Detoxification of xenobiotics in plant cells by glutathione conjugation and vacuolar compartmentalization: A fluorescent assay using monochlorobimane

Abstract

Monochlorobimane (BmCl), a non-fluorescent cell-permeant compound that reacts with glutathione to yield a strong blue fluorescent conjugate bimane-glutathione (Bm-SG), was used to trace the glutathione-dependent detoxification of xenobiotics in plant cells and protoplasts. In BmCl-labelled cells and protoplasts, fluorescence developed rapidly and was quickly concentrated in the vacuole. The rate of fluorescence development was dependent on the concentration of BmCl and the only metabolite formed was the conjugate Bm-SG. The formation of Bm-SG was correlated with a decrease in the amount of intracellular GSH. Compounds which reduced the intracellular levels of GSH severely reduced the formation of Bm-SG. Bm-SG was shown to be transported into isolated vacuoles by an ATP-dependent vanadate-sensitive mechanism. Kinetic analysis of cellular Bm-SG formation implicated both spontaneous conjugation and enzyme catalysis. Our results demonstrate a cellular pathway for xenobiotic detoxification in plants, starting with conjugation to glutathione in the cytoplasm, followed by the transport of the conjugates into the vacuole. This pathway is used to counter the toxic effects of some herbicides and environmental pollutants and overlaps with or parallels the pathway used for the biosynthesis of anthocyanins.