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.

Zinc (Zn) is an essential micronutrient for plant growth. Accordingly, Zn deficiency (2Zn) in agricultural fields is a serious problem, especially in developing regions. Autophagy, a major intracellular degradation system in eukaryotes, plays important roles in nutrient recycling under nitrogen and carbon starvation. However, the relationship between autophagy and deficiencies of other essential elements remains poorly understood, especially in plants. In this study, we focused on Zn due to the property that within cells most Zn is tightly bound to proteins, which can be targets of autophagy. We found that autophagy plays a critical role during 2Zn in Arabidopsis (Arabidopsis thaliana). Autophagy-defective plants (atg mutants) failed to grow and developed accelerated chlorosis under 2Zn. As expected, 2Zn induced autophagy in wild-type plants, whereas in atg mutants, various organelle proteins accumulated to high levels. Additionally, the amount of free Zn 21 was lower in atg mutants than in control plants. Interestingly, 2Zn symptoms in atg mutants recovered under low-light, iron-limited conditions. The levels of hydroxyl radicals in chloroplasts were elevated, and the levels of superoxide were reduced in 2Zn atg mutants. These results imply that the photosynthesis-mediated Fenton-like reaction, which is responsible for the chlorotic symptom of 2Zn, is accelerated in atg mutants. Together, our data indicate that autophagic degradation plays important functions in maintaining Zn pools to increase Zn bioavailability and maintain reactive oxygen species homeostasis under 2Zn in plants.