Quantitative analysis of ABA
ABA concentration in the SEs was determined by gas chromatography-mass spectrometry-selected ion monitoring (GC-MS-SIM), based on Chen et al. (2007, 2008) with some modifications. Fifty SEs from each treatment with two replications were first frozen in liquid N2 and then freeze-dried at -40 °C for 24 h. Dried and ground samples were extracted by 10 mL of 80% (v/v) methanol (MeOH) for 1 h at 4 °C. The supernatant was collected after centrifugation, and the residue was re-extracted, following the same procedures. Ascorbic acid (2.0 mg) and butylated hydroxytoluene (0.4 mg) were added to the combined supernatants, followed by the internal ABA standard. For samples derived from controls and partial-drying treatments, 400 ng [2H6] ABA was added, and for samples from cold-storage treatments, 200 ng [2H6] ABA was used. The supernatant was reduced to 1-2 mL of aqueous residue and adjusted to pH 8.5 with 0.05 M potassium phosphate buffer (ca. 15 mL), then passed through a polyvinylpolypyrrolidone column (ca. 5.0 g). The eluate was partitioned three times against equal volumes (15 mL each) of ethyl acetate (EtOAc). The aqueous (lower) fractions were pooled, adjusted to pH 3 with 0.05 M potassium phosphate buffer (pH 2.0), and then partitioned again with EtOAc three times (3 × 15 mL). The EtOAc (upper) fractions were pooled and filtered through a 0.45 µm filter for high performance liquid chromatography (HPLC) separation.
The HPLC system (Beckman System Gold, USA) was equipped with a Lichrosphere RP-18 column (250 × 4 mm i.d. × 5 µm particle size; Merck, Germany) connected to an LS30 fluorescence spectrophotometer (PerkinElmer, UK) with a 280 nm excitation wavelength and a 355 nm emission wavelength. Gradient elution was programmed at a constant flow-rate of 0.6 mL/min with 30% MeOH containing 0.1% acetic acid (HOAc) (solvent A) and 100% MeOH (solvent B). A linear gradient profile with the following proportions of solvent A was applied (t, %A): (0, 30), (40, 100).
An appropriate fraction from the RP-18 column was collected and further eluted through a Nucleosil N(CH3)2 column (250 × 4.6 mm i.d. × 5 µm particle size; Macherey-Nagel, USA). The elution procedure was maintained for 1 h by 99.9% MeOH containing 0.1% HOAc, at a constant flow-rate of 1.0 mL/min. The purified fraction of ABA was dried under a vacuum and derivatized by adding ethereal diazomethane for methylation and then dried with N2.
The derivatized sample was dissolved in 10 µl hexane, and 1.0 µl of the solution was introduced into the GC-MS system (HP 6890 GC and 5973 MSD) working in the SIM mode and equipped with a DB-1 capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness, J&W Scientific, USA). Operating conditions for GC-MS were similar to the ones described by Nakayama et al. (2001), using helium as a carrier gas at a flow rate of 1.0 mL/min. The interface temperature of the ion source was 250 °C, and the MS source temperature was 200 °C. The electron energy was set at 70 eV and the split/splitless injector was used in the splitless mode at 250 °C. Column temperature was programmed to begin at 60 °C for 2 min, then raised linearly from 60 °C to 210 °C at a rate of 30 °C/min, and then at slower rate of 2 °C/min to 280 °C (for 5 min).
For ABA quantification by SIM, MSD ChemStation (Agilent Technologies, USA) D.03.00.611 software was used to monitor responses to an m/z ratio of 190/194. The responses were integrated and amounts of embryo ABA content were calculated.