Chemstation data analysis application

The flower developmental process from squaring stage to senescence stage was divided into six stages starting at 10:00 AM with 12-h intervals (Fig. 1a). The headspace collection and GC-MS analysis were performed as described previously [46 (link)]. The whole flower of each stage was enclosed in a 500-ml glass bottle with the addition of 1.728 μg ethyl caprate as internal standard. After equilibrium of volatiles for 30 min, a polydimethylsiloxane (PDMS, with 50/30 μm divinylbenzene/Carboxen) fiber (Supelco) was inserted into the bottle to adsorb volatiles for 30 min. Then, trapped floral scent compounds were analyzed by a GC-MS system with Agilent 7890A GC and Agilent 5975C MSD. The instrument was equipped with an Agilent HP-5MS capillary column (30 m × 0.25 mm) and helium as a carrier gas at a constant flow of 1 ml/min. The oven temperature was initially maintained at 40 °C for 2 min, followed by an increase to 250 °C at a rate of 5 °C/min, and held at 250 °C for 5 min. The volatiles were identified by comparing the mass spectra and retention times with authentic standards. Quantification was based on peak areas and the quantity of internal standard using Agilent ChemStation Data Analysis Application. Analysis of variance was performed by SPSS software using Duncan test (P = 0.05).

The emission of MeJA was analyzed using headspace collection and GC-MS method, as described previously [38 (link)]. Briefly, the entire flower at each stage was enclosed in a 500-mL glass bottle with the addition of 1.728 μg of ethyl caprate as the internal standard. After the equilibrium of volatiles and internal standard for 30 min, an SPME fiber (Supelco, Bellefonte, PA, USA) was inserted into the bottle to trap volatile compounds for 30 min. Then, trapped floral volatiles were analyzed using a GC-MS system, as described above. The MeJA isoforms were identified by comparing the retention times and mass spectra with the authentic standard. Quantification was calculated based on the peak area ratio and the quantity of the internal standard using the Agilent ChemStation Data Analysis Application. Analysis of variance was carried out via SPSS 22 software using Tukey’s test (p = 0.05).

The GC–MS system was furnished with an Agilent DB-5MS capillary column (30 m × 0.25 mm), and helium gas was used as a carrier at a persistent flow of 1 mL/min. First, the oven temperature was kept at 40°C for 2 min, increased to 250°C at a rate of 5°C/min, and held for 5 min at 250°C. The identification of floral volatiles was performed by comparing the retention time and mass spectra with authentic standards. The quantification of floral volatiles was measured using the Agilent ChemStation Data Analysis Application based on the peak areas and quantity of internal standard. The floral VOCs were identified by comparing them with mass spectra from the NIST Mass Spectral Library (NIST 08). The identification of compounds was perceived by comparing the mass spectra with NIST 08 at a match factor of ≥80. Linear retention indices (LRIs) of the volatile compounds were measured via an alkane series standard (C7–C40) (Sigma, St. Louis, MO, United States).