Abstract
Berries of Vitis vinifera L. cv. Barbera (disease susceptible) and the interspecific variety Castor (disease resistant) were infected at veraison by the ochratoxin A-producing fungus Aspergillus carbonarius in order to test the stilbene-synthase gene expression by reverse transcriptase-polymerase chain reaction. Stilbene (trans-resveratrol and piceatannol) synthesis was also analyzed. Stilbene-synthase gene expression was induced by the fungus and stilbenes were produced in both varieties, but was significantly higher in Castor. Castor displayed few disease symptoms while Barbera was highly infected.
Stilbenes are low molecular weight phenolics occurring in a number of plant species, including Vitis spp. (Langcake and Price 1976) and have been observed to have beneficial effects on human health (Burns et al. 2000). There is a significant positive correlation between stilbene-synthase (StSy) mRNA levels and stilbene concentration in different grape tissues, and StSy has been identified as the enzyme responsible for the production of these compounds. The StSy gene has also been studied and it belongs to a multigene family in the grapevine genome (Wiese et al. 1994). Stilbenes—including compounds such as trans- and cis-resveratrol, their glucosides (piceid), viniferins, pterostilbene, astringin, and piceatannol (astringinin)—can be expressed constitutively in woody tissue (such as canes, stems, and seeds) or can be present as induced compounds in soft tissue (such as leaves and fruit). In the latter, stilbenes are classified as phytoalexins because of their role in plant defense mechanisms against fungal attacks. Molecules responsible for inducing phytoalexin synthesis in plants are called elicitors: they may be abiotic in nature, such as methyl-jasmonate (Tassoni et al. 2005), and may also be biotic, such as Aspergillus spp. (Bavaresco et al. 2003). The Aspergillus-based elicitors present in the field, particularly in hot and humid climates, are of increasing interest because of their potential role in the production of ochratoxin A (OTA) in grapes. OTA is a carcinogenic toxin in rodents and it possesses teratogenic, immunotoxic, and possibly neurotoxic and genotoxic properties. In 1993 the International Agency for Research on Cancer classified OTA as a possible carcinogenic product for humans, and since 1996 OTA contamination has been detected in wine and grape juice (Zimmerli and Dick 1996). In 2005 the European Commission regulated its content in must and wine (EC regulation 123/2005).
The research presented here examines StSy gene expression in relation to stilbene production after A. carbonarius (Bain.) inoculation on berry tissue. According to previous data (Bavaresco et al. 2003), trans-resveratrol production is positively correlated to OTA berry level. The speculation as to whether there is a link between stilbene elicitation and OTA production was derived from observing unpublished data in a wine survey in which all wines that had high concentrations of OTA also had high concentrations of trans-resveratrol; wines rich in trans-resveratrol were not consistently rich in OTA. According to one study (Jeswal 1998), concurrent administration of grape berry and leaf juice to mice together with OTA significantly reduced the hepatic and renal damage caused by ingestion of this mycotoxin. Perhaps resveratrol has a similar effect in counterbalancing the toxicity of OTA.
Materials and Methods
Genotypes and berry inoculation.
Barbera (a red wine, disease-susceptible V. vinifera cultivar) and Castor (a white wine, disease-resistant interspecific cultivar) were used in this experiment. Sample collection and in vitro inoculation of berries were carried out as described elsewhere (Bavaresco et al. 2003). Briefly, berries were surface-disinfected, punctured, and dipped in a conidial suspension (107 conidia/mL) prepared from a 7-day-old colony of Aspergillus carbonarius (MPVP-A566). After inoculation, berries were transferred onto a sterile metal grate in a box prepared as a moist chamber and incubated for 7 days at 25°C. Noninoculated punctured berries were used as a control. Titratable acidity, pH, and soluble solids were analyzed before inoculation. Macronutrients and trace elements were recorded as described elsewhere (Cottenie 1980).
RNA extraction and reverse-transcriptase-PCR.
Total RNA extraction from 4 g of berry skins (40 g of berries) was carried out according to Moser et al. (2004), except for the initial tissue quantity, since the authors used a lower skin weight. Forward and reverse primers were designed based on the published NCBI sequences of Vitis sp. cv. Norton stilbene-synthase 1 (st1) gene (AF418566 GenBank accession) and of V. vinifera chalcone-synthase (CHS) mRNA (X75969 GenBank accession) (Table 1⇓). Reverse transcriptase (RT)-PCR analysis was performed with the Ready-To-Go RT-PCR Beads kit (Amersham Pharmacia, Bridgeport, PA). Positive and negative controls were used. RT-PCR products were purified with the QIAquick Gel Extraction kit (Qiagen, Hilden, Germany). Sequencing of the produced fragments was carried out at the BMR Genomics sequencing facility (CRIBI, Padova, Italy).
Primers used for RT-PCR analysis of Castor and Barbera berry skins.
