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Olive pulp lipoxygenase regiospecificity and hydroperoxide lyase substrate specificity are important parameters in order to justify the volatile composition of olive oil. A new radiolabelling method to determine simultaneously these properties using only thin layer chromatography steps is described in the present work. The method involves incubation of an enzyme preparation from olive pulp with radiolabelled linoleate, followed by the fractionation of the resulting lipid products, previously treated with 2,4-dinitrophenyl hydrazine, on thin layer chromatography plates coated with polyethylenglycol 400. The results obtained are in agreement with previous studies carried out by other methods.

The hydroperoxide lyase (HPL) pathway for six carbon (C6) volatiles and the allene oxide synthase (AOS) pathway for jasmonates (JAs) share the first part of the pathway. To avoid competition, a separate localization of HPL and AOS might be important. A fusion protein comprising Arabidopsis HPL and green fluorescent protein was transported into chloroplasts, where AOS was located. Arabidopsis harboring β-glucuronidase (GUS) gene downstream of Arabidopsis HPL promoter (pAtHPL::GUS) showed different GUS activity in floral organs compared with that from pAtAOS::GUS. With pAtHPL::GUS, wounding enhanced GUS activity at the periphery of cotyledons; while with pAtAOS::GUS, GUS activity was high in the vasculature. The distribution of the ability to form C6 volatiles correlated with the profile of HPL promoter activity; however, this ability unchanged after wounding. Inconsistency between the AOS promoter activity and JA levels was also evident. Thus, an additional factor should also control the ability to form C6 volatiles and JAs. [ABSTRACT FROM AUTHOR]

Main conclusion: Allene oxide synthases (AOSs) were isolated from liverworts and charophytes. These AOSs exhibited enzymatic properties similar to those of angiosperms but formed a distinct phylogenetic clade. Allene oxide synthase (AOS) and hydroperoxide lyase (HPL) mediate the formation of precursors of jasmonates and carbon-six volatiles, respectively. AOS and HPL utilize fatty acid hydroperoxides and belong to the plant cytochrome P450 74 (CYP74) family that mediates plant defense against herbivores, pathogens, or abiotic stresses. Although members of the CYP74 family have been reported in mosses and other species, the evolution and function of multiple CYP74 genes in plants remain elusive. Here, we show that the liverwort Marchantia polymorpha belongs to a basal group in the evolution of land plants; has two closely related proteins (59 % identity), MpAOS1 and MpAOS2, that are similar to moss PpAOS1 (49 and 47 % identity, respectively); and exhibits AOS activity but not HPL activity. We also found that the green microalgae Klebsormidium flaccidum, consist of multicellular and non-branching filaments, contains an enzyme, KfAOS, that is similar to PpAOS1 (37 % identity), and converts 13-hydroperoxide of linolenic acid to 12-oxo-phytodienoic acid in a coupled reaction with allene oxide cyclase. Phylogenetic analysis showed two evolutionarily distinct clusters. One cluster comprised AOS and HPL from charophytic algae, liverworts, and mosses, including MpAOSs and KfAOS. The other cluster was formed by angiosperm CYP74. Our results suggest that plant CYP74 enzymes with AOS, HPL, and divinyl ether synthase activities have arisen multiple times and in the two different clades, which occurred prior to the divergence of the flowering plant lineage. [ABSTRACT FROM AUTHOR]

2(E)-hexenal is a green note flavour molecule that is widely used in various compositions of aromas, flavours and perfumery. As there is considerable demand for naturally produced aromas this article deals with some aspects of this C6-volatile production with regard to the selection of plant source material and reaction conditions. The following plants were tested for this purpose: runner bean (Phaseolus coccineus), common bean (Phaseolus vulgaris), three (Capsicum annuum) bell pepper varieties, garden cress (Lepidium sativum), green slicing cucumber (Cucumis sativa), mung beans (Vigna mungo) and brown lentils (Lens culinaris). Selection of source material was considered on the basis of 2(E)-hexenal yield and productivity. The common bean leafs were able to produce up to 35 mg of 2(E)-hexenal/kg fresh leaves. [ABSTRACT FROM AUTHOR]

In this study we focused on the development of Cabernet Sauvignon grapes and investigated changes in the activity of alcohol dehydrogenase (ADH) and hydroperoxide lyase (HPL) in different tissues. We sampled grape skin at four, six, seven, eight, nine, 10, 12, 14 and 16 weeks after anthesis; developing flowers when blooming at 0%, 5%, 50%, and 90%; and leaves at two and four weeks before anthesis and at two, four, six, eight, nine, and 10 weeks after anthesis. We also examined the type and fluctuation of volatile contents. ADH activity increased with the development of flowers and grape skins, which led to the increasing of types and concentration of alcohols. Low levels of 9-HPL led to low concentrations of C9 compounds. According to this paper, C6 compounds became abundant with the development of grape berries, while the activity of 13-HPL kept at a low level in the flowers and grape skins. There might have been a high level of 13-HPL activity from the end of flowering until fruit setting that we did not detect. Furthermore, similar C6 and C5 compounds were detected across all tissues, including hexanal, (E)-2-hexenal, (Z)-3-hexenal, (Z)-2-penten-1-ol, (Z)-3-hexen-1-ol, 1-hexanol and 3-hexen-1-ol. Generally speaking, the concentrations of C6 and C5 compounds could be used as the criterion of maturation of the three grape tissues. [ABSTRACT FROM AUTHOR]

Twelve aroma compounds were monitored and quantified by dynamic headspace analysis after their addition in refined olive oil model systems with extra virgin olive oil (EVOO) biophenols to simulate EVOO aroma. The influence of polyphenols on aroma release was studied under simulated mouth conditions by using human saliva, and SPME–GC/MS analysis. While few differences were observed in orthonasal assay (without saliva), interesting results were obtained for retronasal aroma. Biophenols caused generally the lowest headspace release of almost all volatile compounds. However, only ethyl esters and linalool concentrations were significantly lower in retronasal than orthonasal assay. Saliva also caused higher concentration of hexanal, probably due to hydroperoxide lyase (HPL) action on linoleyl hydroperoxides. Epicatechin was compared to EVOO phenolics and the behaviour was dramatically different, likely to be due to salivary protein–tannin binding interactions, which influenced aroma headspace release. These results were also confirmed using two extra virgin olive oils. [ABSTRACT FROM AUTHOR]

