Quantification of trehalose

Quantification of trehalose was performed as described in [55]. Fresh leaves (0.1 g) were frozen in liquid nitrogen and homogenized in a mortar. Two mL of ethanol were added; mixture was boiled for 1 h and ethanol was left to evaporate at 60° in an oven. Five mL of 5 mM sulfuric acid were added and the mixture was centrifuged at 3.200 rpm for 10 min. The supernatant was filtered through 0.2 μm pore PDVF filters and boiled in water for 1 h to hydrolyze sucrose. Once cold, the pH was neutralized with sodium hydroxide, the solution evaporated and the residue was dissolved in distilled water. A sample of 20 μL was analyzed by HPLC (Agilent 1100, CA, USA) with a Sugar Pack1 column (300 mm × 6.5 mm, Waters Corp., MA, USA) at 75 °C and coupled to a refraction index detector at 55 °C. The isocratic elution program consists of 40 min with a mobile phase 0.1% EDTA-calcium and a flux of 0.35 mL min-1 using a pressure of 38 bars. The calibration curve was prepared using trehalose at concentrations ranging 0 to 5 mg mL^− 1.