Allene Oxide Synthase Activity Research Articles

Concentration-dependent impacts of exogenous methyl jasmonate (MeJA) on chlorophyll degradation of apple fruit during ripening

Although jasmonates (JAs) have been proved to regulate degreening of apple fruit, the relevant molecular mechanisms have not yet been clarified. Previous reports revealed that the effects of JAs on degreening of harvested horticultural crops might be dependent on concentration applied. Whether a similar result can be obtained from apple fruit is still unclear. This research explored the impacts of different concentrations of methyl jasmonate (MeJA) treatment on chlorophyll (Chl) degradation of postharvest apple fruit. Apples (Malus × domestica Borkh.) of commercial maturity were treated with 10 or 1500 µM MeJA and then stored at 20 ± 0.5 °C. The results indicated that exogenous 10 µM MeJA delayed degreening and ripening of apple fruit, whereas 1500 µM MeJA treatment had the opposite effects. Treatment with 10 µM MeJA alleviated Chl-oxidation by reducing Chl-peroxidase (POX) activity and reactive oxygen species (ROS) levels in chloroplast of apple peel during ripening, whereas 1500 µM MeJA treatment had the opposite effects. Activities of Magnesium-dechelatase (MDcase) and pheophytinase (PPH) in apple peel were differentially suppressed by 10 µM MeJA treatment while promoted by 1500 µM MeJA treatment during ripening. Application of 10 µM MeJA delayed the peak of endogenous jasmonic acid (JA) content, reduced jasmonate resistant 1 (JAR1) content and allene oxide synthase (AOS) activity, and differentially down-regulated expression of MdNYC3, MdNYE1a, MdPPH1, MdPAO6, MdPAO8, MdRCCR2 and MdMYC2a in apple peel during ripening, whereas opposite results were obtained from the 1500 µM MeJA-treated apple fruit. At the late ripening stage, MdHCAR expression in apple peel was promoted by 10 µM MeJA treatment while reduced by 1500 µM MeJA treatment. Transcript levels of MdNYC1 and MdNOL2 were generally reduced by both treatments at the late ripening stage. Transient overexpression of MdMYC2a accelerated Chl degradation of apple peel by promoting PPH and MDcase activities and enhancing expression of the above Chl catabolic genes (CCGs), except MdHCAR whose expression was down-regulated by it during ripening. The AOS activity, JA and JAR1 contents in apple peel were increased after transient overexpression of MdMYC2a during ripening. These data indicated that the PAO pathway and Chl-oxidation were regulated by JA signal in apple peel during ripening, and exogenous MeJA regulated Chl degradation of apple fruit in a concentration-dependent manner.

Leaf Removal Impacted Jasmonic Acid Metabolism and AsA-GSH in the Roots of Malus baccata (L.) Borkh. under Suboptimal Low Root-Zone Temperatures

In the early growing season in northern China, suboptimal low root-zone temperatures is a common abiotic stress that impairs root function and leaf development in fruit trees. In this study, we investigate the physiological role of leaves in jasmonate metabolism and the capacity of scavenging reactive oxygen species in Malus baccata (L.) Borkh. roots under suboptimal low root-zone temperatures. In the presence of intact leaves, suboptimal low root-zone temperatures significantly increased allene oxide synthase (AOS), jasmonate-resistant 1 (JAR), and jasmonic acid carboxyl methyltransferase (JMT) activities and transcription in jasmonate biosynthesis. Meanwhile, elevated endogenous jasmonic acid (JA), methyl jasmonate (MeJA), and jasmonate-isoleucine (JA-Ile) contents were also observed, as were significantly decreased glutathione reductase and dehydroascorbate reductase activities and AsA/DHA and GSH/GSSG ratios. Conversely, leaf removal substantially reduced AOS, JMT, and JAR activities and transcription at most time points and JA (6–24 h), MeJA (1–24 h), and JA-Ile (1–24 h) levels in roots, affecting key enzymes in the AsA–GSH cycle and the AsA/DHA and GSH/GSSG ratios in response to low-temperature treatment, as a result of a significant increase in malondialdehyde content. Thus, leaves are crucial for jasmonate metabolism in roots under suboptimal low root-zone temperatures, with leaf removal exacerbating root oxidative stress by altering JA signaling and AsA–GSH cycle activity.