In corals a catalase-lipoxygenase fusion protein transforms arachidonic acid to the allene oxide 8R,9-epoxy-5,9,11,14-eicosatetraenoic acid from which arise cyclopentenones such as the prostanoid-related clavulones. Recently we cloned two catalaselipoxygenase fusion protein genes (a and b) from the coral Capnella imbricata, form a being an allene oxide synthase and form b giving uncharacterized polar products (Lõhelaid, H., Teder, T., Tõldsepp, K., Ekins, M., and Samel, N. (2014) PloS ONE 9, e89215). Here, using HPLC-UV, LC-MS, and NMR methods, we identify a novel activity of fusion protein b, establishing its role in cleaving the lipoxygenase product 8R-hydroperoxy-eicosatetraenoic acid into the short-chain aldehydes (5Z)-8-oxo-octenoic acid and (3Z,6Z)-dodecadienal; these primary products readily isomerize in an aqueous medium to the corresponding 6E- and 2E,6Zderivatives. This type of enzymatic cleavage, splitting the carbon chain within the conjugated diene of the hydroperoxide substrate, is known only in plant cytochrome P450 hydroperoxide lyases. In mechanistic studies using 18O labeled substrate and incubations in H2 18O, we established synthesis of the C8-oxo acid and C12 aldehyde with the retention of the hydroperoxy oxygens, consistent with synthesis of a shortlived hemiacetal intermediate that breaks down spontaneously into the two aldehydes. Taken together with our initial studies indicating differing gene regulation of the allene oxide synthase and the newly identified catalase-related hydroperoxide lyase and given the role of aldehydes in plant defense, this work uncovers a potential pathway in coral stress signaling and a novel enzymatic activity in the animal kingdom. [ABSTRACT FROM AUTHOR]

Green leaf volatiles (GLVs), the products of the hydroperoxide lyase (HPL) branch of the oxylipin pathway, play important roles in plant defense responses to both insect pests and pathogens. However, little is known about the role of GLVs in the defense responses of rice to the white-backed planthopper (WBPH, Sogatella furcifera (Horváth)). Here, we found that the rice HPL gene OsHPL3 was upregulated in response to infestation by WBPH adults and nymphs. Using an Agrobacterium-mediated transformation system, we obtained two homozygous lines with an antisense expression vector of OsHPL3 (as- hpl) and a single insertion that reduced the gene's expression by 48.31-52.56 %. Biochemical assays revealed that as- hpl lines had lower wound-induced GLV levels, mainly ( Z)-3-hexenal, ( Z)-3-hexen-1-ol, and ( E)-2-hexenal, but higher JA levels, compared to wild-type (WT) plants. Biological assays showed that WBPH adult females and nymphs preferred to settle and/or oviposit on WT plants, where they survived better than on as- hpl plants. In addition, both male and female WBPH nymphs fed on as- hpl plants performed less well: they developed more slowly, and pairs subsequently laid fewer eggs compared to pairs fed on WT plants. The enhanced resistance of as- hpl plants to WBPH infestation mainly correlated with lower levels of GLVs, especially ( Z)-3-hexenal. Collectively, these results indicated that WBPH infestation may enhance the susceptibility of rice by inducing the release of ( Z)-3-hexenal. [ABSTRACT FROM AUTHOR]

Recently, various biofuels have been synthesized through metabolic engineering approaches to meet the exploding energy demands. Hydrocarbon biofuels, energy-equivalent to petroleum-based fuels, are identified as promising replacements for petroleum. Metabolically engineered Saccharomyces cerevisiae capable of synthesize precursors of medium-chained hydrocarbons is proposed in this study. The hydroperoxide pathway introduced in S. cerevisiae consisted of lipoxygenase (LOX) and hydroperoxide lyase (HPL) from almond, which catalyzes linoleic acid to 3( Z)-nonenal, the precursor for medium-chained hydrocarbon biofuels. Proteomics study showed that 31 proteins displayed different expression levels among four functional strains and most of them were related to carbohydrate metabolism and protein synthesis, suggested prospective capabilities of energy generation and exogenous protein synthesis. Biotransformation efficiency studies carried out by GC-FID were in accordance with the expectations. The highest yield of 3( Z)-nonenal was up to 1.21 ± 0.05 mg/L with the carbon recovery of up to 12.4%. [ABSTRACT FROM AUTHOR]

Highlights: [•] Temperature, light, wounding and water regime regulate olive 13-LOXs and 13-HPL genes. [•] Low temperature and wounding produce an increase in LOX and HPL enzyme activities. [•] C6 volatile compounds were increased after low temperature and wounding stresses. [Copyright &y& Elsevier]

Hydroperoxide lyases (HPLs) play important roles in modulating plant defense by regulating the release of green leaf volatiles (GLVs) and the jasmonic acid (JA) pathway. CsiHPL1-a chloroplast-localized tea gene that encodes HPL-was previously cloned and predicted to be a regulator of plant defense responses. CsiHPL1 was expressed constitutively in transgenic tomato ( Solanum lycopersicum) plants to define its function in plant defense. CsiHPL1 overexpression caused tomato to release more constitutive and wound-induced GLVs [including (Z)-hexenal and (Z)-3-hexen-1-ol]. CsiHPL1 transgenic lines also exhibited lower levels of resistance to the larva of the tomato-chewing herbivore Prodenia litura (Fabricius) but enhanced resistance to the necrotrophic fungus Alternaria alternata f. sp. lycopersici (AAL). Furthermore, transgenic lines exhibited decreased expression levels of JA-related genes ( SlAOS and SlPI-II) induced by P. litura and AAL infection. We thus concluded that constitutive expression of CsiHPL1 can regulate tomato resistance to P. litura and AAL by modulating GLV release and JA gene expression. Application of these results will be helpful in controlling plant defenses against herbivore attack and fungal disease. [ABSTRACT FROM AUTHOR]

