Endoplasmic reticulum oxidoreductin provides resilience against reductive stress and hypoxic conditions by mediating luminal redox dynamics

Dynamic monitoring of the ER luminal glutathione redox potential highlights the role of ER oxidoreductins in defining redox conditions and the interplay between different redox inputs during hypoxia and reductive stress. Oxidative protein folding in the endoplasmic reticulum (ER) depends on the coordinated action of protein disulfide isomerases and ER oxidoreductins (EROs). Strict dependence of ERO activity on molecular oxygen as the final electron acceptor implies that oxidative protein folding and other ER processes are severely compromised under hypoxia. Here, we isolated viable Arabidopsis thaliana ero1 ero2 double mutants that are highly sensitive to reductive stress and hypoxia. To elucidate the specific redox dynamics in the ER in vivo, we expressed the glutathione redox potential (E-GSH) sensor Grx1-roGFP2iL-HDEL with a midpoint potential of -240 mV in the ER of Arabidopsis plants. We found E-GSH values of -241 mV in wild-type plants, which is less oxidizing than previously estimated. In the ero1 ero2 mutants, luminal E-GSH was reduced further to -253 mV. Recovery to reductive ER stress induced by dithiothreitol was delayed in ero1 ero2. The characteristic signature of E-GSH dynamics in the ER lumen triggered by hypoxia was affected in ero1 ero2 reflecting a disrupted balance of reductive and oxidizing inputs, including nascent polypeptides and glutathione entry. The ER redox dynamics can now be dissected in vivo, revealing a central role of EROs as major redox integrators to promote luminal redox homeostasis.