Grape Stilbene Research Articles

Improvement of Stilbene Content in Concord and Isabel Precoce Grapes and Juice through Methyl Jasmonate Application

In this research, the effect of methyl jasmonate (MJ) preharvest treatment was studied on grapes and their juice from two different regions. Concord and Isabel Precoce grapes from south and southeast Brazil received the treatment during two vintages (2017 and 2019). MJ preharvest treatment was able to increase stilbenes in the grape seeds and peels of Isabel Precoce in both regions. The juice elaborated with the treated grapes also has higher stilbene levels, especially piceid, that were detected even after 6 months of storage. Additionally, we performed a small study for this market niche; 189 consumers were interviewed, and it was observed that those with a higher frequency of grape juice consumption were willing to pay 25% more for grape juice with a higher resveratrol content. In conclusion, this work has evidenced the potential of MJ preharvest treatment to improve stilbenes in grapes and their juice, despite regional differences.

Potential Associations among Bioactive Molecules, Antioxidant Activity and Resveratrol Production in Vitis vinifera Fruits of North America.

Grapes (Vitis vinifera L.) are rich in bioactive molecules contributing to health benefits. Consumption of grapes is linked to reduced incidence of cardiovascular diseases. Studies on table grape cultivars are limited although much attention in research was focused on the wine industry. Bioactive effects of grapes as anti-inflammatory, anticarcinogenic, cardioprotective, vasorelaxant, phytoestrogenic and neuroprotective have also been reported. For example, resveratrol is a natural food ingredient present in grapes, with high antioxidant potential. Here we conducted an exploratory study to investigate bioactive molecules, antioxidant activity and the association between constitutive stilbene synthase (STS) gene expression and the resveratrol biosynthesis in selected table grape varieties in North America. The phenolic compounds, fatty acid composition and antioxidant activity of four grape varieties were compared. Red Globe variety was rich in unsaturated fatty acids as well as phenolic compounds such as caffeic acid, quercetin and resveratrol. Meanwhile, the constitutive expression of grape stilbene synthase gene was higher in Flame and Autumn Royal where resveratrol content of these cultivars was relatively low compared to the Red Globe variety. This study shows the potential links in grape antioxidant activity and resveratrol production, but more studies are necessary to show the association.

Three stilbenes make difference to the antifungal effects on ochratoxin A and its precursor production of Aspergillus carbonarius

The present study demonstrated the toxic effects of stilbenes on fungi, which were related to the structures of the stilbenes. Pterostilbene with methoxy had the best antifungal properties, followed by piceatannol, which has a catechol structure, and finally resveratrol. The inhibitory effects of stilbenes at 0.1, 0.2, 0.4, 0.8, 1.6 mM on A. carbonarius mycelia growth and spore germination were assessed by plate inhibition tests and poisoned food technique. Predicted by SPSS software, the IC50 values of resveratrol, piceatannol, and pterostilbene were 5.10, 1.80, and 0.28 mM, respectively. In addition, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that 0.4 mM pterostilbene treatment induced incompleteness of the sporangium and distortion of the mycelial structure. Antitoxic activities of stilbenes were positively correlated with their antifungal activities. 1.6 mM pterostilbene suppressed OTA synthesis better (63.48%) than 1.6 mM piceatannol (25.91%) and 1.6 mM resveratrol (22.98%). Furthermore, in the presence of stilbenes, the examined biosynthetic genes, and regulatory factors like NRPS, PKS, LaeA, HAL, bZIP, and P450 were remarkably downregulated to reduce OTA/OTB production. Increased levels of total stilbenes in grapes after fungal infestation can slow down the increased rate in OTA levels. It indicated stilbenes could be used as naturally safe and efficient compounds in food active packaging or preservatives against OTA in food.

