GC-MS analysis
The plant primary metabolites were analysed by GC-MS as described by
Roessner et al., (2000). 100 mg of freshly collected leaves and roots
were ground finely with liquid nitrogen and extracted with 1.4 ml of
100% methanol containing ribitol as internal standard. The mixture was
kept at 70°C for 15 min and then mixed vigorously with 1.4 ml of water.
Further, the sample was transferred in glass vial (GL-14 Schott Duran)
and centrifuged at 4°C for 15 min at 542 rpm. The upper phase was taken
in a fresh centrifuge tube and evaporated by using Speed Vac drying for
45 min. Finally, the samples were kept in -80°C for storage until
analysis. For derivatization, the residue was dissolved in 80 µ l
of methoxyamine hydrochloride with pyridine at 30°C for 90 min, followed
by 80 µ l of MSTFA (N-methyl-N-(trimethylsilyl)
trifluoroacetamine) and 20 µ l of FAME mix (Sigma, 1 µ g.µ l-1 in hexane) at 30°C for 30 min.
The derivatized sample was analysed on a system LECO-PEGASUS
GCXGC-TOF-MS system (LECO Corporation, USA) equipped with 30 m Rxi-5ms
column with 0.25 mm internal diameter and 0.25 μ m film thickness
(Restek, USA). The ion, interference and source of injection
temperatures were maintained at 200°C, 225°C and, 250°C, respectively.
Chromatographic separation was carried out under following conditions:
isothermal heating at 70˚C for 5 min, followed by 5˚C
min-1 oven temperature ramp to 290˚C and final heating
at 290˚C for 5 min. The flow rate of carrier gas (helium gas) was
adjusted to 1.5 ml. min-1. A volume of 1 µ l
sample was used for analysis and injected in split less mode, and scan
mass range 70 to 600 at 2 scans/ s.
All data sets were generated in the form of NETCDF files from ChromaTOF
software 4.50.8.0 chromatography version (LECO Corporation, USA) GC-MS
and were further exported to MetAlign 3.0 (www.metalign.nl). The MSClust
software was used to adjust signal to noise ratio of ≥2, for baseline
correction, noise estimation and identification of mass peak (ion-wise
mass alignment). Non-repetitive (<3 samples) mass signals were
discarded. The MSClust analysis was performed on the above results for
the reduction of data and compound mass extraction. Further, the MSClust
files were further exported to NISTMS Search v2.2 software for
identifying compounds with the NIST (National Institute of Standard and
Technology) Library and Gӧlm Metabolome Database Library
(http://gmd.mpimp-golm.mpg.de/). Metabolite identity was given based on
the maximum matching factor (MF) value (>700) and least
retention index (RI) value. The intensity of each metabolite value
normalized with internal standard ribitol value. Finally, only annotated
metabolites were taken into consideration for further analysis. Lists of
detected metabolites were given in the supplementary data.