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

Journal of Experimental Botany, May 2011

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.

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

Weirong Xu 0 1 2 3 Yihe Yu 0 1 2 3 Qi Zhou 0 1 2 3 Jiahua Ding 0 1 2 3 Lingmin Dai 0 1 2 3 Xiaoqing Xie 0 1 2 3 Yan Xu 0 1 2 3 Chaohong Zhang 0 1 2 3 Yuejin Wang 0 1 2 3 0 Key laboratory of Agricultural Molecular Biology of Shaanxi Province, Northwest A & F University , Yangling, Shaanxi 712100 , China 1 Key Laboratory of Horticulture Plant Germplasm Utilization in Northwest China, Ministry of Agriculture , Yangling, Shaanxi 712100 , China 2 Abbreviations: ABA , abscisic acid; ABRE, ABA-responsive element; DSRE, defence- and stress-responsive element; ERE, ethylene-responsive element; GA, gibberellin; GARE, gibberellin-responsive element; HRE, hormone-responsive element; HSE, heat stress-responsive element; MeJA, methyl jasmonate; PM, powdery mildew; STS , stilbene synthase. a The Author [2011]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions , please 3 College of Horticulture, Northwest A & F University , Yangling, Shaanxi 712100 , China 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. Introduction Resveratrol is a well-known plant-derived polyphenolic phytoalexin that normally functions as an antimicrobial compound in plants, yet also shows significant healthpromoting effects in animal models, including anti-oxidative, anticancer, anti-inflammatory, neuroprotective, and antiviral properties (Jang et al., 1997; Bernhard et al., 2000; Hsieh et al., 2008). Of the two geometric isomers of resveratrol, cis- and trans-, trans-resveratrol is considered to be more biologically active than cis-resveratrol (Orallo, 2006). Transresveratrol was originally identified as the active ingredient in the roots of the oriental medicinal plant Polygonum cuspidatum (Ko-jo-kon), which has been used for the treatment of a variety of disease conditions such as inflammation, liver disease, and heart disease. Thus far, resveratrol has been identified in >70 plant species (Bavaresco et al., 1999) including grapevine, peanut, rhubarb, false hellebores, blueberries, knotweed, and pine. In nature, resveratrol is synthesized via the well-characterized phenylalanine/polymalonate pathway, the last step of which is catalysed by stilbene synthase (STS). This enzyme belongs to the type III group of the polyketide synthase enzyme superfamily, and is encoded by a multigene family. STS converts one molecule of p-coumaroyl-CoA and three molecules of malonyl-CoA into 3,4,5-trihydroxystilbene or resveratrol (Rupprich and Kindl, 1978). The first grapevine STS was cloned from Vitis vinifera cv. Optima, and functionally characterized in Escherichia coli (Melchior and Kindl, 1990). Recently, 43 genes encoding STS in grapevine have been identified, and 20 of these genes are expressed in response to a pathogen attack, and are therefore considered functional (Jaillon et al., 2007). Grapevine (V. vinifera L.), an agriculturally and economically important fruit crop grown worldwide, is a valuable source of functional compounds, including the intensively studied resveratrol. However, most of the premium V. vinifera cultivars are highly susceptible to powdery mildew (PM) caused by an obligate biotrophic fungus, Uncinula necator (Schw.) Burr. In contrast, some other Vitis species, including Vitis labrusca, Vitis rupestris, Vitis aestivalis, and Muscadinia rotundifolia were reported to have co-evolved with U. necator and to possess various levels of resistance to the pathogen (Boquet et al., 1992). The current research on PM resistance is centred mainly on quantitative trait loci (Pauquet et al., 2001; Barker et al., 2005) and physical barrier-associated defence mechanisms (Ficke et al., 2004). A recent genomewide transcriptome analysis indicated that disease-resistant V. aestivalis responded weakly to PM, whereas the genes encoding key defence components in disease-susceptible V. vinifera were significantly up-regulated in response to PM (Fung et al., 2008). These observations are of importance for further investigations of PM resistance mechanisms in grapevine, which would contribute to a rapid selection of resistant lines. Plants carry different layers of defence, from structural barriers and preformed antimicrobials to adaptive defence mechanisms that encompass race-specific and race nonspecific resistance (Gurr and Rushton 2005). The synthesis of the secondary metabolite, resveratrol phytoalexin, is considered to be one of the adaptive defence mechanisms against pathogen infection (Serazetdinova et al., 2005; Fung et al., 2008). An increasing amount of data shows that the introduction of Vitis STS into heterologous plant species can increase resistance to pathogens (Hain et al., 1993; Fettig and Hess, 1999; Hipskind and Paiva, 2000; Zhu et al., 2004; Jeandet et al., 2010). On the other hand, the choice of the promoter for STS expression is a critical factor for induced resistance by stilbenoids. The promoter most commonly used to overexpress STS genes in a wide range of plants is the strong constitutive CaMV35S promoter (Fischer et al., 1997; Hipskind and Paiva, 2000; Kobayashi et al., 2000; Giorcelli et al., 2004; Giovinazzo et al., 2005; Yu et al., 2005; Liu et al., 2006; Morelli et al., 2006; Yu et al., 2006; Nicoletti et al., 2007; Schwekendiek et al., 2007; Fan et al., 2008; Delaunois et al., 2009). In most cases, this constitutive expression of enzymes involved in the production of antimicrobial metabolites and constitutive activation of disease resistance pathways under the control of CaMV35S promoter, may trigger deleterious side effects that reduce plant vigour and yield (Osusky et al., 2004), by interfering with regeneration, growth, or reproduction of the target plants (Gatz and Lenk, 1998), or by increasing susceptibility to other pathogens (Kim et al., 2006). One solution to these potential problems is the application of pathogen-inducible promoters, as they can eliminate any detrimental effects on growth and development owing to unwanted transgene expression in disease-free conditions (Gurr and Rushton 2005). However, a relatively small number of STS promoters have been extensively studied and only one grapevine Vst1 promoter derived from