Degradation & Condensation

Abstract

Plants respond to adverse environmental conditions through a wide array of physiological and molecular mechanisms. Two such responses are autophagy and the formation of stress granules. Autophagy is a conserved degradative process in which cellular material is engulfed by a double-membrane vesicle, the autophagosome, and delivered to the vacuole for breakdown and recycling. Stress granules, by contrast, are cytoplasmic condensates of proteins and mRNAs that assemble by liquid-liquid phase separation and safeguard their contents during stress. Here, we investigate the regulation of autophagy and the composition of stress granules. Autophagy can be controlled endogenously, through the cell's own machinery. One of the cell's intrinsic regulators of autophagy is the protein ATG8. Its function is governed by the protease ATG4, which both preprocesses ATG8 and later cleaves it from the autophagosomal membrane. This release was long considered a prerequisite for fusion with the vacuole. Contrary to expectation, it proves dispensable in Arabidopsis thaliana, while remaining essential in the green alga Chlamydomonas reinhardtii. Can autophagy also be influenced exogenously? Using a chemical genetics approach, we characterised the chemical autophagy enhancer RMDPi-1 as a competitive inhibitor of the reactive metabolite damage phosphatases (RMDP) from Arabidopsis and Chlamydomonas. Loss of RMDP activity induces autophagy in both organisms, and the accompanying metabolomic accumulation of phosphate sugars and CoA intermediates points to glycation-driven reactive oxygen species as a probable trigger. To support such studies, we developed SPIRO, an affordable, high throughput phenotyping robot that any biologist can build, together with software for the automated analysis of seed germination and root growth. Finally, we develop a refined method for isolating stress granules from scalable Arabidopsis cell cultures, yielding a cleaner proteome enriched in proteins linked to redox balance and ethylene biosynthesis. Together, these findings overturn a long-held assumption about the autophagy machinery, uncover a metabolic route to autophagy induction, and equip the field with new tools to study plant stress responses.