Assessment and optimization of autophagy monitoring methods in Arabidopsis roots indicate direct fusion of autophagosomes with vacuoles.

Autophagy is a degradation pathway that recycles cell materials upon encountering stress conditions or during specific developmental processes. To better understand the physiological roles of autophagy, proper monitoring methods are very important. In mammals and yeast, monitoring of autophagy is often performed with a green fluorescent protein (GFP)-ATG8 fusion protein or with acidotropic dyes such as monodansylcadaverine (MDC) and LysoTracker Red (LTR). To evaluate these monitoring methods, here we examined these systems by inducing autophagy in Arabidopsis thaliana roots as a model for monitoring autophagy in planta. Under carbon- and nitrogen-starved conditions, the number and size of vesicles labeled by GFP-ATG8 was increased for several hours and then gradually decreased to a level higher than that observed before the start of the experiment. We also observed the disappearance of GFP-ATG8-labeled vesicles after treatment with wortmannin, a phosphatidylinositol 3-kinase inhibitor known as an autophagy inhibitor, showing that the GFP-ATG8 transgenic line constitutes an excellent method for monitoring autophagy. These data were compared with plants stained with MDC and LTR. There was no appreciable MDC/LTR staining of small organelles in the root under the induction of autophagy. Some vesicles were eventually observed in the root tip only, but co-localization experiments, as well as experiments with autophagy-deficient atg mutants, provided the evidence that these structures were located in the vacuole and were not manifestly autophagosomes and/or autolysosomes. Extreme caution should therefore be used when monitoring autophagy with the aid of MDC/LTR. Additionally, our observations strongly suggest that autophagosomes fuse directly to vacuoles in Arabidopsis roots.