Hydroperoxide Lyase Research Articles

Plant hydroperoxide-cleaving enzymes (CYP74 family) function as hemiacetal synthases: Structural proof of hemiacetals by NMR spectroscopy

Hydroperoxide lyases (HPLs) of the CYP74 family (P450 superfamily) are widely distributed enzymes in higher plants and are responsible for the stress-initiated accumulation of short-chain aldehydes. Fatty acid hydroperoxides serve as substrates for HPLs; however, details of the HPL-promoted conversion are still incompletely understood. In the present work, we report first time the micropreparative isolation and the NMR structural studies of fatty acid hemiacetal (TMS/TMS), the short-lived HPL product. With this aim, linoleic acid 9(S)‑hydroperoxide (9(S)‑HPOD) was incubated with recombinant melon hydroperoxide lyase (CmHPL, CYP74C2) in a biphasic system of water/hexane for 60 s at 0 °C, pH 4.0. The hexane layer was immediately decanted and vortexed with a trimethylsilylating mixture. Analysis by GC–MS revealed a major product, i.e. the bis-TMS derivative of a hemiacetal which was conclusively identified as 9‑hydroxy‑9‑[(1′E,3′Z)‑nonadienyloxy]‑nonanoic acid by NMR-spectroscopy. Further support for the hemiacetal structure was provided by detailed NMR-spectroscopic analysis of the bis-TMS hemiacetal generated from [13C18]9(S)‑HPOD in the presence of CmHPL. The results obtained provide incontrovertible evidence that the true products of the HPL group of enzymes are hemiacetals, and that the short-chain aldehydes are produced by their rapid secondary chain breakdown. Therefore, we suggest replacing the name “hydroperoxide lyase”, which does not reflect the factual isomerase (intramolecular oxidoreductase) activity, with “hemiacetal synthase” (HAS).

An oriental melon 9-lipoxygenase gene CmLOX09 response to stresses, hormones, and signal substances.

In plants, lipoxygenases (LOXs) play a crucial role in biotic and abiotic stresses. In our previous study, five 13-LOX genes of oriental melon were regulated by abiotic stress but it is unclear whether the 9-LOX is involved in biotic and abiotic stresses. The promoter analysis revealed that CmLOX09 (type of 9-LOX) has hormone elements, signal substances, and stress elements. We analyzed the expression of CmLOX09 and its downstream genes-CmHPL and CmAOS-in the leaves of four-leaf stage seedlings of the oriental melon cultivar "Yumeiren" under wound, hormone, and signal substances. CmLOX09, CmHPL, and CmAOS were all induced by wounding. CmLOX09 was induced by auxin (indole acetic acid, IAA) and gibberellins (GA3); however, CmHPL and CmAOS showed differential responses to IAA and GA3. CmLOX09, CmHPL, and CmAOS were all induced by hydrogen peroxide (H2O2) and methyl jasmonate (MeJA), while being inhibited by abscisic acid (ABA) and salicylic acid (SA). CmLOX09, CmHPL, and CmAOS were all induced by the powdery mildew pathogen Podosphaera xanthii. The content of 2-hexynol and 2-hexenal in leaves after MeJA treatment was significantly higher than that in the control. After infection with P. xanthii, the diseased leaves of the oriental melon were divided into four levels-levels 1, 2, 3, and 4. The content of jasmonic acid (JA) in the leaves of levels 1 and 3 was significantly higher than that in the level 0 leaves. In summary, the results suggested that CmLOX09 might play a positive role in the response to MeJA through the hydroperoxide lyase (HPL) pathway to produce C6 alcohols and aldehydes, and in the response to P. xanthii through the allene oxide synthase (AOS) pathway to form JA.