a disease-susceptible cultivar of V. vinifera cv. Optima, has been successfully used to engineer disease resistance (Hain et al., 1993; Thomzik et al., 1997; StarkLorenzen et al., 1997; Zhu et al., 2004). Recently, the analysis of the role of stilbene induction in artificially infected grapevine leaves revealed that stilbene induced in resistant cultivars does not have the same degree of toxicity against PM infection as it does in susceptible species (Schnee et al., 2008). It is therefore intriguing to question whether the functions of STS genes derived from resistant and susceptible grapevine germplasms, and the molecular process regulating their expression differ significantly in defence reactions to pathogens. Chinese native wild Vitis species represent a valuable genetic resource for grapevine disease resistance breeding (Wang et al., 1995), and particularly the accession Baihe-351 is a highly PM-resistant germplasm, as well as a valuable resource of STS genes. In our previous studies, we demonstrated the isolation of STS cDNA sequences from Chinese wild Vitis pseudoreticulata (Wang et al., 2007) and their expression in susceptible V. vinifera cv. Thompson Seedless (Fan et al., 2008). More recently, we characterized the novel STS promoter from highly PM-resistant Chinese wild V. pseudoreticulata (Xu et al., 2010). In the present study, we focus on the STS gene transcriptional changes associated with U. necator in both resistant Chinese wild V. pseudoreticulata and two susceptible cultivated grapevines, V. vinifera cvs. Carignane and Thompson Seedless. We conducted a comparative analysis of the STS gene genomic sequences and 5#-flanking region identified in Chinese wild V. pseudoreticulata with that of two cultivars. Moreover, we investigated the effects of Alternaria alternata, methyl jasmonate (MeJA), and wounding on these promoter activities. We also analysed the function of the genes encoding STS, in combination with their native promoter in tobacco plants, with the objective of utilizing the identified gene constructs to increase tolerance to pathogens in susceptible plants and/or improve the resveratrol content of food products in the future. Materials and methods Plant materials Chinese wild V. pseudoreticulata accession Baihe-35-1, and V. vinifera cvs. Carignane and Thompson Seedless were cultivated in the Grape Repository of Northwest A & F University, Yangling, Shaanxi, China. Leaves of the three Vitis genotypes were inoculated with U. necator and sampled at each inoculation period of 0, 24, 48, 72, 96, 120, and 144 h. The U. necator-infected leaves were collected from field-grown V. vinifera cv. Cabernet Sauvignon plants, and PM inoculations were carried out in the three Vitis genotypes according to the method previously described (Wang et al., 1995). Six-week-old Nicotiana tabacum cv. NC89 plants were grown in an environmental growth chamber under controlled conditions of 16 h light (12 000 lux)/8 h dark, 65% relative humidity, and a 25 C (day)/21 C (night) temperature cycle. Reverse transcription (RT)-PCR determination of STS gene expression The U. necator-inoculated grapevine leaves were sampled at different time points, and total RNA was extracted from the collected samples by the LiCl precipitation method (Reid et al. 2006). First-strand cDNA was prepared from 5 lg of total RNA in a 20-ll final volume using the oligo(dT) primer and other standard components included with the PrimeScript reverse transcriptase kit (Takara Biotechnology, Dalian, China). The resulting cDNA served as template for PCR amplification of either 1.1 kb of STS gene or 0.2 kb of grapevine elongation factor 1c (EF1c, AF176496), as an internal control. The following primers were used for RT-PCR amplification: 5#-ATGGCTTCAGTTGAGGAA AT-3# and 5#-TTAATTTGTAACCATAGG-3# for STS, 5#-GCGGGCAAGAGATACCTCAA-3# and 5#-TCAATCTGTCTAGGAAAGGAAG-3# for EF1c. Three independent PCR reactions were carried out for each gene, and similar results were obtained. The amplification products were analysed by electrophoresis on a 1% (w/v) agarose gel, quantified using Bio Imaging Systems software (Syngene, Cambridge, UK), and cloned into pMD-19 vector (Takara). Sequencing of the clones containing STS at different time points was performed to determine the amplified sequences (Sangon Biotech Co., Ltd, Shanghai, China). STS genomic clones and sequence analysis Primers specific for full-length STS cDNA were amplified from the genomic DNA of three Vitis genotypes using LA Taq polymerase, incorporating a BamHI site in the forward primer (5#-TAGGATCC ATGGCTTC AGTTGAGGAAAT-3#) and SpeI in the reverse primer (5#-GCACTAGTTTAATTT GTAACCATAGG-3#) to facilitate future cloning. A mixture of cDNAs collected at different inoculation time points from V. pseudoreticulata was used as control. The amplified DNA fragments were cloned to pMD-19 vector, and four resulting plasmids were first subjected to an assay using restriction enzymes BamHI and SpeI, and then sequenced. Homology searches were conducted in the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) database. Chromosomal locations of STS genes were predicted using the BLAT server at the Genoscope Genome Browser (http:// www.genoscope.cns.fr//cgi-bin/vitis/webBlat ). A phylogenetic tree was generated from the aligned deduced amino acid sequences using the maximum-likelihood method and 100 bootstrap replicates as implemented in the phylogeny analysis program, available in the Phylogeny.fr platform (Dereeper et al., 2008). Promoter isolation, cis-regulatory element prediction, and promoterGUS fusion constructs The isolation of STS promoters from V. vinifera cv. Carignane and V. vinifera cv. Thompson Seedless genomes was performed in a procedure similar to that previously described for the VpSTS promoter (Xu et al., 2010).Two primers derived from the regions conserved in VvcSTS and VvtSTS (5#-AATCAGCATAATCAGACTGGTAGACACAGTGGTCG-3# and 5#-CTGTGCCAA TGGCTAGGATGGTGGCCGGACCCTTGG-3#), were designed to amplify the upstream regions of VvcSTS and VvtSTS. The nested PCR fragments were cloned into pMD-19 vector, and sequenced, as well as analysed in the PlantCARE (http:// bioinformatics.psb.ugent.be/webtools/plantcare/html/) database (Lescot et al., 2002). To generate STS promoter-driven GUS constructs, the two promoter regions