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

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.

This research aims to
1. Characterise the plant cysteine oxidase (PCO) family of enzymes and investigate whether they are kinetically suited to act as plant oxygen sensors. This will be achieved by expression and purification of recombinant enzymes and determination of optimal activity conditions, followed by activity and steady state kinetic assays to identify optimal substrates and determination of kinetic parameters for substrates including oxygen by steady state kinetic techniques. Product formation will be determined by matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry for relatively high throughput.
2. Elucidate the molecular mechanism by which the PCOs oxidise Group VII ERFs. This will be achieved by (i) crystallisation and structural determination of members of the PCO family, followed by/in parallel with mutagenesis of key active site residues to confirm important features of the active site by kinetic analysis; (ii) pre-steady state kinetic analysis to identify reaction intermediates (stopped-flow UV-visible spectroscopy, rapid chemical flow and product analysis by mass spectrometry, rapid freeze quench assays and product analysis by electron paramagnetic resonance (EPR)); (iii) static spectroscopy techniques to characterise the nature of the active site (UV-vis, EPR) and inductively coupled plasma mass spectrometry to confirm the presence of Fe.
3. Determine the conservation of oxygen-sensing efficiency amongst different plant species. This will be achieved by obtaining potential wheat, maize and rice PCO homologue sequences and subcloning them into expression vectors, then expressing and purifying enzymes. Their ability to oxidise equivalent peptide substrates (synthesised by solid-phase peptide synthesis) will be investigated by kinetic analysis and mass-spectrometry based product analysis.

I am extremely committed to ensuring that this publicly funded work achieves its maximal broad impact. Modulating the activity of the plant cysteine oxidases with respect to oxygen sensing in Arabidopsis thaliana and other plant species, including crops, has the potential to improve food security in the context of increasing flooding events. The work will be of interest to a range of different scientific communities including:

(i) The basic enzymology community - the PCOs are a novel family of enzymes, likely non-haem Fe oxidases, but as yet uncharacterized structurally or mechanistically. They will be of particular interest to enzymologists with an interest in the kinetics of oxygenases.

(ii) Plant biologists with an interest in tolerance to abiotic stress - the PCOs may be key signaling points for plant responses to hypoxia and thus intervention in their activity may be a means to manipulate hypoxia-tolerance.

(iii) Crop scientists with an interest in generating genetically modified crop species with improved flooding tolerance. Identifying the mechanism(s) for variable tolerance to flooding amongst different crop species (e.g. by different PCO sensitivities to oxygen) and opportunities to modulate this by, for example, introducing variant enzymes, will be of interest to this field.

The translation of this work into manipulating crop tolerance to submergence and flooding events will have a broad impact, including:

(iv) Crop science organisations working to improve crop yields including with respect to coping with climate change, e.g. the International Rice Research Initiative and the National Institute of Agricultural Botany (Cambridge). These groups will be able to apply PCO variants to large scale crop trials and investigate their submergence tolerance alongside potential side-effects.

(iv) The agrochemical industry - chemical targeting of the PCOs may be a viable mechanism to modulate their activity.

(v) Farming communities, politicians and the public - Ultimately, the implications of the project may be of political interest, e.g. improved crop tolerance to flooding could help to fulfil one of the United Nations Millennium Development Goals 'to eradicate extreme poverty and hunger and ensure environmental sustainability', as well as being of direct economic benefit to farmers by improving crop yields, and consumers by maintaining stable crop prices.