Discovering novel components and mechanisms of plant oxygen-sensing

This application describes work that will transform our understanding of the biochemistry of plant oxygen-sensing. Oxygen is a key molecule for aerobic organisms, and oxygen-sensing is a central component of multicellular eukaryote biology. Reduced oxygen levels (hypoxia) due to flooding or waterlogging greatly reduce crop yields, and these important abiotic stresses are increasing in frequency and intensity due to climate change. Recently, in our lab and others, many novel roles for oxygen sensing have been defined in plants. Defining the complete biochemical mechanism of plant oxygen-sensing is an essential prerequisite to providing breeding or biotechnological approaches to stabilise yield in response to flooding and waterlogging. Along with others, we discovered a mechanism of plant oxygen sensing a decade ago, showing that oxygen-required degradation of key regulatory transcription factors controlled plant responses to hypoxia (Gibbs et al Nature 2011). We then showed that genetically enhancing oxygen-sensing in barley increased tolerance to waterlogging (Mendiondo et al Plant Biotechnology Journal 2016), demonstrating that our fundamental biochemical genetic approaches could be translated to address this agricultural problem. The pathway of oxygen-sensing, the PCO N-degron pathway, shares similarity to that of the animal Hypoxia Inducible Factor (HIF) system (that won the 2019 Nobel prize for Medicine and Physiology), including proteasomal destruction of transcription factors following covalent attachment of oxygen via dioxygenase enzymes, though the mechanisms are not related. Unlike the animal HIF system several core mechanisms and components of the plant oxygen-sensing pathway are not resolved. As animals also have an equivalent of the PCO (in animals ADO) N-degron pathway, these aspects are also unresolved in animal biology. This proposal seeks to fill these important knowledge gaps by addressing major inconsistencies between the currently accepted model for plant oxygen sensing and experimental evidence. In the proposed work we will discover new components and mechanisms of the core plant oxygen-sensing system (this will also provide information for the equivalent components and mechanisms of the animal ADO N-degron pathway). By providing new information that will completely redefine the PCO N-degron pathway (that we also showed acts as a mechanism of plant nitric oxide sensing; Gibbs et al Molecular Cell 2014), the work will facilitate the creation of novel resources and approaches to address agronomic problems associated with multiple abiotic stress tolerance, including flooding/waterlogging and salinity/drought. The project will involve a combination of inter-disciplinary experimental approaches spanning synthetic peptide synthesis, Mass Spectrometry, enzymology, genetics and plant physiology, only possible through the proposed collaboration of biologists and chemists. The project therefore provides great potential for novel interdisciplinary training. The proposed work is timely, building on our preliminary data and very recent publications by the project team and others in related fields, and offers the opportunity to resolve all the components of the pathway and defining their functions in this mechanism so essential for plant growth, development and response to environmental stresses.

This proposal aims to define and characterise novel components and mechanisms of plant oxygen-sensing. The project will involve a combination of inter-disciplinary experimental approaches spanning synthetic peptide synthesis, Mass Spectrometry, enzymology, genetics and plant physiology, possible through the proposed collaboration of biologists and chemists. The proposal seeks to fill important knowledge gaps by addressing major inconsistencies between the currently accepted model for plant oxygen-sensing and experimental evidence, and by revealing novel components and mechanisms. With others, we discovered the only known mechanism of plant oxygen-sensing, through the PCO N-degron pathway of ubiquitin-mediated proteolysis, linking the stability of hypoxia gene-regulating transcription factors to environmental oxygen levels (Gibbs et al 2011). We showed that this pathway is important for crop tolerance to waterlogging (Mendiondo et al Plant Biotechnology Journal 2016). Current understanding is that the pathway senses oxygen through dioxygenase addition of molecular oxygen to the amino-terminal cysteine of substrates, and also senses nitric oxide through an unknown mechanism. In three work packages we will define and characterise novel enzymatic components required for cysteine oxidation, define the target and mechanism of nitric oxide sensing, and reveal novel genetic regulators of the pathway. The proposed work is timely, building on our preliminary data and very recent publications by us and others in related fields, and provides the promise of resolving all the components of the pathway and defining their functions in this mechanism, an important prerequisite for biotechnological and breeding approaches to stabilise yield in the face of climate change-related increasing environmental stresses.