Methyl Jasmonate Applications in Viticulture: A Tool to Increase the Content of Flavonoids and Stilbenes in Grapes and Wines

Recently, the interest in methyl jasmonate (MeJ) has increased in viticulture due to its effects on the synthesis of phenolic secondary metabolites in grapes, especially of anthocyanins, flavonols, and stilbenes derivatives, naturally occurring or synthesized, in berries in response to MeJ application to grapevines. These metabolites help to define sensory characteristics of wines by contributing to their color, flavor and mouthfeel properties, and to derive potential beneficial health effects due to their consumption. This review offers an overview of the importance of these phenolic compounds in grape and wine quality, in association with the MeJ supplementation to grapevines, and also considers their natural biosynthesis in grapes. On the other hand, this review describes the adaptation mechanisms induced after the grapevine elicitation. In addition, this report addresses the effects of MeJ over other aspects of Vitis immunity and its association with phenolic compounds and summarizes the recently published reports about the effects of exogenous MeJ applications to grapevines on grape and wine quality.

Stilbenes in grapes and wines of Tannat, Marselan and Syrah from Uruguay

Aim: The aim of the study was to investigate the stilbene composition of grapes and wines of the Vitis vinifera cultivars Tannat, Marselan and Syrah cultivated in Uruguay. The effects of delaying the harvest on stilbene concentrations were determined, and the stability of stilbenes during wine storage was assessed.Methods and results: Stilbene concentrations were determined in the grapes and wines of two vintages (2015 and 2016) and two harvest dates for each cultivar. Vinification was carried out by traditional maceration, and samples of the wines of each vintage were analysed in the period from 3 months after devatting to up to 24 months later. After solid-phase extraction, stilbenes were identified and quantified by HPLC-ESI-MS/MS using a multiple reaction-monitoring approach. In the grape berries, stilbene concentrations were between 1.6 and 7.7 mg/kg, depending on grape cultivar, growing season, and in Syrah, harvest date. In the wines, stilbene concentrations were initially between 0.9 and 5.0 mg/L, being highest in Syrah, lowest in Marselan, and intermediate in Tannat. Stilbene concentrations in the Marselan wines were lower than expected based on stilbene concentrations in the grapes from which they were produced, suggesting poor extraction during winemaking. Total stilbene concentrations remained very stable during the analytical period.Conclusions: Delaying the harvest does not necessarily increase the stilbene content of grapes, but it can do so significantly, as shown for Syrah. For some grape cultivars, such as Marselan, poor extraction of stilbenes during winemaking can limit their concentrations in the resulting wines.Significance and impact of the study: The results of this study show the relevance of grape cultivar, degree of maturity and storage time may have into stilbenes. They provide reference data on the stilbene composition of grapes and wines produced under Uruguayan winegrowing conditions. The high stability of stilbenes during wine storage is relevant for consumers interested in red wine as a source of bioactive compounds.
 In memory of Prof. Isidro Hermosín-Gutiérrez.

The effect of methyl jasmonate and light irradiation treatments on the stilbenoid biosynthetic pathway in Vitis vinifera cell suspension cultures

Grape stilbenes are a well-known family of plant polyphenolics that have been confirmed to have many biological activities in relation to health benefits. In the present study, we investigated the effect of methyl jasmonate (MeJA) elicitor at four different concentrations (25, 50, 100 and 200 μM) in combination or not with high-level light irradiation (10,000 LUX) on a cell line obtained from the pulp of Vitis vinifera cv. Shahani. Our results showed that the stilbene synthesis pathway is inhibited by high-light conditions. A concentration of 50 μM MeJA was optimum for efficient production and high accumulation of total phenolics and total flavonoids as well as total stilbenoids. Furthermore, we showed that there is a significant negative correlation between the production of these metabolites and cell growth. These data provide valuable information for the future scale-up of cell cultures for the production of these very high value compounds in bioreactor system.

Elicitors used as a tool to increase stilbenes in grapes and wines

Two preharvest treatments (methyl jasmonate or cell wall yeast) of grapevines (Monastrell and Tempranillo) were applied during two vintages (2014 and 2015) to check whether these elicitors enhanced stilbene accumulation in berries at the moment of harvest and in the corresponding wines elaborated with them. The main objective was checking the effect of treatment, variety and year on stilbene composition due to the interesting health-related properties of these compounds in both grapes and wines. The results pointed to inter-varietal and inter-annual differences, and that the treatments generally enhanced the stilbene composition of grapes and, particularly, of wines. The increase was more evident in Monastrell variety than in Tempranillo variety and in their wines more than their grapes during 2014 vintage but not during 2015 vintage.

