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신광덕,이한님,정태준 한국분자세포생물학회 2014 Molecules and cells Vol.37 No.5
Autophagy targets cytoplasmic cargo to a lytic compartment for degradation. Autophagy-related (Atg) proteins, including the transmembrane protein Atg9, are involved in different steps of autophagy in yeast and mammalian cells. Functional classification of core Atg proteins in plants has not been clearly confirmed, partly because of the limited availability of reliable assays for monitoring autophagic flux. By using proUBQ10-GFP-ATG8a as an autophagic marker, we showed that autophagic flux is reduced but not completely compromised in Arabidopsis thaliana atg9 mutants. In contrast, we confirmed full inhibition of autophagic flux in atg7 and that the difference in autophagy was consistent with the differences in mutant phenotypes such as hypersensitivity to nutrient stress and selective autophagy. Autophagic flux is also reduced by an inhibitor of phosphatidylinositol kinase. Our data indicated that atg9 is phenotypically distinct from atg7 and atg2 in Arabidopsis, and we proposed that ATG9 and phosphatidylinositol kinase activity contribute to efficient autophagy in Arabidopsis.
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Autophagic flux analysis of Arabidopsis seedlings exposed to salt stress
정혜라,김정훈,신광덕,김지미,이한님,정태준 한국식물학회 2017 Journal of Plant Biology Vol.60 No.2
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.
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