Compositional Changes in Grapes and Leaves as a Consequence of Smoke Exposure of Vineyards from Multiple Bushfires across a Ripening Season.

The negative effects of smoke exposure of grapes in vineyards that are close to harvest are well documented. Volatile phenols in smoke from forest and grass fires can contaminate berries and, upon uptake, are readily converted into a range of glycosylated grape metabolites. These phenolic glycosides and corresponding volatile phenols are extracted into the must and carried through the winemaking process, leading to wines with overtly smoky aromas and flavours. As a result, smoke exposure of grapes can cause significant quality defects in wine, and may render grapes and wine unfit for sale, with substantial negative economic impacts. Until now, however, very little has been known about the impact on grape composition of smoke exposure very early in the season, when grapes are small, hard and green, as occurred with many fires in the 2019-20 Australian grapegrowing season. This research summarises the compositional consequences of cumulative bushfire smoke exposure of grapes and leaves, it establishes detailed profiles of volatile phenols and phenolic glycosides in samples from six commercial Chardonnay and Shiraz blocks throughout berry ripening and examines the observed effects in the context of vineyard location and timing of smoke exposure. In addition, we demonstrate the potential of some phenolic glycosides in leaves to serve as additional biomarkers for smoke exposure of vineyards.

Development and Utilization of a Model System to Evaluate the Potential of Surface Coatings for Protecting Grapes from Volatile Phenols Implicated in Smoke Taint.

Due to the increasing frequency of wildfires in recent years, there is a strong need for developing mitigation strategies to manage the impact of smoke exposure of vines and occurrence of 'smoke taint' in wine. One plausible approach would be to prevent or inhibit the uptake of volatile phenols from smoke into grape berries in the vineyard. In this study we describe a model system we developed for evaluating under controlled conditions the effectiveness of a range of surface coatings (including existing horticultural sprays) for reducing/preventing the uptake of volatile phenols and their subsequent conversion to phenolic glycosides. Grapes were coated with the materials to be tested and then exposed to gaseous phenols, via evaporation from an aqueous solution, in a semi-closed glass container. Analysis of volatile phenols and their glycosidic grape metabolites demonstrated that the treatments typically did not provide any significant protection; in fact, some resulted in higher concentrations of these compounds in the grapes. The highest concentrations of volatile phenols and their glycosides were observed after application of oily, hydrophobic materials, suggesting that these materials may enhance the adsorption or transfer of volatile phenols into grape berries. Therefore, it is important to consider the types of sprays that are being applied in the vineyard before and during smoke events to prevent the potential of exacerbating the uptake of smoke compounds by grape berries.

Uptake and Glycosylation of Smoke-Derived Volatile Phenols by Cabernet Sauvignon Grapes and Their Subsequent Fate during Winemaking.

Wine made from grapes exposed to bushfire smoke can exhibit unpleasant smoky, ashy characters, which have been attributed to the presence of smoke-derived volatile phenols, in free or glycosylated forms. Here we report the uptake and glycosylation of volatile phenols by grapes following exposure of Cabernet Sauvignon vines to smoke, and their fate during winemaking. A significant delay was observed in the conversion of volatile phenols to their corresponding glycoconjugates, which suggests sequestration, the presence of intermediates within the glycosylation pathway and/or other volatile phenol storage forms. This finding has implications for industry in terms of detecting smoke-affected grapes following vineyard smoke exposure. The potential for an in-canopy sprinkler system to mitigate the uptake of smoke-derived volatile phenols by grapes, by spraying grapevines with water during smoke exposure, was also evaluated. While "misting" appeared to partially mitigate the uptake of volatile phenols by grapes during grapevine exposure to smoke, it did not readily influence the concentration of volatile phenols or the sensory perception of smoke taint in wine. Commercial sensors were used to monitor the concentration of smoke particulate matter (PM) during grapevine exposure to low and high density smoke. Similar PM profiles were observed, irrespective of smoke density, such that PM concentrations did not reflect the extent of smoke exposure by grapes or risk of taint in wine. The sensors could nevertheless be used to monitor the presence of smoke in vineyards during bushfires, and hence, the need for compositional analysis of grapes to quantify smoke taint marker compounds.

