GNPS - Metabolite profiling of Arabidopsis WT, ugt76b1, and fmo1 plants during SAR

Experimental Goal: To determine the metabolic profile of Arabidopsis WT, ugt76b1, and fmo1 leaves during systemic acquired resistance.
Methods: Three lower leaves (leaf number 5-7) of 30-32-day-old Col-0, fmo1 and ugt76b1 Arabidopsis plants were infiltrated with 10 mM MgCl2 and a 5e6 cfu/ml suspension of Pst avrRpt2 in 10 mM MgCl2. Forty-eight h later, the three treated lower leaves (local, L) and three untreated upper leaves (distal, D) (leaf number 8-10) were harvested, pooled, respectively, then frozen in liquid nitrogen for metabolic profiling by LC-MS, and triple quadrupole (QQQ)-MS analysis. Plant tissue was harvested, lyophilized to dryness, and homogenized using a ball mill (Retsch MM 400) at 25 Hz for 2 min. Samples were resuspended in 20 ul of 80:20 MeOH:H2O per mg dry tissue and incubated at 4C for 10 min. NHP-Glc and DC-NHP were measured using previously published methods on an Agilent 1260 HPLC coupled to an Agilent 6520 quadrupole time-of-flight electrospray ionization (Q-TOF ESI) mass spectrometer (Chen et al., 2018. PNAS. E4920-E4929). SA and SA-Glc were measured using an Agilent 1290 Infinity II UHPLC coupled to an Agilent 6470 triple quadrupole (QQQ) mass spectrometer. A 1.8 um, 2.1 x 50 mm Zorbax RRHD Eclipse Plus C18 column was used for reverse phase chromatography with mobile phases of A [water with 0.1% formic acid (FA)] and B [acetonitrile (AcN) with 0.1% FA]. The following gradient was used for separation with a flow rate of 0.6 ml/min (percentages indicate percent buffer B): 0-0.2 min (5%), 0.2-4.2 min (5-95%), 4.2-5.2 min (95-100%). The MS was run in negative mode with the following parameters: gas temperature, 250C; gas flow rate, 12 l/min; nebulizer, 25 psig. SA was measured using monitored transitions with the following parameters: Precursor ion, 137.0239; product ions, 93 and 65.1; dwell, 150 ms; fragmentor voltage, 158 V; collision energy, 20 V and 32 V respectively, cell accelerator voltage, 4 V. SA-Glc was measured using monitored transitions with the following parameters: Precursor ion, 299.0767; product ions, 137 and 93; dwell, 150 ms; fragmentor voltage, 158 V; collision energy, 5 V and 20 V respectively, cell accelerator voltage, 4 V.