Characterization of the ufmylation function and mechanism in regulating stress-induced plant pexophagy

Ufmylation is a ubiquitination-like post-translational modification (PTM) system identified in the past decade. In mammals, it has been revealed to be closely associated with DNA repair, autophagy, and endoplasmic reticulum (ER) homeostasis, where its dysfunction causes embryo lethality, serious developmental defects, and affects cancer progression. Ufmylation cascade is revealed to interplay with autophagy, the degradation pathway for bulky cytosolic materials and organelles upon nutrient deprivation or stress induction. In plants, we have identified the E3 ligase of the ufmylation system, Ufl1, as an interactor of the autophagy marker ATG8 under salt stress condition to regulate ERstress associated reticulophagy (ER-phagy). Coordinately, the C53/UFL1/DDRGK1 receptor complex is also proved to regulate plant reticulophagy. We hypothesize that besides reticulophagy, ufmylation plays multiple roles in regulate distinct stress-induced pathways, especially selective autophagy.

The current knowledge of plant selective autophagy receptors and ufmylation is rudimentary, with few known receptors or substrates characterized. Our first aim is to conduct pull-down assays followed by mass spectrometry (MS) using Ufl1 as bait to reveal its candidate interactors as well as the potential plant ufmylome. In our preliminary screening, the presence of canonical Ufl1 interactors in the list strengthens the screening assay. Candidates were then grouped based on their subcellular localization and biological functions. We identified multiple peroxisomal proteins as potential Ufl1 interactors and hypothesized that Ufl1 might function in mediating peroxisome homeostasis under oxidative stresses which potentially involves the autophagy pathway. In this proposal, we have extended and will further conduct our study to verify the interaction of the candidates with Ufl1 or other ufmylation components and investigate the molecular mechanisms by which ufmylation regulates peroxisome homeostasis by pexophagy. This study will establish a clear mechanistic understanding of how ufmylation functions in maintaining cellular homeostasis under stress conditions and provide novel insights into how this process contributes to plant responses to environmental stresses.