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Expanded web feature: High-tech search for mineral-rich wheat grain

Winter 2010

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Consumers and grain processors would benefit from wheat varieties that could be used to make healthier mineral-enriched flour. Pioneering research that combines plant breeding with synchrotron-based x-ray fluorescence analysis is helping to track down novel varieties with the desired properties.

Deficiencies in iron and zinc cause a variety of serious health problems in many parts of the world. According to the World Health Organisation, iron deficiency (anaemia) is estimated to affect 2Bn people worldwide and cause 841,000 deaths are per year. The problem is acute particularly in areas where hookworms and urinary schistosomiasis cause blood-loss, and where diets contain little meat.

Similarly, zinc deficiency is a problem in Africa, the eastern Mediterranean and south-east Asia where it is estimated to contribute toward 406,000 deaths from pneumonia per year – and that’s on top of 176,000 diarrhoea and 207,000 malaria deaths linked to zinc deficiency.

Against the grain

One attractive option for improving the nutritional value of diets is to increase the mineral content of wheat flour. But this is more challenging than it sounds.

Dr Andrew Neal from Rothamsted Research says the location of minerals in the grain and their chemical forms is important. "Minerals are found in the outer (bran) layer and the embryo (germ), but white flour which comprises most of the grain is made from starchy endosperm and is almost devoid of minerals. So, breeding wheat for mineral-enriched white flour requires finding varieties that deposit useable forms of minerals in the endosperm."

Traditionally, staining techniques are used to locate minerals in the different tissues of the grain. But staining is time consuming, a different stain is needed for each mineral, and not all minerals are easily viewed using this approach, nor the fine details of their distribution.

Neal has adopted a radically different approach, which not only locates several different minerals simultaneously, but also provides information about their concentration, complexation (how the metallic elements are arranged) and digestibility.

The new analysis depends on exposing grains to some of the most highly focused and high intensity x-rays on the planet. As the x-rays encounter different minerals in the grains, characteristic fluorescence is emitted that reveal the location and concentration of each mineral.

This isn’t the sort of technology available in plant research facilities. Neal uses an x-ray beam line at the world famous Diamond Light Source, a third generation synchrotron in Oxfordshire (see Super synchrotron).

Pumping iron

Recently, he tested novel wheat lines developed to produce iron-rich endosperm. Genetic modification of the experimental line resulted in an enhanced capability to express ferritin, an easily digested storage form of iron, in the starchy endosperm. And measures of total iron in the grain indicated the lines contained as much as three times the amount of iron as traditional wheat grains. But x-ray analysis showed that much of the extra iron resided in the bran around the crease region of the grain, not in the endosperm at all.

Neal says the issue is why the extra metal is not transported by transfer cells in the crease region into the grain. "It’s like there’s a bottleneck there. If we can understand what’s preventing metals being transferred across the crease and deposited in the endosperm then there’s a very good chance we will succeed."

Neal’s next plan is to image the crease region at much higher resolution to understand iron complexation around the region where minerals are transferred from the maternal plant to the developing grain. Metals are typically transported around plants as organic acids but converted into the more stable phytate (phytic acid) once they cross into the grain.

"The challenge is to keep the extra iron as the organic acid form so it will transfer into the grain" says Neal. "If we could understand the mechanism that would prevent conversion to phytate we would perhaps have a way of solving this bottleneck issue."

And Neal hopes that looking at seeds at a higher resolution will identify in which cell groups the switch from organic acid to phytate occurs. "It’s perfectly possible that a wheat line already exists that transfers a lot more metals across, we just haven’t looked at it yet. That’s why using this technology as a screen could be very powerful."

Inspection gadget

Already Neal’s work has uncovered a few new discoveries, namely that iron isn’t uniformly distributed in the bran. "The further you go away from the embryo the less iron there is. It’s not homogenously distributed but mostly stored near the embryo where it is readily available as the embryo begins to sprout. We assume that’s why, and it would make sense, but it hadn’t been observed before."

Other essential observations will need to be made if more nutritious grains are to become a reality (see Grains of truth). Specifically, if more iron can be packed into a seed, are we sure that there is room and that other elements are not displaced? "That has to be a concern," says Neal. "If you pack the seed with iron are you losing the zinc, nickel or manganese? If you use the staining approach you just can’t tell. This is perhaps the first technology we have that could answer those questions."

