Module 2: Phenolics, Tannins, Anthocyanins

Grape flavonoids derive from the shikimate pathway via phenylalanine. The general phenylpropanoid pathway (PAL, C4H, 4CL) produces 4-coumaroyl-CoA, which condenses with three malonyl-CoA (chalcone synthase, CHS) to naringenin chalcone; chalcone isomerase (CHI) then cyclises to the basic flavonoid skeleton. Flavonoid-3-hydroxylase (F3H), flavonoid-3',5'-hydroxylase (F3'5'H), and dihydroflavonol-4-reductase (DFR) build the dihydroflavonol backbone, which is partitioned at the key branch point.

The Anthocyanin vs. Proanthocyanidin Branch

From leucoanthocyanidin the pathway diverges into two competing branches:

• Anthocyanin branch: leucoanthocyanidin → anthocyanidin (ANS/LDOX) → anthocyanin-3-O-glucoside (UFGT). Expressed in skin of red varieties post-veraison; activated by MYBA1.
• Proanthocyanidin branch: leucoanthocyanidin → (+)-catechin (LAR) or anthocyanidin → (-)-epicatechin (ANR) → condensed tannins. Expressed early in skin and throughout seed development; activated by MYBPA1/2.

The balance between the two branches is set by relative transcription-factor activity: MYBA1/MYBA2 drive the anthocyanin branch while MYBPA1/MYBPA2 (together with bHLH and WD40 partners forming the canonical MBW complex) drive the tannin branch. Simulation 1 integrates the pathway as a system of ODEs and shows how switching the TF balance reroutes flux between skin color and seed tannin.

Boss et al. (1996) first identified a MYB-family transcription factor on chromosome 2 whose expression is necessary and sufficient to activate the final structural genes of anthocyanin biosynthesis (ANS, UFGT) in the berry skin. Kobayashi et al. (2004) and Walker et al. (2007) sequenced the promoter region of VvMybA1 in white and red varieties and showed that white grapes carry a ~10 kbp Gret1 Ty3-like retrotransposon inserted at position - 79 bp upstream of the MybA1transcription start site. The insertion abolishes MybA1 expression; in homozygous ww genotypes no skin anthocyanin is produced and the berry remains green–yellow.

Bud-Sports and Revertants

Most V. vinifera white cultivars (Chardonnay, Riesling, Sauvignon Blanc, Chenin Blanc, Muscat Blanc) share the ww genotype and therefore descend from a single ancestral Gret1 insertion event. Pinot Gris and Pinot Blanc are bud-sports of Pinot Noir in which somatic excision or internal deletion of Gret1 has partly restored MybA1 expression on one chromosome, producing intermediate (grey) or patchy pink pigmentation. The converse event — a new Gret1 insertion into a colored cultivar — is rarer but has been documented in natural populations.

UV-Responsive Transcription

VvMybA1 expression is itself modulated by UV-B: Koyama et al. (2012) showed that sun-exposed clusters accumulate more anthocyanin than shaded clusters, via up-regulation of VvHY5 and downstream MybA1 induction. Berry skin acts as an endogenous UV sunscreen: the same MYB-driven pigments that supply wine color also protect the seed from UV-induced DNA damage, and are under strong photoecological selection.

V. vinifera berries produce six major anthocyanidin skeletons, each glucosylated at the 3-position (most cultivars) and optionally acylated at the 6'':

• Delphinidin-3-glucoside (3 OH on B-ring): bluish, typically 10–20% of skin pigment.
• Cyanidin-3-glucoside (2 OH): cherry red, 5–15%.
• Petunidin-3-glucoside: 10–20%, intermediate.
• Peonidin-3-glucoside (methoxy-cyanidin): orange-red, 10–20%.
• Malvidin-3-glucoside (di-methoxy-delphinidin): deep purple, ~50–60% in ripe Cabernet Sauvignon and most teinturier cultivars.
• Pelargonidin-3-glucoside: orange; only trace amounts in V. vinifera.

pH-Dependent Color Equilibrium

Anthocyanins exist as a pH-dependent equilibrium between four species (Brouillard 1982):

At wine pH (3.3–3.8), ~40% of malvidin-3-glucoside is in the red flavylium form, ~50% is hydrated to the colorless hemiketal, and small amounts of chalcone and quinoidal species contribute yellow and blue tones. Co-pigmentation with flavonols and tannins stabilises the flavylium form and deepens wine color; copigmentation effects can account for 30–50% of the observed absorbance of young red wines.

