Anaerobic lipoxygenase activity from Chlorella pyrenoidosa

The micro-alga Chlorella pyrenoidosa expresses an enzymatic activity that cleaves the 13-hydroperoxide derivatives of linoleic acid [13-hydroperoxy-9(Z),11(E)-octadecadienoic acid, 13-HPOD] and linolenic acid [13-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid, 13-HPOT] into volatile C(5) and non-volatile C(13) oxo-products. This enzymic activity initially was attributed to a hydroperoxide lyase enzyme; however, subsequent studies showed that this cleavage activity is the result of lipoxygenase activity under anaerobic conditions. Headspace analysis of the volatile products by GC/MS showed the formation of pentane when the substrate was 13-HPOD, whereas a more complex mixture of hydrocarbons was formed when 13-HPOT was the substrate. Analysis of the non-volatile cleavage products from 13-HPOD by liquid chromatography/MS indicated the formation of 13-oxo-9(Z),11(E)-tridecadienoic acid (13-OTA) along with the 13-keto-octadecadienoic acid derivative. When the substrate is 13-HPOT, liquid chromatography/MS analysis indicated the formation of 13-OTA as the major non-volatile product. Aldehyde dehydrogenase (AldDH) oxidizes 13-OTA to an omega-dicarboxylic acid, whereas alcohol dehydrogenase (ADH) reduces 13-OTA to an omega-hydroxy carboxylic acid. AldDH and ADH require the oxidized (NAD(+)) and reduced (NADH) forms of the cofactor NAD, respectively. By combining the action of AldDH and ADH into a continuous cofactor-recycling process, it is possible to simultaneously convert 13-OTA to the corresponding omega-dicarboxylic acid and omega-hydroxy carboxylic acid derivatives.     

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.     

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.     

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.     

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.     

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.     

Characterisation of recombinant Hevea brasiliensis allene oxide synthase: effects of cycloxygenase inhibitors, lipoxygenase inhibitors and salicylates on enzyme activity.

Mechanical wounding and jasmonic acid (JA) treatment have been shown to be important factors in controlling laticifer differentiation in Hevea brasiliensis (rubber tree). With the long-term aim of potentially modifying the endogenous levels of JA in H. brasiliensis by gene transfer, we describe in this paper the molecular cloning of a H. brasiliensis allene oxide synthase (AOS) cDNA and biochemical characterisation of the recombinant AOS (His(6)-HbAOS) enzyme. The AOS cDNA encodes a protein with the expected motifs present in CYP74A sub-group of the cytochrome P450 super-family of enzymes that metabolise 13-hydroperoxylinolenic acid (13-HPOT), the intermediate involved in JA synthesis. The recombinant H. brasiliensis AOS enzyme was estimated to have a high binding affinity for 13-HPOT with a K(m) value of 4.02+/-0.64 microM. Consistent with previous studies, mammalian cycloxygenase (COX) and lipoxygenase (LOX) inhibitors were shown to significantly reduce His(6)-HbAOS enzyme activity. Although JA had no effect on His(6)-HbAOS, salicylic acid (SA) was shown to significantly inhibit the recombinant AOS enzyme activity in a dose dependent manner. Moreover, it was demonstrated that SA, and various analogues of SA, acted as competitive inhibitors of His(6)-HbAOS when 13-HPOT was used as substrate. We speculate that this effect of salicylates on AOS activity may be important in cross-talking between the SA and JA signalling pathways in plants during biotic/abiotic stress.     

