A Bioluminescent Reporter System for Real‐Time Monitoring of the Unfolded Protein Response in Plants

The unfolded protein response (UPR) is a critical mechanism for maintaining endoplasmic reticulum (ER) homeostasis under stress. Here, we developed a bioluminescent reporter system, AtbZIP60-LUC, in Arabidopsis to dynamically monitor ER stress by coupling IRE1-mediated splicing of bZIP60 mRNA to firefly luciferase (LUC) expression. Under ER stress, IRE1 removes a 23-bp sequence from bZIP60u, producing a spliced bZIP60s transcript in-frame with LUC, enabling luciferin-dependent luminescence. Transgenic AtbZIP60-LUC lines exhibited specificity for canonical ER stressors (heat, DTT, tunicamycin) but not osmotic stressors (NaCl, mannitol), confirmed by bioluminescence, qPCR, and immunoblotting. Time-course assays revealed rapid LUC induction by DTT (peak at 1 h) and delayed activation by tunicamycin (peak at 1-2 h), followed by signal decline, reflecting adaptive UPR dynamics. Heat stress optimization identified 38°C as optimal, inducing robust LUC expression after 2-3 h without compromising viability, while 42°C caused irreversible damage. Genetic validation in ire1a ire1b mutants abolished LUC induction, confirming IRE1 dependency, whereas constitutive UPR activation via maize 16-kDa γ-zein (16γz) overexpression triggered LUC expression without stress. Extending this system to tobacco and tomato, we engineered NbbZIP60-LUC and SlbZIP60-LUC, which similarly responded to heat (38°C), DTT, tunicamycin, and ER-localized protein aggregation (16γz, zeolin) in transient and stable assays. This work establishes bZIP60-LUC as versatile, non-invasive tools for real-time UPR monitoring in plants, offering insights into ER stress dynamics and enabling cross-species studies of stress adaptation mechanisms.