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Micronutrient malnutrition is widespread, especially in poor populations across the globe, and iron deficiency anemia is one of the most prevalent forms of micronutrient deficiencies. Iron deficiency anemia has severe consequences for human health, working ability, and quality of life. Several interventions including iron supplementation and food fortification have been attempted and met with varied degrees of success. Rice, which is a staple food for over half of the world’s population, is an important target crop for iron biofortification. The genetic variability of iron content in the rice germplasm is very narrow, and thus, conventional breeding has not been successful in developing high iron rice varieties. Therefore, genetic engineering approaches have targeted at increasing iron uptake, translocation, and storage in the rice endosperm. We previously reported that  AtIRT1 , when expressed together with  AtNAS1  and  PvFERRITIN  ( PvFER ) in high-iron (NFP) rice, has a synergistic effect of further increasing the iron concentration of polished rice grains. We have now engineered rice expressing  AtIRT1, AtNAS1 , and  PvFER  as a single locus gene cassette and compared the resulting lines with transgenic lines expressing  AtIRT1  and  PvFER  gene cassettes. We also evaluated the efficacies of the  MsENOD12B  and native  AtIRT1  promoters for the expression of  AtIRT1  in rice in both types of gene cassettes, and found the native  AtIRT1  promoter to be a better choice for driving the  AtIRT1  expression in our biofortification strategy. All the single insertion transgenic lines have significant increases of iron concentration, both in polished and unpolished grains, but the concerted expression of  AtIRT1, AtNAS1 , and  PvFER  resulted to be a more effective strategy in achieving the highest iron increases of up to 10.46 μg/g dry weight. Furthermore, the transformed high iron lines grew better under iron deficiency growth conditions and also have significantly increased grain zinc concentration. Together, these rice lines have nutritionally relevant increases in polished grain iron and zinc concentration necessary to support human health.

Enhanced Grain Iron Levels in Rice Expressing an IRON-REGULATED METAL TRANSPORTER, NICOTIANAMINE SYNTHASE, and FERRITIN Gene Cassette