Acylation and Stability

Coumaroylated and acetylated anthocyanins (e.g. malvidin-3-(6''-p-coumaroyl)-glucoside) are more stable than their non-acylated counterparts by a factor of 2–5 against hydrolysis, and dominate the pigment of aged red wines. Acylation at the 6''-OH protects the glycosidic linkage and favors intramolecular stacking that shields the flavylium chromophore from nucleophilic attack by water.

Condensed tannins, also known as proanthocyanidins (PAs), are oligomeric and polymeric flavan-3-ols: chains of (+)-catechin and (-)-epicatechin monomers linked by C4→C8 (and occasionally C4→C6) bonds. Degree of polymerization (DP) ranges from 2 (dimers, e.g. B1–B4) to ~30 in the polymer fraction. Grape skins produce procyanidin-rich tannins with mean DP (mDP) ~8–10; seed tannins have lower mDP (3–5) but much higher galloylation and strong bitterness.

Hydrolysable vs. Condensed Tannins

Hydrolysable tannins (ellagitannins, gallotannins) are based on a glucose core with galloyl or ellagoyl esters. These are major constituents of oak wood and are extracted into wine during barrel aging; V. vinifera berries themselves contain only trace hydrolysable tannins. The dominant native grape tannin is thus condensed (flavan-3-ol).

Evolution of Tannin Structure During Ripening

Tannin biosynthesis proceeds mainly pre-veraison (Adams 2006); during Stages II and III tannin mass is relatively constant, but tannin structure changes: (i) mDP rises as monomers extend existing polymers, (ii) proportion of trihydroxylated subunits (epigallocatechin, EGC) in skin tannins increases, (iii) non-covalent binding to skin polysaccharides (mannoproteins, pectin) increases, and (iv) oxidative coupling to anthocyanins begins. These changes modify the tannin extraction behavior during vinification and the subsequent stability of wine color and mouth-feel.

Astringency is the tactile (not gustatory) sensation of dryness, roughness, and puckering in the mouth. It is mediated primarily by binding of condensed tannins to salivary proline-rich proteins (PRPs), which constitute ~70% of total salivary protein by mass. Tannin-PRP complexes aggregate and precipitate, coating the oral mucosa and reducing lubrication.

Proline-Rich Proteins

PRPs (acidic, basic, glycosylated) have polyproline-II secondary structures with exposed amide bonds and proline ring surfaces that stack efficiently with aromatic flavan-3-ol rings via hydrophobic + H-bond interactions. Tannins with higher mDP bind more tightly because they can engage multiple proline sites simultaneously (avidity effect). Pascal et al. (2007) measured dissociation constants in the range 50–500 mg/L (CAT eq.) depending on mDP.

Gawel 1997 Mouth-Feel Wheel

Gawel (1997) introduced a formal sensory vocabulary for tannin-driven mouth-feel: grainy, silky, velvety, dry, chalky, pucker, coarse, fine. The sensation scales cooperatively with tannin concentration; Simulation 2 models this as a Hill dose-response of bound PRP fraction multiplied by mDP (which proxies for precipitation propensity).

Polyphenol-Protein Precipitation

Above a threshold tannin load, PRP-tannin aggregates nucleate into soluble colloids, grow, and eventually precipitate. The aggregate’s size scales with tannin mass, explaining why very-high-tannin wines (Madiran, Nebbiolo, vintage Bordeaux) feel harsher in the mouth: larger aggregates are mechanically more perceptible.

