Functional characterization of Iron Regulator Sensor (IRS) proteins in plants

Iron is an essential mineral in our food. Therefore children are encouraged to eat iron-rich green vegetables such as spinach advertised in Popeye cartoons. Plant foods are the primary source of our dietary iron which they obtain directly from the soil. Plants are extremely efficient at mining the soil for iron, but they will take up no more than they require for their own needs. This is because iron, in its free form, is extremely toxic. As soon as iron enters the cell, it is bound by chaperone proteins to carry iron safely around the cell and then immediately incorporate it in enzymes to function as catalysts. Some iron can also be stored in specific proteins or cell compartments. Balancing the uptake, use and storage of iron is called homeostasis. Central to maintaining homeostasis is an iron sensor, which signals to regulators of gene expression to alter the levels of transporters (and other iron homeostasis proteins). Iron sensors have been described in bacteria, in yeast and in mammals. However, we still do not know how iron is sensed in plants, or which proteins function as iron sensors. However, we do know that the expression of iron homeostasis genes is very tightly regulated in plants, so there must be a very sensitive Fe sensor.
Extensive analyses of gene expression networks in plants grown under iron-deficient conditions has turned up three candidate proteins for the iron sensor. The proteins are related in sequence but are present in different tissues. What makes them good candidates is their pattern of regulation within the iron regulatory network, their similarity to an iron-sensing protein in mammals, and several iron binding protein domains. In the proposed project we would like to confirm the role of these proteins, named IRS1, IRS2 and BTS, in iron homeostasis. First, we will measure iron binding to the separate protein domains, what form the iron is in and how strong the binding is. A relatively weak binding constant is expected in sensor proteins. We will also try and elucidate how the proteins orchestrate downstream signalling events. They are likely to interact with other proteins, and we will use established methods to identify these interaction partners.
To feed the growing world population, it has been suggested that we should all eat less meat. However, if we cut down on this rich source of iron, this could lead to iron deficiency anaemia, which is already common among girls and women in the UK and widespread in the developing world. Plants can provide enough iron if our diet is rich in green vegetables and pulses. This is not always the case, therefore it would be beneficial to increase the amount of iron in staple foods such as wheat (for bread) and potatoes. The only way to do this, is to trick the plant in taking up more iron than it needs, and enable a large iron storage capacity. For this, we need first find the iron sensor, so we can alter its sensitivity and signalling capacities.

Iron (Fe) is an essential element for most organisms because of its versatility as a catalyst and oxygen carrier. We obtain most of our iron from plants, which take it up from the soil. The uptake process is tightly regulated because free intracellular Fe is toxic. Changes in gene expression associated with Fe deficiency are well characterized in many plant species, and a number of Fe-regulated transcription factors have been identified, however it is not known how intracellular Fe levels are sensed. Transcriptomics network analysis by Jorge Rodriguez-Celma has identified a small family of three proteins that are likely candidates for the plant Fe sensor. This is based on co-regulation with other Fe homeostasis genes and homology to the recently identified FBXL5 Fe-sensing protein in mammals. Interestingly, additional Fe-binding domains are present in the plant proteins. The proposed project aims to characterize the metal binding properties of the plant proteins using advanced spectroscopy techniques. We will also carry out a yeast two-hybrid screen for interacting proteins that are likely to be down-stream targets of the signalling pathway. Potential candidates will be confirmed by co-immunoprecipitation, plus we will test if the interaction depends on the iron concentration. The signalling mechanism will be further studied using Arabidopsis mutant lines. Our goal is to unravel the Fe-sensing mechanism such that it can, in the future, be exploited to improve crop growth and bio-available iron levels for human nutrition.

It is estimated that up to 2 billion people - over 30% of the world's population - are anaemic, many due to iron deficiency (www.who.int). Lack of iron causes fatigue, lower immunity and lost productivity. In the UK, approximately 20% of women aged 11-64 have low serum ferritin levels (Heath & Fairweather-Tait, 2002 Best Pract Res Clin Haematol. 15:225-41; National Diet and Nutrition Survey Report 2012) and are therefore at risk for developing iron deficiency anaemia (IDA). Indeed, IDA is common in pregnant women, which is treated with iron sulphate pills but these have undesirable side effects.
Plant-based foods provide up to 90% of our iron intake, even in diets with a large proportion of meat (Bruggraber et al, 2012 Br J Nutr. 108:2221-8). Cereals and wheat flours are usually fortified with iron salts or iron filings. Although this is not expensive, natural species of iron are likely to be more bio-available. Increasing the iron content of crops, in particular cereals, is the aim of several international research programmes (e.g. HarvestPlus). Currently, increases in the order of 3-6% have been achieved (Bashir K et al, 2013 Front Plant Sci. 4:15), which is little. The major issue is that plants are genetically programmed to maintain iron homeostasis. However, if we could manipulate the regulatory mechanism, the iron content in specific plant organs can be increased, e.g. in seeds or tubers. For this, we first need to identify the iron-sensing proteins and elucidate the complete Fe signalling pathway, which we aim to address in this research proposal.

The research might benefit (i) plant breeders (ii) nutritionists and health professionals (iii) policy makers and regulators and, finally (iv) society as a whole, in particular women aged 11 - 64.

(i) Plant breeders
The candidate iron-sensing proteins that are the focus of this research proposal are present in all higher plant species. Therefore, our findings based on the model species Arabidopsis will be transferable to any other crop. Modern breeding using sequence-based markers is accelerating the introduction of new varieties without the need for genetic modification. Commercial plant breeders will be involved to introduce desirable traits, such as high iron content, into modern crop varieties. The John Innes Centre has contacts with major plant breeding companies in the UK. Dr Balk has contacts with Wherry & Son, a large breeder of pulses who provide peas for a JIC Innovation funded project (Oct 2014 - March 2015), and with Limagrain, King's Lynn.
Dr Balk is also involved in a project funded by HarvestPlus, together with Cristobal Uauy, Dale Sanders and Tony Miller, to identify iron and zinc transporters in wheat (September 2013 - ). Wheat also has homologs of the candidate iron-sensing proteins, which could be an additional target for manipulating iron levels.

(ii) Nutritionists and health professionals
Dr Balk is co-investigator on a BBSRC-DRINC2 project headed by Prof Peter Shewry at Rothamsted Research, to investigate the bio-availability of forms of non-heme iron using an in-vitro system of intestinal cells (Sept 2014 - August 2017). Ultimately, this information is important for companies that need to know the iron content for labelling purposes, or for industries that add iron to food products (e.g. breakfast cereals). The proposed project on the iron sensing mechanism in plants will yield mutant lines that accumulate iron. Because of our participation in the DRINC2 project, we can immediately test what species the accumulated iron is, and if this is bio-available.
Dr Balk is also a member of the "Metals in Biology" Network in Biotechnology and Bio-energy, an excellent platform to communicate results of the proposed project.

(iii) Policy makers and regulators
Crops with increased iron content will concern policy makers and regulators, to decide on labeling, but also to introduce high-iron vegetables in menu choices for institutions and schools.