Localisation of iron in wheat grain using high resolution secondary ion mass spectrometry Katie L. Moore a, * , Fang-Jie Zhao b , Cristina S. Gritsch b , Paola Tosi b , Malcolm J. Hawkesford b , Steve P. McGrath b , Peter R. Shewry b , Chris R.M. Grovenor a a Department of Materials, University of Oxford, Parks Road, Oxford OX1 3PH, UK b Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK article info Article history: Received 15 June 2011 Received in revised form 30 October 2011 Accepted 9 November 2011 Keywords: Iron Wheat Secondary ion mass spectrometry Phytate abstract Insufficient iron (Fe) is one of the most prevalent micronutrient deficiencies in humans, with billions of people affected. Cereal grains are an important source of Fe for humans but the bioavailability of Fe in cereals is generally low. Information regarding the cellular and sub-cellular localisation of Fe in wheat grain will aid optimising nutrient delivery for human health. In this study high resolution secondary ion mass spectrometry (NanoSIMS) was used to map the distribution of Fe in the aleurone layer and in the endosperm of immature wheat grain. Iron was shown to be localised strongly in the phytin globoids in the aleurone cells and to a lesser extent in the cytoplasm around the starch granules in the endosperm. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Iron (Fe) deficiency is one of the most common nutritional disor- ders in the world, affecting an estimated 2 billion people (WHO, 2002; Zimmermann and Hurrell, 2007), and is particularly prevalent in developing countries where cereals form a major part of the diet (WHO, 2002). Iron deficiency can affect resistance to infection, cognitive development and pregnancy (Mayer et al., 2008). Cereal grains are important components of strategies to alleviate micro- nutrient deficiencies as they are the primary staple food for the human diet (Brinch-Pedersen et al., 2007). Although Fe is present in cereal grains, its bioavailability is generally very low (Zimmermann and Hurrell, 2007). The amount of phytic acid, the main storage form of phosphorus in cereals, is considered to control the bioavailability of Fe since phytic acid strongly chelates metal ions including Fe, Zn and Ca (Ockenden et al., 2004) forming insoluble complexes which cannot be digested or absorbed by humans due to the lack of intestinal phytase enzymes (Abebe et al., 2007; Persson et al., 2009). It has been recently demonstrated using size exclusion chromatography (SEC)-ICP-MS that Fe in barley embryo is mainly bound to myo-inositol-1,2,3,4,5,6- hexakisphosphate; IP6 (Persson et al., 2009). Cereal grain mutants with low phytic acid contents are a possible strategy to increase Fe intake in developing countries; however it has been estimated that the phytic acid content would need to be reduced by >90% to give a sufficient increase in Fe intake (Zimmermann and Hurrell, 2007). Phytic acid is mainly present in phytin globoids inside the protein storage vacuoles in the aleurone cells of cereal grains and Fe also mainly accumulates in this tissue (Mazzolini et al., 1985; Ockenden et al., 2004), accounting for 70% of the total iron in mature barley grains (Brinch-Pedersen et al., 2007). However, the bulk analysis techniques used to determine the localisation of Fe are limited in their resolution and give no details of Fe distribution at the sub- cellular level. Recently synchrotron techniques have been increas- ingly used for the study of element localisation in cereal grains. Synchrotron low-energy X-ray fluorescence has been used to inves- tigate the sub-cellular distribution of important elements in wheat aleurone (Regvar et al., 2011). Lombi et al. (2011) used synchrotron X- Ray fluorescence to map the distribution of Fe, Cu, Zn and other micronutrients in barley grain with a resolution of 1.25 mm, a major improvement in resolution compared to bulk analysis techniques using milling fractions. The authors found Fe to be highly localised to the aleurone layer but with some also localised to the endosperm in regions near the aleurone layer. However, the sub-cellular distribu- tion pattern of Fe in the aleurone and endosperm remains unclear. High resolution secondary ion mass spectrometry (SIMS) allows the detection of trace elements in biological samples at the cellular and sub-cellular scales. This is a technical challenge for most analytical instruments because of the difficulty in achieving the necessary combination of sensitivity and resolution. SIMS imaging also allows the detection of the lighter elements and molecular * Corresponding author. Tel.: þ44 (0) 1865 273766; fax: þ44 (0) 1865 273789. E-mail address: katie.moore@materials.ox.ac.uk (K.L. Moore). Contents lists available at SciVerse ScienceDirect Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs 0733-5210/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2011.11.005 Journal of Cereal Science 55 (2012) 183e187