ATG8 delipidation is not universally critical for autophagy in plants

Autophagy, a critical recycling process in eukaryotic cells, maintains homeostasis by degrading and reusing cellular components. While extensively studied for its intricacy and elegance, aspects of this process remain understudied. In our recent study, we reveal how a serendipitous discovery revealed new insights on the role of autophagy-related gene 8 (ATG8) processing in autophagosome formation in two green plant species, the unicellular alga Chlamydomonas reinhardtii and the multicellular dicotyledon Arabidopsis thaliana.

Figure 1. A, An overview of the role of ATG8 and ATG4 in autophagosome formation. B, ATG4 truncates ATG8 by cleaving off a C-terminal peptide, exposing the critical glycine residue for lipidation. After autophagosome maturation, ATG4 delipidates ATG8, thus removing it from the autophagosome membrane.
To enable selective recycling of cellular components through autophagy, material destined for degradation is engulfed by double-membraned vesicles called autophagosomes. Nascent autophagosomes are decorated with ATG8 proteins in a process called ATG8 lipidation. For lipidation to occur, the ATG8 protein must be cleaved by the dedicated protease ATG4, thereby exposing a glycine residue which is required for lipidation. Before fusing with the vacuole for delivery of its cargo, lipidated ATG8 is removed (delipidated) from the outer autophagosomal membrane, again by ATG4. In fungi and animals, both lipidation and delipidation of ATG8 are essential steps in the autophagy pathway.
Our study began with a chance finding: an Arabidopsis atg4 mutant line expressing a pre-cleaved form of ATG8 (thereby bypassing the need for initial ATG4 processing) exhibited a wild-type-like phenotype. This highly unexpected observation was initially attributed to experimental error. However, after a long and careful examination, where we combined cell biology, biochemistry, reverse genetics, and plant phenotyping to confirm the veracity of our results, we arrived at the conclusion that ATG8 delipidation is indeed not required for autophagosome maturation, autophagic flux, or stress tolerance in Arabidopsis, thus revealing a heretofore unique autophagy mechanism in higher plants.
Building on this result, we became curious about how evolutionary pressures have shaped autophagy mechanisms in green organisms, from unicellular algae to vascular plants. To test whether our findings in Arabidopsis applied to unicellular green algae, we turned to Chlamydomonas reinhardtii. We found that unlike Arabidopsis, Chlamydomonas depends on ATG8 delipidation for autophagosome formation and autophagic flux, consistent with its simplified set of ATG genes. Moreover, when we analyzed Arabidopsis ATG8 orthologs (Arabidopsis has nine of them) under nitrogen starvation we observed distinct functional roles, which suggests an evolutionary rationale for the expansion of the ATG8 gene family in higher plants that likely enables fine-tuned autophagic responses to various environmental stressors.

Figure 2. Overview of the influence of ATG8 delipidation on autophagosome processing in yeast, plants, and mammals after incorporating the findings of our study.
This study underscores the value of examining biological processes across diverse organisms to uncover both conserved principles and lineage-specific adaptations. By investigating ATG8 processing, we hope to inspire deeper exploration into the evolutionary dynamics of autophagy and its potential applications in plant biology. Our findings raise compelling questions for future research: Why has ATG8 delipidation become dispensable in some plant lineages? What evolutionary forces drove the diversification of the ATG8 family in higher plants? And could these insights guide efforts towards engineering autophagy pathways for improved crop resilience under stress?
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