The iron-regulated control network of nutrient uptake in plants

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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.