The "heterolytic hydroperoxide lyase" is an isomerase producing a short-lived fatty acid hemiacetal

To elucidate the reaction mechanism of hydroperoxide lyase (HPL), the enzyme from guava (Psidium guajava) fruits, was incubated for 10-60 s at 0 degrees C with 13-HPOT. The products were rapidly extracted and derivatized by trimethylsilylation. Two trapping products, namely the trimethylsilyl ether/ester derivatives of the hemiacetal 12-(1'-hydroxy-3'-hexenyloxy)-9,11-dodecadienoic acid and the enol (9Z,11E)-12-hydroxy-9,11-dodecadienoic acid, were detected by gas chromatography-mass spectrometry (GC-MS) analyses. The structural assignments were supported by mass spectra recorded for (a) hydrogenated products; (b) products biosynthesized from [9,10,12,13,15,16] 13-HPOT or [(18)O(2)]13-HPOT; (c) chemically prepared reference compounds. Kinetic experiments showed that the hemiacetal and enol were both unstable and transiently appearing compounds (half-lives, ca. 20 s and 2 min, respectively). Hemiacetal and enol biosynthesized from [(18)O(2)]13-HPOT retained two and one (18)O atoms, respectively, whereas no (18)O was incorporated from [(18)O]water. The data demonstrated that: (1) the true enzymatic product formed from 13-HPOT in the presence of HPL is a short-lived hemiacetal; (2) the hemiacetal spontaneously dissociates into (3Z)-hexenal and the unstable enol form of (9Z)-12-oxo-9-dodecenoic acid; (3) the enzymatic isomerization of 13-HPOT into the hemiacetal occurs homolytically.     

Role of structure and pH in cyclization of allene oxide fatty acids: implications for the reaction mechanism

Incubations of allene oxide synthases of flax or maize with the E,E-isomers of the 13- and 9-hydroperoxides of linoleic acid (E,E-13- and E,E-9-HPOD, respectively) at pH 7.5 afforded substantial yields of trans-disubstituted cyclopentenones. Under the conditions used, (Z,E)-HPODs were converted mainly into -ketols and afforded only trace amount of cyclopentenones. These findings indicated that changing the double bond geometry from Z to E dramatically increased the rate of formation of the pericyclic pentadienyl cation intermediate necessary for electrocyclization of 18:2-allene oxides and thus the yield of cyclopentenones. The well-known cyclization of the homoallylic allene oxide (12,13-EOT) derived from -linolenic acid 13-hydroperoxide (E,Z-13-HPOT) into cis-12-oxo-10,15-phytodienoic acid was suppressed at pH below neutral and was not observable at pH 4.5. In contrast, cyclization of the allene oxide ((9E)-12,13-EOD) derived from (E,E)-13-HPOD was slightly favoured at low pH. The finding that the cyclizations of 12,13-EOT and (9E)-12,13-EOD were differently affected by changes in pH suggested that the mechanisms of cyclization of these allene oxides are distinct.     

The “heterolytic hydroperoxide lyase” is an isomerase producing a short-lived fatty acid hemiacetal

To elucidate the reaction mechanism of hydroperoxide lyase (HPL), the enzyme from guava (Psidium guajava) fruits, was incubated for 10–60 s at 0 °C with 13-HPOT. The products were rapidly extracted and derivatized by trimethylsilylation. Two trapping products, namely the trimethylsilyl ether/ester derivatives of the hemiacetal 12-(1′-hydroxy-3′-hexenyloxy)-9,11-dodecadienoic acid and the enol (9Z,11E)-12-hydroxy-9,11-dodecadienoic acid, were detected by gas chromatography-mass spectrometry (GC-MS) analyses. The structural assignments were supported by mass spectra recorded for (a) hydrogenated products; (b) products biosynthesized from [9,10,12,13,15,16] 13-HPOT or [¹⁸O₂]13-HPOT; (c) chemically prepared reference compounds. Kinetic experiments showed that the hemiacetal and enol were both unstable and transiently appearing compounds (half-lives, ca. 20 s and 2 min, respectively). Hemiacetal and enol biosynthesized from [¹⁸O₂]13-HPOT retained two and one ¹⁸O atoms, respectively, whereas no ¹⁸O was incorporated from [¹⁸O]water. The data demonstrated that: (1) the true enzymatic product formed from 13-HPOT in the presence of HPL is a short-lived hemiacetal; (2) the hemiacetal spontaneously dissociates into (3Z)-hexenal and the unstable enol form of (9Z)-12-oxo-9-dodecenoic acid; (3) the enzymatic isomerization of 13-HPOT into the hemiacetal occurs homolytically.     

