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1.1 General Introduction

In this chapter we begin by providing an overview of the biochemistry of phytoalexin synthesis in Vitis vinifera and the significance of these molecules in plant physiology. We then discuss the concentrations of these molecules in wines, the main dietary source of phytoalexins produced by Vitis vinifera. Our goal in this first chapter is to cultivate an appreciation for red wine polyphenols that extends beyond their effects in mammalian cells.

1.2 Grapevine Phytoalexins

Phytoalexins are secondary metabolites produced by plants in response to biotic and abiotic stressors. Across plant species, an enormous range of phytoalexins is produced, and these generally have antifungal and antimicrobial activities (Ahuja et al. 2012). Phytoalexins, and related compounds termed phytoanticipins (constitutively produced secondary metabolites with similar properties), have been studied for their possible beneficial effects on human health. Amongst these molecules are biologically active compounds with the potential to be further developed into functional food ingredients or dietary supplements (Boue et al. 2009). In the grapevine Vitis vinifera, the predominant phytoalexins produced in response to stress belong to a family of compounds called stilbenes (Fig. 1.1), which are synthesized from the amino acid phenylalanine.

Fig. 1.1
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Basic carbon skeleton structure of a stilbene

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One of the main stilbenes produced in grapevines is trans-resveratrol (trans-­3,5,4'-trihydroxystilbene), which has been the subject of intensive investigation over the past two decades owing to its apparent health promoting properties. Although red wines are perhaps the best known dietary sources of resveratrol and its derivatives, these molecules are produced by a variety of phylogenetically diverse plant species, including peanut (Arachis hypogaea), Japanese knotweed (Fallopia japonica), sorghum (Sorghum bicolor), and Pinus and Picea species (Parage et al. 2012). In instances where food or beverages are produced from these plants the biologically active phytoalexins are present in the consumed product, sometimes in negligible amounts and occasionally at concentrations sufficient to perhaps elicit biological responses.

1.3 Stilbene Synthesis

Trans-resveratrol is the initial stilbene product of p-coumaroyl-CoA and three molecules of malonyl-CoA in a reaction catalyzed by stilbene synthase (full pathway shown in Fig. 1.2). While most plants are capable of producing malonyl-CoA and CoA esters of cinnamic acid, the ability to synthesize stilbenes is limited to only a few plant species (reviewed in Chong et al. 2009). In Vitis vinifera, stilbene synthase is encoded by between 20 and 40 stilbene synthase genes, which is considerably more genetic diversity within this gene family than has been found in any other plant species (Chong et al. 2009; Parage et al. 2012). Trans-resveratrol serves as a precursor molecule that is further converted into a wide variety of compounds including piceid, pterostilbene, and viniferins by glycosylation, methoxylation, and oxidative oligomerization, respectively (Fig. 1.3). Some of these compounds, particularly piceid, are present at ­significantly higher concentrations than resveratrol in grapevine leaves (Boso et al. 2012), grape skins (Romero-Perez et al. 2001; Bavaresco et al. 2007), grape juices (Romero-Perez et al. 1999), and wines (Mark et al. 2005; Moreno-Labanda et al. 2004; Naugler et al. 2007).

Fig. 1.2
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Synthesis of trans-resveratrol from p-coumaroyl-CoA and malonyl-CoA by stilbene synthase

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Fig. 1.3
figure 00013

Trans-resveratrol is a precursor molecule that may be converted into piceid, pterostilbene, and viniferins by glycosylation, methoxylation, and oxidative oligomerization, respectively