Effect of Micro-Perforated Film Packing on Fatty Acid-Derived Volatile Metabolism of “Red Globe” Table Grapes

The present study was aimed at understanding the effect of micro-perforated polyethylene film packing (MPFP) on the metabolism of fatty acid-derived volatiles and physicochemical attributes in “Red Globe” table grapes under storage at 0 °C for 50 days. Physicochemical properties evaluated in grapes were total soluble solids (TSS), texture profile, decay incidence, fatty acid content, and aroma-related enzyme activities including lipoxygenase (LOX), hydroperoxide lyase (HPL), alcohol dehydrogenase (ADH), and volatiles in grape peel and flesh. Aroma-related genes (VvLOX, VvHPL, and VvADH) were also evaluated. Grapes treated with MPFP was found to retain the texture profile and decay incidence was reduced (17.4% of reduction compared with control) significantly (P ≤ 0.05). Linoleic acid (LA), the main substrate of LOX/HPL pathway, was the highest in table grapes packed in MPFP, indicating the use of MPFP was able to preserve LA compound. Micro-perforated film packing upregulated VvLOX and VvHPL, downregulated VvADH, increased LOX and HPL activities, and inhibited ADH activity, enhancing C6/C9 compounds. The results indicated that MPFP system was the best choice to improve aroma quality of “Red Globe” table grapes in comparison to polyethylene film packing (PFP) and control (no packing).

Contribution of abscisic acid to aromatic volatiles in cherry tomato (Solanum lycopersicum L.) fruit during postharvest ripening

Fruit aroma development depends on ripening. Abscisic acid (ABA) has been reported to be involved in the regulation of tomato fruit ripening. In the present study, the effects of exogenous ABA on aromatic volatiles in tomato fruit during postharvest ripening were studied. The results showed that exogenous ABA accelerated color development and ethylene production as well as the accumulation of carotenoids, total phenolics and linoleic acid in tomato fruit during ripening. Moreover, exogenous ABA increased the accumulation of volatile compounds such as 1-peten-3-one (2.06-fold), β-damascenone (1.64-fold), benzaldehyde (3.29-fold) and benzyl cyanide (4.15-fold); induced the expression of key genes implicated in the biosynthesis pathways of aromatic volatiles, including TomloxC, HPL, ADH2, LeCCD1B and SlBCAT1 (the values of the log2 fold changes ranged from −3.02 to 2.97); and promoted the activities of lipoxygenase (LOX), hydroperoxide lyase (HPL) and alcohol dehydrogenase (ADH). In addition, the results of promoter analyses revealed that cis-acting elements involved in ABA responsiveness (ABREs) exist in 8 of the 12 key genes involved in volatile biosynthesis, suggesting that ABA potentially affects aromatic volatile emissions via the regulation of gene expression profiles.

“Biosynthesis of volatile compounds by hydroperoxide lyase enzymatic activity during virgin olive oil extraction process”

The approach of this research was to describe the biochemical transformations of minor components of virgin olive oil with high impact on its sensory quality, specifically “volatile compounds” during its extraction process. For this purpose `Picual´, `Arbequina´ and `Hojiblanca´ cultivars were processed at three different harvesting times. Their volatile profiles and levels of enzymatic activity of “hydroperoxide lyase”, responsible for the biosynthesis of C6 aldehydes related to “green and fruity notes”, were monitored and identified in mesocarp, paste before kneading and paste after kneading based on the catalytic action of crude enzyme extracts. Both of them, volatiles and HPL, were analyzed by incubating with their corresponding substrates and reaction products formed were analyzed by Solid Phase Microextraction-Gas Chromatography and Mass Spectrometry. The results obtained in the present research can be useful in order to develop new markers biochemical whereby improved aroma quality in olive breeding programs or in the design of new protocol of VOO extraction.

Grapevine fatty acid hydroperoxide lyase generates actin-disrupting volatiles and promotes defence-related cell death.

