Hp 6890 gas chromatograph

The chemical components of EOs were determined by GC–MS. EO samples were diluted in hexane (5 μl/ml)), injected in a split mode at a ratio of 1:35 and analysed using an Agilent HP-6890 Gas Chromatograph coupled with Agilent HP-5973 mass selective detector equipped with a BPX5 fused silica column (30 m × 0.25 mm ID, 0.25 μm film thickness). The GC column temperature was programmed from 50 to 300 °C via a ramp of 3 °C/min, and maintained at 300 °C for additional 3 min. The carrier gas was helium with a flow rate of 0.5 ml/min. The MS source was adjusted to 220 °C, and a mass range of m/z 40–500 was recorded acquiring all mass spectra in EI mode. The chromatogram visualization and determining the peak area integration was performed with Agilent Chemstation software (Agilent Technologies, Waldbronn, Germany). EOs chemical constituents were identified based on search in MS database using Mass Spectral Library (NIST 05), an in-house retention index library of MS spectra of volatile organic compounds, and comparison of spectra with MS data reported in literature (Sparkman 2005 (link)).

GC-MS analyses were performed using a HP-5MS column (30 m × 0.25 mm × 0.25 µm, Agilent, Santa Clara, CA, USA). The following temperature program was employed: 60 °C (1 min)/6 °C·min⁻¹/150 °C (1 min)/12 °C·min⁻¹/280 °C (5 min). ¹H and ¹³C NMR spectra were recorded on a 400 or 500 MHz spectrometer (Bruker, Billerica, MA, USA) and the chemical shift scale was based on internal tetramethylsilane. All the chromatographic separations were carried out on silica gel. Chiral GC analyses of compounds 1 (as acetyl derivatives obtained by treatment with acetic anhydride in pyridine) and 3 were performed on a Chirasil DEX CB (25 m × 0.25 mm × 0.25 μm, Chrompack, Agilent, Santa Clara, CA, USA) column, installed on HP 6890 gas chromatograph (Agilent, Santa Clara, CA, USA); (a) compound 1 45 °C/1.0 °C·min⁻¹/65 °C (1 min)/50 °C·min⁻¹/180 °C (5 min): (3S,4S)-1 t_R = 15.5 min, (3S,4R)-1 t_R = 16.0 min, (3R,4R)-1 t_R = 17.3 min, (3R,4S)-1 t_R = 17.7 min; (b) compound 3: 55 °C/0.8 °C·min⁻¹/67 °C (1 min)/90 °C min⁻¹/180 °C·2 min), (R)-3 t_R = 5.0 min, (S)-3 t_R = 5.5 min.

Samples taken at the different time points were extracted by two-phase separation using 0.4 ml of methyl t-butyl ether (MTBE) as solvent; the organic phase was dried over anhydrous Na₂SO₄ and analyzed by GC/MS.
GC/MS analyses were performed on an Agilent HP 6890 gas chromatograph equipped with a 5973 mass detector and an HP-5-MS column (30 m × 0.25 mm × 0.25 μm, Agilent), employing the following temperature program: 60 °C (1 min)/6 °C min⁻¹/150 °C (1 min)/12 °C min⁻¹/280 °C (5 min). The end products of the biotransformations were identified by GC/MS analysis, using authentic commercial samples as reference compounds: (i) cyclohexenone t_R = 5.40 min m/z 96 (M⁺, 33), 81 (19), 68 (100); cyclohexanone t_R = 4.65 min m/z 98 (M⁺, 47), 83 (13), 55 (100); cyclohexanol t_R = 4.45 min m/z 100 (M⁺, 2), 82 (35), 57 (100), (ii) α-methylcinnamaldehyde t_R = 14.7 min m/z 146 (M⁺, 64), 145 (100), 117 (79), 91 (43); α-methylcinnamyl alcohol t_R = 15.5 min m/z 148 (M⁺, 50), 115 (63), 91 (100); α-methyldihydrocinnamyl alcohol t_R = 13.7 min m/z 150 (M⁺, 12), 117 (62), 91 (100); (iii) methyl cinnamate t_R = 16.03 min m/z 162 (M⁺, 58), 131 (100), 103 (72); cinnamyl alcohol t_R = 12.80 min m/z 134 (M⁺, 53), 115 (65), 92 (100); phenylpropanol t_R = 12.36 min m/z 136 (M⁺, 21), 117 (100), 91 (84).