C5 volatile compounds, derived from fatty acids, are among the most important contributors to consumer liking of fresh tomatoes. Despite their important roles in flavour, the genes responsible for C5 volatile synthesis have yet to be identified. This work shows that their synthesis is catalysed in part by a 13-lipoxygenase (LOX), TomloxC, the same enzyme responsible for synthesis of C6 volatiles. C5 synthesis is independent of hydroperoxide lyase (HPL); moreover, HPL knockdown significantly increased C5 volatile synthesis. This LOX-dependent, HPL-independent pathway functions in both fruits and leaves. Synthesis of C5 volatiles increases in leaves following mechanical wounding but does not increase in response to infection with Xanthomonas campestris pv. vesicatoria. Large reductions in C5 and C6 volatiles in antisense TomloxC knockdown plants were observed but those reductions did not alter the development of disease symptoms, indicating that these volatiles do not have an important defensive function against this bacterial pathogen. [ABSTRACT FROM PUBLISHER]

Abstract: Synthesis of the aroma compounds of virgin olive oil (VOO) occurs through the lipoxygenase (LOX) pathway comprising mainly the actuation of LOX and hydroperoxide lyase (HPL) enzymes. The aim of this work was to determine whether the cleavage of polyunsaturated fatty acid hydroperoxides catalyzed by HPL is a limiting factor for the biosynthesis of VOO volatile compounds during the oil extraction process. For this purpose, HPL activity and the availability of substrates for this activity were modified during the oil extraction process from olive fruits of cultivars Arbequina and Picual, which give rise to oils with quantitatively different volatile profiles. Experimental data suggest that the HPL enzyme activity is just slightly limited during the oil extraction process in both cultivars, being this limitation apparently more significant during the processing of Arbequina fruits than of Picual fruits. However, this difference in HPL limitation seems to be more related to the differences in the amount of hydroperoxides produced in each cultivar than to the level of HPL activity during the olive processing. [Copyright &y& Elsevier]

Highlights: [•] Chitosan hybrid hydrogel was a suit carrier matrix for HPL immobilization. [•] The introduction of hydrophobic spacer-arm was benefit to the immobilization of HPL. [•] Product yield of 2(E)-hexenal by immobilized HPL was higher than the free enzyme. [•] The amount of enzyme demanded in the catalytic reaction was lower for immobilized HPL. [Copyright &y& Elsevier]

full-length cDNA coding for hydroperoxide lyase (CsHPL) was isolated from cucumber fruits of No. 26 (Southern China type) and No.14-1 (Northern China type), which differed significantly in fruit flavor. The deduced amino acid sequences of CsHPL from both lines show the same and significant similarity to known plant HPLs and contain typical conserved domains of HPLs. The recombinant CsHPL was confirmed to have 9/13-HPL enzymatic activity. Gene expression levels of CsHPL were measured in different organs, especially in fruits of different development stages of both lines. The HPL activities of fruit were identified basing on the catalytic action of crude enzyme extracts incubating with 13-HPOD (13-hydroperoxy-(9Z,12E)-octadecadienoic acid) and 13-HPOD + 9-HPOD (9-hydroperoxy-(10E,12Z)-octadecadienoic acid), and volatile reaction products were analyzed by GC-MS (gas chromatography-mass spectrometry). CsHPL gene expression in No. 26 fruit occurred earlier than that of total HPL enzyme activity and 13-HPL enzyme activity, and that in No. 14-1 fruit was consistent with total HPL enzyme activity and 9-HPL enzyme activity. 13-HPL enzyme activities decreased significantly and the 9-HPL enzyme activities increased significantly with fruit ripening in both lines, which accounted for the higher content of C6 aldehydes at 0-6 day post-anthesis (dpa) and higher content of C9 aldehydes at 9-12 dpa. [ABSTRACT FROM AUTHOR]

Polymers based on renewable resources have become increasingly important. The natural functionalization of fats and oils enables an easy access to interesting monomeric building blocks, which in turn transform the derivative biopolymers into high-performance materials. Unfortunately, interesting building blocks of medium-chain length are difficult to obtain by traditional chemical means. Herein, a biotechnological pathway is established that could provide an environmentally suitable and sustainable alternative. A multiple enzyme two-step one-pot process efficiently catalyzed by a coupled 9 S-lipoxygenase ( St-LOX1, Solanum tuberosum) and 9/13-hydroperoxide lyase ( Cm-9/13HPL, Cucumis melo) cascade reaction is proposed as a potential route for the conversion of linoleic acid into 9-oxononanoic acid, which is a precursor for biopolymers. Lipoxygenase catalyzes the insertion of oxygen into linoleic acid through a radical mechanism to give 9 S-hydroperoxy-octadecadienoic acid (9 S-HPODE) as a cascade intermediate, which is subsequently cleaved by the action of Cm-9/13HPL. This one-pot process afforded a yield of 73 % combined with high selectivity. The best reaction performance was achieved when lipoxygenase and hydroperoxide lyase were applied in a successive rather than a simultaneous manner. Green leaf volatiles, which are desired flavor and fragrance products, are formed as by-products in this reaction cascade. Furthermore, we have investigated the enantioselectivity of 9/13-HPLs, which exhibited a strong preference for 9 S-HPODE over 9 R-HPODE. [ABSTRACT FROM AUTHOR]

Highlights: [•] The interrelation of CYP74 enzymes was tested by single point mutations. [•] The mutant forms of tobacco and flax divinyl ether synthases (DESs) were prepared. [•] Mutation sites V379F and E292G belong to the ERR-triad and I-helix domains. [•] Both mutations converted DESs into allene oxide synthases (AOSs). [•] DES to AOS conversions caused by single point mutations are described for the first time. [ABSTRACT FROM AUTHOR]

Abstract: Expression of dissatisfaction with tomato aroma prompted us to lead this study on the impact of domestic storage conditions on volatile compounds. Two storage modalities (20 and 4°C) and two cultivars (Levovil and LCx) were used. Volatile compounds were analysed by gas chromatography–mass spectrometry detection after accelerated solvent extraction. Physical characteristics, lipoxygenase activity, hydroperoxide lyase activity; linoleic acid and linolenic acid were monitored. Storing tomatoes at 4°C induced a drastic loss in volatiles, whatever their biosynthetic origin. After 30days at 4°C, the concentration of volatiles had decreased by 66%. Reconditioning for 24h at 20°C was able to recover some aroma production after up to 6days storage at 4°C. Volatile degradation products arising from carotenoids and amino acids increased when tomatoes were kept at 20°C, while lipid degradation products did not vary. Storing tomatoes at fridge temperature, even for short durations, was detrimental for their aroma. This should be taken into account to formulate practical advice for consumers. [Copyright &y& Elsevier]