Improvement of grape and wine phenolic content by foliar application to grapevine of three different elicitors: Methyl jasmonate, chitosan, and yeast extract

Phenolic compounds play a key role in grape and wine organoleptic properties, being therefore a key parameter in wine quality. Elicitor application constitutes an interesting field of research since it is indirectly involved in the accumulation of phenolic compounds. The aim of this study was to compare the effect of the application of three different elicitors on both grape and wine phenolic content. Methyl jasmonate, chitosan, and a commercial yeast extract were applied to the canopy at veraison and one week later. Results showed that foliar treatments carried out with methyl jasmonate and yeast extract achieved the best results, increasing grape and wine anthocyanin content when compared to the control. Moreover, the application of the yeast elicitor also enhanced grape stilbene content. In contrast, the chitosan treatment did not have a substantial impact on the phenolic compounds. The results of this study indicate that methyl jasmonate and yeast extract applications could be a simple practice to increase grape and wine phenolic content.

GENETIC AND PHYSIOLOGICAL BASES OF HEALTHY ATTRIBUTES OF GRAPES AND WINE

Grapes and wine are claimed to have beneficial effects on human health, due mainly to their high polyphenolic content. Polyphenols include several classes of compounds regarded mostly as effective antioxidants in humans when assumed in the diet (e.g., hydroxycinnamic acids, flavonols, anthocyanins, tannins (proanthocyanidins) and stilbenes). The latter deserve particular attention due to their crucial function in plants; stilbene phytoalexins accumulate in the soft tissues of the vine and they are produced by the phenylpropanoid pathway. Stilbene synthase (STS) is the key enzyme of the above pathway, and it produces resveratrol, the basic monomer which can be glycosylated, hydroxylated, methylated or converted into more complex compounds (dimers, trimers, tetramers). According to the grapevine genome sequence, STS is encoded by a gene family of many highly similar paralogues, and recent data account for their expression in different grapevine organs. At the berry (fruit) level, stilbenes are produced by the skin after the occurrence of a biotic or abiotic stress, and by the seeds as constitutive compounds. Berry skin is the main available source of stilbenes from grape and wine consumption. Thus it is crucial to deeply understand mechanisms of biotic and abiotic elicitors of stilbene phytoalexins in order to increase their production. Study of metabolomics allows identification of a large array of stilbenes in grapes and wine that could have major implications for health. Under the same elicitation pressure, the final concentration of stilbenes in grapes and wine is affected by decisions applied for viticultural practices and, in the case of wine, for winemaking (i.e., oenological factors). The most recent health claims for resveratrol are discussed, including the effect in extending the lifespan of organisms (human beings, too) by triggering the Sir/SIRT genes.

Comment on Stilbene Levels in Grape Cane of Different Cultivars in Southern Chile: Determination by HPLC-DAD-MS/MS Method

Health benefits of trans-resveratrol and other stilbenes in grapes, must, and wine have been pointed out by numerous authors. Less attention has been paid to the presence of stilbene derivatives in viticultural residues, such as grape canes. The present work reports the first results of a systematic study of stilbene levels in different grape varieties and cultivation areas in Chile, to evaluate their potential as an alternative source of bioactive stilbenes. In all cane samples, the predominant stilbene is trans-resveratrol, followed by e-viniferin and piceatannol. In canes of Pinot noir up to 5590 ± 172 mg kg–1 of trans-resveratrol and up to 6915 ± 175 mg kg–1 of total stilbenes were detected. The observed concentrations of stilbenes in canes of Pinot noir from southern Chile until now are higher than those reported previously for this red variety. However, the highest concentration of total stilbenes observed in the analyzed samples was in the canes of white variety Gewurztraminer with 7857 ± 498 mg kg...

