Volatile science? Metabolic engineering of terpenoids in plants

Terpenoids are important for plant survival and also possess biological properties that are beneficial to humans. Here, we describe the state of the art in terpenoid metabolic engineering, showing that significant progress has been made over the past few years. Subcellular targeting of enzymes has demonstrated that terpenoid precursors in subcellular compartments are not as strictly separated as previously thought and that multistep pathway engineering is feasible, even across cell compartments. These engineered plants show that insect behavior is influenced by terpenoids. In the future, we expect rapid progress in the engineering of terpenoid production in plants. In addition to commercial applications, such transgenic plants should increase our understanding of the biological relevance of these volatile secondary metabolites.     

Volatile Compounds Formed on Roasting dl-α-Alanine with d-Glucose

The mixture of dl-α-alanine and d-glucose was roasted at 250° for about an hour in nitrogen atmosphere. From the volatile condensates were isolated and identified alkyl and acyl pyrroles, alkyl pyrrole-2-aldehydes, furfuryl pyrroles, alkyl pyrazines and furanic compounds. The identification of these compounds was based on the spectroscopic methods (MS, GC-MS and IR) and gas chromatographic analysis.     

Volatile compounds from the symbiotic system Azolla filiculoides‐Anabaena azollae bacteria

Abstract

Azolla filiculoides is an aquatic pteridophyte that may be used as animal food, biofertilizer and phytoremediation. Its volatile composition was never studied although several phytochemical analyses were performed. The volatile composition of A. filiculoides grown outdoors in a pond at the Botanical Garden of Lisbon University (BGLU) or in culture conditions as well as the effect of different harvesting times and the storage type were evaluated. The volatiles isolated by hydrodistillation and distillation‐extraction were analysed by gas chromatography and gas chromatography‐mass spectrometry. The oil of all the A. filiculoides samples studied affords a yellowish colour and an unpleasant odour in a yield of 0.01% (v/fw). Alcohols, aldehydes, alkanes and ketones dominated the culture samples, while aldehydes, alcohols, terpenoids and alkanes represented the main volatiles of the BGLU samples. Some quantitative differences were detected in seasonal and type of storage (fresh, dry or frozen at ?20°C) studies of A. filiculoides from the BGLU. The BGLU and culture volatiles showed qualitative differences: 2‐ethyl‐1‐hexanol was only identified in the fern culture, whereas acetophenone, pentylfuran, acetylpyridine and 2‐octanone were only detected in BGLU samples. The dendrogram showed two distinct clusters (culture and BGLU samples). The possible biological origin and bioactivity of some of the volatile compounds is discussed.

Abbreviations: FID, flame ionization detector; i.d., internal diameter; v/fw, volume by fresh weight; GC, gas chromatography; GC‐MS, gas chromatography‐mass spectrometry; u, unified atomic mass unit     

Volatile flavour components of cashew ‘apple’ (Anacardium occidentale)

An essence of fresh cashew ‘apple’, obtained by well-established procedures and possessing the characteristic aroma of that fruit, was analysed by GC and by GC/MS using both EI and CI techniques. The fruit produced a very small quantity of aroma volatiles (ca 3.6 μ/kg fresh fruit), much less than that obtained from most similar tropical fruits. Five aldehydes comprised ca 26% of the sample, but terpene hydrocarbons (ca 38%) provided the major group of compounds, consisting of four monoterpenes (ca 34%) and three sesquiterpenes (ca 4%). Important constituents of the essence included hexanal, car-3-ene, limonene, trans-hex-2-enal and benzaldehyde. Car-3-ene (24.3%) was the major constituent, and it was observed that during some recent analyses of tropical fruits this relatively uncommon aroma volatile had been identified in three out of four fruits studied of Venezuelan origin.     

Volatile compounds of β-diketonates of dioxouranium(VI) with tetrahydropyran and tetrahydrothiophene

Complexes of the type [UO₂(β-diket)₂·nB] where β-diket=CF₃COCHCOR (R=CF₃, CH₃, C₄H₃S or C₆H₅ and denoted by HFAA, TFAA, TTA or BTA, respectively), B=tetrahydropyran (THP) and tetrahydrothiophene (THT) and n= 0.5–2.0, have been synthesized and characterised by infrared, ¹H NMR and mass spectral techniques. Vapour pressure of UO₂(HFAA)₂·THP was measured by the transpiration method between the temperature range of 323–391 K. The enthalpy of vaporisation of UO₂(HFAA)₂·THP was estimated to be 18.4 kcal/mol.     

