*2.11. Metabolite Extraction and Enrichment Analysis*

Metabolite quantification for *A. thaliana* samples (treated and control) was carried out using the method described by Lisec et al. (2006) [52], with minor changes. Briefly, 200 mg of fresh leaves from all four samples of *A. thaliana* treated with 0.25 mM, 0.5 mM and 1.0 mM rohitukine concentrations and the untreated control were excised using a fine pair of scissors and crushed in liquid nitrogen to a fine powder. Afterwards, 3 mL precooled HPLC-grade methanol (100%) was added and the homogenate was transferred to a glass vial. Then, 100 μL of ribitol (2 mg/10 mL) (Sigma-Aldrich, St. Louis, MI, USA) was added as an internal standard, followed by incubation at 70 ◦C for 10 min with continuous shaking. After incubation, the homogenate was centrifuged for 10 min at 15,000 rpm in the cold centrifuge; the supernatant was collected and 1.5 mL chloroform and 3 mL precooled autoclaved double-distilled water was added and slight vortexed. The upper polar and lower nonpolar layers were transferred to new well-labelled glass vials and dried over a rotary evaporator. The dried samples were then derivatized by adding 80 μL of 20 mg/mL Methoxy amine hydrochloride (Sigma-Aldrich USA), prepared in pyridine, to each tube, followed by incubation at 37 ◦C for 2 h. N, O-Bis (trimethylsilyl) trifluoroacetamide (BSTFA) (derivatization-grade) (Sigma-Aldrich USA) was added and another incubation at 37 ◦C took place for 1 h. After incubation, the mixture was transferred to suitable GC vials with inserts and analysed using GC–MS.

The sample was analysed using a GC–MS 4000 system (Varian, Crawley, UK) equipped with a Supelco capillary column (15 m × 60.32 mm × 60.25 m). A 1 μL volume of derivatized sample was injected; the injection temperature was 230 ◦C, the interface was set to

150 ◦C and the ion source was adjusted to 250 ◦C. The program of gradient temperature was: initial temperature of 40 ◦C for 6 min, +10 ◦C/min up to 300 ◦C and hold at 300 ◦C for 6 min. Mass spectrometry was determined by the full-scan method, ranging from 35 to 780 (*m*/*z*). The metabolites were identified by comparison of mass spectra with the NIST17 library using the molecular ion masses (*m*/*z*) of the fragments and retention time indexes (RI). The downstream analysis of each metabolite was carried out with the peak area of each metabolite taken as source data, using the MetaboAnalyst 5.0 online tool [53].
