Glutathione and glutathione-S-transferase activities of the vacuoles of the beet (Beta vulgaris L.) roots

Abstract

275 The central vacuole of the plant cell, which occu� pies up to 80% of the cell volume, performs multiple functions, including utilization of metabolic products by means of redox reactions (1). The reactive oxygen species (ROSs) are involved into numerous redox pro� cesses in cells of aerobic organisms. There is every rea� son to believe that, as in other cell compartments, the amount of ROS in the vacuoles should be controlled strictly during metabolite oxidation or in the course of spontaneous processes. What mechanisms underlie this control is yet to be clarified. In animal and plant cells, most of ROSs are known to be utilized with involvement of glutathione and glutathionedepen� dent enzymes, which interact either immediately with ROS or in the glutathione redox system and the ascor� bate-glutathione cycle (2). The restored glutathione (GSH) and GSHdependent enzymes, as well as the components of the ascorbate-glutathione cycle, were detected in various cell structures, such as plastids, mitochondria, peroxisomes, and the cytosol (2, 3). Little is known about the possible localization of glu� tathione and glutathione transferases in vacuoles (3-6). For example, a protein highly homologous to microsomal glutathioneStransferase (GST) (EC 2.5.1.12) has been detected in the vacuolar con� tents by the method of proteomic analysis of the tono� plast (4-6). This glutathionedependent enzyme is well known to be concentrated in cytosol, mainly near the cell membranes; it was also found in chloroplasts and microsomes. By conjugation with GSH, this enzyme restores the hydrophobic hydroperoxides and metabolizes toxic products of peroxidation of lipids and other exogenous and endogenous electrophilic compounds. GST is believed to be an important com� ponent of the antioxidant protection in animal and plant cells (2). Therefore, according to the main pur� pose of our study, which was to identify the compo� nents of the antioxidant system in plant cell vacuoles, the following priorities were set: analysis of the vacuole fractions for the glutathioneStransferase activity and glutathione identification. Our analysis demonstrated a glutathioneS� transferase activity in vacuoles isolated from the beet (Beta vulgaris L.) roots; in vacuoles, glu� tathione may be present not only in conjugates with xenobiotics, as it was previously believed, but also in a free state. Roots of beet (Beta vulgaris L.) of Bordeaux variety were selected in the period of dormancy (stored at 4°C). Both healthy roots (without any external signs of damage) and roots with pronounced signs of dis� eases, such as grey mold (caused by Botrytis cinerea Pers. ex Fr.) and phomosa (Phoma betae Frank), were studied.