Rational engineering of the Arabidopsis thaliana plant cysteine oxidase 4 active site can reduce biochemical activity...

The increase in flooding events due to climate change is having devastating effects on food security. Plants can respond to submergence by reducing their energy requirements and switching their metabolism from oxidative phosphorylation to fermentation. This response is promoted by group VII ethylene response factors (ERF-VIIs) whose stability is dependent on oxygen availability; in normoxia, plant cysteine oxidase (PCO) enzymes catalyze oxidation of ERF-VII N-terminal Cys residues to trigger ERF-VII degradation via the Cys/Arg-N-degron pathway. In hypoxia, reduced PCO activity prevents ERF-VII degradation, enabling acclimation to anaerobic conditions. We have proposed previously that reducing PCO activity by enzyme engineering could enhance ERF-VII stabilization and improve submergence tolerance. Here, we describe the rational design and detailed in vitro analysis of a range of PCO variants based on the most active PCO isoform in Arabidopsis thaliana, AtPCO4. We introduced single substitutions of specific amino acids in the active site and analysed their impact on enzyme catalysis, substrate affinity and iron binding capacity. We selected two variants with minimally or severely reduced activity (C173A and Y183F, respectively) and investigated their effect in an Arabidopsis model. Following submergence, survival and recovery were both improved in plants expressing the AtPCO4 variants compared to AtPCO4 wildtype. This proof-of-concept study demonstrates the potential to engineer just one amino acid in PCOs to improve flood resilience in plants and has implications for enhancing submergence tolerance in agronomically important crop species.