CRISPR/Cas9-mediated disruption of ALLENE OXIDE SYNTHASE results in defective 12-oxo-phytodienoic acid accumulation and reduced defense against spider mite (Tetranychus urticae) in liverwort (Marchantia polymorpha).

Allene oxide synthase (AOS) is a key enzyme involved in the biosynthesis of 12-oxo-phytodienoic acid (OPDA) and jasmonic acid and plays an important role in plant defense against herbivore attacks. In the liverwort, Marchantia polymorpha, we previously identified cytosol-type MpAOS1 and chloroplast-type MpAOS2 that show AOS activities. However, there is no direct evidence to show the subcellular localization of MpAOSs and their contribution to plant defense via OPDA production in M. polymorpha. In this study, we generated M. polymorpha mutants, with the MpAOS1 and MpAOS2 genes disrupted via CRISPR/Cas9-mediated genome editing; the loss of OPDA production was analyzed in double-knockout mutants. On AOS mutants, the survival rate and oviposition of spider mites (Tetranychus urticae) increased relative to those on wild-type plants. Overall, these findings suggest that defense systems via OPDA-signaling pathways in response to spider mites have been established in M. polymorpha.

Oxylipin biosynthesis in spikemoss Selaginella moellendorffii: Identification of allene oxide synthase (CYP74L2) and hydroperoxide lyase (CYP74L1)

Nonclassical P450s of the CYP74 family catalyse the secondary conversions of fatty acid hydroperoxides to bioactive oxylipins in plants. The model organism, spikemoss Selaginella moellendorffii Hieron, possesses at least ten CYP74 genes of novel J, K, L, and M subfamilies. The cloning of three CYP74L genes and catalytic properties of recombinant proteins are described in the present work. The CYP74L1 possessed mainly hydroperoxide lyase (HPL) activity towards the 13(S)-hydroperoxide of α−linolenic acids (13-HPOT) and nearly equal HPL and allene oxide synthase (AOS) activities towards the 13(S)-hydroperoxide of linoleic acids (13-HPOD). The 9-hydroperoxides were poor substrates for CYP74L1 and led to the production of mainly the α−ketols (AOS products) and minorities of HPL and epoxyalcohol synthase (EAS) products. The CYP74L2 possessed the AOS activity towards all tested hydroperoxides. CYP74L3 possessed low HPL/EAS activity. Besides, the aerial parts of S. moellendorffii plants possessed complex oxylipins patterns including divinyl ethers, epoxyalcohols, and 12-oxo-phytodienoic acid. Characterization of the CYP74L enzymes and oxylipin pattern updates the knowledge on the complex oxylipin biosynthetic machinery in the surviving oldest taxa of vascular plants.

ALLENE OXIDE SYNTHASE and HYDROPEROXIDE LYASE, Two Non-Canonical Cytochrome P450s in Arabidopsis thaliana and Their Different Roles in Plant Defense.

The channeling of metabolites is an essential step of metabolic regulation in all living organisms. Multifunctional enzymes with defined domains for metabolite compartmentalization are rare, but in many cases, larger assemblies forming multimeric protein complexes operate in defined metabolic shunts. In Arabidopsis thaliana, a multimeric complex was discovered that contains a 13-lipoxygenase and allene oxide synthase (AOS) as well as allene oxide cyclase. All three plant enzymes are localized in chloroplasts, contributing to the biosynthesis of jasmonic acid (JA). JA and its derivatives act as ubiquitous plant defense regulators in responses to both biotic and abiotic stresses. AOS belongs to the superfamily of cytochrome P450 enzymes and is named CYP74A. Another CYP450 in chloroplasts, hydroperoxide lyase (HPL, CYP74B), competes with AOS for the common substrate. The products of the HPL reaction are green leaf volatiles that are involved in the deterrence of insect pests. Both enzymes represent non-canonical CYP450 family members, as they do not depend on O2 and NADPH-dependent CYP450 reductase activities. AOS and HPL activities are crucial for plants to respond to different biotic foes. In this mini-review, we aim to summarize how plants make use of the LOX2–AOS–AOC2 complex in chloroplasts to boost JA biosynthesis over volatile production and how this situation may change in plant communities during mass ingestion by insect pests.

