A stress-inducible resveratrol O-methyltransferase involved in the biosynthesis of pterostilbene in grapevine

Stilbenes are considered the most important phytoalexin group in grapevine (Vitis vinifera) and they are known to contribute to the protection against various pathogens. The main stilbenes in grapevine are resveratrol and its derivatives and, among these, pterostilbene has recently attracted much attention due both to its antifungal and pharmacological properties. Indeed, pterostilbene is 5 to 10 times more fungitoxic than resveratrol in vitro and recent studies have shown that pterostilbene exhibits anticancer, hypolipidemic, and antidiabetic properties. A candidate gene approach was used to identify a grapevine resveratrol O-methyltransferase (ROMT) cDNA and the activity of the corresponding protein was characterized after expression in Escherichia coli. Transient coexpression of ROMT and grapevine stilbene synthase in tobacco (Nicotiana benthamiana) using the agroinfiltration technique resulted in the accumulation of pterostilbene in tobacco tissues. Taken together, these results showed that ROMT was able to catalyze the biosynthesis of pterostilbene from resveratrol both in vitro and in planta. ROMT gene expression in grapevine leaves was induced by different stresses, including downy mildew (Plasmopara viticola) infection, ultraviolet light, and AlCl(3) treatment.     

Expression pattern, genomic structure, and promoter analysis of the gene encoding stilbene synthase from Chinese wild Vitis pseudoreticulata

The gene encoding stilbene synthase (STS) plays a central role in many biochemical and physiological actions, and its metabolite resveratrol possesses broad-spectrum resistance to pathogens, as well as diverse pharmacological properties, notably an anticancer effect. Here, we report the expression analysis of the gene encoding STS and its promoter function from a powdery mildew (PM)-resistant Chinese wild Vitis pseudoreticulata, and compare it with two PM-susceptible cultivated grapevines, Vitis vinifera cvs. Carignane and Thompson Seedless. We show an unusual expression pattern of STS in V. pseudoreticulata, which differs markedly from that of the cultivated species. Sequence comparisons reveal that the genomic DNA sequences encoding STS in the three grapevines are highly conserved, but a novel residue mutation within the key motif of STS is solely present in V. pseudoreticulata. Moreover, the STS promoter in V. pseudoreticulata displays a significantly different structure from that found in the two V. vinifera. The three promoter-driven GUS differential expression patterns in transformed tobacco plants induced with Alternaria alternata, methyl jasmonate, and wounding indicated that the structurally different STS promoter of V. pseudoreticulata is responsible for its specific regulatory function. We also demonstrate that the expression of STS genes from their native promoters are functional in transformed tobacco and retain pathogen inducibility. Importantly, the genomic DNA-2 of V. pseudoreticulata under its native promoter shows good induction and the maximum level of resveratrol content. These findings further our understanding of the regulation of STS expression in a resistant grapevine and provide a new pathogen-inducible promoter system for the genetic improvement of plant disease resistance.     

Identification of pterostilbene as a phytoalexin from Vitis vinifera leaves

An inducible antifungal compound in grapevine leaves (Vitis vinifera L., cv Cabernet-Sauvignon) has been identified as trans-pterostilbene (3,5-dimethoxy-4′-hydroxy stilbene). It is only a minor component of the phytoalexin response of V. vinifera but its antifungal activity is relatively high by comparison with resveratrol and the viniferins, stress metabolites which have been identified previously in grapevine. Methods for the quantitative analysis of pterostilbene, resveratrol, ε- and α-viniferins by HPLC are described.     

Ectopic expression of VpALDH2B4, a novel aldehyde dehydrogenase gene from Chinese wild grapevine (Vitis pseudoreticulata), enhances resistance to mildew pathogens and salt stress in Arabidopsis

Aldehyde dehydrogenases (ALDHs) catalyze the irreversible oxidation of a broad spectrum of reactive aldehydes to their corresponding carboxylic acids. Although the proteins have been studied from various organisms and at different growth stages in plants, their potential roles in pathogen infection have not been examined. Here we isolated and functionally characterized a pathogen-inducible ALDH gene (VpALDH2B4) from Chinese wild grapevine Vitis pseudoreticulata accession Baihe-35-1. When transiently expressed in Arabidopsis leaves, VpALDH2B4 was found to be localized in mitochondria. Escherichia coli expressed GST-VpALDH2B4 exhibited ALDH activity in vitro and was capable of utilizing malondialdehyde (MDA), acetaldehyde and glyceraldehydes as its substrate. Over-expression of VpALDH2B4 in Arabidopsis resulted in hypersensitive response-like cell death, enhanced resistance to downy mildew and powdery mildew presumably via the SA-signaling pathway. The same Arabidopsis transgenic plants also showed enhanced tolerance to salt stress, which is accompanied by less MDA accumulation and upregulation of the stress-responsive superoxide dismutase activity. Taken together, our results suggest that VpALDH2B4 and perhaps its orthologous genes may be involved in responses of plants to stresses imposed by both biotrophic pathogens and high salinity conditions.     

