Abscisic acid

Three grams of pulp tissues was extracted with 40 ml of 80% (v/v) methanol at –20 °C for 18 h. The methanol extracts were then centrifuged at 10 000 g for 20 min, and the pellet was extracted twice with 20 ml of 80% methanol at –20 °C for 2 h. The supernatants were subsequently combined and dried under vacuum, and the residue was dissolved in 10 ml of 0.02 M phosphate assay buffer (pH 8.0) and extracted three times with 10 ml petroleum ether. The organic phase was removed, and the pH of the aqueous phase was adjusted to 8.0, followed by the addition of 0.2 g of insoluble polyvinylpolypyrrolidone. After stirring for 15 min at 0 °C, polyvinylpolypyrrolidone was removed through vacuum filtration. The mixed liquid was adjusted to pH 3.0 and subsequently extracted three times with ethyl acetate. The ethyl acetate phase was dried under vacuum and dissolved in 1 ml 50% methanol (v/v). The ABA content was determined via HPLC (Agilent Technologies 1200) using a 4.8×150 mm C18 column (Agilent Technologies), at a flow rate of 0.8 ml min⁻¹. Elution was performed using both solvent A (0.8% (v/v) glacial acetic acid) and solvent B (100% methanol). We employed (±)-abscisic acid (Sigma-Aldrich, St Louis, MO, USA) as the standards for determination at 260 nm. Three replicates were conducted for each sample.