of V. vinifera cv. Carignane and V. vinifera cv. Thompson Seedless were amplified using primers PVvc-P-F (5#CGGGATCCGCTAATGATTCCAAATTCTAAAT-3#), PVvtP-F (5#-CGGGATCCCTCCAAGCTGGTAGTGGGATCAC-3#) and PVv-P-R 5#-TTGGTTCTGCAGGGATGCTAGATACGTAATGAA-3), which include BamHI and PstI sites in the flanking regions of the two promoter sequences. The amplified products were digested with BamHI and PstI, and cloned into the corresponding sites of pC0380::GUS (Xu et al., 2010) to generate PvvcSTS::GUS and PvvtSTS::GUS, respectively. The construct CaMV35S::GUS (Xu et al., 2010) was used as positive control. Generation of chimeric constructs A 1.7-kb BamHIPstI fragment containing the VpSTS promoter was isolated from PvpSTS::GUS, and inserted into the same sites of pCAMBIA1380 to generate pCPvpSTS. The VpSTS cDNA-1, cDNA-2, gDNA-1, and gDNA-2 fragments were amplified with primers containing BglII and SpeI sites and protective bases in the flanking regions. The PCR product was verified by DNA sequencing, digested with BglII and SpeI, and then ligated in BglIISpeI sites of pCPvpSTS to produce PvpSTS::VpSTS C-1, PvpSTS::VpSTS C-2, PvpSTS::VpSTS G-1, and PvpSTS::VpSTS G-2, respectively. The vector PvvcSTS::VvcSTS G-1 (or PvvcSTS::VvcSTS G-2) was created by the ligation of the BglIISpeI (or NcoIBglII) fragment including the 1.5-kb genomic sequence of the VvcSTS1 (or VvcSTS2) gene combined with the 2.0-kb VvcSTS promoter isolated from PvvcSTS::GUS into the corresponding sites of pCAMBIA1390. The PvvtSTS::VvtSTS G-1 (or PvvtSTS::VvtSTS G-2) vector contains a 1.5-kb NcoIBglII (or BglIISpeI) fragment of the VvtSTS1 (or VvtSTS2) genomic sequence under control of the VvtSTS promoter isolated from PvvtSTS::GUS by BamHI/PstI digestion and subcloned into the same sites of pCAMBIA1380. These expression constructs were then introduced into Agrobacterium tumefaciens strain GV3101 by electroporation. Transient expression assays in tobacco plants Agrobacterium-mediated transient transformation was conducted on fully expanded leaves that were still attached to the intact tobacco plants according to the protocol described previously (Sparkes et al., 2006). Biotic and abiotic treatments Fungal infection, wounding, or MeJA treatment, was conducted on tobacco 48 h after agroinfiltration. For fungal inoculation, A. alternata f. sp. lycopersici was grown on potato dextrose broth medium containing 3% sucrose at 28 C for 3 d. Spores were collected by flooding the dish with tap water containing 2% (w/v) sucrose, and filtered with a three-layer gauze. The concentration of conidia was adjusted to 13105 spores/ml, and the spore suspension was sprayed onto the abaxial epidermis of infiltrated leaves. Mockinoculated tobacco leaves were sprayed with 2% (v/v) sucrose solution only. Leaves were sampled at indicated times for quantitative real-time PCR analysis, or 24 h after A. alternata inoculation for GUS assay. Mechanical wounding was performed by creating three wounds along leaf veins using a sterile inoculating needle. The wounded tobacco leaves and controls were sampled for GUS assays 24 h after treatment. MeJA was dissolved in 0.1% ethanol to a final concentration of 100 lM and added to an aqueous solution containing the wetting agent Tween 20 at 0.05% (v/v). The MeJA solution was sprayed onto the upper surfaces of the infiltrated or wild-type tobacco leaves, whereas 0.05% Tween solution was sprayed onto the control leaves. The MeJA-sprayed and mocksprayed leaves were harvested for GUS assays 24 h after treatment. Real-time PCR analysis GUS transcript levels of N. tabacum leaves inoculated or uninoculated with A. alternata were quantified by two-step real-time PCR using an iCycler iQ5 thermal cycler (Bio-Rad, Hercules, CA, USA), and a SYBR Premix ExTM TaqII kit (Takara). Total RNA was isolated from tobacco leaf samples using an RNAsimple Total RNA kit (Tiangen Biotech Co., Ltd, Beijing, China), and was adjusted to 500 ng for reverse transcription. The reactions were carried out in triplicate in 96-well plates (25 ll/well) in a mixture containing 12.5 ll of 23SYBR Premix ExTM TaqII, 1 ll of 10 lM each primer, 1 ll of template cDNA and 9.5 ll of ddH2O. Twostep real-time PCR reactions were performed under the following conditions: 95 C for 10 s, followed by 40 cycles of denaturation at 94 C for 15 s, annealing and extension at 64.8 C for 30 s, and data acquisition at 64.8 C for 15 s for 60 cycles. Tobacco ubiquitin (Ubi, U66264) validated by a Ct difference statistical approach, showed good linearity with a correlation coefficient of 0.937, which was used as reference control. GUS transcript levels were calculated using the standard curve method and normalized against the tobacco ubiquitin gene. The following primers were used for real-time PCR amplifications: 5#-ATTATGCGGGCAACGTCTGGTATCAG-3# and 5#-CATCGGCTTC AAATGGCGTAGC-3# for GUS, 5#-ATGAACGCTGGCGGCATGCTTA-3# and 5#-AGATCTGC ATTCCTCCCCTCAGCTA-3# for Ubi. GUS activity assay Histochemical and quantitative GUS assays were carried out according to the procedure of Jefferson (1987). Fluorescence of the methyl umbelliferone products was quantified using a Hitachi 850 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). Protein concentrations were measured with the proteindye binding assay (Bradford, 1976) using a Nicolet Evolution 300 UV-VIS spectrophotometer (Thermo Electron Corp., Madison, WI, USA) with BSA as the standard. HPLC analysis Leaves of wild-type and transformed N. tabacum plants were inoculated with either A. alternata or mock inoculated, then incubated for 24 h. Trans-resveratrol was extracted and quantified using HPLC by measuring its maximum absorbance at a wavelength of 306 nm. Tobacco leaves (1.0 g) were collected and ground to a fine powder in liquid nitrogen, then transferred to a tube containing 15 ml of 80% methanol. Samples were extracted for 30 min by ultrasound-assisted extraction, followed by incubation at 4 C for 10 h. The samples were centrifuged for 15 min at 5500 rpm at room temperature and the supernatants were evaporated to a final volume of ;3 ml at 25 C using a rotary evaporator RE-52AA (Shanghai Jinpeng Analytical Instruments Co. Ltd, Shanghai, China) at 40% rotor speed. Three additional extractions were carried out with 10, 5, and 5 ml each of ethyl acetate. The ethyl acetate phases