James F. Harbertson

Modulation of flavonoid biosynthetic pathway genes and anthocyanins due to virus infection in grapevine (Vitis vinifera L.) leaves

BackgroundSymptoms of grapevine leafroll disease (GLRD) in red-fruited wine grape (Vitis vinifera L.) cultivars consist of green veins and red and reddish-purple discoloration of inter-veinal areas of leaves. The reddish-purple color of symptomatic leaves may be due to the accumulation of anthocyanins and could reflect an up-regulation of genes involved in their biosynthesis.ResultsWe examined six putative constitutively expressed genes, Ubiquitin, Actin, GAPDH, EF1-a, SAND and NAD5, for their potential as references for normalization of gene expression in reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). Using the geNorm program, a combination of two genes (Actin and NAD5) was identified as the stable set of reference genes for normalization of gene expression data obtained from grapevine leaves. By using gene-specific RT-qPCR in combination with a reliable normalization factor, we compared relative expression of the flavonoid biosynthetic pathway genes between leaves infected with Grapevine leafroll-associated virus 3 (GLRaV-3) and exhibiting GLRD symptoms and virus-free green leaves obtained from a red-fruited wine grape cultivar (cv. Merlot). The expression levels of these different genes ranged from two- to fifty-fold increase in virus-infected leaves. Among them, CHS3, F35H, F3H1, LDOX, LAR1 and MybA1 showed greater than 10-fold increase suggesting that they were expressed at significantly higher levels in virus-infected symptomatic leaves. HPLC profiling of anthocyanins extracted from leaves indicated the presence of cyanidin-3-glucoside and malvidin-3-glucoside only in virus-infected symptomatic leaves. The results also showed 24% higher levels of flavonols in virus-infected symptomatic leaves than in virus-free green leaves, with quercetin followed by myricetin being the predominant compounds. Proanthocyanidins, estimated as total tannins by protein precipitation method, were 36% higher in virus-infected symptomatic leaves when compared to virus-free green leaves.ConclusionsThe results, the first example to our knowledge, showed that modulation of the flavonoid biosynthetic pathway occurred in GLRaV-3-infected leaves of a red-fruited wine grape cultivar (cv. Merlot) leading to de novo synthesis of two classes of anthocyanins. These anthocyanins have contributed to the expression of reddish-purple color of virus-infected grapevine leaves exhibiting GLRD symptoms.

Regulated Deficit Irrigation Alters Anthocyanins, Tannins and Sensory Properties of Cabernet Sauvignon Grapes and Wines

Four regulated deficit irrigation (RDI) regimes were applied to Cabernet Sauvignon grapes, which were analyzed for phenolics and also made into wine over three consecutive growing seasons. Relative to an industry standard regime (IS), yield was reduced over the three years by 37% in a full-deficit (FD) regime and by 18% in an early deficit (ED) regime, whereas no yield reduction occurred with a late deficit (LD) regime. Relative to IS, skin anthocyanin concentration (fresh weight basis) was 18% and 24% higher in ED and FD, respectively, whereas no effect was seen in LD. Seed tannin concentration was 3% and 8% higher in ED and FD, respectively, relative to the other two RDI regimes, whereas seed tannin content (amount per berry) was higher in IS than in FD. There were no practically relevant effects on the basic chemistry of the wines. The finished wines showed concentrations of tannins and anthocyanins that generally mirrored observed differences in skin and seed phenolic concentrations, although these were amplified in FD wines. Descriptive sensory analysis of the 2008 wines showed that FD wines were the most saturated in color, with higher purple hue, roughness, dryness and harshness, followed by ED wines, whereas IS and LD wines were less saturated in color and with higher brown and red hues. Overall, FD and ED seemed to yield fruit and wine with greater concentrations of phenolics than IS and LD, with the additional advantage of reducing water usage. However, these apparent benefits need to be balanced out with reductions in crop yields and potential long-term effects associated with pre-véraison water deficits.

