Thermal conversions of trimethylsilyl peroxides of linoleic and linolenic acids

The trimethylsilyl (TMS) peroxides/esters of the fatty acid hydroperoxides (9S,10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid (9-HPOD) and (9Z,11E,13S,15Z)-13-hydroperoxy-9,11,15-octadecatrienoic acid (13-HPOT) were subjected to gas chromatography-mass spectrometry and products formed by thermal rearrangements were identified. The main products were decadienals and the TMS derivatives of 13-oxo-9,11-tridecadienoic acid, epoxyalcohols, hemiacetals, and ketodienes. Oxy radicals as well as epoxyallylic radicals served as intermediates in the formation of these compounds. The thermal TMS peroxide conversions documented provided biomimetic models for enzymatic conversions of fatty acid hydroperoxides and also offered a method to generate an array of oxylipin derivatives of value as reference compounds in GC-MS studies.     

Peroxidase-like activity of lipoxygenases: different substrate specificity of potato 5-lipoxygenase and soybean 15-lipoxygenase and particular affinity of vitamin E derivatives for the 5-lipoxygenase.

Potato 5-lipoxygenase (5-PLO) catalyzes the reduction of 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13-HPOD) in the presence of vitamin E. I mol of vitamin E is required to consume 2 mol of 13-HPOD. The mechanism of the 5-PLO-catalyzed oxidation of vitamin E by 13-HPOD is similar to that previously established for the soybean 15-lipoxygenase (L-1)-catalyzed oxidation of phenidone by 13-HPOD, and seems to involve a one-electron reduction of the O-O bond of 13-HPOD. 5-PLO and L-1 exhibit very different substrate specificities and pH profiles for their peroxidase-like activity. Actually, among the 20 compounds containing various reducible functions and the 10 derivatives of vitamin E which have been studied, only four products containing hydrophobic long chains, ascorbic acid 6-palmitate, the trolox esters of octanol and undecanol, and vitamin E exhibit high peroxidase-like activities for 5-PLO. On the contrary, much more compounds, even not very hydrophobic, are good substrates for the peroxidase-like activity of L-1.     

Fatty acid hydroperoxides biotransformation by potato tuber cell-free extracts

Cleavage of 13-HPOD, 13-HPOT, 9-HPOD and 9-HPOT by potato tuber cell-free extracts was investigated. 13-HPOD and 13-HPOT enzymes were degraded almost completely while 9-HPOD and 9-HPOT were partially transformed. GC-MS analysis of the volatile compounds formed during the reactions revealed that (Z)-3 hexenal, (E)-2-hexenal, pentenols and dimers of pentene were obtained from 13-HPOT while from 13-HPOD hexanal and pentan-1-ol were formed. No volatile was found when 9-HPO isomers were used as substrate, but colneleic acid was produced. When Triton X-100 was omitted in the extraction buffer, only pentenols and dimers of pentene were identified from 13-HPOT and pentan-1-ol from 13-HPOD. Our results reveal that potato tubers that contain Lox, which forms mainly 9-HPO, are able to metabolise the four HPO isomers. Moreover, 13-HPO cleaving activities are due to two distinct enzymatic systems based on, respectively, homolytic and heterolytic mechanisms. The fact that oxygenation of reaction medium dramatically decreases the amount of product resulting from homolytic cleavage strengthens the hypothesis of an anaerobic reaction due to Lox.     

Purification, stabilization and characterization of tomato fatty acid hydroperoxide lyase

