Plant Biotechnology Journal (2009) 7, pp. 2–12

doi: 10.1111/j.1467-7652.2008.00377.x

Review article Bertrand Review Resveratrol article Delaunois engineering in plants Blackwell Oxford, Plant PBI © 1467-7652 1467-7644 XXX 2008 Biotechnology UK Blackwell Publishing Publishing Journal Ltd et al.Ltd

Molecular engineering of resveratrol in plants Bertrand Delaunois1,2, Sylvain Cordelier2, Alexandra Conreux1, Christophe Clément2 and Philippe Jeandet1,* 1

Laboratory of Oenology and Applied Chemistry, Research Unit ‘Vines and Wines of Champagne – Stress and Environment’, UPRES EA 2069, Faculty of Sciences,

University of Reims, PO Box 1039, 51687 Reims cedex 02, France 2

Laboratory of Plant Stress, Defences and Reproduction, Research Unit ‘Vines and Wines of Champagne – Stress and Environment’, UPRES EA 2069, Faculty of

Sciences, University of Reims, PO Box 1039, 51687 Reims cedex 02, France

Summary Received 25 June 2008; revised 27 August 2008; accepted 31 August 2008 *Correspondence (fax +333 91 33 40; e-mail [email protected])

The grapevine phytoalexin resveratrol, the synthesis of which is achieved by stilbene synthase (STS), displays a wide range of biological effects. Most interest has centred, in recent years, on STS gene transfer experiments from grapevine to the genome of numerous plants. This work presents a comprehensive review on plant molecular engineering with the STS gene. Gene and promoter options are discussed, namely the different promoters used to drive the transgene, as well as the enhancer elements and/or heterologous promoters used to improve transcriptional activity in the transformed lines. Factors modifying transgene expression and epigenetic modifications, for instance transgene copy number, are also presented. Resveratrol synthesis in plants, together with that of its glucoside as a

Keywords: biological activity, genetic transformation, human health,

result of STS expression, is described, as is the incidence of these compounds on plant metabolism and development. The ectopic production of resveratrol can lead to

phytoalexins, plant–microbe

broad-spectrum resistance against fungi in transgenic lines, and to the enhancement

interactions, resveratrol, stilbenes,

of the antioxidant activities of several fruits, highlighting the potential role of this compound

stilbene synthase.

in health promotion and plant disease control.

Introduction Phytoalexins are low-molecular-weight defensive substances produced by plants in response to infection (Jeandet et al., 2002). The term phytoalexin derives from Greek and means ‘warding-off agents in plants’. This elegant concept was proposed after deliberating two important phenomena in plant pathology. The first is the active response of the plant cell to infection, and the second is the acquisition of resistance by plants after exposure to microorganisms. Phytoalexins produced by grapevine in response to biotic and abiotic stress factors have attracted considerable attention over the last two decades. One of the most significant components of the phytoalexin response in Vitaceae is the synthesis of the stilbene compound resveratrol (Langcake and Pryce, 1977; Jeandet et al., 1997, 2002), which is also found in several other fruits and vegetables, including cranberries, blueberries, mulberries, peanuts and jackfruit. Resveratrol displays a wide range of biological effects, notably as a

2

cardioprotective, antitumour or neuroprotective agent, as well as an antifungal or antibacterial compound (Adrian et al., 1997; Adrian and Jeandet, 2006; Anekonda, 2006; King et al., 2006; Athar et al., 2007; Barger et al., 2008; Saiko et al., 2008). Resveratrol has been proposed to extend the lifespan of lower and higher organisms, and there is a growing body of evidence to show that resveratrol may have potential therapeutic applications in vivo (Baur and Sinclair, 2006). The large amounts of resveratrol in grapes and wine have focused attention on its biological functions. Stilbene phytoalexins are formed via the phenylalanine/polymalonate route (Langcake and Pryce, 1977; Jeandet et al., 2002; Hall and Yu, 2008). The last step of this biosynthesis pathway is catalysed by stilbene synthase (STS; EC 2.3.1.95). STS is encoded by a multigene family, comprising the well-characterized resveratrol-forming STS genes from grapevine and the pinosylvin-forming STS genes from pine. Because of resveratrol’s capacity to confer disease resistance in grapevine, and given its clinically useful biological properties, © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd

