Chemical wax extraction

After removal of epicuticular wax crystals, berries were extracted with chloroform for 5 min in ultra-sonic. Extracts were washed three times with deionized water to remove hydrophilic organic compounds like sugars. As was described previously, the grape berry wax consists of two fractions, the cyclic triterpenoids and a mixture of long chain aliphatics [26] which were separated with different solvents, to prevent overlying peaks in the gas chromatographic analyzing procedure (S2 Fig). After evaporation to dryness under nitrogen, the residue was dispensed three times in methanol to dissolve the more polar triterpenoid wax fraction. The methanol extracts were filtered and the remaining residue containing the more apolar, aliphatic wax components was dissolved in chloroform. N-tetracosane was added to both fractions as internal standard.