Delving into the seeds using synchrotron light is a time consuming process. The images take 15-20 hours each, and in a typical four-day visit to the synchrotron Neal has to choose between gathering in depth data on a limited number of grains or superficial information on a number of different lines. "You can’t do an awful lot in 4 days, even working 24 hours a day" he says. "To realise the true potential of this technique it would be nice to have regular access."

Although disappointed by this and other studies which showed that iron in the new grains was not in the form of ferritin but was in the less digestible and more common phytate form, Neal remains upbeat about the technology. "It is early days for this approach, but already we have shown it can screen-out unsuitable lines early on, and prevent breeders wasting investment in them."

Grains of truth

Neal’s work at Diamond is funded by BBSRC, but his ferritin-expressing wheat lines were provided by work initiated by the Healthgrain project.

The Healthgrain programme aims to improve the health of European consumers by increasing the nutritional content of whole grains and products derived from them. Studies have shown that balanced intake of whole grains and cereal dietary fibre can protect against chronic diseases such as cardiovascular disease and type 2 diabetes.

The Healthgrain project winds up in 2010

Work under one of the programme’s 4 modules has screened 200 cereal lines, primarily of bread-making wheat (Triticum aestivum), but that also included 50 other lines including barley, rye, durum and oats.

Neal is not the only scientist with an interest in the high-tech imaging of food-related structures. Clare Mills, Leader of the Food Structure and Health programme at the Institute of Food Research (IFR, an institute of BBSRC), is also a work package leader on the Healthgrain project. She says that combining spatial information with information on chemical composition is extremely powerful. "It gives an insight into how the structural compartmentalisation of different components in plants foods affects the nutritional quality of what we eat," she says.

In terms of the nutritional value of food such an approach is important in understanding how processes like milling, which fractures the grains, can lead to the loss of useful compounds. "You may lose nutrients you want to keep, or keep things you don’t want to keep," she says. "Structure is a very important part of understanding how food gives health benefits.

The health-protective compounds in grains, in addition to dietary fibre, include folates, tocopherols and tocotrienols, other vitamins, trace elements and minerals as well as a range of phytochemicals such as lignans, phenolic acids, alkylresorcinols, phytosterols. These compounds are concentrated in the outer layers of the grain, and are thus removed in production of white wheat flour.

The €16M programme brings together 43 organisations from 15 European countries from 2005-2010 to develop ways to produce cereal foods containing more of these protective compounds, reveal the physiological mechanisms that underpin their role in prevention of metabolic diseases, and gauge consumers’ acceptance of wholegrain products.

Super synchrotron

The Diamond Light Source synchrotron in Oxfordshire is one of the newest and most advanced particle accelerators in the world. Electrons are accelerated to near the speed of light and the high energy x-rays produced, called synchrotron light, are focused into beamline experimental stations and pass through the target object which, at Diamond, can be as small as a crystallised molecule or as large as an aircraft engine. It means that this 45,000 square metre construction is really one of the most powerful microscopes in the world.

The Diamond Light Source became fully operational in 2007. Image: DLS

The advantages of using the synchrotron to analyse seeds are that little sample preparation required and simultaneous collection of data on multiple elements is possible. The x-rays are so highly focused that chemical complex (how the elements are arranged in molecules) details are revealed.

This is possible by extended x-ray absorption fine structure spectroscopy (EXAFS). Electrons are lost when x-rays strike the sample but before they are lost they interact with the local molecular structure which can reveal how the elements are organised; is iron in the form of phytate or ferritin, for example. It means that two properties of x-rays are exploited in synchrotrons systems and EXAFS can reveal important details related to the bioavailability and digestibility of the iron present because the body can absorb more iron from ferritin than from phytates.

Phase I construction (£263M) was funded by the Science and Technologies Facilities council and the Wellcome trust. BBSRC has since contributed around £2M for phase II funding of additional beamlines, in addition to the Diamond Fellowship that has brought Professor So Iwata’s expertise to the Diamond synchrotron community to unravel the structures of membrane proteins – key targets for drug discovery (see video feature ' Beautiful biology from particle physics').

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