BACKGROUND Hydroperoxide lyase is the key enzyme in lipoxygenase pathway producing green-note flavours and has potential value for the flavour additive industry. So far, only a low yield of green-note flavours produced by hydroperoxide lyase has been achieved, primarily because of its instability. The aim of this study was to stabilise hydroperoxide lyase from Amaranthus tricolor leaves by immobilisation and investigate the characteristics of immobilised enzyme in comparison with free and native membrane-bound enzyme. RESULTS A maximum activity of 2.85 ± 0.1 U g−1 (wet) ceramic hydroxyapatite and a yield of 80% were obtained under optimised coupling conditions. The optimal reaction pH was 6.0, 6.0 and 7.5 for free, membrane-bound and immobilised enzyme respectively, while the optimal reaction temperature was 30, 35 and 35 °C respectively. Thermal and operational stability of immobilised enzyme were substantially enhanced. However, a higher substrate diffusion resistance was imposed after immobilisation, as evidenced by the Km value of immobilised enzyme being higher than that of free and membrane-bound enzyme. CONCLUSION Ceramic hydroxyapatite was a candidate for the immobilisation of hydroperoxide lyase from A. tricolor leaves. The stability of hydroperoxide lyase was substantially improved after immobilisation on this substrate. © 2012 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Biocatalysis with hydroperoxide lyase (HPL) in extracts from Penicillium camemberti, in neat organic solvent media has been investigated. The effects of reaction conditions including organic solvent mixtures, initial water activity ( aw) and reaction temperature as well as the effect of the lyoprotectants, KCl and dextran 1 kDa, on HPL activity were studied. The addition of KCl to the enzymatic extract (70:1 protein, w/w) prior to lyophilization, enhanced HPL activity 6.53-fold. In contrast, the presence of dextran at a ratio of 8:1 decreased the enzymatic activity. Using hexane as the reaction medium, with an initial aw of 0.1 and 0.5, the HPL specific activity was determined to be as 6.3 and 65.9 nmol converted 10-HPOD/mg protein/min, for the enzymatic extract without and with KCl present, respectively. Although HPL enzymatic extract with KCl showed a relatively low optimum reaction temperature (45°C) compared to 55°C without KCl, it exhibited a 2.51- and 2.78-fold higher thermal stability at 60 and 80°C, respectively. The kinetic results indicated that the highest HPL catalytic efficiency, Vmax/ Km, of 6.58 × 10−2 mL/mg protein/min, was obtained in the presence of KCl. [ABSTRACT FROM AUTHOR]

Abstract: The performance of a 13-hydroperoxide lyase from guava, an enzyme of the CYP74 family, which is of interest for the industrial production of saturated and unsaturated C6-aldehydes and their derivatives, was improved by directed evolution. Four rounds of gene shuffling and random mutagenesis improved the functional expression in E. coli by offering a 15-fold higher product yield factor. The increased product yield factor relates to an improved total turnover number of the variant enzyme, which also showed higher solubility and increased heme content. Thermal stability was also dramatically improved even though there was no direct selection pressure applied for evolving this trait. A structure based sequence alignment with the recently solved allene oxide synthase of Arabidopsis thaliana showed that most amino acid alterations occurred on the surface of the protein, distant of the active site and often outside of secondary structures. These results demonstrate the power of directed evolution for improving a complex trait such as the total turnover number of a cytochrome P450, a critical parameter for process performance that is difficult to predict even with good structural information at hand. [Copyright &y& Elsevier]

Fatty acid hydroperoxide lyase (HPL) is a membrane protein, member of the lipoxygenase pathway, which holds a central role in plant defense. Green bell pepper fatty acid hydroperoxide lyase, overexpressed in Escherichia coli, was purified and solubilized in two different non ionic detergents, Triton X-100 and dodecyl maltoside (DM). DM is considered to be more useful compared to Triton X-100, as it allows characterization of the protein with spectroscopic techniques, for which Triton X-100 was inapplicable. Circular dichroism demonstrated that HPL's secondary structure in DM consists of 13.53 % α-helix, 32.73 % β-sheet, 21.76 % turn and 31.13 % unordered. [ABSTRACT FROM AUTHOR]

Plants produce short-chain aldehydes and hydroxy fatty acids, which are important industrial materials, through the lipoxygenase pathway. Based on the information that lipoxygenase activity is up-regulated in tobacco leaves upon infection with tobacco mosaic virus ( TMV), we introduced a melon hydroperoxide lyase ( CmHPL) gene, a tomato peroxygenase ( SlPXG) gene and a potato epoxide hydrolase ( StEH) into tobacco leaves using a TMV-based viral vector system to afford aldehyde and hydroxy fatty acid production. Ten days after infiltration, tobacco leaves infiltrated with CmHPL displayed high enzyme activities of 9- LOX and 9- HPL, which could efficiently transform linoleic acid into C9 aldehydes. Protein extracts prepared from 1 g of CmHPL-infiltrated tobacco leaves (fresh weight) in combination with protein extracts prepared from 1 g of control vector-infiltrated tobacco leaves (as an additional 9- LOX source) produced 758 ± 75 μg total C9 aldehydes in 30 min. The yield of C9 aldehydes from linoleic acid was 60%. Besides, leaves infiltrated with SlPXG and StEH showed considerable enzyme activities of 9- LOX/ PXG and 9- LOX/ EH, respectively, enabling the production of 9,12,13-trihydroxy-10( E)-octadecenoic acid from linoleic acid. Protein extracts prepared from 1 g of SlPXG -infiltrated tobacco leaves (fresh weight) in combination with protein extracts prepared from 1 g of StEH-infiltrated tobacco leaves produced 1738 ± 27 μg total 9,12,13-trihydroxy-10( E)-octadecenoic acid isomers in 30 min. The yield of trihydroxyoctadecenoic acids from linoleic acid was 58%. C9 aldehydes and trihydroxy fatty acids could likely be produced on a larger scale using this expression system with many advantages including easy handling, time-saving and low production cost. [ABSTRACT FROM AUTHOR]

