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

Abstract

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 in the presence of a cysteine protease inhibitor. The autolysosomes in BY-2 cells are located in 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 the structures stained with FM4–64, which likely correspond to autolysosomes, was seen although at lower level than in wild type roots.

Keywords::

• autophagy
• Arabidopsis
• endocytosis
• vacuole
• autolysosome/autophagosome
• cysteine protease
• protease inhibitor