Analysis of thiol compounds and amino acids
For thiol compounds analysis, approximately 100 mg of fresh seedling tissues (a homogenous mixture of root and shoot) was collected in a 1.5 ml microcentrifuge tube containing 1 ml of extraction buffer (6.3 mM diethylenetriamine pentacetic acid mixed with 0.1% trifluoroacetic acid). The extracts were used for derivatization and analysis of thiol compounds – Cysteine, γ-EC, GSH, and PCs as described in Minochaet al . 2008. For amino acid analysis, a homogeneous mixture (shoot and root) of 100 mg fresh tissue was collected in a 1.5 ml microcentrifuge tube containing 1ml of 5% (v/v) perchloric acid and stored at -20o C. The samples were prepared and analyzed via HPLC as per Minocha and Long (2004) with minor modifications described in (Majumdar et al . 2018).
Analysis of 5-Oxoproline (5-OP)For 5-OP analysis, samples were homogenized in 80% chilled ethanol. After vortexing briefly, the samples were centrifuged at 13000 rpm for 15 mins. Supernatants were transferred to new tubes and evaporated under vacuum at RT for 16 to 24 hours. Samples were then lyophilized and re-dissolved in 10 mM ammonium acetate. Samples were processed with LC-MS-MS (UPLC/Xevo TQD QQQ-MS, Waters Corporation, Milford, MA) using the protocol from (Eckstein et al . 2008) with minor modifications. Briefly, Synregi 4µ Hydro-RP 80R analytical column (Phenomenex, Torrance, CA) was used at a flow rate of 0.3ml/min, with a total run time of 7 minutes per sample.