Abstract: Following a promising preliminary study concerning sugar beet leaves valorization and to fulfill the high demand in natural C6-aldehydes flavors, an efficient biocatalytic reaction was developed to synthesize (2E)-hexenal at large scale. As major product of the lipoxygenase enzyme, 13-HPOT was converted by sugar beet hydroperoxide lyase extracted from leaves or expressed by recombinant Escherichia coli strains. With the adaptation of a fed-batch substrate addition and a continuous extraction of volatiles, 3.46mM and 1.37mM of C6-aldehydes were produced with the native hydroperoxide lyase at respectively 2 and 100L scale. Furthermore, higher molar productivity of green leaf volatiles was reached with recombinant hydroperoxide lyase (5.5mM at 2L scale) while no others side products from the lipoxygenase pathway were formed. Once purified, these natural aromas are suitable for food and beverage preparations, perfumes or cosmetic products. [Copyright &y& Elsevier]

The western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), is one of the most destructive pests of corn in North America and is currently invading Europe. The two major invasive outbreaks of rootworm in Europe have occurred, in North-West Italy and in Central and South-Eastern Europe. These two outbreaks originated from independent introductions from North America. Secondary contact probably occurred in North Italy between these two outbreaks, in 2008. We used 13 microsatellite markers to conduct a population genetics study, to demonstrate that this geographic contact resulted in a zone of admixture in the Italian region of Veneto. We show that i) genetic variation is greater in the contact zone than in the parental outbreaks; ii) several signs of admixture were detected in some Venetian samples, in a Bayesian analysis of the population structure and in an approximate Bayesian computation analysis of historical scenarios and, finally, iii) allelic frequency clines were observed at microsatellite loci. The contact between the invasive outbreaks in North-West Italy and Central and South-Eastern Europe resulted in a zone of admixture, with particular characteristics. The evolutionary implications of the existence of a zone of admixture in Northern Italy and their possible impact on the invasion success of the western corn rootworm are discussed. [ABSTRACT FROM AUTHOR]

(2E)-Hexenal is widely used in flavors and perfumes; however, it is mainly derived from chemical synthesis. In this study, hydroperoxide lyase (HPL) naturally originated from Amaranthus tricolor was used to catalyze the reaction to generate (2E)-hexenal from 13-hydroperoxy-9Z, 11E, 15 Z-octadecatrienoic acid (13-HPOT), and salt-adding steam distillation was used for the separation of (2E)-hexenal. A maximum yield of (2E)-hexenal that reached 1,156.4 mg L was obtained with a high substrate concentration (40 mM 13-HPOT) under the following conditions: 10 min, pH 7.5, 20 °C, HPL 16 U mL, butylated hydroxytoluene (BHT) 1 mM, and dithiothreitol (DTT) 15 mM. Then the separation of (2E)-hexenal was conducted by salt-adding steam distillation. It was found that AlCl had the greatest effect on the separation of (2E)-hexenal, followed by CaCl and NaCl. The distillate yield with the addition of AlCl was 93.2 %, while the distillate yield without the addition of salt was 81.2 %. The distillate concentration with AlCl was 6,464.2 mg L, while the distillate concentration without the addition of salt was 4,490.3 mg L. The addition of salt improved the efficiency of the steam distillation. This study was greatly meaningful for providing a green method to the large-scale production of (2E)-hexenal. [ABSTRACT FROM AUTHOR]

As important signal molecules, jasmonates (JAs) and green leaf volatiles (GLVs) play diverse roles in plant defense responses against insect pests and pathogens. However, how plants employ their specific defense responses by modulating the levels of JA and GLVs remains unclear. Here, we describe identification of a role for the rice HPL3 gene, which encodes a hydroperoxide lyase (HPL), OsHPL3/CYP74B2, in mediating plant-specific defense responses. The loss-of-function mutant hpl3-1 produced disease-resembling lesions spreading through the whole leaves. A biochemical assay revealed that OsHPL3 possesses intrinsic HPL activity, hydrolyzing hydroperoxylinolenic acid to produce GLVs. The hpl3-1 plants exhibited enhanced induction of JA, trypsin proteinase inhibitors and other volatiles, but decreased levels of GLVs including ( Z)-3-hexen-1-ol. OsHPL3 positively modulates resistance to the rice brown planthopper [BPH, Nilaparvata lugens (Stål)] but negatively modulates resistance to the rice striped stem borer [SSB, Chilo suppressalis (Walker)]. Moreover, hpl3-1 plants were more attractive to a BPH egg parasitoid, Anagrus nilaparvatae, than the wild-type, most likely as a result of increased release of BPH-induced volatiles. Interestingly, hpl3-1 plants also showed increased resistance to bacterial blight ( Xanthomonas oryzae pv. oryzae). Collectively, these results indicate that OsHPL3, by affecting the levels of JA, GLVs and other volatiles, modulates rice-specific defense responses against different invaders. [ABSTRACT FROM AUTHOR]

C6 compounds are the major fraction of the volatile profiles of grape berries, contributing the typical 'green' aroma to the grape and wine. Hydroperoxide lyase (HPL) catalyzes the cleavage of fatty acid hydroperoxides to produce C6 compounds. Two hypothetical genes, VvHPL1 and VvHPL2 were cloned from grape berries ( Vitis vinifera L. Cabernet Sauvignon). Bioinformatics analysis revealed that the proteins encoded by these two genes both belong to subfamily of cytochrome P450 and contain typical conserved domains of HPLs, and have high identity with HPLs from other plants. Prokaryotically-expressed VvHPL1 and VvHPL2 with thioredoxin-6xHis-fusion partner were confirmed to have enzymatic activity. VvHPL1 is specific for 13-HPOD (T) producing C6 aldehydes with relatively higher activity and VvHPL2 catalyzes the cleavage of both 9- and 13-hydroperoxides producing C6 aldehydes and C9 aldehydes respectively. Analysis of real time-PCR showed that VvHPL2 was highly expressed in the leaves and the flowers of the grapes, while relatively low transcript abundance was detected in the berries, tendril and stems; VvHPL1 had high expression in all detected tissues. During grape berry development, the expression of these two isogenes presented similar trends with a rapid increase after veraison and a decrease at full-ripen stage, which roughly corresponded to the accumulation of their volatile products. These data lay an essential foundation for further study on the accumulation and control of C6 volatiles in grape berries. [ABSTRACT FROM AUTHOR]

