Citation: Yanagisawa, M.; Chuong, S.D.X. Chloroplast Envelopes Play a Role in the Formation of Autophagy-Related Structures in Plants. Plants 2023, 12, 443. https:// doi.org/10.3390/plants12030443 Academic Editors: Li’na Yin and Xiaomin Liu Received: 27 December 2022 Revised: 12 January 2023 Accepted: 16 January 2023 Published: 18 January 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). plants Article Chloroplast Envelopes Play a Role in the Formation of Autophagy-Related Structures in Plants Makoto Yanagisawa and Simon D. X. Chuong * Department of Biology, University of Waterloo, Waterloo, ON N2L 3G1, Canada * Correspondence: schuong@uwaterloo.ca Abstract: Autophagy is a degradation process of cytoplasmic components that is conserved in eu- karyotes. One of the hallmark features of autophagy is the formation of double-membrane structures known as autophagosomes, which enclose cytoplasmic content destined for degradation. Although the membrane source for the formation of autophagosomes remains to be determined, recent studies indicate the involvement of various organelles in autophagosome biogenesis. In this study, we examined the autophagy process in Bienertia sinuspersici: one of four terrestrial plants capable of performing C 4 photosynthesis in a single cell (single-cell C 4 species). We demonstrated that narrow tubules (stromule-like structures) 30–50 nm in diameter appear to extend from chloroplasts to form the membrane-bound structures (autophagosomes or autophagy-related structures) in chlorenchyma cells of B. sinuspersici during senescence and under oxidative stress. Immunoelectron microscopic analysis revealed the localization of stromal proteins to the stromule-like structures, sequestering portions of the cytoplasm in chlorenchyma cells of oxidative stress-treated leaves of B. sinuspersici and Arabidopsis thaliana. Moreover, the fluorescent marker for autophagosomes GFP-ATG8, colo- calized with the autophagic vacuole maker neutral red in punctate structures in close proximity to the chloroplasts of cells under oxidative stress conditions. Together our results implicate a role for chloroplast envelopes in the autophagy process induced during senescence or under certain stress conditions in plants. Keywords: autophagy; autophagosome; autophagic vacuole; chloroplast envelopes; stromules 1. Introduction Autophagy is a process responsible for the degradation of cytosolic and organellar materials for nutrient recycling and the removal of undesirable components in eukaryotes. Although there are several types of autophagy, two main autophagic pathways have been described in plants: microautophagy and macroautophagy [1,2]. Microautophagy involves the direct engulfing of cytoplasmic contents by the invagination of the tonoplast, followed by their release inside the vacuole. Macroautophagy is a process where a portion of cytoplasm, including organelles, is sequestered into a double- or multi-membrane structure called an autophagosome. Subsequently, the autophagosome is transported into lysosome in mammals or vacuole in yeast and plants for hydrolysis and degradation by proteases. Finally, the degraded products, such as amino acids, are reallocated and recycled [for plant autophagy, see reviews: [3–7]. In plants, autophagy is induced under nutrient-limiting conditions such as carbon and nitrogen starvation [8–11], during developmental events such as senescence [12–14], and in response to oxidative stress and pathogen attacks [15–17]. Autophagy observed under normal growth conditions has been suggested to serve as a housekeeping role [18,19]. Evidence connecting the relationship between autophagosome and autophagic vacuole biogenesis has been suggested in previous studies. For example, provacuoles formed tubule structures sequestering portions of cytoplasm, which eventually led to the formation of autophagic vacuoles that have been documented in the root meristematic cells of Euphorbia Plants 2023, 12, 443. https://doi.org/10.3390/plants12030443 https://www.mdpi.com/journal/plants