Iron fortification of rice seeds through activation of the nicotianamine synthase gene

Abstract

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

Nutritionally Enhanced Staple Food Crops

“…More importantly, the transgenic rice plants containing AtNAS and Pvferritin genes increased seed-Fe concentration by sixfold, with no yield penalty but such plants were earlier to flower, and that the Fe in the endosperm of the transgenic rice lines accumulated in spots, most probably as a consequence of spatially restricted ferritin accumulation (Wirth et al 2009). Biofortifying rice with NAS alone or in combination with ferritin has great potential in combating global human Fe deficiency in people dependent on rice for their sustenance (Lee et al 2009;Zheng et al 2010). Mugineic acid family phytosiderophores (MAs) play an important role in the uptake of Fe from the soil and Fe transport within the plant in graminaceous plants.…”

Iron Uptake and Loading into Rice Grains

“…However, a similar approach using HvNAS1 in combination with soybean ferritin did not increase the seed Fe content (Masuda et al 2009). Activation tagging for OsNAS3 also resulted in increased Fe accumulation in roots, shoots, and seeds (Lee et al 2009b). OsNAS3-activated plants grown on a paddy field accumulated three times more Fe than WT plants and 9.6 times more NA in their seeds.…”

Activation of Rice nicotianamine synthase 2 (OsNAS2) Enhances Iron Availability for Biofortification

“…We have previously shown that grains from activation-tagged lines of OsNAS3 have 2.9-fold more Fe (Lee et al, 2009b). Furthermore, the hemoglobin levels in anemic mice fed with seeds from those transgenic plants recover to normal readings when animals are placed on that diet for two weeks.…”

“…Fe-bioavailability using anemic mice was tested as previously described (Lee et al, 2009b). After three weeks of weaning for pathogen-free female Balb/c mice, one group was fed with AIN-93DIET (45 mg Fe kg -1 ) as a control diet (CD) and the second group was given an iron-depleted (ID) diet (modified AIN-93G diet containing 3 mg Fe kg -1 ).…”

“…Mice fed with OsNAS2-D1 seeds recover more quickly from anemia To evaluate the bioavailability of Fe when complexed with NA, we used mouse feeding experiments that applied our previously established system of tests with seeds from OsNAS3-activation-tagged plants (Lee et al, 2009b). In the current study, we assayed Fe-bioavailability in OsNAS2-D1 seeds.…”