Fatty acid hydroperoxides can generate short-chained volatile aldehydes that may participate in plant defence. A grapevine hydroperoxide lyase (VvHPL1) clustering to the CYP74B class was functionally characterized with respect to a role in defence. In grapevine leaves, transcripts of this gene accumulated rapidly to high abundance in response to wounding. Cellular functions of VvHPL1 were investigated upon heterologous expression in tobacco BY-2 cells. A C-terminal green fluorescent protein (GFP) fusion of VvHPL1 was located in plastids. The overexpression lines were found to respond to salinity stress or the bacterial elicitor harpin by increasing cell death. This signal-dependent mortality response was mitigated either by addition of exogenous jasmonic acid or by treatment with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases. By feeding different substrates to recombinantly expressed enzyme, VvHPL1 could also be functionally classified as true 13-HPL. The cognate products generated by this 13-HPL were cis-3-hexenal and trans-2-hexenal. Using a GFP-tagged actin marker line, one of these isomeric products, cis-3-hexenal, was found specifically to elicit a rapid disintegration of actin filaments. This response was not only observed in the heterologous system (tobacco BY-2), but also in a grapevine cell strain expressing this marker, as well as in leaf discs from an actin marker grape used as a homologous system. These results are discussed in the context of a role for VvHPL1 in a lipoxygenase-dependent signalling pathway triggering cell death-related defence that bifurcates from jasmonate-dependent basal immunity.

A Comparison of the Effects of FATTY ACID DESATURASE 7 and HYDROPEROXIDE LYASE on Plant-Aphid Interactions.

The spr2 mutation in tomato (Solanum lycopersicum), which disrupts function of FATTY ACID DESATURASE 7 (FAD7), confers resistance to the potato aphid (Macrosiphum euphorbiae) and modifies the plant’s C6 volatile profiles. To investigate whether C6 volatiles play a role in resistance, HYDROPEROXIDE LYASE (HPL), which encodes a critical enzyme in C6 volatile synthesis, was silenced in wild-type tomato plants and spr2 mutants. Silencing HPL in wild-type tomato increased potato aphid host preference and reproduction on 5-week old plants but had no influence on 3-week old plants. The spr2 mutation, in contrast, conferred strong aphid resistance at both 3 and 5 weeks, and silencing HPL in spr2 did not compromise this aphid resistance. Moreover, a mutation in the FAD7 gene in Arabidopsis thaliana also conferred resistance to the green peach aphid (Myzus persicae) in a genetic background that carries a null mutation in HPL. These results indicate that HPL contributes to certain forms of aphid resistance in tomato, but that the effects of FAD7 on aphids in tomato and Arabidopsis are distinct from and independent of HPL.

Effects of MAP Treatment on Aroma Compounds and Enzyme Activities in Flat Peach during Storage and Shelf Life

Cold storage is used to delay the senescence of peaches, but it can also lead to internal browning and aroma loss. Modified atmosphere packaging (MAP) has been reported to inhibit the internal browning and prolong the storage time. Four MAP treatments in the present study were set as follows: I: O2 1% to 3%, CO2 3% to 5%, and N2 92% to 96%; II: O2 3% to 5%, CO2 3% to 5%, and N2 90% to 94%; III: O2 6% to 8%, CO2 3% to 5%, and N2 87% to 91%; and control (CK): O2 21%, CO2 0.03%, and N2 79%. The concentration of sugars, acids, aroma compounds, superoxide radical (O2−), hydrogen peroxide (H2O2), and malondialdehyde (MDA), as well as the activities of enzymes, such as superoxide dismutase (SOD), peroxidase (POD), lipoxygenase (LOX), hydroperoxide lyase (HPL), alcohol dehydrogenase, and alcohol O-acyltransferase (AAT) activities, were investigated. The results revealed that MAP, especially for treatment II, could inhibit the loss of flavors such as sugars, acids, and aroma compounds; maintain higher SOD and POD activities; and inhibit the accumulation of O2−, H2O2, and MDA during shelf life after storage at low temperature for 30 days. It could also inhibit the LOX and HPL activities at low temperature, but maintain higher LOX and HPL activities during shelf life. These findings indicated that treatment II could prolong the storage time to 30 days and shelf life for 3 days; maintain the higher content of sugars, acids, and aroma compounds; protect the cell membrane from oxidative injury; and inhibit internal browning during cold storage and shelf life.

