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18th International Symposium on Iron Nutrition and Interaction in Plants
Madrid – May 30-June 3 2016
LOCALIZATION OF METALS AND METAL LIGANDS IN RICE SEEDS OVEREXPRESSING NICOTIANAMINE
SYNTHASE AND/OR BARLEY NICOTIANAMINE AMINO TRANSFERASE
Pablo Diaz-Benito1*, Beatriz Fernández2, Raviraj Banakar3, Sara Rodríguez2, Paul Christou3,4, Rosario
Pereiro2, Javier Abadía1, Ana Álvarez-Fernández1
1Dept. of Plant Nutrition, Estación Experimental de Aula Dei (CSIC), Avda. Montañana 1005, 50059
Zaragoza, Spain; 2Dept. of Physical & Analytical Chemistry, University of Oviedo, Julian Clavería, 33006
Oviedo, Spain; 3Dept. of Plant Production and Forestry Science, ETSEA, University of Lleida-Agrotecnio
Center, Av. Alcalde Rovira Roure, 191, 25198 Lleida, Spain; 4Institució Catalana Recerca i Estudis
Avançats, Passeig Lluís Companys 23, 08010 Barcelona, Spain
*[email protected]
Biofortification of rice seeds with micronutrients has the potential to mitigate deficiencies of Fe and Zn in
humans [1]. Increasing the synthesis of the metal ligands nicotianamine (NA) and deoxymugineic acid (DMA)
in rice plants leads to increases in seed Fe and Zn [1], and high levels of NA in rice seeds enhance intestinal
Fe and Zn absorption [2]. Since the high metal content embryo and bran parts of the rice seed are removed
during processing (polishing), the localization of the accumulation of NA, DMA and metals in biofortified
seeds is relevant. Laser ablation (LA) inductively coupled plasma (ICP) mass spectrometry (MS) provides
spatial localization of elements in tissues, with a resolution in the µm range, and allows for an accurate
absolute elemental quantification [3,4]. In this study, we used LA-ICP-MS to localize and quantify different
elements (e.g., Fe, Zn) in 60 µm-thick longitudinal sections of rice seeds from WT and transgenic lines overexpressing rice nicotianamine synthase (OsNAS1) and/or barley nicotianamine amino transferase
(HvNAATB) genes. The embryo and endosperm were also excised and analyzed for NA and DMA using
liquid chromatography (HPLC) coupled to high-resolution time-of-flight (TOF) MS, and for micronutrients by
ICP-MS. In WT seeds, the concentrations of DMA were 14 and 25 µg g-1 DW in endosperm and embryo,
respectively, whereas NA was 6 µg g-1 DW in the embryo but was not detected in the endosperm. In the
endosperm, the overexpression of OsNAS1 caused the appearance of NA and increases in DMA (8-fold), Fe
(2-fold) and Zn (2-fold) concentrations, whereas in the embryo it caused increases in NA (9-fold), DMA (3fold) and Fe (2-fold) concentrations. In contrast, the overexpression of HvNAATB did not affect the
concentrations of ligands and metals neither in endosperm nor embryo. The simultaneous overexpression of
OsNAS1 and HvNAATB increased significantly the DMA and Fe concentrations both in the endosperm (14fold for DMA and 3-fold for Fe) and the embryo (8-fold for DMA and 2-fold for Fe). Regarding metal
localization, the most relevant result was the preferential accumulation of Fe in transfer cells and root
primordia in lines overexpressing OsNAS1 and HvNAATB, and in leaf and root primordia in lines
overexpressing OsNAS1. Results support the roles of NA and DMA as key ligands for increasing Fe and Zn
concentrations in rice seeds, and suggest that each ligand may promote Fe mobilization in different embryo
tissues.
Keywords: Endosperm, embryo, laser ablation-inductively coupled plasma-mass spectrometry,
micronutrients, phytosiderophores.
REFERENCES
[1] Bashir K, Takahashi R, et al., 2013 Front Plant Sci 4:15; [2] Clemens S, 2014 Plant Sci 225:52; [3]
Wu B, Becker JS, 2012 Metallomics 4:403; [4] Basnet P, Amarasiriwardena D, et al., 2014 Environ Pollut
195:148.
ACKNOWLEDGEMENTS: Supported by the Spanish Ministry of Economy and Competitiveness (projects
AGL2012-31988 and AGL2013-42175-R, co-financed with FEDER) and the Aragón Government (group
A03). P.D.-B. was supported by a MINECO-FPI contract.
Preferred Presentation format: ORAL
Selected Sessions: Iron fortification of crops for a better human nutrition.
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