Abstract Old Yellow Enzymes (OYEs) are a widely distributed family of ene -reductases that were first described in a Saccharomyces cerevisiae ferment. In plants, cis -12- oxo -phytodienoic acid ( cis -OPDA) reductase (OPR) is the best studied OYE. In Arabidopsis thaliana , the peroxisomal AtOPR3 was characterized as the major OPDA reductase, which generates 3- oxo -2-(2-pentenyl)-cyclopentane-1-octanoic acid in the jasmonic acid (JA) biosynthesis. In Atopr3 lines, only small amounts of JA are detectable after wounding. Here, we describe an OPR-like enzyme (named BnOPR) from the gram-positive Brevibacillus nitrificans . The sequence was identified in an early version of the Physcomitrium patens genome and is assumed to be a contamination by a bacterium growing in association with P. patens . In complementation experiments with an Atopr3 line, we demonstrate that expression of BnOPR, fused with a peroxisomal targeting signal, rescues the male infertile phenotype and increases JA and JA-Ile levels. The catalytic parameters of BnOPR were determined for a set of substrates, including cis -OPDA and prednisone. Interestingly, B. nitrificans, B. brevis , and Paenibacillus physcomitrellae were shown to have a positive effect on P. patens growth. Highlight The bacterial enzyme BnOPR rescues the male infertile phenotype of Atopr3 plants.

Oxylipins are oxygenated products of fatty acids proposed to exert a regulatory role in cnidarian-dinoflagellate symbiosis; however, this has not been investigated in detail. We integrated physiological measurements and molecular phenotyping with comparative transcriptome mining to examine how the symbiotic cnidarian model, the sea anemone Aiptasia (i.e. Exaiptasia diaphana), and its dinoflagellate symbiont Breviolum minutum respond to symbiosis and thermal stress. We performed lipidomics in combination with the quantification of oxylipins, including octadecanoids, eicosanoids, and docosanoids derived from C18, C20, and C22 fatty acids, respectively, and reconstructed their putative biosynthetic routes through cross-phylogenetic protein sequence homology. Relative to aposymbiotic, symbiotic anemones were enriched with omega-3 fatty acids and downstream octadecanoids of symbiont origin, consistent with inter-partner metabolite flux. Cytochrome P450-derived eicosanoids and docosanoids increased up to 300-fold in symbiotic versus aposymbiotic anemones. Under elevated temperature, anemones showed minor changes in their physiology and lipid profiles; however, the symbiont fraction displayed multiple signatures of stress. In comparison, aposymbiotic anemones showed a 50% reduction in protein abundance as well as structural and storage lipids, while simultaneously accumulating oxylipins linked to inflammation and oxidative stress. Our findings report novel oxylipins that have not been previously observed in dinoflagellates. We identified regulatory pathways that are conserved across cnidarians and higher metazoans, advancing our understanding of cnidarian-dinoflagellate symbiosis and its response to warming climate. We are targeting specific oxylipins and signalling pathways for further research that may aid molecular intervention strategies for selective breeding and assisted evolution to enhance coral resilience in warming oceans.

Oxylipins are oxygenated products of fatty acids that are being increasingly studied due to their role in multiple physiological processes. Investigations to date have focused on the 20-carbon eicosanoids and 22-carbon docosanoids. However, more recently, interest has grown into the 18-carbon octadecanoids. A significant obstacle in the study of these compounds is a lack of authentic standards for functional studies as well as for development of methods for their quantification. We developed a fast and simple one-step synthetic strategy to produce mono-hydroxylated metabolites based on the photosensitized oxidation of 18-carbon polyunsaturated fatty acids (PUFAs). Four different PUFAs including α-linolenic acid (ALA, ω3), γ-linolenic acid (GLA, ω6), stearidonic acid (SDA, ω3), and octadecapentaenoic acid (ODPA, ω3) were photooxidized in the presence of methylene blue and oxygen under exposure to light from a halogen lamp. The uncommon PUFA ODPA was prepared from docosahexaenoic acid (DHA, ω3) in a 6-step synthesis with 30% overall yield. The hydroperoxide products were reduced with sodium borohydride and the mono-hydroxylated octadecanoids were separated by HPLC. Product identification was performed by GC-MS. The final purities of isolated products ranged from 80% to 98%, with oxidation of non-terminal double bonds being preferred. It is likely that this approach could be extended to PUFAs of varying chain length, suggesting that photosensitized oxidation could be employed to rapidly prepare hydroperoxides from multiple unsaturated fatty acids. As interest in oxylipins continues to increase, this approach will be useful for large-scale preparation of multiple standards for the study of these new compounds.

