Autophagic flux analysis of Arabidopsis seedlings exposed to salt stress

Abstract

In plant cells, autophagy is required for efficient recycling of cytoplasmic macromolecules in vacuoles. It was previously shown that autophagy-deficient mutants also exhibited hypersensitivity to various abiotic stresses, such as salt, osmotic changes, heat, drought, and oxidative damage. However, it has not been clearly determined whether autophagy is induced or inhibited by these environmental stressors. Using the GFP-ATG8 (green fluorescent protein fused to AUTOPHAGY-RELATED PROTEIN 8) processing assay and confocal microscopy, we assessed autophagic flux of Arabidopsis seedlings exposed to salt stress. Treatment with 150 mM NaCl resulted in an increase in the processing of GFP-ATG8. Notably, the effects of concanamycin A, an inhibitor of vacuolar proton pumps, on GFP-ATG8 processing indicated that the apparent increase in GFP-ATG8 processing by salt-induced stress was due to inefficient vacuolar degradation of the GFP moiety processed from GFP-ATG8. Salt and osmotic stresses did not increase the abundance of autophagic vesicles in the root cells. Although NaCl, KCl, and mannitol did not greatly inhibit the vacuolar trafficking of GFP-ATG8, LiCl partially inhibited autophagy. These data indicated that NaCl stress neither increases nor substantially inhibits autophagic flux. Our work illustrates the importance of autophagic flux analysis to assess the effect of abiotic stresses on plant autophagy.