Abstract
The effect of an exogenous grape berry application of methyl jasmonate (MeJA) on the enhancement of the in vivo resveratrol production was tested. MeJA is a plant volatile that acts as an important intracellular regulator mediating diverse developmental processes and defense responses. A treatment with MeJA spray solution was performed on clusters of Vitis vinifera cv. Barbera grafted onto 3309 C rootstock under drought stress condition and regular water supply and at fruit set, veraison, and ripening. Cumulative MeJA treatments significantly increased berry resveratrol and ε-viniferin production at ripening. Water stress did not affect stilbene synthesis.
Grapevine (Vitis vinifera L.) has been cultivated for centuries in temperate climates as a source of fresh fruit, raisins, wine, and distilled beverages. The economic importance of grapevine as a crop plant makes V. vinifera a good model system to study the improvement of the nutraceutical properties of food products. Nutraceutical (nutritional + pharmaceutical) is a term that describes products that are naturally occurring substances (e.g., vitamins, amino acids, herbals) or formulations of these substances (Kharb and Singh 2004). Resveratrol (trans-3,5,4′-trihydroxystilbene), a phenolic compound, is of interest because it is claimed to be the active compound in red wines that contributes to a reduction in heart diseases and that influences cancer chemoprevention and phytoestrogen activities (Burns et al. 2000). Resveratrol in wine is basically due to the contact between must and berry skins during maceration: red wine generally contains higher resveratrol levels than white wine; cluster-stem fragments from pressing can also increase final resveratrol levels. This stilbene is synthesized both as a constitutive in woody organs (stems, roots, and seeds) and as an induced compound in soft tissues (leaves and berry skins) in response to biotic and abiotic stresses, acting as a phytoalexin (Langcake and McCarthy 1979).
Numerous experimental trials have recently been conducted with different elicitors to promote resveratrol synthesis in grapevine berries, such UV irradiation (Cantos et al. 2003) and benzothiadiazole (Iriti et al. 2004), and in cell suspension cultures (for biotechnological purposes) cyclodextrins (Morales et al. 1998), Na-orthovanadate, and jasmonates, to which methyl jasmonate (MeJA) and its less active acid, jasmonic acid, refer (Tassoni et al. 2005). MeJA was initially identified from f lowers of Jasminum grandif lorum (Demole et al. 1962) and is ubiquitously distributed in the plant kingdom (Hamberg and Gardner 1992). Jasmonates are synthesized in plants via the octadecanoid pathway. Cellular organelles such as plastids or peroxisomes are regarded as the primary site(s) of jasmonic acid biosynthesis; in addition, a cytoplasmic pathway has also been described. Jasmonic acid is then catabolized further to form numerous conjugates and its volatile counterpart MeJA (Hamberg and Gardner 1992). This latter compound acts as an important intracellular regulator and a diffusible intercellular signal transducer mediating diverse developmental processes and defense responses, becoming a strong candidate for resistance inference (Seo et al. 2001). Sustained exposure of both grapevine leaves and cell-suspension cultures to MeJA sprays have induced hypersensitive cell death (Repka et al. 2004). Moreover, airborne (gaseous) in vitro MeJA applications on leaves and berries have recently been performed, showing increased stilbene synthesis (Larronde et al. 2003). Further to these in vitro results, in the present study the effect of an exogenous grape berry application of MeJA on the enhancement of the in vivo resveratrol production was tested. At the same time (since no data are reported in literature) the effect of drought stress was tested as additional potential elicitor of the in vivo berry resveratrol production.
Materials and Methods
Cluster treatments.
A non-phytotoxic dosage of MeJA was sprayed on clusters of V. vinifera cv. Barbera, grafted on 3309 C rootstock and grown in pot (30-L vol, 60% sand, 30% soil, 10% acidic peat) at fruit set, veraison, and ripening (45 days after veraison). At each growth stage, three spray treatments were performed (at day 1, 3, and 5) on the clusters (T) with 10 mM MeJA (dissolved in 100% ethanol), whereas the controls (NT) were obtained by spraying only ethanol. Each treatment was carried out on three replicates. All samples were collected at day 8, after all treatments. The cumulative effect of MeJA application on stilbene production during the previous growth stage/s was also considered. Moreover, the experimental plan provided for water stress (WS) and relative control (C). Water stress was applied ~48 hr before MeJA treatments (leaf water potential ψ = −13.6 bar, measured by the Schollander pressure bomb), while the control plants were irrigated to keep the soil near field capacity and leaf water potential (ψ) at approximately −5 bar.
Standards.
trans-Resveratrol (trans-3,4′,5-trihydroxy-stilbene) and trans-piceatannol (trans-3,3′,4,5′-tertrahydroxy-stilbene) standards were purchased from Sigma (St. Louis, MO); cis-resveratrol was obtained by trans-resveratrol exposure to UV rays (λ = 366 nm) for 40 min (90% of conversion); ε-viniferin (dimer of trans-resveratrol) was kindly supplied by G. Hoos (formerly BFA für Rebenzüchtung Geilweilerhof, Siebeldingen, Germany). The purity of each stilbene was verified by HPLC and the identity was confirmed according a published method (Mattivi et al. 1995). Working standards of stilbenes were prepared by dissolving them in methanol and diluting the solution with acetonitrile/water (40:60; v:v).
