Extraction of Cuticular Waxes and Gas Chromatography-Mass Spectrometry (GC–MS) Analysis

Approximately 100 mg of leaves and stems of 6-week-old wild-type (WT) and transgenic Arabidopsis plants were removed. The sample is gently moistened with distilled water and then the tissue surface is tenderly drained with filter paper, after which they were placed in a reaction flask. Cuticular waxes were extracted described by immersing tissues for 1 min in 2 mL of chloroform containing 10 μg of tetracosane as an internal standard. The extracts were dried under a gentle stream of nitrogen gas. The dried wax residues were derivatized by adding 20 μL of silylation reagent (BSTFA) and 20 μL of pyridine and incubated for 40 min at 70°C. The chromatographic column used in the experiment adopts a universal (30 m × 0.25 mm × 0.25 μm) hp-5MS capillary column with polydimethylsiloxane as the stationary phase. The chromatographic conditions were the inlet temperature of 280°C, the column flow rate of 2 mL/min and a constant flow rate; the Aux-2 temperature of 280°C, helium as the carrier gas, sample injection without splitting and the injection volume of 1 μL. The mass spectrometry program is set to electron impact ionization mode, energy 70 eV, mass scanning range 50 ∼ 600 amu and full scanning mode. The temperature rise process is to hold at 50°C for 1 min, raise the temperature to 170°C at 20°C/min, and hold for 2 min; then raise the temperature to 300°C at 5°C/min and hold for 8 min. The ion peaks of various wax components are detected by GC–MS (GCMS-QP2010, Shimadzu, Agilent). According to the mass spectrum library to search and determine and compare the FID peak area data, the single wax compound was quantified relative to the internal standard. Three biological replicates were used for each sample. The specific methods used were described by Chen et al. (2011) and Liu et al. (2012).