Disclosing the Molecular Basis of the Postharvest Life of Berry in Different Grapevine Genotypes.

The molecular events that characterize postripening grapevine berries have rarely been investigated and are poorly defined. In particular, a detailed definition of changes occurring during the postharvest dehydration, a process undertaken to make some particularly special wine styles, would be of great interest for both winemakers and plant biologists. We report an exhaustive survey of transcriptomic and metabolomic responses in berries representing six grapevine genotypes subjected to postharvest dehydration under identical controlled conditions. The modulation of phenylpropanoid metabolism clearly distinguished the behavior of genotypes, with stilbene accumulation as the major metabolic event, although the transient accumulation/depletion of anthocyanins and flavonols was the prevalent variation in genotypes that do not accumulate stilbenes. The modulation of genes related to phenylpropanoid/stilbene metabolism highlighted the distinct metabolomic plasticity of genotypes, allowing for the identification of candidate structural and regulatory genes. In addition to genotype-specific responses, a core set of genes was consistently modulated in all genotypes, representing the common features of berries undergoing dehydration and/or commencing senescence. This included genes controlling ethylene and auxin metabolism as well as genes involved in oxidative and osmotic stress, defense responses, anaerobic respiration, and cell wall and carbohydrate metabolism. Several transcription factors were identified that may control these shared processes in the postharvest berry. Changes representing both common and genotype-specific responses to postharvest conditions shed light on the cellular processes taking place in harvested berries stored under dehydrating conditions for several months.

Towards a scientific interpretation of the terroir concept: plasticity of the grape berry metabolome.

BACKGROUND: The definition of the terroir concept is one of the most debated issues in oenology and viticulture. The dynamic interaction among diverse factors including the environment, the grapevine plant and the imposed viticultural techniques means that the wine produced in a given terroir is unique. However, there is an increasing interest to define and quantify the contribution of individual factors to a specific terroir objectively. Here, we characterized the metabolome and transcriptome of berries from a single clone of the Corvina variety cultivated in seven different vineyards, located in three macrozones, over a 3-year trial period. RESULTS: To overcome the anticipated strong vintage effect, we developed statistical tools that allowed us to identify distinct terroir signatures in the metabolic composition of berries from each macrozone, and from different vineyards within each macrozone. We also identified non-volatile and volatile components of the metabolome which are more plastic and therefore respond differently to terroir diversity. We observed some relationships between the plasticity of the metabolome and transcriptome, allowing a multifaceted scientific interpretation of the terroir concept. CONCLUSIONS: Our experiments with a single Corvina clone in different vineyards have revealed the existence of a clear terroir-specific effect on the transcriptome and metabolome which persists over several vintages and allows each vineyard to be characterized by the unique profile of specific metabolites.

Beyond Volatile Phenols: An Untargeted Metabolomic Approach to Revealing Additional Markers of Smoke Taint in Grapevines (Vitis vinifera L.) cv. Merlot.

When bushfires occur near wine regions, vineyards are frequently exposed to environmental smoke, which can negatively affect grapes and wine. For evaluating the severity of smoke exposure, volatile phenols and their glycosides are commonly used as biomarkers of smoke exposure. While critical to refining smoke taint diagnostics, few studies have comprehensively assessed the compositional impact of smoke exposure of grapes. In this study, Merlot grapevines were exposed to smoke post-véraison, with grapes being sampled both pre-smoke exposure and repeatedly post-smoke exposure, for analysis by liquid chromatography-high-resolution mass spectrometry. Volatile phenol glycosides were detected in control and smoke-affected grapes at ≤22 µg/kg and up to 160 µg/kg, respectively. The metabolite profiles of control and smoke-affected grapes were then compared using an untargeted metabolomics approach and compounds differentiating the sample types tentatively identified. The results demonstrate the presence of novel phenolic glycoconjugates as putative metabolites from environmental smoke together with stress-related grapevine metabolites and highlight the need to further characterize the consequences of grapevine smoke exposure with respect to the regulation of abiotic stress and plant defense mechanisms.