Branching off the same 4-coumaroyl-CoA precursor that feeds chalcone synthase, the grapevine stilbene synthase (STS, VvSTS family, ~48 paralogs) condenses 4-coumaroyl-CoA with three malonyl-CoA to produce trans-resveratrol (3,5,4'-trihydroxy-trans-stilbene). The STS family is strongly induced by Botrytis cinerea and Plasmopara viticola infection, by UV-C exposure, and by mechanical wounding; resveratrol and its derivatives (viniferins, pterostilbene, piceid) act as phytoalexins that inhibit fungal growth.

The French Paradox, Carefully

Renaud and de Lorgeril (1992) proposed that the relatively low rate of coronary heart disease in southern France despite a high intake of saturated fat (“French paradox”) might be partly explained by red-wine polyphenols. Subsequent in vitro work on resveratrol showed anti-oxidant, anti-inflammatory, and sirtuin-activating properties. However, red wine contains only 1–15 mg/L of resveratrol, and human dose-response studies have consistently failed to reproduce in vitro findings at dietary doses. Pragmatic conclusion: the “health benefits of red wine” narrative remains scientifically controversial and is not a public-health recommendation.

Phenolic extraction from skin and seed during fermentation is one of the central levers available to the winemaker. Major tools:

• Pre-fermentation cold soak (4–14 °C, 3–7 days): extracts water-soluble anthocyanins and low-MW tannins before ethanol accumulation.
• Cap management (punch-down, pump-over, delestage): regulates contact between liquid and skin/seed cap; higher intensity favors tannin extraction.
• Extended maceration post-fermentation (1–4 weeks): promotes polymerization of anthocyanin-tannin-polysaccharide adducts and softening of perceived tannin.
• Lees aging: dead-yeast polysaccharides (mannoproteins) bind tannin and reduce astringency; widely used in sur-lie Sauvignon Blanc and Chardonnay.
• Barrel aging (French or American oak): introduces hydrolysable tannins (ellagitannins) and accelerates anthocyanin-tannin co-oxidation.

Anthocyanin-Tannin Polymerization

During wine aging, anthocyanins react with tannin flavan-3-ol monomers via two main routes: direct condensation producing type-A bridge pigments (vitisins) and acetaldehyde-mediated coupling producing ethyl-bridged flav-anth pigments. The resulting polymeric pigments are more stable against pH change, oxidation, and SO2 bleaching than native monomeric anthocyanins, which explains the shift from purple-red to brick-red in bottle-aged red wines. After ~5–10 years, free monomeric anthocyanins are essentially gone and all wine color is carried by polymeric pigments.

Flavonols (quercetin, kaempferol, myricetin, isorhamnetin) accumulate in sun-exposed skin as a direct response to UV-B stress, via the VvHY5-MYBF1-FLS regulatory axis. Shaded clusters have flavonol loads only a fraction of sun-exposed clusters. Flavonols are important copigments that stabilise the anthocyanin flavylium cation in wine, so canopy management has measurable downstream effects on wine color intensity and longevity.

UV-B and Skin Chemistry

The action spectrum of UV-B-induced flavonoid synthesis peaks at ~310 nm, the region filtered by stratospheric ozone. In high-altitude or high-UV vineyards (Mendoza, Elqui Valley, Wachau) flavonol concentrations can reach 200 mg/kg skin; in deep canopy interiors they fall below 10 mg/kg. Leaf-removal (leaf pulling, “effeuillage”) is the standard viticultural tool to adjust cluster UV exposure.

Methoxypyrazine Photo-Degradation

UV also degrades 3-isobutyl-2-methoxypyrazine (IBMP) and related compounds by photo-oxidation. In Cabernet Sauvignon and Sauvignon Blanc, which produce high IBMP levels, leaf pulling on the east-facing side of the canopy reduces the “green pepper” aroma by 50–70% without excessive exposure that would bleach anthocyanins.

Modern red-wine enology exploits two distinct solvents for phenolic extraction: water (pre-fermentation) and ethanol (during and after fermentation). Water preferentially extracts anthocyanins and small low-MW tannins; ethanol is required to extract high- MW tannins and seed phenolics effectively. Pre-fermentation cold soak at 4–14 °C for 3–7 days was popularised in Burgundy by Guy Accad in the 1980s and is now standard in most premium red-wine cellars.