Fatty acid hydroperoxide lyase (HPO-lyase) was purified 300-fold from tomatoes. The enzymatic activity appeared to be very unstable, but addition of Triton X100 and beta-mercaptoethanol to the buffer yielded an active enzyme that could be stored for several months at -80 degrees C. The enzyme was inhibited by desferoxamine mesylate (desferal), 2-methyl-1,2-di-3-pyridyl-1-propanone (metyrapone), nordihydroguaiaretic acid (NDGA), n-propyl gallate and butylated hydroxyanisole, suggesting the involvement of free radicals in the reaction mechanism and the existence of a prosthetic group in the active center. However, no heme group could be demonstrated with the methods commonly used to identify heme groups in proteins. Only 13-hydroperoxides from linoleic acid (13-HPOD) and alpha-linolenic acid (alpha-13-HPOT) were cleaved by the tomato enzyme, with a clear preference for the latter substrate. The pH-optimum was 6.5, and for concentrations lower than 300 microM a typical Michaelis-Menten curve was found with a K(m) of 77 microM. At higher alpha-13-HPOT concentrations inhibition of the enzyme was observed, which could (at least in part) be attributed to 2E-hexenal. A curve of the substrate conversion as a function of the enzyme concentration revealed that 1 nkat of enzyme activity converts 0.7 mumol alpha-13-HPOT before inactivation. Headspace analysis showed that tomato HPO-lyase formed hexanal from 13-HPOD and 3Z-hexenal from alpha-13-HPOT. A trace of the latter compound was isomerized to 2E-hexenal. In addition to the aldehydes, 12-oxo-9Z-dodecenoic acid was found by GC/MS analysis. To a small extent, isomerization to 12-oxo-10E-dodecenoic acid occurred.     

Photolysis of unsaturated fatty acid hydroperoxides. 4. Fatty acid products from the aerobic decomposition of methyl 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate dissolved in cyclohexane

In the presence of oxygen, UV-irradiation of a solution of methyl 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate (13-HPOD) in cyclohexane leads to a broad pattern of reaction products of which a trihydroxyene, seven epoxyhydroxides, four hydroxydienes, four epoxyhydroperoxides, six oxodienes and an epoxycyclohexylene were identified as the main components. Two oxodienes having a (Z)-double bond adjacent to the carbonyl group and the epoxycyclohexylene are reported for the first time. In contrast to results published recently for the UV-degradation of the 13-HPOD in methanol, the decomposition of the 13-HPOD in cyclohexane results in the formation of the 9-HPOD by a rearrangement of the hydroperoxy group. Consequently the reaction products are formed as mixtures of positional isomers. The reaction pathways leading to the identified compounds are discussed.     

The novel pathway for ketodiene oxylipin biosynthesis in Jerusalem artichoke (Helianthus tuberosus) tubers

The new route of the plant lipoxygenase pathway, directed specifically towards the ketodiene formation, was detected during in vitro experiments with Jerusalem artichoke (Helianthus tuberosus) tubers. Through this pathway (9Z,11E,13S)-13-hydroperoxy-9,11-octadecadienoic acid (13-HPOD) is reduced to corresponding 13-hydroxy acid (13-HOD), which is in turn dehydrogenated into ketodiene (9Z,11E,13S)-13-oxo-9,11-octadecadienoic acid (13-KOD). Dehydrogenation of 13-HOD into 13-KOD was not dependent on the presence of either NAD or NADP, but was strongly dependent on the presence of oxygen. Under anoxic conditions, 13-HOD dehydrogenation was blocked, but addition of 2,6-dichlorophenolindophenol restored it. Sulfite addition fully suppressed the aerobic dehydrogenation of 13-HOD. Hydrogen peroxide is a by-product formed by the enzyme along with 13-KOD. These data suggest that the ketodiene biosynthesis in H. tuberosus tubers is catalyzed by flavin dehydrogenase. (9S,10E,12Z)-9-Hydroxy-10,12-octadecadienoic acid (9-HOD) is dehydrogenated by this enzyme as effectively as 13-HOD, while alpha-ketol, (9Z)-12-oxo-13-hydroxy-9-octadecenoic acid, and ricinoleic acid did not act as substrates for dehydrogenase. The enzyme was soluble and possessed a pH optimum at pH 7.0-9.0. The only 13-HOD dehydrogenase known so far was detected in rat colon. However, unlike the H. tuberosus enzyme, the rat dehydrogenase is NAD-dependent.     

