53 1. Introduction Grape (Vitis vinifera L.) is an economically important fruit crop, served fresh and used for wine production. Grape is one of the most studied fruit crops, given that the grape genome sequence is available (Jaillon et al., 2007; Velasco et al., 2007). Grape skin contains several phe- nolic compounds, such as anthocyanin, resveratrol, and catechin, which are important not only for resistance to biotic and abiotic stresses but also for berry qualities such as color, astringency, and human health benefits (Kader, 2002; Steyn, 2009). Resveratrol, a stilbenoid accumulat- ing in the grape berry, is a key compound in the “French paradox” (Renaud and De Lorgeril, 1992) and is attracting attention in medicine and food science. ATP binding cassette (ABC) transporters are well- known transporters responsible for secondary metabolite accumulation in plants (Yazaki, 2006). They form a large gene family and are found in all living organisms (Rea, 2007). Plants have much larger numbers of ABC trans- porters than animals or microorganisms: Arabidopsis and rice have more than 120 ABC proteins (Rea, 2007; Yazaki et al., 2009; Kretzschmar et al., 2011). ABC transporters have a transmembrane domain (TMD) and a nucleotide-binding domain (NBD) compris- ing ATP-binding Walker A and B motifs (Martinoia et al., 2002). ABC transporters are classified into eight subfami- lies (ABCA-H) according to their structure and sequence similarity. Half-size ABC transporters contain one TMD and one NBD, whereas full-size ABC transporters contain two repeats of the structure of half-size ABC transporters, two TMDs and two NBDs (Verrier et al., 2008). The substrate specificity of ABC transporters is broad, and plant ABC transporters have been reported to trans- port various compounds, such as secondary metabolites, heavy metals, lipids, chlorophyll catabolites, xenobiotics, and plant hormones (Rea, 2007; Yazaki et al., 2009). ABC transporters show different localizations, such as the plas- ma membrane, vacuole, ER, Golgi apparatus, mitochon- drion, and peroxisome; subcellular localizations of ABC transporters in the same subfamily are not always the same (Yazaki et al., 2009; Kretzschmar et al., 2011). The ABCG subfamily is a major ABC transporter sub- family. It contains both half-size transporters, called the Molecular cloning and characterization of ABCG/PDR- type ABC transporter in grape berry skin M. Suzuki 1, 2, M. Jasinski3,4, E. Martinoia5, R. Nakabayashi6, M. Suzuki6, K. Saito6, 7, K. Shiratake1 (*) 1 Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan. 2 National Institute of Vegetables and Tea Science, Minaminakane, Taketoyo, Aichi 470-2351, Japan. 3 Department of Biochemistry and Biotechnology, Poznań University of Life Sciences, Dojazd, 60-637 Poznań, Poland. 4 Institute of Bioorganic Chemistry, Polish Academy of Science, Noskowskiego 61-704 Poznań, Poland. 5 University of Zurich, Institute of Plant Biology, Zollikerstrasse CH-8008 Zurich, Switzerland. 6 RIKEN Center for Sustainable Resource Science, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan. 7 Graduate School of Pharmaceutical Sciences, Chiba University, Inohana, Chuo-ku, Chiba 260-8675, Japan. Key words: full-size ABCG transporter, gene expression, grape, resveratrol, UV. Abstract: Grape (Vitis vinifera L.) skin contains the phenolic compound resveratrol which is important not only for re- sistance to biotic and abiotic stresses but also for human health. However, little is known about resveratrol transport in plant cells. ABC (ATP binding cassette) transporters are well-known transporters responsible for secondary metabolite accumulation in plants. Previous reports speculated that the full-size ABCG transporter pleiotropic drug-resistant (PDR) is involved in resveratrol transport in fungi and plants. In this paper, all full-size ABCG transporters found in the grape genome database are listed and focus is placed on VvABCG44/VvPDR14 as a candidate resveratrol transporter. The full- length cDNA of VvABCG44 was cloned by RT-PCR using mRNAs extracted from grape berry skin. VvABCG44 expres- sion was induced by UV irradiation, and the expression pattern of VvABCG44 in various grape organs was similar to that of stilbene synthase (STS), a key enzyme in resveratrol synthesis. Resveratrol content in grape berry skin increased after UV irradiation. These results suggest that VvABCG44 functions as a resveratrol transporter in grape. Adv. Hort. Sci., 2014 28(2): 53-63 (*) Corresponding author: shira@agr.nagoya-u.ac.jp Received for publication 31 March 2014 Accepted for publication 16 June 2014 54 white-brown complex (WBC) subfamily, and full-size transporters, called the pleiotropic drug-resistant (PDR) subfamily. One of the best studied full-size ABCG sub- family members is yeast PDR5 (Lamping et al., 2010; Prasad and Goffeau, 2012). PDR5 is an exporter in yeast plasma membrane and is associated with multidrug re- sistance (Decottignies and Goffeau, 1997; Golin et al., 2007). In plants, full-size ABCG transporters have been reported to transport phytoalexins (Banasiak et al., 2013), abscisic acid (ABA) (Kang et al., 2010), strigolactone (Kretzschmar et al., 2012), and other compounds. A strain of the plant pathogenic fungus Botrytis cinerea, lacking a full-size ABCG transporter BcatrB, is sensitive to resveratrol (Schoonbeek et al., 2001). On the other hand, after treatment with an elicitor, cyclodextrin, grape culture cells accumulated resveratrol and full-size ABCG transport- er genes were induced in the cells (Zamboni et al., 2009). These results suggest that full-size ABCG transporters are associated with resveratrol transport in fungi and plants. In this study, we listed all full-size ABCG transporters (PDRs) found in the grape genome database and focused on VvABCG44/VvPDR14 as a candidate resveratrol trans- porter. We cloned the full-length cDNA of VvABCG44 and determined its gene expression in various organs and after UV irradiation. VvABCG44 expression was induced by UV irradiation and the expression pattern of VvABCG44 in various grape organs was similar to that of stilbene syn- thase (STS), a key enzyme in resveratrol synthesis. To the best of our knowledge, this is the first report of an ABCG transporter in grape. 