Lipid mediators are potent biomolecules that may help to gain specific information about biological processes. However, due to incomplete functional annotations and hence interpretive challenges these molecules are hardly considered in molecular profiling experiments. We hypothesized that correlating lipid mediators extracted from tissues with corresponding proteomics data and the corresponding Gene Ontology terms characteristic of specific tissue types could enable functional annotation of these lipids. Thirteen organs and tissues from sheep ( Ovis aries ) were analyzed using mass spectrometry-based untargeted proteomics and lipidomics. In total, 4717 proteins and 166 free fatty acids, oxylipins, lysolipids, endocannabinoids, and bile acids were catalogued. This included the previously uncharacterized oxylipin 4-hydroxy-eicosatetraenoic acid (4-HETE), whose identity was confirmed by chemical synthesis. Co-expression and clustering analyses validated our hypothesis, successfully reproducing known and suggesting novel lipid mediator functions. Tissue levels of polyunsaturated fatty acids (PUFAs) correlated with pro-resolving mediators and, unexpectedly, with the cellular protein synthesis and folding machinery. These findings suggest that PUFAs and their derivatives support protein synthesis fidelity and efferocytosis, both essential contributors for the effective resolution of inflammation. ### Competing Interest Statement The authors have declared no competing interest.

The octadecanoids are a broad class of lipids consisting of the oxygenated products of 18-carbon fatty acids. Originally referring to production of the phytohormone jasmonic acid, the octadecanoid pathway has been expanded to include products of all 18-carbon fatty acids. Octadecanoids are formed biosynthetically in mammals via cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) activity, as well as nonenzymatically by photo- and autoxidation mechanisms. While octadecanoids are well-known mediators in plants, their role in the regulation of mammalian biological processes has been generally neglected. However, there have been significant advancements in recognizing the importance of these compounds in mammals and their involvement in the mediation of inflammation, nociception, and cell proliferation, as well as in immuno- and tissue modulation, coagulation processes, hormone regulation, and skin barrier formation. More recently, the gut microbiome has been shown to be a significant source of octadecanoid biosynthesis, providing additional biosynthetic routes including hydratase activity (e.g., CLA-HY, FA-HY1, FA-HY2). In this review, we summarize the current field of octadecanoids, propose standardized nomenclature, provide details of octadecanoid preparation and measurement, summarize the phase-I metabolic pathway of octadecanoid formation in mammals, bacteria, and fungi, and describe their biological activity in relation to mammalian pathophysiology as well as their potential use as biomarkers of health and disease.

The present work reports the detection and cloning of a new CYP74 clan gene of the European lancelet (Branchiostoma lanceolatum) and the biochemical characterization of the recombinant protein CYP440A19. CYP440A19 possessed epoxyalcohol synthase (EAS) activity towards the 13-hydroperoxides of linoleic and α-linolenic acids, which were converted into oxiranylcarbinols, i.e., (11S,12R,13S)-11-hydroxy-12,13-epoxy derivatives. The conversion of 9-hydroperoxides produced distinct products. Linoleic acid 9(S)-hydroperoxide (9-HPOD) was mainly converted into 9,14-diol (10E,12E)-9,14-dihydroxy-10,12-octadecadienoic acid and macrolactone 9(S),10(R)-epoxy-11(E)-octadecen-13(S)-olide. In addition, (8Z)-colneleic acid was formed. Brief incubations of the enzyme with 9-HPOD in a biphasic system of hexane–water enabled the isolation of the short-lived 9,10-epoxydiene (9S,10R,11E,13E)-9,10-epoxy-11,13-octadecadienoic acid. The structure and stereochemistry of the epoxyalcohols, macrolactone, (8Z)-colneleic acid (Me), and 9,10-epoxydiene (Me) were confirmed by 1H-NMR, 1H-1H-COSY, 1H-13C-HSQC, and 1H-13C-HMBC spectroscopy. Macrolactone and cis-9,10-epoxydiene are novel products. The 9-hydroperoxide of α-linolenic acid was mainly converted into macrolactone 9(S),10(R)-epoxy-11(E),15(Z)-octadecadiene-13(S)-olide and a minority of divinyl ethers, particularly (8Z)-colnelenic acid. The versatility of enzyme catalysis, as well as the diversity of CYP74s and other enzymes involved in oxylipin biosynthesis, demonstrates the complexity of the lipoxygenase pathway in lancelets.