Impacts of Grapevine Leafroll Disease on Fruit Yield and Grape and Wine Chemistry in a Wine Grape (Vitis vinifera L.) Cultivar.

Grapevine leafroll disease (GLD) is an economically important virus disease affecting wine grapes (Vitis vinifera L.), but little is known about its effect on wine chemistry and sensory composition of wines. In this study, impacts of GLD on fruit yield, berry quality and wine chemistry and sensory features were investigated in a red wine grape cultivar planted in a commercial vineyard. Own-rooted Merlot vines showing GLD symptoms and tested positive for Grapevine leafroll-associated virus 3 and adjacent non-symptomatic vines that tested negative for the virus were compared during three consecutive seasons. Number and total weight of clusters per vine were significantly less in symptomatic relative to non-symptomatic vines. In contrast to previous studies, a time-course analysis of juice from grapes harvested at different stages of berry development from symptomatic and non-symptomatic vines indicated more prominent negative impacts of GLD on total soluble solids (TSS) and berry skin anthocyanins than in juice pH and titratable acidity. Differences in TSS between grapes of symptomatic and non-symptomatic vines were more pronounced after the onset of véraison, with significantly lower concentrations of TSS in grapes from symptomatic vines throughout berry ripening until harvest. Wines made from grapes of GLD-affected vines had significantly lower alcohol, polymeric pigments, and anthocyanins compared to corresponding wines from grapes of non-symptomatic vines. Sensory descriptive analysis of 2010 wines indicated significant differences in color, aroma and astringency between wines made from grapes harvested from GLD-affected and unaffected vines. The impacts of GLD on yield and fruit and wine quality traits were variable between the seasons, with greater impacts observed during a cooler season, suggesting the influence of host plant × environment interactions on overall impacts of the disease.

Glucose, fructose and sucrose increase the solubility of protein–tannin complexes and at high concentration, glucose and sucrose interfere with bisulphite bleaching of wine pigments

Wines were modified with increasing sugar concentrations and decreasing tannin concentrations and analysed by a combination of protein precipitation and bisulphite bleaching. Increasing sugar concentration decreased the precipitation of tannin and protein-precipitable polymeric pigments (PPP). The use of a hydrogen bond disruptor (urea) to reduce protein-tannin and protein-pigment complex formation showed that the effect of sugar concentration occurred by increasing the solubility of the tannin-protein complex, not by interfering with protein-tannin complex formation. By increasing the solubility of pigment-protein complexes, non-protein-precipitable polymeric pigments (nPPP) appeared to increase. There was also an increase in total polymeric pigments at each tannin concentration with increasing glucose and sucrose concentration, indicating that sugar concentration might also affect bisulphite bleaching of wine pigments. While a significant effect of sugar concentration on tannin-protein complex solubility was observed, these effects were greatest at sugar concentrations far in excess of normal wine making conditions. Under normal wine making conditions, sugar concentration will have a negligible effect on protein-precipitable tannin, PPP and nPPP concentrations.

Improvement of BSA Tannin Precipitation Assay by Reformulation of Resuspension Buffer

Analysis of protein precipitable wine tannins has become more commonplace due to the simplicity of the method and the strong association between tannin content and perception of astringency. The traditional protein precipitation method for wine measures tannins and polymeric pigments using 5% w/v sodium dodecyl sulfate (SDS) and the alkaline buffer triethanolamine (TEA) pH 9.4 to dissolve the tannin-protein precipitate and support the colorimetric reaction with ferric chloride. However, this method results in a high background absorbance presumably due to the alkaline pH of the resuspension buffer, which has been shown to oxidize phenolics. Experimentation with several buffer formulations showed that pairing TEA with urea instead of SDS allowed formulation of buffers at lower pH. Urea-TEA buffers at pH 7 and 8 showed significantly lower background absorbance and drift over time as well as a significantly greater amount of tannin recovered. These trends in tannin content and iron reactive phenolics were maintained across a wide range of diluted samples (100–1200 mg/L catechin equivalents). Using a TEA-urea buffer at pH 7 or 8 may improve on previous methods of protein tannin precipitation by increasing yield of tannins from the wine and decreasing the background absorbance and drift.

