Laura Dalle Carbonare

Oxygen availability underpins energy production in multicellular organisms, yet internal oxygen gradients arise in both plants and animals. Plants sense these variations via the plant cysteine oxidase branch of the N-degron pathway, which regulates the stability of key transcription factors. Originally linked to metabolic control, this pathway recently emerged as a development regulator. While the shoot apical meristem was shown to be hypoxic, the oxygen dynamics of organs originating from this low-oxygen niche remain unknown. Here, we show that developing leaves form a spatiotemporal oxygen gradient that is sensed through the oxygen-sensing machinery. This pathway integrates local oxygen availability to regulate leaf morphogenesis: Early hypoxia restricts cell expansion, whereas subsequent distal-to-proximal oxygenation depletes group VII ethylene response factors enabling specialized cell-fate acquisition and controlling proliferation. Our findings reveal that oxygen acts as a positional cue in normal growth, guiding developmental trajectories. This highlights opportunities to harness oxygen gradients and sensing to direct plant form and function.

Oxygen availability underpins energy production in multicellular organisms, yet internal oxygen gradients arise in both plants and animals. Plants sense these variations via the PLANT CYSTEINE OXIDASE branch of the N-degron pathway, which regulates the stability of key transcription factors. Originally linked to metabolic control, this pathway recently emerged as a development regulator. While the shoot apical meristem was shown to be hypoxic, the oxygen dynamics of organs originating from this low-oxygen niche remain unknown. Here we show that developing leaves form a spatiotemporal oxygen gradient that is sensed through the oxygen sensing machinery. This pathway integrates local oxygen availability to regulate leaf morphogenesis: early hypoxia restricts cell expansion, while subsequent distal-to-proximal oxygenation enables specialized cell fates acquisition. Our findings reveal that oxygen acts as a positional cue in normal growth, guiding developmental trajectories. Our work highlights opportunities to harness oxygen gradients or sensing to direct plant form and function.

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.

Summary: Plants sense reduced oxygen availability (hypoxia) through Plant Cysteine Oxidases (PCOs). Reduced PCO activity in hypoxia, as seen during submergence, stabilises Group VII Ethylene Response Factors (ERFVIIs), master regulators of adaptive metabolic and anatomic responses. Equally important is timely arrest of these responses upon reoxygenation, assumed to occur through ERFVII degradation. Reoxygenation involves reactive oxygen species (ROS) production. Here, we report that instead of degradation, reoxygenation results in ERFVII nuclear stabilisation, an effect mimicked by direct H ₂ O ₂ treatment. Interestingly, typical hypoxia marker genes are repressed while genes involved in ROS homeostasis and oxidative stress protection are upregulated. Using in planta , heterologous and biochemical assays, we reveal that ROS-related ERFVII stabilisation is caused by PCO inactivation. Stabilised ERFVIIs are retained at hypoxia-responsive promoters but become repressors. Our findings suggest that by responding to both oxygen and ROS, PCOs coordinate ERFVII stability to regulate timely responses to damaging fluctuations in oxygen availability.

Limited aeration that is caused by tissue geometry, diffusion barriers, high elevation, or a flooding event poses major challenges to plants and is often, but not exclusively, associated with low oxygen. These processes span a broad interest in the research community ranging from whole plant and crop responses, post‐harvest physiology, plant morphology and anatomy, fermentative metabolism, plant developmental processes, oxygen sensing by ERF‐VIIs, gene expression profiles, the gaseous hormone ethylene, and O₂ dynamics at cellular resolution. The International Society for Plant Anaerobiosis (ISPA) gathers researchers from all over the world contributing to understand the causes, responses, and consequences of limited aeration in plants. During the 14th ISPA meeting, major research progress was related to the evolution of O₂ sensing mechanisms and the intricate network that balances low O2 signaling. Here, the work moved beyond flooding stress and emphasized novel underexplored roles of low O2 and limited aeration in altitude adaptation, fruit development and storage, and the vegetative development of growth apices. Regarding tolerance towards flooding, the meeting stressed the relevance and regulation of developmental plasticity, aerenchyma, and barrier formation to improve internal aeration. Additional newly explored flood tolerance traits concerned resource balance, senescence, and the exploration of natural genetic variation for novel tolerance loci. In this report, we summarize and synthesize the major progress and future challenges for low O₂ and aeration research presented at the conference.

Significance: The plant cysteine oxidases (PCOs) have been identified as oxygen-sensing enzymes in plants, controlling hypoxia-dependent processes, including adaptive responses to flooding. As such, they are potential targets for engineering plants with enhanced flood tolerance. To approach this in a rational manner requires an understanding of how PCO structure relates to their ability to trigger hypoxic adaptation. We report the structures of two PCOs from Arabidopsis and show that the effects of mutagenesis of key amino acids at their active site are seen both in vitro and, importantly, in planta. This work provides a platform for further efforts to manipulate PCO structure and function to improve the ability of crops to withstand future climate extremes.

N-terminal cysteine oxidases (NCOs) are enzymes that use molecular oxygen to oxidize the amino-terminal cysteine of specific proteins, thereby initiating the proteolytic N-degron pathway and thus conferring them oxygen-dependent instability. To expand the characterization of the plant family of NCOs (PCOs), we performed a phylogenetic analysis across different plant taxa in terms of sequence similarity and transcriptional regulation. Based on this survey, we propose a distinction of PCOs into two main groups: A-type and B-type sequences. A-type PCOs are conserved across all plant species and are generally unaffected at the mRNA level by oxygen availability. Instead, B-type PCOs differentiated in spermatophytes to acquire specific amino acid features and transcriptional regulation in response to hypoxia. Both groups of PCO proteins possess the ability to destabilize Cys-initiating proteins. Indeed, the inactivation of two A-type PCOs in Arabidopsis thaliana , PCO4 and PCO5, is sufficient to activate, at least partially, the anaerobic response in young seedlings, whereas the additional removal of B-type PCOs leads to a stronger induction of anaerobic genes and impairs plant growth and development. Our results show that both PCO types are required to regulate the anaerobic response in angiosperm. Therefore, while it is possible to distinguish two clades within the PCO family, separated by both amino acid features and transcriptional regulation, we conclude that they both contribute to restrain the anaerobic transcriptional program in normoxic conditions and together generate a molecular switch to toggle the hypoxic response in Arabidopsis. One sentence summary: Hypoxic induction of Plant Cysteine Oxidases has been acquired and fixed in seed plants by ancestor proteins able to initiate the proteolysis of Cys-initiating protein substrates by the Arg/N-degron pathway.