Product specificity of fungal 8R- and 9S-dioxygenases of the peroxidase-cyclooxygenase superfamily with amino acid derivatized polyenoic fatty acids

Pathogenic fungi express fatty acid dioxygenases (DOX) fused to cytochromes P450 with diol or allene oxide synthase activities. The orientation of the fatty acids in the active sites of DOX was investigated with amino acid conjugates of 18:3n-3 and 18:2n-6. 9S-DOX-allene oxide synthase (AOS) oxidized the Gly, Ile, and Trp derivatives at C-9, which suggests that these conjugates enter the substrate recognition site with the omega end in analogy with fatty acids bound to cyclooxygenases and coral 8R-lipoxygenase (8R-LOX). In contrast, 7,8-diol synthases (7,8-LDS), 5,8-LDS, and 8R-DOX-AOS oxidized the Gly conjugates in most case only to small amounts of metabolites, but with retention of hydrogen abstraction at C-8 and relatively minor hydrogen abstraction at C-11. The Ile and Trp conjugates were not oxidized at C-8, and often insignificantly at C-9/C-13. The 8-DOX domains of these enzymes likely position the carboxyl group of substrates at the end of the active site in analogy with plant α-DOX and 9-LOX. Tyr radicals of the 9S-DOX and 8R-DOX domains catalyze antarafacial hydrogen abstraction and oxygen insertion in 18:3n-3. This occurs by abstraction of the proR and proS hydrogens at C-11 and C-8, respectively, in agreement with different “head to tail” orientation in the active site.

Identification and Analysis of Genes Involved in the Jasmonate Pathway in Response to Ethephon and 1-Methylcyclopropene during the Ripening of Apple Fruit

Fruit ripening is a complex process involving many physiological changes and the dynamic interplay between different phytohormones. In addition to ethylene, jasmonates (JAs) have also been demonstrated to play an important role in the regulation of fruit ripening. However, the mechanisms underlying the interaction between these two pathways during fruit ripening are unknown. In recent years, research has been conducted to illustrate the effects of JAs on the ethylene biosynthesis and signaling pathway, but little is known regarding the effects of ethylene on JA biosynthesis and the signaling pathway during fruit ripening. Herein, we aimed to evaluate the effects of ethylene on JA biosynthesis in ripening apple (Malus ×domestica) fruit and on the expression of key genes involved in the JA biosynthesis and the signaling pathway. For this purpose, we treated apple fruit with ethephon and 1-methylcyclopropene (1-MCP) at commercial maturity. Our data indicated that endogenous JA content and allene oxide synthase (AOS) activity were reduced by ethephon treatment at the early ripening stage, whereas they were enhanced by 1-MCP treatment at the late ripening stage. Quantitative real-time polymerase chain reaction (PCR) analysis revealed that the expression profiles of three AOS genes (MdAOS2, MdAOS3, and MdAOS5) and two lipoxygenase (LOX) genes (MdLOX22 and MdLOX28) showed similar trends with the change of AOS activity in all groups during fruit ripening. The expression of MdLOX21 and MdLOX23 was in accordance with the change of ethylene production on ripening, and it was positively regulated by ethylene, whereas the opposite effect was observed for MdLOX39 expression. The transcription of MdLOX310 and MdLOX61 appeared unaffected by ethylene during fruit ripening. Three jasmonate ZIM-domain (JAZ) genes (MdJAZ9, MdJAZ10, and MdJAZ18) were differentially upregulated by ethephon treatment whereas being downregulated by 1-MCP treatment during fruit ripening. Expression of MdJAZ13 and MdJAZ14 was downregulated at the early ripening stage by both treatments. Our results suggested regulating roles of ethylene on the JA biosynthesis and signaling pathway during fruit ripening and senescence.

