Group VII ethylene response factors (ERF-VIIs) regulate transcriptional adaptation to flooding-induced hypoxia in plants. ERF-VII stability is controlled in an O₂-dependent manner by the Cys/Arg branch of the N-end rule pathway whereby oxidation of a conserved N-terminal cysteine residue initiates target degradation. This oxidation is catalyzed by plant cysteine oxidases (PCOs), which use O₂ as cosubstrate to generate Cys-sulfinic acid. The PCOs directly link O₂ availability to ERF-VII stability and anaerobic adaptation, leading to the suggestion that they act as plant O₂ sensors. However, their ability to respond to fluctuations in O₂ concentration has not been established. Here, we investigated the steady-state kinetics of Arabidopsis thaliana PCOs 1-5 to ascertain whether their activities are sensitive to O₂ levels. We found that the most catalytically competent isoform is AtPCO4, both in terms of responding to O₂ and oxidizing AtRAP2.2/2,12 (two of the most prominent ERF-VIIs responsible for promoting the hypoxic response), which suggests that AtPCO4 plays a central role in ERF-VII regulation. Furthermore, we found that AtPCO activity is susceptible to decreases in pH and that the hypoxia-inducible AtPCOs 1/2 and the noninducible AtPCOs 4/5 have discrete AtERF-VII substrate preferences. Pertinently, the AtPCOs had K_m(O2)^app values in a physiologically relevant range, which should enable them to sensitively react to changes in O₂ availability. This work validates an O₂-sensing role for the PCOs and suggests that differences in expression pattern, ERF-VII selectivity, and catalytic capability may enable the different isoforms to have distinct biological functions. Individual PCOs could therefore be targeted to manipulate ERF-VII levels and improve stress tolerance in plants.

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