DETOXIFICATION OF XENOBIOTICS IN PLANT-CELLS BY GLUTATHIONE CONJUGATION AND VACUOLAR COMPARTMENTALIZATION - A FLUORESCENT ASSAY USING MONOCHLOROBIMANE

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-dependen t detoxification of xenobiotics in plant cells and protoplasts, In BmC l-labelled cells and protoplasts, fluorescence developed rapidly and w as quickly concentrated in the vacuole, The rate of fluorescence devel opment was dependent on the concentration of BmCl and the only metabol ite formed was the conjugate Bm-SG. The formation of Bm-SG was correla ted with a decrease in the amount of intracellular GSH, Compounds whic h reduced the intracellular levels of GSH severely reduced the formati on 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 e nzyme catalysis, Our results demonstrate a cellular pathway for xenobi otic detoxification in plants, starting with conjugation to glutathion e in the cytoplasm, followed by the transport of the conjugates into t he 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.