CROPNUT: increasing iron in cereals

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Most modern wheat varieties, although excellent providers of carbohydrates, are poor sources of mineral micronutrients. Levels of the micronutrients iron and zinc are especially low in the endosperm, which is used to make white flour. Conversely grains contain relatively high levels of the anti-nutrient phytate. Therefore, the Food Standards Agency requires all milled flour sold in the UK to be fortified with iron salts or iron powder. A much more sustainable method is biofortification, whereby plants are induced to translocate more minerals into edible parts. We have recently developed a high-iron wheat line by overexpressing a vacuolar iron transporter using an endosperm-specific promoter (Connorton et al, manuscript in preparation). The sequence are from wheat itself (cisgenic). Iron in the white flour fraction is increased 3-fold to 16 - 17 ppm, which would remove the legal requirement for fortification. However, we actually do not fully understand why this particular strategy is so successful whereas other strategies have only marginally increased iron and zinc levels. Here we propose to use the high-iron wheat line as a tool to understand how iron is transported into the grain and further distributed to the aleurone, endosperm and embryo. We will study changes in gene expression as a consequence of the increased iron flux, and map the route of iron through the different tissue and cell types using isotope studies and NanoSIMS imaging (with Dr Katie Moore, Manchester University). We will also investigate if the increased iron is due to increased uptake by the roots or increased remobilization from senescing leaves. In addition, we will investigate bioavailability of the iron for human nutrition in the white flour fractions and how this is affected by food processing, such as baking bread(with Paul Sharp, King's College London). This knowledge will be used to design non-GM approaches to increase the mineral content of cereals.

The iron levels in the new high-iron wheat line are 16 - 17 mg/kg in white flour, well above the upper limit of natural variation (~ 12 mg/kg) and in line with the legal requirement for chemical fortification (16.5 mg/kg). So far, we do not see any negative impact on plant growth or yield. The work to date has been carried out in the Fielder cultivar, but this trait could now in principle be bred into modern commercial wheat varieties and remove the requirement for post-milling chemical fortification. We have contacted several potential UK stakeholders and received interested responses from the baking company Warburtons and Marks & Spencers (see letters of support).

One obstacle to more widespread acceptance is that the successful high-iron line, though not transgenic, is by definition genetically modified (GM). The approach taken was what is called cisgenic: we used a wheat promoter to change the timing and levels of expression of a wheat gene. So the sequences that were transformed are from the same species, The manipulations that are required cannot currently be achieved by non-GM methods. Although the cisgenic line is of interest to countries that do accept GM crops (e.g. India) most UK stake holders, and also the international maize and wheat improvement centre CIMMYT (see letter of support) are hesitant to use this line in their breeding programmes. We therefore seek to replicate the striking phenotype seen in this line through non-GM means.

Now that we know that a dramatic increase in iron and zinc in the endosperm of cereal grains is technically possible, it should be possible to design other, non-GM strategies to meet the same goal. For this we need to better understand what is changed in the high-iron line with regards to iron and zinc transport. We suspect that increased accumulation of iron into the vacuole of the endosperm has triggered other changes in gene expression, such as increased uptake by the roots and/or increased remobilization from senescing leaves, while lowering the saturation point of regulatory mechanisms. Though overexpressing genes in wheat through non-GM means is not currently feasible we do have a population of TILLING lines available with single nucleotide polymorphisms (SNPs) in potential genes of interest that will severely disrupt their function. These lines are not classed as GM and so through crossing with commercial wheat varieties high iron traits associated with these SNPs can be incorporated into existing breeding programmes. An alternative approach is CRISPR, which is also not classed as GM, and could be used to simultaneously knock down the function of multiple genes. The iron sensing and regulatory machinery of wheat would be excellent targets for these approaches.