The Production of Volatile Compounds by Yeasts Isolated from Small Brazilian cachaça distilleries

Summary The production of volatile compounds by 24 strains of Saccharomyces cerevisiae and one strain each of Candida apicola, C. famata, C. guilliermondii, Hanseniaspora occidentalis, Pichia subpelicullosa and Schizosaccharomyces pombe was evaluated with respect to the production of cacha?a. They were isolated from small cacha?a distilleries (27), industrial cacha?a distilleries (2) and one sugarcane alcohol distillery, and tested in synthetic medium for the production of acetaldehyde, ethyl acetate, propanol, isobutanol, isoamyl alcohol, acetic acid and glycerol. The Saccharomyces strains showed a narrow range of variation in the production of such compounds, near 50% of the average of each volatile compound concentration. Principal component analysis showed the separation of the strains into six groups, and acetic acid production was the variable of greatest impact in the differentiation of the strains. The strains of S. pombe formed a distinct group (Group 2), and the strains of C. apicola and H. occidentalis formed a joint group (Group 6) as did Sc13 and Sc4 (Group 4). Group 1 was formed exclusively of S. cerevisiae. The closest non-Saccharomyces strains were C. apicola and H. occidentalis, with a similarity index of about 0.95. The strain P. subpelliculosa showed general characteristics more similar to those of the S. cerevisiae strains than to the non-Saccharomyces strains.     

Volatile metabolomic signature of bladder cancer cell lines based on gas chromatography–mass spectrometry

Introduction

Recent studies provide a convincing support that the presence of cancer cells in the body leads to the alteration of volatile organic compounds (VOCs) emanating from biological samples, particularly of those closely related with tumoral tissues. Thus, a great interest emerged for the study of cancer volatilome and subsequent attempts to confirm VOCs as potential diagnostic biomarkers.

Objectives

The aim of this study was to determine the volatile metabolomic signature of bladder cancer (BC) cell lines and provide an in vitro proof-of-principle that VOCs emanated into the extracellular medium may discriminate BC cells from normal bladder epithelial cells.

Methods

VOCs in the culture media of three BC cell lines (Scaber, J82, 5637) and one normal bladder cell line (SV-HUC-1) were extracted by headspace-solid phase microextraction and analysed by gas chromatography-mass spectrometry (HS-SPME/GC–MS). Two different pH (pH 2 and 7) were used for VOCs extraction to infer the best pH to be used in in vitro metabolomic studies.

Results

Multivariate analysis revealed a panel of volatile metabolites that discriminated cancerous from normal bladder cells, at both pHs, although a higher number of discriminative VOCs was obtained at neutral pH. Most of the altered metabolites were ketones and alkanes, which were generally increased in BC compared to normal cells, and alcohols, which were significantly decreased in BC cells. Among them, three metabolites, namely 2-pentadecanone, dodecanal and γ-dodecalactone (the latter only tentatively identified), stood out as particularly important metabolites and promising volatile biomarkers for BC detection. Furthermore, our results also showed the potential of VOCs in discriminating BC cell lines according to tumour grade and histological subtype.

Conclusions

We demonstrate that a GC–MS metabolomics-based approach for analysis of VOCs is a valuable strategy for identifying new and specific biomarkers that may improve BC diagnosis. Future studies should entail the validation of volatile signature found for BC cell lines in biofluids from BC patients.

