Determination and characterization of cysteine, glutathione and phytochelatins (PC_(2-6)) in Lolium perenne L. exposed to Cd stress under ambient and elevated carbon dioxide using HPLC with fluorescence detection

摘要

Metal-binding thiols, involved in detoxification mechanisms in plant and other organism under heavy metal stress, are receiving more and more attentions, and various methods have been developed to determine related thiols such as cysteine (Cys), glutathione (GSH) and phytochelatins (PCs). In present study, an HPLC method was established for simultaneous determination of Cys GSH and PC_(2-6) after treatment with disulfide reductant of tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and thiolyte reagent of monobromobimane (mBBr). The separation of thiol derivatives was performed on an Agilent Zorbax Eclipse XDB-C18 column (4.6mm×30mm, 1.8μm) with a linear gradient elution of 0.1% (v/v) trifluoroacetic acid (TFA)-acetonitrile (ACN) at 0.8mLmin-1. The temperature of the column was maintained at 25°C. The excitation and emission wavelengths were set at 380 and 470nm, respectively. The thiol derivatives were well separated in 19min, and the total analysis time was 30min. The established method was proved selective, specific and reproducible, and could be applicable to determine Cys, GSH and PC_(2-6) and to evaluate their roles in detoxification mechanisms in Cd-treated Lolium perenne L. under ambient and elevated carbon dioxide (CO_2). It was found that the total SH contents and proportions of thiols in roots and shoots were dependent on Cd concentration, whereas the total SH contents decreased and the proportions of thiols altered without significance at elevated CO_2 level.