Part V — Chapter 15

Flavonoids & Anthocyanins

The flavonoid pathway branches from the phenylpropanoid route to produce UV-absorbing flavonols, pH-dependent anthocyanin pigments, and polymerised condensed tannins — with over 9,000 known structures.

Flavonoid Pathway: From Chalcone to Anthocyanins

Chalcone Synthase (CHS) — Gateway Enzyme

Chalcone synthase (CHS) is a type III polyketide synthase that catalyses the first committed step of flavonoid biosynthesis. It uses a Cys-His-Asn catalytic triad and performs sequential decarboxylative condensation of three malonyl-CoA extender units onto p-coumaroyl-CoA, then intramolecular cyclisation to produce naringenin chalcone.

CHS Reaction Stoichiometry

\( \text{p-coumaroyl-CoA} + 3\,\text{malonyl-CoA} \xrightarrow{\text{CHS}} \text{naringenin chalcone} + 4\,\text{CoA} + 3\,\text{CO}_2 \)

Each malonyl-CoA provides a 2-carbon unit via decarboxylation; the starter unit (p-coumaroyl-CoA) provides the B-ring and propanoid chain.

Chalcone Isomerase (CHI)

CHI catalyses the stereospecific intramolecular ring closure of chalcone to (2S)-flavanone. The reaction is \( 10^7 \)-fold faster than the spontaneous non-enzymatic isomerisation and is essential for chiral specificity.

\( \text{naringenin chalcone} \xrightarrow{\text{CHI}} \text{(2S)-naringenin} \)

Pathway Branching: Flavonols vs Anthocyanins

Toward Anthocyanins

F3H (flavanone 3-hydroxylase)

Naringenin → DHK

2-ODD dioxygenase, Fe²⁺ and 2-oxoglutarate dependent. Adds 3-OH to flavanone ring.

F3'H / F3'5'H (CYP450 hydroxylases)

B-ring hydroxylation

F3'H gives eriodictyol/DHQ (pink-red); F3'5'H gives pentahydroxy substrate (blue-violet).

DFR (dihydroflavonol 4-reductase)

DHK/DHQ/DHM → leucoanthocyanidins

NADPH-dependent. Stereospecific (3R,4S). Rate-determining for anthocyanin flux.

ANS / LDOX (anthocyanidin synthase)

Leucoanthocyanidin → anthocyanidin

2-ODD dioxygenase; requires Fe²⁺, O₂, and 2-oxoglutarate. Oxidative desaturation.

UFGT (UDP-glucose:flavonoid 3-O-glucosyltransferase)

Anthocyanidin → anthocyanin

Glycosylation stabilises the anthocyanidin; vacuolar import follows via MATE transporters.

Competing Branches

FLS (flavonol synthase)

DHK → kaempferol; DHQ → quercetin

2-ODD dioxygenase. Competes with DFR for dihydroflavonol substrates. UV-B inducible.

IFS (isoflavone synthase)

Liquiritigenin → genistein

CYP93C; aryl ring migration (1,2-aryl shift). Legume-specific; phytoestrogens.

LAR (leucoanthocyanidin reductase)

Leucoanthocyanidin → (2R,3S)-flavan-3-ol

NADPH-dependent. Initiates condensed tannin polymer (proanthocyanidin) formation.

ANR (anthocyanidin reductase)

Anthocyanidin → (2R,3R)-epicatechin

NADPH-dependent; alternative entry to condensed tannins; seed coat browning.

Transcriptional Regulation (MBW complex)

Structural genes are co-regulated by the MBW complex: MYB (R2R3-MYB transcription factor) + bHLH + WD-repeat protein. In Arabidopsis: PAP1/2 (MYB), TT8 (bHLH), TTG1 (WD40) activate DFRA, ANS, UFGT. UV-B activates HY5 which directly induces CHS.

pH-Dependent Anthocyanin Color

Anthocyanins are unique among plant pigments in their pH-dependent structural transformations. The coloured flavylium cation (AH⁺) predominates at pH < 3 (red), the purple quinoidal base (A) at pH 5–6, and the colourless carbinol pseudobase (B) above pH 7.

pH < 3

Flavylium (AH⁺)

Red colour. Protonated oxonium ion. Flowers of Pelargonium and Dahlia. Vacuolar pH ~4.

pH ~5

Quinoidal (A)

Purple/blue colour. Deprotonation of 7-OH. Co-pigmentation with flavonols enhances blue colour.

pH > 7

Carbinol (B)

Colourless. Nucleophilic attack of water at C-2. Anthocyanins bleach at neutral-alkaline pH.

