.