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Autophagosomes arise in yeast and animals from the sealing of a cup-shaped double-membrane precursor, the phagophore. The concerted action of about 30 evolutionarily conserved autophagy related (ATG) proteins lies at the core of this process. However, the mechanisms allowing phagophore generation and its differentiation into a sealed autophagosome are still not clear in detail, and very little is known in plants. This is due in part to the scarcity of structurally informative, real-time imaging data of ATG proteins at the phagophore site. Among these, the ATG5 complex directs anchoring of ATG8 to the phagophore, an event required for membrane expansion. Detailed real-time and 3D imaging of ATG5, ATG8, and an ER marker at the expanding phagophore allowed us to propose a model for autophagosome formation in plants. This model implies tight connections of the growing phagophore with the outer face of the cortical endoplasmic reticulum and prompts new questions on the mechanism of autophagosome biogenesis.

Autophagosomes are the organelles responsible for macroautophagy and arise, in yeast and animals, from the sealing of a cup-shaped double-membrane precursor, the phagophore. How the phagophore is generated and grows into a sealed autophagosome is still not clear in detail, and unknown in plants. This is due, in part, to the scarcity of structurally informative, real-time imaging data of the required protein machinery at the phagophore formation site. Here we find that in intact living Arabidopsis tissue, autophagy-related protein ATG5, which is essential for autophagosome formation, is present at the phagophore site from early, sub-resolution stages and later defines a torus-shaped structure on a flat cisternal early phagophore. Movement and expansion of this structure are accompanied by the underlying endoplasmic reticulum, suggesting tight connections between the two compartments. Detailed real-time and 3D imaging of the growing phagophore are leveraged to propose a model for autophagosome formation in plants.

Autophagy is a catabolic process used by eukaryotic cells to maintain or restore cellular and organismal homeostasis. A better understanding of autophagy in plant biology could lead to an improvement of the recycling processes of plant cells and thus contribute, for example, towards reducing the negative ecological consequences of nitrogen-based fertilizers in agriculture. It may also help to optimize plant adaptation to adverse biotic and abiotic conditions through appropriate plant breeding or genetic engineering to incorporate useful traits in relation to this catabolic pathway. In this review, we describe useful protocols for studying autophagy in the plant cell, taking into account some specificities of the plant model. [ABSTRACT FROM AUTHOR]

Recently, a number of diverse correlative light and electron microscopy (CLEM) protocols have been developed for several model organisms. However, these CLEM methods have largely bypassed plant cell research, with most protocols having little application to plants. Using autophagosome identification as a biological background, we propose and compare two CLEM protocols that can be performed in most plant research laboratories, providing a good compromise that preserves fluorescent signals as well as ultrastructural features. These protocols are based on either the adaptation of a high pressure fixation/GMA acrylic resin embedding method, or on the Tokuyasu approach. Both protocols suitably preserved GFP fluorescence while allowing the observation of cell ultrastructure in plants. Finally, the advantages and disadvantages of these protocols are discussed in the context of multiscale imaging of plant cells. [ABSTRACT FROM AUTHOR]