Summary The MADS box transcription factor RIN is a global regulator of fruit ripening. However, the direct targets modulated by RIN and the mechanisms underlying the transcriptional regulation remain largely unknown. Here we identified 41 protein spots representing 35 individual genes as potential targets of RIN by comparative proteomic analysis of a rin mutant in tomato fruits. Gene expression analysis showed that the mRNA level of 26 genes correlated well with the protein level. After examining the promoter regions of the candidate genes, a variable number of RIN binding sites were found. Five genes ( E8, TomloxC, PNAE, PGK and ADH2) were identified as novel direct targets of RIN by chromatin immunoprecipitation. The results of a gel mobility shift assay confirmed the direct binding of RIN to the promoters of these genes. Of the direct target genes, TomloxC and ADH2, which encode lipoxygenase (LOX) and alcohol dehydrogenase, respectively, are critical for the production of characteristic tomato aromas derived from LOX pathway. Further study indicated that RIN also directly regulates the expression of HPL, which encodes hydroperoxide lyase, another rate-limiting enzyme in the LOX pathway. Loss of function of RIN causes de-regulation of the LOX pathway, leading to a specific defect in the generation of aroma compounds derived from this pathway. These results indicate that RIN modulates aroma formation by direct and rigorous regulation of expression of genes in the LOX pathway. Taken together, our findings suggest that the regulatory effect of RIN on fruit ripening is achieved by targeting specific molecular pathways. [ABSTRACT FROM AUTHOR]

A cDNA encoding hydroperoxide lyase (HPL) was isolated from Solanum tuberosum, cloned into pQE-30 vector, and expressed in E. coli. The recombinant protein was purified by nickel affinity chromatography and showed an approximate molecular weight of 54 kDa by SDS-PAGE analysis, which was similar to the predicted value based on the putative amino acid sequences (53.9 kDa). 13-Hydroperoxy-linolenic acid (13-HPOT) was the preferred substrate for the enzyme compared with 13-hydroperoxy-linoleic acid (13-HPOD). The corresponding volatile products were 2(E)-hexenal and n-hexanal tested by headspace-gas chromatography, respectively. The enzyme was optimally active at 25 °C and pH 6.5. The K, V, and the catalytic efficiency ( V/ K) for 13-HPOT were 56.6 μM, 71.3 units/mg, and 1.26 units/mg · μM, respectively. Activity of the recombinant potato HPL increased when Triton X-100, sodium chloride, or potassium chloride was added in the reaction mixture, while calcium chloride decreased activity of the recombinant enzyme. [ABSTRACT FROM AUTHOR]

Green notes are substances that characterize the aroma of freshly cut grass, cucumbers, green apples, and foliage. In plants, they are synthesized by conversion of linolenic or linoleic acid via the enzymes lipoxygenase (LOX) and hydroperoxide lyase (HPL) to short-chained aldehydes. Current processes for production of natural green notes rely on plant homogenates as enzyme sources but are limited by low enzyme concentration and low specificity. In an alternative approach, soybean LOX2 and watermelon HPL were overexpressed in Saccharomyces cerevisiae. After optimization of the expression constructs, a yeast strain coexpressing LOX and HPL was applied in whole cell biotransformation experiments. Whereas addition of linolenic acid to growing cultures of this strain yielded no products, we were able to identify high green note concentrations when resting cells were used. The primary biotransformation product was 3(Z)-hexenal, a small amount of which isomerized to 2(E)-hexenal. Furthermore, both aldehydes were reduced to the corresponding green note alcohols by endogenous yeast alcohol dehydrogenase to some extent. As the cosolvent ethanol was the source of reducing equivalents for green note alcohol formation, the hexenal/hexenol ratio could be influenced by the use of alternative cosolvents. Further investigations to identify the underlying mechanism of the rather low biocatalyst stability revealed a high toxicity of linolenic acid to yeast cells. The whole cell catalyst containing LOX and HPL enzyme activity described here can be a promising approach towards a highly efficient microbial green note synthesis process. [ABSTRACT FROM AUTHOR]

Plant oxylipins are involved in defense responses against pathogens or herbivores. Each oxylipin compound exerts its distinctive physiological function. The ability of many legumes to fix nitrogen with rhizobia gives them special importance in natural environments and agriculture. It has been postulated that oxylipins are somehow related to symbiosis; however, this is still a controversial issue. In this study, we isolated five genes at the branching point of the oxylipin pathway in Lotus japonicus, and their biochemical functions were identified as allene oxide synthases (AOSs), 13-hydroperoxide lyase (13HPL), and 9/13-HPLs. When the leaves were mechanically wounded, AOS and 9/13HPL were upregulated in leaves and roots, respectively, from which their implications in wound response were suggested. When the plants were inoculated with rhizobia, no big change in the expression levels of genes was found. When high N was supplied to the nodulated plants, the number of nodules decreased, and simultaneously, AOS in the leaves was downregulated. Significance of AOS in response to the N status in the plants was suggested. [ABSTRACT FROM PUBLISHER]

