Arabidopsis glutathione reductase 2 is indispensable in plastids, while mitochondrial glutathione is safeguarded by additional reduction and transport systems

<jats:title>Summary</jats:title> <jats:p> <jats:list list-type="bullet"> <jats:list-item> <jats:p> A highly negative glutathione redox potential ( <jats:italic>E</jats:italic> <jats:sub>GSH</jats:sub> ) is maintained in the cytosol, plastids and mitochondria of plant cells to support fundamental processes, including antioxidant defence, redox regulation and iron−sulfur cluster biogenesis. Out of two glutathione reductase (GR) proteins in Arabidopsis, GR2 is predicted to be dual‐targeted to plastids and mitochondria, but its differential roles in these organelles remain unclear. </jats:p> </jats:list-item> <jats:list-item> <jats:p> We dissected the role of GR2 in organelle glutathione redox homeostasis and plant development using a combination of genetic complementation and stacked mutants, biochemical activity studies, immunogold labelling and <jats:italic>in vivo</jats:italic> biosensing. </jats:p> </jats:list-item> <jats:list-item> <jats:p> Our data demonstrate that GR2 is dual‐targeted to plastids and mitochondria, but embryo lethality of <jats:italic>gr2</jats:italic> null mutants is caused specifically in plastids. Whereas lack of mitochondrial GR2 leads to a partially oxidised glutathione pool in the matrix, the ATP‐binding cassette (ABC) transporter ATM3 and the mitochondrial thioredoxin system provide functional backup and maintain plant viability. </jats:p> </jats:list-item> <jats:list-item> <jats:p>We identify GR2 as essential in the plastid stroma, where it counters GSSG accumulation and developmental arrest. By contrast a functional triad of GR2, ATM3 and the thioredoxin system in the mitochondria provides resilience to excessive glutathione oxidation.</jats:p> </jats:list-item> </jats:list> </jats:p>