Localization of iron in rice grain using synchrotron X-ray fluorescence microscopy and high resolution secondary ion mass spectrometry
Introduction
Rice is the staple food for billions of people in developing countries of South Asia, Sub-Saharan Africa and Latin America and contributes more than 50% of total caloric intake in many of these countries (Khush, 2005). The concentration and bioavailability of iron (Fe) is generally low in rice grain and, as a result, rice-based diets often result in human Fe deficiency. Iron deficiency is the most common micronutrient deficiency in the world and affects more than two billion people. Symptoms of Fe deficiency include impaired immune function and mental development in children, increased risk of child and maternal mortality, and Fe-deficiency anaemia. Whilst Fe supplements and fortification of manufactured rice products with Fe fortificants, such as ferrous sulphate, have traditionally been used to increase Fe intakes in rice diets, the development of biofortified rice with increased Fe accumulation in polished grain presents a more sustainable and economical means of increasing Fe intakes in rice-based diets over the long term (Bouis et al., 2011).
Iron homeostasis is tightly controlled in plants throughout germination, vegetative growth and reproductive cycles. Excess Fe can result in Fe toxicity through the formation of hydroxyl radicals in the presence of free ferrous ions during the Fenton reaction. It is therefore important that Fe remains complexed to chelating agents – nicotianamine (NA), deoxymugineic acid (DMA), ferritin, citrate and phytate – in plant tissues to avoid toxicity and maintain cellular iron homeostasis. In vegetative tissues, iron complexes are often sequestered into chloroplasts, as is the case for Fe-ferritin, or into vacuoles, as is the case with Fe-phytate (Regvar et al., 2011). Within rice grain, protein storage vacuoles (PSVs) of the embryo and aleurone are important storage sites for Fe, nutrients and enzymes required for germination. Iron-phytate complexes have been found to be present as globoids in the PSV of embryo, scutellum and provascular tissues of the rice grain, with levels increasing during grain development (Wada and Lott, 1997, Yoshida et al., 1999). While Fe is relatively abundant throughout the aleurone, scutellum and embryonic tissues of rice grain, little Fe is present in the endosperm (Hansen et al., 2012).
Several different techniques have been employed to examine the distribution of Fe in rice grain. Histochemical stains such as Perl's Prussian Blue have demonstrated Fe localisation in outer grain tissues believed to comprise the aleurone layer (Choi et al., 2007, Prom-u-thai et al., 2003) but the semi-quantitative colourimetric stains are not sufficiently sensitive to detect the low Fe concentrations present in endosperm. Synchrotron X-ray fluorescence microscopy (XFM) is a more sensitive analytical technique that enables fine mapping of Fe and other micronutrients across the entire grain surface (Lombi et al., 2009, Takahashi et al., 2009). Several studies employing this technique have demonstrated that Fe is present in outer layers of rice endosperm albeit at low concentrations (Iwai et al., 2012, Meharg et al., 2008). In the outer endosperm tissues of rice, Fe does not appear to co-localize with P and is therefore unlikely to be bound to phytate (Johnson et al., 2011).
High resolution secondary ion mass spectrometry (NanoSIMS) has emerged as a highly sensitive technique for the localisation of trace elements in biological samples at cellular and subcellular scales (Moore et al., 2012). NanoSIMS is capable of simultaneous high lateral resolution, high mass resolution and high sensitivity analysis allowing subcellular localisation of elements and has been used to determine the localisation of arsenic in rice grain (Moore et al., 2010), high-resolution imaging of nickel distribution in leaf tissue of the hyperaccumulator species Alyssum lesbiacum (Smart et al., 2010), in situ mapping of nitrogen uptake in the rhizosphere (Clode et al., 2009) and Fe localisation in wheat grain (Moore et al., 2012). Here we describe the use of XFM and NanoSIMS to map the distribution of Fe, zinc (Zn) and other nutritionally relevant elements in the aleurone and subaleurone layers of grain from wild-type and an iron-enriched line of rice, to further investigate Fe localization within grain tissues.
Section snippets
Plant material
Rice (Oryza sativa L. ssp. japonica cv. Nipponbare) plants in wild-type (WT) and an OsNAS2 overexpressing background were germinated on moist filter paper before transfer to 15 cm (1 L capacity) containers of University of California (UC) potting mix in a glasshouse maintained at 28 °C day, 24 °C night, 12 h light/dark. The OsNAS2 overexpressing line (OE-OsNAS2D) came from the Oryza sativa nicotianamine synthase (OsNAS) gene overexpression collection described in Johnson et al. (2011) and
Embryo and aleurone
The XFM tri-colour maps for manganese (Mn), Fe and Zn reported in Figs. 1A and 2A indicate that the overall distribution of these micronutrients in WT and OE-OsNAS2D grain is similar even though the intensity of the Fe signal is substantially higher in OE-OsNAS2D grain. Iron and Zn are clearly co-localized in the scutellum and in most other regions of the embryo. This distribution is similar to that reported for rice by Takahashi et al. (2009) and for barley by Lombi et al. (2011). These
Conclusions
This two-pronged study using XFM and NanoSIMS technology to probe Fe localization at the tissue and subcellular levels clearly demonstrates that a portion of Fe in both WT and Fe-biofortified rice grain is accumulated in the subaleurone/outer endosperm layers in a manner that is not associated with phytic acid (P). We hypothesize that Fe within these layers may be bound to the metal cation chelator NA, particularly as the Fe localization profile was similar in grain from both WT and an
Acknowledgements
This research was undertaken using the XFM beamline at the Australian Synchrotron, Victoria, Australia and supported by grants from the Australian Research Council (LP0883746) and the HarvestPlus Challenge Program. K. L. Moore was supported by an EPSRC Doctoral Training Account studentship. The authors would like to thank Professor Chris Grovenor for providing access to the NanoSIMS.
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- 1
These authors contributed equally to this work.
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Present address: Center for Desert Agriculture, Division of Biological and Environmental Sciences and Engineering, 4700 King Abdullah University of Science and Technology, Thuwal 23955-6900, Saudi Arabia.