Quantification of Soluble Sugars in Leaf Samples

Soluble sugars (sucrose, fructose, glucose, and sorbitol) were determined in approximately 400 mg of powdered frozen leaf material, based on the method previously described [63 (link)] with alterations as in [64 (link)]. Briefly, the samples were homogenized in 4 mL of cold H₂O with 50 mg of polyvinylpolypyrrolidone, left to extract for 20 min on ice at 100 rpm and centrifuged (12,000× g, 5 min, 4 °C). The supernatant was boiled to denature the proteins (3 min), placed on ice (6 min) and centrifuged again. The obtained clear solution was then filtered (0.45 µm, nylon) before the injection of a 50 μL aliquot into an HPLC system equipped with a refractive index detector (Model 2414, Waters, Milford, MA, USA). The separation of sugars was performed using a SugarPak 1 column (300 × 6.5 mm, Waters) at 90 °C, with H₂O as the eluent (containing 50 mg EDTA-Ca L⁻¹ H₂O) and a flow rate of 0.5 mL min⁻¹ for 22 min. Standard curves were used for the quantification of each sugar.