Beatrice Giuntoli

Hydrogen sulfide (H2S) is a gasotransmitter molecule that regulates essential plant biological processes. In the context of climate change and increasing flooding events, its role in plant responses to oxygen (O2) deficiency has gained significant attention. Accumulating evidence indicates that H2S exerts beneficial effects under hypoxia by modulating antioxidant defenses, mitochondrial function, and hormonal signaling, mainly through its ability to refine protein activity via the post-translational modification (PTM) of persulfidation. Here, we discuss how this PTM is linked to the gasotransmitter molecule nitric oxide (NO) and reactive oxygen species (ROS), and how they can act synergistically or antagonistically, influencing the cell redox state and survival. Additionally, we examine how H2S turnover is intrinsically linked to O2 availability, acting as a conserved mediator of low O2 responses in different domains of life. By integrating biochemical insights with molecular signaling, we underscore the potential of H2S in enhancing crop resilience to low O2-related stresses, such as submergence and waterlogging.

Understanding plant molecular responses to flooding is crucial for strategies to increase resilience. Plants respond to submergence-induced low oxygen (hypoxia) through decreased plant cysteine oxidase (PCO) activity, which stabilizes group VII ethylene response factors (ERFVIIs), master regulators of metabolic and anatomic acclimation responses ^1–4 . Rapid reoxygenation on desubmergence induces a burst of reactive oxygen species (ROS) generation and metabolic reconfiguration ^5,6 ; however, how plants mitigate this post-hypoxic stress to facilitate submergence recovery has remained unknown. Here we report that ERFVIIs are also important in post-submergence recovery, remaining stable upon reoxygenation through ROS-mediated PCO inhibition. Stabilized ERFVIIs are retained at hypoxia-responsive promoters, becoming repressors of typical hypoxia marker genes but upregulators of genes involved in ROS homeostasis and oxidative stress protection. Our findings suggest that PCOs and ERFVIIs integrate signals from both oxygen and ROS to coordinate ERFVII stability through submergence-induced hypoxia and desubmergence stress to promote plant survival and recovery.

Vascular plants and metazoans use selective proteolysis to control responses to hypoxia, although through distinct biochemical mechanisms. The reason for this divergence is puzzling, since the molecular components necessary for both strategies are conserved. To explore the alternative scenario where plants and animals respond to hypoxia through the same mechanisms, we engineer a three-components system aimed to target proteins for degradation in an oxygen dependent manner in Arabidopsis thaliana . When used to control transcription, the synthetic system partially restores hypoxia responsiveness in oxygen-insensitive mutants. Additionally, we demonstrate its potential to regulate growth under flood-induced hypoxia. Our work highlights the use of synthetic biology to reprogramme signalling pathways, providing insights into the evolution of oxygen sensing and offering tools for crop improvement under stress conditions.

Plants face hypoxic conditions either chronically, as particular tissues are characterized by fluctuating or stable low oxygen levels, or acutely, when flooded. In vascular plants, transcriptional adaptive responses to hypoxia are rapidly mounted by Ethylene Response Factors VII (ERFVIIs), regulated by Plant Cysteine Oxidases (PCOs) through the cysteine branch of the N-degron pathway (Cys-NDP) for oxygen sensing. However, this relatively simple regulatory circuit, consisting of both constitutively expressed as well as hypoxia-inducible ERFVIIs and PCOs, interacts with diverse signaling cues and pathways invoked by hypoxia. To understand the share of the PCO-mediated oxygen sensing mechanism in the production of hypoxia responses, we insulated the PCO/ERFVII circuit from Arabidopsis thaliana and adapted it to Saccharomyces cerevisiae . Using a reporter gene to monitor the output of the circuit allowed us to compare the speed and amplitude of response to hypoxia in the engineered yeast and the source organism. Hypoxia triggered ERFVII stabilization both in Arabidopsis and yeast, leading to a similarly fast transcriptional response that was however larger in plants. A simple hypoxia-inducible feedback loop improved the amplitude of response in yeast, demonstrating the importance of this regulation in the endogenous PCO/ERFVII circuit. Finally, computational modeling of the yeast circuit enabled us to identify promoter competition and presence of hypoxia-inducible PCOs as key parameters that shape early hypoxia responses in plant cells.

In plants, iron homeostasis and oxygen metabolism are strictly related, indeed several Fe‐requiring enzymes catalyze reactions that also involve O ₂ as a reagent, product, entry or end point of the pathway. Oxygen sensing itself relies on the Fe‐dependent enzymes Plant cysteine oxidases. However, the impact of iron deficiencies on the response to hypoxic stresses has not been investigated so far. PCOs channel the ERFVII ethylene‐responsive factors into a proteasomal N‐degron pathway that connects hypoxia‐inducible responses to the stabilization of the ERFVII transcription factors, which act as master regulators of plant hypoxic transcription. Here, we investigated the interplay between low oxygen and Fe‐deficiency stresses in A. thaliana . PCO activity in vivo was inferred from the expression of hypoxia marker genes and from the activity of a genetically encoded reporter of ERFVII protein stability. Our results highlight that Fe deprivation can elicit hypoxia‐like responses depending on its severity. Moreover, evidence from the pentuple erfVII mutant indicate that the ERFVIIs take part to the responses to chronic Fe‐deficiency and fine‐tune nutrient content to the shoot of submerged plants growing on moderately Fe‐deficient substrates. This work expands the known functions of the ERFVII factors and provides new information to understand plant responses to combined environmental stresses.

