Engineering a TurboID-based proximity labeling tool to track the dynamic secretomes under salt stress

Secretory proteins are essential for plant development and environmental adaptation, yet selectively profiling cargoes transported through the conventional endoplasmic reticulum (ER)-Golgi pathway remains technically challenging because apoplastic secretomes contain proteins from multiple secretion routes. We developed an ER-anchored TurboID proximity labeling method to selectively enrich proteins entering the conventional secretory pathway in Arabidopsis thaliana. Quantitative proteomics under control and salt stress conditions identified dynamic secretome changes and showed that most enriched proteins carry N-terminal signal peptides, supporting the utility of this approach for profiling conventional secretory proteins. Using this method, we found that sodium chloride (NaCl) stress remodels the conventional secretome by enhancing the secretion of a subset of cell wall-associated proteins. We further identified Apoplastic EDS1-Dependent 3 (AED3) as a salt-induced secretory protein, confirmed its ER-Golgi-dependent secretion, and demonstrated its role in salt tolerance. Overall, this study provides a useful approach for pathway-specific profiling of the conventional plant secretome, enabling the discovery of dynamic secretory proteins involved in plant environmental responses.