Tissue-specific stilbene accumulation is an early response to wounding/grafting as revealed by using spatial and temporal metabolomics.

Grafting is widely used in horticulture. Shortly after grafting, callus tissues appear at the graft interface and the vascular tissues of the scion and rootstock connect. The graft interface contains a complex mix of tissues, we hypothesised that each tissue has its own metabolic response to wounding/grafting and accumulates different metabolites at different rates. We made intact and wounded cuttings and grafts of grapevine, and then measured changes in bulk flavonoid, phenolic acid and stilbenoid concentration and used metabolite imaging to study tissue-specific responses. We show that some metabolites rapidly accumulate in specific tissues after grafting, for example, stilbene monomers accumulate in necrotic tissues surrounding mature xylem vessels. Whereas other metabolites, such as complex stilbenes, accumulate in the same tissues at later stages. We also observe that other metabolites accumulate in the newly formed callus tissue and identify genotype-specific responses. In addition, exogenous resveratrol application did not modify grafting success rate, potentially suggesting that the accumulation of resveratrol at the graft interface is not linked to graft union formation. The increasing concentration of complex stilbenes often occurs in response to plant stresses (via unknown mechanisms), and potentially increases antioxidant activity and antifungal capacities.

Highlighting the varietal origin of eugenol in Armagnac wine spirits from Baco blanc, a hybrid grape variety.

We report on the first evidence of a varietal origin of eugenol as a molecular marker in Baco blanc, one of the grape varieties used to produce Armagnac. Eugenol was identified and quantified by HS-SPME-GC-MS. For two separate vintages, the concentrations found in monovarietal wine spirits made from Baco blanc, were, on average, 10 times higher than those in other Vitis varieties, ranging from 31.0 to 174.7 µg/L. Investigations were carried out to quantify eugenol in the wines used for distillation, in the musts and finally in several parts of the plant. For all matrices over both vintages, it was confirmed that eugenol is much more abundant in Baco blanc than in Ugni blanc and Folle blanche. Moreover, enzymatic hydrolysis made it possible to release a significant quantity of eugenol from precursors, demonstrating that more than 90% of eugenol is bound in the must and in the grape berry pulp.

Pesticide contamination in an intensive insect predator of honey bees.

Pesticides used for plant protection can indirectly affect target and non-target organisms and are identified as a major cause of insect decline. Depending on species interactions, pesticides can be transferred into the environment from plants to preys and predators. While the transfer of pesticides is often studied through vertebrate and aquatic exposure, arthropod predators of insects may represent valuable bioindicators of environmental exposure to pesticides. A modified QuEChERS extraction coupled with HPLC-MS/MS analysis was used to address the question of the exposure to pesticides of the invasive hornet Vespa velutina, a specialist predator of honey bees. This analytical method allows the accurate quantification of nanogram/gram levels of 42 contaminants in a sample weight that can be obtained from single individuals. Pesticide residues were analyzed in female workers from 24 different hornet nests and 13 different pesticides and 1 synergist, piperonyl butoxide, were identified and quantified. In 75 % of the explored nests, we found at least one compound and in 53 % of the positive samples we could quantify residues ranging from 0.5 to 19.5 ng.g-1. In this study, hornets from nests located in sub-urban environments were the most contaminated. Pesticide residue analysis in small and easy to collect predatory insects opens new perspectives for the study of environmental contamination and the transfer of pesticides in terrestrial trophic chains.

Investigation into mousy off-flavor in wine using gas chromatography-mass spectrometry with stir bar sorptive extraction.

Three major compounds, 2-acetyl-1-pyrroline (APY), 2-acetyltetrahydropyridine (ATHP) and 2-ethyltetrahydropyridine (ETHP), have been identified as responsible for the mousy off-flavor in wines, although to date quantification data reported in the literature are limited. A simple method for simultaneous quantitation, by SBSE-GC-MS, of these N-heterocyclic compounds was developed. Both previously reported tautomers of ATHP, 2-acetyl-1,4,5,6-tetrahydropyridine and 2-acetyl-3,4,5,6-tetrahydropyridine were identified. The limits of detection and quantification of the method were determined in white, rosé and red wines and are lower than previously published concentrations in spoiled wine. ETHP was detected in almost all wines produced with limited use of SO2. ATHP was detected in almost all wines suspected of mousiness whereas APY was only detected in few cases. This method will provide a support for further studies aimed at understanding the phenomena that influence the occurrence of mousy off-flavor and the oenological parameters that modulate its expression.

Methyl Salicylate, an Odor-Active Compound in Bordeaux Red Wines Produced without Sulfites Addition.