Flash Détente and Thermovinification

For high-volume production and for salvaging Botrytis-affected or underripe fruit, thermovinification (heating must to 70 °C for 30 min before fermentation) or flash détente (heating to 80 °C followed by vacuum-induced cell rupture) rapidly solubilise skin anthocyanins and inactivate laccase and polyphenol oxidase. The resulting wines have high color density but often reduced structural complexity compared to traditional vinification.

Carbonic Maceration

In carbonic maceration (Beaujolais Nouveau style), whole intact berries are placed under a CO2 atmosphere. Intracellular fermentation by berry enzymes produces ~2% v/v ethanol within 5–10 days, extracting color and volatile esters (isoamyl acetate, ethyl cinnamate) that give the characteristic banana-bubblegum aroma. Only when berries rupture does classical yeast fermentation begin. Carbonic maceration produces low-tannin, high-color, fruit-forward wines for early drinking.

Saignee (Bleeding)

A fraction of juice is drawn off early in fermentation to concentrate the remaining skin-to-juice ratio and produce a rose or saignée wine as a by-product. This practice increases the anthocyanin and tannin concentration of the main red wine without changing its sugar level or fermentation duration.

Boss et al. (1996) published the first systematic quantification of transcript levels for every structural gene in the V. viniferaanthocyanin pathway (PAL, CHS, CHI, F3H, DFR, LDOX/ANS, UFGT) across berry development in Shiraz. Their key finding was that all structural genes remain at baseline expression until veraison, at which point they rise in near-synchrony over ~5 days. Later work (Deluc 2007, Boss & Davies 2009) showed that the synchronous induction is coordinated by the MBW complex: MYB (MybA1 for anthocyanin, MybPA1 for tannin) + bHLH (MYC-like) + WD40 (TTG1 ortholog) assemble on promoters of the late pathway genes and jointly activate transcription.

Chromatin-Level Regulation

Recent ChIP-seq studies of grape berry chromatin (Guo 2016) have revealed that VvMybA1 itself is under H3K27me3 Polycomb-mediated repression pre-veraison. Demethylation at veraison removes the repressive mark and allows MybA1 expression, placing the ultimate decision to ignite ripening at the level of chromatin remodelling rather than transcription-factor binding alone. This explains the sharp, switch-like onset of veraison: once the chromatin context permits MybA1 expression, the MBW complex self-assembles autocatalytically.

The phenolic profile of a wine is the integrated outcome of (i) the structural genes of the flavonoid pathway that are cultivar-specific, (ii) the transcription-factor switches (MYBA1, MYBPA1/2, MYBF1, HY5) that are modulated by veraison, sunlight, and biotic stress, (iii) the enological levers of extraction and aging that determine which phenolics actually reach the glass, and (iv) the biochemistry of tannin-protein binding that determines how those phenolics are perceived in the mouth. No other single class of molecules so completely links vineyard decisions to wine style.

After bottling, red wine continues to evolve chemically: monomeric anthocyanins react with tannin monomers and with each other to form polymeric pigments, generating the characteristic brick-red colour of mature wines. Over 5–10 years, free monomeric malvidin-3-glucoside drops by a factor of >10, and most of the visible pigment is carried by acetaldehyde-bridged flav-anth, pyranoanthocyanins (vitisins A and B), and polymeric pigment fractions. Tannin mean degree of polymerization rises, but the tannin becomes less astringent as larger aggregates precipitate as sediment.

Sulfite and Stability

Free SO2 (30–50 mg/L) protects wine against oxidation by forming SO3H adducts with acetaldehyde and anthocyanins. Over bottle aging, free SO2 depletes and the wine becomes vulnerable to oxidative browning (brown hue, acetaldehyde / oxidised aldehyde aromas). Moderate oxygen exposure, however, is required to drive the polymerization chemistry and build complexity: this is the rationale for barrel aging and the micro-oxygenation technology widely used in modern red-wine production.