2. Materials and Methods Plant material and treatments Vitis vinifera “Pinot Noir” was harvested in the vine- yards of the AZUMI Apple Corporation in Nagano Pre- fecture and of Nagoya University in Aichi Prefecture, Ja- pan. For molecular cloning and gene expression analysis, young leaves, mature leaves, tendrils, stems, seeds, pulp, and berry skin were harvested in June and July. For mo- lecular cloning, the skin of the berries after UV irradiation was used. For UV irradiation and ABA treatment analyses, the grape berry clusters were harvested in June and July, before the veraison stage. UV irradiation and ABA treat- ment were performed as described below. Berry clusters were irradiated using a UV-C lamp (253.7 nm, GL-15, TOSHIBA, Japan) at a 50-cm distance for 1 h. Control samples (dark) were covered with a box and placed beside the sample receiving UV irradiation. For RNA extraction, the skin of the berries was collected immediately after UV irradiation. For measurement of res- veratrol content, after UV irradiation, berry clusters were maintained for 23 h in the dark at room temperature and then the skins of the berries were collected. Berry clusters were sprayed with 960 mM ABA con- taining 0.05% (v/v) Tween 20 and maintained in the dark at room temperature for 48 h. Control samples (water) were sprayed with water containing 0.05% (v/v) Tween 20 and placed beside the ABA-treated samples. After treat- ment, the skins of the berries were collected. Three biological replicates were assayed for each treat- ment. Identification of full-size ABCG transporter genes in the 12× version 1 of Vitis vinifera genome Full-size ABCG transporters in grape were searched with BLAST (Basic Local Alignment Search Tool) at NCBI (http://www.ncbi.nlm.nih.gov/) against the pre- dicted protein sequence dataset of the 12× version 1 (v1) of CRIBI (http://genomes.cribi.unipd.it/grape/) using the NpPDR1 protein sequence (CAC40990) as a query. Be- cause the average full-size ABCG protein comprises 1,400 amino acids (Rea, 2007), only sequences comprising more than 400 amino acids were taken into account. These no- menclatures were represented according to Çakır and Kılıçkaya (2013). The sequences corresponding to full- size ABCG transporters confirmed that there was at least one PDR motif. Molecular cloning of VvABCG44 The genome sequence corresponding to the partial cDNA sequence of a grape full-size ABCG transporter [tentative consensus sequence- TC76318, the grape gene index data- base (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain. pl?gudb=grape)], induced by cyclodextrin (Zamboni et al., 2009), was searched in the NCBI database (http://www.ncbi. nlm.nih.gov/) by BLAST. A genome sequence (accession number AM449250.2), provided by the IASMA Research Center (http://genomics.research.iasma.it/), was matched. The open reading frame (ORF) of the gene was predicted by Softberry (http://linux1.softberry.com/berry.phtml) and primers to amplify the entire ORF were designed (Table 1). Total RNA was extracted from the berry skin by hot borate method (Wan and Wilkins, 1994). The full-length cDNA of VvABCG44 was amplified using the PrimeScript High Fidelity RT-PCR kit (TaKaRa, Japan) according to the manufacturer’s instructions. Three motifs (Walker A, Walker B, and ABC signature) were confirmed according to van den Brûle and Smart (2002). TMD was predicted by PHD (NPS@) (Rost and Sander, 1993, 1994). Sequence data of VvABCG44 have been deposited in DDBJ under accession number AB910387. Gene expression analysis Total RNA from grape tissues was extracted by the technique described above. Total RNA was reverse-tran- scribed using a PrimeScript RT reagent Kit with gDNA eraser (perfect real-time) (TaKaRa) according to the man- ufacturer’s recommendations. Transcript levels were determined by quantitative RT- PCR using SYBER Premix EX Taq II (perfect real-time) (TaKaRa) and Thermal Cycler Dice Real Time System TP800 (TaKaRa) software ver. 3.00D. Primers for VvAB- CG44, STS, and actin are shown in Table 1. Reaction con- ditions for thermal cycling were as follows: after enzyme 55 activation at 95°C for 10 s, amplification was performed in a two-step PCR with 40 cycles of 5 s at 95°C for denatur- ation and 30 s at 60°C for annealing/extension. Transcript levels were calculated using a standard curve, and normal- ized against actin as described by Reid et al. (2006). All reactions were performed in triplicate with three biologi- cal replicates. Measurement of resveratrol content Extraction of resveratrol (CAS number 501-36-0) and its analysis using an LC-Q-TOF/MS system equipped with an ESI interface (HPLC: Waters Acquity UPLC system; MS: Waters Xevo G2 Q-Tof , Waters, Germany) were performed according to Tamura et al. (2014). Iden- tification, determination, and semi-quantification were compared with a 100 μM chemical reference standard. 10-camphorsulfonic acid was used as the internal con- trol. Three samples of biological replicates were divided into two aliquots and a total of six samples were analyzed for each treatment. 