     

Volatile Components Formed by Thermal Degradation of Nondialyzable Melanoidins Prepared from Sugar–Amino Acid Reaction Systems

The nondialyzable melanoidins prepared from glucose-glycine (I), xylose-glycine (II) and glucose-β-alanine (III) reaction systems were pyrolyzed at 350°C for 0.5 ~ 2hr and the volatile pyrolyzates were investigated. To trap the volatile compounds, cold trapping and Tenax GC trapping methods were used. Identification of these compounds was made by gas chromatographymass spectrometry using a glass capillary column. The volatile components of I and II were qualitatively similar to each other. Major components of volatile pyrolyzates of I and II were identified as methyl acetate, five furans, toluene, two pyrroles, two pyridines and acetic acid. The major volatile components of III were methyl acetate, five furans, two 2-cyclopentenones, pyrrole, two pyridines and acetic acid.     

Do Insectivorous Birds use Volatile Organic Compounds from Plants as Olfactory Foraging Cues? Three Experimental Tests

Some insectivorous birds orient towards insect‐defoliated trees even when they do not see the foliar damage or the herbivores. There are, however, only a few studies that have examined the mechanisms behind this foraging behaviour. Previous studies suggest that birds can use olfactory foraging cues (e.g. volatile organic compounds (VOCs) emitted by defoliated plants), indirect visual cues or a combination of the two sensory cues. VOCs from insect‐defoliated plants are known to attract natural enemies of herbivores, and researchers have hypothesized that VOCs could also act as olfactory foraging cues for birds. We conducted three experiments across a range of spatial scales to test this hypothesis. In each experiment, birds were presented with olfactory cues and their behavioural responses or foraging outcomes were observed. In the first experiment, two different VOC blends, designed to simulate the volatile emissions of mountain birch (Betula pubescens ssp. czerepanovii) after defoliation by autumnal moth (Epirrita autumnata) larvae, were used in behavioural experiments in aviaries with pied flycatchers (Ficedula hypoleuca). The second experiment was a field‐based trial of bird foraging efficiency; the same VOC blends were applied to mountain birches, silver birches (B. pendula) and European white birches (B. pubescens) with plasticine larvae attached to the trees to serve as artificial prey for birds and provide a means to monitor predation rate. In the third experiment, the attractiveness of silver birch saplings defoliated by autumnal moth larvae versus intact controls was tested with great tits (Parus major) and blue tits (Cyanistes caeruleus) in an aviary. Birds did not orient towards either artificial or real trees with VOC supplements or towards herbivore‐damaged saplings when these saplings and undamaged alternatives were hidden from view. These findings do not support the hypothesis that olfactory foraging cues are necessary in the attraction of birds to herbivore‐damaged trees.     

Volatile constituents of Erodium cicutarium (L.) L’ Hérit. (Geraniaceae)

Essential oils from Erodium cicutarium were obtained by hydrodistillation (samples consisting of entire plants (ec1), leaves and stems (ec2)) and analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS), resulting in a total of 177 components being identified. The essential oils were of a very similar chemical composition and consisted mainly of aliphatic compounds and their derivatives. Fatty acids and fatty acid derived compounds were the most common, 51.3% (ec1) and 60.1% (ec2), followed by carotenoid derived compounds, 12.6% (ec1) and 20.2% (ec2), and then terpenoids, 14.9% (ec1) and 14.2% (ec2). The main constituents in the oils were hexadecanoic acid, 22.8% (ec2) and 35.9% (ec1) and hexahydrofarnesyl acetone, 10.8% (ec2) and 11.6% (ec1). The results obtained differ markedly from those previously reported for the same species.     

Heterologous Production of Methionine-γ-Lyase from Brevibacterium linens in Lactococcus lactis and Formation of Volatile Sulfur Compounds