Photolysis of unsaturated fatty acid hydroperoxides. 3. Products from the aerobic decomposition of methyl 13(S)-hydroperoxy-9(Z),11(E-octadecadienoate dissolved in methanol

In the presence of oxygen, UV-irradiation of a solution of methyl 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoate (13-HPOD) in methanol yields stereoisomers of methyl 9,13-dihydroxy-10-methoxy-11-(E)-octadecenoate and methyl 9,13-dihydroxy-12-methoxy-10(E)-octadecenoate as major products. The reaction pathway to the products was established by photolysis experiments with labeled 13-HPOD and with intermediates of the reaction route. When methanol was substituted by water in the reaction system, the corresponding trihydroxyoctadecenoic acids were formed. This was confirmed by aerobic photolysis of 13-HPOD (free acid) dissolved in water. Threo and erythro methyl 12, 13-epoxy-11-hydroxy-9(Z)-octadecenoates belong to the minor compounds formed during aerobic photolysis of the 13-HPOD in methanol. Labeling experiments indicated that the threo compound resulted mainly from a rearrangement of the 13-HPOD while the erythro compound is mainly formed via secondary hydroperoxidation.     

Oxylipin biosynthesis in spikemoss Selaginella moellendorffii: Molecular cloning and identification of divinyl ether synthases CYP74M1 and CYP74M3

Nonclassical P450s of CYP74 family control the secondary conversions of fatty acid hydroperoxides to bioactive oxylipins in plants. At least ten genes attributed to four novel CYP74 subfamilies have been revealed by the recent sequencing of the spikemoss Selaginella moellendorffii Hieron genome. Two of these genes CYP74M1 and CYP74M3 have been cloned in the present study. Both recombinant proteins CYP74M1 and CYP74M3 were active towards the 13(S)-hydroperoxides of α-linolenic and linoleic acids (13-HPOT and 13-HPOD, respectively) and exhibited the activity of divinyl ether synthase (DES). Products were analyzed by gas chromatography–mass spectrometry. Individual oxylipins were purified by HPLC and finally identified by their NMR data, including the ¹H NMR, 2D-COSY, HSQC and HMBC. CYP74M1 (SmDES1) specifically converted 13-HPOT to (11Z)-etherolenic acid and 13-HPOD to (11Z)-etheroleic acid. CYP74M3 (SmDES2) turned 13-HPOT and 13-HPOD mainly to etherolenic and etheroleic acids, respectively. CYP74M1 and CYP74M3 are the first DESs detected in non-flowering plants. The obtained results demonstrate the existence of the sophisticated oxylipin biosynthetic machinery in the oldest taxa of vascular plants.     

Epoxyalcohol synthase activity of the CYP74B enzymes of higher plants

The CYP74B subfamily of fatty acid hydroperoxide transforming cytochromes P450 includes the most common plant enzymes. All CYP74Bs studied yet except the CYP74B16 (flax divinyl ether synthase, LuDES) and the CYP74B33 (carrot allene oxide synthase, DcAOS) are 13-hydroperoxide lyases (HPLs, synonym: hemiacetal synthases). The results of present work demonstrate that additional products (except the HPL products) of fatty acid hydroperoxides conversion by the recombinant StHPL (CYP74B3, Solanum tuberosum), MsHPL (CYP74B4v1, Medicago sativa), and CsHPL (CYP74B6, Cucumis sativus) are epoxyalcohols. MsHPL, StHPL, and CsHPL converted the 13-hydroperoxides of linoleic (13-HPOD) and α-linolenic acids (13-HPOT) primarily to the chain cleavage products. The minor by-products of 13-HPOD and 13-HPOT conversions by these enzymes were the oxiranyl carbinols, 11-hydroxy-12,13-epoxy-9-octadecenoic and 11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid. At the same time, all enzymes studied converted 9-hydroperoxides into corresponding oxiranyl carbinols with HPL by-products. Thus, the results showed the additional epoxyalcohol synthase activity of studied CYP74B enzymes. The 13-HPOD conversion reliably resulted in smaller yields of the HPL products and bigger yields of the epoxyalcohols compared to the 13-HPOT transformation. Overall, the results show the dualistic HPL/EAS behaviour of studied CYP74B enzymes, depending on hydroperoxide isomerism and unsaturation.     