Effect of 1-MCP on the production of volatiles and biosynthesis-related gene expression in peach fruit during cold storage

As an effective ethylene action inhibitor, 1-Methylcyclopropene (1-MCP) can delay fruit ripening and senescence. However, little is known about the effect of 1-MCP on peach fruit aroma-related volatiles and their biosynthesis-related gene expression during cold storage. Peaches (cv. Xiahui 6) were treated with 1-MCP (10 μL L−1 for 12 h) to investigate the influence of 1-MCP on aromatic volatile biosynthesis and involved genes expression during cold storage. 1-MCP treatment significantly decreased ethylene production and delayed the ripening and senescence of peach at cold storage. A lower production of esters was found in 1-MCP treated fruit, while there were higher alcohol acyltransferase (AAT) activity and PpaAAT1/2 expression in treated fruit during the early storage. The performance patterns of both aldehydes and alcohols were different between 1-MCP and control fruit accompanied by lower lipoxygenase (LOX), alcohol dehydrogenase (ADH), hydroperoxide lyase (HPL) activities in accordance with the expression of PpaLOX3, PpaADH1 and PpaHPL1 in 1-MCP treated fruit compared with the control in the late storage. All in all, the role of 1-MCP on the volatiles and its related genes expression in peach fruits was discussed, and 1-MCP affected the formation of aroma volatile by functioning in the expression of pivotal enzyme genes including LOX, HPL, ADH and AAT.

Double function hydroperoxide lyases/epoxyalcohol synthases (CYP74C) of higher plants: identification and conversion into allene oxide synthases by site-directed mutagenesis

The CYP74C subfamily of fatty acid hydroperoxide transforming enzymes includes hydroperoxide lyases (HPLs) and allene oxide synthases (AOSs). This work reports a new facet of the putative CYP74C HPLs. Initially, we found that the recombinant CYP74C13_MT (Medicago truncatula) behaved predominantly as the epoxyalcohol synthase (EAS) towards the 9(S)-hydroperoxide of linoleic acid. At the same time, the CYP74C13_MT mostly possessed the HPL activity towards the 13(S)-hydroperoxides of linoleic and α-linolenic acids. To verify whether this dualistic behaviour of CYP74C13_MT is occasional or typical, we also examined five similar putative HPLs (CYP74C). These were CYP74C4_ST (Solanum tuberosum), CYP74C2 (Cucumis melo), CYP74C1_CS and CYP74C31 (both of Cucumis sativus), and CYP74C13_GM (Glycine max). All tested enzymes behaved predominantly as EAS toward 9-hydroperoxide of linoleic acid. Oxiranyl carbinols such as (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acids were the major EAS products. Besides, the CYP74C31 possessed an additional minor 9-AOS activity. The mutant forms of CYP74C13_MT, CYP74C1_CS, and CYP74C31 with substitutions at the catalytically essential domains, namely the “hydroperoxide-binding domain” (I-helix), or the SRS-1 domain near the N-terminus, showed strong AOS activity. These HPLs to AOSs conversions were observed for the first time. Until now a large part of CYP74C enzymes has been considered as 9/13-HPLs. Notwithstanding, these results show that all studied putative CYP74C HPLs are in fact the versatile HPL/EASs that can be effortlessly mutated into specific AOSs.

Identification of differentially expressed genes in flower, leaf and bulb scale of Lilium oriental hybrid \u2018Sorbonne\u2019 and putative control network for scent genes