Red wines produced without the addition of any SO2 are currently the source of a new consumer trend. The first characterization approaches regarding these specific wines were devoted to sensory studies that highlighted differences according to the use of SO2 during winemaking. The goal of this paper is to extend our knowledge of such aromatic specificities. Examining experimental wines produced with and without the addition of SO2, aroma fractionation, gas chromatography coupled with olfactometry, and mass spectrometry were applied to identify compounds at the origin of the specificities of these wines. Thus, we identified methyl salicylate as being impacted by the use of SO2 during the winemaking process. Studying a large number of commercial Bordeaux red wines, methyl salicylate was significantly quantified at a higher content in wines without added SO2. A sensory approach revealed a significant impact of methyl salicylate on wines without the sulfite aroma, particularly concerning fruity aromas and wine freshness.

Identifying early metabolite markers of successful graft union formation in grapevine.

Grafting is an important horticultural technique used for many crop species. However, some scion/rootstock combinations are considered as incompatible due to poor graft union formation and subsequently high plant mortality. The early identification of graft incompatibility could allow the selection of non-viable plants before planting and would have a beneficial impact on research and development in the nursery sector. In general, visible phenotypes of grafted plants (size, root number, etc.) are poorly correlated with grafting success, but some studies have suggested that some polyphenols could be used as markers of graft incompatibility several months or years after grafting. However, much of the previous studies into metabolite markers of grafting success have not included all the controls necessary to unequivocally validate the markers proposed. In this study, we quantified 73 primary and secondary metabolites in nine hetero-grafts and six homo-grafted controls 33 days after grafting at the graft interface and in both the scion and rootstock woody tissues. Certain biomarker metabolites typical of a high stress status (such as proline, GABA and pallidol) were particularly accumulated at the graft interface of the incompatible scion/rootstock combination. We then used correlation analysis and generalized linear models to identify potential metabolite markers of grafting success measured one year after grafting. Here we present the first attempt to quantitatively predict graft compatibility and identify marker metabolites (especially asparagine, trans-resveratrol, trans-piceatannol and α-viniferin) 33 days after grafting, which was found to be particularly informative for homo-graft combinations.

Minty aroma compounds in red wine: Development of a novel automated HS-SPME-arrow and gas chromatography-tandem mass spectrometry quantification method.

A novel automated method was developed for the quantitative determination of nine terpenoids that could contribute to the minty notes of red wine bouquet. The method couples headspace SPME-Arrow extraction with GC-MS/MS analysis. PDMS/DVB fiber was chosen for the extraction and an ionization energy of 30 eV permitted to optimize the analyte detection. The optimal sample preparation consists of a two-fold dilution of the wine sample with addition of 4 g of sodium chloride while the most suitable extraction conditions take place at 50 °C for 1 h. The method shows good linearity, intraday variations between 2 and 25%, interday variations between 7 and 23% and recoveries between 80 and 119%. The method exhibits the required low detection (between 3 and 60 ng/L) and quantification (between 6 ng/L and 200 ng/L) limits. These limits have permitted the quantification of the pool of minty terpenoids in fourteen red Bordeaux wines.

Characterization of Stilbene Composition in Grape Berries from Wild Vitis Species in Year-To-Year Harvest.

Asian and American Vitis species possess a strong potential for crossbreeding programs, owing to their several resistance properties. Stilbenes are phenolic compounds present in grape berries and are well-known for their main role as phytoalexins and resistance to biotic stresses in plants. However, their identification and quantification in grape berries from wild Vitis remains unexplored. A mass spectrometry multiple reaction monitoring method combined with the analysis of pure standards allowed for the unambiguous characterization of 20 stilbenes in the grape berry skin extracts of nine native Vitis species and one cultivated Vitis vinifera species (cv. Cabernet Sauvignon). A main occurrence of monomeric (Z-piceid, E-piceid, E-isorhapontin, and E-astringin), dimeric (E-ε-viniferin, Z-ε-viniferin, and pallidol), and oligomeric (isohopeaphenol and r-viniferin) stilbenes was highlighted. Some stilbenes were clearly characterized for the first time in grape berries, such as the dimers ampelopsin A, E-vitisinol C, and parthenocissin A as well as the tetramers r2-viniferin and r-viniferin. Stilbene composition and content varied widely among several Vitis species and vintage years.

Resveratrol transformation in red wine after heat treatment.

