Manipulating plant oxygen sensing through NCO substitution reveals trade-offs between growth and flooding tolerance

Abstract

The oxygen-dependent degradation of Ethylene Response Factors VII (ERFVIIs) 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 catalysing the oxidation of an N-terminal Cys 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 characterisation 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 differences in enzyme architecture that appear to affect the enzymes capacity to regulate hypoxia responses in plants. Notably, NCO efficiency in oxidising 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 it is possible to enhance plant resilience under fluctuating oxygen conditions through enzyme engineering and precision breeding. Significance statementControl of low oxygen responses to improve crop flooding tolerance is a long-sought objective of molecular plant breeders. The oxygen-dependent oxidation of N-terminal cysteines in transcription factors is thought to be a key step in modulating the transcriptional response to hypoxia. In this study, we tested this hypothesis by substituting endogenous N-terminal cysteine dioxygenases with homologues from different species characterized by highly divergent sequences, structures, and kinetic properties. We show that indeed these variations effectively and predictably influence gene transcription in plants exposed to hypoxia, thereby affecting their tolerance to submergence. However, we also demonstrate that, unexpectedly, these substitutions impact plant growth and development under aerobic conditions, revealing a trade-off between flooding stress resilience and biomass accumulation or yield.