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Phytoalexins are produced in grapevines constitutively at low levels and are present in root, stem, leaf, and grape tissues (Wang et al. 2010). However, production and accumulation of phytoalexins within leaf and grape tissues is strongly induced by stressors such as UV light, heavy metals, ozone, and fungal infection (Jeandet et al. 2002). The expression of at least 20 stilbene synthase genes is induced by inoculation with the downy mildew fungus Plasmopara viticola (reviewed in Chong et al. 2009), and increased abundance of stilbene synthase protein on the leaf epidermis following UV exposure has been demonstrated using immunolocalization (Pan et al. 2009). Trans-resveratrol levels measured in leaf tissue vary with Vitis vinifera variety, but in all varieties they are increased substantially (up to 30-fold) within several days of inoculation with P. viticola (Boso et al. 2012). Like trans-­resveratrol, the production of pterostilbene, piceid, and viniferins is also strongly enhanced by stress. Pterostilbene levels increase from undetectably low levels to a few μmol/mg free weight within several days following infection with P. viticola (Boso et al. 2012), in parallel with an approximately fivefold increase in the expression of resveratrol O-methyltransferase, the enzyme catalyzing trans-pterostilbene synthesis from trans-resveratrol (Schmidlin et al. 2008). Piceid, ε-viniferin, and δ-viniferin undergo similar increases of up to 100-fold within 3 days following P. viticola infection. Although many of the measurements of polyphenol levels following infection have been made in grapevine leaves, similar increases have been shown also to occur in grape skins, which is relevant to the abundance of these molecules in red wines (Romero-Perez 2001; Montero et al. 2003).

1.4 Roles of Stilbenes in Grapevines

Stilbene production in plants is a central component of their response to stress. Resistance of Vitis species to fungal infection is generally correlated with their ability to produce stilbene phytoalexins (Douillet-Breuil et al. 1999; Schnee et al. 2008; Malacarne et al. 2011). Exogenous application of trans-resveratrol to grape berries is also an effective means of enhancing their resistance to fungal infection (Gonzalez Urena et al. 2003). Transgenic expression of stilbene synthase genes in non grapevine species is being utilized to enable stilbene production and in turn capture the potent antifungal properties of these molecules (Thomzik et al. 2001; Coutos-­Thevenot et al. 2001; Zhu et al. 2004; Liu et al. 2011).

Indeed, there is good experimental evidence for potent antibacterial, antifungal, and anti-nematodal activities of trans-resveratrol and its derivatives (Chong et al. 2009). In laboratory experiments, trans-resveratrol inhibits P. viticola growth and development, and exhibits similar suppressive activity against a number of other fungal pathogens, including Cladosporium cucumerinum, Botrytis cinerea, Oidium tuckeri, Pyricularia oryzae, and Sphaeropsis sapinea at concentrations that are roughly equivalent to those produced by grapevine (reviewed in Jeandet et al. 2002). Pterostilbene is actually a more potent inhibitor of fungal growth than trans-­resveratrol (Jeandet et al. 2002), though it may not accumulate to levels required for this activity in grape skin. ε-viniferin has antifungal activity similar to that of pterostilbene, suggesting that trans-resveratrol may be the least effective as an antifungal agent of this family of stilbene compounds.

1.5 Approaches to Increasing Stilbene Levels in Grapevines

Increased stilbene synthesis in Vitis vinifera is beneficial on two levels: (1) elevated stilbene levels, particularly in grape skin, will lead to higher concentrations of these apparent health-promoting compounds in wine, and (2) the increased stilbene concentrations will impart greater resistance to environmental stressors. Strategies to achieve this goal are therefore the focus of much research.

Application of exogenous stressors may be used to promote the endogenous production of trans-resveratrol and its derivatives in grapevine leaves and grapes, and this is of interest as a means of enhancing the levels of these compounds in red wines. One approach to enhancing stilbene levels in grapes has been to stimulate the signalling pathways involved in upregulating stilbene production. The plant hormone jasmonic acid is an effective inducer of phenylalanine ammonia lyase and stilbene synthase expression that significantly enhances stilbene synthesis. Repeated application of methyljasmonate to growing vines substantially increases the levels of trans-resveratrol and ε-viniferin in berries (Larronde et al. 2003; Vezzulli et al. 2007). In grapevine cell cultures, both methyljasmonate, and also cyclodextrins, stimulate stilbene synthesis (Lijavetzky et al. 2008; Zamboni et al. 2009). Interestingly, brief exposure to anoxia also appears to stimulate trans-resveratrol synthesis in harvested grapes (Jimenez et al. 2007). Thus, several non-genomic approaches have proven effective in enhancing stilbene levels in wine grapes and wine grape cells.

