Use of protease inhibitors for detecting autophagy in plants.

In cultured tobacco (BY-2) cells, autophagy seems to be induced under nutrient-starvation conditions, whereas in root cells from Arabidopsis and barley, it occurs constitutively though is activated under nutrient starvation conditions. In both cases, protease inhibitors such as E-64, E-64c, antipain, and leupeptin block autophagy at the step of degradation of the cytoplasm enclosed in lysosomes/vacuoles, and cause the accumulation of autolysosomes (lysosomes containing parts of the cytoplasm) and/or of many cytoplasmic inclusions in the central vacuoles. Both types of autophagy are inhibited by 3-methyladenine, which is known as a potent inhibitor of autophagy in mammalian cells. Thus, using protease inhibitors and 3-methyladenine provides us with a method useful for analyzing autophagy in plant cells. This chapter describes protocols for detecting autophagic compartments in BY-2 cells and in the root-tip cells of Arabidopsis and barley by microscopy.