3. Results Zamboni et al. (2009) reported the partial sequence of a grape full-size ABCG gene that was induced by cyclodex- trin. To obtain the full-length cDNA clone of the gene, we searched the genome sequence corresponding to the gene and successfully amplified a full-length cDNA using prim- ers designed from the genome sequence data. The gene was designated VvABCG44 or VvPDR14. VvABCG44 had a 4,350 bp coding region and was predicted to encode a protein of 1,450 amino acids (Fig. 1A) with two TMDs and two NBDs (Fig. 1B). A phylogenetic tree of plant full-size ABCG transporters including VvABCG44 and all full-size ABCG transporters in Arabidopsis (Fig. 2) shows that NtPDR1 (BAD07483), NpPDR1 (CAC40990), and MtABCG10 (AES68070) are the closest homologues to VvABCG44. NtPDR1 (Crouzet et al., 2013) and NpPDR1 (Jasiński et al., 2001) were re- ported to transport diterpenes including sclareol, whereas MtABCG10 (Banasiak et al., 2013) transported isofla- vonoids. A close homologue of VvABCG44, SpTUR2 (O24367) (van den Brûle and Smart, 2002), transported sclareol. Other close homologues transport different com- pounds; AtABCG40 (AAF71978) (Kang et al., 2010) and PaPDR1 (JQ292812) (Kretzschmar et al., 2012) transport ABA and strigolactone, respectively. The substrate range of VvABCG44 homologues is very broad, and it is not easy to identify the substrate of VvABCG44. To determine the tissues in which VvABCG44 is ex- pressed, quantitative RT-PCR analyses were performed Table 1 - Primers used in this study Primer name Purpose Primer sequence(5’-3’) Take2_Forward Cloning CAC CAT GGC GAC GGC TGA AAT TTA TAR AG Take2_Reverse Cloning TCG CCT TTG GAA GTT CAA TGC VvPDR14_exp_Fw Gene expression TAG GAG TGG TTG CAG CTG TG VvPDR14_exp_Rv Gene expression TTT TGC TCC GTG TGA CTT CTT VvSTS_exp_Fw Gene expression GGG TCA CTA AGA GCG AGC AC VvSTS_exp_Rv Gene expression GCT CCT CAA GCA TTT CTT CG VvACT_Fw Gene expression TCC TGT GGA CAA TGG ATG GA VvACT_Rv Gene expression CTTGCA TCC CTC AGC ACC TT Fig. 2 - Phylogenetic tree of all full-size ABCGs in Arabidopsis, VvAB- CG44, and characterized full-size ABCGs from various plant species. NpPDR1 and NpPDR2 from Nicotiana plumbaginifo- lia, NtPDR1 and NtPDR3 from tobacco, SpTUR2 from Spiro- della polyrhiza, OsABCG36 and OsABCG43 from rice, Hv- ABCG31 from barley, PaPDR1 from Petunia, Lr34 from wheat, and GmPDR12 from soybean. The neighbor-joining tree was constructed with MEGA5 (Tamura et al., 2011). 56 (Fig. 3). The highest expression of VvABCG44 was ob- served in mature leaves, which was 9.6 times higher than that in young leaves. VvABCG44 expression in tendril and stem was higher than that in young leaves, but was not as high as that in mature leaves. VvABCG44 expression was relatively low in the grape berryVvABCG44, where it was highest in the skin and lowest in seeds. We also de- termined the gene expression of stilbene synthase (STS), a key enzyme in resveratrol synthesis. The expression pat- tern of STS in various grape organs is similar to that of VvABCG44 (Fig. 3), suggesting a relationship between VvABCG44 and resveratrol synthesis. A Fig. 1 - Nucleotide sequence, amino acid sequence, and topology of VvABCG44 A: Nucleotide sequence of VvABCG44 and the deduced amino acid sequence. Walker A motifs are underlined. ABC signature motifs are boxed. Walker B motifs are double underlined. Transmembrane domains are dotted-lined. Arrows indicate the primers for quantitative PCR analysis. B: Putative topology of VvABCG44. The protein is composed of two halves and each half harbors TMD (gray boxes) and NBD (dashed lines), which contains an ABC signature and Walker A and B motifs. B 57 Later, we determined the induction of VvABCG44 in grape berry skin by UV irradiation and by ABA treatment. Expression of VvABCG44 was upregulated 2.7 times by UV irradiation, and the STS gene was strongly induced by UV ir- radiation. Furthermore, resveratrol content in the grape berry skin increased 159 times after 23 h of incubation following UV irradiation (Fig. 4). These results suggest a relationship between VvABCG44 and resveratrol accumulation. On the other hand, the expression of VvABCG44 was not induced by ABA treatment in the grape berry skin (Fig. 5). 4. Discussion and Conclusions There are few reports of grape ABC transporters, al- though comprehensive analyses, such as transcriptomics and proteomics, report the expression of ABC transport- ers in grape. Recently, Çakır and Kılıçkaya (2013) iden- tified all ABC proteins using whole genome sequencing with 12× coverage and Francisco et al. (2013) identified an ABCC transporter of grape as a vacuolar anthocyanin Fig. 3 - Gene expression of STS andVvABCG44 in various grape or- gans [YL ( young leaves), ML; (mature leaves), stem, tendrils, seeds, pulp, and skin]. mRNA levels of STS and VvABCG44 were detected by quantitative PCR. Actin was used as an inter- nal control. Each value represents mean ± SE of three indepen- dent experiments. Fig. 4 - Expression of STS,VvABCG44 and resveratrol content in the grape berry skin after UV irradiation. mRNA levels of STS and VvABCG44 were detected by quantitative PCR. Actin was used as an internal control. Each value represents mean ± SE of three independent experiments. Resveratrol content was assayed by LC-ESI-Q-TOF/MS system in negative ion mode. Each value represents mean ± SE of six independent measurements. Fig. 5 - Expression of VvABCG44 in the grape berry skin after ABA treatment. mRNA level of VvABCG44 was detected by quan- titative PCR. Actin was used as an internal control. Each value represents mean ± SE of three independent experiments. 58 transporter. To the best of our knowledge, this is the only characterized full-size ABC transporter in grape. Why have such few full-size ABC transporters been studied? This is because of the difficulty in cloning full- length cDNA encoding full-size ABC transporters, par- ticularly full-size ABCG transporters. One of the reasons for this difficulty is the very large size (ca. 4,000 bp) of full-size ABCG transporter cDNAs. Another reason is the frequently observed low growth rate of Escherichia coli harboring full-size ABCG transporter cDNA. The reason for this low growth rate of E. coli is unclear. Therefore, few or no full-length cDNA clones encoding full-size ABCG transporters are found in public cDNA databases or resources, and should be cloned. Although 15 full-size ABCG transporters are present in Arabidopsis (van den Brûle and Smart, 2002), only five of them, AtABCG40, AtABCG37, AtABCG36, AtABCG32, and AtABCG30, have been characterized (Campbell et al., 2003; Lee et al., 2005; Ito and Gray, 2006; Kobae et al., 2006; Stein et al., 2006; Kim et al., 2007; Badri et al., 2009; Strader