were combined, followed by three-time separation with 25 ml of 3% NaHCO3 and 25 ml of deionized water, respectively. The purified ethyl acetate phases were evaporated to dryness under a vacuum, resuspended in 3 ml of methanol, and stored at 80 C before analysis. The extracts were filtered through a 0.45 lm membrane filter and analysed in a Shimadzu LC-2010 HPLC system (Shimadzu Corp, Kyoto, Japan) equipped with an Agilent Zorbax SB-C18 column (5 lm, 4.63250 mm). Chromatographic separation was performed using a solvent system of water with 0.5% (v/v) formic acid (A) and acetonitrile (B), with a linear gradient of 1018% B over 08 min, 18% B over 810 min, 1825% B over 1015 min, 2535% B over 1518 min, 35% B over 1825 min, and 3570% B over 2530 min. The flow rate was maintained at 1 ml min 1 and the elution was monitored by using a photodiode array detector (200600 nm). HPLC grade trans-resveratrol (Sigma-Aldrich Inc., St Louis, MO, USA) was dissolved in methanol and used as standard sample. The concentrations of trans-resveratrol extracted were measured using the external standard method. Calibration curves were established by plotting the area of peaks against different concentrations of trans-resveratrol. Expression patterns of PM-responsive STS genes in both resistant and susceptible Vitis genotypes Changes in STS gene expression were investigated by the development of U. necator on inoculated leaves of V. pseudoreticulata, V. vinifera cv. Carignane, and V. vinifera cv. Thompson Seedless. In susceptible Vitis cultivars, no induction of STS transcript (0 h) from V. vinifera cv. Thompson Seedless (VvtSTS) was found in the mockinoculated leaves (Fig. 1B), and low level expression of STS transcript (0 h) from V. vinifera cv. Carignane (VvcSTS) was detected in the mock-inoculated leaves (Fig. 1C). Transcripts of VvcSTS increased dramatically from 0 to 48 h post-inoculation (hpi), decreased from 48 to 72 hpi, increased again from 72 to 96 hpi, and then decreased again from 96 to 144 hpi (Fig. 1B). A slight difference was observed in VvtSTS expression patterns compared with the VvcSTS gene, which showed stable mRNA accumulation from 48 to 96 hpi, and an increase in transcripts from 120 to 144 hpi (Fig. 1C). In contrast, mRNA accumulation of STS from Chinese wild V. pseudoreticulata (VpSTS) showed a specific expression profile (Fig. 1A), similar to our previous results (Xu et al., 2010). VpSTS is expressed early and strongly but with rapid degradation of the mRNA produced from 0 to 72 hpi, followed by rapid mRNA accumulation from 72 to 144 hpi. To explore and extend these observations, the single bands of all transcripts were cloned and 20 different recombinant clones were sequenced. All clones containing STS from V. vinifera cv. Carignane were identical and involved in one STS expression at different time courses, whereas the clones containing STS from V. vinifera cv. Thompson Seedless were also determined with one STS gene that was slightly different from the former. Interestingly, the expression of STS from Chinese wild V. pseudoreticulata accession Baihe-35-1 at different inoculation times included two STS genes. Genomic structure, chromosomal localization, and phylogenetic relationship of STS genes in grapevine Genomic sequences encoding STS genes derived from V. pseudoreticulata, V. vinifera cv. Carignane, and V. vinifera cv. Thompson Seedless have been determined. Restriction enzyme analysis, confirmed by sequencing of the genomic clones, revealed that the isolated clones of the STS gene in PM-resistant and PM-susceptible Vitis genotypes could be classified into two types (Fig. 2A). One, represented by three electrophoretic bands, was characterized with the same STS gene sequences in the four clones from each Vitis geneotype, and the corresponding sequence was designated gDNA-1 or cDNA-1. The other, represented by two electrophoretic bands, was also characterized with identical STS gene sequences, and designated gDNA-2. To further ascertain the two distinct types, two clones derived from each type were used for restriction enzyme analysis. The results indicated that a BamHI site was found in both VpSTS cDNA-1 (C-1) at position 678682 bp and VpSTS gDNA-1 (G-1) at position 10331037 bp, but a SpeI site was present in VvcSTS gDNA-2 (G-2) and VvtSTS gDNA-1 (G-1) at position 490494 bp. The presence of these two sites generated two types of electrophoresis band, which suggests some diversity in the genomic complexity of STS genes in the three Vitis genotypes. The genomic fragments encompassing VpSTS G-1 (GenBank accession no. FJ830329), VpSTS G-2 (GenBank accession no. FJ830330), VvcSTS G-1 (GenBank accession no. FJ851182), VvcSTS G-2 (GenBank accession no. FJ851183), VvtSTS G-1 (GenBank accession no. FJ851184) and VvtSTS G-2 (GenBank accession no. FJ851185), vary in size from 1534 to 1539 bp, but each consists of two exons split by one intron. A comparison of the six STS genes shows that they are 9499% homologous to the previously identified STS genes from grapevine plants at the nucleotide and amino acid levels. Homology searches of the genome sequence of V. vinifera cv. Pinot Noir clone PN40024 were conducted with the six cloned STS nucleotide sequences. The genome analysis revealed that their homologues in Pinot Noir (98.599.3% sequence similarity) were present as multiple copies, mapping on chromosome 16 (Fig. 2B). Moreover, the genetic distribution showed that VpSTS G-1 (VpseSTS1) and VvtSTS G-2 (VthoSTS2), and VpSTS G-2 (VpseSTS2) and VvcSTS G-1 (VcarSTS1), are clustered at two loci, which are positioned in the opposite direction to another locus, including VvcSTS G-2 (VcarSTS2) and VvtSTS G-1 (VthoSTS1). Similar distributions were also observed on the 13-kb fragment containing three linked STS genes (Wiese et al., 1994). Detailed analysis of STS-deduced amino acid sequences among Vitis plants (Fig. 3A) revealed that the motif GVLFGFGPGLT, which is the family signature sequence for stilbene and chalcone synthases (Fliegmann et al., 1992), is highly conserved without any deviations. However, a serine (S)-to-phenylalanine (F) exchange within the IPNSAGAIAGN motif, which is specific for STS (Wiese et al., 1994), was observed solely in the predicted amino acid sequence of the VpSTS G-2 gene. A phylogenetic tree of the putative