Sensory Impact of Extended Maceration and Regulated Deficit Irrigation on Washington State Cabernet Sauvignon Wines

Irrigation practices such as regulated deficit irrigation (RDI) and winemaking practices such as extended maceration have been experimentally evaluated from a chemical perspective but their impacts on sensory composition and interactive effects merit scientific attention. This study evaluated the sensory impact of extended maceration applied to Cabernet Sauvignon grapes sourced from a vineyard subjected to four RDI treatments: replenishment of 100%, 70%, and 25% of full-vine crop evapotranspiration (ETc) from fruit set until veraison followed by 100% ETc until harvest (labeled 100% ETc, 70% ETc, and 25% ETc, respectively) and 25% ETc from fruit set to veraison followed by 100% ETc from veraison to harvest (labeled 25/100% ETc). Each RDI treatment was replicated four times (n = 4) and made into wine, with two replicates designated as controls (10 day skin contact) and two as extended maceration (30 day skin contact). Wines were evaluated by descriptive analysis with a trained panel (n = 15) and chemical and sensory data were correlated using canonical correlation analysis. Wine-perceived saturation and purple component ratings were highest in 25% ETc wines and were highly correlated with the concentration of flavonols, malvidin- and delphinidin-derivatives, and small polymeric pigments. Fruit-based aroma descriptors were highest in the 25/100% ETc and 70% ETc wines. Extended maceration increased perceived astringency and bitterness, which were in turn correlated with the concentration of flavan-3-ol and oligomeric proanthocyanidins. These results suggest that moderate RDI protocols such as 70% ETc and 25/100% ETc impact positively the fruity aroma component (black and red fruit), whereas extended maceration lowered fruity aromas, possibly due to the masking effect of the oxidized character perceived in these wines.

Influence of Fruit Maturity, Maceration Length, and Ethanol Amount on Chemical and Sensory Properties of Merlot Wines

Selected winemaking conditions were applied to fruit with ~20.3 and ~24.9 Brix over two seasons. Merlot grapes were harvested 33 (2011) and 34 (2012) days apart. At each harvest, half of the must was adjusted to emulate the other harvest’s soluble solids content to evaluate the effect of ethanol (EtOH) on phenolic extraction at different fruit maturities. Additionally, two maceration lengths of 10 days (control) and 30 days (extended maceration; EM) were tested. Control wines had significantly higher anthocyanin content, saturation, and red color component, whereas EM wines had enhanced tannin extraction from seeds, lower anthocyanin content, lower saturation, higher hue, and higher large polymeric pigment content. EtOH differences up to 2.7% (v/v) showed no significant effect on tannin and anthocyanin extraction, suggesting a minor role of this solvent under standard winemaking conditions. The later harvest date had a prevailing and positive effect on the sensory profile of the wines over winemaking factors such as maceration length and EtOH treatments. Wines from the early harvest fruit were defined by fresh vegetal character, acidity, and low color saturation. Wines from the late harvest fruit were defined by viscous mouthfeel, sweet taste, and fruit-derived aromas. Extended maceration shifted the sensory profile toward higher astringency, lighter and yellower color components, and cooked vegetal aromas. Chaptalization of early harvest fruit to 25 Brix shifted the sensory profile from cooked and fresh vegetable characters toward sweet taste, alcoholic, floral, chocolate/caramel attributes, astringency, and viscous mouthfeel. Overall, unripe fruit and the application of extended maceration had a negative impact on the sensory profile of the wines, whereas chaptalization of unripe fruit yielded wines with an improved sensory profile.