The conversion of methionine to volatile sulfur compounds (VSCs) is of great importance in flavor formation during cheese ripening and is the focus of biotechnological approaches toward flavor improvement. A synthetic mgl gene encoding methionine-γ-lyase (MGL) from Brevibacterium linens BL2 was cloned into a Lactococcus lactis expression plasmid under the control of the nisin-inducible promoter PnisA. When expressed in L. lactis and purified as a recombinant protein, MGL was shown to degrade l-methionine as well as other sulfur-containing compounds such as l-cysteine, l-cystathionine, and l-cystine. Overproduction of MGL in recombinant L. lactis also resulted in an increase in the degradation of these compounds compared to the wild-type strain. Importantly, gas chromatography-mass spectrometry analysis identified considerably higher formation of methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) in reactions containing either purified protein, whole cells, or cell extracts from the heterologous L. lactis strain. This is the first report of production of MGL from B. linens in L. lactis. Given their significance in cheese flavor development, the use of lactic acid bacteria with enhanced VSC-producing abilities could be an efficient way to enhance cheese flavor development.Methionine (Met) catabolism plays a major role in cheese flavor development. Met is believed to be the precursor of numerous diverse and quantitatively minor volatile sulfur compounds (VSCs) (38) which make important contributions to the overall flavor that are typical to different cheeses (7). Most of these compounds are derived from the degradation of Met to methanethiol (MTL), giving rise to a variety of compounds such as dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), and S-methylthioesters (41).The Met biosynthetic and catabolic pathways leading to MTL vary among bacteria (36), as do the enzymes involved and the amount of MTL produced during cheese ripening (15). The most direct pathway occurs via l-methionine γ-elimination of Met to MTL, α-ketobutyrate, and ammonia. This l-methionine γ-elimination activity has been shown to be quite efficient in Brevibacterium linens (21), while its presence has been suggested in several other cheese surface bacteria such as Micrococcus luteus, Arthrobacter sp., Corynebacterium glutamicum, and Staphylococcus equorum (9). In B. linens, this activity is catalyzed by an l-methionine-γ-lyase (MGL), which has been previously purified and characterized (16). Moreover, disruption of the mgl gene encoding the enzyme has been shown to almost eliminate this strain''s considerable capacity to produce VSCs (2). In lactococci, which produce only limited amounts of VSCs (15), this reaction is catalyzed by cystathionine lyases (β- or γ-) which are responsible for the simultaneous deamination and demethylthiolation of Met to MTL (1, 11, 18, 22); recently, a new C-S lyase (YtjE) with α,γ-elimination activity that degrades Met into MTL has been characterized in our laboratory (30). Unfortunately, C-S lyases display relatively low activities toward Met, limiting their capacities to produce VSCs. Another route for Met catabolism involving a two-step mechanism initiated by an aminotransferase has also been identified in lactococci (8, 23) and other cheese-ripening bacteria (3, 9), which leads to the formation of α-keto-γ-methylthiobutyric acid which is subsequently converted to MTL; this route, however, produces only limited amounts of MTL.In recent years, numerous studies have pursued the control and/or diversification of VSCs primarily by means of using selected cheese-ripening microorganisms or combinations of them (4, 7, 25). A few studies have focused on engineering lactic acid bacteria (LAB) with enhanced VSC-producing abilities by increasing cystathionine lyase activities (22, 27). In this respect, a Lactococcus lactis strain engineered to overproduce cystathionine β-lyase was shown to produce larger quantities of VSCs with Met as a substrate compared to the wild-type strain (22). However, this study also showed no significant difference in VSC production between the wild-type strain and a cystathionine β-lyase-knockout variant, implying that other enzymes may play a more prominent role in the conversion of Met.The aim of this study was to develop LAB with improved capability to produce MTL and other related VSCs as a more efficient strategy to enhance cheese flavor development. We describe the bioengineering of food-grade L. lactis strains to produce MGL, which has been reported to play a major role in the catabolism of Met to MTL in B. linens, a good producer of VSCs (2). The enzyme activity of the recombinant MGL was confirmed, and its role in the production of VSCs by recombinant L. lactis was also investigated.     

Volatile compounds associated to the loss of astringency in persimmon fruit revealed by untargeted GC–MS analysis