Smoky Characters in Wine: Distinctive Flavor or Taint?

The frequency of wildfires has significantly increased in recent years, posing concerns for many grapegrowers and winemakers. Exposure of grapes to smoke can result in wines with notable smoky notes, which in severe cases are described as "smoke tainted". However, smoky aromas in wine are not a priori quality defects but may be considered desirable in some styles of wines, as also widely found and appreciated in many spirits. In this perspective, we summarize recent research on sources and assessment of smoky sensory attributes in wine and provide an outlook on opportunities for managing excessive smoky characters.

MStractor: R Workflow Package for Enhancing Metabolomics Data Pre-Processing and Visualization.

Untargeted metabolomics experiments for characterizing complex biological samples, conducted with chromatography/mass spectrometry technology, generate large datasets containing very complex and highly variable information. Many data-processing options are available, however, both commercial and open-source solutions for data processing have limitations, such as vendor platform exclusivity and/or requiring familiarity with diverse programming languages. Data processing of untargeted metabolite data is a particular problem for laboratories that specialize in non-routine mass spectrometry analysis of diverse sample types across humans, animals, plants, fungi, and microorganisms. Here, we present MStractor, an R workflow package developed to streamline and enhance pre-processing of metabolomics mass spectrometry data and visualization. MStractor combines functions for molecular feature extraction with user-friendly dedicated GUIs for chromatographic and mass spectromerty (MS) parameter input, graphical quality-control outputs, and descriptive statistics. MStractor performance was evaluated through a detailed comparison with XCMS Online. The MStractor package is freely available on GitHub at the MetabolomicsSA repository.

Compositional Changes in Smoke-Affected Grape Juice as a Consequence of Activated Carbon Treatment and the Impact on Phenolic Compounds and Smoke Flavor in Wine.

An increase in bushfires and wildfires globally and consequent smoke exposure of grapevines has seen an elevated need for remediation options to manage the impact of smoke taint in the wine industry. Two commercially available activated carbons (PS1300 and CASPF) were evaluated at 1, 2, and 4 g/L with juice from smoke-affected Pinot Noir and Chardonnay grapes. PS1300 and CASPF treatments removed up to 75 and 92% of the phenolic glycosides in the smoke-affected Pinot Noir rosé juice, respectively, and both carbons removed virtually all (i.e., 98-99%) of the phenolic glycosides in the smoke-affected Chardonnay juice at the highest dose rate (4 g/L). The free volatile phenols in the wines were similarly lower in concentration following treatment. Sensory analysis confirmed that the wines made from carbon fined juice had reduced smoke aroma and flavor compared to those from the nontreated controls. However, desirable sensory properties such as color and fruity attributes were also negatively affected by the treatment. The dose rate should be optimized in industry practice to find a balance between reducing the intensity of smoke-related sensory attributes while maintaining or enhancing positive attributes.

Factors Contributing to Interindividual Variation in Retronasal Odor Perception from Aroma Glycosides: The Role of Odorant Sensory Detection Threshold, Oral Microbiota, and Hydrolysis in Saliva.

Glycosides are sugar conjugates of aroma compounds that are found in many fruits and vegetables, and while glycosides are non-volatile, they can release flavor during eating, through enzyme hydrolysis from oral microbiota. Recently, a range of sensory phenotypes for glucoside perception have been observed, reflecting interindividual variation in response to precursors of floral and smoky flavors, geranyl glucoside and guaiacyl glucoside. To understand this variation and investigate the role of oral microbiota on in vitro hydrolysis of glucosides in saliva, metagenomic screening was conducted using individuals representing the range of sensory phenotypes for geranyl and guaiacyl glucosides. In parallel, sensory retronasal detection thresholds for geranyl glucoside, guaiacyl glucoside, and the volatile odorants geraniol and guaiacol were determined. Oral microbial communities correlated with hydrolysis of glucosides in saliva, but the relationship did not extend to sensory phenotypes. Overall, the retronasal detection threshold of the volatile odorants studied was the main factor determining sensory phenotype.

