Targeting intracellular transport combined with efficient uptake and storage significantly increases grain iron and zinc levels in rice.

Rice, a staple food for more than half of the world population, is an important target for iron and zinc biofortification. Current strategies mainly focus on the expression of genes for efficient uptake, long-distance transport and storage. Targeting intracellular iron mobilization to increase grain iron levels has not been reported. Vacuole is an important cell compartment for iron storage and the NATURAL RESISTANCE ASSOCIATED MACROPHAGE PROTEIN (NRAMP) family of transporters export iron from vacuoles to cytosol when needed. We developed transgenic Nipponbare rice lines expressing AtNRAMP3 under the control of the UBIQUITIN or rice embryo/aleurone-specific 18-kDa Oleosin (Ole18) promoter together with NICOTIANAMINE SYNTHASE (AtNAS1) and FERRITIN (PvFER), or expressing only AtNRAMP3 and PvFER together. Iron and zinc were increased close to recommended levels in polished grains of the transformed lines, with maximum levels when AtNRAMP3, AtNAS1 and PvFER were expressed together (12.67 μg/g DW iron and 45.60 μg/g DW zinc in polished grains of line NFON16). Similar high iron and zinc levels were obtained in transgenic Indica IR64 lines expressing the AtNRAMP3, AtNAS1 and PvFER cassette (13.65 μg/g DW iron and 48.18 μg/g DW zinc in polished grains of line IR64_1), equalling more than 90% of the recommended iron increase in rice endosperm. Our results demonstrate that targeting intracellular iron stores in combination with iron and zinc transport and endosperm storage is an effective strategy for iron biofortification. The increases achieved in polished IR64 grains are of dietary relevance for human health and a valuable nutrition trait for breeding programmes.