Alternative oxidase and ethylene form a positive feed-forward loop in mitochondrial retrograde signaling

To dissect the ANAC017 mitochondrial retrograde signaling pathway, we identified 2-oxoglutarate and Fe(II)-dependent oxygenase (OGO) as being induced by perturbation of mitochondrial function with antimycin A (AA), but not by high light. A forward genetic screen was implemented using the OGO promoter fused to firefly luciferase to identify regulators of OGO, thereby distinguishing regulators of mitochondrial perturbation from those that also impact chloroplast function. A mutant termed rog1 (regulator of OGO 1) was identified as encoding mitochondrial alternative oxidase 1a (AOX1a). To understand how AOX1a affects OGO expression, we investigated ethylene production in rog1 (aox1a) mutant lines and found that it was significantly reduced. Importantly, ethylene production could be restored by expression of AOX1c, indicating that alternative oxidase activity in general, rather than AOX1a specifically, is required for ethylene production. Ethylene production was also constitutively induced in ANAC017 overexpression lines. Metabolite profiling of aox1a lines treated with AA revealed pronounced perturbations in folate, methionine, and ascorbate-glutathione cycle metabolites, significant reductions in adenosine triphosphate (ATP) and adenosine diphosphate (ADP) levels, and alterations in the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH):nicotinamide adenine dinucleotide phosphate (NADP) ratio. Taken together, these results reveal a central role for AOX in maintaining hormone production and reduction-oxidation (redox) balance under mitochondrial perturbation through key metabolites previously characterized as important in abiotic stress responses. These findings establish AOX as an essential component required for a variety of abiotic and biotic stress responses.