Linking gene expression and oenological traits: Comparison between Torulaspora delbrueckii and Saccharomyces cerevisiae strains.

Wine fermentations typically involve the yeast Saccharomyces cerevisiae. However, many other yeast species participate to the fermentation process, some with interesting oenological traits. In this study the species Torulaspora delbrueckii, used occasionally in mixed or sequential fermentation with S. cerevisiae to improve wine sensory profile, was investigated to understand the physiological differences between the two. Next generation sequencing was used to characterize the transcriptome of T. delbrueckii and highlight the different genomic response of these yeasts during growth under wine-like conditions. Of particular interest were the basic differences in the glucose fermentation pathway and the formation of aromatic and flavour compounds such as glycerol, esters and acetic acid. Paralog genes were missing in glycolysis and glycerol biosynthesis in T. delbrueckii. Results indicate the tendency of T. delbrueckii to produce less acetic acid relied on a higher expression of alcoholic fermentation related genes, whereas acetate esters were influenced by the absence of esterases, ATF1-2. Additionally, in the Δbap2 S. cerevisiae strain, the final concentration of short branched chain ethyl esters (SBCEEs) was related to branched chain amino acid (BCAA) uptake. In conclusion, different adaption strategies are apparent for T. delbrueckii and S. cerevisiae yeasts, an understanding of which will allow winemakers to make better use of such microbial tools to achieve a desired wine sensory outcome.

Vineyard Soil Microbiome Composition Related to Rotundone Concentration in Australian Cool Climate 'Peppery' Shiraz Grapes.

Soil microbial communities have an integral association with plants and play an important role in shaping plant nutrition, health, crop productivity and product quality. The influence of bacteria and fungi on wine fermentation is well known. However, little is known about the role of soil microbes, other than microbial pathogens, on grape composition or their role in vintage or site (terroir) impacts on grape composition. In this study, we used an amplicon sequencing approach to investigate the potential relationships between soil microbes and inherent spatial variation in grape metabolite composition - specifically, the concentration of the 'impact aroma compound' rotundone in Shiraz grapes (Vitis vinifera L.) grown in a 6.1 ha vineyard in the Grampians region of Victoria, Australia. Previous work had demonstrated temporal stability in patterns of within-vineyard spatial variation in rotundone concentration, enabling identification of defined 'zones' of inherently 'low' or 'high' concentration of this grape metabolite. 16S rRNA and ITS region-amplicon sequencing analysis of microbial communities in the surface soils collected from these zones indicated marked differences between zones in the genetic diversity and composition of the soil bacterial and fungal microbiome. Soils in the High rotundone zone exhibited higher diversity of bacteria, but lower diversity of fungi, compared to the soils in the Low rotundone zone. In addition, the network analysis of the microbial community in the High rotundone zone soils appeared well structured, especially with respect to the bacterial community, compared to that in the Low rotundone zone soils. The key differences in the microbial community structure between the rotundone zones are obvious for taxa/groups of both bacteria and fungi, particularly for bacteria belonging to Acidobacteria-GP4 and GP7, Rhizobiales, Gaiellaceae, Alphaproteobacteria and the Nectriaceae and Tremellaceae families of fungi. Although mulching in some parts of the vineyard caused changes in bacterial and fungal composition and overall microbial catabolic diversity and activity, its effects did not mask the rotundone zone-based variation. This finding of a systematic rotundone zone-based variation in soil microbiomes suggests an opportunity to bring together understanding of microbial ecology, plant biochemistry, and viticultural management for improved management of grape metabolism, composition and wine flavor.

Understanding Yeast Impact on 1,1,6-Trimethyl-1,2-dihydronaphthalene Formation in Riesling Wine through a Formation-Pathway-Informed Hydrolytic Assay.