Wheat, an essential crop for global food security, is well adapted to a wide variety of soils. However, the gene networks shaping different root architectures remain poorly understood. We report here that dosage differences in a cluster of monocot-specific 12-OXOPHYTODIENOATE REDUCTASE genes from subfamily III (OPRIII) modulate key differences in wheat root architecture, which are associated with grain yield under water-limited conditions. Wheat plants with loss-of-function mutations in OPRIII show longer seminal roots, whereas increased OPRIII dosage or transgenic over-expression result in reduced seminal root growth, precocious development of lateral roots and increased jasmonic acid (JA and JA-Ile). Pharmacological inhibition of JA-biosynthesis abolishes root length differences, consistent with a JA-mediated mechanism. Transcriptome analyses of transgenic and wild-type lines show significant enriched JA-biosynthetic and reactive oxygen species (ROS) pathways, which parallel changes in ROS distribution. OPRIII genes provide a useful entry point to engineer root architecture in wheat and other cereals.

In plants, jasmonate signaling regulates a wide range of processes from growth and development to defense responses and thermotolerance. Jasmonates, such as jasmonic acid (JA), (+)-7-iso-jasmonoyl-l-isoleucine (JA-Ile), 12-oxo-10,15(Z)-phytodienoic acid (OPDA), and dinor-12-oxo-10,15(Z)-phytodienoic acid (dn-OPDA), are derived from C18 (18 Carbon atoms) and C16 polyunsaturated fatty acids (PUFAs), which are found ubiquitously in the plant kingdom. Bryophytes are also rich in C20 and C22 long-chain polyunsaturated fatty acids (LCPUFAs), which are found only at low levels in some vascular plants but are abundant in organisms of other kingdoms, including animals. The existence of bioactive jasmonates derived from LCPUFAs is currently unknown. Here, we describe the identification of an OPDA-like molecule derived from a C20 fatty acid (FA) in the liverwortMarchantia polymorpha(Mp), which we term (5Z,8Z)-10-(4-oxo-5-((Z)-pent-2-en-1-yl)cyclopent-2-en-1-yl)deca-5,8-dienoic acid (C20-OPDA). This molecule accumulates upon wounding and, when applied exogenously, can activate known Coronatine Insensitive 1 (COI1) –dependent and –independent jasmonate responses. Furthermore, we identify a dn-OPDA–like molecule (Δ4-dn-OPDA) deriving from C20-OPDA and demonstrate it to be a ligand of the jasmonate coreceptor (MpCOI1–Mp Jasmonate-Zinc finger inflorescence meristem domain [MpJAZ]) inMarchantia. By analyzing mutants impaired in the production of LCPUFAs, we elucidate the major biosynthetic pathway of C20-OPDA and Δ4-dn-OPDA. Moreover, using a double mutant compromised in the production of both Δ4-dn-OPDA and dn-OPDA, we demonstrate the additive nature of these molecules in the activation of jasmonate responses. Taken together, our data identify a ligand of MpCOI1 and demonstrate LCPUFAs as a source of bioactive jasmonates that are essential to the immune response ofM. polymorpha.