six STS proteins together with those previously described in other Vitis species was constructed (Fig. 3B), and diverges into two major clusters. Cluster I comprises six STS members from which the six Vitis genotypes were derived; four were resistant to PM (V. labrusca, V. aestivalis cv. Norton, V. riparia and V. rotundifolia), and two were susceptible (V. vinifera cv. Optima and V. vinifera cv. Pinot Noir). Cluster II contains 12 STS members, of which five were derived from three PM-resistant Vitis genotypes (V. pseudoreticulata, V. riparia, and V. rotundifolia), and seven were derived from six PM-susceptible Vitis genotypes (V. vinifera cv. Carignane, V. vinifera cv. Thompson Seedless, V. aestivalis cv. Norton, V. vinifera cv. Pinot Noir, V. vinifera cv. Cabernet Sauvignon, and V. vinifera cv. Jing Yu). Structurally different STS upstream region in PMresistant Chinese wild V. pseudoreticulata To elucidate whether the differential expression patterns of VpSTS, VvcSTS, and VvtSTS genes correlate with the regulation of their promoters, we comparatively analysed genomic sequences upstream from VpSTS (GenBank accession no. FJ605484), VvcSTS (GenBank accession no. GU269272), and VvtSTS (GenBank accession no. GU269273). Compared with the 1772-bp VpSTS promoter sequence (Xu et al., 2010), the isolated upstream regions of VvcSTS and VvtSTS are 2024 and 2149 bp in length, respectively. Sequence alignment revealed that the VpSTS promoter was 53.0% and 51.7% identical to the VvcSTS and VvtSTS promoters, respectively, whereas 93.4% homology was found between the latter two. Detailed analysis of the cis-regulatory elements occurrring in three promoters revealed that these elements were classified into two groups according to their responsive functions: hormone-responsive elements (HREs), and defence and stress-responsive elements (DSREs). HREs consisted of abscisic acid (ABA)-responsive elements (ABRE), gibberellin (GA)-responsive elements (GARE motif and P box), auxin-responsive elements (AuxRR core), MeJA-responsive elements (TGACG motif), and ethylene-responsive elements (EREs) as shown in Table 1. DSREs consist of a variety of factors including a fungal elicitor-responsive element (Box-W1), a heat stressresponsive element (HSE), a low-temperature-responsive element (LTR), a MYB binding site (CCAAT box), a wounding-responsive element (WUN motif), a defence and stress-responsive element (TC-rich repeats), and Name of cis element Sequence Number of cis elements Function Reference V. pseudoreticulata V. vinifera cv. V .vinifera cv. Carignane Thompson Seedless a high transcription level-related element (5UTR Py-rich stretch) (Table 1). Cis-regulatory elements located on the VvcSTS promoter are identical to those on the VvtSTS promoter, except for an extra MYBHv1 binding site (CCAAT box). However, the VpSTS promoter displayed some key differences in the composition or distribution of putative cis-acting elements compared with the above two promoters (Fig. 4AC). Further comparison of the DSREs revealed that there are two W-box motifs in V. vinifera cv. Carignane and V. vinifera cv. Thompson Seedless STS promoters, as opposed to only one in the V. pseudoreticulata STS promoter. Of particular interest is a TC-rich repeat (ATTAGAGAAT), which is uniquely found at position 752 of the VpSTS promoter sequence. The TC-rich repeat sequence is opposite and complementary to the sequence ATTCTCTAAC reported in Table 1. Analysis of STS promoter activity in response to pathogen To investigate further whether the structurally different VpSTS promoter contributes to the differential transcriptional regulation of expression, the three promoterGUS fusion constructs (Fig. 5A) were analysed in an Agrobacterium-mediated transient tobacco expression system using quantitative real-time PCR assays. Apart from high pathogen-inducible activity for the VvtSTS promoter, GUS expression patterns are highly similar, and even identical, in the tobacco leaves transformed with either a PvvcSTS::GUS or a PvvtSTS::GUS chimeric construct (Fig. 5C, D). They also showed low and comparable basal expression levels among transformed tobacco leaves at 0 h, and then transiently increased in the A. alternata-inoculated leaves at 8 h. After 8 h of treatment, GUS transcripts of these two promoter constructs decreased to relatively low levels in inoculated plants compared with uninoculated plants. However, another peak in GUS mRNA levels in transformed tobacco leaves elicited with A. alternata occurred in VvcSTS and VvtSTS promoter constructs at 16 to 32 h. The VvcSTS and VvtSTS promoters responded strongly to A. alternata infection, and the highest level of their GUS transcripts accumulated at 40 h compared with other assayed time points. In contrast, GUS transcripts driven by the VpSTS promoter showed a unique kinetic and dynamic property in response to fugal infection (Fig. 5B). A basal GUS mRNA level even before inoculation was also detected in infiltrated tobacco leaves with VpSTS promoter constructs. In tobacco leaves transformed with the VpSTS promoter construct, GUS transcript accumulation displayed a partial similarity in grapevinePM interaction. In contrast to the early time stages (016 h), at 24 h after A. alternata inoculation, the VpSTS promoter construct showed strongly increased GUS transcript levels, followed by a gradually decreasing trend until the final sampling point at 48 h. Analysis of mockinoculated samples showed that in tobacco leaves infiltrated with the VpSTS promoter construct there was only a limited effect on GUS transcript levels at various time intervals (Fig. 5B). Verification of pathogen-responsive STS promoters by quantitative GUS assay The quantitative real-time PCR data showed kinetic and dynamic changes of promoter-driven GUS genes in response to A. alternata at the transcriptional level. In addition to translation levels, GUS activities derived from these promoters were also tested in the same transformed tobacco system. Six-week-old tobacco plants (Fig. 6A) were infiltrated with three promoter fusion constructs and cultured for 48 h, followed by inoculation with A. alternata. The responses of GUS genes under three promoters were initially analysed by histochemical GUS staining 24 h after pathogen inoculation. As shown in Fig. 6B, compared with