Resveratrol engineering in plants 3

Figure 1 Chemical structures of the most common stilbene phytoalexins: 1 and 3, transand cis-piceid; 2, trans-resveratrol. Glc, glucosyl (C6H11O5). Ring A results from the condensation of three malonyl-CoA units. Ring B originates from the p-coumaroyl-CoA moiety. R1–R4, functional groups attached to the different compounds.

most interest has centred, in recent years, on STS gene transfer from grapevine to the genome of numerous plants, with the objective of increasing their tolerance to pathogenic microorganisms and/or improving the nutritional quality of food products through the expression of resveratrol in plants incapable of synthesizing this compound. Studies have been published describing STS forms isolated from grapevine and their use for plant genetic transformation. This review explores recent research on the transfer of STS-encoding genes to different crops.

STS and the biosynthesis of resveratrol Although phytoalexins display a huge chemical diversity, phytoalexins produced by grapevine constitute a rather restricted group of molecules belonging to the stilbene family (Langcake and Pryce, 1977; Jeandet et al., 2002). The chemical structure of stilbene phytoalexins is based on a polyphenol-type stilbene, so-called resveratrol (trans-3,5,4′trihydroxystilbene), and on its 3-O-β-D-glucoside derivative, known as piceid (Figure 1) (Waterhouse and LamuelaRaventos, 1994). Stilbene phytoalexins are formed via the well-characterized phenylalanine/polymalonate biosynthetic pathway, the last step of which is catalysed by STS. STS belongs to the huge type III group of the polyketide synthase enzyme superfamily, a class of enzymes which carry out iterative condensation reactions with malonyl-coenzyme A (malonyl-CoA) as a substrate (Shen and Hutchinson, 1993; Austin and Noel, 2003). STS produces simple stilbene phytoalexins in a single enzymatic reaction, with CoA-esters of cinnamic acid derivatives (p-coumaroyl-CoA in the case of resveratrol or dihydrocinnamoyl-CoA in the case of dihydropinosylvin, a phytoalexin from pine) and three malonyl-CoA units as starting blocks (Schröder et al., 1988; Jeandet et al., 2002;

Hall and Yu, 2008). There are three sequential condensation reactions with malonyl-CoA units, followed by ring closure of a tetraketide intermediate (Ferrer et al., 1999; Austin and Noel, 2003). STS, as well as chalcone synthases (CHSs), which are key enzymes of the flavonoid biosynthesis pathway, contains a single and essential cysteine residue, Cys164, which most probably represents the active site of the enzyme (Jez and Noel, 2000; Jez et al., 2000; Suh et al., 2000). STS was first purified from cell suspension cultures of Arachis hypogea (Schoeppner and Kindl, 1984). It is encoded by a multigene family comprising, notably, the resveratrolforming STS genes from grapevine (pSV21, pSV25, pSV696 and pSV368; Melchior and Kindl, 1991; Vst1, Vst2, Vst3 and StSy; Wiese et al., 1994) and the pinosylvin-forming STS genes from pine (PST-1, PST-2, PST-3, PST-4 and PST-5; PreisigMüller et al., 1999). Three novel STS genes (pdsts1, pdsts2 and pdsts3) have been isolated more recently from the roots of Pinus densiflora (Kodan et al., 2001, 2002), as well as a new STS gene from Vitis riparia cv. Gloire de Montpellier (Goodwin et al., 2000). There is, at present, only one STS gene described in monocotyledonous plants, the SbSTS1 gene isolated from sorghum (Yu et al., 2005). STS genes were grouped according to their responsiveness to external signals, including abiotic stresses or biotic signals originating from fungal cells (Brehm et al., 1999; Preisig-Müller et al., 1999). STS cDNA and genomic clones have been described from Scots pine (Fliegmann et al., 1992), groundnut (Lanz et al., 1990) and grapevine (Melchior and Kindl, 1990; Sparvoli et al., 1994).

