Agilent 5977a mass spectrometry detector

Arabidopsis seedlings (Wt and pgr5 mutant) reached the bolting stage after approximately 40 days of culture and were then transferred to glass containers containing 40 mL of ddH₂O [27 (link)]. After 3 days of culture in the glass containers, culture media containing exudates were passed through 0.45-μm filter membranes. Approximately 35 mL of the solution was freeze-dried, dissolved in 500 μL of 80% methanol, and derivatized with Bis (trimethylsilyl) trifluoroacetamide and 1% chlorotrimethylsilane. The derivatized samples were analyzed by gas chromatography/mass spectroscopy GC-MS (Agilent 7890B gas chromatographic system coupled to an Agilent 5977A Mass Spectrometry Detector (Agilent, USA)). The potential involvement of all identified exudates in Arabidopsis biochemical pathways was subsequently determined by reference to the KEGG database [52 (link)].

LrSRRPs were treated with methanolic HCl (1 M) for 16 h and 5 μg of myo-inositol added as internal standard. Silver carbonate (~50 mg) was added to the solution, followed by 100 μL acetic anhydride and the reactions were incubated at room temperature for 16 h in the dark. Lipids were removed by three washes with heptane and the remaining methanolic phase was dried under a gentle nitrogen flow. Tri-Sil HTP reagent (200 μL) (ThermoFischer Scientific, Loughborough, UK) was added to the dried sample and the reaction was incubated at 80°C for 30 min. The solution was dried under nitrogen and 1 mL of hexane was used to extract sugars by sonication for 15 min. The samples were transferred to clean vials, dried and dissolved in dichloromethane (100 μL) before injection onto the GC–MS. The samples were analyzed on an Agilent 7890B GC–MS system paired with an Agilent 5977 A mass spectrometry detector (Agilent, UK), using a BPX70 column (SGE Analytical Science, Australia). Helium was used as the carrier gas. The inlet was maintained at 220°C, 12.9 psi, and 23 mL/min flow. The injection volume was 1 μL in split mode (1:20). The oven temperature increased initially from 100°C to 120°C over 5 min, followed by a second increase from 120°C to 230°C over 40 min.