Terroir and variety: Two key factors for obtaining stilbene-enriched grapes

Grapes and wine are among the main sources of bioactive stilbenes in the diet. The stilbene composition of grapes depends on both variety and terroir. The effect of terroir on grape stilbenes has not been widely studied. In this research the study of the stilbene content (resveratrol, piceatannol and viniferins) in four different red grape varieties (Syrah, Merlot, Cabernet sauvignon and Pinot noir) and from four different Andalusian terroirs (Jerez, Cabra, Cadiar and Ronda) was carried out during the 2009 vintage. In addition, stilbene induction capacity was studied in each terroir and variety after ultraviolet-C (UVC) postharvest treatment. Data were analysed by multivariate statistical methods. Principal component analysis applied to the oenological parameters and stilbene data classified varieties into two terroir groups satisfactorily: Jerez and Cabra on the one hand, and Ronda and Cadiar on the other hand. Terroir effect is stronger than variety on stilbene induction capacity after UVC treatment. Syrah from Cabra stood out due to both the high basal (13.07mg/kg fw) and induced (33.14mg/kg fw) stilbene concentration attained. Therefore, it can be affirmed that Cabra and Syrah are the most suitable terroir and variety, respectively, for obtaining a stilbene-enriched wine.

Focusing on bioactive compounds in grapes: stilbenes in Uvalino cv.

Phenolic compounds belonging to the group of stilbenes have recently received a particular attention as bioactive compounds because of their role in plant physiology and for their antioxidant properties. Monomer stilbenes in grapes are mainly present as glycosides. The aim of this work was the identification and quantification of monomer stilbenes (as both free and glycosilated form) in Uvalino grapes, a grapevine variety known for its significant content in stilbenes. Their content in grapes was determined during ripening and overripening stages, in comparison with other phenolic compounds. Piceid (cis and trans form), piceatannol (cis and trans form), cis resveratrol and trans-pterostilbene were identified and quantified in grape skin. Piceid was the most abundant stilbene as both cis and trans forms. As for anthocyanins, the synthesis and the stilbenes accumulation started during veraison, and then, the content of these molecules remained constant with the exception of piceid, whose content slightly increased until harvest. Therefore, stilbenes accumulation seems not to be affected by anthocyanins synthesis. On the contrary, proanthocyanidins, flavonols and hydroxycinnamyl tartrates decreased during ripening. When withering was applied to grapes as a post-harvest treatment, the content of anthocyanins, proanthocyanidins and of the most abundant stilbenes increased, while flavonols and hydroxycinnamyl tartrates decreased. The results suggest that degradation processes during withering are probably weaker or absent in the case of stilbenes, anthocyanins and proanthocyanidins.

An innovative approach to grape metabolomics: stilbene profiling by suspect screening analysis

Suspect screening analysis is a targeted metabolomics approach in which identification of compounds relies on specific available information such as their molecular formula and isotopic pattern. This method was applied to the study of grape metabolomics with an UPLC/MS high-resolution Q-TOF mass spectrometer (nominal resolution 40,000) coupled with a Jet Stream ionization source. The present paper describes the detailed qualitative and quantitative study of grape stilbenes, the principal polyphenols associated with the beneficial effects of drinking wine. For identification of compounds, a new database was expressly constructed from the molecular information of potential metabolites of grape and wine from the literature and other electronic databases. Currently, GrapeMetabolomics contains about a thousand putative grape compounds. If untargeted analysis of a sample provides identification of a new compound with a sufficiently confident score, it is added to the database. Thus, by increasing the number of samples studied, GrapeMetabolomics can be expanded. This method is effective for identification of the molecular formulae of several hundred metabolites in two runs (positive and negative ionization) with minimal sample preparation, and can also be used to analyse some single classes of compounds involved in cell and tissue metabolism. With this approach, a total of 18 stilbene derivatives was identified in two grape samples (Raboso Piave and Primitivo) on the basis of accurate mass measurements and isotopic patterns, and identification was confirmed by MS/MS analysis. The approach can also potentially be applied to the metabolomics of other plant varieties.

Resveratrol Biosynthesis Up-Regulates the Ascorbate/Glutathione Pathway in Transgenic Tomato Fruit

