2.4 Measurement of phytohormones.
The concentrations of JA and salicylic acid (SA) in wheat seedlings were measured using the methods developed in our laboratory (Dong Li et al., 2020). Briefly, wheat seedlings powder (100 mg) was extracted with 1mL of solution (80%:19%:1% V/V/V, methanol: water: formic acid). The solutions were used ultrasound for 10 min and then centrifuged at 12000 rpm for 10 min. All supernatants were blown dry with nitrogen and made a volume of 100µL with 80% methanol in water. The specific parameters of JA and SA were supplied in Table S1.
DIMBOA was measured using the method reported by Kong et al (Kong et al., 2018). The frozen wheat leaves were ground, and the powder (100 mg) was extracted with 1.0 mL methanol containing 1% formic acid. The mixture solutions were using ultrasound for 5min, then overnight extracted in a refrigerator at 4℃. The next day, the treated samples used ultrasound for 5min at 4℃, then centrifuged at 10,000 rpm for 5min. The above method was repeated three times and all supernatants were collected. The supernatants were blown dry with nitrogen gas and made up the final volume at 1mL (methanol: water: acetic acid=59:40:1,V/V/V). The solutions were filtered with a 0.22μm nylon syringe. Acetonitrile and formic acid water (containing 0.1% formic acid, V: V = 38:62) were used as mobile phases A and B for isocratic elution on a reversed-phase C18 column (4.6 mm × 150 mm, 5μm), respectively. The flow rate was 1.0 mL/min at a wavelength of 280nm on an HPLC-1260 instrument equipped with a UV detector.
The determination of melatonin content was modified based on Li et al (M. Q. Li et al., 2016). In short, wheat leaves (100mg) were ground and then added 1mL solution (acetonitrile: hexane=80:20, V: V). The mixture was violently shacked for 5min. It was used ultrasound for 30min at 30℃ and centrifuged for 5min at 12000rpm. They were collected by repeating the twice operating method. All supernatants were blown dry with nitrogen and the residues were extracted by 1mL solution. These solutions were purified in a 2mL centrifuge tube containing 20mg MWCNT. After that, it was shacked for 2min and centrifuged for 1min at 10000rpm. The concentration was filtered with a 0.22μm nylon syringe. The experiment was analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) equipped with an electrospray ionization (ESI) source operating in positive mode (Agilent 6410B triple quadrupole). Acetonitrile and formic acid water (contain 1% formic acid) were used as mobile phases A and B for isocratic elution, respectively. The injection volume was 5μL and the flow rate was 0.2 mL/min. Other optimized information was applied in Table S1.