Stilbene extraction and quantification.
trans-Resveratrol, piceatannol, and trans-piceid were extracted from whole berries (excluding seeds), and high-performance liquid chromatography (HPLC) analysis was carried out according to Bavaresco et al. (2003). Briefly, about 10 g of fresh berries, without seeds, was crushed and soaked with 30 mL methanol/water (95:5; v:v). After filtration, evaporation in vacuo, and phase partitioning, the stilbenes were analyzed by HPLC on a PerkinElmer 200 system (Perkin-Elmer, Waltham, MA) equipped with an ISS 200 sampling system and a FP-1520 fluorescence detector (JASCO, Tokyo, Japan) set at 330 nm excitation and 374 nm emission. JASCO Borwin 1.5 software and a personal computer were used for data storage and evaluation (JASCO Borwin, Great Dunmow, UK). A LiChrospher RP-18 column (5 μm particle size, 125 x 4 mm i.d.; Merck KgaA, Darmstadt, Germany) was used at ambient temperature, using gradient elution with acetonitrile/aqueous 2% acetic acid (from 8 to 52% of acetonitrile in 15 min), at 1 mL/min. Injection volume was 20 μL. Amounts of trans-resveratrol standard between 8 and 100 picograms were injected, while for piceatannol and trans-piceid the amounts injected were between 0.4 and 10 picograms. Quantification was on the basis of peak areas using the Borwin PC software.
Statistical analysis.
There were six repetitions per each variable combination (inoculated and control berries; Castor and Barbera genotypes). ANOVA analysis was performed with R software (http://cran.r-project.org).
Results and Discussion
The inoculation trial was carried out in vitro at 25°C according to Bavaresco et al. (2003), who found that this temperature was the most suitable for fungal growth. Aspergillus carbonarius was tested because it is considered to be the key fungus involved in the OTA contamination of grapes. The veraison growth stage was chosen as the time of inoculation because trans-resveratrol production is higher at veraison compared with other stages (such as ripening) and berry conditions after fungal attack are conducive to fungal development. Moreover, only punctured berries were considered for both infection and control treatments, since puncturing results in significantly higher trans-resveratrol concentrations than when berries are infected without puncturing (Bavaresco et al. 2003).
In order to perform the StSy gene expression analysis, genotypes differing for their genetic resistance to fungus diseases were chosen. At veraison, fruit quality parameters of the two tested genotypes were different as follows for Barbera and Castor, respectively: pH of 2.31 and 2.63; titratable acidity of 38.6 g/L and 21.5 g/L; soluble solids of 10.5 Brix and 12.3 Brix. Mineral element composition of the berries was also different between genotypes, particularly for nitrogen (% DW 0.50 and 0.89) and boron (mg kg−1 DW 9.5 and 21.4), for Barbera and Castor, respectively.
The RT-PCR analysis of StSy gene expression found a highly homologue (96%) sequence to the st1 gene coding region of Vitis sp. cv. Norton (AF418566 GenBank accession) in both the inoculated Castor and Barbera, sized 1134 and 1127 bp, respectively. Because the StSy gene belongs to a multigenic family, the AF418566 GenBank accession was used as a reference for primer design, as the st1 gene reacts to fungus elicitation with a much higher expression level in berry tissue compared with other genes of the same family (Hou et al. 2002). The StSy gene expression was clearly induced after fungus inoculation, since it was absent in control berries (Figure 1⇓). This result was consistent with the chemical data, since trans-resveratrol was not produced in control berries.
RT-PCR analysis with StSy1/StSy2 primers on Castor (C) and Barbera (B) berry skins (two technical replicates), inoculated (i) and control (c). M: ladder 100 bp. trans-Resveratrol average concentrations are reported.
However, only the inoculated Castor berries produced significantly higher resveratrol concentrations (5% level) than the control. Moreover, trans-resveratrol concentration in inoculated Castor berries (1.19 μg g−1 FW) was significantly (5% level) higher than in inoculated Barbera berries (0.04 μg g−1 FW), while A. carbonarius infection symptoms were by sight remarkably lower in Castor compared with Barbera. A significant (5% level) increase in piceatannol production was observed in inoculated Castor berries (0.36 μg g−1 FW), compared with its control, while trans-piceid was not detected in any genotype. These data further confirm that stilbene synthesis in berry tissue is regulated by an inducible system.
Finally, a sequence (89% homology) corresponding to a coding region of the CHS3 gene of Vitis vinifera cv. Cabernet Sauvignon (AB066274 GenBank accession) was isolated. This gene was expressed in both genotypes and treatments, inoculated and control. For red and white grapes, veraison corresponds to the beginning of anthocyanin and flavonol synthesis, respectively, which are upstream of the CHS enzyme. The amplification of the partial CHS gene (366 bp with CHS1/CHS3 primers), instead of the complete gene (CHS1/CHS2 primers), may have been due to differences between the studied genotypes in the annealing site of the CHS2 primer.
Conclusion
Results indicate that stilbene production induced by A. carbonarius is related to StSy gene expression. Results also demonstrate that trans-resveratrol acts as a phytoalexin, providing Castor with a higher disease resistance, and add information to previous studies that used different fungi (Bavaresco et al. 1997).
Footnotes
Acknowledgments: The authors thank A. Pietri and T. Bertuzzi from the Istituto di Scienze degli Alimenti e Nutrizione for their contribution to the project.
- Received May 2006.
- Revision received July 2006.
- Copyright © 2007 by the American Society for Enology and Viticulture
Vol 58 Issue 1
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