Irreversible inactivation of soybean lipoxygenase-1 by hydrophobic thiols

Soybean lipoxygenase-1 is inactivated by micromolar concentrations of the following hydrophobic thiols: 1-octanethiol, 12(S)-mercapto-9(Z)-octadecenoic acid (S-12-HSODE), 12(R)-mercapto-9(Z)-octadecenoic acid (R-12-HSODE), and 12-mercaptooctadecanoic acid (12-HSODA). In each case, inactivation is time-dependent and not reversed by dilution or dialysis. Inactivation requires 13-hydroperoxy-9(Z),11(E)-octadecadienoic acid (13-HPOD), which suggests that it is specific for the ferric form of the enzyme. Lipoxygenase catalyzes an oxygenation reaction on each of the aforementioned thiols, as judged by the consumption of O(2). These reactions also require 13-HPOD. 1-Octanethiol is converted to 1-octanesulfonic acid, which was identified by GC/MS of its methyl ester. The rates of oxygen uptake for R- and S-12-HODE are about 5- and 2.5-fold higher than the rate with 1-octanethiol. The stoichiometries of inactivation imply that inactivation occurs on approximately 1 in 18 turnovers for 12-HSODA, 1 in 48 turnovers for 1-octanethiol, 1 in 63 turnovers for S-12-HSODE, and 1 in 240 turnovers for R-12-HSODE. These data imply that close resemblance to lipoxygenase substrates is not a crucial requirement for either oxidation or inactivation. Under the conditions of our experiments, inactivation was not observed with several more polar thiols: mercaptoethanol, dithiothreitol, L-cysteine, glutathione, N-acetylcysteamine, and captopril. The results imply that hydrophobic thiols irreversibly inactivate soybean lipoxygenase by a mechanism that involves oxidation at sulfur.     

Characterization of a C-5,13-Cleaving Enzyme of 13(S)-Hydroperoxide of Linolenic Acid by Soybean Seed

An activity was found in mature soybean seeds (Glycine max L. cv Century) that cleaved 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid (13S-HPOT) into 13-oxo-9(Z),11(E)-tridecadienoic acid and two isomeric pentenols, 2(Z)-penten-1-ol and 1-penten-3-ol. Isomeric pentene dimers were also produced and were presumably derived from the combination of two pentene radicals. 13(S)-Hydroperoxy-9(Z),11(E)-octadecadienoic acid (13S-HPOD) was, by contrast, a poor substrate. Activity with 13S-HPOT increased 24-fold under anaerobic conditions reminiscent of a similar anaerobic promoted reaction of 13S-HPOD catalyzed by lipoxygenase (LOX) in the presence of linoleic acid. However, prior to ion-exchange chromatography, cleavage activity did not require linoleic acid. After separation by gel filtration followed by ion-exchange chromatography, cleavage activity was lost but reappeared in the presence of either linoleic acid or dithiothreitol. Under these conditions cleavage activity was coincident with the activity of types 1 and 2 LOX. LOX inhibitors suppressed the cleavage reaction in a manner similar to inhibition of LOX activity. Heat-generated alkoxyl radicals derived from either 13S-HPOT or 13S-HPOD afforded similar products and yields of 13-oxo-9(Z),11(E)-tridecadienoic acid compared to the enzymic reaction. The product 1-penten-3-ol may be the precursor of the "raw-bean" volatile ethylvinylketone.     

Soybean lipoxygenase-1 enzymically forms both (9S)- and (13S)-hydroperoxides from linoleic acid by a pH-dependent mechanism

Soybean lipoxygenase-1 produces a preponderance of two chiral products from linoleic acid, (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid and (9S)-(10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid. The former of these hydroperoxides was generated at all pH values, but in the presence of Tween 20, the latter product did not form at pH values above 8.5. As the pH decreased below 8.5, the proportion of (9S)-hydroperoxide increased linearly until at pH 6 it constituted about 25% of the chiral products attributed to enzymic action. Below pH 6, lipoxygenase activity was barely measurable, and the hydroperoxide product arose mainly from autoxidation and possibly non-enzymic oxygenation of the pentadienyl radical formed by the enzyme. The change in percent enzymically formed 9-hydroperoxide between pH 6.0 and 8.5 paralleled the pH plot of a sodium linoleate/linoleic acid titration. It was concluded that the (9S)-hydroperoxide is formed only from the nonionized carboxylic acid form of linoleic acid. Methyl esterification of linoleic acid blocked the formation of the (9S)-hydroperoxide by lipoxygenase-1, but not the (13S)-hydroperoxide. Since the hydroperoxydiene moieties of the (9S)- and (13S)-hydroperoxides are spatially identical when the molecules are arranged head to tail in opposite orientations, it is suggested that the carboxylic acid form of the substrate can arrange itself at the active site in either orientation, but the carboxylate anion can be positioned only in one orientation. These observations, as well as others in the literature, suggest and active-site model for soybean lipoxygenase-1.     