Stilbene extraction and quantification.
trans-Resveratrol, cis-resveratrol, piceatannol, and ε-viniferin were extracted from the berries according to Bavaresco et al. (2005). HPLC was consistent with previously reported conditions (Bavaresco et al. 2005). Quantification of stilbenes was done on the basis of peak areas using Chemstation software (Agilent, Waldbronn, Germany). Data were expressed on the basis of berry fresh weight (μg/g FW), with each value the mean of three replicates. Data were calculated as the difference between the recorded value at day 8 and the recorded value before spray treatments, at day 0. At fruit set and veraison, the studied stilbenes were absent in the berries collected at day 0.
Statistical analysis.
ANOVA with interactions was performed with “R” software (http://cran.r-project.org). Growth stage (fruit set, veraison, ripening), MeJA treatment status (single, cumulative, control), and water status (water stress, control) were considered as variables. Means were compared by using the Tukey’s test (5% level).
Results and Discussion
Interactions between the growth stage and MeJA treatment factors were significant (5% level); results for all combinations are shown in Figure 1⇓. The application of a water stress did not significantly influence the induced stilbene synthesis (data not shown). At ripening, but not at veraison, trans-resveratrol synthesis was significantly increased by the cumulative MeJA applications. cis-Resveratrol was detected at each MeJA treatment status only during ripening when the cumulative MeJA treatments increased its concentration, even if nonsignificant over the control. At veraison a low concentration of ε-viniferin was detected only after both the single and the cumulative MeJA applications, whereas at ripening it was present under all conditions and it was significantly elicited by both the single and the cumulative MeJA treatments. Few MeJA treatments on grapevine cell suspension cultures and leaves have been reported (Tassoni et al. 2005, Repka et al. 2004). However, airborne (gaseous) MeJA applications on berries have recently been performed in vitro (Larronde et al. 2003); here, first results of the in vivo liquid spray MeJA application on berry clusters are provided. Barbera was selected for its importance among red grape varieties in north Italy. Within the jasmonate class, MeJA was chosen as elicitor because it has recently been demonstrated that jasmonate acid is less efficient in promoting endogenous resveratrol accumulation (Tassoni et al. 2005).
Berry stilbene production at fruit set, veraison, and ripening, related to MeJA single (sin-T) or cumulative treatment (cum-T) and relative controls (sin-NT and cum-NT). Bars represent standard deviations.
Given the resveratrol property to act as phytoalexin after elicitation, the increased berry stilbene synthesis after MeJA treatment is consistent with the ability of jasmonates to induce plant defense responses against a group of pathogens (for review, see Reymond and Farmer 1998), mechanical or herbivorous arthropod-driven wounding (Délano-Frier et al. 2004), and to induce systemic defenses that have a direct negative impact on insect oviposition and survivorship (Omer et al. 2000). Therefore, since stilbenes (as induced compounds) are related to vine resistance against pathogens such as Botrytis cinerea (Langcake and McCarthy 1979, Bavaresco et al. 1997), Plasmopara viticola (Langcake 1981), Uncinula necator (Romero-Pérez et al. 2001), Rhizopus stolonifer (Sarig et al. 1997), and Aspergillus carbonarius (Bavaresco et al. 2003), these results suggest that the MeJA treatment could potentially provide an increased resistance to a broad range of diseases, although that has not been tested.
Drought conditions were included in the experiment in order to study the interaction with an abiotic stress. According to previous data, an abiotic stress such as soil lime conditions was related to an enhancement of stilbene berry concentration (Bavaresco et al. 2005), while the present data suggest no effect of drought stress. Further investigations are required to control whether or not stilbene-synthase gene expression is affected by drought stress.
In the present study, cumulative MeJA treatments significantly increased the berry trans-resveratrol production at ripening. In addition, at this growth stage ε-viniferin was significantly elicited by the cumulative MeJA treatments and by the single treatment. Given the antioxidant and anticarcinogenetic activity of resveratrol, MeJA application also enhanced nutraceutical grape properties. Moreover, since ethanol was used as a base for applying the MeJA treatments, side effects on berry development should be taken into account. It has been reported that an enhancement of the anthocyanin accumulation occurred after spraying Cabernet Sauvignon grapes with 5% ethanol at veraison (El Kereamy et al. 2002) and that an ethanol application at the dose of 10% increased berry weight and slightly enhanced berry color (Chervin et al. 2005). In the present study, the 100% ethanol application on berry cluster could have affected berry size, berry color, and aroma, although all compounds involved and responsible for these traits were not chemically analyzed. Therefore the MeJa treatment could present a negative side effect consisting of the potential change in color and aroma, even if it enhanced stilbene production in berries. As ethanol is a proper solvent of many chemical compounds, the precise effects of 100% ethanol application could be the object of future studies.
Finally, since MeJA is a strong candidate for airborne signals that mediate interplant communication for defense responses (Farmer and Ryan 1990), the positive effects of a MeJA treatment in the field might be enhanced compared to this trial where treated clusters were at a three-plant distance, although this has not yet been tested.
Conclusion
MeJA treatment significantly promoted the berry resveratrol and ε-viniferin production at veraison and ripening, providing a potential enhancement of nutraceutical grape properties. Many aspects related to jasmonates remain unknown and should add considerably to interest in the jasmonate pathway. Further investigation of gas-phase signaling by volatile MeJA within the plant may contribute to the general knowledge of the biology of volatile regulators of gene expression.
Footnotes
Acknowledgments: The authors are grateful to Giuseppe Bruzzi and Pasquale Presutto (lab crew) for their technical support and to Nello Bagni (Università di Bologna) for transferring information about treatments with methyl jasmonate.
- Received December 2006.
- Revision received April 2007.
- Copyright © 2007 by the American Society for Enology and Viticulture
Literature Cited
Vol 58 Issue 4
Jump to section
Related Articles
Cited By...
More from this TOC section
Similar Articles