BackgroundLily is an economically important plant, with leaves and bulbs consisting of overlapping scales, large ornamental flowers and a very large genome. Although it is recognized that flowers and bulb scales are modified leaves, very little is known about the genetic control and biochemical differentiation underlying lily organogenesis and development. Here we examined the differentially expressed genes in flower, leaf and scale of lily, using RNA-sequencing, and identified organ-specific genes, including transcription factors, genes involved in photosynthesis in leaves, carbohydrate metabolism in bulb scales and scent and color production in flowers.ResultsOver 11Gb data were obtained and 2685, 2296, and 1709 differentially expressed genes were identified in the three organs, with 581, 662 and 977 unique DEGs in F-vs-S, L-vs-S and L-vs-F comparisons. By functional enrichment analysis, genes likely to be involved in biosynthetic pathways leading to floral scent production, such as 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 3-ketoacyl-CoA thiolase (KAT), hydroperoxide lyase (HPL), geranylgeranyl pyrophosphate (GGPP) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HDS) and terpene synthase (TPS), and floral color genes, such as dihydroflavonol 4-reductase (DFR), chalcone synthase (CHS), chalcone isomerase (CHI), flavonol synthase (FLS) were identified. Distinct groups of genes that participate in starch and sucrose metabolism, such as sucrose synthase (SS), invertase (INV), sucrose phosphate synthase (SPS), starch synthase (SSS), starch branching enzyme (SBE), ADP-glucose pyrophosphorylase (AGP) andβ-amylase (BAM) and photosynthesis genes (Psa, Psb, Pet and ATP) were also identified. The expression of six floral fragrance-related DGEs showed agreement between qRT-PCR results and RPKM values, confirming the value of the data obtained by RNA-seq. We obtained the open reading frame of the terpene synthase gene from Lilium ‘Sorbonne’, designated LsTPS, which had 99.55% homology to transcript CL4520.Contig5_All. In addition, 54, 48 and 50 differently expressed transcription factor were identified by pairwise comparisons between the three organs and a regulatory network for monoterpene biosynthesis was constructed.ConclusionsAnalysis of differentially expressed genes in flower, leaf and bulb scale of lily, using second generation sequencing technology, yielded detailed information on lily metabolic differentiation in three organs. Analysis of the expression of flower scent biosynthesis genes has provided a model for the regulation of the pathway and identified a candidate gene encoding an enzyme catalyzing the final step in scent production. These digital gene expression profiles provide a valuable and informative database for the further identification and analysis of structural genes and transcription factors in different lily organs and elucidation of their function.

Structural and functional insights into the reaction specificity of catalase-related hydroperoxide lyase: A shift from lyase activity to allene oxide synthase by site-directed mutagenesis.

Two highly identical fusion proteins, an allene oxide synthase-lipoxygenase (AOS-LOX) and a hydroperoxide lyase-lipoxygenase (HPL-LOX), were identified in the soft coral Capnella imbricata. Both enzymes initially catalyze the formation of 8R-hydroperoxy-eicosatetraenoic acid (8R-HpETE) from arachidonic acid by the C-terminal lipoxygenase (LOX) domain. Despite the fact that the defined catalytically important residues of N-terminal catalase-related allene oxide synthase (cAOS) domain are also conserved in C. imbricata hydroperoxide lyase (cHPL), their reaction specificities differ. In the present study, we tested which of the amino acid substitutions around the active site of cHPL are responsible for a control in the reaction specificity. The possible candidates were determined via comparative sequence and structural analysis of the substrate channel and the heme region of coral cAOSs and C. imbricata cHPL. The amino acid replacements in cHPL—R56G, ME59-60LK, P65A, F150L, YS176-177NL, I357V, and SSSAGE155-160PVKEGD—with the corresponding residues of cAOS were conducted by site-directed mutagenesis. Although all these mutations influenced the catalytic efficiency of cHPL, only F150L and YS176-177NL substitutions caused a shift in the reaction specificity from HPL to AOS. The docking analysis of P. homomalla cAOS with 8R-HpETE substrate revealed that the Leu150 of cAOS interacts with the C5-C6 double bond and the Leu177 with the hydrophobic tail of 8R-HpETE. We propose that the corresponding residues in cHPL, Phe150 and Ser177, are involved in a proper coordination of the epoxy allylic radical intermediate necessary for aldehyde formation in the hydroperoxide lyase reaction.