Resveratrol is a well-known wine constituent. Its concentration can vary according to the cultivar choice and the winemaking process. Due to its phenolic structure, resveratrol could be transformed under high temperature or oxidative conditions, leading to the formation of various derivatives including oligomers. Hence, the goal of this study is to investigate the presence of these derivatives in wine. In the first stage, hemisynthesis of oligomeric stilbenes was achieved from resveratrol in ethanol by oxidative coupling using metals. Four de novo synthetized resveratrol derivatives were identified by MS and NMR spectroscopy including two new molecules, oxistilbenin F and oxistilbenin G. In the second stage, analysis of red wine after heat treatment by LC-MS confirmed the presence of some of these compounds in wine. Finally, the anti-inflammatory effects of the compounds were evaluated by studying their ability to prevent lipopolysaccharide (LPS)-induced upregulation of nitric oxide (NO) and reactive oxygen species (ROS) production in RAW 264.7 macrophage cell line.

Polyphenol Profiles of Just Pruned Grapevine Canes from Wild Vitis Accessions and Vitis vinifera Cultivars.

Grapevine canes are an abundant byproduct of the wine industry. The stilbene contents of Vitis vinifera cultivars have been largely studied, but little is known about the stilbene contents of wild Vitis accessions. Moreover, there have only been few studies on the quantification of other phenolic compounds in just pruned grapevine canes. In our study, we investigated the polyphenol profile of 51 genotypes belonging to 15 Vitis spp. A total of 36 polyphenols (20 stilbenes, 6 flavanols, 7 flavonols, and 3 phenolic acids) were analyzed by high-performance liquid chromatography coupled with a triple quadrupole mass spectrometer. Our results suggest that some wild Vitis accessions could be of interest in terms of the concentration of bioactive polyphenols and that flavanols contribute significantly to the antioxidant activity of grapevine cane extracts. To the best of our knowledge, this is the most exhaustive study of the polyphenolic composition of grapevine canes of wild Vitis spp.

Metabolite profiling during graft union formation reveals the reprogramming of primary metabolism and the induction of stilbene synthesis at the graft interface in grapevine.

BACKGROUND: Grafting with rootstocks is essential for the culture of many perennial fruit crops and is increasing being used in the production of annual fruits and vegetables. Our previous work based on microarrays showed that transcripts encoding enzymes of both primary and secondary metabolism were differentially expressed during graft union formation in both homo-grafts (a genotype grafted with itself) and hetero-grafts (two different genotypes grafted together). The aim of this study was to profile primary and secondary metabolites, and quantify the activity of phenylalanine ammonia lyase (PAL) and neutral invertase (NI) in the scion and rootstock tissues and the graft interface of homo and hetero-grafts of grapevine 1 month after grafting. Table-top grafting was done on over-wintering stems (canes) of grapevine and the graft interface tissues (containing some woody stem tissues and callus) were compared to the surrounding rootstock and scion tissues. The objective was to identify compounds involved in graft union formation and hetero-grafting responses. RESULTS: A total of 54 compounds from primary and secondary metabolism (19 amino acids, five primary and 30 secondary compounds metabolites) and the activity of two enzymes were measured. The graft interface was associated with an increase in the accumulation of the branched-chain amino acids, basic amino acids, certain stilbene compounds and higher PAL and NI activity in comparison to the surrounding woody stem tissues. Some amino acids and stilbenes were identified as being accumulated differently between the graft interfaces of the scion/rootstock combinations in a manner which was unrelated to their concentrations in the surrounding woody stem tissues. CONCLUSIONS: This study revealed the modification of primary metabolism to support callus cell formation and the stimulation of stilbene synthesis at the graft interface, and how these processes are modified by hetero-grafting. Knowledge of the metabolites and/or enzymes required for successful graft union formation offer us the potential to identify markers that could be used by nurseries and researchers for selection and breeding purposes.

Dual solid-phase and stir bar sorptive extraction combined with gas chromatography-mass spectrometry analysis provides a suitable tool for assaying limonene-derived mint aroma compounds in red wine.

A novel analytical method was developed for quantitative determination of eight limonene-derived monoterpenes responsible for the mint aroma in red wine. As these aromatic compounds are present at trace levels, a new dual extraction approach was proposed, combining solid-phase extraction (SPE) and stir bar sorptive extraction (SBSE), followed by gas chromatography-mass spectrometry analysis. The various parameters affecting the efficiency of extracting the analytes from wine samples in both the SPE and SBSE steps were first investigated, to determine the best compromise for the simultaneous analysis of the compounds studied. Following preliminary optimization of the dilution factor, phase ratio, and methanol content in the SBSE sample, cartridge sorbent mass, type of solvent, elution volume, and wine sample volume in the pre-concentration SPE step were studied. Highest response values were obtained when a 90 mL wine sample was extracted on a 500 mg SPE C18 cartridge and eluted with 1.5 mL methanol. The wine extract was then diluted in 10 mL water to obtain a final methanol content of 15% before the SBSE step. Good linearity, repeatability, reproducibility, accuracy and the required low detection and quantification limits were obtained under the conditions described, making this SPE-SBSE combination a suitable, powerful tool for routine analysis of the selected limonene-derived mint aroma compounds in large series of wine samples. Finally, the validated method was applied to 15 commercial red Bordeaux wines, aged from 3 to 23 years. Most of the compounds studied, present within the ng.L-1 range, were easily quantified for the first time in wine.