Proanthocyanidins (Condensed Tannins)

Flavan-3-ol units (catechin, epicatechin) polymerise via C4→C8 or C4→C6 bonds to form condensed tannins (degree of polymerisation 2–50). They precipitate proteins, act as feeding deterrents, and confer astringency in wine and tea. Seed coats accumulate proanthocyanidins via the ANR/LAR branches; the TT locus (transparent testa) mutants of Arabidopsis lack proanthocyanidins and have golden-yellow (transparent) seed coats.

\( n \times \text{flavan-3-ol} \xrightarrow{\text{LAC/PRX oxidation}} \text{proanthocyanidin} \, (\text{DP}\,2{-}50) \)

Python: Anthocyanin Color as a Function of pH

Simulate anthocyanin absorbance spectra (cyanidin-3-glucoside) at different pH values using equilibrium structural forms, and model the complete flavonoid-to-anthocyanin pathway flux.

Python

script.py128 lines

import numpy as np
import matplotlib
matplotlib.use('Agg')
import matplotlib.pyplot as plt

# Model anthocyanin absorbance spectra and color as a function of pH
# Anthocyanins exist as 4 equilibrium forms: flavylium cation (AH+), quinoidal base (A),
# carbinol pseudobase (B), and chalcone (C)
# At low pH: AH+ dominates (red, lambda_max ~520 nm)
# At intermediate pH: A quinoidal forms (purple, lambda_max ~560 nm)
# At high pH: B/C forms (colourless/yellow)

# Model cyanidin-3-glucoside (most common anthocyanin) pH response
pH_vals = np.linspace(1, 10, 500)
wavelengths = np.linspace(380, 720, 340)

# pKa values for cyanidin-3-glucoside
pKa1 = 2.6   # AH+ -> A (quinoidal)
pKa2 = 4.5   # AH+ -> B (hydration, pseudo-base formation)

# Fraction of each form
def fractions(pH, pKa1, pKa2):
    Ka1 = 10**(-pKa1)
    Ka2 = 10**(-pKa2)
    H = 10**(-pH)
    # Simplified: treat as two competing equilibria
    fAH = H**2 / (H**2 + Ka1 * H + Ka1 * Ka2)
    fA = Ka1 * H / (H**2 + Ka1 * H + Ka1 * Ka2)
    fB = Ka1 * Ka2 / (H**2 + Ka1 * H + Ka1 * Ka2)
    return fAH, fA, fB

fAH_arr, fA_arr, fB_arr = fractions(pH_vals, pKa1, pKa2)

# Simulate absorbance spectrum at different pH values
def gaussian(x, mu, sigma, height):
    return height * np.exp(-0.5 * ((x - mu) / sigma)**2)

def spectrum_at_pH(pH_val):
    fAH, fA, fB = fractions(pH_val, pKa1, pKa2)
    # AH+ (flavylium): red, lambda_max ~ 520 nm
    spec = fAH * gaussian(wavelengths, 520, 28, 1.0)
    # A (quinoidal): purple, lambda_max ~ 560 nm
    spec += fA * gaussian(wavelengths, 560, 32, 0.85)
    # Minor UV absorption (all forms)
    spec += 0.15 * gaussian(wavelengths, 280, 20, 1.0)
    # B form: faint yellow / colourless
    spec += fB * gaussian(wavelengths, 415, 25, 0.3)
    return spec

fig, axes = plt.subplots(2, 2, figsize=(13, 9))
fig.patch.set_facecolor('#0f172a')
for ax in axes.flat:
    ax.set_facecolor('#1e293b')
    ax.tick_params(colors='#94a3b8')
    ax.spines['bottom'].set_color('#475569')
    ax.spines['left'].set_color('#475569')
    ax.spines['top'].set_visible(False)
    ax.spines['right'].set_visible(False)

# Plot 1: Absorbance spectra at pH 2, 4, 6, 8
ph_colors = {2.0: '#ef4444', 4.0: '#a855f7', 6.0: '#3b82f6', 8.0: '#6b7280'}
for ph_val, col in ph_colors.items():
    spec = spectrum_at_pH(ph_val)
    axes[0, 0].plot(wavelengths, spec, color=col, linewidth=2.5,
                    label=f'pH {ph_val}')
axes[0, 0].set_xlabel('Wavelength (nm)', color='#94a3b8', fontsize=9)
axes[0, 0].set_ylabel('Absorbance (a.u.)', color='#94a3b8', fontsize=9)
axes[0, 0].set_title('Anthocyanin Absorbance Spectra vs pH', color='#e2e8f0', fontsize=10)
axes[0, 0].legend(fontsize=8, facecolor='#1e293b', edgecolor='#475569', labelcolor='#e2e8f0')
axes[0, 0].axvspan(620, 720, alpha=0.05, color='red')
axes[0, 0].axvspan(490, 560, alpha=0.05, color='green')
axes[0, 0].axvspan(430, 490, alpha=0.05, color='blue')