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.     

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.     

Epoxyalcohol synthase of Ectocarpus siliculosus. First CYP74-related enzyme of oxylipin biosynthesis in brown algae

Enzymes of CYP74 family play the central role in the biosynthesis of physiologically important oxylipins in land plants. Although a broad diversity of oxylipins is known in the algae, no CYP74s or related enzymes have been detected in brown algae yet. Cloning of the first CYP74-related gene CYP5164B1 of brown alga Ectocarpus siliculosus is reported in present work. The recombinant protein was incubated with several fatty acid hydroperoxides. Linoleic acid 9-hydroperoxide (9-HPOD) was the preferred substrate, while linoleate 13-hydroperoxide (13-HPOD) was less efficient. α-Linolenic acid 9- and 13-hydroperoxides, as well as eicosapentaenoic acid 15-hydroperoxide were inefficient substrates. Both 9-HPOD and 13-HPOD were converted into epoxyalcohols. For instance, 9-HPOD was turned primarily into (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acid. Both epoxide and hydroxyl oxygen atoms of the epoxyalcohol were incorporated mostly from [¹⁸O₂]9-HPOD. Thus, the enzyme exhibits the activity of epoxyalcohol synthase (EsEAS). The results show that the EsEAS isomerizes the hydroperoxides into epoxyalcohols via epoxyallylic radical, a common intermediate of different CYP74s and related enzymes. EsEAS can be considered as an archaic prototype of CYP74 family enzymes.     

Overexpression of the Tomato 13-Lipoxygenase Gene TomloxD Increases Generation of Endogenous Jasmonic Acid and Resistance to Cladosporium fulvum and High Temperature

Expression of the tomato gene encoding 13-lipoxygenase,TomloxD, is stimulated by wounding, pathogen infection, jasmonate, and systemin, but its role during growth and development of tomato (Lycopersicon Spp.) remains unclear. To assess the physiological role of TomloxD, we produced transgenic tomato plants with greatly increased TomloxD content using sense constructs under the control of the CaMV 35S promoter. Overexpression of TomloxD in transgenic tomatoes led to a marked increase in the levels of lipoxygenase activity and content of endogenous jasmonic acid (JA), which suggested that TomloxD can use α-linolenic acid as a substrate to produce (13S)-hydroperoxyoctadecatrienoic acid (13-HPOT); the 13-HPOT produced appears to be metabolized further to synthesize JA. Real-time RT-PCR revealed that the expression levels of defense genes LeHSP90, LePR1, LePR6 and LeZAT in the transformants were higher than those in non-transformed plants. Assay for resistance to pathogenic fungus and high temperature stresses suggested that transgenic plants harboring TomloxD were more tolerant to Cladosporium fulvum and high temperature stress than non-transformed tomato plants. The data presented here indicate clearly that TomloxD is involved in endogenous JA synthesis and tolerance to biotic and abiotic stress. The tomloxD gene has potential applications in engineering cropping plants that are resistant to biotic and/or abiotic stress factors.     

Inhibition of soybean lipoxygenase 1 by N-alkylhydroxylamines

Micromolar concentrations of N-octylhydroxylamine dramatically increase the induction period in the conversion of linoleic acid to 13(S)-hydroperoxy-cis-9,trans-11-octadecadienoic acid (13-HPOD) catalyzed by soybean lipoxygenase 1. The induction period produced by N-octylhydroxylamine is abolished by 13-HPOD but not by the corresponding hydroxy acid. Addition of a catalytic amount of lipoxygenase to a mixture of 13-HPOD and N-octylhydroxylamine results in consumption of approximately 1 mumol of 13-HPOD/mumol of N-octylhydroxylamine present. These results can be explained by a model in which 13-HPOD oxidizes the enzyme from an inactive ferrous form to an active ferric form, as proposed by previous workers, and N-octylhydroxylamine reduces the enzyme back to the ferrous form. Consistent with this model, the ESR signal at g = 6.1 characteristic of ferric lipoxygenase is rapidly abolished by N-octylhydroxylamine and can be regenerated by 13-HPOD. These results provide additional support for earlier proposals that ferric lipoxygenase is the catalytically active form and also establish a novel method of inhibiting enzymes in this class. The octyl group of N-octylhydroxylamine appears to contribute to binding near the iron, since hydroxylamine and N-methylhydroxylamine do not extend the induction period. In the n-RNHOH series, activity passes through an optimum at R = decyl.     