Abstract: Hexanal, Z-3-hexenal, E-2-hexenal, hexanol, and Z-3-hexenol are major tomato (Solanum lycopersicum L.) volatile aroma compounds derived from oxygenation of unsaturated fatty acids. Chilling and heating may suppress production of these C6 volatiles. The objective of this research was to determine the effects of chilling or heating on gene expression and enzyme activity of lipoxygenase (LOX), hydroperoxide lyase (HPL), and alcohol dehydrogenase (ADH), which catalyze key steps in C6 volatile production via the oxylipin pathway. ‘Tasti-Lee’ and ‘Sanibel’ tomatoes, harvested at different stages of fruit development, were ripened to full (red) ripe stage at 20°C. Fruit were then treated by either chilling (5°C for 5d) or heating (52°C hot water for 15min), then cooled with 23°C tap water to 25°C and placed at 20°C for 4d, or held continuously at 20°C as the control. Both chilling and heating reduced C6 aldehyde and alcohol aroma volatiles immediately after treatment, and the levels of aldehydes did not fully recover after 4d at 20°C. Chilling down-regulated expression of TomloxA, B, and C, but not D; however, it increased total LOX activity. Chilling also down-regulated HPL and ADH expression immediately after treatment, but, after 4d at 20°C, both genes were up-regulated compared to the control. HPL activity in chilled tomatoes was reduced, but recovered to control levels after 4d at 20°C. ADH activity in chilled fruit decreased after 4d at 20°C. On the other hand, heating greatly up-regulated TomloxB and C expression, even after 4d at 20°C, and slightly down-regulated TomloxA and D, while increasing overall LOX activity. Heating up-regulated both HPL and ADH, and that effect persisted for 4d at 20°C. However, heating reduced the activities of HPL for 4d at 20°C and ADH immediately after treatment. The results indicate that heating and chilling regulate C6 volatile production by different mechanisms. Chilling-induced inhibition of C6 volatile production may be due to down-regulation of gene expression, and subsequent reduction of HPL and ADH enzyme activities in the oxylipin pathway. Heating-inhibition of C6 volatile production, however, does not appear to be due to down-regulation of gene expression, but HPL and ADH activities were briefly suppressed. [Copyright &y& Elsevier]

Abstract: The profiles of non-volatile oxylipins of pea (Pisum sativum) seedlings were examined by gas chromatography–mass spectrometry after in vitro incubation with α-linolenic acid. The 13-lipoxygenase/hydroperoxide lyase (HPL) products were predominant in the leaves, while the roots possess both 13- and 9-HPL products. Allene oxide synthase (AOS) and divinyl ether synthase (DES) products were not detected in the leaves or in the roots of any age. The HPL cascade produces a diversity of oxylipins, including the compounds (2E)-4-hydroxy-traumatic, (10E)-9,12-dihydroxy-10-dodecenoic and 9,12-dihydroxydodecanoic acids, as well as (2E)-4-hydroxy-2-nonenoic acid, which has not yet been detected in plants. Oxylipin patterns were altered by infection, water deficit, as well as by plant age. Infection caused the specific strong accumulation of azelaic (nonane-1,9-dioic) acid in the leaves. The azelaic acid content in the aged (14 and 18day-old) leaves was significantly higher than in the younger leaves. Water deficit induced the accumulation of (2E)-4-hydroxy-2-nonenoic acid and (2E)-traumatic acid in the roots. Results demonstrate that: (1) the HPL cascade is the predominant branch of the lipoxygenase pathway in pea seedlings; (2) the HPL products may have the regulatory role both in growth control and adaptation. [Copyright &y& Elsevier]

Abstract: A combined theoretical and experimental study highlights the reaction mechanism of allene oxide synthase (AOS) and its possible link to hydroperoxide lyase (HPL) pathway. A previously published study (Lee et al., Nature 455 (2008) 363) has shown that the F137 residue is of central importance in differentiating between the AOS and HPL pathways after initial identical steps. In the experimental part of this study, we show that wild-type AOS from Arabidopsis or rice in fact produces both AOS and HPL products in a ratio of about 80:15, something that was found only in trace amounts before. Theoretical calculations successfully map the whole AOS pathway with 13(S)-hydroperoxy linolenic and linoleic acid as substrates. Subsequent calculations investigated the effects of in silico F137L mutation at the suggested diverging point of the two pathways. The results show that QM/MM calculations can reasonably reproduce three out of four experimentally available cases, and confirm that the pathways are energetically very close to each other, thus making a switch from one path to other plausible under different circumstances. [Copyright &y& Elsevier]

Hydroperoxide lyase (HPL) was extracted from amaranth tricolor leaves using Triton X-100, and purified to electrophoretic homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic interaction chromatography and hydroxyapatite chromatography. The purified HPL preparation consisted of a single band and spot with a molecular mass of about 55 kDa as shown in SDS-PAGE and 2-D PAGE, respectively; the isoelectric point was found to be about 5.4. The maximum activity of the enzyme was observed at pH 6.0 and 25 °C, respectively. The HPL showed higher activity against 13-hydroperoxy-linolenic acid compared to 13-hydroperoxy-linoleic acid. K value for 13-hydroperoxy-linolenic acid was 62.7 μM, and the corresponding V was 178.5 μM min. The activity of HPL was significantly inhibited by nordihydroguaiaretic acid, HgCl and 2(E)-hexenal but not by EDTA and hexanal. [ABSTRACT FROM AUTHOR]

Abstract: Fatty acid hydroperoxide lyase (HPL) is a member of the cytochrome P450 family acting on fatty acid hydroperoxides in many organisms. The active green bell pepper HPL, cloned and expressed in the yeast Yarrowia lipolytica, was purified by immobilized metal-ion affinity chromatography (IMAC) in the presence of 2% of Triton X-100R. The secondary structure prediction by bioinformatics servers of HPL was realized by ANTHEPROT software, using the GOR, DPM and Predator methods. The theoretical results which are average values obtained from three different calculation methods showed 33% α-helix, 18% β-sheet, 7% turn and 42% coil. On the other hand, the secondary structure approach of the purified active HPL (specific activity of 2.94U/mg protein) was realized by differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy, and showed 13% α-helix, 29% β-sheet, 5% turn and 53% random coil. [Copyright &y& Elsevier]

A pre-infestation of the white-backed planthopper (WBPH), Sogatella furcifera Horváth, conferred resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae ( Xoo) in rice ( Oryza sativa L.) under both laboratory and field conditions. The infestation of another planthopper species, the brown planthopper (BPH) Nilaparvata lugens Stål, did not significantly reduce the incidence of bacterial blight symptoms. A large-scale screening using a rice DNA microarray and quantitative RT-PCR revealed that WBPH infestation caused the upregulation of more defence-related genes than did BPH infestation. Hydroperoxide lyase 2 ( OsHPL2), an enzyme for producing C6 volatiles, was upregulated by WBPH infestation, but not by BPH infestation. One C6 volatile, ( E)-2-hexenal, accumulated in rice after WBPH infestation, but not after BPH infestation. A direct application of ( E)-2-hexenal to a liquid culture of Xoo inhibited the growth of the bacterium. Furthermore, a vapour treatment of rice plants with ( E)-2-hexenal induced resistance to bacterial blight. OsHPL2-overexpressing transgenic rice plants exhibited increased resistance to bacterial blight. Based on these data, we conclude that OsHPL2 and its derived ( E)-2-hexenal play some role in WBPH-induced resistance in rice. [ABSTRACT FROM AUTHOR]