and Bartel, 2009; Kang et al., 2010; Kim et al., 2010; Růz̆ic̆ka et al., 2010; Bessire et al., 2011; Underwood and Somerville, 2013; Xin et al., 2013). In other plant species, only two full-size ABCG transporters (OsABCG36, OsABCG43) in rice (Moons, 2003; Oda et al., 2011) and five full-size ABCG transporters (NpPDR1, NpPDR2, NtPDR1, NtPDR3, and ABCG5/PDR5) in to- bacco family plants have been studied (Jasinski et al., 2001; Sasabe et al., 2002; Schenke et al., 2003; Ducos et al., 2005; Stukkens et al., 2005; Trombik et al., 2008; Bul- treys et al., 2009; Navarre et al., 2011; Bienert et al., 2012; Seo et al., 2012; Crouzet et al., 2013). In this study, we successfully cloned the full-length cDNA of VvABCG44 using the primers designed from the grape genome sequence data, using a high-grade enzyme for PCR reactions and optimized E. coli culture conditions (culture at lower temperature and in higher volume). This appears to be the first report of a grape full-size ABCG transporter. Two different data sets of grape genome sequences have been disclosed to the public. First, the Pinot Noir clone ENTAV115 was released by an Italian group, IASMA Re- search Center (http://genomics.research.iasma.it/) (Velasco et al., 2007). We used this information for cDNA clon- ing of VvABCG44. Second, the Pinot Noir-derived inbred PN40024 was sequenced by the French-Italian public con- sortium (Jaillon et al., 2007) (http://www.genoscope.cns. fr/externe/GenomeBrowser/Vitis/). The latter data set was updated from 8× to 12× and is now widely used. Recently the 12× version1(v1) has been made available by an Italian group, CRIBI (Grimplet et al., 2012) (http://genomes.cribi. unipd.it/grape/). Therefore, we used v1 to find all full-size ABCG transporters in the grape genome (Table 2). Fifteen and 23 full-size ABCG transporters were found in Arabidopsis (van den Brûle and Smart, 2002) and rice (Moons, 2008), respectively. In the grape genome data, we found 34 full-size ABCG transporters (Table 2, Fig. 6). This number is much larger than that in Arabidopsis and rice, suggesting a diversity of roles of full-size ABCG transporters in grape. As mentioned above, substrate and subcellular localization of full-size ABCG transporter cannot be determined from sequence similarity. However, full-size ABCG transporters are responsible for transport of secondary metabolites, plant hormones, cutins, and heavy metals (Fig. 2) and should have an important role in grape berry. Recent reports showed that plant full-size ABCG trans- porters, transport plant hormones or their precursors, such Fig. 6 - Phylogenetic tree of all full-size ABCGs in Arabidopsis and VvABCG44. A neighbor-joining tree was constructed with MEGA5 (Tamura et al., 2011). 59 Ta bl e 2 - Fu ll- si ze A B C G t ra ns po rt er s in g ra pe ( V iti s vi ni fe ra ). C ol um ns c on ta in t he V it is V in if er a 12 × V 1 ID , c hr om os om e lo ca tio n, p ro te in l en gt h, P D R s ig na tu re s, a nn ot at ed d es cr ip tio n by T ai r1 0, pr ot ei n ac ro ny m ( N am e) a nd V it is V in if er a 12 × V 0 ID f or e ac h ge ne a re g iv en 12 X V 1 ID C hr om os om e lo ca tio n Pr ot ei n PD R si gn at ur es ** D es cr ip tio n of T ai r1 0 Sa nc he z- H G N C ** * 1 2X V 0 ID C hr St ra nd St ar t En d LL LG PP G LD SS T G LD A R A - A G I c od e Sh or t d es cr ip tio n Su bf am ily n am e Su bf am ily n am e V IT _1 1s 00 16 g0 45 40 11 + 38 25 50 6 38 37 07 9 14 22 + + + AT 2G 26 91 0. 1 pl ei ot ro pi c dr ug re si st an ce 4 V vP D R 1 V vA B C G 31 G SV IV T0 10 15 45 60 01 V IT _1 1s 00 16 g0 45 90 11 - 38 91 36 7 38 98 72 7 14 78 + + - AT 1G 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 V vP D R 2 V vA B C G 32 G SV IV T0 10 15 46 10 01 V IT _0 9s 00 02 g0 35 50 9 + 32 29 01 2 32 42 58 2 64 9 + + - AT 1G 15 21 0. 1 pl ei ot ro pi c dr ug re si st an ce 7 V vP D R 3 V vA B C G 33 G SV IV T0 10 16 99 10 01 V IT _0 9s 00 02 g0 35 60 9 + 32 42 58 3 32 44 57 4 42 7 - - + AT 3G 16 34 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 V vP D R 4 V vA B C G 34 G SV IV T0 10 16 99 20 01 V IT _0 9s 00 02 g0 35 80 9 + 32 46 54 4 32 52 73 4 69 1 + + - AT 3G 16 34 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 V vP D R 5 V vA B C G 35 G SV IV T0 10 16 99 30 01 V IT _0 9s 00 02 g0 36 30 9 - 33 18 73 2 33 27 35 4 14 11 + + + AT 1G 59 87 0. 1 A B C -2 a nd P la nt P D R A B C -ty pe V vP D R 6 V vA B C G 36 G SV IV T0 10 16 99 80 01 V IT _0 9s 00 02 g0 36 40 9 - 33 28 21 2 33 36 62 6 14 94 + + + AT 3G 16 34 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 V vP D R 7 V vA B C G 37 G SV IV T0 10 16 99 90 01 V IT _0 9s 00 02 g0 53 60 9 - 50 99 14 6 51 14 84 9 14 90 + + + AT 1G 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 V vP D R 8 V vA B C G 38 G SV IV T0 10 17 18 40 01 V IT _0 9s 00 02 g0 53 70 9 - 51 15 50 5 51 22 76 0 14 22 + + + AT 1G 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 V vP D R 9 V vA B C G 39 G SV IV T0 10 17 18 50 01 V IT _0 9s 00 02 g0 54 00 9 - 51 46 16 7 51 60 09 0 15 65 + + + AT 1G 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 V vP D R 10 V vA B C G 40 G SV IV T0 10 17 18 70 01 V IT _0 9s 00 02 g0 54 10 9 - 51 69 12 5 51 76 18 9 14 38 + + + AT 1G 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 V vP D R 11 V vA B C G 41 G SV IV T0 10 17 18 80 01 V IT _0 9s 00 02 g0 54 90 9 - 52 16 53 6 52 23 50 7 12 80 + + + AT 1G 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 V vP D R 12 V vA B C G 42 G SV IV T0 10 17 19 60 01 V IT _0 9s 00 02 g0 55 30 9 - 52 59 17 5 52 66 31 4 14 60 + + + AT 1G 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 V vP D R 13 V vA B C G 43 G SV IV T0 10 17 19 80 01 V IT _0 9s 00 02 g0 55 60 * 9 - 52 81 29 6 52 88 25 5 14 55 + + + AT 1G 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 V vP D R 14 V vA B C G 44 G SV IV T0 10 17 20 10 01 V IT _0 9s 00 02 g0 55 70 9 - 52 94 43 7 53 01 67 7 14 55 + + + AT 1G 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 V vP D R 15 V vA B C G 45 G SV IV T0 10 17 20 20 01 V IT _0 9s 00 02 g0 55 90 9 - 53 16 14 4 53 23 42 0 14 55 + + + AT 1G 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 V vP D R 16 V vA B C G 46 G SV IV T0 10 17 20 40 01 V IT _0 9s 00 02 g0 56 00 9 - 53 36 09 0 53 43 69 9 14 51 + + + AT 1G 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 V vP D R 16 V vA B C G 46 G SV IV T0 10 17 20 40 01 V IT _0 5s 00 20 g0 06 80 5 + 25 48 76 2 25 57 92 1 14 38 + + + AT 3G 53 48 0. 