HYPOXIA-SENSING IN PLANTS: THE ROLE OF THE PLANT CYSTEINE OXIDASES

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Worldwide population growth in the next half century will need to be supported by an increase in food production. This needs to take place against the backdrop of an increase in global flooding events as the effects of climate change intensify, resulting in reduced crop yields. Identifying ways to improve crop tolerance to flooding will help address the grand challenge of food security. A major consequence of flooding is reduced oxygen availability (hypoxia), such that plants have to reconfigure their metabolism to generate energy for survival at the expense of growth. This hypoxic response is driven by a set of transcription factors, the Group VII ethylene response factors (ERFs). The Group VII ERFs are negatively regulated by oxygen: oxidation at N-terminal cysteine residues targets them for degradation, whereas in hypoxia their levels are maintained thus enabling the hypoxic response. A set of enzymes has recently been discovered that catalyse this oxidation in an oxygen-dependent manner, termed the plant cysteine oxidases (PCOs). The PCOs may therefore be key plant oxygen sensors. This hypoxic response mechanism has similarities with the equivalent mechanism in animals, whereby levels of the Hypoxia-Inducible transcription Factor (HIF) are regulated in an oxygen-dependent manner by key oxygen-sensing enzymes, the HIF hydroxylases.A comprehensive understanding of the structural, functional and kinetic features of the HIF hydroxylases (in which I have extensive expertise) is facilitating manipulation of the hypoxic response in humans for therapeutic advantage (e.g. inhibitors in clinical trials to treat anaemia). The recent identification of the PCOs, combined with my expertise in oxygen-sensing enzymes and the pressing need to address global food security issues means that the time is right to undertake detailed characterisation of the structural and mechanistic features of the PCOs, to identify ways in which their activity may be manipulated to improve plant hypoxia tolerance during flooding. We will conduct biochemical, biophysical, structural and kinetic assays to understand how the PCOs interact with oxygen and characterise their capacity to act as oxygen sensors (i.e. how enzyme activity correlates with oxygen concentration). There are 5 PCO isoforms and 5 Group VII ERFs, thus part of our characterisation will dissect the roles of each PCO with respect to different substrates (including oxygen). We will thoroughly probe the active site of the enzymes to understand their catalytic mechanisms. This will include substituting amino acids to modify activity, particularly with respect to oxygen. These experiments will initially be conducted on PCOs from the model species Arabidopsis thaliana; variant PCOs with altered characteristics will be introduced into this species to investigate whether bespoke mutations confer altered hypoxia tolerance.One of the most important aspects of this work will be to investigate equivalent oxygen-sensing systems in crop species. Rice, wheat and maize all possess putative PCO homologues, which we will investigate biochemically to determine (i) whether they also have the potential to act as oxygen-sensing enzymes in these species, (ii) whether their activity regulates levels of hypoxia-responsive transcription factors, and (iii) to identify mechanisms to alter their oxygen sensitivity. We will work with plant/crop biologists to translate our findings in planta.Excitingly, this work has significant potential to identify mechanisms to alter hypoxia sensing in plants, and therefore to improve flood tolerance, via modulating levels of Group VII ERFs. Interestingly, the rice Group VII ERF SUB1A is stable even in normoxia, conferring flood tolerance. This suggests that altering PCO activity, genetically or chemically, may be a viable strategy to address food security amongst increased floods. We will undertake the basic bioscience to underpin strategies in this direction.