High resolution volatile profiling (67 compounds identified) of fruits from 12 persimmon cultivars was established and used to characterize the different astringency types of persimmon fruit before and after deastringency treatment. Analysis of the volatile profile of fruit enables us to differentiate between cultivars that at the moment of harvest produced non-astringent fruit (Pollination Constant Non Astringent—PCNA-type) from astringent ones (non-PCNA-type). Fruit failing to accumulate astringent compounds naturally (PCNA fruit) showed high levels of 3(2H)-benzofuranone, while this compound was not detected in any astringent type fruit (non-PCNA). In addition to this, PCNA cultivars also showed at harvest higher accumulation of benzeneacetaldehyde and lipid-derived aldehydes (hexanal, heptanal, octanal and decanal) than non-PCNA fruit. The application of postharvest deastringency treatment to all non-PCNA cultivars resulted on an important insolubilization of tannins. In general the CO₂-treatment enhanced the levels of acetaldehyde, however those cultivars showing high levels of dihydrobenzofuran at harvest did not present an increment of acetaldehyde. In contrast, all non-PCNA cultivars exhibited an important accumulation of lipid-derived aldehydes due to CO₂-treatment. Therefore, we propose that lipid-derived aldehydes (mainly decanal, octanal and heptanal) may be playing a role in the astringency loss. Our results suggest that 3(2H)-benzofuranone, benzeneacetaldehyde and lipid-derived aldehydes could be used as markers for both natural and artificial loss of astringency.     

Composition,Antibiofilm, and Antibacterial Potential of Volatile Oils from Geopropolis of Different Stingless Bees’ Species**

We aimed to characterize and investigate the antibacterial potential of the native stingless bees geopropolis volatile oils (VO) for the search of potentially new bioactive compounds. Geopropolis samples from Melipona bicolor schencki, M. compressipes manaosensis, M. fasciculata, M. quadrifasciata, M. marginata and M. seminigra merrillae were collected from hives in South Brazil. VO were obtained by hydrodistillation and characterised by gas chromatography coupled to mass spectrometry (GC/MS). Antimicrobial activity was assessed by microplate dilution method. The lowest MIC against cell walled bacteria was 219±0 μg mL⁻¹ from M. quadrifasciata geopropolis VO with Staphylococcus aureus. The M. b. schencki geopropolis VO minimal inhibition concentration (MIC) was 424±0 μg mL⁻¹ against all the mycoplasma strains evaluated. Fractionation resulted in the reduction of 50 % of the MIC value from the original oil. However, its compounds’ synergism seems to be essential to this activity. Antibiofilm assays demonstrated 15.25 % eradication activity and 13.20 % inhibition of biofilm formation after 24 h for one subfraction at 2× its MIC as the best results found. This may be one of the essential mechanisms by which geopropolis VOs perform their antimicrobial activity.     

Induced Release of a Plant-Defense Volatile ‘Deceptively’ Attracts Insect Vectors to Plants Infected with a Bacterial Pathogen

Transmission of plant pathogens by insect vectors is a complex biological process involving interactions between the plant, insect, and pathogen. Pathogen-induced plant responses can include changes in volatile and nonvolatile secondary metabolites as well as major plant nutrients. Experiments were conducted to understand how a plant pathogenic bacterium, Candidatus Liberibacter asiaticus (Las), affects host preference behavior of its psyllid (Diaphorina citri Kuwayama) vector. D. citri were attracted to volatiles from pathogen-infected plants more than to those from non-infected counterparts. Las-infected plants were more attractive to D. citri adults than non-infected plants initially; however after feeding, psyllids subsequently dispersed to non-infected rather than infected plants as their preferred settling point. Experiments with Las-infected and non-infected plants under complete darkness yielded similar results to those recorded under light. The behavior of psyllids in response to infected versus non-infected plants was not influenced by whether or not they were carriers of the pathogen. Quantification of volatile release from non-infected and infected plants supported the hypothesis that odorants mediate psyllid preference. Significantly more methyl salicylate, yet less methyl anthranilate and D-limonene, was released by infected than non-infected plants. Methyl salicylate was attractive to psyllids, while methyl anthranilate did not affect their behavior. Feeding on citrus by D. citri adults also induced release of methyl salicylate, suggesting that it may be a cue revealing location of conspecifics on host plants. Infected plants were characterized by lower levels of nitrogen, phosphorus, sulfur, zinc, and iron, as well as, higher levels of potassium and boron than non-infected plants. Collectively, our results suggest that host selection behavior of D. citri may be modified by bacterial infection of plants, which alters release of specific headspace volatiles and plant nutritional contents. Furthermore, we show in a laboratory setting that this apparent pathogen-mediated manipulation of vector behavior may facilitate pathogen spread.     