levels in mock-inoculated leaves, GUS expression was greatly increased in A. alternata-inoculated leaves. None of the wild-type tobacco leaves inoculated with A. alternata or mock inoculated showed any detectable GUS expression, whereas high GUS activity was found in pathogen-inoculated or mock-inoculated leaves infiltrated with the CaMV35S::GUS construct. It is important to note that there were no obvious disease symptoms such as necrosis spots or mould layers on the surface of infected leaves 24 h after inoculation, but clearly inducible expression of the GUS gene was detected in the inoculated leaves with different promoter fusion constructs. Furthermore, the histochemical data were confirmed by GUS fluorogenic quantitative assay (Fig. 6C). GUS expression driven by the VvtSTS promoter in response to A. alternata was very high, with a 16.9-fold induction (one-side paired t test; P<0.01) over mock-inoculated leaves. In the leaves containing the VvcSTS promoter fusion construct, GUS expression induction was ;3.47-fold (P<0.01) higher within 24 h of pathogen infection compared with control leaves. Although VpSTS responded to A. alternata with only a 2.35-fold (P<0.01) higher inducibility over mock-inoculated leaves, relatively high GUS activity was clearly observed in uninoculated controls. It is also noteworthy that relatively low background expression level was observed in wild-type tobacco leaves infected by pathogen or mock infected, whereas no pathogen-induced GUS activity was found in either A. alternata-inoculated or mock-inoculated leaves transformed with the CaMV35S::GUS construct. Effects of wounding and MeJA treatments on STS promoter activity Comparative analysis of cis-regulatory elements of three STS promoters revealed that a WUN motif and an MeJAresponsive motif were present in the VvcSTS and VvtSTS promoters, but absent in the VpSTS promoter (Fig. 4, Table.1). Therefore, the effects of these two elements that might be involved in wound- and MeJA-responsive induction, were determined in three promoters. GUS assays displayed that both VvcSTS and VvtSTS were evidently induced by wounding treatment (Fig. 6D). VvtSTS promoterdriven GUS activity was ;2.81-fold (P<0.01) higher than in unwounded leaves. The response of the VvcSTS promoter to the wounding treatment was lower than that of the VvtSTS promoter, with wounding-inducible GUS activity being only 1.65-fold (P<0.01) higher compared with controls. In contrast, mechanical wounding did not induce but rather repressed GUS activity in the VpSTS promoter. With regard to MeJA treatment, GUS expression was detected in all MeJA-sprayed and mock-treated leaves 24 h after MeJA treatments (Fig. 6E). The VpSTS promoter was slightly activated by MeJA treatment with a 1.19-fold (P<0.5) higher inducible GUS activity compared with controls. VvcSTS promoter-driven GUS expression increased by ;2.02-fold (P<0.01) in MeJA-treated leaves, while VvtSTS promoter induction was induced 3.52-fold (P<0.01) relative to mock-treated leaves. In both wounding and MeJA treatments, wild-type leaves and those transformed with the CaMV35S::GUS construct showed no obvious inducible GUS activity compared with controls (Fig. 6D, E). Functional characterization of transiently expressed STS in tobacco To further investigate the possibility that these putative genes are functional, we analysed the production of stilbenoids in tobacco lines transiently transformed with different constructs (Fig. 7A). Using such transformed tobacco lines, we specifically quantified the resultant trans-resveratrol after A. alternata inoculation by HPLC. As shown in the chromatograms, no trans-resveratrol was detected in extracts from wild-type tobacco leaves that were either mock inoculated (Fig. 7B) or inoculated with A. alternata (Fig. 7C), whereas trans-resveratrol accumulation at various levels was observed in transformed lines, e.g. in PvpSTS::VpSTS G-2 transformed tobacco that was mock inoculated (Fig. 7D) or inoculated with A. alternata (Fig. 7E). Analysis of transresveratrol content revealed that A. alternata elicitation of resveratrol production was much stronger than in controls. The maximum content of trans-resveratrol with 2.81 lg g 1 fresh weight (FW), was detected in the transformed tobacco leaves infiltrated with the PvpSTS::VpSTS G-2 construct 24 h after infection, followed by the PvvcSTS::VvcSTS G-1, PvvcSTS::VvcSTS G-2, PvvtSTS::VvtSTS G-2, PvvtSTS::VvtSTS G-1, PvpSTS::VpSTS C-1, PvpSTS::VpSTS C-2, and PvpSTS::VpSTS G-1 constructs (Fig. 7F). In this study, we compared the expression of STS genes and their promoters in three grapevine genotypes, V. pseudoreticulata, V. vinifera cv. Carignane, and V. vinifera cv. Thompson Seedless, of which the former is highly resistant to U. necator and the latter two are susceptible to infection. Comparative analysis of STS gene transcripts from these three Vitis genotypes revealed that the expression patterns of PM-induced STS genes in PM-resistant V. pseudoreticulata are significantly different from those of PM-susceptible V. vinifera, which suggests some specific physiological roles for Chinese wild V. pseudoreticulata in response to U. necator attack. Analysis of these STS genomic sequences and promoter activities combined with transient expression in a heterologous system allowed us to obtain functional characteristics of them in a short time. We provide evidence that the structurally specific promoter responsible for the PM-associated STS gene expression patterns may play an important role in the novel expression patterns in resistant Chinese wild V. pseudoreticulata. PM-induced STS expression is different in V. pseudoreticulata and V. vinifera Expression of the STS gene leading to two waves in the accumulation of the corresponding mRNAs has been well documented in grapevine cell suspensions induced with cell walls of Botrytis cinerea (Liswidowati et al., 1991) or Phytophthora cambivora (Wiese et al., 1994), or in UVirradiated leaves grown in vitro (Borie et al., 2004). Similar STS transcript profiles after ozone exposure in Scots pine (Pinus sylvestris) have also been observed (Zinser et al., 2000). Here we identify a novel PM-induced STS expression pattern in a resistant accession of Chinese wild V. pseudoreticulata. The expression of VpSTS is down-regulated by