Octadecanoids are broadly defined as oxylipins (i.e., lipid mediators) derived from 18-carbon fatty acids. In contrast to the well-studied eicosanoids, there is a lack of analytical methods for octadecanoids, hampering further investigations in the field. We developed an integrated workflow combining chiral separation by supercritical fluid chromatography (SFC) and reversed-phase liquid chromatography (LC) coupled to tandem mass spectrometry detection for quantification of a broad panel of octadecanoids. The platform includes 70 custom-synthesized analytical and internal standards to extend the coverage of the octadecanoid synthetic pathways. A total of 103 octadecanoids could be separated by chiral SFC and complex enantioseparations could be performed in <13 min, while the achiral LC method separated 67 octadecanoids in 13.5 min. The LC method provided a robust complementary approach with greater sensitivity relative to the SFC method. Both methods were validated in solvent and surrogate matrix in terms of linearity, lower limits of quantification (LLOQ), recovery, accuracy, precision, and matrix effects. Instrumental linearity was good for both methods (R2 > 0.995) and LLOQ ranged from 0.03 to 6.00 ng/mL for SFC and 0.01 to 1.25 ng/mL for LC. The average accuracy in the solvent and surrogate matrix ranged from 89 to 109% in SFC and from 106 to 220% in LC, whereas coefficients of variation (CV) were <14% (at medium and high concentrations) and 26% (at low concentrations). Validation in the surrogate matrix showed negligible matrix effects (<16% for all analytes), and average recoveries ranged from 71 to 83%. The combined methods provide a platform to investigate the biological activity of octadecanoids and expand our understanding of these little-studied compounds.

Octadecanoids are broadly defined as oxylipins (i.e., lipid mediators) derived from 18-carbon fatty acids. In contrast to the well-studied eicosanoids, there is a lack of analytical methods for octadecanoids, hampering further investigations in the field. We developed an integrated workflow combining chiral separation by supercritical fluid chromatography (SFC) and reversed-phase liquid chromatography (LC) coupled to tandem-MS detection for quantification of a broad panel of octadecanoids. The platform included 70 custom-synthesized analytical and internal standards to extend the coverage of the octadecanoid synthetic pathways. A total of 103 octadecanoids could be separated by chiral SFC and complex enantioseparations could be performed in <13 minutes, while the achiral LC method separated 67 species in 13.5 minutes. The LC method provided a robust complementary approach with greater sensitivity relative to the SFC method. Both methods were validated in solvent and surrogate matrix in terms of linearity, lower limits of quantification (LLOQ), recovery, accuracy, precision, and matrix effects. Instrumental linearity was good for both methods (R2>0.995) and LLOQ ranged from 0.03-6.00 ng/mL for SFC and 0.01-1.25 ng/mL for LC. The average accuracy in solvent and surrogate matrix ranged from 89-109% in SFC and from 106-220% in LC, whereas coefficients of variation (CV) were <14% (at medium and high concentration) and 26% (at low concentration). Validation in surrogate matrix showed negligible matrix effects (<16% for all analytes) and average recoveries ranged from 71-83%. The combined methods provide a platform to investigate the biological activity of octadecanoids and expand our understanding of these little studied compounds.

BACKGROUND:The major mast cell prostanoid PGD2 is targeted for therapy of asthma and other diseases, because the biological actions include bronchoconstriction, vasodilation and regulation of immune cells mediated by three different receptors. It is not known if the alternative to selectively inhibit the biosynthesis of PGD2 affects release of other prostanoids in human mast cells.OBJECTIVES:To determine the biochemical consequences of inhibition of the hematopoietic prostaglandin D synthase (hPGDS) PGD2 in human mast cells.METHODS:Four human mast cell models, LAD2, cord blood derived mast cells (CBMC), peripheral blood derived mast cells (PBMC) and human lung mast cells (HLMC), were activated by anti-IgE or ionophore A23187. Prostanoids were measured by UPLC-MS/MS.RESULTS:All mast cells almost exclusively released PGD2 when activated by anti-IgE or A23187. The biosynthesis was in all four cell types entirely initiated by COX-1. When pharmacologic inhibition of hPGDS abolished formation of PGD2 , PGE2 was detected and release of TXA2 increased. Conversely, when the thromboxane synthase was inhibited, levels of PGD2 increased. Adding exogenous PGH2 confirmed predominant conversion to PGD2 under control conditions, and increased levels of TXB2 and PGE2 when hPGDS was inhibited. However, PGE2 was formed by non-enzymatic degradation.CONCLUSIONS:Inhibition of hPGDS effectively blocks mast cell dependent PGD2 formation. The inhibition was associated with redirected use of the intermediate PGH2 and shunting into biosynthesis of TXA2 . However, the levels of TXA2 did not reach those of PGD2 in naïve cells. It remains to determine if this diversion occurs in vivo and has clinical relevance.