Changes in Expression of GABAA α4 Subunit mRNA in the Brain under Anesthesia Induced by Volatile and Intravenous Anesthetics

We investigated changes in levels of GABA_A receptor α4 subunit mRNA in the mouse brain after administration of volatile or i.v. anesthetic, by performing quantitative RT-PCR. We also performed immunohistochemical assays for c-fos-like protein. During deep anesthesia (which was estimated by loss of righting reflex) after administration of propofol, levels of GABA_A receptor α4 subunit mRNA in the hippocampus, striatum and diencephalons were significantly greater than those observed after administration of pentobarbital, midazolam or GOI (5.0% isoflurane and 70% nitrous oxide in oxygen). Under incomplete anesthesia, levels of GABA_A receptor α4 subunit mRNA were significantly increased by midazolam in all brain regions, and were significantly increased by pentobarbital in the posterior cortex and striatum. Expression of GABA_A receptor α4 subunit mRNA closely correlated with expression of c-fos-like protein. These results indicate that the GABA_A receptor α4 subunit plays an important role in regulating the anesthetic stage of i.v. anesthetics.     

Insights into Distinct Modulation of α7 and α7β2 Nicotinic Acetylcholine Receptors by the Volatile Anesthetic Isoflurane

Nicotinic acetylcholine receptors (nAChRs) are targets of general anesthetics, but functional sensitivity to anesthetic inhibition varies dramatically among different subtypes of nAChRs. Potential causes underlying different functional responses to anesthetics remain elusive. Here we show that in contrast to the α7 nAChR, the α7β2 nAChR is highly susceptible to inhibition by the volatile anesthetic isoflurane in electrophysiology measurements. Isoflurane-binding sites in β2 and α7 were found at the extracellular and intracellular end of their respective transmembrane domains using NMR. Functional relevance of the identified β2 site was validated via point mutations and subsequent functional measurements. Consistent with their functional responses to isoflurane, β2 but not α7 showed pronounced dynamics changes, particularly for the channel gate residue Leu-249(9′). These results suggest that anesthetic binding alone is not sufficient to generate functional impact; only those sites that can modulate channel dynamics upon anesthetic binding will produce functional effects.     

Effects of Shading on the Synthesis of Volatile Organic Compounds in ‘Marselan’ Grape Berries (Vitis vinifera L.)

Journal of Plant Growth Regulation - Light is a vital environmental factor that can affect the synthesis of volatile organic compounds (VOCs) in grape berries. However, the mechanism through which...     

Volatile Anesthetics,Not Intravenous Anesthetic Propofol Bind to and Attenuate the Activation of Platelet Receptor Integrin αIIbβ3

Background

In clinical reports, the usage of isoflurane and sevoflurane was associated with more surgical field bleeding in endoscopic sinus surgeries as compared to propofol. The activation of platelet receptor αIIbβ3 is a crucial event for platelet aggregation and clot stability. Here we studied the effect of isoflurane, sevoflurane, and propofol on the activation of αIIbβ3.

Methods

The effect of anesthetics on the activation of αIIbβ3 was probed using the activation sensitive antibody PAC-1 in both cell-based (platelets and αIIbβ3 transfectants) and cell-free assays. The binding sites of isoflurane on αIIbβ3 were explored using photoactivatable isoflurane (azi-isoflurane). The functional implication of revealed isoflurane binding sites were studied using alanine-scanning mutagenesis.

Results

Isoflurane and sevoflurane diminished the binding of PAC-1 to wild-type αIIbβ3 transfectants, but not to the high-affinity mutant, β3-N305T. Both anesthetics also impaired PAC-1 binding in a cell-free assay. In contrast, propofol did not affect the activation of αIIbβ3. Residues adducted by azi-isoflurane were near the calcium binding site (an important regulatory site termed SyMBS) just outside of the ligand binding site. The mutagenesis experiments demonstrated that these adducted residues were important in regulating integrin activation.

Conclusions

Isoflurane and sevoflurane, but not propofol, impaired the activation of αIIbβ3. Azi-isoflurane binds to the regulatory site of integrin αIIbβ3, thereby suggesting that isoflurane blocks ligand binding of αIIbβ3 in not a competitive, but an allosteric manner.