Engineering reduced activity in the oxygen-sensing Arabidopsis thaliana plant cysteine oxidase 4 enzyme results in improved flood resilience

Plant cysteine oxidases (PCOs) are O ₂ -sensing enzymes that play an important role in plant responses to low oxygen (hypoxia). PCO-catalysed dioxygenation of the N-terminal Cys of substrates, including Group VII Ethylene Response Factors (ERVIIs), targets them for degradation via the Cys/Arg N-degron pathway, however these substrates are stabilized in hypoxia due to reduced PCO activity. When plants are flooded, submergence-induced hypoxia results in ERFVII-mediated upregulation of hypoxia responsive genes that reconfigure plant metabolism and allow short-term resilience to the conditions. However, the increasing frequency and duration of flood events requires strategies to improve plant flood resilience, particularly amongst agronomic crops. One possibility is to prolong the stability of ERFVIIs by engineering the PCOs to catalyse their oxidation less efficiently. We report a structure-guided kinetic and biophysical investigation of Arabidopsis thaliana PCO4 that reveals residues important for substrate-binding and catalysis. We subsequently selected At PCO4 variants Y183F and C173A, with severe and mild impacts on At PCO4 activity, respectively, to complement Arabidopsis pco1pco2pco4pco5 plants and investigate their impact on submergence resilience. Both variants appeared to be beneficial for survival and recovery after 2.5 and 3.5 days of dark submergence when compared to control plants, indicating that engineering PCOs can be used as a strategy to improve flood tolerance in plants.