Melon13-lipoxygenase CmLOX18 may be involved in C6 volatiles biosynthesis in fruit

To better understand the function role of the melon CmLOX18 gene in the biosynthesis of C6 volatiles during fruit ripening, we biochemically characterized CmLOX18 and identified its subcellular localization in transgenic tomato plants. Heterologous expression in yeast cells showed that the molecular weight of the CmLOX18 protein was identical to that predicted, and that this enzyme possesseed lipoxygenase activity. Linoleic acid was demonstrated to be the preferred substrate for the purified recombinant CmLOX18 protein, which exhibited optimal catalytic activity at pH 4.5 and 30 °C. Chromatogram analysis of the reaction product indicated that the CmLOX18 protein exhibited positional specificity, as evidenced by its release of only a C-13 oxidized product. Subcellular localization analysis by transient expression in Arabidopsis protoplasts showed that CmLOX18 was localized to non-chloroplast organelles. When the CmLOX18 gene was transgenically expressed in tomato via Agrobacterium tumefaciens-mediated transformation, it was shown to enhance expression levels of the tomato hydroperoxide lyase gene LeHPL, whereas the expression levels of six TomLox genes were little changed. Furthermore, transgenic tomato fruits exhibited increases in the content of the C6 volatiles, namely hexanal, (Z)-3-hexanal, and (Z)-3-hexen-1-ol, indicating that CmLOX18 probably plays an important role in the synthesis of C6 compounds in fruits.

The hydroperoxide lyase branch of the oxylipin pathway protects against photoinhibition of photosynthesis.

This study describes a new role for hydroperoxide lyase branch of oxylipin biosynthesis pathway in protecting photosynthetic apparatus under high light conditions. Lipid-derived signaling molecules, oxylipins, produced by a multi-branch pathway are central in regulation of a wide range of functions. The two most known branches, allene oxide synthase (AOS) and 13-hydroperoxide lyase (HPL) pathways, are best recognized as producers of defense compounds against biotic challenges. In the present work, we examine the role of these two oxylipin branches in plant tolerance to the abiotic stress, namely excessive light. Towards this goal, we have analyzed variable chlorophyll fluorescence parameters of intact leaves of Arabidopsis thaliana genotypes with altered oxylipin profile, followed by examining the impact of exogenous application of selected oxylipins on functional activity of photosynthetic apparatus in intact leaves and isolated thylakoid membranes. Our findings unequivocally bridge the function of oxylipins to photosynthetic processes. Specifically, HPL overexpressing lines display enhanced adaptability in response to high light treatment as evidenced by lower rate constant of photosystem 2 (PS2) photoinhibition and higher rate constant of PS2 recovery after photoinhibition. In addition, exogenous application of linolenic acid, 13-hydroperoxy linolenic acid, 12-oxophytodienoic acid, and methyl jasmonate individually, suppresses photochemical activity of PS2 in intact plants and isolated thylakoid membranes, while application of HPL-branch metabolites-does not. Collectively these data implicate function of HPL branch of oxylipin biosynthesis pathway in guarding PS2 under high light conditions, potentially exerted through tight regulation of free linolenic acid and 13-hydroperoxy linolenic acid levels, as well as competition with production of metabolites by AOS-branch of the oxylipin pathway.

Isolation and characterization of 9-lipoxygenase and epoxide hydrolase 2 genes: Insight into lactone biosynthesis in mango fruit (Mangifera indica L.)

Uniqueness and diversity of mango flavour across various cultivars are well known. Among various flavour metabolites lactones form an important class of aroma volatiles in certain mango varieties due to their ripening specific appearance and lower odour detection threshold. In spite of their biological and biochemical importance, lactone biosynthetic pathway in plants remains elusive. Present study encompasses quantitative real-time analysis of 9-lipoxygenase (Mi9LOX), epoxide hydrolase 2 (MiEH2), peroxygenase, hydroperoxide lyase and acyl-CoA-oxidase genes during various developmental and ripening stages in fruit of Alphonso, Pairi and Kent cultivars with high, low and no lactone content and explains their variable lactone content. Study also covers isolation, recombinant protein characterization and transient over-expression of Mi9LOX and MiEH2 genes in mango fruits. Recombinant Mi9LOX utilized linoleic and linolenic acids, while MiEH2 utilized aromatic and fatty acid epoxides as their respective substrates depicting their role in fatty acid metabolism. Significant increase in concentration of δ-valerolactone and δ-decalactone upon Mi9LOX over-expression and that of δ-valerolactone, γ-hexalactone and δ-hexalactone upon MiEH2 over-expression further suggested probable involvement of these genes in lactone biosynthesis in mango.

