The iron-regulated control network of nutrient uptake in plants

植物养分吸收的铁调节控制网络

• 批准号: BB/V015095/1
• 负责人: Janneke Balk
• 金额: $ 71.94万
• 依托单位: John Innes Centre
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FV015095%2F1
• 关键词: iron; regulated; control; network; nutrient

Iron and zinc are essential micronutrients for most forms of life. Our bodies require large amounts of iron for haemoglobin molecules in the blood, but also for muscle, brain and liver function. Zinc is important for many enzyme functions. Iron and zinc enter the food chain through plants, which are extremely good at mining the soil for these minerals, as they need it for their own growth and development. While the main actors (encoded by genes) involved in the uptake, transport and storage of iron and zinc have been identified over the past decades, how these processes are regulated is far from understood. Such understanding is important in order to manipulate different aspects of iron management, for example to increase iron in plant foods (Balk et al. 2019 Nutr Bull) or improve crop yield.Here we propose a 3-year research project to study the regulation of iron and zinc uptake in plants, with a focus on proteins that control the levels and thus activity of key regulators (transcription factors) which repress or activate mineral uptake genes. The proteins of interest have iron/zinc-binding motifs on one end, and a so-called ubiquitin E3 ligase domain on the other end. Ubiquitin generally serves as a tag to label proteins for degradation, and the E3 ligase helps moving ubiquitin to a specific target protein. In our recent publication we showed that two of those E3 ligases function in the roots, and that an important degradation target is the transcription factor FIT, which activates genes involved in iron uptake. Plants lacking the E3 ligases accumulate 2 to 3-fold more iron in all tissues, including the seeds, and are also able to grow on toxic levels of zinc. Our main question is how metal binding to one end of the protein influences the ligase activity of the other half of the protein. Preliminary data confirmed that iron binding stabilised the protein in vitro. What is not clear is whether iron binding simply results in a stably folded protein, or whether the absence of iron, or substitution by zinc, leads to self-ubiquitination and degradation. There is also the interesting observation that the proteins in the roots have 2 iron-binding motifs, but the one in the shoot has 3 iron-binding motifs. Could this difference be important in sensing the amount of iron in the cell, and thus setting the threshold at which the ligases are activated? Moreover, based on findings for a distantly related protein in humans, we suspect that oxygen and reactive oxygen species can modify the oxidation state of the iron and thus affect protein stability, which would explain some of the contradictory findings in the literature.In the proposed project, we will expand the set of protein targets of the E3 ligases, as suggested by gene expression data (Objective 1). These targets will serve as 'read outs' in addition to FIT, to measure E3 ligase activity in intact plants. To investigate the effect of oxygen on the iron-binding ligases, we will first conduct studies on the isolated protein domains. In particular, we will use advanced spectroscopy to see if oxygen, or reactive oxygen species, bind directly, and whether the folding of the protein domain is affected (Objective 2). These 'in vitro' studies will then be extended to experiments in plants, using transiently produced E3 ligase (Objective 3). Finally, by altering the number of metal-binding motifs and playing with the iron and zinc concentrations in the medium, we can test whether this changes the sensing threshold (Objective 4).Together, the detailed biochemical investigation combined with experiments on whole plants should elucidate the working mechanism of the evolutionary conserved iron-binding E3 ligases and how they can be manipulated to enhance the iron content of plant foods.

铁和锌是大多数生命形式必需的微量营养素。我们的身体需要大量的铁来维持血液中的血红蛋白分子，也需要大量的铁来维持肌肉、大脑和肝脏的功能。锌对许多酶的功能都很重要。铁和锌通过植物进入食物链，植物非常善于从土壤中开采这些矿物质，因为它们自身的生长和发育需要这些矿物质。虽然在过去的几十年里，参与铁和锌的摄取、运输和储存的主要参与者（由基因编码）已经被确定，但这些过程是如何被调节的还远未被理解。这种理解对于操纵铁管理的不同方面非常重要，例如增加植物性食物中的铁（Balk等人，2019 Nutr Bull）或提高作物产量。在此，我们提出了一个为期3年的研究项目，研究植物对铁和锌的吸收调节，重点研究控制抑制或激活矿物质吸收基因的关键调节因子（转录因子）的水平和活性的蛋白质。感兴趣的蛋白质一端具有铁/锌结合基元，另一端具有所谓的泛素E3连接酶域。泛素通常作为标记蛋白质降解的标签，E3连接酶有助于将泛素移动到特定的靶蛋白。在我们最近发表的文章中，我们展示了其中两种E3连接酶在根中起作用，并且一个重要的降解目标是转录因子FIT，它激活了参与铁摄取的基因。缺乏E3连接酶的植物在包括种子在内的所有组织中积累了2到3倍的铁，并且也能够在有毒的锌水平下生长。我们的主要问题是金属与蛋白质一端的结合如何影响蛋白质另一半的连接酶活性。初步数据证实，铁结合在体外稳定了蛋白质。目前尚不清楚的是，铁结合是否仅仅导致了稳定折叠的蛋白质，还是缺铁或被锌取代导致了自泛素化和降解。还有一个有趣的观察是，根部的蛋白质有2个铁结合基序，但茎部的蛋白质有3个铁结合基序。这种差异在感知细胞中铁的数量，从而设定连接酶被激活的阈值方面是否很重要？此外，基于对人类远亲蛋白的发现，我们怀疑氧气和活性氧可以改变铁的氧化状态，从而影响蛋白质的稳定性，这可以解释文献中一些相互矛盾的发现。在拟建的项目中，我们将根据基因表达数据扩大E3连接酶的蛋白靶标集（目标1）。除了FIT之外，这些靶标将作为“读出”，以测量完整植物中的E3连接酶活性。为了研究氧对铁结合连接酶的影响，我们将首先对分离的蛋白质结构域进行研究。特别是，我们将使用先进的光谱来观察氧或活性氧是否直接结合，以及蛋白质结构域的折叠是否受到影响（目标2）。这些“体外”研究随后将扩展到植物实验，使用瞬时产生的E3连接酶（目标3）。最后，通过改变金属结合基序的数量和改变介质中的铁和锌浓度，我们可以测试这是否会改变感应阈值（目标4）。总之，详细的生化研究结合整个植物的实验，将阐明进化保守的铁结合E3连接酶的工作机制，以及如何操纵它们来提高植物性食物中的铁含量。

项目成果

Genetic basis of the historical iron-accumulating dgl and brz mutants in pea

豌豆历史铁积累dgl和brz突变体的遗传基础

• DOI: 10.1111/tpj.16514
• 发表时间: 2023
• 期刊: The Plant Journal
• 作者: Harrington S