The iron-regulated control network of nutrient uptake in plants

铁和锌是大多数生命形式所必需的微量营养素。我们的身体需要大量的铁来形成血液中的血红蛋白分子，同时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，植物非常善于开采土壤中的这些矿物质，因为它们需要这些矿物质来促进自身的生长和发育。虽然在过去的几十年中，已经确定了参与铁和锌的吸收，运输和储存的主要行为者（由基因编码），但这些过程是如何调节的还远未了解。这样的理解对于操纵铁管理的不同方面是重要的，例如增加植物性食物中的铁（Balk et al.2019 Nutr Bull）或提高作物产量。在这里，我们提出了一个为期3年的研究项目，研究植物对铁和锌吸收的调节，重点是控制水平的蛋白质，从而抑制或激活矿物质吸收基因的关键调节因子（转录因子）的活性。感兴趣的蛋白质在一端具有铁/锌结合基序，在另一端具有所谓的泛素E3连接酶结构域。泛素通常用作标记蛋白质以进行降解的标签，并且E3连接酶帮助将泛素移动到特定的靶蛋白。在我们最近的出版物中，我们表明其中两种E3连接酶在根中起作用，并且一个重要的降解靶点是转录因子FIT，它激活参与铁吸收的基因。缺乏E3连接酶的植物在所有组织（包括种子）中积累2至3倍的铁，并且也能够在有毒水平的锌下生长。我们的主要问题是金属如何结合到蛋白质的一端影响蛋白质的另一半的连接酶活性。初步数据证实，铁结合在体外稳定蛋白质。目前尚不清楚的是，铁结合是否仅仅导致稳定折叠的蛋白质，或者铁的缺乏或锌的取代是否导致自我泛素化和降解。还有一个有趣的观察结果是，根中的蛋白质有2个铁结合基序，而茎中的蛋白质有3个铁结合基序。这种差异在感知细胞中铁的含量，从而设定连接酶被激活的阈值方面是否很重要？此外，基于在人类中的一个远亲蛋白质的研究结果，我们怀疑氧和活性氧可以改变铁的氧化态，从而影响蛋白质的稳定性，这将解释一些矛盾的研究结果在literature.In拟议的项目，我们将扩大E3连接酶的蛋白质靶点，如基因表达数据所建议的（目标1）。除了FIT之外，这些靶标将用作“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学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”研究将扩展到植物实验中，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，并与培养基中的铁和锌的浓度，我们可以测试这是否会改变传感阈值（目标4）。总之，详细的生物化学研究结合整个植物的实验应该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食物的铁含量。

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.