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

未来半个世纪的全球人口增长将需要粮食产量的增加来支撑。这需要在全球洪灾事件增加的背景下进行，因为气候变化的影响加剧，导致作物减产。找出提高作物对洪水的耐受性的方法，将有助于应对粮食安全这一重大挑战。淹水的一个主要后果是氧气供应减少(缺氧)，以至于植物不得不重新调整自己的新陈代谢，以牺牲生长为代价来产生生存所需的能量。这种低氧反应是由一组转录因子驱动的，即第七组乙烯反应因子(ERF)。第VII族ERF受到氧的负调控：N端半胱氨酸残基的氧化作用使其降解，而在低氧条件下，它们的水平保持不变，从而能够进行低氧反应。最近发现了一组以氧依赖的方式催化这种氧化的酶，称为植物半胱氨酸氧化酶(PCOS)。因此，PCOS可能是关键的植物氧气传感器。这种低氧反应机制与动物中的同等机制有相似之处，在动物中，低氧诱导转录因子(HIF)的水平受关键的氧感应酶HIF羟基酶以氧依赖的方式调节。全面了解HIF羟化酶的结构、功能和动力学特征(我在这方面有丰富的专业知识)有助于操纵人类的低氧反应以获得治疗优势(例如，在临床试验中治疗贫血的抑制剂)。最近发现的PCOS，加上我在氧气感应酶方面的专业知识，以及解决全球粮食安全问题的迫切需要，意味着现在是时候对PCOS的结构和机制特征进行详细描述，以确定可以通过哪些方式操纵它们的活性，以提高植物在淹水期间的耐缺氧能力。我们将进行生化、生物物理、结构和动力学分析，以了解PCOS如何与氧相互作用，并表征它们作为氧传感器的能力(即酶活性如何与氧浓度相关)。有5个PCO亚型和5个第VII类ERF，因此我们的部分特征将剖析每个PCO相对于不同底物(包括氧)的作用。我们将深入研究这些酶的活性部位，以了解它们的催化机制。这将包括取代氨基酸来改变活性，特别是与氧有关的活性。这些实验将首先在模式物种拟南芥的PCOS上进行；特征发生改变的变异PCOS将被引入到该物种中，以研究定制的突变是否具有改变的低氧耐受性。这项工作的一个最重要的方面将是研究作物物种中同等的氧气感应系统。水稻、小麦和玉米都有可能的PCO同源物，我们将对其进行生化研究，以确定(I)它们是否也具有作为氧敏感酶的潜力，(Ii)它们的活性是否调节低氧响应转录因子的水平，以及(Iii)确定它们改变其氧敏感性的机制。我们将与植物/作物生物学家合作，将我们的发现转化为PLAN。令人兴奋的是，这项工作具有重大的潜力，可以通过调节第VII组ERF的水平来确定改变植物对低氧的感知的机制，从而提高抗洪能力。有趣的是，水稻VIIERF SUB1A族即使在常氧条件下也是稳定的，赋予了抗洪能力。这表明，在洪灾加剧的情况下，改变PCO的遗传或化学活性可能是解决粮食安全问题的可行战略。我们将开展基础生物科学，以支持这一方向的战略。

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.