Abstract
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.
Highlights
Iron deficiency is one of the most prevalent micronutrient deficiencies, affecting around two billion people globally (WHO, 2016)
Shoots and roots generally showed the expected expression of Arabidopsis thaliana IRT1 gene (AtIRT1) and AtNAS1 but not PvFERRITIN, consistent with the choice of promoters, the activity of the promoters was variable among different independent lines (Figure 2)
Both MsENOD12B and AtIRT1 promoters are active in root hair and epidermal cells (Terada et al, 2001; Cailliatte et al, 2010), and it cannot be excluded that the AtIRT1 expression specificities in Figure 2 were biased by proportional differences in the amount of root hair cells in the collected root samples
Summary
Iron deficiency is one of the most prevalent micronutrient deficiencies, affecting around two billion people globally (WHO, 2016). In order to control and prevent iron deficiency anemia, the WHO and UNICEF recommend intervention strategies including increased iron intake via dietary diversification, supplementation, and/or fortification, overall improved nutritional status by controlling other nutritional deficiencies as well as appropriate control of infectious diseases (WHO, 2001). Rice is a staple food of more than half of the world’s population but contains only small amounts of micronutrients, including dietary iron. Polished grains of widely grown rice varieties provide only around 2 μg/g of iron (Bouis et al, 2011) and rice is an important target crop for iron biofortification with recommended polished grain iron concentration of up to 15 μg/g dry weight (DW; Bouis et al, 2011)
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