Genetic transformation of a fruit-specific,highly expressed stilbene synthase gene from Chinese wild Vitis quinquangularis

Planta - The stilbene synthase gene VqSTS6, from Chinese wild type Vitis quinquangularis accession Danfeng-2, increases the resveratrol content and pathogen resistance of transgenic plants of V....     

Pterostilbene production by microorganisms expressing resveratrol O-methyltransferase

Pterostilbene (3,5-dimethoxy-4′-hydroxyl-trans-stilbene)—a derivative of resveratrol—is a natural dietary compound and the primary antioxidant component in berries. Pterostilbene has significant advantages over resveratrol in bioavailability, half-life in the body, cellular uptake, oral absorption and metabolic stability. Here, we expressed the resveratrol O-methyltransferase (ROMT) gene (VvROMT) from grape (Vitis vinifera) in Escherichia coli and Saccharomyces cerevisiae and confirmed its specific ability to catalyze the production of pterostilbene from resveratrol. By co-expressing an additional two genes from the resveratrol biosynthetic pathway—4-coumarate CoA-ligase (4CL) and stilbene synthase (STS)—a large amount of pterostilbene was produced, with a trace amount of pinostilbene detected. To understand the molecular basis of the catalytic activity, four key amino acid residues were identified in a 3D-model of VvROMT and mutagenized and assayed for augmented catalytic activity. Our results demonstrate the potential utility of the engineered microorganisms for pterostilbene production and provide protein engineering targets that will hopefully lead to increased activity of the ROMT enzyme.     

In planta production of the highly potent resveratrol analogue pterostilbene via stilbene synthase and O-methyltransferase co-expression

Resveratrol and related stilbenes are thought to play important roles in defence responses in several plant species and have also generated considerable interest as nutraceuticals owing to their diverse health-promoting properties. Pterostilbene, a 3,5-dimethylether derivative of resveratrol, possesses properties similar to its parent compound and, additionally, exhibits significantly higher fungicidal activity in vitro and superior pharmacokinetic properties in vivo. Recombinant enzyme studies carried out using a previously characterized O-methyltransferase sequence from Sorghum bicolor (SbOMT3) demonstrated its ability to catalyse the A ring-specific 3,5-bis-O-methylation of resveratrol, yielding pterostilbene. A binary vector was constructed for the constitutive co-expression of SbOMT3 with a stilbene synthase sequence from peanut (AhSTS3) and used for the generation of stably transformed tobacco and Arabidopsis plants, resulting in the accumulation of pterostilbene in both species. A reduced floral pigmentation phenotype observed in multiple tobacco transformants was further investigated by reversed-phase HPLC analysis, revealing substantial decreases in both dihydroquercetin-derived flavonoids and phenylpropanoid-conjugated polyamines in pterostilbene-producing SbOMT3/AhSTS3 events. These results demonstrate the potential utility of this strategy for the generation of pterostilbene-producing crops and also underscore the need for the development of additional approaches for minimizing concomitant reductions in key phenylpropanoid-derived metabolites.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) with a novel stilbene synthase gene from Chinese wild <Emphasis Type="Italic">Vitis pseudoreticulata</Emphasis>

A novel stilbene synthase gene (STS), cloned from Chinese wild Vitis pseudoreticulata (W. T. Wang) and responsible for synthesis of the phytoalexin resveratrol in grapevine, was successfully transferred into V. vinifera L. cv. Thompson Seedless via Agrobacterium tumefaciens-mediated transformation. Using transformation procedures developed in the present study, 72% GFP-positive germinated embryos were produced with about 38% of transformed embryos regenerated into normal plantlets. Integration of the STS gene into the transgenic plants was verified by PCR and Southern blot analysis. Expression of the STS gene was detected by high performance liquid chromatography (HPLC), which showed that the resveratrol concentration in the transgenic plants was 5.5 times higher than that in non-transformed control plants. Chaohong Fan and Ni Pu contributed equally to this work.     

In vitro tolerance to Botrytis cinerea of grapevine 41B rootstock in transgenic plants expressing the stilbene synthase Vst1 gene under the control of a pathogen-inducible PR 10 promoter.

Resveratrol is a major phytoalexin in grapevine but its synthesis in response to phytopathogen attack decreases with grape berry ripening. A chimeric gene combining an alfalfa PR 10 promoter and Vst1 (Vitis stilbene synthase 1) gene was introduced into the genome of 41B rootstock. Transgenic plants were analysed for resveratrol production in leaves infected with Botrytis using an in vitro test. Among the 50 transgenic lines analysed, some exhibited a production lower than the non-transgenic control, but others accumulated resveratrol from 5-100-fold. Moreover, in the latter clones, symptoms were highly reduced in response to infection. These results were a good indication that the combination of a pathogen-inducible promoter and a defence gene may increase tolerance against fungi in grapevine. The efficacy of this approach should be further tested by experiments conducted in the vineyard.     