Study of Substituted Ester Formation in Red Wine by the Development of a New Method for Quantitative Determination and Enantiomeric Separation of Their Corresponding Acids.

A new method was developed for quantifying substituted acids including, where applicable, their various unexplored enantiomeric forms. A new step was added to acids' usual quantification methods, consisting of extraction, derivatization to methyl esters, and gas chromatography analysis: preliminary extraction was performed at basic pH to eliminate ethyl esters, thus avoiding their transesterification during derivatization. Quantitation and enantiomeric distribution of some substituted esters and their corresponding acids were established in 31 commercial Bordeaux red wines from 0 to 20 years old. A strong positive correlation was observed between the age of wine and levels of ethyl 2-methylpropanoate, ethyl 3-methylbutanoate, ethyl 2-methylbutanoate, ethyl (3R)-3-hydroxybutanoate, both enantiomeric forms of ethyl 2-hydroxy-3-methylbutanoate, and ethyl (2S)-2-hydroxy-4-methylpentanoate, but not ethyl (3S)-3-hydroxybutanoate. However, the standard deviations of average concentrations for the corresponding substituted acids were so large that only few correlations between concentrations and age were observed. Concentrations of (2S)-2-hydroxy-3-methylbutanoic acid and (2S)-2-hydroxy-4-methylpentanoic acid increased slightly over time, while (2R)-2-hydroxy-4-methylpentanoic acid levels decreased slightly with the age. Variations in the ratio of substituted ethyl esters to their corresponding acids over time detected thanks to these analytical advances suggested that, in general, acids were continuously esterified during aging.

Quantification of three galloylglucoside flavour precursors by liquid chromatography tandem mass spectrometry in brandies aged in oak wood barrels.

Vanillin-(6'-O-galloyl)-ß-glucopyranoside (VGG), 3,4,5-trimethoxyphenyl-(6'-O-galloyl)-ß-glucopyranoside (TMPGG), and (6R,9R)-3-oxo-α-ionol-9-O-(6'-O-galloyl)-ß-glucopyranoside (macarangioside E) were identified as aroma precursors in oak wood. An LC-MS/MS method was developed and validated to quantify these three galloylglucoside compounds in brandies aged in oak barrels. The detection system consisted of a triple quadrupole mass analyser operating in multiple reaction monitoring (MRM) mode. For the first time, vanillin-ß-d-xylopyranoside (VX) was synthesised for use as an internal standard. The detection limits (48 µg L(-1) for VGG, 52 µg L(-1) for TMPGG, and 19 µg L(-1) for macarangioside E) were low enough to quantify these aroma precursors in spirits without any sample preparation.

Multi-residue off-flavour profiling in wine using stir bar sorptive extraction-thermal desorption-gas chromatography-mass spectrometry.

A multi-residue method (MRM) for the detection and quantification of eight compounds responsible for off-flavours in wine using stir bar sorptive extraction (SBSE) followed by thermal desorption (TD) and gas chromatography-mass spectrometry (GC-MS) analysis is presented. The extraction and desorption conditions were optimised in order to get the best compromise for the simultaneous analysis of the eight target solutes, belonging to different chemical classes. The analytical conditions enable the quantification of the solutes below their respective organoleptic perception thresholds in wine. The method displayed good linearity over the concentration ranges explored in wine as well as excellent repeatability (RSD below 6%) and good reproducibility (RSD below 24%). The developed methodology was applied to the analysis of several wines and showed good agreement with the results collected with headspace solid-phase microextraction (HS-SPME) or liquid-liquid extraction (LLE) followed by GC-MS or electron capture detection (ECD). Good correlation was also found between the analytical and sensory results.

Electronically varied quinazolinaps for asymmetric catalysis.

The synthesis and resolution of electronically varied axially chiral Quinazolinaps is reported. These ligands bear different aryl groups on the donor phosphorus atom and were synthesised as part of our investigations into electronic effects within this ligand class. A diastereomerically pure palladacycle of one ligand was characterised by X-ray crystallography. The application of these Quinazolinaps to the rhodium-catalysed hydroboration of vinylarenes resulted in enantioselectivities of up to 92%. Their application to the palladium-catalysed allylic alkylation of 1,3-diphenylprop-2-enyl acetate resulted in conversions of up to 99% and enantioselectivities of up to 94%.