Production of resveratrol in transgenic plants: gene and promoter options The genetic transformation of plants with STS genes has led to interesting developments (Fischer and Hain, 1994; Hain

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Plant Biotechnology Journal, 7, 2–12

4 Bertrand Delaunois et al.

and Grimming, 2000), and is still being studied intensively. The first gene transfer experiments were performed with a complete STS gene from Arachis hypogea introduced into tobacco (Hain et al., 1990), leading to resveratrol accumulation after induction with short-wavelength ultraviolet (UV) light, a well-known elicitor of resveratrol synthesis (Jeandet et al., 1997). It has also been shown that the transfer of two grapevine STS genes, Vst1 and Vst2, in tobacco confers a higher resistance to Botrytis cinerea infection (Hain et al., 1993). This latter study constituted the first report of disease resistance resulting from foreign phytoalexin expression in a novel plant. Since this elegant, pioneering work, STS genes have been transferred to a number of crops, including rice (Stark-Lorenzen et al., 1997), tomato (Thomzik et al., 1997; Giovinazzo et al., 2005; Morelli et al., 2006; Nicoletti et al., 2007), alfalfa (Hipskind and Paiva, 2000), kiwifruit (Kobayashi et al., 2000), apple (Szankowski et al., 2003; Rühmann et al., 2006), Arabidopsis (Yu et al., 2006), aspen (Seppänen et al., 2004), white poplar (Giorcelli et al., 2004), pea (Richter et al., 2006), hop (Schwekendiek et al., 2007), papaya (Zhu et al., 2004), lettuce (Liu et al., 2006), oilseed rape (Hüsken et al., 2005), barley and wheat (Leckband and Lörz, 1998; Fettig and Hess, 1999; Liang et al., 2000; Serazetdinova et al., 2005), banana (Vishnevetsky et al., 2005), Rehmannia (Lim et al., 2005) and grapevine (Coutos-Thévenot et al., 2001; Fan et al., 2008) (Table 1). In grapevine, one of the plant species in which the largest amounts of resveratrol are found naturally, genome sequencing has revealed a large array of STS genes, with 43 genes identified and 20 of these being shown to be expressed, thus suggesting the importance of stilbene metabolism for this species (Jaillon et al., 2007). To date, Vst1 and Stsy genes have been the most common genes used for plant transformation (Table 1). Other STS-encoding genes have also been used, notably the AhRS gene from Arachis hypogea (Hain et al., 1990; Hipskind and Paiva, 2000), the SbSTS1 gene from Sorghum bicolor (Yu et al., 2005, 2006) and an STS-encoding gene from Parthenocissus henryana (Liu et al., 2006). To increase the levels of stilbene production, some investigators have used chimeric genes or a combination of two STSencoding genes, based on Vst1 and Vst2 (Fischer et al., 1997; Fettig and Hess, 1999). The Vst1 expression rate has been shown to be six-fold higher than the expression rate of Vst2 in wheat (Serazetdinova et al., 2005). The same observation was made in Vitis species, in which transcript levels of Vst2 were 10- to 100-fold higher than those of Vst1 (Wiese et al., 1994). Vst2 transcripts accumulated significantly later than those of Vst1, but remained detectable over a longer period of time (Serazetdinova et al., 2005). In most cases, the