The occurrence in Riesling wine of the potent odorant 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN) is dependent upon vineyard and winemaking conditions, and TDN can have a prominent impact on the attributes of a wine after years in a bottle. As such, immediately assessing the impact of vineyard or winery treatments on future TDN formation requires forced creation of the aroma compound under non-wine-like conditions from other precursors. Here, we use a Box-Behnken approach and known TDN end points in commercial wines to optimize the conditions (pH, temperature, and time) of a "total TDN" hydrolytic assay for Riesling wine, which was intended to not interfere with yeast-derived formation pathways. The new assay (75 °C, pH 1.7, and 60 min) was used to determine the role of industry-relevant commercial yeasts as well as novel hybrid yeast strains on total TDN concentrations in young Riesling wines. While significant differences were observed between some yeasts, the impact of defoliation as a viticultural intervention outweighed yeast effects, suggesting that elevated TDN concentrations in wine are likely due to grape growing conditions and cannot be readily reduced or compensated for in the winery.

Rapid isolation of red wine polymeric polyphenols by solid-phase extraction.

A rapid technique for the isolation of polymeric polyphenols from red wine has been developed and validated. A copolymer reversed-phase SPE cartridge was utilized in conjunction with predominantly organic eluents to provide three phenolic fractions from red wine without the need for sample pretreatment. The first fraction contained the bulk of the monomeric and oligomeric phenolic material, while the second and third fractions contained the polymeric polyphenolic compounds, as determined by HPLC analysis. The two polymeric polyphenolic fractions differed in their solubility and extent of pigmentation, and the differences appeared to be related to wine age. This method contrasted with other available fractionation techniques because the interfering, nonpolymeric material can be removed in a single wash fraction, while the polymeric material is separated into two distinct fractions based on their diverse physicochemical properties. It is anticipated that the rapid access to discrete polymeric fractions afforded by this method will be of benefit in furthering the understanding of red wine polymeric polyphenols.

From wine to pepper: rotundone, an obscure sesquiterpene, is a potent spicy aroma compound.

An obscure sesquiterpene, rotundone, has been identified as a hitherto unrecognized important aroma impact compound with a strong spicy, peppercorn aroma. Excellent correlations were observed between the concentration of rotundone and the mean 'black pepper' aroma intensity rated by sensory panels for both grape and wine samples, indicating that rotundone is a major contributor to peppery characters in Shiraz grapes and wine (and to a lesser extent in wine of other varieties). Approximately 80% of a sensory panel were very sensitive to the aroma of rotundone (aroma detection threshold levels of 16 ng/L in red wine and 8 ng/L in water). Above these concentrations, these panelists described the spiked samples as more 'peppery' and 'spicy'. However, approximately 20% of panelists could not detect this compound at the highest concentration tested (4000 ng/L), even in water. Thus, the sensory experiences of two consumers enjoying the same glass of Shiraz wine might be very different. Rotundone was found in much higher amounts in other common herbs and spices, especially black and white peppercorns, where it was present at approximately 10000 times the level found in very 'peppery' wine. Rotundone is the first compound found in black or white peppercorns that has a distinctive peppery aroma. Rotundone has an odor activity value in pepper on the order of 50000-250000 and is, on this criterion, by far the most powerful aroma compound yet found in that most important spice.

Aroma Precursors in Grapes and Wine: Flavor Release during Wine Production and Consumption.

Pioneering investigations into precursors of fruity and floral flavors established the importance of terpenoid and C13-norisoprenoid glycosides to the flavor of aromatic wines. Nowadays flavor precursors in grapes and wine are known to be structurally diverse, encompassing glycosides, amino acid conjugates, odorless volatiles, hydroxycinnamic acids, and many others. Flavor precursors mainly originate in the grape berry but also from oak or other materials involved in winemaking. Flavors are released from precursors during crushing and subsequent production steps by enzymatic and nonenzymatic transformations, via microbial glycosidases, esterases, C-S lyases, and decarboxylases, and through acid-catalyzed hydrolysis and chemical rearrangements. Flavors can also be liberated from glycosides and amino acid conjugates by oral microbiota. Hence, it is increasingly likely that flavor precursors contribute to retronasal aroma formation through in-mouth release during consumption, prompting a shift in focus from identifying aroma precursors in grapes to understanding aroma precursors present in bottled wine.

