The assembly of iron-sulphur proteins in germinating seeds

Seeds contain large amount of carbon and minerals to help the germinating seedling put down a root and fold out its first leaves, before it can obtain its own water, nutrition and energy. This fascinating process of seedling establishment is underpinned by dramatic biochemical changes invisible to the naked eye. Storage lipids are broken down and converted to sugars and protein, while the photosynthetic machinery and green pigments are rapidly assembled. In this proposal we will investigate how one important nutrient, iron (Fe), is mobilized from its stores in seed and is incorporated into the catalytic sites of Fe enzymes.
Iron is an essential mineral that functions predominantly in electron transfer and catalytic reactions. Its chemical versatility is enhanced by how the iron interacts with the enzyme, such as in a ring structure in haem, or together with inorganic sulphide in Fe-S clusters. The latter form of Fe is most dominant in plants, and therefore an important source of human iron nutrition. Because free Fe and sulphide are toxic, the assembly of Fe-S clusters needs to be tightly controlled, involving proteins that carefully handle the Fe and S, and deliver it safely to the newly produced enzymes without inadvertent release of the intermediates. At least 30 proteins have so far been identified that are involved in Fe-S cluster assembly in plants. The proteins have been assigned to three different pathways, located in different parts of the cell: the mitochondria, chloroplasts and cytosol. While the former two are fairly well studied because the pathways are similar in bacteria, our knowledge on the cytosolic pathway is very limited. This pathway is essential for a range of critical Fe-S enzymes, however, such as a key enzyme in seedling lipid mobilization (aconitase), and more than 20 enzymes involved in DNA replication, repair and regulation.
In the past years, we have started to characterize a protein, NBP35, that is thought to play a key role in the Fe-S assembly pathway in the cytosol. In addition, we have evidence that the sulphide is provided by the mitochondria. In the next few years we would like to find the source of iron and how it is delivered to NBP35, and identify other proteins that function together with NBP35.
To investigate the source of iron, we will extend our initial studies on iron transporter mutants, and analyse how they are affected in the activity of Fe-S enzymes in different cell compartments. Since little is known about the mobilization of Fe in germinating seedlings, we will also compile a data set with changes in Fe distribution over day 1 - 10, and compare this to changes in protein and transcript levels of genes that play a role in handling or regulating Fe. The systems approach will be combined with a molecular-biochemical study of S and Fe delivery to NBP35.
To find direct interaction partners of NBP35, we will precipitate the protein from cell extract with specific antibodies. Proteins that co-precipitate will be identified using mass spectrometry, and their interaction with NBP35 will be further characterized in so called yeast-2-hybrid assays. Last but not least, any genes of interest that come out of the systems- or protein-interaction studies will be further investigated using mutant analyses. This will reveal whether the proteins indeed function in cytosolic Fe-S cluster assembly.
Knowledge on the mobilization of Fe in seeds and how it enters cofactor biosynthesis pathways is important for improving germination success rates and crop yields. This knowledge will also be important for expressing Fe-S proteins like nitrogenase in plants. This project links in with research to improve seeds as a source for human Fe nutrition.

Seeds and grains store iron that is rapidly mobilized upon germination for incorporation into an array of essential enzymes. As catalytic sites, iron-sulphur (Fe-S) clusters are much more common in plants than haem and other Fe cofactors, but much less is known about their biosynthesis. The assembly of Fe-S clusters in plants takes place in three cell compartments, the mitochondria, plastids and cytosol. During germination, high activities of mitochondrial electron transfer complexes and a transient burst in cytosolic aconitase depend on Fe-S clusters and their rapid assembly. This is followed by expression of the Fe-S rich photosynthetic machinery from day 5. In this proposal, we will focus on the as yet obscure cytosolic pathway, in finding the source of iron, and how S and Fe are delivered and assembled on the cytosolic scaffold protein NBP35. Following germination from day 1 to 10, we will carry out a time course of subcellular Fe pools, and link this to changes in expression of abundant Fe-S enzymes and cofactor assembly genes. Genome-wide co-expression analysis will be carried out to identify novel candidates involved in the assembly process. S and Fe delivery to NBP35 will be studied by monitoring its conformational changes in vivo and in vitro, while critical amino acids for S and Fe binding to NBP35 will be studied by point mutagenesis in vitro. To identify interactions partners of NBP35 we will carry out co-immunoprecipitation and yeast-2-hybrid studies. The various candidate genes for cytosolic Fe-S assembly will then be subjected to functional characterization studies using Arabidopsis mutants.
The outcome of this study is basic knowledge on the transport and utilization of Fe in germinating seedlings, which is important for crop yield, human nutrition and plant biotechnology, for example to express transgenic Fe-S enzymes such as nitrogenase.

1. Who might benefit from this research?

In addition to academic beneficiaries, the research might benefit plant breeders, farmers, nutritionists and health professionals. Also, in the long term, the health service and patients may benefit.

2. How might they benefit from this research?

Plant breeders and farmers
Iron is an important element for plant growth, because plants need sufficient iron for photosynthesis and other processes. The iron is used in the form of haem or iron-sulphur clusters. Therefore, knowledge of the biochemical pathway for assembling iron-sulphur clusters can be relevant for plant specialists.
We have recently discovered an isozyme that is critical for germination in the model plant Arabidopsis. The enzyme is called aconitase_3 (ACO3), which needs a bound iron-sulphur cluster for its catalytic function. The ACO3 gene is specifically upregulated during seed germination, and drives the conversion of seed storage lipids into sugars and other carbon compounds (Hooks and Balk, submitted). We found that the activity of ACO3 is dependent on the cytosolic iron-sulphur cluster assembly pathway. Both the specific role of aconitase during germination, as well as the cluster assembly pathway are thought to be conserved in crop plants. Knowledge of this process may lead to agronomic or genetic improvements to seed germination, for example by supplementing iron, or increasing the activity of ACO3.

Nutritionists and health professionals
Non-heme iron from plant foods is an important source of iron nutrition (Reddy, Hurrell and Cook, 2006 J Nutr. 136, 576-581). Therefore, knowledge of the levels of non-heme iron in plant foods are important for nutritional epidemiology studies (Rickard et al 2009 Br J Nutr. 102, 1678-85). Ultimately, this information is important for companies that need to know the Fe content for labelling on food products, or add Fe to food products (e.g. cereals). The information could also inform menu choices in institutions and schools.

Health service and patients
Our recent study on the IND1 gene in iron-sulphur cluster assembly is a good example of the wider, but unexpected, impact that fundamental research may have. We were the first to characterize this gene and showed that it is required for the assembly of respiratory complex I (Bych et al 2008 EMBO J 27, 1736-46). Subsequently, this gene was included in a high-throughput exon sequencing effort to identify the genetic basis of 100 patients with complex I deficiency, the most frequent class of mitochondrial diseases. One of cases turned out to be due to mutations in the human IND1 gene (Calvo et al 2010 Nature Genetics). The IND1 gene may now be included in more routine genetic screens for complex I deficiency across the globe.
Moreover, the associated genetic polymorphism is found in 1 in 150 Europeans, and may be an important determinant in the onset of Parkinson's disease, which has clear links with complex I deficiencies later in life (Tucker et al 2011 Hum. Mutat. In press).
The IND1 gene and its molecular function is related to NBP35 in the proposed research project. The human homologues of three other proteins in the same pathway are under investigation by Simon Boulton's group at Cancer Research UK.