expression of STS-encoding genes in transformed plants led to stilbene accumulation and/or pathogen resistance, confirming that such transgenes are functional in foreign species. In practice, the modulation of gene expression is mainly controlled by the promoter chosen to drive the transgene. To date, STS-encoding genes for plant transformation have been expressed under the control of a limited number of promoters, in particular the well-characterized constitutive promoter pCaMV35S, its own stress-responsive promoter pVst1, the fungus-inducible promoter pPR10.1 or the tissuespecific promoter p-nap (Table 1). The most commonly used promoter to over-express a transgene in plants is the strong constitutive pCaMV35S promoter (Fischer et al., 1997). As expected, the pCaMV35S promoter triggered strong and constitutive stilbene accumulation in most studies, but, as a consequence, caused a drastic depletion of the endogenous pools of precursors. In grapevine, the pVst1 promoter is induced either by biotic factors (pathogens, elicitors) or abiotic stresses (wounding, UV light) (Adrian et al., 1996; Jeandet et al., 1997; Breuil et al., 1999; Commun et al., 2003; Aziz et al., 2006). Most importantly, this inducible promoter leads to stilbene accumulation without interfering with secondary biosynthetic pathways (Hain et al., 1990, 1993; Thomzik et al., 1997). The pVst1 promoter has been used successfully in several studies to produce resveratrol, suggesting that it is regulated by common transcriptional factors present in most plant species. It provides a rapid and strong accumulation of both STS transcripts and their products following inoculation with phytopathogenic fungi (Hain et al., 1990; Stark-Lorenzen et al., 1997; Thomzik et al., 1997; Zhu et al., 2004). In contrast with constitutive promoters, the choice of an inducible promoter thus appears to be a promising way to maintain precursor pool levels, while allowing strong stilbene accumulation at the infection site. To improve transcriptional activity, STS-encoding gene expression has been optimized by the use of enhancer elements (Leckband and Lörz, 1998; Serazetdinova et al., 2005) and/or heterologous promoters (Fischer et al., 1997; Hipskind and Paiva, 2000; Kobayashi et al., 2000; CoutosThévenot et al., 2001; Hüsken et al., 2005). A combination of the pVst1 promoter with the 35S enhancer element (listed as 35S-4 fold in Table 1) can lead to higher expression of the transgene (Leckband and Lörz, 1998; Serazetdinova et al., 2005) without affecting promoter inducibility or specific expression patterns (Leckband and Lörz, 1998). The use of a chimeric promoter, e.g. a fusion between the alfalfa pPR10.1 promoter and the pVst1 promoter, may also allow a high expression of the transgene linked to an increased production

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Plant Biotechnology Journal, 7, 2–12

Arachis hypogea STS

Medicago sativa L.

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Plant Biotechnology Journal, 7, 2–12 CaMV35S

Vst1

Vitis pseudoreticulata STS

Vitis vinifera L.

CaMV35S CaMV35S

StSy

StSy

Vst1

CaMV35S

Vst1 and Vst2

Lycopersicon

Vst1

Vst1

StSy

Vst1

Borkh.

esculentum Mill.

Vst1

Malus domestica

gene

ms PR10.1

pSV25

Actinidia deliciosa

CaMV35S

SbSTS1

CaMV35S

CaMV35S

Vst1 (+35S-4 fold)

Arabidopsis thaliana L.

gene (AhRS)

Vst1

Hordeum vulgare L.

Vst1 (+35S-4 fold)

promoter

Maize ubiquitin

Chimeric STS gene

Vst1 and Vst2

Vst1 (+35S-4 fold)

CaMV35S

Chimeric STS gene

Vst1

Vst1

Vst1 and Vst2

Triticum aestivum L.

Stress-induced promoter

Arachis hypogea STS gene

Nicotiana tabacum L.

