Plant is a catabolic process where cellular components such as protein aggregates and dysfunctional organelles are degraded and recycled to maintain homeostasis and facilitate stress resilience. Autophagy relies on a double-membrane vesicle called the autophagosome, which delivers cellular cargo to the vacuole for degradation. The Arabidopsis reporter line is a valuable tool widely used to visualize and quantify autophagosomes via microscopy and monitor autophagic degradation via immunoblotting. Consistent assessment of autophagic activity requires standardized protocols for sample preparation, imaging, and data analysis. Here, we present protocols for monitoring autophagy in Arabidopsis seedlings expressing GFP-ATG8, including treatments to induce or inhibit autophagic flux, as well as imaging and image analysis procedures. These methods enable reliable evaluation of autophagic activity and can be adapted for diverse experimental conditions and genotypes. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Growth of Arabidopsis seedlings Basic Protocol 2: Activation of autophagy in Arabidopsis seedlings by abiotic stresses Basic Protocol 3: Inhibition of vacuolar degradation by concanamycin A treatment Basic Protocol 4: Quantification of GFP-ATG8-labeled autophagosomes in Arabidopsis seedlings via microscopy Basic Protocol 5: Analysis of autophagic degradation of GFP-ATG8 via immunoblotting.