Impact of Grape Maturity and Ethanol Concentration on Sensory Properties of Washington State Merlot Wines

Chaptalization and saignée-watering back were used to investigate the effects of modifying ethanol concentrations on sensory properties of Merlot wines made from fruit harvested at different maturities. Fruit was harvested on three dates with soluble solids concentrations of 20, 24, and 28 Brix. At each harvest, one-third of the must was fermented at the natural soluble solids concentration, and the other two-thirds were manipulated to match the other harvests’ Brix, leading to wine ethanol concentrations of 11.6, 14.0, and 16.2% for each of the three soluble solids concentrations. Both fruit maturity and ethanol concentration had significant effects on the chemical and sensory profiles of the wines. Wine physical viscosity increased with increasing ethanol concentrations, and wine color was positively impacted, with higher ethanol concentrations favoring the formation of polymeric pigments leading to darker wines. Wines made from unripe fruit (20 Brix) were characterized by green flavors and sour taste in the sensory analysis, whereas wines made from overripe fruit (28 Brix) were described as fruity and sweet. Manipulations targeting the adjustment of ethanol had a greater effect on wine sensory properties than fruit maturity: wines made from ripe (24 Brix) or overripe fruit adjusted to low ethanol concentrations were described similarly to wines made from unripe fruit, and wines made from unripe or ripe fruit adjusted to high ethanol concentrations were described similarly to wines made from overripe fruit. The results of this study demonstrate that both ethanol concentration and manipulations to achieve desired ethanol concentrations have a large influence on wine chemistry and sensory properties, and suggest that wine ethanol concentration is more important for the sensory profiles of wines than is fruit maturity at harvest.

Cofermentation of Syrah with Viognier: Evolution of Color and Phenolics during Winemaking and Bottle Aging

Cofermentation of Syrah with additions of 5, 10, and 20% (by weight) of the white variety Viognier was followed during winemaking and up to 580 days postpressing to assess the effect of Viognier additions on the chromatic and phenolic composition of the wines. Additions of 10 and 20% Viognier lowered most of the chromatic parameters. At day 580, no differences were observed for tannins and iron reactive phenolics among the wines. Analysis of anthocyanins and flavonols uncovered no differences between 100% Syrah and 5% Viognier wines but the 20% Viognier wines had lower concentrations of anthocyanins and flavonols, suggesting possible dilution of these phenolics. These results suggest that additions of Viognier at the rates studied here neither improve the phenolic composition nor enhance the color stability of the final wines.

Deficit Irrigation Alters Grapevine Growth, Physiology, and Fruit Microclimate

A deficit irrigation trial was conducted with field-grown Cabernet Sauvignon grapevines in the Columbia Valley of southeastern Washington. Four irrigation regimes were applied in four replicated blocks to replace various fractions of crop evapotranspiration (ETc) between fruit set and harvest. These treatments were designated ET100 (100% ETc), ET70 (70% ETc), ET25 (25% ETc), and ET25/100 (25% ETc before veraison and 100% ETc thereafter). Leaf water status and gas exchange, canopy growth and microclimate, and yield formation were evaluated over three years. Despite yearly variation in growing season temperatures, irrigation treatment effects were consistent among years. Overall, deficit irrigation did not enhance water-use efficiency. The ET100 and ET70 regimes rarely differed in vine physiology and performance. The ET25 regime, however, strongly limited gas exchange and led to a decline in vine capacity and productivity, suggesting that this degree of water deficit was economically unsustainable. In addition, this treatment was associated with small berries on small clusters, very high fruit-zone sunlight exposure, and elevated cluster temperature. The ET25/100 regime was generally intermediate in vine physiology, growth, and yield components. This treatment resulted in open canopies and small berries without the penalty in vine capacity and yield that was incurred with ET25. Potential effects of water deficit on fruit composition may be related to altered canopy size and microclimate, in addition to decreased berry size.