Promoter

Gene

Plant species

stage of ripening and fruit samples

0.42–126 depending on the

trans-, cis-piceid

0.1–1.2

4–53

chlorogenic acid contents

Differences in rutin, naringenin and

properties

Enhancement of natural antiradical

activity

and increase in total antioxidant

Antioxidant primary metabolism

and Alternaria solani

No resistance to Botrytis cinerea

infestans

Resistance to Phytophthora

compounds

–

No influence on other phenolic

23–62 for UV-irradiated fruit

–

Under investigation

cinerea

In vitro resistance to Botrytis

No resistance to Botrytis cinerea

–

Resistance to Phoma medicaginis

Resistance to Botrytis cinerea

and Septoria nodorum

Resistance to Puccinia recondita

–

Resistance to Botrytis cinerea

sterility

Altered flower morphology, male

Resistance to Botrytis cinerea

–

Biological activity

3–7 for non-UV-irradiated fruit;

–

2.6

20–182

584

0.5–20

–

35–190

2

–

50–290

400

–

(μg/g FW)

Stilbene concentration

trans-, cis-Resveratrol and

piceid

trans-Resveratrol and trans-

piceid

trans-Resveratrol and trans-

Resveratrol

trans-Piceid

glycoside

Unknown resveratrol

Resveratrol

Resveratrol

trans-Piceid

trans- and cis- Piceid

trans-Piceid

–

compounds

Unknown derivative stilbene

Resveratrol

–

Resveratrol

trans-Resveratrol

trans-Resveratrol

Biochemical output

Nicoletti et al. (2007)

Morelli et al. (2006)

Giovinazzo et al. (2005)

Thomzik et al. (1997)

Rühmann et al. (2006)

Szankowski et al. (2003)

Fan et al. (2008)

Coutos-Thévenot et al. (2001)

Kobayashi et al. (2000)

Yu et al. (2005, 2006)

Hipskind and Paiva (2000)

Leckband and Lörz (1998)

Serazetdinova et al. (2005)

Fettig and Hess (1999)

Liang et al. (2000)

Leckband and Lörz (1998)

Fischer et al. (1997)

Hain et al. (1993)

Hain et al. (1990)

Reference

Table 1 Stilbene synthase genes and promoters used to genetically transform plants, and the resulting effects on stilbene levels, resistance to pathogens and antioxidant activities

Resveratrol engineering in plants 5

Vst1

StSy

Vst1

Vst1

Vst1

Pisum. Sativum L.

Populus alba L.

Carica papaya L.

Brassica napus L.

Humulus lupulus L.

CaMV35S

p-nap

Vst1

CaMV35S

Vst1

CaMV35S

cis-resveratrol

trans-astringin, trans- and

unknown stilbene cis-isomer,

trans- and cis-Piceid,

Resveratrol glucoside

Resveratrol glucoside

trans- and cis-Piceid

compounds

resveratrol glucoside

Occurrence of two

trans-Resveratrol

Resveratrol and piceid

22–116

–

490–560

361–616

54

309–615

0.53–5.2

56.4

STS

Parthenocissus henryana

CaMV35S

–

(μg/g FW)

Stilbene concentration

Lactuca sativa L.

AhRS3

Rehmannia glutinosa

Vst1

Biochemical output

Up to 650 with stress treatment

Vst1

Oryza sativa L.

Promoter

Libosch.

Gene

Plant species

Table 1 Continued.

palmivora

acids

Higher amounts of flavonoids and

of sinapate esters

piceid rate content and reduction

Food quality improvement: high

Schwekendiek et al. (2007)

Hüsken et al. (2005)

Zhu et al. (2004)

Seppänen et al. (2004) Resistance to Phytophthora

Giorcelli et al. (2004) Melampsora pulcherrima

Richter et al. (2006)

Liu et al. (2006)

Lim et al. (2005)

(1997)

Stark-Lorenzen et al.

Reference

No in vitro resistance to

–

Effect on Hela cell morphology

Resistance to Fusarium oxysporum

Antioxidant capabilities

Resistance to Pyricularia oryzae?