Investigation of 'stone fruit' aroma in Chardonnay, Viognier and botrytis Semillon wines.

Despite numerous studies, the identity of the compounds that are responsible for 'stone fruit' aroma in wine has not been conclusively established. This study focussed on wine varieties that often display peach and apricot characters, such as Chardonnay, Viognier and botrytis-affected sweet Semillon wines. Wines with high and low 'stone fruit' aroma were evaluated by gas chromatography-olfactometry-mass spectrometry (GC-O-MS) using extracts representative of the aroma of the wine in a glass. No aroma-active zone was described as 'stone fruit' aroma across all three wine varietals. However, for the individual varieties, terpenes, such as linalool and geraniol, in the Viognier wines, several esters in the Chardonnay wines, and γ-nonalactone in the botrytis Semillon were associated with 'stone fruit' aroma. Notably, this is the first study assessing the aroma profile of Viognier wine by GC-O. In addition, an extension study of Viognier grape monoterpene profiles clarified its classification as an aromatic variety.

Analysis, potency and occurrence of (Z)-6-dodeceno-γ-lactone in white wine.

(Z)-6-Dodeceno-γ-lactone is a potent aroma compound that has been little studied and its prevalence in wines is unknown. An efficient stable isotope dilution assay was developed using a simple, direct immersion solid-phase microextraction and gas chromatography-tandem mass spectrometry method suitable for routine use with a low ng/L limit of quantitation. Using this method, 99 out of 104 young white wines analysed were found to contain detectable (Z)-6-dodeceno-γ-lactone. The highest concentrations were found in Riesling and Viognier wines. (Z)-6-Dodeceno-γ-lactone was found to have an aroma detection threshold of 700 ng/L in a neutral white wine. This study established that (Z)-6-dodeceno-γ-lactone is widely present in Australia's most popular white wine varieties, but generally at concentrations below its aroma detection threshold.

Volatile Compounds Related to 'Stone Fruit' Aroma Attributes in Viognier and Chardonnay Wines.

A 'stone fruit' aroma is important in many white wine varieties and styles, but little is known about the chemical basis of this wine aroma attribute. A set of Viognier and Chardonnay wines that featured 'stone fruit' aroma attributes were selected by a panel of wine experts. The selected wines were characterized by sensory descriptive analysis and detailed volatile chemical composition analyses. This comprehensive data also allowed Viognier wine to be profiled for the first time. By partial least-squares regression, several esters and fatty acids and benzaldehyde were indicated as contributing to the 'peach' attribute; however, a reconstitution sensory study was unsuccessful in mimicking this attribute. A mixture of γ-lactones, monoterpenes, and aldehydes were positively correlated to the 'apricot' aroma, which were generally higher in the Viognier wines. Reconstitution studies confirmed that the monoterpenes linalool, geraniol, and nerol were the most important compounds for the mixture being perceived as having an 'apricot' aroma.

High throughput analysis of red wine and grape phenolics-adaptation and validation of methyl cellulose precipitable tannin assay and modified Somers color assay to a rapid 96 well plate format.

The methyl cellulose precipitable (MCP) tannin assay and a modified version of the Somers and Evans color assay were adapted to high-throughput (HTP) analysis. To improve efficiency of the MCP tannin assay, a miniaturized 1 mL format and a HTP format using 96 well plates were developed. The Somers color assay was modified to allow the standardization of pH and ethanol concentrations of wine samples in a simple one-step dilution with a buffer solution, thus removing inconsistencies between wine matrices prior to analysis and allowing for its adaptation to a HTP format. Validation studies showed that all new formats were efficient, and results were reproducible and analogous to the original formats.