The influence of high-intensity pulsed electric field (HIPEF) parameters, namely, pulse frequency, pulse width, and polarity on tomato juice lipoxygenase (LOX) and hydroperoxide lyase (HPL) activities was studied using a response surface methodology. Samples were subjected to square-shaped pulses of 35 kV/cm for 1000 μs, with pulse width ranging from 1 to 7 μs at frequencies from 50 to 250 Hz, either in monopolar or bipolar mode. Tomato LOX was more resistant to HIPEF than HPL within the range of assayed conditions. An increase in frequency or pulse width resulted in a decrease of both residual LOX ( RALOX) and HPL ( RAHPL) activities. The lowest RALOX (81%) was observed when tomato juice was treated at 250 Hz for 7 μs in bipolar mode. Moreover, the same conditions led to the highest HPL activity reduction ( RAHPL = 10%). A validation of the predictive models determined that 2nd-order expressions were accurate enough to fit the experimental results. [ABSTRACT FROM AUTHOR]

Abstract: The existence of CYP5, CYP8A, and the CYP74 enzymes specialized for reaction with fatty acid peroxide substrates presents opportunities for a “different look” at the catalytic cycle of the cytochrome P450s. This review considers how the properties of the peroxide-metabolizing enzymes are distinctive, and how they tie in with those of the conventional monooxygenase enzymes. Some unusual reactions of each class have parallels in the other. As enzyme reactions and P450 structures emerge there will be possibilities for finding their special properties and edging this knowledge into the big picture. [Copyright &y& Elsevier]

The article presents a study which develops a volatile system suited for comprehensive screening of soybean volatiles. The study established an effective screening system with the use of automated headspace sampler connected to gas chromatography. It states that by using the screening system five primary volatile compounds were examined within 12 minutes. The study isolated four soybean varieties which manifest unique components of volatile compounds through employing the screening system.

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. [Display omitted] • The coding sequences of Selaginella moellendorffii CYP74L genes have been cloned. • The recombinant CYP74L1 possesses mainly hydroperoxide lyase activity. • The recombinant CYP74L2 exhibits allene oxide synthase activity. • CYP74Ls append the complex picture of oxylipin biosynthesis in oldest land plants. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Circular dichroism (CD) spectroscopy of secondary structure conformation of the purified green bell pepper hydroperoxide lyase (HPL), cloned in the yeast Yarrowia lipolytica, was investigated. The CD spectra of HPL in iso-octane, obtained at 60°C, in the presence of the reducing agent dithiothreitol showed dramatic increase in α-helix content. The enzyme conformation remained unchanged over a range of pH values of 5.0–7.0. Using 13-hydroperoxide of linoleic acid (13-HPOD) as substrate, the biocatalysis of HPL in organic solvent media, including chloroform, dichloromethane, hexane, iso-octane, octane and toluene, was investigated. The results indicated an increase in HPL activity in the biphasic hexane medium. [Copyright &y& Elsevier]

Abstract: An enzymatic extract from Penicillium camemberti, containing lipoxygenase (LOX) and hydroperoxide lyase (HPL) activities, was immobilized on oxirane acrylic beads, Eupergit C and Eupergit C250L-iminodiacetic acid (IDA). The optimum pH for LOX activity was determined to be 4.0 and 6.0 for the free enzyme extract and 6.0 for the immobilized one, whereas that for the HPL activity was 4.0 and 6.0 for the immobilized and free extracts. The optimal reaction temperature for LOX activity was 30 and 55°C for the free and immobilized enzyme extracts, respectively, whereas the HPL activity showed its optima at 45 and 30°C, for the free and immobilized extracts, respectively. The immobilization of the enzymatic extract dramatically enhanced the thermostability of LOX and HPL activities. In term of enzymatic stability, the lyophilized immobilized extract showed that its HPL activity at 4°C was more stable than that of LOX. The results indicated a decrease and an increase in enzyme efficiency for LOX and HPL activity, respectively, upon immobilization. [Copyright &y& Elsevier]

Hydroperoxide lyase activity was found in sugar beet leaves. Its optimum pH and temperature were, respectively, 6.7 and 22°C. Under these conditions, conversion of linolenic acid 13-hydroperoxide to cis-3-hexenal with a maximum yield of 80% was reached after only 2 min. The stability of cis-3-hexenal was improved by acidifying the reaction medium. Based on these studies, a bioprocess producing green-note aldehydes in a laboratory-scale was achieved. [ABSTRACT FROM AUTHOR]

The production of aroma volatile compounds and standard quality attributes, in addition to lipoxygenase (LOX), hydroperoxide lyase (HPL), pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH) and alcohol o-acyltransferase (AAT) activities, were assessed during maturation of ‘Pink Lady®’ apples. Low production of aroma volatiles was observed in early harvested fruit, which gradually increased as ripeness approached. Hexyl acetate, hexyl 2-methylbutanoate, hexyl hexanoate, hexyl butanoate, 2-methylbutyl acetate and butyl acetate were prominent within the blend of volatiles produced by fruit throughout maturation. Multivariate analysis showed these compounds had the highest influence on differentiation of maturity stages, indicating that aroma volatile emission is an important factor for definition of fruit ripeness, which suggests production of these esters might be useful as an index of maturity. No large variations in AAT activity were found throughout the experimental period despite increasing ester emission, suggesting the enhancement of ester production by ‘Pink Lady®’ apples at ripening arises mainly from greater availability of substrates. Increased LOX activity was observed at later stages of fruit development, and the possible role of this enzyme activity on enhanced capacity for aroma volatile biosynthesis in more mature fruit is discussed. [Copyright &y& Elsevier]