15-hydroxyeicosatetraenoic acid (15-HETE) is an arachidonic acid derived lipid mediator which can originate both from 15-lipoxygenase (15-LOX) activity and cyclooxygenase (COX) activity. The enzymatic source determines the enantiomeric profile of the 15-HETE formed. 15-HETE is the most abundant arachidonic acid metabolite in the human lung and has been suggested to influence the pathophysiology of asthma. Mast cells are central effectors in asthma, but there are contradictory reports on whether 15-HETE originates from 15-LOX or COX in human mast cells. This prompted the current study where the pathway of 15-HETE biosynthesis was examined in three human mast cell models; the cell line LAD2, cord blood derived mast cells (CBMC) and tissue isolated human lung mast cells (HLMC). Levels and enantiomeric profiles of 15-HETE and levels of the downstream metabolite 15-KETE, were analyzed by UPLC-MS/MS after stimulation with anti-IgE or calcium ionophore A23187 in the presence and absence of inhibitors of COX isoenzymes. We found that 15-HETE was produced by COX-1 in human mast cells under these experimental conditions. Unexpectedly, chiral analysis showed that the 15(R) isomer was predominant and gradually accumulated, whereas the 15(S) isomer was metabolized by the 15-hydroxyprostaglandin dehydrogenase. We conclude that during physiological conditions, i.e., without addition of exogenous arachidonic acid, both enantiomers of 15-HETE are produced by COX-1 in human mast cells but that the 15(S) isomer is selectively depleted by undergoing further metabolism. The study highlights that 15-HETE cannot be used as an indicator of 15-LOX activity for cellular studies, unless chirality and sensitivity to pharmacologic inhibition is determined.

Several methods are available for elongation of fatty acid acyl chains. The present paper describes adaptation to the fatty acid field of a previously published protocol for manganese-based Wurtz type coupling of alkyl bromides. 22-Bromo-3(Z),6(Z),9(Z),12(Z),15(Z),18(Z)-docosahexaene, easily prepared from 4(Z),7(Z),10(Z),13(Z),16(Z),19(Z)-docosahexaenoic acid, was coupled to homologous omega-bromoesters by stirring for 4 hours at 40 degrees C in the presence of manganese powder, a nickel catalyst and terpyridine. This afforded in yields of 70-75% a series of omega 3-hexaenoates of chain lengths of 32-40 carbons. The corresponding fatty acids of >98% purity were obtained following saponification and final purification. By using methyl [2,2,3,3,4,4-H-2(6)]10-bromodecanoate as coupling partner it was possible to prepare a very long chain fatty acid in isotopically labeled form, i.e., [2,2,3,3,4,4-H-2(6)]14(Z),17(Z),20(Z),23(Z),26(Z),29(Z)-dotriacontahexaenoic acid. Also prepared were the monounsaturated long chain fatty acids 15(Z)-octadecenoic acid and 15(Z)-tetracosenoic acid. Very long chain fatty acids have been isolated from retina and other tissues and are of biological relevance. The methodology described will assist in further analytical and biological studies in this field.