Hydroperoxide Lyase and Other Hydroperoxide-Metabolizing Activity in Tissues of Soybean, Glycine max

Hydroperoxide lyase (HPLS) activity in soybean (Glycine max) seed/seedlings, leaves, and chloroplasts of leaves required detergent solubilization for maximum in vitro activity. On a per milligram of protein basis, more HPLS activity was found in leaves, especially chloroplasts, than in seeds or seedlings. The total yield of hexanal from 13(S)-hydroperoxy-cis-9,trans-11-octadecadienoic acid (13S-HPOD) from leaf or chloroplast preparations was 58 and 66 to 85%, respectively. Because of significant competing hydroperoxide-metabolizing activities from other enzymes in seed/seedling preparations, the hexanal yields from this source were lower (36-56%). Some of the products identified from the seed or seedling preparations indicated that the competing activity was mainly due to both a hydroperoxide peroxygenase and reactions catalyzed by lipoxygenase. Different HPLS isozyme compositions in the seed/seedling versus the leaf/chloroplast preparations were indicated by differences in the activity as a function of pH, the K_m values, relative V_max with 13S-HPOD and 13(S)-hydroperoxy-cis-9,trans-11,cis-15-octadecatrienoic acid (13S-HPOT), and the specificity with different substrates. With regard to the latter, both seed/seedling and chloroplast HPLS utilized the 13S-HPOD and 13S-HPOT substrates, but only seeds/seedlings were capable of metabolizing 9(S)-hydroperoxy-trans-10,cis-12-octadecadienoic acid into 9-oxononanoic acid, isomeric nonenals, and 4-hydroxynonenal. From 13S-HPOD and 13S-HPOT, the products were identified as 12-oxo-cis-9-dodecenoic acid, as well as hexanal from 13S-HPOD and cis-3-hexenal from 13S-HPOT. In seed preparations, there was partial isomerization of the cis-3 or cis-9 into trans-2 or trans-10 double bonds, respectively.     

An Enzymatic Conversion of Lipoxygenase Products by a Hydroperoxide Lyase in Blue-Green Algae (Oscillatoria sp.)

An enzyme has been isolated from blue-green algae Oscillatoria sp. which utilizes the product, 13-hydroperoxy-9, 11-octadecadienoic acid (13-HPOD), of lipoxygenase for its substrate. This enzyme, termed hydroperoxide lyase, converts the conjugated diene 13-hydroperoxide of linoleic acid to 13-oxotrideca-9, 11-dienoic acid. The structure of the latter has been determined by ultraviolet spectroscopy and mass spectrometry. 9-HPOD is not a substrate for this enzyme. The hydroperoxide lyase from Oscillatoria sp. has a maximum of activity at pH 6.4 and 30°C. The molecular weight of the enzyme was estimated at 56,000. The enzyme was not inhibited by BW 755C, but was inhibited by molecules containing more than one hydroxyl group. Quercetin was found to be the best inhibitor of the enzyme activity. The purified hydroperoxide lyase from Oscillatoria sp. showed an apparent K_m of 7.4 micromolar and a V_max of 35 nanomoles per minute per milligram of protein for 13-HPOD. An enzymatic pathway for the biogenesis of oxodienoic acid from linoleic acid is proposed. This involves the sequential activity of lipoxygenase and hydroperoxide lyase enzymes.     

Production of hexanal from hydrolyzed sunflower oil by lipoxygenase and hydroperoxid lyase enzymes

Hexanal was produced from hydrolyzed sunflower oil in two steps: 1) 13-hydroperoxy-9-(Z),11(E)-octadecadienoic acid (13-HPOD) was formed from linoleic acid (100 mM) by soybean lipoxygenase-1 isoenzyme (Lox-1) with O₂, the reaction resulted 68.7 mM 13-HPOD with a yield of 72%. 2) 13-HPOD (15 mM) was cleaved by spinach leaf hydroperoxide lyase resulting 8.2 mM hexanal (54% yield). Hexanal was isolated from the reaction mixture by repeated steam distillation.     