Leaf samples (1 g), ground with liquid nitrogen, were sequentially extracted with hexane, diethyl ether, and methanol. For the determination of trehalose, raffinose, and myo-inositol, the residue obtained after extraction in diethyl ether was further extracted with methanol and dried under vacuum at 90 °C. The pellets were resuspended in 0.5 cm³ pyridine, and the metabolites were derivatized by adding 0.05 cm³ N,O-bis(trimethylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane. Extracts were analyzed with an HP 6890 Gas Chromatograph equipped with a mass selective detector MSD 5973 (Agilent Technologies, Santa Clara, CA, USA), with an electronic autosampler 7693A ALS system, electronic pressure control, and split/splitless injector (Agilent Technologies, Santa Clara, CA, USA). The injector worked in a split 1:50 mode at 250 °C. The volume of the sample introduced into the injector was 1 μL. The transfer line temperature was 280 °C. Separation was performed on HP-5ms (30 m × 0.25 mm; 0.25 μm film thickness) fused silica column, with a helium flow rate of 1 cm³ min^–1 [1 (link)].

Application of 10 mM DIECA was used once a day for two days (DIECA treatment 1). On the third day, leaf samples were collected for the measurements of endogenous fatty acids, glutathione, and plant hormones. Fatty acid analysis was measured as described by Li et al., (2016), using HP6890 gas chromatograph (Agilent Technologies). Leaf tissues were dried in an oven at 45℃ for 60 hr and then ground into a powder; 200 mg of powder for each sample was placed in a screw capped glass vial. Glutathione and plant hormones, including JA, IAA, SA, BR, gibberellins (GA3 and GA4), dihydrozeatin riboside (DHZR), zeatin riboside (ZR), indolepropionic acid (IPA), and abscisic and acid (ABA), were measured from leaf tissues as described by Cao, Li, Chen, Liu, and Li (2016), and Zhao et al., (2006), with slightly modification  by using different internal reference and antibodies. Leaf cell death response was observed at 7 dpi by trypan blue staining as described previously (Koch & Slusarenko, 1990). ROS was estimated using DAB staining solution (0.1 g DAB, 100 ml distilled water, KOH adjusted to pH = 5.8) to stain infected leaves for 8 hr at 28℃ and then 100% ethanol was used to depigment infected leaves for 1 day.

¹H NMR (400 MHz), ¹³C NMR (100 MHz), and 2D-NMR spectra were acquired on an AVANCE III FT-NMR spectrometer (Bruker BioSpin, Rheinstetten, Germany) equipped with a 5 mm BBFO probe. The chemical shifts were referenced to residual ¹H or ¹³C signals of the solvents: acetone-d₆ (δ_H 2.05; δ_C 29.84) or methanol-d₄ (δ_H 3.31; δ_C 49.00). The UV spectra were acquired using a V-550 spectrometer (Jasco, Tokyo, Japan); the samples for UV spectroscopy were dissolved in MeOH. LC-MS and LC-MS/MS were performed with a 3200 QTRAP LC/MS/MS system (SCIEX, Framingham, MA, USA) coupled with a Prominence UFLC system (Shimadzu Co., Kyoto, Japan). The FAB-MS were recorded with a JMS-BU25 mass spectrometer (Jeol, Tokyo, Japan) in the negative ion mode; glycerol was used as the matrix and argon was used as the FAB gas. Polyethylene glycol was used as an internal standard for HRMS analysis. GC-MS was performed with a JMS-BU25 mass spectrometer coupled with an HP6890 gas chromatograph (Agilent Technologies, Santa Clara, CA, USA). Preparative HPLC was carried out with PU-980 HPLC pumps and an MD-910 photodiode array detector (Jasco).