Analysis of Sugar Compounds in Leaf Samples

High Pressure Liquid Chromatography (HPLC) was used for the analysis of sugars (pectins, sucrose, fructose, and glucose) [85 (link),114 (link)]. Briefly, 100 mg of leaf samples powdered with liquid nitrogen were added to 1 mL of distilled H₂O. Samples were homogenized by Ultra-Turrax^® T-25 basic (IKA^®-Werke GmbH & Co. KG, Staufen im Breisgau, Germany), centrifuged at 3000 RCF for 5 min; the supernatants were transferred to 2 mL Eppendorf^® tubes and then centrifuged again at 12,000 RCF for 5 min (Eppendorf^® Microcentrifuge 5415D, Hamburg, Germany). Samples were filtered (0.45 µm) and 20 µL of each extract was injected into a Waters Sugar-Pak I ion exchange column (6.5 × 300 mm) at a temperature of 90 °C. The mobile phase consisted of MilliQ H₂O (pH 7) with a flow of 0.3 mL min⁻¹. The overall duration of the separation was 30 min. Identification of components was done using a Waters 2410 refractive index detector by comparing the retention times with those of reference standards. The experiment was conducted in three technical replicates for each sample. Finally, the mean and standard deviation were calculated. To verify the significance of the data obtained, the t-test (* p ≤ 0.05, ** p ≤ 0.01) were carried out.