Biological activity

6 Bertrand Delaunois et al.

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Plant Biotechnology Journal, 7, 2–12

Resveratrol engineering in plants 7

of resveratrol on fungal infection, reaching 5–100-fold the levels found in non-transgenic leaves (Coutos-Thévenot et al., 2001). Another promoter option is the choice of tissue-specific promoters that can be used to limit stilbene accumulation and its potent detrimental effects against targeted organs, such as, for example, male sterility induced by the overexpression of STS in tobacco flowers (Fischer et al., 1997). Hüsken et al. (2005) have used the p-nap seed-specific napin promoter to specifically induce stilbene production in oilseed rape seeds and, in combination with a sinapate glucosyltransferase gene extinction strategy, to deplete sinapate esters. The choice of the promoter for STS expression should be performed depending on the expected results. When the main objective is the enhancement of plant resistance against phytopathogens through phytoalexin production, the choice of a strong constitutive promoter or a pathogen-inducible promoter may be indicated. When the improvement of food products is sought, a tissue-specific or inducible promoter would be a better option. The main disadvantages of using a strong constitutive promoter could be precursor storage depletion and the possible occurrence of post-transcriptional gene silencing, leading to transgene extinction and a subsequent lack of stilbene production. Limiting STS expression and resveratrol synthesis to infection time and location will probably reduce the negative effects of resveratrol accumulation on plant morphology or fertility.

Variability of transgene expression and epigenetic modifications In addition to promoter characteristics, transcription levels can be affected by transgene insertion events, leading, in some cases, to sequence modification of the inserted DNA (Lim et al., 2005), recombination into repeated sequences or multiple copy insertions (Laufs et al., 1999). Most of the high stilbene-accumulating transgenic lines developed so far carry only one copy of the transgene, whereas lower stilbeneaccumulating lines typically contain two or three copies (Hüsken et al., 2005; Richter et al., 2006). Inversion and translocation events may also occur after sequence rearrangement in the transgene (Laufs et al., 1999) or the surrounding regions (Gelvin, 1998), which may lead to transgene expression variability. In white poplar, a high transgene copy number correlating with low levels of transcripts suggests that gene silencing occurs in these lines (Giorcelli et al., 2004). Transcriptional and/or post-transcriptional gene silencing (Jakowitsch et al., 1999; Pickford and Cogoni, 2003) may reduce expression levels when the number of copies is high. Furthermore, epigenetic promoter modifications and

endogenous trans- or cis-acting elements can modify transcriptional kinetics and the localization of transgene expression (Fojtova et al., 2003). In some cases, and although a constitutive promoter was used to drive transgene expression, stilbene accumulation has been reported to be tissue- and development-specific (Hipskind and Paiva, 2000; Schwekendiek et al., 2007), which could be a result of regulation by endogenous cis and trans transcriptional factors. The two most commonly used genetic transformation technologies, particle bombardment and Agrobacteriummediated methods, have successfully been applied to produce STS-expressing plants. However, as epigenetic modifications may occur and lead to expression variability, influencing stilbene synthesis levels, it may be more appropriate to select plants with a single gene insertion, and thus the use of Agrobacterium-mediated transformation, leading to lower transgene insertion numbers, should preferentially be chosen.

Stilbene production and spatiotemporal distribution Qualitative and quantitative comparisons between different transgenic plants synthesizing resveratrol and related stilbenes are difficult, because these compounds are usually assayed using different analytical methods. Such analyses have shown various stilbene levels and spatiotemporal distributions, leading to a huge variability in terms of relative amounts for the different forms. The glycosylation of polyphenolic compounds, in particular, commonly occurs in plants both to protect cells from their potential toxic effects and to protect resveratrol from oxidation and enzymatic degradation (Jeandet et al., 1997; Hipskind and Paiva, 2000). The occurrence of glycosylated resveratrol forms in planta shows that free resveratrol is first synthesized, before being glycosylated by endogenous glycosyltransferases. In transgenic plants expressing STSencoding genes, qualitative analysis reveals the presence of resveratrol glycosylated derivatives. Both free resveratrol and its glycosylated forms have been detected (Giovinazzo et al., 2005). Piceid, a predominant component of the phytoalexin response, occurs in different plant species transformed with STS genes (Table 1) (Kobayashi et al., 2000; Giorcelli et al., 2004; Hüsken et al., 2005; Lim et al., 2005; Yu et al., 2005, 2006; Morelli et al., 2006; Rühmann et al., 2006; Nicoletti et al., 2007; Schwekendiek et al., 2007). The stilbene content also strongly depends on the plant species, probably because of different endogenous pools of enzymes or precursors, as well as differences in secondary metabolism pathways.

