Abstract
Under nutrient starvation conditions, cells promote autophagy and recycle cellular constituents. It was shown that autophagy is induced not only by nutrient deprivation but also by salt and osmotic stresses in Arabidopsis, and that the activation of autophagy by nutrient deprivation and a salt stress occurs through a signal pathway involving reactive oxygen speicies. In this study, we have examined whether or not autophagy is really induced by salt and osmotic stresses in tobacco cells, which are large enough to observe autophagy more clearly. Autophagosomes, which are formed during autophagy were visualized by expressing a fusion protein of Atg8 and GFP. In this study, we chemically fixed cells and tried to quantify autophagy by measuring the total number of autophagosomes using a confocal laser microscope.Almost all cells died within 24 hours when they were treated with the same concentration of NaCl or mannitol as for Arabidopsis root cells. So we first determined the highest concentrations of NaCl and mannitol that BY-2 cells can survive. At present, we are examining the levels of autophagy activated by each stress and the effect of a NADPH oxidase inhibitor on the activation.
