The most widespread dietary problem in the world is mineral deficiency. We used the nicotianamine synthase (NAS) gene to increase mineral contents in rice grains. Nicotianamine (NA) is a chelator of metals and a key component of metal homeostasis. We isolated activation-tagged mutant lines in which expression of a rice NAS gene, OsNAS3, was increased by introducing 35S enhancer elements. Shoots and roots of the OsNAS3 activationtagged plants (OsNAS3-D1) accumulated more Fe and Zn. Seeds from our OsNAS3-D1 plants grown on a paddy field contained elevated amounts of Fe (2.9-fold), Zn (2.2-fold), and Cu (1.7-fold). The NA level was increased 9.6-fold in OsNAS3-D1 seeds. Analysis by size exclusion chromatography coupled with inductively coupled plasma mass spectroscopy showed that WT and Os-NAS3-D1 seeds contained equal amounts of Fe bound to IP6, whereas OsNAS3-D1 had 7-fold more Fe bound to a low molecular mass, which was likely NA. Furthermore, this activation led to increased tolerance to Fe and Zn deficiencies and to excess metal (Zn, Cu, and Ni) toxicities. In contrast, disruption of OsNAS3 caused an opposite phenotype. To test the bioavailability of Fe, we fed anemic mice with either engineered or WT seeds for 4 weeks and measured their concentrations of hemoglobin and hematocrit. Mice fed with engineered seeds recovered to normal levels of hemoglobin and hematocrit within 2 weeks, whereas those that ate WT seeds remained anemic. Our results suggest that an increase in bioavailable mineral content in rice grains can be achieved by enhancing NAS expression. activation tagging ͉ bioavailability ͉ hemoglobin ͉ metal homeostasis

Summary The visual deficiency symptoms developing on plants constitute the ultimate manifestation of suboptimal nutrient supply. In classical plant nutrition, these symptoms have been extensively used as a tool to characterise the nutritional status of plants and to optimise fertilisation. Here we expand this concept by bridging the typical deficiency symptoms for each of the six essential macronutrients to their molecular and physiological functionalities in higher plants. We focus on the most recent insights obtained during the last decade, which now allow us to better understand the links between symptom and function for each element. A deep understanding of the mechanisms underlying the visual deficiency symptoms enables us to thoroughly understand how plants react to nutrient limitations and how these disturbances may affect the productivity and biodiversity of terrestrial ecosystems. A proper interpretation of visual deficiency symptoms will support the potential for sustainable crop intensification through the development of new technologies that facilitate automatised management practices based on imaging technologies, remote sensing and in‐field sensors, thereby providing the basis for timely application of nutrients via smart and more efficient fertilisation.

The increasing prevalence of iron (Fe) and zinc (Zn) deficiencies in human populations worldwide has stressed the need for more information about the distribution and chemical speciation of these elements in cereal products. In order to investigate these aspects, barley grains were fractionated into awns, embryo, bran and endosperm and analysed for Fe and Zn. Simultaneously, phosphorus (P) and sulfur (S) were determined since these elements are major constituents of phytic acid and proteins, respectively, compounds which are potentially involved in Fe and Zn binding. A novel analytical method was developed in which oxygen was added to the octopole reaction cell of the ICP-MS. This approach greatly improved the sensitivity of sulfur, measured as (48)SO(+). Simultaneously, Fe was measured as (72)FeO(+), P as (47)PO(+), and Zn as (66)Zn(+), enabling sensitive and simultaneous analysis of these four elements. The highest concentrations of Zn, Fe, S and P were found in the bran and embryo fractions. Further analysis of the embryo using SEC-ICP-MS revealed that the speciation of Fe and Zn differed. The majority of Fe co-eluted with P as a species with the apparent mass of 12.3 kDa, whereas the majority of Zn co-eluted with S as a 3 kDa species, devoid of any co-eluting P. Subsequent ion pairing chromatography of the Fe/P peak showed that phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate: IP(6)) was the main Fe binding ligand, with the stoichiometry Fe(4)(IP(6))(18). When incubating the embryo tissue with phytase, the enzyme responsible for degradation of phytic acid, the extraction efficiency of both Fe and P was doubled, whereas that of Zn and S was unaffected. Protein degradation on the other hand, using protease XIV, boosted the extraction of Zn and S, but not that of Fe and P. It is concluded that Fe and Zn have a different speciation in cereal grain tissues; Zn appears to be mainly bound to peptides, while Fe is mainly associated with phytic acid.

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