1 pl ei ot ro pi c dr ug re si st an ce 9 V vP D R 17 V vA B C G 47 G SV IV T0 10 17 67 60 01 V IT _0 6s 00 04 g0 65 60 6 + 72 84 90 1 72 97 72 4 12 74 + + + AT 2G 29 94 0. 1 pl ei ot ro pi c dr ug re si st an ce 3 V vP D R 18 V vA B C G 48 G SV IV T0 10 24 74 30 01 V IT _1 4s 00 60 g0 04 70 14 + 43 97 01 44 86 96 14 49 + + - AT 3G 53 48 0. 1 pl ei ot ro pi c dr ug re si st an ce 9 V vP D R 19 V vA B C G 49 G SV IV T0 10 31 31 40 01 V IT _0 6s 00 61 g0 14 90 6 - 1, 9E +0 7 1, 9E +0 7 14 55 + + + AT 2G 36 38 0. 1 pl ei ot ro pi c dr ug re si st an ce 6 V vP D R 20 V vA B C G 50 G SV IV T0 10 31 37 70 01 V IT _0 6s 00 61 g0 14 80 6 - 1, 9E +0 7 1, 9E +0 7 14 61 + + + AT 2G 36 38 0. 1 pl ei ot ro pi c dr ug re si st an ce 6 V vP D R 21 V vA B C G 51 G SV IV T0 10 31 37 80 01 V IT _0 6s 00 61 g0 14 70 6 - 1, 9E +0 7 1, 9E +0 7 11 23 + + + AT 1G 66 95 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 1 V vP D R 22 V vA B C G 52 G SV IV T0 10 31 38 00 01 V IT _0 8s 00 07 g0 37 10 8 + 1, 8E +0 7 1, 8E +0 7 14 52 + + + AT 2G 36 38 0. 1 pl ei ot ro pi c dr ug re si st an ce 6 V vP D R 23 V vA B C G 53 G SV IV T0 10 33 80 40 01 V IT _1 3s 00 74 g0 06 60 13 - 88 18 11 3 88 27 87 4 14 73 + + - AT 2G 36 38 0. 1 pl ei ot ro pi c dr ug re si st an ce 6 V vP D R 24 V vA B C G 54 G SV IV T0 10 34 74 10 01 V IT _1 3s 00 74 g0 06 80 13 - 88 59 78 6 88 67 04 7 14 77 + + - AT 1G 66 95 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 1 V vP D R 25 V vA B C G 55 G SV IV T0 10 34 74 50 01 V IT _1 3s 00 74 g0 06 90 13 - 88 76 00 0 88 83 07 8 13 79 + + - AT 2G 36 38 0. 1 pl ei ot ro pi c dr ug re si st an ce 6 V vP D R 26 V vA B C G 56 G SV IV T0 10 34 74 60 01 V IT _1 3s 00 74 g0 07 00 13 - 88 97 68 8 89 04 96 5 14 81 + + - AT 2G 36 38 0. 1 pl ei ot ro pi c dr ug re si st an ce 6 V vP D R 27 V vA B C G 57 G SV IV T0 10 34 74 80 01 V IT _0 4s 00 08 g0 42 30 4 - 35 96 68 3 36 05 45 2 14 22 + + - AT 2G 26 91 0. 1 pl ei ot ro pi c dr ug re si st an ce 4 V vP D R 28 V vA B C G 58 G SV IV T0 10 35 71 50 01 V IT _0 4s 00 08 g0 47 90 4 - 42 27 01 7 42 34 51 8 14 37 + + + AT 1G 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 V vP D R 29 V vA B C G 59 G SV IV T0 10 35 78 00 01 V IT _0 4s 00 08 g0 48 20 4 + 42 58 54 1 42 65 24 1 14 20 + + + AT 1G 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 V vP D R 30 V vA B C G 60 G SV IV T0 10 35 78 40 01 V IT _0 4s 00 08 g0 48 30 4 + 42 82 42 5 42 86 09 4 76 4 + + - AT 1G 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 V vP D R 31 V vA B C G 61 G SV IV T0 10 35 78 50 01 V IT _0 4s 00 08 g0 48 40 4 + 42 86 95 4 42 95 63 1 11 20 - - + AT 1G 15 52 0. 1 pl ei ot ro pi c dr ug re si st an ce 1 2 V vP D R 32 V vA B C G 62 G SV IV T0 10 35 78 60 01 V IT _0 6s 00 80 g0 00 40 6 + 2E +0 7 2E +0 7 15 07 + + - AT 2G 36 38 0. 1 pl ei ot ro pi c dr ug re si st an ce 6 V vP D R 33 V vA B C G 63 G SV IV T0 10 36 18 40 01 * V IT _0 9s 00 02 g0 55 60 s ho w n w ith b ol d le tte rs is c or re sp on de d to V vA B C G 44 . ** PD R s ig na tu re s w er e re po rt ed b y va n de n B rû le a nd S m ar t ( 20 02 ). ** * S an ch ez -F er na nd ez a nd H G N C s ub fa m ily n am es w er e re po rt ed b y Ç ak ır a nd K ılı çk ay a (2 01 3) . 60 as ABA (Kang et al., 2010), strigolactone (Kretzschmar et al., 2012), and auxin (Ruzicka et al., 2010). One of the closest homologues of VvABCG44, AtABCG40, was re- ported to transport ABA (Kang et al., 2010) (Fig. 2). In grape berry, ABA accumulates just before maturation, called “veraison”, and ABA works as a trigger of berry maturation (Coombe and Hale, 1973; Davies et al., 1997). After veraison, both sugar and anthocyanin accumulate considerably in the grape berry (Coombe, 1992; Davies et al., 1997; Deluc et al., 2007). In this study, we determined VvABCG44 induction by ABA in the berry skin before ve- raison. However, no induction was observed (Fig. 5). Many plant full-size ABCG transporters have been sug- gested to be associated with biotic and abiotic stress resis- tance, particularly resistance against pathogens, and some have been observed to transport secondary metabolites that function as phytoalexins (Fig. 2). Therefore, VvABCG44 is considered to be associated with biotic and abiotic stress re- sistance and transports phytoalexins. VvABCG44 was first found as an elicitor-induced gene in grape culture cells and the induction of VvABCG44 corresponded to resveratrol ac- cumulation in the cells (Zamboni et al., 2009). It is known that UV irradiation induces resveratrol ac- cumulation in the grape berry skin (Douillet-Breuil et al., 1999; Adrian et al., 2000; Versari et al., 2001; Takayanagi et al., 2004). Therefore, we determined the effect of UV irradiation on VvABCG44 expression together with STS expression, a key enzyme for resveratrol synthesis and resveratrol accumulation. A clear induction of VvABCG44 by UV irradiation, though not large compared with that of STS expression and resveratrol accumulation, VvABCG44 was observed (Fig. 4). A similar pattern in the gene expres- sion of VvABCG44 and STS in various grape organs was also observed (Fig. 3). These results suggest a relationship between VvABCG44 and resveratrol accumulation. Close homologues of VvABCG44, NtPDR1 (Crouzet et al., 2013), NpPDR1 (Jasiński et al., 2001), MtABCG10 (Banasiak et al., 2013), SpTUR2 (van den Brûle and Smart, 2002), AtABCG40 (Kang et al., 2010), and PaPDR1 (Kretzschmar et al., 2012), transport diterpenoids, isofla- vonoids, ABA, and strigolactones (Fig. 2). These functions are surprising because their molecular structures are com- pletely different. Resveratrol is a compound