There is a growing interest in producing food plants with increased amounts of antioxidants because of their potential health benefits. Polyphenolic secondary metabolites, such as flavonoids and stilbenes, have been investigated for their significant antioxidant activity, important both for plant physiology and human nutrition. With the aim to verify whether the synthesis of resveratrol in tomato fruit affected the redox status of transformed tomato tissues, in the present work we took advantage of the availability of two different transgenic tomato lines in which a grape stilbene synthase gene was expressed under a constitutive or a tissue specific promoter, respectively. The induction of resveratrol synthesis in tomato affected the redox status of transformed tomato fruit. In particular, ascorbate and glutathione pool increased, significantly and proportionally to the amount of the resveratrol synthesised in transformed tissues. Noteworthy, the increase in ascorbate and glutathione pool paralleled with a significant increase in the activities of the main enzymes involved in redox homeostasis, i.e. catalase, ascorbate peroxidase as well the enzymes responsive for ascorbate recycling. Furthermore, lipoxygenase activity and levels of polyunsaturated fatty acids hydroperoxides, were reduced in fruit tissues from transgenic tomato lines. The synthesis of resveratrol, together with the global redox status had an impact on the total antioxidant activity of transgenic fruits. Again the total antioxidant capability increased proportionally to the amount of synthesised resveratrol with the most remarkable increase recorded from the lipophilic-resveratrol containing-fraction of transgenic fruits. Overall, our results pointed a higher nutritional value of resveratrol synthesizing tomato fruits.

APPLICATION OF BTH AND METHYL JASMONATE DURING THE RIPENING OF GRAPES (VITIS VINIFERA L.) AND ITS EFFECTS ON THE STILBENE CONTENT: PRELIMINARY RESULTS

Pre-harvest treatments of grapevine (‘Monastrell’) with the plant defence response elicitor benzothiadiazole (BTH, 0.3 mM) and methyl jasmonate (10 mM), an important growth regulator, were applied to check whether these compounds could enhance stilbene accumulation in berries at the moment of harvest, due to the interesting health properties of the stilbenes in grapes and wines. Both compounds were sprayed on clusters at veraison. Both compounds enhanced the level of stilbenes in grapes, the highest increase being found with the use of methyl jasmonate. Since a highest presence of stilbenes in grapes could be a protection against mould infection, the application of these compounds could, not only improve the healthiness of grapes, but also limit the use of fungicides.

Stilbene Levels in Grape Cane of Different Cultivars in Southern Chile: Determination by HPLC-DAD-MS/MS Method

Health benefits of trans-resveratrol and other stilbenes in grapes, must, and wine have been pointed out by numerous authors. Less attention has been paid to the presence of stilbene derivatives in viticultural residues, such as grape canes. The present work reports the first results of a systematic study of stilbene levels in different grape varieties and cultivation areas in Chile, to evaluate their potential as an alternative source of bioactive stilbenes. In all cane samples, the predominant stilbene is trans-resveratrol, followed by ε-viniferin and piceatannol. In canes of Pinot noir up to 5590 ± 172 mg kg(-1) of trans-resveratrol and up to 6915 ± 175 mg kg(-1) of total stilbenes were detected. The observed concentrations of stilbenes in canes of Pinot noir from southern Chile until now are higher than those reported previously for this red variety. However, the highest concentration of total stilbenes observed in the analyzed samples was in the canes of white variety Gewürztraminer with 7857 ± 498 mg kg(-1). Preliminary results indicate that these levels can evolve if canes are left for some months on the vineyard after pruning, observing an increase during the first 2 months and a decrease after this period.

Over-expression of a grape stilbene synthase gene in tomato induces parthenocarpy and causes abnormal pollen development

A novel strategy to induce parthenocarpy in tomato fruits by the induction of resveratrol biosynthesis in flower tissues was exploited. Two transgenic tomato lines were considered: a higher resveratrol-producing (35SS) line, constitutively expressing a grape stilbene synthase cDNA, and a lower resveratrol-producing (LoxS) line, expressing stilbene synthase under a fruit-specific promoter. The expression of the stilbene synthase gene affected flavonoid metabolism in a different manner in the transgenic lines, and in one of these, the 35SS line, resulted in complete male sterility. Resveratrol was synthesised either in 35SS or LoxS tomato flowers, at an even higher extent (about 8-10 times) in the former line. We further investigated whether stilbene synthase expression may have resulted in impaired naringenin accumulation during flower development. In the 35SS flowers, naringenin was significantly impaired by about 50%, probably due to metabolic competition. Conversely, the amount of glycosylated flavonols increased in transgenic flowers, thereby excluding the diminished production of flavonols as a reason for parthenocarpy in tomato. We further investigated whether resveratrol synthesis may have resulted changes to pollen structure. Microscopic observations revealed the presence of few and abnormal flake-like pollen grains in 35SS flowers with no germination capability. Finally, the analysis of coumaric and ferulic acids, the precursors of lignin and sporopollenin biosynthesis, revealed significant depletion of these compounds, therefore suggesting an impairment in structural compounds as a reason for pollen ablation. These overall outcomes, to the best of our knowledge, reveal for the first time the major role displayed by resveratrol synthesis on parthenocarpy in tomato fruits.

