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1. Systematic Transmission Electron Microscopy-Based Identification of Cellular Degradation Machinery

   https://doi.org/doi: https://doi.org/10.1101/2021.09.26.461841  

   Neikirk, K.; Vue, Z.; Katti, P.; Shao, J.; Christensen, T.; Garza-Lopez, E; Palavino-Maggio, C.B.; Ponce, J.; Alghanem, A.; Vang, L.; et al (January 2021, bioRxiv)

   Autophagosomes and lysosomes work in tandem to conduct autophagy, an intracellular degradation system which is crucial for cellular homeostasis. Altered autophagy contributes to the pathophysiology of various diseases, including cancers and metabolic diseases. Although many studies have investigated autophagy to elucidate disease pathogenesis, specific identification of the various components of the cellular degradation machinery remains difficult. The goal of this paper is to describe an approach to reproducibly identify and distinguish subcellular structures involved in autophagy. We provide methods that avoid common pitfalls, including a detailed explanation for how to distinguish lysosomes and lipid droplets and discuss the differences between autophagosomes and inclusion bodies. These methods are based on using transmission electron microscopy (TEM), capable of generating nanometer-scale micrographs of cellular degradation components in a fixed sample. In addition to TEM, we discuss other imaging techniques, such as immunofluorescence and immunogold labeling, which can be utilized for the reliable and accurate classification of cellular organelles. Our results show how these methods may be employed to accurately quantify the cellular degradation machinery under various conditions, such as treatment with the endoplasmic reticulum stressor thapsigargin or the ablation of dynamin-related protein 1. 

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2. Autophagic degradation of membrane-bound organelles in plants

   https://doi.org/10.1042/BSR20221204  

   Wang, Jiaojiao; Zhang, Qian; Bao, Yan; Bassham, Diane C. (January 2023, Bioscience Reports)

   Abstract Eukaryotic cells have evolved membrane-bound organelles, including the endoplasmic reticulum (ER), Golgi, mitochondria, peroxisomes, chloroplasts (in plants and green algae) and lysosomes/vacuoles, for specialized functions. Organelle quality control and their proper interactions are crucial both for normal cell homeostasis and function and for environmental adaption. Dynamic turnover of organelles is tightly controlled, with autophagy playing an essential role. Autophagy is a programmed process for efficient clearing of unwanted or damaged macromolecules or organelles, transporting them to vacuoles for degradation and recycling and thereby enhancing plant environmental plasticity. The specific autophagic engulfment of organelles requires activation of a selective autophagy pathway, recognition of the organelle by a receptor, and selective incorporation of the organelle into autophagosomes. While some of the autophagy machinery and mechanisms for autophagic removal of organelles is conserved across eukaryotes, plants have also developed unique mechanisms and machinery for these pathways. In this review, we discuss recent progress in understanding autophagy regulation in plants, with a focus on autophagic degradation of membrane-bound organelles. We also raise some important outstanding questions to be addressed in the future. 

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3. Analysis of Autophagy Under Abiotic Stress in Arabidopsis Seedlings Expressing the GFP‐ATG8 Autophagosome Marker

   https://doi.org/10.1002/cpz1.70166  

   Agbemafle, William; Jayasinghe, Vishadinie; Bassham, Diane C (June 2025, Current Protocols)

   Abstract Plant autophagy 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 GFP‐ATG8 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 

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4. Coordination and Crosstalk between Autophagosome and Multivesicular Body Pathways in Plant Stress Responses

   https://doi.org/10.3390/cells9010119  

   Wang, Mengxue; Li, Xifeng; Luo, Shuwei; Fan, Baofang; Zhu, Cheng; Chen, Zhixiang (January 2020, Cells)

   null (Ed.)

   In eukaryotic cells, autophagosomes and multivesicular bodies (MVBs) are two closely related partners in the lysosomal/vacuolar protein degradation system. Autophagosomes are double membrane-bound organelles that transport cytoplasmic components, including proteins and organelles for autophagic degradation in the lysosomes/vacuoles. MVBs are single-membrane organelles in the endocytic pathway that contain intraluminal vesicles whose content is either degraded in the lysosomes/vacuoles or recycled to the cell surface. In plants, both autophagosome and MVB pathways play important roles in plant responses to biotic and abiotic stresses. More recent studies have revealed that autophagosomes and MVBs also act together in plant stress responses in a variety of processes, including deployment of defense-related molecules, regulation of cell death, trafficking and degradation of membrane and soluble constituents, and modulation of plant hormone metabolism and signaling. In this review, we discuss these recent findings on the coordination and crosstalk between autophagosome and MVB pathways that contribute to the complex network of plant stress responses. 

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5. Studying plant autophagy: challenges and recommended methodologies

   https://doi.org/10.1007/s44307-023-00002-8  

   Qi, Hua; Wang, Yao; Bao, Yan; Bassham, Diane C.; Chen, Liang; Chen, Qin-Fang; Hou, Suiwen; Hwang, Inhwan; Huang, Li; Lai, Zhibing; et al (December 2023, Advanced Biotechnology)

   Abstract In plants, autophagy is a conserved process by which intracellular materials, including damaged proteins, aggregates, and entire organelles, are trafficked to the vacuole for degradation, thus maintaining cellular homeostasis. The past few decades have seen extensive research into the core components of the central autophagy machinery and their physiological roles in plant growth and development as well as responses to biotic and abiotic stresses. Moreover, several methods have been established for monitoring autophagic activities in plants, and these have greatly facilitated plant autophagy research. However, some of the methodologies are prone to misuse or misinterpretation, sometimes casting doubt on the reliability of the conclusions being drawn about plant autophagy. Here, we summarize the methods that are widely used for monitoring plant autophagy at the physiological, microscopic, and biochemical levels, including discussions of their advantages and limitations, to provide a guide for studying this important process. 

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