Separation of enzymatic functions and variation of spin state of rice allene oxide synthase-1 by mutation of Phe-92 and Pro-430

Rice allene oxide synthase-1 mutants carrying F92L, P430A or F92L/P430A amino acid substitution mutations were constructed, recombinant mutant and wild type proteins were purified and their substrate preference, UV–vis spectra and heme iron spin state were characterized. The results show that the hydroperoxide lyase activities of F92L and F92L/P430A mutants prefer 13-hydroperoxy substrate to other hydroperoxydienoic acids or hydroperoxytrienoic acids. The Soret maximum was completely red-shifted in P430A and F92L/P430A mutants, but it was partially shifted in the F92L mutant. ESR spectral data showed that wild type, F92L and P430A mutants occupied high and low spin states, while the F92L/P430A mutant occupied only low spin state. The extent of the red shift of the Soret maximum increased as the population of low spin heme iron increased, suggesting that the spectral shift reflects the high to low transition of heme iron spin state in rice allene oxide synthase-1. Relative to wild type allene oxide synthase-1, the hydroperoxide lyase activities of F92L and F92L/P430A are less sensitive to inhibition by imidazole with (13S or 9S)-hydroperoxydienoic acid as substrate and more sensitive than wild type with (13S)-hydroperoxytrienoic acid as substrate. Our results suggest that hydroperoxydienoic acid is the preferred substrate for the hydroperoxide lyase activity and (13S)-hydroperoxytrienoic acid is the preferred substrate for allene oxide synthase activity of allene oxide synthase-1.

Discovery of a linoleate 9S-dioxygenase and an allene oxide synthase in a fusion protein of Fusarium oxysporum

Fusarium oxysporum is a devastating plant pathogen that oxidizes C₁₈ fatty acids sequentially to jasmonates. The genome codes for putative dioxygenase (DOX)-cytochrome P450 (CYP) fusion proteins homologous to linoleate diol synthases (LDSs) and the allene oxide synthase (AOS) of Aspergillus terreus, e.g., FOXB_01332. Recombinant FOXB_01332 oxidized 18:2n-6 to 9S-hydroperoxy-10(E),12(Z)-octadecadienoic acid by hydrogen abstraction and antarafacial insertion of molecular oxygen and sequentially to an allene oxide, 9S(10)-epoxy-10,12(Z)-octadecadienoic acid, as judged from nonenzymatic hydrolysis products (α- and γ-ketols). The enzyme was therefore designated 9S-DOX-AOS. The 9S-DOX activity oxidized C₁₈ and C₂₀ fatty acids of the n-6 and n-3 series to hydroperoxides at the n-9 and n-7 positions, and the n-9 hydroperoxides could be sequentially transformed to allene oxides with only a few exceptions. The AOS activity was stereospecific for 9- and 11-hydroperoxides with S configurations. FOXB_01332 has acidic and alcoholic residues, Glu⁹⁴⁶-Val-Leu-Ser⁹⁴⁹, at positions of crucial Asn and Gln residues (Asn-Xaa-Xaa-Gln) of the AOS and LDS. Site-directed mutagenesis studies revealed that FOXB_01332 and AOS of A. terreus differ in catalytically important residues suggesting that AOS of A. terreus and F. oxysporum belong to different subfamilies. FOXB_01332 is the first linoleate 9-DOX with homology to animal heme peroxidases and the first 9-DOX-AOS fusion protein.

