2.4. Protein extraction

Fruit samples were freeze-dried and crushed using a mortar containing liquid N₂ to obtain a fine powder. Proteins were extracted in parallel through a modified version of the phenol extraction method (Saravanan and Rose, 2004). Thus, 1 g of fine powder of each sample added with 0.01 g of polyvinylpyrrolidone was resuspended in a buffer consisting in 0.7 M sucrose, 0.1 M KCl, 0.5 M Tris-HCl (pH 8.8), 50 mM EDTA, 40 mM DL-dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, plus 30 µL of protease inhibitors cocktail for plant tissues (Sigma-Aldrich, USA). Three independent biological replicates were analyzed in comparison for each cultivar. The mixtures were homogenized with T10 basic Ultra-Turrax™ (IKA, Staufen, Germany) with 5 cycles (20,000 rpm) of 10 sec, and 20-sec pauses on ice. Afterward, an equal volume of saturated phenol in 0.5 M Tris-HCl, pH 8.1 (Sigma-Aldrich) was added, and the solution was incubated on a shaker for 20 min, at 4°C. The samples were centrifuged at 10,000 g for 10 min, at 4°C. The phenolic phase was recovered carefully to avoid contact with the interphase and poured into a new tube. This phenol phase was then back-extracted with 3 mL of resuspension buffer. All samples were shaken for 3 min and then vortexed. The phenolic phase was further recovered by centrifugation at 10000 g for 10 min, at 4°C. Proteins were precipitated by adding six vol of ice-cold 0.1 M ammonium acetate in methanol, overnight, at -20°C. Protein pellets were then washed three times with ice-cold 0.1 M ammonium acetate in methanol, and three times with ice-cold acetone. Protein pellets were air-dried and then solubilized in 600 μL of 8 M urea, 50 mM triethylammonium bicarbonate (TEAB), pH 8.5. Samples were vortexed, sonicated in ultrasonic bath for 5 min, and left on a shaker at 22°C, overnight. Samples were centrifuged at 12,000 rpm for 30 min, at 22°C, and protein concentration was determined on the supernatant using the Pierce BCA Protein assay kit™ (Thermo Scientific, Rockford, IL, USA), according to manufacturer’s instructions. The purity and overall quality of protein extracts were evaluated with Laemmli buffer-including SDS-PAGE (Laemmli, 1970). Electrophoresis was conducted using a SE600 Chroma™ device (Hoefer, Holliston, USA). The gel was then stained with Coomassie Brilliant Blue G-250 (Bio-Rad, Hercules, CA).