Activation and Stabilization of Olive Recombinant 13-Hydroperoxide Lyase Using Selected Additives.

The stabilization of olive recombinant hydroperoxide lyases (rHPLs) was investigated using selected chemical additives. Two rHPLs were studied: HPL full-length and HPL with its chloroplast transit peptide deleted (matured HPL). Both olive rHPLs are relatively stable at 4°C, and enzyme activity can be preserved (about 100% of the rHPL activities are maintained) during 5weeks of storage at -20 or at -80°C in the presence of glycerol (10%, v/v). Among the additives used in this study, glycine (2.5% w/v), NaCl (0.5M), and Na2SO4 (0.25M) provided the highest activation of HPL full-length activity, while the best matured HPL activity was obtained with Na2SO4 (0.25M) and NaCl (1M). Although the inactivation rate constants (k) showed that these additives inactivate both rHPLs, their use is still relevant as they strongly increase HPL activity. Results of C6-aldehyde production assays also showed that glycine, NaCl, and Na2SO4 are appropriate additives and that NaCl appears to be the best additive, at least for hexanal production.

Efficient Biochemical Cascade for Accessing Green Leaf Alcohols

The biotechnological production of six-carbon membered green leaf aldehydes and alcohols appreciated for their fruity, green-grassy odors in fragrances and flavors exploits the 13-lipoxygenase pathway found in higher plants. Homogenized seeds, fruits, or wild-type microorganisms have traditionally served as catalysts used in a one-pot reaction. However, many of these catalysts can be advantageously replaced by enzymes produced in engineered microorganisms for improved process performance. Substituting guava fruits by an engineered 13-hydroperoxide lyase produced in E. coli was recently shown to provide increased efficiency for cleaving fatty acid 13-hydroperoxides into green leaf aldehydes. Replacing baker’s yeast by a cosubstrate-dependent recombinant ketoreductase was found to offer superior productivity and chemo-selectivity in the reduction of hexenals to the corresponding alcohols. Finally, combining both cellular catalysts at reasonable loading in a simple one-pot cascade reaction offered the green ...

Gene-Metabolite Networks of Volatile Metabolism in Airen and Tempranillo Grape Cultivars Revealed a Distinct Mechanism of Aroma Bouquet Production.

Volatile compounds are the major determinants of aroma and flavor in both grapes and wine. In this study, we investigated the emission of volatile and non-volatile compounds during berry maturation in two grape varieties (Airén and Tempranillo) throughout 2010 and 2011. HS-SPME coupled to gas chromatography and mass spectrometry was applied for the identification and relative quantitation of these compounds. Principal component analysis was performed to search for variability between the two cultivars and evolution during 10 developmental stages. Results showed that there are distinct differences in volatile compounds between cultivars throughout fruit development. Early stages were characterized in both cultivars by higher levels of some apocarotenoids such as β-cyclocitral or β-ionone, terpenoids (E)-linalool oxide and (Z)-linalool oxide and several furans, while the final stages were characterized by the highest amounts of ethanol, benzenoid phenylacetaldehyde and 2-phenylethanol, branched-amino acid-derived 3-methylbutanol and 2-methylbutanol, and a large number of lipid derivatives. Additionally, we measured the levels of the different classes of volatile precursors by using liquid chromatography coupled to high resolution mass spectrometry. In both varieties, higher levels of carotenoid compounds were detected in the earlier stages, zeaxanthin and α-carotene were only detected in Airén while neoxanthin was found only in Tempranillo; more variable trends were observed in the case of the other volatile precursors. Furthermore, we monitored the expression of homolog genes of a set of transcripts potentially involved in the biosynthesis of these metabolites, such as some glycosyl hydrolases family 1, lipoxygenases, alcohol dehydrogenases hydroperoxide lyases, O-methyltransferases and carotenoid cleavage dioxygenases during the defined developmental stages. Finally, based on Pearson correlation analyses, we explored the metabolite-metabolite fluctuations within VOCs/precursors during the berry development; as well as tentatively linking the formation of some metabolites detected to the expression of some of these genes. Our data showed that the two varieties displayed a very different pattern of relationships regarding the precursor/volatile metabolite-metabolite fluctuations, being the lipid and the carotenoid metabolism the most distinctive between the two varieties. Correlation analysis showed a higher degree of overall correlation in precursor/volatile metabolite-metabolite levels in Airén, confirming the enriched aroma bouquet characteristic of the white varieties.