Association between accumulation of allene oxide synthase activity and development of resistance against downy mildew disease of pearl millet

The present study was aimed at understanding the possible association of allene oxide synthase (AOS), an enzyme implicated in the octadecanoid pathway during the pearl millet-downy mildew interaction. AOS 13-HPOT (13-hydroperoxy-9,11,15-octadecatrienoic acid) metabolizing activity assays assessed in various pearl millet cultivars with differential resistances against downy mildew revealed a positive correlation between cultivar resistance levels and AOS activities. Furthermore, the involvement of AOS in response to downy mildew was demonstrated by induction of AOS activity in both susceptible and resistant pearl millet cultivars during Sclerospora graminicola infection with higher induction observed in the resistant cultivar. Consistently, western blot analysis and tissue-blot immunoassay demonstrated the remarkable increase in AOS protein accumulation in the incompatible interaction. In addition, the tissue-blot immunoassay also showed the compartmentalization of AOS in the epidermis and vascular bundles of pearl millet seedlings. Expression analysis of a putative PgAOS1 gene revealed a marked difference in accumulation of PgAOS1 transcripts between contrasting plants, with pathogen-induced higher accumulation of the transcripts observed only in the resistant cultivar; a result which is in agreement with pathogen-induced AOS level and activity, indicating that PgAOS1 plays an important role in regulation of AOS level and activity in pearl millet upon S. graminicola infection. Our findings suggest an important role for AOS in regulation of responses to downy mildew disease in pearl millet. The differential AOS activities can potentially be used for selection of new disease-resistant pearl millet varieties, and the identified AOS-encoding gene(s) as genetic resource for development of enhanced downy mildew-resistant cultivars.

Expression of Fusion Proteins of Aspergillus terreus Reveals a Novel Allene Oxide Synthase

Aspergilli oxidize C18 unsaturated fatty acids by dioxygenase-cytochrome P450 fusion proteins to signal molecules involved in reproduction and host-pathogen interactions. Aspergillus terreus expresses linoleate 9R-dioxygenase (9R-DOX) and allene oxide synthase (AOS) activities in membrane fractions. The genome contains five genes (ATEG), which may code for a 9R-DOX-AOS fusion protein. The genes were cloned and expressed, but none of them oxidized 18:2n-6 to 9R-hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HPODE). ATEG_02036 transformed 9R-HPODE to an unstable allene oxide, 9(R),10-epoxy-10,12(Z)-octadecadienoic acid. A substitution in the P450 domain (C1073S) abolished AOS activity. The N964V and N964D mutants both showed markedly reduced AOS activity, suggesting that Asn(964) may facilitate homolytic cleavage of the dioxygen bond of 9R-HPODE with formation of compound II in analogy with plant AOS (CYP74) and prostacyclin synthase (CYP8A1). ATEG_03992 was identified as 5,8-linoleate diol synthase (5,8-LDS). Replacement of Asn(878) in 5,8-LDS with leucine (N878L) mainly shifted ferryl oxygen insertion from C-5 toward C-6, but replacements of Gln(881) markedly affected catalysis. The Q881L mutant virtually abolished the diol synthase activity. Replacement of Gln(881) with Asn, Glu, Asp, or Lys residues augmented the homolytic cleavage of 8R-HPODE with formation of 10-hydroxy-8(9)-epoxy-12(Z)-octadecenoic acid (erythro/threo, 1-4:1) and/or shifted ferryl oxygen insertion from C-5 toward C-11. We conclude that homolysis and heterolysis of the dioxygen bond with formation of compound II in AOS and compound I in 5,8-LDS are influenced by Asn and Gln residues, respectively, of the I-helices. AOS of A. terreus appears to have evolved independently of CYP74 but with an analogous reaction mechanism.

