Overexpression of rice nicotianamine synthase 2 and soybean ferritin H1 upregulates iron acquisition genes

Rice endosperm lacks essential micronutrients, notably iron. Iron deficiency leads to iron deficiency anemia, which causes morbidity, impaired mental development, and death. At IRRI [International Rice Research Inst., Los Baños, Laguna, Philippines], rice transgenic events with high iron concentration in starchy endosperm harboring selected iron homeostasis genes are being developed and characterized. The effects of selected transgenes on the expression of endogenous iron homeostasis genes are being examined to gain a deeper understanding of the mechanism of iron homeostasis. Results could help identify candidate genes for engineering high-endosperm-iron rice transgenics. Gene expression at the reproductive stage in the flag leaf and seed endosperm in milky, dough, and mature stages in the high-iron (12-18 ppm polished endosperm iron) transgenic event IR64-IRS274 (Trijatmiko et al., in preparation) expressing 35Spro::OsNAS2 amd GluA2pro::SoyFERH1 and its wild-type parent IR64, were determined by quantitative real-time PCR. The 31 targets are endogenous genes for iron uptake/translocation (OsDMAS, OsFRO, OsIRT2, OsNAS, OsNAAT, OsTOM, and OsYSL), storage (OsFER), compartmentalization (OsVIT1, VIT2, and OsNRAMP), regulation (OsIRO), divalent metal transport (OsZIP), and the two transgenes (OsNAS2) and (SoyFERH1). Fold change in gene expression relative to IR64 was calculated using the 2-CT method (Livak and Schmittgen, 2001). Expression data (Cp values) were normalized with the Geometric mean of two rice endogenous reference genes triose phosphate isomerase and tumor protein homolog. Expression of the iron acquisition genes increased in all tissues examined, most notably in milky endosperm for YSL4, YSL15, YSL18, FR02, and IR02, indicating their importance in endosperm iron acquisition. These high expression levels, however, were not sustained through seed maturity. Overexpression of these genes throughout seed development, using appropriate promoters, could result in higher polished rice endosperm iron content that satisfies human dietary iron requirements.