Tea green leafhopper, Empoasca vitis, chooses suitable host plants by detecting the emission level of (3Z)-hexenyl acetate.

Green leaf volatiles (GLVs) have been reported to play an important role in the host-locating behavior of several folivores that feed on angiosperms. However, next to nothing is known about how the green leafhopper, Empoasca vitis, chooses suitable host plants and whether it detects differing emission levels of GLV components among genetically different tea varieties. Here we found that the constitutive transcript level of the tea hydroperoxide lyase (HPL) gene CsiHPL1, and the amounts of (Z)-3-hexenyl acetate and of total GLV components are significantly higher in tea varieties that are susceptible to E. vitis (Enbiao (EB) and Banzhuyuan (BZY)) than in varieties that are resistant to E. vitis (Changxingzisun (CX) and Juyan (JY)). Moreover, the results of a Y-tube olfactometer bioassay and an oviposition preference assay suggest that (Z)-3-hexenyl acetate and (Z)-3-hexenol offer host and oviposition cues for E. vitis female adults. Taken together, the two GLV components, (Z)-3-hexenol and especially (Z)-3-hexenyl acetate, provide a plausible mechanism by which tea green leafhoppers distinguish among resistant and susceptible varieties. Future research should be carried out to obtain the threshold of the above indices and then assess their reasonableness. The development of practical detection indices would greatly improve our ability to screen and develop tea varieties that are resistant to E. vitis.

The activity of HYDROPEROXIDE LYASE 1 regulates accumulation of galactolipids containing 12-oxo-phytodienoic acid in Arabidopsis.

Arabidopsis produces galactolipids containing esters of 12-oxo-phytodienoic acid (OPDA) and dinor-12-oxo-phytodienoic acid (dnOPDA). These lipids are referred to as arabidopsides and accumulate in response to abiotic and biotic stress. We explored the natural genetic variation found in 14 different Arabidopsis accessions to identify genes involved in the formation of arabidopsides. The accession C24 was identified as a poor accumulator of arabidopsides whereas the commonly used accession Col-0 was found to accumulate comparably large amounts of arabidopsides in response to tissue damage. A quantitative trait loci analysis of an F2 population created from a cross between C24 and Col-0 located a region on chromosome four strongly linked to the capacity to form arabidopsides. Expression analysis of HYDROPEROXIDE LYASE 1 (HPL1) showed large differences in transcript abundance between accessions. Transformation of Col-0 plants with the C24 HPL1 allele under transcriptional regulation of the 35S promoter revealed a strong negative correlation between HPL1 expression and arabidopside accumulation after tissue damage, thereby strengthening the view that HPL1 competes with ALLENE OXIDE SYNTHASE (AOS) for lipid-bound hydroperoxide fatty acids. We further show that the last step in the synthesis of galactolipid-bound OPDA and dnOPDA from unstable allene oxides is exclusively enzyme-catalyzed and not the result of spontaneous cyclization. Thus, the results presented here together with previous studies suggest that all steps in arabidopside biosynthesis are enzyme-dependent and apparently all reactions can take place with substrates being esterified to galactolipids.