title: 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.
authors: Graduate School of Science and Engineering, Saitama University, Saitama, Japan.
authors: Yuumi
authors: Y
authors: Oh-ye
authors: Yuko
authors: Y
authors: Inoue
authors: Yuji
authors: Y
authors: Moriyasu
mesh_terms: D017360:Arabidopsis / Q000378:metabolism*; D001343:Autophagy; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry; D004705:Endocytosis; D007930:Leucine / Q000031:analogs & derivatives* / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D018517:Plant Roots / Q000166:cytology* / Q000378:metabolism; D011726:Pyridinium Compounds / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods; D014617:Vacuoles / Q000378:metabolism
chemical_list: D003470:Culture Media; C092350:FM 4-64; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D007930:Leucine; C108192:aloxistatin