8R-Lipoxygenase-catalyzed synthesis of a prominent cis-epoxyalcohol from dihomo-γ-linolenic acid: a distinctive transformation compared with S-lipoxygenases

Conversion of fatty acid hydroperoxides to epoxyalcohols is a well known secondary reaction of lipoxygenases, described for S-specific lipoxygenases forming epoxyalcohols with a trans-epoxide configuration. Here we report on R-specific lipoxygenase synthesis of a cis-epoxyalcohol. Although arachidonic and dihomo-γ-linolenic acids are metabolized by extracts of the Caribbean coral Plexaura homomalla via 8R-lipoxygenase and allene oxide synthase activities, 20:3ω6 forms an additional prominent product, identified using UV, GC-MS, and NMR in comparison to synthetic standards as 8R,9S-cis-epoxy-10S-erythro-hydroxy-eicosa-11Z,14Z-dienoic acid. Both oxygens of (18)O-labeled 8R-hydroperoxide are retained in the product, indicating a hydroperoxide isomerase activity. Recombinant allene oxide synthase formed only allene epoxide from 8R-hydroperoxy-20:3ω6, whereas two different 8R-lipoxygenases selectively produced the epoxyalcohol.A biosynthetic scheme is proposed in which a partial rotation of the reacting intermediate is required to give the observed erythro epoxyalcohol product. This characteristic and the synthesis of cis-epoxy epoxyalcohol may be a feature of R-specific lipoxygenases.

Linolenate 9R‐Dioxygenase and Allene Oxide Synthase Activities of Lasiodiplodia theobromae

Jasmonic acid (JA) is synthesized from linolenic acid (18:3n-3) by sequential action of 13-lipoxygenase, allene oxide synthase (AOS), and allene oxide cyclase. The fungus Lasiodiplodia theobromae can produce large amounts of JA and was recently reported to form the JA precursor 12-oxophytodienoic acid. The objective of our study was to characterize the fatty acid dioxygenase activities of this fungus. Two strains of L. theobromae with low JA secretion (~0.2mg/L medium) oxygenated 18:3n-3 to 5,8-dihydroxy-9Z,12Z,15Z-octadecatrienoic acid as well as 9R-hydroperoxy-10E,12Z,15Z-octadecatrienoic acid, which was metabolized by an AOS activity into 9-hydroxy-10-oxo-12Z,15Z-octadecadienoic acid. Analogous conversions were observed with linoleic acid (18:2n-6). Studies using [11S-(2)H]18:2n-6 revealed that the putative 9R-dioxygenase catalyzed stereospecific removal of the 11R hydrogen followed by suprafacial attack of dioxygen at C-9. Mycelia from these strains of L. theobromae contained 18:2n-6 as the major polyunsaturated acid but lacked 18:3n-3. A third strain with a high secretion of JA (~200mg/L) contained 18:3n-3 as a major fatty acid and produced 5,8-dihydroxy-9Z,12Z,15Z-octadecatrienoic acid from added 18:3n-3. This strain also lacked the JA biosynthetic enzymes present in higher plants.

Linoleate 9 R-dioxygenase and allene oxide synthase activities of Aspergillus terreus

Oxygenation of linoleic acid by Aspergillus terreus was studied with LC–MS/MS. 9( R) -Hydroperoxy-10( E),12( Z)-octadecadienoic acid (9 R-HpODE) was identified along with 10( R)-hydroxy-8( E),12( Z)-octadecadienoic acid and variable amounts of 8( R)-hydroxy-9( Z),12( Z)-octadecadienoic acid. 9 R-HpODE was formed from [11 S- 2H]18:2 n − 6 with loss of the deuterium label, suggesting antarafacial hydrogen abstraction and oxygenation. Two polar metabolites were identified as 9-hydroxy-10-oxo-12( Z)-octadecenoic acid (α-ketol) and 13-hydroxy-10-oxo-11( E)-octadecenoic acid (γ-ketol), likely formed by spontaneous hydrolysis of an unstable allene oxide, 9( R),10-epoxy-10,12( Z)-octadecadienoic acid. α-Linolenic acid and 20:2 n − 6 were oxidized to hydroperoxy fatty acids at C-9 and C-11, respectively, but α- and γ-ketols of these fatty acids could not be detected. The genome of A. terreus lacks lipoxygenases, but contains genes homologous to 5,8-linoleate diol synthases and linoleate 10 R-dioxygenases of aspergilli. Our results demonstrate that linoleate 9 R-dioxygenase linked to allene oxide synthase activities can be expressed in fungi.