belonging to the stilbenoids and both stilbenoids and flavonoids belong to the phenylpropanoids. The closest homologue of VvAB- CG44, MtABCG10, transports isoflavonoids (Banasiak et al., 2013). Although no direct evidence of stilbenoid trans- port activity of full-size ABCG transporter has been re- ported, it was observed that B. cinerea, lacking a full-size ABCG transporter, BcatrB, was more sensitive to resve- ratrol than the wild-type strain (Schoonbeek et al., 2001). This result suggests that BcatrB is an exporter of resvera- trol in B. cinerea. It can be concluded that VvABCG44 may work as a resveratrol transporter in grape. We attempted to express VvABCG44 in yeast lacking eight ABC transporters (Kang et al., 2010) and measure resveratrol transport activity. However, this attempt was unsuccessful because heterologous expression of plant full-size ABCG transporters is difficult not only in E. coli but also in yeast. We identified 34 full-size ABCG transporters in the grape genome, including VvABCG44. It is assumed they transport key compounds for plant growth and stress resis- tance, including secondary metabolites, plant hormones, cutins, and heavy metals, and have important roles in grape. Further study on full-size ABCG transporters in grape is warranted. Acknowledgements We thank Mr. Hiroya Saito and Mr. Chiharu Uchikata at AZUMI Apple Corporation for supplying grape berries. We also thank Mr. Yutaka Nishikawa and Mr. Kenji Wada of Mie Prefecture Agricultural Research Institute and Dr. Takafumi Tezuka of Nagoya University for advice on UV treatment of grape berries. We also thank Mr. Tetsuya Mori at RIKEN CSRS for technical assistance. This work was supported by the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Indus- try from Bio-oriented Technology Research Advancement Institution (BRAIN) and by Grant-in-Aids for Scientific Research from The Japan Society for the Promotion of Science (JSPS). References ADRIAN M., JEANDET P., DOUILLET-BREUIL A.C., TES- SON L., BESSIS R., 2000 - Stilbene content of mature Vitis vinifera berries in response to UV-C elicitation. - J. Agric. Food Chem., 48: 6103-6105. BADRI D.V., QUINTANA N., EL KASSIS E.G., KIM H.K., CHOI Y.H., SUGIYAMA A., VERPOORTE R., MARTI- NOIA E., MANTER D.K., VIVANCO J.M., 2009 - An ABC transporter mutation alters root exudation of phytochemicals that provoke an overhaul of natural soil microbiota. - Plant Physiol., 151: 2006-2017. BANASIAK J., BIALA W., STASZKÓW A., SWARCEWICZ B., KEPCZYNSKA E., FIGLEROWICZ M., JASINSKI M., 2013 - A Medicago truncatula ABC transporter belonging to subfamily G modulates the level of isoflavonoids. - J. Exp. Bot., 64: 1005-1015. BESSIRE M., BOREL S., FABRE G., CARRAÇA L., EFRE- MOVA N., YEPHREMOV A., CAO Y., JETTER R., JAC- QUAT A.C., MÉTRAUX J.P., NAWRATH C., 2011 - A member of the PLEIOTROPIC DRUG RESISTANCE family of ATP binding cassette transporters is required for the for- mation of a functional cuticle in Arabidopsis. - Plant Cell., 23: 1958-1970. BIENERT M.D., SIEGMUND S.E.G., DROZAK A., TROM- BIK T., BULTREYS A., BALDWIN I.T., BOUTRY M., 2012 - A pleiotropic drug resistance transporter in Nicotiana tabacum is involved in defense against the herbivore Man- duca sexta. - Plant J., 72(5): 745-757. BULTREYS A., TROMBIK T., DROZAK A., BOUTRY M., 2009 - Nicotiana plumbaginifolia plants silenced for the 61 ATP-binding cassette transporter gene NpPDR1 show in- creased susceptibility to a group of fungal and oomycete pathogens. - Mol. Plant Pathol., 10: 651-663. ÇAKIR B., KILIÇKAYA O., 2013 - Whole-genome survey of the putative ATP-binding cassette transporter family genes in Vitis vinifera. - PLoS ONE, 8:e78860. CAMPBELL E.J., SCHENK P.M., KAZAN K., PENNINCKX I.A.M.A., ANDERSON J.P., MACLEAN D.J., CAMMUE B.P.A., EBERT P.R., MANNERS J.M., 2003 - Pathogen- responsive expression of a putative ATP-binding cassette transporter gene conferring resistance to the diterpenoid sclareol is regulated by multiple defense signaling pathways in Arabidopsis. - Plant Physiol., 133: 1272-1284. CHEN G., KOMATSUDA T., MA J.F., NAWRATH C., POUR- KHEIRANDISH M., TAGIRI A., HU Y.-G., SAMERI M., LI X., ZHAO X., LIU Y., LI C., MA X., WANG A., NAIR S., WANG N., MIYAO A., SAKUMA S., YAMAJI N., ZHENG X., NEVOF E., 2011 - An ATP-binding cassette subfamily G full transporter is essential for the retention of leaf water in both wild barley and rice. - Proc. Natl. Acad. Sci. USA, 108: 12354-12359. COOMBE B.G., 1992 - Research on development and ripening of the grape berry. - Am. J. Enol. Vitic., 43: 101-110. COOMBE B.G., HALE C.R., 1973 - The hormone content of ripening grape berries and the effects of growth substance treatments. - Plant physiol., 51: 629-634. CROUZET J., ROLAND J., PEETERS E., TROMBIK T., DU- COS E., NADER J., BOUTRY M., 2013 - NtPDR1, a plasma membrane ABC transporter from Nicotiana tabacum, is in- volved in diterpene transport. - Plant Mol Biol., 82: 181-192. DAVIES C., BOSS P.K., ROBINSON S.P., 1997 - Treatment of grape berries, a nonclimacteric fruit with a synthetic auxin, retards ripening and alters the expression of developmental- ly regulated genes. - Plant Physiol., 115: 1155-1161. DECOTTIGNIES A., GOFFEAU A., 1997 - Complete inventory of the yeast ABC proteins. - Nat. Genet., 15: 137-145. DELUC L.G., GRIMPLET J., WHEATLEY M.D., TILLETT R.L., QUILICI D.R., OSBORNE C., SCHOOLEY D.A., SCHLAUCH K.A., CUSHMAN J.C., CRAMER G.R., 2007 - Transcriptomic and metabolite analyses of Cabernet Sau- vignon grape berry development. - BMC genomics, 8: 429. DOUILLET-BREUIL A.C., JEANDET P., ADRIAN M., BES- SIS R., 1999 - Changes in the phytoalexin content of various Vitis spp. in response to ultraviolet C elicitation. - J. Agric. Food Chem., 47: 4456-4461. DUCOS E., FRAYSSE S., BOUTRY M., 2005 - NtPDR3, an iron-deficiency inducible ABC transporter in Nicotiana taba- cum. - FEBS Lett., 579: 6791-6795. EICHHORN H., KLINGHAMMER M., BECHT P., TENHAK- EN R., 2006 - Isolation of a novel ABC-transporter gene from soybean induced by salicylic acid. - J. Exp. Bot., 57: 2193-2201. FRANCISCO R.M., REGALADO A., AGEORGES A., BUR- LA B.J., BASSIN B., EISENACH C., ZARROUK O., VI- ALET S., MARLIN T., CHAVES. M., MARTINOIA E., NAGYA R., 2013 - ABCC1, an ATP binding cassette protein from grape berry, transports anthocyanidin 3-O-Glucosides. - Plant Cell., 25: 1840-1854. GOLIN J., KON Z.N., WU C.