Factors affecting the initial rate of lipoxygenase catalysis

The rate of peroxidation of linoleic acid by soybean type-1 lipoxygenase was studied under conditions which assured that the substrate was present as a monomolecular solution and that the first 5% of the reaction was observed. In order to achieve this, the kinetics were carried out at pH 10.0 in borate buffer using linoleic acid and enzyme concentrations of less than 75 μM and 0.2 nM respectively. The initial rate was increased by the presence of added product (13-hydroperoxy-9(Z),11(E)-octadecadienoic acid) in the substrate solutions in a concentration dependent and saturatable fashion. Product analogues lacking the hydroperoxide group (13-hydroxy-9(Z),11(E)-octadecadienoic acid and 13-methoxy-9(Z),11(E)-octadecadienoic acid) did not evoke this rate enhancing effect. These compounds reduced the initial rate when preincubated with enzyme prior to mixing with substrate. The results indicated that the chemical reactivity of the product was a necessary requirement for its activating effect on the enzyme.     

Hidden stereospecificity in the biosynthesis of divinyl ether fatty acids

Incubations of [8(R)-2H]9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid, [14(R)-2H]13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid and [14(S)-2H]13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid were performed with preparations of plant tissues containing divinyl ether synthases. In agreement with previous studies, generation of colneleic acid from the 8(R)-deuterated 9(S)-hydroperoxide was accompanied by loss of most of the deuterium label (retention, 8%), however, the opposite result (98% retention) was observed in the generation of 8(Z)-colneleic acid from the same hydroperoxide. Formation of etheroleic acid and 11(Z)-etheroleic acid from the 14(R)-deuterated 13(S)-hydroperoxide was accompanied by loss of most of the deuterium (retention, 7-8%), and, as expected, biosynthesis of these divinyl ethers from the corresponding 14(S)-deuterated hydroperoxide was accompanied by retention of deuterium (retention, 94-98%). Biosynthesis of omega5(Z)-etheroleic acid from the 14(R)- and 14(S)-deuterated 13(S)-hydroperoxides showed the opposite results, i.e. 98% retention and 4% retention, respectively. The experiments demonstrated that biosynthesis of divinyl ether fatty acids from linoleic acid 9- and 13-hydroperoxides takes place by a mechanism that involves stereospecific abstraction of one of the two hydrogen atoms alpha to the hydroperoxide carbon. Furthermore, a consistent relationship between the absolute configuration of the hydrogen atom eliminated (R or S) and the configuration of the introduced vinyl ether double bond (E or Z) emerged from these results. Thus, irrespective of which hydroperoxide regioisomer served as the substrate, divinyl ether synthases abstracting the pro-R hydrogen generated divinyl ethers having an E vinyl ether double bond, whereas enzymes abstracting the pro-S hydrogen produced divinyl ethers having a Z vinyl ether double bond.     

Cytochrome P450-dependent metabolism of oxylipins in tomato. Cloning and expression of allene oxide synthase and fatty acid hydroperoxide lyase

Allene oxide synthase (AOS) and fatty acid hydroperoxide lyase (HPL) are plant-specific cytochrome P450s that commit fatty acid hydroperoxides to different branches of oxylipin metabolism. Here we report the cloning and characterization of AOS (LeAOS) and HPL (LeHPL) cDNAs from tomato (Lycopersicon esculentum). Functional expression of the cDNAs in Escherichia coli showed that LeAOS and LeHPL encode enzymes that metabolize 13- but not 9-hydroperoxide derivatives of C(18) fatty acids. LeAOS was active against both 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid (13-HPOT) and 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid, whereas LeHPL showed a strong preference for 13-HPOT. These results suggest a role for LeAOS and LeHPL in the metabolism of 13-HPOT to jasmonic acid and hexenal/traumatin, respectively. LeAOS expression was detected in all organs of the plant. In contrast, LeHPL expression was predominant in leaves and flowers. Damage inflicted to leaves by chewing insect larvae led to an increase in the local and systemic expression of both genes, with LeAOS showing the strongest induction. Wound-induced expression of LeAOS also occurred in the def-1 mutant that is deficient in octadecanoid-based signaling of defensive proteinase inhibitor genes. These results demonstrate that tomato uses genetically distinct signaling pathways for the regulation of different classes of wound responsive genes.