The Cys N-degron pathway as sensor of complex environmental stresses in A. thaliana.

In Arabidopsis thaliana, molecular responses under low oxygen conditions are primarily regulated by the Cys N-degron pathway. In this pathway, a family of transcription factors known as ETHYLENE RESPONSIVE FACTOR VII (ERFVII) is selectively degraded based on oxygen availability through the activity of PLANT CYSTEINE OXIDASES (PCOs). PCOs, which belong to the dioxygenase superfamily, rely on molecular oxygen and ferrous iron (Fe2+) as cofactor for their enzymatic activity. Under normoxic conditions, PCOs oxidize the free N-terminal cysteine residue of ERFVII, targeting them for degradation. In contrast, during oxygen deprivation, PCO activity is inhibited, leading to the stabilization of ERFVII proteins. Under such condition, ERFVII migrate into the nucleus where induce the expression of HYPOXIA RESPONSIVE GENES (HRGs), leading to various adaptations, including metabolic and morphological modifications.However, in submerged conditions, plants face challenges beyond oxygen deprivation. These include nutritional deficiencies, such as changes in iron availability, due to alteration in the soil properties. Additionally, gaseous molecules like ethylene, nitric oxide (NO), and hydrogen sulfide (H₂S) accumulate locally, as their diffusion is impeded by water coverage. This accumulation can alter the activity of proteins directly involved in the oxygen-sensing. This study aims to investigate the impact of more complex environmental conditions on the activity of the Cys N-degron pathway in A. thaliana. In the first part we explored how iron deficiency influences PCO activity and, consequently, the anaerobic responses. In the second part we characterized the effects of H₂S on the anaerobic response, examining its interaction with oxygen-sensing proteins and its underlying molecular mechanisms. We demonstrated that PCOs require only small amounts of iron to function efficiently. Under mild iron deficiency, PCO activity is not significantly affected, and no hypoxia-like response is induced. However, under severe iron deficiency, the hypoxic responses are significantly induced. Specifically, we reported that severe iron deprivation inhibits PCO activity, resulting in the stabilization of ERFVII proteins such as RAP2.12 and RAP2.3 and the subsequent activation of anaerobic gene expression. This finding suggests the high affinity of PCOs for iron and underscores the intricate regulatory mechanisms governing hypoxic responses. We also identified for the first time a signaling role for H₂S in the hypoxic response. Our results reveal that H₂S inhibits PCOs activity through a post-translational modification (PTM) known as persulfidation. This modification selectively suppresses PCOs activity, thereby facilitating an efficient anaerobic response. Furthermore, we demonstrated that mutants deficient in endogenous H₂S production exhibit a reduced anaerobic response, highliting the importance of this signalling molecule during oxygen deprivation. This study describes H₂S as a novel component in the molecular mechanism of oxygen sensing and highlights its pivotal role in modulating the anaerobic responses.