Another experimental method being used to increase stilbene levels is transgenic engineering to promote the synthesis of trans-resveratrol and its derivatives in plant species that do not naturally produce these molecules. Since all plant species produce the immediate precursors of trans-resveratrol, 4-coumaroyl-CoA and malonyl-­CoA, transgenic overexpression of stilbene synthase alone is sufficient to instigate trans-resveratrol synthesis. Transgenic lines overexpressing stilbene synthase and/or O-methyltransferase have been engineered in a number of plant species, including tomato (Solanum lycopersicum), Arabidopsis thaliana, and tobacco ( Nicotiana tabacum ). Typically, when just stilbene synthase is overexpressed, the predominant polyphenol accumulating is trans-piceid rather than trans-resveratrol (Liu et al. 2006; Ingrosso et al. 2011). In transgenic plants co-expressing stilbene synthase and O-methyltransferase, pterostilbene accumulates as the major product, again with relatively low levels of trans-resveratrol (Rimando et al. 2012; Xu et al. 2012). Although these studies indicate that engineered plants could be used to produce stilbenes, it is interesting to note that there is evidence that excessive levels of these molecules interferes also with normal plant development (Ingrosso et al. 2011). The application of transgenic engineering to enhance stilbene levels is an active and developing area of research.

1.6 Stilbenes in Wines

Although resveratrol and its derivative molecules are found in a variety of wines, they are typically present at much higher levels in red wines due to a long fermentation process that includes contact with the grape skins. This allows the highly hydrophobic stilbene compounds to be extracted from grape skin into the forming ethanol. Although red wines are a relatively rich dietary source of resveratrol and its derivatives, the absolute concentrations of these compounds are nonetheless low, ranging from undetectable to about 63 μM (Stervbo et al. 2007; Dourtoglou et al. 1999; Naugler et al. 2007). Levels of resveratrol vary with wine variety, with relatively high levels identified in some Pinot Noir and Merlot wines (Stervbo et al. 2007). Regional variation is also evident, and indeed environmental stresses unique to a given region are likely to affect resveratrol and stilbene production in grape skin, and therefore the levels present in wines produced from these grapes.

There has been a great deal of focus on the levels of resveratrol alone in red wines; however, it is important to note that some resveratrol derivatives are actually present at higher levels than resveratrol itself in red wines. For example, in studies of Hungarian (Mark et al. 2005), Spanish (Moreno-Labanda et al. 2004), and Canadian (Naugler et al. 2007) wines, piceid levels were found to be as much as tenfold higher than those of resveratrol. Piceid is also present at higher concentrations than resveratrol in grape juices (Romero-Perez et al. 1999) and cocoa (Hurst et al. 2008). Several resveratrol oligomers similarly accumulate to quite high levels in grapevine leaves (Boso et al. 2012), particularly following fungal elicitation. However, the levels of ε-viniferin (Adrian et al. 2000a, b), hopeaphenol (Boutegrabet et al. 2011), and pallidol (Naugler et al. 2007) in red wines appear to be generally lower than those of resveratrol and piceid. Pterostilbene levels are particularly low in grape berries and in red wines (e.g., Adrian et al. 2000a, b; Boso et al. 2012). Thus, resveratrol and piceid appear to be the major stilbenes present in red wines.

1.7 Conclusions

Grapevines produce a variety of stilbene molecules in response to both biotic and abiotic stresses. Identification of the signalling pathways and genes involved has led to strategies for targeting these with the goal of enhancing stilbene production both in Vitaceae and other plant species. Many of these strategies have now been shown to be quite effective at boosting the concentrations of resveratrol and its derivatives in wine grapes and instigating their production in other plant species. Given the accumulating evidence for applications of these compounds in human health contexts, ongoing research in this area is likely to yield increasingly high resveratrol grapes for wine production. Recent work has indicated that red wine supplemented with up to 200 mg/L resveratrol is palatable and stable (Gaudette & Pickering 2011), and therefore, high resveratrol wines should be an effective means of increasing dietary intake of resveratrol and its stilbene derivatives.