Roots frequently encounter low oxygen (hypoxia) from soil compaction or water saturation and must adapt to this stress. We investigated how root tip cells sense hypoxia and adjust the meristem epigenome to activate genes that promote tolerance and growth under oxygen limitation. In Arabidopsis root tips, hypoxia tolerance was linked to increased trimethylation of histone H3 at lysine 4 (H3K4me3). We also found that group 7 demethylases (KDM7s) are directly inhibited by hypoxia, and that genetic inactivation of KDM7s, like hypoxia, induces expression of genes essential for meristem survival under oxygen deprivation. We propose that KDM7s function as root-specific oxygen sensors that prime and support hypoxia tolerance.

Hydrogen sulfide (H₂S) is a gaseous molecule historically regarded as toxic. Nevertheless, increasing evidence has brought to light important physiological roles in both animals and plants. In plants, H₂S is involved in environmental and developmental responses, such as stomatal closure and seed germination, and in tolerance mechanisms to different stress conditions like salinity, drought and waterlogging. In this study, we report a function of H₂S as a modulator of hypoxic responses in Arabidopsis thaliana . A combination of biochemical and genetic evidence demonstrates that H₂S inhibits the activity of Plant Cysteine Oxidases, the molecular sensors of oxygen, through protein persulfidation to modulate hypoxia-associated responses. Furthermore, we show that H₂S physiology contributes to responses to low oxygen, as disturbing H ₂ S production impaired activation of hypoxia-responsive genes and submergence tolerance. Overall, this work introduces H₂S as signalling modulator in plant hypoxic responses and adds a regulatory layer to the plant oxygen-sensing mechanism.

Plants face hypoxic conditions either chronically, as particular tissues are characterized by fluctuating or stable low oxygen levels, or acutely, when flooded. In vascular plants, transcriptional adaptive responses to hypoxia are rapidly mounted by Ethylene Response Factors VII (ERFVIIs), regulated by Plant Cysteine Oxidases (PCOs) through the cysteine branch of the N-degron pathway (Cys-NDP) for oxygen sensing. However, this relatively simple regulatory circuit, consisting of both constitutively expressed as well as hypoxia-inducible ERFVIIs and PCOs, interacts with diverse signalling cues and pathways invoked by hypoxia. To understand the share of the PCO-mediated oxygen sensing mechanism in the production of hypoxia responses, we insulated the PCO/ERFVII circuit from Arabidopsis thaliana and adapted it to Saccharomyces cerevisiae . Using a reporter gene to monitor the output of the circuit allowed us to compare the speed and amplitude of response to hypoxia in the engineered yeast and the source organism. Hypoxia triggered ERFVII stabilization both in Arabidopsis and yeast, leading to a similarly fast transcriptional response that was however larger in plants. A simple hypoxia-inducible feedback loop improved the amplitude of response in yeast, demonstrating the importance of this regulation in the endogenous PCO/ERFVII circuit. Finally, computational modelling of the yeast circuit enabled us to identify promoter competition and presence of hypoxia-inducible PCOs as key parameters that shape early hypoxia responses in plant cells. Significance Statement: We report the design, testing and optimisation of a synthetic molecular switch that activates gene expression in response to hypoxia in the yeast Saccharomyces cerevisiae . This is based on enzymes that consume molecular oxygen to regulate the stability of transcription factors in plant cells. By generating such a hybrid molecular device, we were able to demonstrate the efficacy of this hypoxia response strategy independently of the many ancillary components that affect gene regulation in plant cells. In this way, we were able to assess its activation dynamics, characterised by similarly fast induction of gene expression in both yeast and plants. Our approach also revealed the requirement of interlocked feedback loops to achieve the magnitude of gene induction measured in plants.

In plants, Fe homeostasis and O ₂ metabolism are strictly related, indeed several Fe-requiring enzymes catalyze reactions that also involve oxygen, as a reagent, product, entry or end point of the metabolic pathway in which the enzyme takes part. Oxygen sensing itself relies on Fe-dependent enzymes, the Plant cysteine oxidase (PCO) family of 2-OG independent thiol dioxygenases. PCOs are responsible for the degradation of ERFVII ethylene-responsive factors through a proteasomal N-degron pathway that connects hypoxia-inducible responses to the stabilization of the ERFVII transcription factors. Here, we investigated the interplay between low oxygen and Fe-deficiency stresses in A. thaliana . We used plants expressing a genetically encoded reporter of ERFVII protein stability and measured the expression of anaerobic genes to infer PCO activity in vivo . Our results highlight that Fe deprivation can elicit hypoxia-like responses depending on its severity. To test the involvement the ERFVII factors further, we examined the response of a pentuple erfVII mutant to Fe-deficiency stress, individually or combined with low oxygen. Our data indicate that the ERFVIIs might take part to the acclimation to chronic Fe deficiency by acting as positive regulators of starvation-responsive genes. Moreover, our results suggest that the ERFVIIs fine-tune nutrient mobilization to the shoots of submerged plants growing on moderately Fe-deficient substrates. This work expands the known functions of the ERFVII factors and provides new information to understand plant responses to combined environmental stresses.

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.