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Plant Biotechnology Journal, 7, 2–12

8 Bertrand Delaunois et al.

In the case of plants transformed with the pCaMV35S promoter, resveratrol contents range from 0.1 to 615 μg/g fresh weight (FW), which is comparable with the levels found in either stress-induced Vitis leaves or in grape skins, where the highest resveratrol amounts are generally detected (Jeandet et al., 1997; Adrian et al., 2000). Genetic modification with adequate regulatory sequences allows stilbene production at levels similar to those observed in natural stilbene-producing plants. The pCaMV35S promoter seems to drive stilbene levels higher than those produced with inducible promoters (Table 1). In most cases, however, the correlation between STS-encoding gene expression and stilbene content is unclear (Serazetdinova et al., 2005). With regard to the amounts of stilbenes detected in different studies, no common trend can be found, which possibly may be explained by both the plant species and the specific genetic constructs used for transformation. In tomato and apple fruit, the free to glycosylated resveratrol ratio (or the piceid level) naturally depends on the fruit ripening stage (Giovinazzo et al., 2005; Rühmann et al., 2006), these two compounds accumulating differentially in fruit parts at the mature stage (Nicoletti et al., 2007). Such variations may be a result of different endogenous β-glucosidase expression patterns. Moreover, stilbene production may depend on the age of the organ, as reported in transgenic tobacco and kiwi, in which young leaves produce higher stilbene amounts than do older leaves (Hain et al., 1993; Kobayashi et al., 2000). More than the plant species considered, the stilbene levels and forms in transgenic plants depend on the tissues or organs used as source material, as well as on the development and fruit ripening stages.

Impact of resveratrol accumulation on plant metabolism and development Generally, stilbene accumulation does not trigger any detrimental effect on plant development, growth, morphology or fertility, and no influence of newly synthesized stilbenes has been reported on other secondary metabolite pools (Hipskind and Paiva, 2000; Rühmann et al., 2006; Schwekendiek et al., 2007). For example, constitutive expression of an STS transgene in oilseed rape does not lead to any negative effect with regard to seed yield or oil content (Hüsken et al., 2005). Likewise, no differences in the flavonoid or acid content have been observed in transgenic hop (Schwekendiek et al., 2007). By contrast, STS-encoding gene expression under the control of the pCaMV35S constitutive promoter triggers, in some cases, an altered flower morphology, colour modification

and male sterility in tobacco and petunia (Fischer et al., 1997), or in conifers (Höfig et al., 2006). Other studies have reported a reduction in fertility for some transgenic wheat lines, although other lines show a normal development. Such events may be explained by the possible transgene insertion site, such as, for instance, insertions within a gene involved in male fertility for sterile lines (Fettig and Hess, 1999). Sterility may also be linked to a competition for substrates between STS and endogenous CHS, considering that fertility can be restored by exogenous flavonol addition in tobacco (Fischer et al., 1997). STS and CHS are polyphenolic pathway enzymes that both use p-coumaroyl-CoA and malonyl-CoA as substrates to synthesize, respectively, resveratrol and naringenin chalcone, a key compound in the flavonoid, tannin and anthocyanidin biosynthesis pathways. It is reasonable to assume that the expression of an exogenous STS may lead to substrate competition with CHS, which may, in turn, decrease CHS activity or other related pathways. Such competition between the two enzymes may also explain the lack of correlation observed between STS activity and resveratrol accumulation in transgenic white poplar (Giorcelli et al., 2004), and the negligible accumulation of piceid in redpigmented apple fruit synthesizing anthocyanins (Rühmann et al., 2006).