Reprogramming of fatty acid and oxylipin synthesis in rhizobacteria-induced systemic resistance in tomato

The rhizobacterium Pseudomonas putida BTP1 stimulates induced systemic resistance (ISR) in tomato. A previous work showed that the resistance is associated in leaves with the induction of the first enzyme of the oxylipin pathway, the lipoxygenase (LOX), leading to a faster accumulation of its product, the free 13-hydroperoxy octadecatrienoic acid (13-HPOT), 2 days after Botrytis cinerea inoculation. In the present study, we further investigated the stimulation of the oxylipin pathway: metabolites and enzymes of the pathway were analyzed to understand the fate of the 13-HPOT in ISR. Actually the stimulation began upstream the LOX: free linolenic acid accumulated faster in P. putida BTP1-treated plants than in control. Downstream, the LOX products 13-fatty acid hydroperoxides esterified to galactolipids and phospholipids were more abundant in bacterized plants than in control before infection. These metabolites could constitute a pool that will be used after pathogen attack to produce free fungitoxic metabolites through the action of phospholipase A2, which is enhanced in bacterized plants upon infection. Enzymatic branches which can use as substrate the fatty acid hydroperoxides were differentially regulated in bacterized plants in comparison to control plants, so as to lead to the accumulation of the most fungitoxic compounds against B. cinerea. Our study, which is the first to demonstrate the accumulation of an esterified defense metabolite during rhizobacteria-mediated induced systemic resistance, showed that the oxylipin pathway is differentially regulated. It suggests that this allows the plant to prepare to a future infection, and to respond faster and in a more effective way to B. cinerea invasion.     

Wound response in passion fruit (<Emphasis Type="Italic">Passiflora</Emphasis> f. <Emphasis Type="Italic">edulis flavicarpa</Emphasis>) plants: gene characterization of a novel chloroplast-targeted allene oxide synthase up-regulated by mechanical injury and methyl jasmonate

The induction of a chloroplast-localized 13-lipoxygenase (13-LOX) in passion fruit leaves in response to methyl jasmonate (MeJa) was previously reported. Since allene oxide synthase (AOS) is a key cytochrome P450 enzyme in the oxylipin pathway leading to AOS-derived jasmonates, the results above led in turn to an investigation of AOS in our model plant. Spectrophotometric assays showed that 24 h exposure of MeJa caused a high increase in 13-hydroperoxy linolenic acid (13-HPOT) metabolizing activity in leaf tissue. Western analysis using polyclonal antibodies against tomato AOS strongly indicate that, at least a part of the 13-HPOT metabolizing capacity can be attributed to AOS activity. We cloned the cDNA from a novel AOS encoding gene from passion fruit, named PfAOS. The 1,512 bp open reading frame of the AOS–cDNA codes a putative protein of 504 amino acid residues containing a chloroplast target sequence. Database comparisons of the deduced amino acid sequence showed highest similarity with dicot AOSs. Immunocytochemistry analysis showed the compartmentalization of AOS in chloroplasts of MeJa treated leaves, corroborating the predicted subcellular localization. Northern analysis showed that AOS gene expression is induced in leaf tissue in response to mechanical wounding and exposure to MeJa. In addition, such treatments caused an increase in papain inhibitor(s) in leaf tissue. Taken together, these results indicate that PfAOS may play an important role in systemic wound response against chewing insect attack. Furthermore, it can be useful as a tool for understanding the regulation of jasmonates biosynthesis in passion fruit.