Quantitative in vivo measurement of glutathione in Arabidopsis cells

A new, non-destructive assay is described to quantify cytoplasmic glutathio ne (GSH) levels in vivo in single cells or populations of cells from Arabid opsis suspension cultures. Cytoplasmic GSH was labelled with monochlorobima ne (MCB) in situ to give a fluorescent GSH-bimane (GSB) conjugate. At low ( 10-100 muM) concentrations of MCB, labelling was mediated by a glutathione S-transferase, which confers specificity for GSH. HPLC analysis of MCB-labe lled low molecular-weight thiols showed that the assay measures the total G SH pool, including the oxidized glutathione. The progress curve for the lab elling could be described using Michaelis-Menten kinetics with an apparent K-M of 40 mum and V-max of 470 mu mol I-cyt(-1) min(-1). There was no evide nce for de novo synthesis of GSH during the labelling period of 2 h, sugges ting that control of GSH synthesis is not mediated by feedback control of g amma -glutamylcysteine synthetase in this system. The total cellular level of GSH was calculated from the plateau value of the progress curve, after a ppropriate calibration, as 830-942 nmol g(-1) FW. The volume fraction of cy toplasm was measured from serial optical sections of bimane-labelled cells collected by confocal laser scanning microscopy (CLSM) with excitation 442 nm, or two-photon laser scanning microscopy (TPLSM) with excitation 770 nm. A value of 42 +/- 3% cytoplasm was determined by manual segmentation, and a value of 37 +/- 2% using stereological techniques. Using these figures, v alues for cytoplasmic [GSH] were estimated to be between 2.7 +/- 0.3 and 3. 2 +/- 0.3 mm for cell populations. In addition, measurement of GSH levels i n individual cells using CLSM and TPLSM gave values of 3.0 +/- 0.5 and 3.5 +/- 0.7 mM, respectively.