Biological benefits of resveratrol synthesis in transgenic plants From a practical viewpoint, the ectopic production of resveratrol can lead to broad-spectrum resistance against fungi in transgenic plants. Since the first successful gene transfer experiment was performed with a complete STS gene from Vitis vinifera to tobacco, to confer resistance to B. cinerea (Hain et al., 1993), STS genes have been transferred to numerous plants. In most studies, different transgenic plant species over-expressing an STS-encoding gene, and accumulating resveratrol or derivatives, exhibit an increased resistance to attacks by various phytopathogenic microorganisms (Table 1). The disease resistance of plants may partly depend on their ability to rapidly produce high stilbene amounts following infection. However, disease symptoms in transgenic lines are often only partially reduced (Hain et al., 1993; Thomzik et al., 1997; Hipskind and Paiva, 2000; Serazetdinova et al., 2005) and, in some cases, fungal infection is not completely prevented and pathogen growth is slowed but not stopped (Zhu et al., 2004). Pathogendependent resistance has also been reported (Thomzik et al., 1997; Serazetdinova et al., 2005), and variations in resistance have been observed for the same pathogen in different

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Plant Biotechnology Journal, 7, 2–12

Resveratrol engineering in plants 9

transgenic lines of the same species (Serazetdinova et al., 2005). In a few cases, STS-expressing plants have not shown any sign of resistance to pathogens (Kobayashi et al., 2000), even when the transformed plant is producing high stilbene amounts (Giorcelli et al., 2004). However, several studies have reported an enhancement of antioxidant activities in transgenic plants over-expressing stilbene-encoding genes (Giovinazzo et al., 2005; Rühmann et al., 2006). In transgenic tomato, resveratrol synthesis increased the global antioxidant properties of the fruit, as well as its ascorbate/glutathione content (Giovinazzo et al., 2005). Resveratrol accumulation also induced a two-fold increase in antioxidant activity in transgenic tomato fruit, and a correlation was found between resveratrol concentrations and antioxidant activities in ripe and unripe fruits (Morelli et al., 2006). Another study on STS heterologous expression in oilseed rape, combined with sinapate glucosyltransferase gene shutdown, has been reported to decrease strongly the undesirable sinapate ester contents, thus improving the forage quality of the modified plant (Hüsken et al., 2005).

Conclusion Some interesting and important developments may be expected from plant transformation with STS. For example, promising results have been obtained with STS-encoding genes in transgenic plants, confirming that disease resistance can arise from foreign phytoalexin expression. Increasing the resistance of plants to phytopathogenic microorganisms thus provides an alternative mechanism to the use of pesticides and fungicides. In addition, plant molecular engineering with resveratrol may lead to food products comprising edible legumes, cereals or fruits, which can be ingested, with their potential clinical benefits, by humans. The recent literature also suggests the potential of genetically modified microorganisms as an alternative mechanism for increasing resveratrol amounts in food products, as this compound can be synthesized directly in recombinant bacteria or yeasts, such as Escherichia coli (Watts et al., 2006) and Saccharomyces cerevisiae (Beekwilder et al., 2006; Zhang et al., 2006). These observations are of interest for the food industry, which could produce resveratrol in large quantities in biofermentators. Transgenic yeasts expressing a gene for a glycosyl hydrolase have also been reported to increase the resveratrol content in wine (Gonzalez-Candelas et al., 2000) or the wine-related antioxidant content (Becker et al., 2003). Taken together, these results suggest the general relevance of STS-encoding sequences as a tool for engineering disease

resistance and the nutritional quality of agricultural crops and food products.

Acknowledgements The authors thank the Région Champagne Ardenne and the Comité Interprofesionnel du Vin de Champagne for financial support of the PhD Thesis of B.D.

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