Detecting autophagy in Arabidopsis roots by membrane-permeable cysteine protease inhibitor E-64d and endocytosis tracer FM4-64

Autophagy is the process by which cells degrade their own components in lysosomes or vacuoles. Autophagy in tobacco BY-2 cells cultured in sucrose-free medium takes place in formed autolysosomes, which accumulate around the nucleus in the presence of a cysteine protease inhibitor. Autolysosomes in BY-2 cells are located on the endocytotic pathway and thus can be stained with fluorescent endocytosis marker FM4-64. In the present study, in order to detect autophagy in the root cells of Arabidopsis, we incubated root tips from Arabidopsis seedlings in culture medium containing the membrane-permeable cysteine protease inhibitor E-64d and FM4-64, and examined whether autolysosomes stained with FM4-64 are accumulated. The results suggest that autophagy accompanying the formation of autolysosomes also occurs in Arabidopsis root cells. Such autophagy appeared to occur constitutively in the root cells in nutrient-sufficient culture medium. Even in atg5 mutants in which an autophagy-related gene is disrupted, accumulation of structures stained with FM4-64, which likely correspond to autolysosomes, was seen although at lower levels than in wild-type roots.