Improved tolerance toward fungal diseases in transgenic Cavendish banana (Musa spp. AAA group) cv. Grand Nain

The most devastating disease currently threatening to destroy the banana industry worldwide is undoubtedly Sigatoka Leaf spot disease caused by Mycosphaerella fijiensis. In this study, we developed a transformation system for banana and expressed the endochitinase gene ThEn-42 from Trichoderma harzianum together with the grape stilbene synthase (StSy) gene in transgenic banana plants under the control of the 35S promoter and the inducible PR-10 promoter, respectively. The superoxide dismutase gene Cu,Zn-SOD from tomato, under control of the ubiquitin promoter, was added to this cassette to improve scavenging of free radicals generated during fungal attack. A 4-year field trial demonstrated several transgenic banana lines with improved tolerance to Sigatoka. As the genes conferring Sigatoka tolerance may have a wide range of anti-fungal activities we also inoculated the regenerated banana plants with Botrytis cinerea. The best transgenic lines exhibiting Sigatoka tolerance were also found to have tolerance to B. cinerea in laboratory assays.

Induction of stilbenes in grapes by UV-C: Comparison of different subspecies of Vitis

Bioactive products enriched in stilbenes are considered of potential future interest, and the main sources of stilbenes in human diet are grapes. Postharvest ultraviolet C (UV-C) treatment was used to induce stilbene biosynthesis in grapes of three varieties of Vitis vinifera sylvestris, seven of Vitis vinifera sativa, and two Hybrid Direct Producers (HDPs). Stilbenes have been identified by UPLC-DAD-TQD and quantified by HPLC-DAD, and cluster analyses have been performed to classify subspecies by their stilbene profile. After UV-C treatment, the Syrah variety reached a maximum of 25 mg kg− 1 f.w. of total stilbenes in the 2008 vintage, and in the statistical analysis, this variety stood out from the other Vitis varieties tested. In 2008, varieties belonging to the sylvestris group and Vitis vinifera sativa Merlot also presented high stilbene production; however, the expected concentration in the HDPs was not obtained. In this study, postharvest UV-C treatment has been applied to obtain stilbene-enriched grapes from several subspecies of Vitis. The most suitable raw material for bioactive stilbene-enriched products, such as nutraceutics and wines, has been established. This is an essential prerequisite to scaling up the process. Such stilbene-enriched products are known to have added-value as a result of the confirmed positive bioactivity of these compounds, and are gaining market acceptance.

Differential expression of a cell wall-localized peroxidase isoenzyme capable of oxidizing 4-hydroxystilbenes during the cell culture of grapevine (Vitis vinifera cv. Airen and Monastrell)

Differential expression and localization of peroxidase isoenzymes capable of oxidizing 4-hydroxystilbenes was studied during establishment of callus cultures from Vitis vinifera ‘Airen’ (anthocyanin-non accumulating) and ‘Monastrell’ (anthocyanin-accumulating) berries. Callus formation from mesocarp tissues was accompanied by differential expression of several peroxidase isoenzyemes located in cell walls, among which only peroxidase isoenzyme A1 was capable of oxidizing 4-hydroxystilbene to any great extent. Likewise, grape cell cultures were capable of accumulating the grape stilbene phytoalexin, resveratrol. However, e-viniferin, the most powerful phytoalexin in grapevines and previously considered as the product of peroxidase-mediated oxidative coupling of two resveratrol moieties, was only detectable in trace amounts. Since grapevine suspension cell cultures were unable to produce H2O2 as revealed by the luminol test, H2O2 production by the cultured cells appears to be one of the main factors which limits resveratrol oxidation in the cell walls of grapevine cells cultured in suspension.