Nicotiana attenuataSIPK, WIPK, NPR1, and Fatty Acid-Amino Acid Conjugates Participate in the Induction of Jasmonic Acid Biosynthesis by Affecting Early Enzymatic Steps in the Pathway

Wounding and herbivore attack elicit the rapid (within minutes) accumulation of jasmonic acid (JA) that results from the activation of previously synthesized biosynthetic enzymes. Recently, several regulatory factors that affect JA production have been identified; however, how these regulators affect JA biosynthesis remains at present unknown. Here we demonstrate that Nicotiana attenuata salicylate-induced protein kinase (SIPK), wound-induced protein kinase (WIPK), nonexpressor of PR-1 (NPR1), and the insect elicitor N-linolenoyl-glutamate [corrected] (18:3-Glu) participate in mechanisms affecting early enzymatic steps of the JA biosynthesis pathway. Plants silenced in the expression of SIPK and NPR1 were affected in the initial accumulation of 13-hydroperoxy-linolenic acid (13-OOH-18:3) after wounding and 18:3-Glu elicitation by mechanisms independent of changes in 13-lipoxygenase activity. Moreover, 18:3-Glu elicited an enhanced and rapid accumulation of 13-OOH-18:3 that depended partially on SIPK and NPR1 but was independent of increased 13-lipoxygenase activity. Together, the results suggested that substrate supply for JA production was altered by 18:3-Glu elicitation and SIPK- and NPR1-mediated mechanisms. Consistent with a regulation at the level of substrate supply, we demonstrated by virus-induced gene silencing that a wound-repressed plastidial glycerolipase (NaGLA1) plays an essential role in the induction of de novo JA biosynthesis. In contrast to SIPK and NPR1, mechanisms mediated by WIPK did not affect the production of 13-OOH-18:3 but were critical to control the conversion of this precursor into 12-oxo-phytodienoic acid. These differences could be partially accounted for by reduced allene oxide synthase activity in WIPK-silenced plants.

Cloning and Functional Analysis of an Allene Oxide Synthase inPhyscomitrella patens

Jasmonic acid (JA) is a plant hormone that plays important roles in a large number of processes in stress adaptation and development in flowering plants. A search of genome database indicated the existence of allene oxide synthase (AOS), an enzyme of JA biosynthesis, in Physcomitrella patens, a model plant among mosses. In this study, the presence of JA was detected in P. patens. The recombinant AOS of P. patens, which was overexpressed in Escherichia coli, showed AOS activity. These data suggest that the octadecanoid pathway also exists in P. patens.

Evidence for an Ionic Intermediate in the Transformation of Fatty Acid Hydroperoxide by a Catalase-related Allene Oxide Synthase from the Cyanobacterium Acaryochloris marina

Allene oxides are reactive epoxides biosynthesized from fatty acid hydroperoxides by specialized cytochrome P450s or by catalase-related hemoproteins. Here we cloned, expressed, and characterized a gene encoding a lipoxygenase-catalase/peroxidase fusion protein from Acaryochloris marina. We identified novel allene oxide synthase (AOS) activity and a by-product that provides evidence of the reaction mechanism. The fatty acids 18.4omega3 and 18.3omega3 are oxygenated to the 12R-hydroperoxide by the lipoxygenase domain and converted to the corresponding 12R,13-epoxy allene oxide by the catalase-related domain. Linoleic acid is oxygenated to its 9R-hydroperoxide and then, surprisingly, converted approximately 70% to an epoxyalcohol identified spectroscopically and by chemical synthesis as 9R,10S-epoxy-13S-hydroxyoctadeca-11E-enoic acid and only approximately 30% to the 9R,10-epoxy allene oxide. Experiments using oxygen-18-labeled 9R-hydroperoxide substrate and enzyme incubations conducted in H2(18)O indicated that approximately 72% of the oxygen in the epoxyalcohol 13S-hydroxyl arises from water, a finding that points to an ionic intermediate (epoxy allylic carbocation) during catalysis. AOS and epoxyalcohol synthase activities are mechanistically related, with a reacting intermediate undergoing a net hydrogen abstraction or hydroxylation, respectively. The existence of epoxy allylic carbocations in fatty acid transformations is widely implicated although for AOS reactions, without direct experimental support. Our findings place together in strong association the reactions of allene oxide synthesis and an ionic reaction intermediate in the AOS-catalyzed transformation.