-P., MARTELLO J., HANSON L., SUPERNAVAGE S., AMBUDKAR S.V., SAUNA Z.E., 2007 - Complete inhibition of the Pdr5p multidrug efflux pump ATPase activity by its transport substrate clotrimazole suggests that GTP as well as ATP may be used as an energy source. - Biochemistry, 46: 13109-13119. GRIMPLET J., VAN HEMERT J., CARBONELL-BEJERANO P., DÍAZ-RIQUELME J., DICKERSON J., FENNELL A., PEZZOTTI M., MARTÍNEZ-ZAPATER J.M., 2012 - Com- parative analysis of grapevine whole-genome gene predic- tions, functional annotation, categorization and integration of the predicted gene sequences. - BMC Res Notes, 5: 213. ITO H., GRAY W.M., 2006 - A gain-of-function mutation in the Arabidopsis pleiotropic drug resistance transporter PDR9 confers resistance to auxinic herbicides. - Plant physiol., 142: 63-74. JAILLON O., AURY J.M., NOEL B., POLICRITI A., CLEPET C., CASAGRANDE A., CHOISNE N., AUBOURG S., VI- TULO N., JUBIN C., VEZZI A., LEGEAI F., HUGUENEY P., DASILVA C., HORNER D., MICA E., JUBLOT D., POULAIN J., BRUYÈRE C., BILLAULT A., SEGURENS B., GOUYVENOUX M., UGARTE E., CATTONARO F., ANTHOUARD V., VICO V., DEL FABBRO C., ALAUX M., DI GASPERO G., DUMAS V., FELICE N., PAIL- LARD S., JUMAN I., MOROLDO M., SCALABRIN S., CANAGUIER A., LE CLAINCHE I., MALACRIDA G., DURAND E., PESOLE G., LAUCOU V., CHATELET P., MERDINOGLU D., DELLEDONNE M., PEZZOTTI M., LECHARNY A., SCARPELLI C., ARTIGUENAVE F., PÈ M.E., VALLE G., MORGANTE M., CABOCHE M., ADAM-BLONDON A.F., WEISSENBACH J., QUÉTIER F., WINCKER P., FRENCH-ITALIAN PUBLIC CONSOR- TIUM FOR GRAPEVINE GENOME CHARACTERIZA- TION, 2007 - The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. - Na- ture, 449: 463-467. JASIŃSKI M., STUKKENS Y., DEGAND H., PURNELLE B., MARCHAND-BRYNAERT J., BOUTRY M., 2001 - A plant plasma membrane ATP binding cassette-type transporter is involved in antifungal terpenoid secretion. - Plant Cell., 13: 1095-1107. KADER A.A., 2002 - Fruits in the global market, pp. 1-16. - In: KNEE M. (ed.) Fruit quality and its biological basis. Shef- field Academic Press, Sheffield, UK, pp. 279. KANG J., HWANG J.U., LEE M., KIM Y.Y., ASSMANN S.M., MARTINOIA E., LEE Y., 2010 - PDR-type ABC transporter mediates cellular uptake of the phytohormone abscisic acid. - Proc. Natl. Acad. Sci. USA, 107: 2355-2360. KIM D.Y., BOVET L., MAESHIMA M., MARTINOIA E., LEE Y., 2007 - The ABC transporter AtPDR8 is a cadmium extru- sion pump conferring heavy metal resistance. - Plant J., 50: 207-218. KIM D.Y., JIN J.Y., ALEJANDRO S., MARTINOIA E., LEE Y., 2010 - Overexpression of AtABCG36 improves drought and salt stress resistance in Arabidopsis. - Physiol Plant., 139: 170-180. KOBAE Y., SEKINO T., YOSHIOKA H., NAKAGAWA T., MARTINOIA E., MAESHIMA M., 2006 - Loss of AtPDR8, a plasma membrane ABC transporter of Arabidopsis thali- ana, causes hypersensitive cell death upon pathogen infec- tion. - Plant Cell Physiol., 47: 309-318. KRETZSCHMAR T., BURLA B., LEE Y., MARTINOIA E., 62 NAGY R., 2011 - Functions of ABC transporters in plants. - Essays Biochem., 50: 145-160. KRETZSCHMAR T., KOHLEN W., SASSE J., BORGHI L., SCHLEGEL M., BACHELIER J.B., REINHARDT D., BOURS R., BOUWMEESTER H.J., MARTINOIA E., 2012 - A petunia ABC protein controls strigolactone-dependent symbiotic signalling and branching. - Nature, 483: 341-344. LAMPING E., BARET P.V., HOLMES A.R., MONK B.C., GOFFEAU A., CANNON R.D., 2010 - Fungal PDR trans- porters: Phylogeny, topology, motifs and function. - Fungal Genet. Biol., 47(2): 127-142. LEE M., LEE K., LEE J., NOH E., LEE Y., 2005 - AtPDR12 contributes to lead resistance in Arabidopsis. - Plant Physi- ol., 138: 827-836. MARTINOIA E., KLEIN M., GEISLER M., BOVET L., FOR- ESTIER C., KOLUKISAOGLU U., MÜLLER-RÖBER B., SCHULZ B., 2002 - Multifunctionality of plant ABC trans- porters- more than just detoxifiers. - Planta, 214: 345-355. MOONS A., 2003 - Ospdr9, which encodes a PDR-type ABC transporter, is induced by heavy metals, hypoxic stress and redox perturbations in rice roots. - FEBS Lett., 553, 370- 376. MOONS A., 2008 - Transcriptional profiling of the PDR gene family in rice roots in response to plant growth regulators, redox perturbations and weak organic acid stresses. - Planta, 229: 53-71. NAVARRE C., SALLETS A., GAUTHY E., MAÎTREJEAN M., MAGY B., NADER J., PETY DE THOZÉE C., CROUZET J., BATOKO H., BOUTRY M., 2011 - Isolation of heat shock-induced Nicotiana tabacum transcription promoters and their potential as a tool for plant research and biotech- nology. - Transgenic Res., 20: 799-810. ODA K., OTANI M., URAGUCHI S., AKIHIRO T., FUJI- WARA T., 2011 - Rice ABCG43 is Cd inducible and con- fers Cd tolerance on yeast. - Biosci. Biotechnol., Biochem., 75(6): 1211-1213. PRASAD R., GOFFEAU A., 2012 - Yeast ATP-binding cassette transporters conferring multidrug resistance. - Annu. Rev. Microbiol., 66: 39-63. REA P.A., 2007 - Plant ATP-binding cassette transporters. - Annu. Rev. Plant. Biol., 58: 347-375. REID K.E., OLSSON N., SCHLOSSER J., PENG F., LUND S.T., 2006 - An optimized grapevine RNA isolation proce- dure and statistical determination of reference genes for real-time RT-PCR during berry development. - BMC Plant Biol., 6: 27. RENAUD S., DE LORGERIL M., 1992 - Wine, alcohol, plate- lets, and the French paradox for coronary heart disease. - Lancet, 339: 1523-1526. ROST B., SANDER C., 1993 - Prediction of protein secondary structure at better than 70% accuracy. - J. Mol. Biol., 232: 584-599. ROST B., SANDER C., 1994 - Combining evolutionary infor- mation and neural networks to predict protein secondary structure. - Proteins, 19: 55-72. RŮŽIČKA K., STRADER L.C., BAILLY A., YANG H., BLAKESLEE J., ŁANGOWSKI L., NEJEDLÁ E., FUJITA H., ITOH H., SYŌNO K., HEJÁTKO J., GRAY W.M., MAR- TINOIA E., GEISLER M., BARTEL B., MURPHY A.S., FRIML J., 2010 - Arabidopsis PIS1 encodes the ABCG37 transporter of auxinic compounds including the auxin pre- cursor indole-3-butyric acid. - Proc. Natl. Acad. Sci. USA, 107: 10749-10753. SASABE M., TOYODA K., SHIRAISHI T., INAGAKI Y., ICHINOSE Y., 2002 - cDNA cloning and characterization of tobacco ABC transporter: NtPDR1 is a novel elicitor-re- sponsive gene. - FEBS lett., 518: 164-168. SCHENKE D., SASABE M., TOYODA K., INAGAKI Y.