LC-MS analysis
The endogenous levels of phytohormones were quantified by LC-MS using a protocol from Pan et al., (2010) with some modifications. Freshly harvested leaves and roots of control and salt-treated plants were finely ground to powder with liquid nitrogen. Approximately 100 mg of powdered tissue was suspended in 500 µ l of extraction solvent (2-Propanol:H2O:HCl, 2:1:0.002, v/v/v) and kept in thermomixer at 4°C at 500 rpm for 30 min. The above step was repeated with addition of 1 ml ice cold dichloromethane. The resulting mixture was centrifuged at 4°C 13000g for 5 min and collected in fresh centrifuge tube. The sample tubes were placed in speed vac to evaporate extra solvent for 45 min. The final residue was dissolved in 70µ l of ice cold methanol followed by centrifugation at 13000g for 5 min. Finally, samples were taken into a transfer vial and were analysed by using Exactive™Plus Orbitrap mass spectrometer (Thermo Fisher, USA) coupled with UPLC (Waters, Milford, MA, USA).
LC-MS analysis was performed on an Aquity UPLC™ System equipped with quaternary pump, and auto-sampler to perform hormone analysis. For analysis, we used following conditions: capillary temperature 350˚C, sheath gas flow (N2) 35 (arbitrary units), AUX gas flow rate (N2) 10 (arbitrary units), collision gas (N2) 4 (arbitrary units) and capillary voltage 4.5 kV under ultrahigh vacuum 4e-10 mbar. Chromatographic separation was carried out in a Hyperreal GOLD C18 (Thermo Scientific) column (2.1×75 mm, 2.7 μ M). Formic acid (0.1%, v/v) and acetonitrile with 0.1% formic acid were used as mobile phase A and B, respectively. A gradient elution program was performed using two solvents system, solvent A and B chromatographic run for 9 min at 20˚C. Hormones such as ABA, GA, JA and SA were quantified by using all ion fragmentation (AIF) mode (m/z 50-450) with positive heated electrospray ionization (ESI) in negative ion mode. Similarly, hormones zeatin, IAA, indolebutyric acid (IBA) and methyljasmonate (MeJA) were measured using Turbo Ion spray source in positive ion mode. All phytohormones were quantified by using standard calibration curves.