PM infection at the early stage, but up-regulated in late stages, displaying a unique expression pattern (Fig. 1A). Moreover, a highly expressed VpSTS transcript was detected in mock-inoculated leaves (0 h), which may possess a higher level of resistance to U. necator. The transcriptional changes in STS expression found in resistant V. pseudoreticulata may point to the post-infection resistance mechanisms described in other wild Vitis species, including the accumulation of reactive oxygen species, pathogenesis-related proteins, antimicrobial compounds, peroxidases, and hypersensitive response activation (Kortekamp et al., 1998; Kortekamp and Zyprian, 2003; Kortekamp, 2006; Allegre et al., 2007; Unger et al., 2007; Diez-Navajas et al., 2008). It is therefore, important to understand whether the early and significant VpSTS transcript accumulation in leaves before pathogen attack is an important defence recognition signal marker, and contributes to the early interruption of PM development in this resistant Vitis genotype, thus conferring total resistance. In the susceptible cultivars, VvcSTS transcript accumulation showed the typical profile with two peaks (Fig. 1B), whereas VvtSTS had a slightly different transcription profile (Fig. 1C) from the expression pattern of VvcSTS. We therefore assume that this kind of accumulation pattern in susceptible grapevine varieties may act as a passive defence reaction to cope with pathogen attack, and may confer only partial resistance. In addition, whether the two VpSTS-expressing genes observed in Chinese wild V. pseudoreticulata are functionally equivalent in the secondary metabolite pathway or whether they are compatible with either transcriptionally active or repressed genes, remains to be determined. Novel transcriptional changes in the VpSTS gene are most likely caused by the structurally different VpSTS promoter Our results from the homology analysis of genomic nucleotide and deduced amino acid sequences demonstrated that the six cloned STS genes share highly homologous sequences, and possess similar gene structure. High-quality draft grapevine genome sequencing (Jaillon et al., 2007; Velasco et al., 2007) makes it possible to characterize the STS gene family and obtain further information on the six STS genomic clones obtained from different grapevine genotypes. By means of the informative genome analysis and phylogenetic data, we have been able to gain an understanding of the evolutionary lineages of STS genes. Although these grapevine STS genes formed two distinct clusters, there was no significant association between PMresistant and PM-susceptible Vitis genotypes. These data indicated that the differential expression patterns of PMresponsive STS genes could not be the main result of the sequence similarity, presumably due to the high homology of genomic sequences among different Vitis genotypes. It is possible that the differential responsiveness of STS gene expression following PM infection is associated with differences in the upstream regulatory regions of the STS genes. To test this possibility, we compared the upstream regions of STS genes isolated from three Vitis genotypes. Surprisingly, the VpSTS promoter showed low sequence similarity to the other promoters, and the constitution and distribution of cis-elements within the VpSTS promoter were significantly different from those of the two cultivars of V. vinifera. The W box in the three promoters has been previously described as the binding site for WRKY transcription factors that function in positively or negatively regulating the transcriptional plant defence response (Eulgem and Somssich, 2007). However, the combined effect of two W-box motifs in the induction of the plant defence pathway remains to be elucidated. Interestingly, V. pseudoreticulata only contains one TC-rich repeat in its STS promoter, compared with its counterparts. Such sequences were previously described in tobacco as cisregulatory elements involved in defence and stress responsiveness (Diaz-De-Leon et al., 1993). It is possible that genetic variation in these putative elicitor-responsive elements and other stress-responsive elements may differentially regulate transcription in V. pseudoreticulata and V. vinifera. This idea is consistent with previous observations that the promoter sequence of the Pinus sylvestris gene PST-5 is distinctly different from other STS genes, and leads to a different expression profile (Preisig-Muller et al., 1999). Similarly, two adjacent grapevine STS genes possess very similar coding regions, but differ substantially in their promoter regions, exhibiting striking differences in elicited grapevine cell cultures (Wiese et al., 1994). Taken together, our results indicate that the structurally different STS promoter sequence obtained from V. pseudoreticulata may be closely associated with the unique expression patterns of PM-induced STS genes. The specific VpSTS promoter is strictly regulated by pathogen Grapevine Vst promoter, which is isolated from susceptible V. vinifera cv. Optima, has been shown to be activated in response to biotic (Liswidowati et al., 1991; Bais et al., 2000; Keller et al., 2003) and abiotic stress, including UV irradiation (Leckband and Lorz 1998) and wounding treatment (Jeandet et al., 1997). However, a recent study by our research group found that the VpSTS promoter is only 56.4% identical to the Vst promoter (Xu et al., 2010), indicating that the features of the phenylpropanoid pathway may be functionally different. Hence, in the current study, we comparatively analysed whether the VpSTS promoter, and two STS promoters derived from susceptible V. vinifera cultivars, possess different regulation patterns in response to biotic and abiotic elicitations, particularly in pathogeninduced scenarios. Real-time PCR data suggested that GUS expression patterns were similar when driven by VvcSTS and VvtSTS promoters in a heterologous system involving A. alternata infection. However, these patterns differed significantly from that of the VpSTS promoter (Fig. 5B), which confirms its specfic function in differential expression patterns after elicitation with fungi. These transcriptional results were further confirmed by GUS histochemical and quantitative assays, in which similar expression patterns in response