Esi source

Each sample consisted of 10 (±1) mg freeze-dried, ground plant material of 4-week-old rosettes, with 8 biological replicates per genotype, of plants that had been grown in randomized complete block design at 23°C SD. SA was extracted twice with 400 μl 20% methanol (LCMS-grade) / 0.1% hydrofluoroalkane by 5 min ultrasonic extraction, followed by 20 min incubation on ice, and removal of solids by centrifugation for 10 min at 13,500 g. 320 μl supernatant were removed after each extraction step and combined in a new vial. A third extraction step with 400 μl of 100% methanol (conditions see above) was performed and the supernatant was combined with the previous ones. The total volume was split in half before drying in a speed vac. For analysis of the conjugated and free salicylic acid the pellets were redissolved in 30 μl 50% methanol / 0,1% formic acid. Ultra Performance Liquid Chromatography Mass Spectrometry (UPLC-MS) analysis was performed on a Waters Acquity UPLC system coupled to a SYNAPT G2 QTOF mass spectrometer equipped with an ESI-Source (Waters Corporation, Milford, MA) at the University of Tübingen—ZMBP Analytics Laboratory. MassLynx v4.1 was used to control the LCMS system and TargetLynx (Waters Corporation) to perform data integration.

The same UPLC system, column and solvents were used for quantification with UPLC-TQ-MS/MS. The LC elution program was the same as given in the ‘UPLC-TOF and data analysis conditions’ section.
Quantitative detections were performed on a Xevo-TQ-MS equipped with an ESI source (Waters). The conditions of TQ-MS/MS were set as follows: capillary voltage, 3.5 kV; source temperature, 120°C; desolvation temperature, 400°C; cone gas flow, 30 l h^–1; desolvation gas flow, 800 l h^–1; and argon collision gas pressure, 3 × 10^–3 mbar. The precursor ion, product ion and their optimal MRM parameters are shown in Table 2.

The growth of Mycobacterial cells and induction for gene expression were done as mentioned above. The cells were grown to an exponential phase, collected at specified time intervals, and poured into tubes containing 1M formic acid. The tubes were immediately frozen in liquid nitrogen and stored at -80°C. The frozen cell samples were thawed at 37 ± 0.5°C for 30 min and immediately placed in an ice bath with mild vortexing at intervals of ~30min. Thawed cells were centrifuged at 7000 X g for 10 mins. The supernatant was collected and dialyzed using a 100 Da cut off dialysis tubing (Spectrum), against 100 volumes of deionized water overnight. The dialysate was then subjected to ESI-MS spectral analysis using the XEVO G2 XS Q-TOF mass spectrometer equipped with an ESI source (Waters) operating in the negative ESI mode with a flow rate of 5μl/min.

The HR-ESI-MS spectra were obtained on a Waters Xevo G2 Q-TOF mass spectrometer equipped with a Spray™ ESI source in both the positive and negative ion mode. NMR spectra were recorded in CDCl₃, CD₃OD, or DMSO-d₆ on a Bruker Avance III 400 Mz spectrometer at room temperature. Optical rotation was measured by an Anton Paar MCP 100 polarimeter. UV and ECD spectra were acquired on a JASCO J-810 spectropolarimeter. Preparative HPLC (high-performance liquid chromatography) separations for purification were carried out on an Agilent 1260 system with a semi-preparative chromatographic column (COSMOSIL 5C₁₈-MS-II, 5PFP, SL-II, 250 mm × 10 mm). Materials for column chromatography (CC) included silica gel, ODS (octadecylsilyl), and Sephadex LH-20.