# Plot 2: Fraction of each form vs pH
axes[0, 1].fill_between(pH_vals, fAH_arr, alpha=0.6, color='#ef4444', label='Flavylium (AH+) - red')
axes[0, 1].fill_between(pH_vals, fA_arr, alpha=0.6, color='#8b5cf6', label='Quinoidal (A) - purple')
axes[0, 1].fill_between(pH_vals, fB_arr, alpha=0.6, color='#94a3b8', label='Carbinol (B) - colourless')
axes[0, 1].set_xlabel('pH', color='#94a3b8', fontsize=9)
axes[0, 1].set_ylabel('Mole fraction', color='#94a3b8', fontsize=9)
axes[0, 1].set_title('pH-Dependent Anthocyanin Equilibrium Forms', color='#e2e8f0', fontsize=10)
axes[0, 1].legend(fontsize=7.5, facecolor='#1e293b', edgecolor='#475569', labelcolor='#e2e8f0')
axes[0, 1].axvline(x=pKa1, color='#475569', linestyle=':', alpha=0.8)
axes[0, 1].axvline(x=pKa2, color='#475569', linestyle=':', alpha=0.8)
axes[0, 1].text(pKa1 + 0.05, 0.75, f'pKa1={pKa1}', color='#64748b', fontsize=8)
axes[0, 1].text(pKa2 + 0.05, 0.55, f'pKa2={pKa2}', color='#64748b', fontsize=8)

# Plot 3: Flavonoid pathway flux (arbitrary units)
enzymes = ['CHS', 'CHI', 'F3H', 'F3 5H', 'DFR', 'ANS', 'UFGT']
flux = [100, 95, 80, 60, 55, 50, 48]
colors3 = ['#a3e635', '#4ade80', '#34d399', '#22d3ee', '#60a5fa', '#a78bfa', '#f472b6']
bars = axes[1, 0].bar(enzymes, flux, color=colors3, edgecolor='#475569', alpha=0.85)
axes[1, 0].set_xlabel('Enzyme', color='#94a3b8', fontsize=9)
axes[1, 0].set_ylabel('Relative Flux (%)', color='#94a3b8', fontsize=9)
axes[1, 0].set_title('Flavonoid Pathway Flux to Anthocyanins', color='#e2e8f0', fontsize=10)
for bar, val in zip(bars, flux):
    axes[1, 0].text(bar.get_x() + bar.get_width()/2, bar.get_height() + 1,
                    str(val), ha='center', va='bottom', color='#e2e8f0', fontsize=8)

# Plot 4: Effect of pH on visible color (650nm / 520nm ratio as proxy)
color_ratio = []
for pH_val in pH_vals:
    spec = spectrum_at_pH(pH_val)
    idx_520 = np.argmin(np.abs(wavelengths - 520))
    idx_560 = np.argmin(np.abs(wavelengths - 560))
    idx_415 = np.argmin(np.abs(wavelengths - 415))
    total = spec[idx_520] + spec[idx_560] + 0.001
    color_ratio.append(spec[idx_560] / total)

axes[1, 1].plot(pH_vals, color_ratio, color='#a78bfa', linewidth=2.5)
axes[1, 1].fill_between(pH_vals, color_ratio, alpha=0.2, color='#8b5cf6')
axes[1, 1].set_xlabel('Vacuolar pH', color='#94a3b8', fontsize=9)
axes[1, 1].set_ylabel('Purple/Red absorbance ratio', color='#94a3b8', fontsize=9)
axes[1, 1].set_title('Color Shift: Red (low pH) to Purple (high pH)', color='#e2e8f0', fontsize=10)
axes[1, 1].axvspan(1, 3, alpha=0.1, color='red')
axes[1, 1].axvspan(4.5, 6.5, alpha=0.1, color='purple')
axes[1, 1].text(1.5, 0.6, 'Red', color='#ef4444', fontsize=10, fontweight='bold')
axes[1, 1].text(4.8, 0.55, 'Purple', color='#a855f7', fontsize=10, fontweight='bold')
axes[1, 1].text(7.0, 0.2, 'Colourless', color='#64748b', fontsize=9, fontweight='bold')

fig.suptitle('Anthocyanin Color Chemistry: pH-Dependent Equilibria & Spectra',
             color='#e2e8f0', fontsize=12, fontweight='bold')
plt.tight_layout()
plt.savefig('output.png', dpi=110, bbox_inches='tight',
            facecolor='#0f172a', edgecolor='none')
print("pH of maximum red (flavylium):", round(pH_vals[np.argmax(fAH_arr)], 2))
print("pH of max quinoidal (purple):", round(pH_vals[np.argmax(fA_arr)], 2))
print("At pH 7: colourless fraction:", round(float(fractions(7, pKa1, pKa2)[2]), 3))

Click Run to execute the Python code

Code will be executed with Python 3 on the server

← Ch 14: Alkaloid Biosynthesis Part VI: Carbohydrates →