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CROPNUT: increasing iron in cereals

ReferenceBB/P019072/1
Principal Investigator / Supervisor Dr Janneke Balk
Co-Investigators /
Co-Supervisors		 Dr James Connorton, Professor Cristobal Uauy
Institution University of East Anglia
DepartmentBiological Sciences
Funding typeResearch
Value (£) 476,570
StatusCompleted
TypeResearch Grant
Start date 01/10/2017
End date 31/03/2021
Duration42 months

Abstract

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.

Summary

Deficiencies in iron and zinc are global health issues, which are currently addressed by supplements and fortification programmes. In the developing world, iron supplements are an integral part of aid programmes and combatting anaemia is a major priority of the World Health Organization. Closer to home, low serum iron levels are prevalent in ~5% of females aged 11 - 64 (National Diet and Nutrition Survey 2012) correlating with low iron intake. To combat iron deficiency, all flours milled in the UK are chemically fortified with iron salts or iron powder up to 16.5 mg/kg (UK Flour Regulations 1998). The natural variation of iron in modern bread wheat is limited within the range of 6 - 13 mg/kg in white flour. Fertilization experiments have shown that extra iron is not taken up by plants. Therefore, increasing iron and zinc levels using genetic methods, known as biofortification, is the most sustainable approach to increase mineral levels in our diet which is dominated by a few staple crops. The limited variation in iron levels in bread wheat has been attributed to a number of factors. Over centuries, crop varieties have been selected for yield, at the cost of micronutrient content. Moreover, polyploid crops such as wheat are genetically buffered: gene variants that could change a certain trait are masked by other copies of the same gene, which makes it hard to select novel traits. In addition, iron levels are tightly regulated by plants to prevent over-accumulation of this metal that is toxic in its free ionic form. And last but not least, cereals have not evolved to put large amounts of minerals into the starchy endosperm, the part of the grain that we prefer to eat. In a very successful collaboration between the Balk and Uauy labs, we have recently found that, against expectations, iron and zinc levels in white flour from wheat or barley can be increased 3- and 2-fold, respectively. Element analysis showed that the iron levels of white flour were 16 - 17 mg/kg, similar to the legal requirement for fortification, and higher still in wholemeal flour. This was achieved by a cis-genics approach: wheat plants were genetically modified but the sequences are from wheat itself. We placed an endosperm-specific regulatory sequence in front of a wheat iron transporter. Our results show that, in principle, plants can direct much more iron and zinc to the endosperm than they do naturally. Moreover, there does not seem to be any major negative effect on growth. While the timely overexpression of the vacuolar iron transporter works remarkably well in boosting iron and zinc levels in grain, we do not yet understand why this strategy is so successful. After all, the up-regulated gene is a simple transporter and not a regulator. If we draw an analogy to traffic flow, increasing the number of lanes on the M25 does not per se improve traffic flow. Access roads, junctions and so on all need to be widened to increase traffic and prevent congestion. Also, we found that the transporter is specific for iron and cannot transport zinc. So why are zinc levels increased? To exploit our findings for biofortification, we will investigate the molecular and cell biological changes that underlie increased mineral transfer in the high-iron wheat line. We will also investigate what the source of iron and zinc is, for example if the plants take up more iron from the soil or whether the iron is more efficiently remobilized from other parts of the plant. We will then use this information to develop non-GM strategies to increase iron and zinc in wheat and other cereals. The bioavailability of the iron and zinc will be tested by offering digested white flour and bread to cultured intestinal cells. Taken together, these studies will greatly enhance our knowledge on nutrient transport, provide us with novel and non-GM strategies to increase the nutritional quality of wheat and give us a way to assess their impact on human nutrition.

Impact Summary

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 from the National Association of British and Irish Millers (NABIM) (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 theroots 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.
Committee Research Committee B (Plants, microbes, food & sustainability)
Research TopicsCrop Science, Plant Science
Research PriorityX – Research Priority information not available
Research Initiative X - not in an Initiative
Funding SchemeX – not Funded via a specific Funding Scheme
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