to A. alternata infection were displayed. Wounding led to a high induction of VvtSTS promoter activity, with the VvcSTS promoter also being activated by wounding treatment. In addition, similar results were found in both the VvtSTS and VvcSTS promoters with regard to MeJA responsiveness. These results indicate that mechanical wounding and MeJA treatments may function similarly to exogenous stimuli that elicit promoter activity. In contrast, the VpSTS promoter was induced by a downregulated expression pattern in response to wounding treatment, while MeJA treatment seemed to have no significant effect on the VpSTS promoter. The combined results indicate that the VpSTS promoter may be strictly regulated, being primarily responsive to pathogens but not responsive to stress factors. Hence, the VpSTS promoter may be a feasible candidate to drive the expression of elicitors in the hypersensitive response or to drive antifungal proteins to engineer fungal resistance in transgenic plant species. The combination of VpSTS G-2 and VpSTS promoter leads to increased production of trans-resveratrol Interactions between tobacco and fungal pathogen A. alternata have been well documented in identifying the genes involved in pathogen resistance (Shi et al., 2000; Brandwagt et al., 2002). We used this model system to investigate the role of the putative STS genes in the resveratrol biosynthetic pathway. Moreover, the successful application of the Agrobacterium-mediated transient expression technique for studying foreign gene expression (Kapila et al., 1997) allowed us to rapidly obtain the functional information for these genes. Resveratrol, the best-characterized stilbenoid, is the major product of STS enzyme activity in vitro (Yu et al., 2005). The first gene transfer experiment was described with the Arachis hypogea STS gene introduced into tobacco, leading to resveratrol accumulation after UV induction (Hain et al., 1993). It has also been shown that both grapevines and peanuts can accumulate elevated levels of this stilbene following pathogen inoculation and elicitor treatment (Schroder et al., 1988; Wiese et al., 1994). To provide further functional evidence for the six putative STS genes, we examined the expression levels of these genes under their native promoters in the same tissue under similar conditions. We observed that the cloned STS genes are functionally expressed in tobacco leaves and retain pathogen inducibility. Our HPLC analyses showed that trans-resveratrol was detected in different transformed tobacco leaves with variable expression levels. More importantly, we found that the highest comparative yields of trans-resveratrol were detected in PvpSTS::VpSTS G-2transformed tobacco leaves. Although quantitative GUS assays demonstrated that the pathogen-inducible activity of the VpSTS promoter was less effective than that of the VvtSTS promoter, a relatively high level of trans-resveratrol was detected in transformed tobacco leaves using a chimeric gene construct involving fusion of the VpSTS promoter and genomic DNA-2. A possible explanation for this discrepancy could be the co-regulation occurring between the genomic sequence itself and the cis-regulatory elements in the promoter region. However, we could not exclude that the enhanced effect may be the result of the novel sequence variation due to the Ser- to- Phe exchange in the STS conserved motif IPNSAGAIAGN in the predicted protein sequence of the VpSTS G-2 gene. The sequence context (IPNSAGAIAGN) around Ser250, is considered a relatively highly conserved region specific to STS, which distinguishes it from the IPDSAGAIAGD motif in chalcone synthases (Wiese et al., 1994). There are no reports on the relationship between the function of the gene encoding STS and the key mutation in this signature sequence. However, a mutation within the leucine-rich repeat domain of the Arabidopsis disease resistance gene RPS5 that can confer bacterial and downy mildew resistance was previously identified (Warren et al., 1998). Therefore, further research is required to investigate the relationship between the new mutation within the key motif and the function of the VpSTS G-2 gene with regard to its role in an active pathogen resistance process. In conclusion, the current findings of differential gene expression in different Vitis genotypes in response to U. necator infection expand our understanding of the molecular mechanisms in grapevinepathogen interaction. Moreover, a new grapevine stilbene synthase promoter from Chinese wild Vitis species, designated PvpSTS, has been characterized whose structure is significantly different from that of its counterparts in V. vinifera. The structurally different promoter directs a specific expression profile of the gene encoding stilbene synthase in V. pseudoreticulata. Our work also highlights linkages between gene and promoter, and provides a new pathogen-inducible promoter candidate for genetic engineering of plant disease resistance. Acknowledgements The authors thank Carlo Nuss (USDA, ARS, Beltsville, MD, USA) for critical reading of the manuscript. This work was supported by the National Natural Science Foundation of China for Functional analysis of stilbene synthase genes and its specific promoter isolated from Chinese wild Vitis pseudoreticulata (Grant no. 30971972). Allegre M, Daire X, Heloir MC, Trouvelot S, Mercier L, Adrian M, Pugin A. 2007. Stomatal deregulation in Plasmopara viticola-infected grapevine leaves. New Phytologist 173, 832840. Bais AJ, Murphy PJ, Dry IB. 2000. The molecular regulation of stilbene phytoalexin biosynthesis in Vitis vinifera during grape berry development (corrigendum, vol 27, 723). Australian Journal of Plant Physiologyogy 27, 425433. Baker SS, Wilhelm KS, Thomashow MF. 1994. The 5-region of Arabidopsis thaliana cor15a has cis-acting elements that confer cold-, drought- and ABA-regulated gene expression. Plant Molecular Biology 24, 701713. 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Weirong Xu, Yihe Yu, Qi Zhou, Jiahua Ding, Lingmin Dai, Xiaoqing Xie, Yan Xu, Chaohong Zhang, Yuejin Wang. Expression pattern, genomic structure, and promoter analysis of the gene encoding stilbene synthase from Chinese wild Vitis pseudoreticulata, Journal of Experimental Botany, 2011, 2745-2761, DOI: 10.1093/jxb/erq447