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

Abstract

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 (EGSH) sensor Grx1-roGFP2iL-HDEL with a midpoint potential of -240 mV in the ER of Arabidopsis plants. We found EGSH values of -241 mV in wild-type plants, which is less oxidizing than previously estimated. In the ero1 ero2 mutants, luminal EGSH was reduced further to -253 mV. Recovery to reductive ER stress induced by dithiothreitol, was delayed in ero1 ero2. The characteristic signature of EGSH 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.

Mitochondrial alternative NADH dehydrogenases NDA1 and NDA2 promote survival of reoxygenation stress in Arabidopsis by safeguarding photosynthesis and limiting ROS generation

“…S10). Multiwell plate reader‐based monitoring of seedlings exposed to changing O 2 as introduced previously (De Col et al ., 2017; Wagner et al ., 2019; Ugalde et al ., 2022) revealed reduction in the sensor in the first hour of the near‐anoxia treatment (set to 0.1% O 2 ), indicating H 2 O 2 production at lower rates. This phase was followed by a steady increase of sensor oxidation indicating elevated rates of H 2 O 2 production or lower rates of H 2 O 2 scavenging under near‐anoxia.…”

“…Cytosolic and mitochondrial H 2 O 2 dynamics were assessed using the genetically encoded biosensor roGFP2‐Orp1, stably expressed in Col‐0 wild‐type (WT) background (Nietzel et al ., 2019). A multiwell plate reader‐based fluorimetric assay was used similar to the approaches described previously (De Col et al ., 2017; Wagner et al ., 2019; Ugalde et al ., 2022) to analyze in vivo sensor responses during anoxia and reoxygenation. For more details, see Methods S4.…”

The role of glutathione in periplasmic redox homeostasis and oxidative protein folding inEscherichia coli

“…For normalization, 1 mM AT-2 and 10 mM DTT were added to the cells achieving full oxidation or reduction of the probe. For calculation, equation [1] The redox potential of roGFP-iL in the periplasm was calculated as previously described (Ugalde et al, 2022;Xie et al, 2020) using the Nernst equation [2]:…”

Early detection of late blight in potato by whole‐plant redox imaging

“…High spatiotemporal‐resolution‐monitoring of glutathione redox potential ( E GSH ) can be achieved using redox‐sensitive green fluorescent proteins (roGFPs; Meyer et al., 2007; Meyer & Dick, 2010), which carry two engineered cysteine residues that form an intramolecular disulfide bridge that impacts its fluorescence characteristics (Dooley et al., 2004; Hanson et al., 2004). roGFP‐based biosensors are effective tools for exploring redox dynamics in subcellular compartments at high spatiotemporal resolution in plants (Bratt et al., 2016; Gutscher et al., 2008; Jiang et al., 2006; Lampl et al., 2022; Meyer, 2008; Meyer & Dick, 2010; Nietzel et al., 2019; Schwarzländer et al., 2016; Ugalde et al., 2021; Ugalde et al., 2022). Genetically encoded biosensors have also been introduced into crop plants, including potato (Chamusco et al., 2022; Hipsch et al., 2021; Huang et al., 2014; Rattanawong et al., 2021), and spatially resolved mapping of stress‐induced long‐term redox perturbations has recently been demonstrated by whole‐plant imaging of potato plants expressing roGFP2 in their chloroplasts (Hipsch et al., 2021).…”