Pupylation-based proximity labeling reveals APC/C-mediated ubiquitination and degradation of PEROXIN11C in soybean abiotic stress responses

Proximity-dependent labeling coupled with mass spectrometry (PL-MS) is a powerful approach for mapping low-affinity, transient protein-protein interactions (PPIs). While widely employed in animal systems, its application in plants has been restricted primarily to TurboID/miniTurbo-based systems due to the limited enzyme compatibility. To address this, we optimized the pupylation-based interaction tagging (Pup-IT) with a dexamethasone-inducible expression strategy in Nicotiana benthamiana and soybean (Glycine max). Using Pup-IT, we mapped the interactome of the soybean anaphase-promoting complex/cyclosome (APC/C) and identified the peroxisomal membrane protein GmPEX11C as a substrate. GmAPC/C ubiquitinates GmPEX11C at lysine 116, targeting it for proteasomal degradation. In addition, Gmilpa1/apc8 mutant exhibited enhanced salt and drought tolerance, whereas knockdown of GmPEX11C via RNA interference abolishes this stress-resistant phenotype. Our study establishes Pup-IT as a useful tool for interrogating PPIs in planta and uncovers a mechanistic link between APC/C-mediated ubiquitination and abiotic stress adaptation in soybean, offering potential avenues for crop improvement.