Transcriptional analysis of NCO-complemented Arabidopsis thaliana 4pco mutants seedlings
The oxygen dependent degradation of Ethylene Response Factors VII, ERFVII, through the N degron pathway is central to regulating the transcriptional responses to hypoxia in vascular plants. Plant Cysteine Oxidases, PCOs, control this step by catalyzing the oxidation of an N-terminal cysteine residue exposed by ERFVIIs. In the present study, we investigated the functional impact of replacing Arabidopsis PCOs with diverse N-terminal cysteine oxidases, NCOs, from across the three eukaryotic kingdoms, hypothesizing that structural and kinetic differences may influence gene regulation of ERFVII targets under hypoxia and thus impact stress tolerance. Combining structural analyses, in vitro biochemical characterization and in planta complementation assays, we observed that not all tested NCOs are functionally equivalent to the endogenous PCOs. In fact, despite the remarkable conservation of catalytic motifs, we identified key divergences in enzyme architecture that affect the enzyme's capacity to regulate hypoxia responses in plants. Notably, NCO efficiency in oxidizing ERFVII peptides inversely correlated with hypoxic gene expression under aerobic conditions and enhanced submergence survival, suggesting that partial ERFVII stabilization primes plants to cope with hypoxia. However, enhanced basal expression of hypoxia responsive genes in turn correlated negatively with development and biomass accumulation, pointing to a trade-off between growth and stress resilience. Our findings demonstrate that tuning NCO activity can reshape the transcriptional and physiological hypoxia response, suggesting solutions to enhance plant resilience under fluctuating oxygen conditions through enzyme engineering and precision breeding.