Engineering prolyl hydroxylase-dependent proteolysis enables the orthogonal control of hypoxia responses in plants

Vascular plants and metazoans use selective proteolysis of transcription factors to control the adaptive responses to hypoxia, although through distinct biochemical mechanisms. The reason for this divergence is puzzling, especially when considering that the molecular components necessary to establish both strategies are conserved across the two kingdoms. To explore an alternative evolutionary scenario where plants sense hypoxia as animals do, we engineered a three-components system aimed to target proteins for degradation in an oxygen dependent manner in Arabidopsis thaliana. Applying the synthetic biology framework, we produced a hypoxia-responsive switch independent of endogenous pathways. When applied to control transcription, the synthetic system partially restored hypoxia responsiveness in oxygen-insensitive mutants. Additionally, we demonstrated its potential to regulate growth under flood-induced hypoxia. Our work highlights the use of synthetic biology to reprogram signalling pathways in plants, providing insights into the evolution of oxygen sensing and ofering tools for crop improvement under stress conditions. Overall design: One-week-old transgenic Arabidopsis lines expressing HIFODD-?RAP2.12-HA in an erfVII background were grown under normoxia and then exposed to hypoxic atmospheres of 1% or 0.1% O2 for 2 h in controlled chambers. Following treatment, plants and parallel normoxic controls were harvested for downstream molecular analyses to assess hypoxia-responsive changes in gene expression and signaling.