Background: The major mast cell prostanoid PGD2 is an interesting target for therapeutic intervention because of its influence on bronchoconstriction, vasodilation and eosinophilic recruitment. We investigated the biochemical impact of inhibition of PGD2 biosynthesis in four human mast cell models. Methods: Mast cells (LAD2, cord blood derived mast cells (CBMC), peripheral blood mast cells (PBMC) and human lung mast cells (HLMC)) were activated by anti-IgE or ionophore A23187. Expression of COX-1, COX-2 and hematopoietic prostaglandin D synthase (hPGDS) was examined by western blot. The formation of different prostanoids was measured by UPLC-MS/MS, with release of PGD2 also measured by enzyme immunoassay. Pathways of biosynthesis were determined by pharmacological interventions and addition of the prostanoid precursor PGH2. Results: The biosynthesis of PGD2 and other prostanoids was exclusively catalyzed by COX-1. During inhibition of hPGDS, cell activation was associated with an increase in PGE2 and the TXA2 metabolite TXB2. Combined inhibition of hPGDS and thromboxane synthase led to a greater PGE2 increase. Furthermore, addition of PGH2 while inhibiting hPGDS also increased levels of TXB2 and PGE2. TXB2 was produced only in the presence of cells, whereas PGE2 was formed readily by non-enzymatic degradation. Conclusion: Mast cell production of prostanoids is COX-1 dependent. Downstream inhibition of the major prostanoid producing enzyme hPGDS will lead to shunting of PGH2 to form other prostanoids. Physiologically, the high hPGDS activity prevents the formation of other prostanoids in mast cells.

Trihydroxyoctadecenoic acids (TriHOMEs) are linoleic acid-derived lipid mediators reported to be dysregulated in obstructive lung disease. In contrast to many other oxylipins, TriHOME biosynthesis in humans is still poorly understood. The association of TriHOMEs with inflammation prompted the current investigation into the ability of human granulocytes to synthesize the 16 different 9,10,13-TriHOME and 9,12,13-TriHOME isomers and of the TriHOME biosynthetic pathway. Following incubation with linoleic acid, eosinophils and (to a lesser extent) the mast cell line LAD2, but not neutrophils, formed TriHOMEs. Stereochemical analysis revealed that TriHOMEs produced by eosinophils predominantly evidenced the 13(S) configuration, suggesting 15-lipoxygenase (15-LOX)-mediated synthesis. TriHOME formation was blocked following incubation with the 15-LOX inhibitor BLX-3887 and was shown to be largely independent of soluble epoxide hydrolase and cytochrome P450 activities. TriHOME synthesis was abolished when linoleic acid was replaced with 13-HODE, but increased in incubations with 13-HpODE, indicating the intermediary role of epoxy alcohols in TriHOME formation. In contrast to eosinophils, LAD2 cells formed TriHOMEs having predominantly the 13(R) configuration, demonstrating that there are multiple synthetic routes for TriHOME formation. These findings provide for the first-time insight into the synthetic route of TriHOMEs in humans and expand our understanding of their formation in inflammatory diseases.

Lipid droplets (LDs) have classically been viewed as seed storage particles, yet they are now emerging as dynamic organelles associated with developmental and stress responses. Nevertheless, their involvement in plant immunity has still been little studied. Here, we found LD accumulation in Arabidopsis thaliana leaves that induced a hypersensitive response (HR) after Pseudomonas infection. We established a protocol to reproducibly isolate LDs and to analyze their protein content. The expression of GFP fusion proteins in Nicotiana benthamiana and in transgenic Arabidopsis lines validated the LD localization of glycerol-3-phosphate acyltransferase 4 (GPAT4) and 8 (GPAT8), required for cutin biosynthesis. Similarly, we showed LD localization of α-dioxygenase1 (α-DOX1) and caleosin3 (CLO3), involved in the synthesis of fatty acid derivatives, and that of phytoalexin-deficient 3 (PAD3), which is involved in camalexin synthesis. We found evidence suggesting the existence of different populations of LDs, with varying protein contents and distributions. GPAT4 and GPAT8 were associated with LDs inside stomata and surrounding cells of untreated leaves, yet they were mainly confined to LDs in guard cells after bacterial inoculation. By contrast, α-DOX1 and PAD3 were associated with LDs in the epidermal cells of HR-responding leaves, with PAD3 mostly restricted to cells near dead tissue, while CLO3 had a more ubiquitous distribution. As such, the nature of the proteins identified, together with the phenotypic examination of selected mutants, suggests that LDs participate in lipid changes and in the production and transport of defense components affecting the interaction of plants with invading pathogens.

