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.