Determinants governing the CYP74 catalysis: Conversion of allene oxide synthase into hydroperoxide lyase by site-directed mutagenesis

Bioinformatics analyses enabled us to identify the hypothetical determinants of catalysis by CYP74 family enzymes. To examine their recognition, two mutant forms F295I and S297A of tomato allene oxide synthase LeAOS3 (CYP74C3) were prepared by site-directed mutagenesis. Both mutations dramatically altered the enzyme catalysis. Both mutant forms possessed the activity of hydroperoxide lyase, while the allene oxide synthase activity was either not detectable (F295I) or significantly reduced (S297A) compared to the wild-type LeAOS3. Thus, both sites 295 and 297 localized within the “I-helix central domain” (“oxygen binding domain”) are the primary determinants of CYP74 type of catalysis.

A lipoxygenase-divinyl ether synthase pathway in flax ( Linum usitatissimum L.) leaves

Incubation of linoleic acid with an enzyme preparation from leaves of flax ( Linum usitatissimum L.) led to the formation of a divinyl ether fatty acid, i.e. (9 Z,11 E,1′ Z)-12-(1′-hexenyloxy)-9,11-dodecadienoic [(ω5 Z)-etheroleic] acid, as well as smaller amounts of 13-hydroxy-9( Z),11( E)-octadecadienoic acid. The 13-hydroperoxide of linoleic acid afforded the same set of products, whereas incubations of α-linolenic acid and its 13-hydroperoxide afforded the divinyl ether (9 Z,11 E,1′ Z,3′ Z)-12-(1′,3′-hexadienyloxy)-9,11-dodecadienoic [(ω5 Z)-etherolenic] as the main product. Identification of both divinyl ethers was substantiated by their UV, mass-, 1H NMR and COSY spectral data. In addition to the 13-lipoxygenase and divinyl ether synthase activities demonstrated by these results, flax leaves also contained allene oxide synthase activity as judged by the presence of endogenously formed (15 Z)- cis-12-oxo-10,15-phytodienoic acid in all incubations.

Response of Endogenous Salicylic Acid and Jasmonates to Mechanical Wounding in Pea Leaves

The roles of on endogenous jasmonates (JAs) and salicylic acid (SA) in wounding response were investigated. Pea ( Pisum sativum L.) seedlings were treated with three different methods including mechanical wounding, JAs application, and SA application. The contents of endogenous JAs and SA, as well as the activities of the related enzymes were detected by enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC), and spectrophotometer, respectively. The results showed that endogenous JA rapidly accumulated within 30 min after wounding. The increase in the activities of both lipoxygenase (LOX) and allene oxide synthase (AOS) lagged behind JAs burst. A second slight increase in JAs level was observed at 24 h after wounding treatment, and at the same time point, higher activities of LOX and AOS were also detected. Endogenous free SA content decreased accompanied with JAs burst. Effects of exogenous JA application were similar to those of wounding treatment on endogenous SA level and phenylalanine ammonia lyase (PAL) activity, whereas exogenous SA application led to the significant inhibition of LOX and AOS activities and the decrease of endogenous JAs level at the early stage of treatment. It is thus suggested that JAs burst and SA decrease in early response to wounding may constitute an important mechanism by which plant starts the related defense reaction and adapts to wounding stress.