Fusarium oxysporum f. sp. tulipae (FOT) secretes (+)-7-iso-jasmonoyl-(S)-isoleucine ((+)-JA-Ile) to the growth medium together with about 10 times less 9,10-dihydro-(+)-7-iso-JA-Ile. Plants and fungi form (+)-JA-Ile from 18:3n-3 via 12-oxophytodienoic acid (12-OPDA), which is formed sequentially by 13S-lipoxygenase, allene oxide synthase (AOS), and allene oxide cyclase (AOC). Plant AOC does not accept linoleic acid (18:2n-6)-derived allene oxides and dihydrojasmonates are not commonly found in plants. This raises the question whether 18:2n-6 serves as the precursor of 9,10-dihydro-JA-Ile in Fusarium, or whether the latter arises by a putative reductase activity operating on the n-3 double bond of (+)-JA-Ile or one of its precursors. Incubation of pentadeuterated (d5 ) 18:3n-3 with mycelia led to the formation of d5 -(+)-JA-Ile whereas d5 -9,10-dihydro-JA-Ile was not detectable. In contrast, d5 -9,10-dihydro-(+)-JA-Ile was produced following incubation of [17,17,18,18,18-2 H5 ]linoleic acid (d5 -18:2n-6). Furthermore, 9(S),13(S)-12-oxophytoenoic acid, the 15,16-dihydro analog of 12-OPDA, was formed upon incubation of unlabeled or d5 -18:2n-6. Appearance of the α-ketol, 12-oxo-13-hydroxy-9-octadecenoic acid following incubation of unlabeled or [13 C18 ]-labeled 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid confirmed the involvement of AOS and the biosynthesis of the allene oxide 12,13(S)-epoxy-9,11-octadecadienoic acid. The lack of conversion of this allene oxide by AOC in higher plants necessitates the conclusion that the fungal AOC is distinct from the corresponding plant enzyme.

RationaleJasmonates are formed from 12‐oxo‐10,15(Z)‐phytodienoic acid (12‐OPDA) in plants and also from 12‐oxo‐10‐phytoenoic acid (12‐OPEA) in fungi. Collision‐induced dissociation (CID) of [M‐H]− generates characteristic product anions at m/z 165 [C11H17O]−. Our goal was to investigate the structure and mode of formation of this anion by CID of 12‐OPDA, 12‐OPEA, and 12‐oxophytonoic acid (12‐OPA).MethodsWe investigated the CID of the [M‐H]−, [M‐H‐CO2]−, and [M‐H‐H2O]− anions using electrospray ionization and MS/MS analysis of 12‐OPDA, 12‐OPEA, and 12‐OPA, and compared the results with the data obtained with the corresponding compounds labeled with 2H at C‐6 and C‐7 and with structural and side chain analogs.ResultsCID of [6,6,7,7‐2H4]12‐OPEA and [6,6‐2H2]12‐OPDA ([M‐H]− and [M‐H‐CO2]−) showed that one or two 2H atoms were transferred to anions at m/z 165 as judged by the signal intensities of m/z 165 + 1 or 165 + 2, respectively. CID of [6,6‐2H2]‐ and [6,6,7,7‐2H4]‐12‐OPA ([M‐H]− and [M‐H‐CO2]−) yielded the loss of H2 from the cyclopentanone and displayed the transfer of one 2H atom in analogy to 12‐OPEA. In contrast, CID of [6,6,7,7‐2H4]12‐OPEA and [6,6,7,7‐2H4]12‐OPA [M‐H‐H2O]− demonstrated the transfer of two 2H atoms (m/z 165 + 2). All spectra obtained by CID of [6,6,7,7‐2H4]12‐OPDA and [6,6,7,7‐2H4]12‐oxo‐9(13),15(Z)‐phytodienoic acid showed that one or two additional 2H atoms could be transferred to this anion at m/z 167 of [6,6‐2H2]12‐OPDA due to isotope scrambling.ConclusionsCID of 12‐OPDA and 12‐OPEA generates cyclopentanone enolate anions at m/z 165 by charge‐driven hydride transfer as a common mechanism and by bond cleavage between C‐7 and C‐8 of the carboxyl side chains with either gain or loss of a hydrogen atom.