Production of highly bioactive resveratrol analogues pterostilbene and piceatannol in metabolically engineered grapevine cell cultures

Grapevine stilbenes, particularly trans‐resveratrol, have a demonstrated pharmacological activity. Other natural stilbenes derived from resveratrol such as pterostilbene or piceatannol, display higher oral bioavailability and bioactivity than the parent compound, but are far less abundant in natural sources. Thus, to efficiently obtain these bioactive resveratrol derivatives, there is a need to develop new bioproduction systems. Grapevine cell cultures are able to produce large amounts of easily recoverable extracellular resveratrol when elicited with methylated cyclodextrins and methyl jasmonate. We devised this system as an interesting starting point of a metabolic engineering‐based strategy to produce resveratrol derivatives using resveratrol‐converting enzymes. Constitutive expression of either Vitis vinifera resveratrol O‐methyltransferase (VvROMT) or human cytochrome P450 hydroxylase 1B1 (HsCYP1B1) led to pterostilbene or piceatannol, respectively, after the engineered cell cultures were treated with the aforementioned elicitors. Functionality of both gene products was first assessed in planta by Nicotiana benthamiana agroinfiltration assays, in which tobacco cells transiently expressed stilbene synthase and VvROMT or HsCYP1B1. Grapevine cell cultures transformed with VvROMT produced pterostilbene, which was detected in both intra‐ and extracellular compartments, at a level of micrograms per litre. Grapevine cell cultures transformed with HsCYP1B1 produced about 20 mg/L culture of piceatannol, displaying a sevenfold increase in relation to wild‐type cultures, and reaching an extracellular distribution of up to 45% of total production. The results obtained demonstrate the feasibility of this novel system for the bioproduction of natural and more bioactive resveratrol derivatives and suggest new ways for the improvement of production yields.     

核基质结合区对转爬山虎芪合酶基因烟草中产生白藜芦醇的作用

采用核基质结合区(MARs)来提高转芪合酶基因(STS)烟草(Nicotianatabacum L.)中白藜芦醇产物的含量.MARs是细胞中能与核基质特异紧密结合的DNA片段,体外结合实验表明克隆自酵母的MARs序列能特异地与烟草核基质结合.芪合酶是白藜芦醇生物合成中的关键酶,用RT-PCR方法从川鄂爬山虎(Parthenocissus henryana(Hemsl.)Diels et Gilg)中克隆了与葡萄芪合酶基因有较高同源性的芪合酶编码区,将其置于CaMV35SΩ强启动子下,分别构建两侧带有MARs及不含MARs序列的表达载体,通过农杆菌介导转化烟草.Northern blot及HPLC等分析表明STS基因已整合至烟草染色体中并正常转录,且表达的外源芪合酶在烟草中可催化其底物合成白藜芦醇产物.与对照相比,MARs的存在使转芪合酶基因烟草中白藜芦醇的含量平均提高了约一倍.MARs在转芪合酶基因植物中的应用也为获得抗病性更强、白藜芦醇含量更高、更保健的转基因果蔬的研究奠定了基础.     

Resveratrol acts as a natural profungicide and induces self-intoxication by a specific laccase

The grapevine (Vitis) secondary metabolite resveratrol is considered a phytoalexin, which protects the plant from Botrytis cinerea infection. Laccase activity displayed by the fungus is assumed to detoxify resveratrol and to facilitate colonization of grape. We initiated a functional molecular genetic analysis of B. cinerea laccases by characterizing laccase genes and evaluating the phenotype of targeted gene replacement mutants. Two different laccase genes from B. cinerea were characterized, Bclcc1 and Bclcc2. Only Bclcc2 was strongly expressed in liquid cultures in the presence of either resveratrol or tannins. This suggested that Bclcc2, but not Bclcc1, plays an active role in the oxidation of both resveratrol and tannins. Gene replacement mutants in the Bclcc1 and Bclcc2 gene were made to perform a functional analysis. Only Bclcc2 replacement mutants were incapable of converting both resveratrol and tannins. When grown on resveratrol, both the wild type and the Bclcc1 replacement mutant showed inhibited growth, whereas Bclcc2 replacement mutants were unaffected. Thus, contrary to the current theory, BcLCC2 does not detoxify resveratrol but, rather, converts it into compounds that are more toxic for the fungus itself. The Bclcc2 gene was expressed during infection of B. cinerea on a resveratrol-producing host plant, but Bclcc2 replacement mutants were as virulent as the wild-type strain on various hosts. The activation of a plant secondary metabolite by a pathogen introduces a new dimension to plant-pathogen interactions and the phytoalexin concept.