Comparative analyses of glutathione system of vacuoles and leucoplasts isolated from the storage parenchyma cells of dormant red beetroots (Beta vulgaris L.).

ABSTRACT

The role of glutathione in the plant vacuole is still being debated. In the present paper, the redox state of glutathione and the activity of glutathione S-transferase (GST, E 2.5.1.18) in the vacuole compared to those in leucoplast have been studied. Organelles were isolated from dormant red beet (Beta vulgaris L.) taproots. Two generally used approaches have been applied to quantitatively assess the content of glutathione. Initially, levels of glutathione were measured in isolated organelles after labeling with monochlorobimane (MCB) and imaging with the use of confocal laser scanning microscopy. However, there are factors limiting the specificity of this method, because of which the resulting concentrations of vacuolar GSH have been underestimated. Another approach used was HPLC, which allows to simultaneously quantify the reduced glutathione (GSH) and glutathione disulfide (GSSG). The concentration of the total glutathione (GSHt) and GSSG in vacuoles determined with the aid of HPLC-UV was higher in comparison to that in the leucoplasts. The reduction potential (Eh) for the glutathione couple in the vacuoles was more positive (-163 mV), than that in plastids (-282 mV). The relatively rapid increase in fluorescence in the isolated vacuoles and plastids during MCB-labeling has indicated to the contribution of GSTs, since the conjugation of GSH to bimane is catalysed by these enzymes. The GST activity in the vacuoles has been assessed to be quite high compared to that of leucoplasts. The number of isoforms of GSTs also differed markedly in vacuoles and plastids. Collectively, our findings suggest the idea that the glutathione accumulated by central vacuole seems to contribute to the redox processes and to the detoxification, which can take place in this compartment.