-S., SHIRAISHI T., ICHINOSE Y., 2003 - Genomic structure of the NtPDR1 gene, harboring the two miniature inverted- repeat transposable elements, NtToya1 and NtStowaway101. - Genes & Genet. Syst., 78: 409-418. SCHOONBEEK H., DEL SORBO G., DE WAARD M.A., 2001 - The ABC transporter BcatrB affects the sensitivity of Botrytis cinerea to the phytoalexin resveratrol and the fungi- cide fenpiclonil. - Mol. Plant Microbe Interact., 14: 562-571. SEO S., GOMI K., KAKU H., ABE H., SETO H., NAKATSU S., NEYA M., KOBAYASHI M., NAKAHO K., ICHINOSE Y., MITSUHARA I., OHASHI Y., 2012 - Identification of natural diterpenes that inhibit bacterial wilt disease in to- bacco, tomato and Arabidopsis. - Plant Cell Physiol., 53: 1432-1444. STEIN M., DITTGEN J., SÁNCHEZ-RODRÍGUEZ C., HOU B.-H., MOLINA A., SCHULZE-LEFERT P., LIPKA V., SOMERVILLEA S., 2006 - Arabidopsis PEN3/PDR8, an ATP binding cassette transporter, contributes to nonhost re- sistance to inappropriate pathogens that enter by direct pen- etration. - Plant Cell, 18: 731-746. STEYN W.J., 2009 - Prevalence and Functions of anthocyanins in fruits, pp. 86-105. - In: WINEFIELD C., K. DAVIES, and K. GOULD (eds.). Anthocyanins. Biosynthesis, functions, and applications. Springer-Verlag, New York, USA, pp. 336. STRADER L.C., BARTEL B., 2009 - The Arabidopsis PLEIO- TROPIC DRUG RESISTANCE8/ABCG36 ATP binding cas- sette transporter modulates sensitivity to the auxin precursor indole-3-butyric acid. - Plant Cell., 21(7): 1992-2007. STUKKENS Y., BULTREYS A., GREC S., TROMBIK T., VANHAM D., BOUTRY M., 2005 - NpPDR1, a pleiotro- pic drug resistance-type ATP-binding cassette transporter from Nicotiana plumbaginifolia, plays a major role in plant pathogen defense. - Plant physiol., 139: 341-352. TAKAYANAGI T., OKUDA T., MINE Y., YOKOTSUKA K., 2004 - Induction of resveratrol biosynthesis in skins of three grape cultivars by ultraviolet irradiation. - J. Japan. Soc. Hort. Sci., 73: 193-199. TAMURA K., PETERSON D., PETERSON N., STECHER G., NEI M., KUMAR S., 2011 - MEGA5: molecular evolution- ary genetics analysis using maximum likelihood, evolution- ary distance, and maximum parsimony methods. - Mol. Biol. Evol., 28: 2731-2739. TAMURA M., TSUJI Y., KUSUNOSE T., OKAZAWA A., KAMIMURA N., MORI T., NAKABAYASHI R., HISHI- YAMA S., FUKUHARA Y., HARA H., SATO-IZAWA K., MURANAKA T., SAITO K., KATAYAMA Y., FUKUDA M., MASAI E., KAJTA S., 2014 - Successful expression of a novel bacterial gene for pinoresinol reductase and its effect on lignan biosynthesis in transgenic Arabidopsis thaliana. - Appl. Microbiol. Biotechnol., in press. TROMBIK T., JASINSKI M., CROUZET J., BOUTRY M., 2008 - Identification of a cluster IV pleiotropic drug resis- 63 tance transporter gene expressed in the style of Nicotiana plumbaginifolia. - Plant Mol. Biol., 66: 165-175. UNDERWOOD W., SOMERVILLE S.C., 2013 - Perception of conserved pathogen elicitors at the plasma membrane leads to relocalization of the Arabidopsis PEN3 transporter. - Proc. Natl. Acad. Sci. USA, 110: 12492-12497. VAN DEN BRÛLE S., MÜLLER A., FLEMING A.J., SMART C.C., 2002 - The ABC transporter spTUR2 confers resistance to the antifungal diterpene sclareol. - Plant J., 30: 649-662 VAN DEN BRÛLE S., SMART C.C., 2002 - The plant PDR family of ABC transporters. - Planta, 216: 95-106. VELASCO R., ZHARKIKH A., TROGGIO M., CARTWRIGHT D.A., CESTARO A., PRUSS D., PINDO M., FITZGERALD L.M., VEZZULLI S., REID J., MALACARNE G., ILIEV D., COPPOLA G.,WARDELL B., MICHELETTI D., MA- CALMA T., FACCI M., MITCHELL J.T., PERAZZOLLI M., ELDREDGE G., GATTO P., OYZERSKI R., MORET- TO M., GUTIN N., STEFANINI M., CHEN Y., SEGALA C., DAVENPORT D., DEMATTÈ L., MRAZ A., BATTILANA J., STORMO K., COSTA F., TAO Q., SI-AMMOUR A., HARKINS T., LACKEY A., PERBOST C., TAILLON B., STELLA A., SOLOVYEV V., FAWCETT J.A., STERCK L., VANDEPOELE K., GRANDO S.M., TOPPO S., MOSER C., LANCHBURY J., BOGDEN R., SKOLNICK M., SGA- RAMELLA V., BHATNAGAR S.K., FONTANA P., GUTIN A., VAN DE PEER Y., SALAMINI F., VIOLA R., 2007 - A high quality draft consensus sequence of the genome of a heterozygous grapevine variety. - PLoS ONE, 2(12): e1326. VERRIER P.J., BIRD D., BURLA B., DASSA E., FORESTIER C., GEISLER M., KLEIN M., KOLUKISAOGLU U., LEE Y., MARTINOIA E., MURPHY A., REA P.A., SAMUELS L., SCHULZ B., SPALDING E.J., YAZAKI K., THEODOU- LOU F.L., 2008 - Plant ABC proteins - a unified nomencla- ture and updated inventory. - Trends Plant Sci., 13: 151-159. VERSARI A., PARPINELLO G.P., TORNIELLI G.B., FER- RARINI R., GIULIVO C., 2001 - Stilbene compounds and stilbene synthase expression during ripening, wilting, and UV treatment in grape cv. Corvina. - J. Agric. Food Chem., 49: 5531-5536. WAN C.Y., WILKINS T.A., 1994 - A modified hot borate meth- od significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.). - Anal. Biochem., 223(1): 7-12. XIN X.F., NOMURA K., UNDERWOOD W., HE S.Y., 2013 - Induction and suppression of PEN3 focal accumulation dur- ing Pseudomonas syringae pv. tomato DC3000 infection of Arabidopsis. - Mol. Plant Microbe Interact., 26: 861-867. YAZAKI K., 2006 - ABC transporters involved in the transport of plant secondary metabolites. - FEBS Lett., 580: 1183-1191. YAZAKI K., SHITAN N., SUGIYAMA A., TAKANASHI K., 2009 - Cell and molecular biology of ATP-binding cassette proteins in plants. - Int. Rev. Cell Mol. Biol., 276: 263-299. ZAMBONI A., GATTO P., CESTARO A., PILATI S., VIOLA R., MATTIVI F., MOSER C., VELASCO R. 2009 - Grape- vine cell early activation of specific responses to DIMEB, a resveratrol elicitor. - BMC genomics, 10: 363.