Main conclusion

Expression ofbHLH104-GFPdriven by theMYB72promoter improves plants’ tolerance to Fe deficiency and increases seed Fe concentrations.

Iron (Fe) deficiency causes reduced crop yield and quality. In humans, Fe deficiency is directly associated with Fe-deficiency anemia. Therefore, breeding Fe-deficiency tolerant and Fe-enriched plants are an ideal approach to deal with these problems. Here, different strategies were explored to generate Fe-deficiency tolerant and Fe-enriched plants. Unexpectedly, the overexpression of Fe-deficiency responsive genes (IRT1, MYB72, and bHLH100) resulted in enhanced sensitivity to Fe deficiency, including leaf chlorosis and short roots under Fe-deficiency conditions. Next, three different types of Fe-deficiency responsive promoters (Pro_IRT1, Pro_MYB72, and Pro_bHLH100) were used to drive the expression of bHLH104-GFP fusion gene in Arabidopsis. Pro_IRT1:bHLH104-GFP plants showed the enhanced sensitivity to Fe deficiency on Fe-deficient media and the reduced fertility in alkaline soil. In contrast, Pro_bHLH100:bHLH104-GFP plants displayed a slight tolerance to Fe deficiency and Pro_MYB72:bHLH104-GFP plants had a significant advantage in growth in alkaline soil, including increased root length, chlorophyll, and biomass. Further analysis revealed that the expression of Fe-deficiency responsive genes was dramatically upregulated in both Pro_MYB72:bHLH104-GFP and Pro_bHLH100:bHLH104-GFP plants under Fe-deficiency conditions. When grown in alkaline soil, Pro_MYB72:bHLH104-GFP plants greatly improved the seed yield and Fe concentration. These results are fundamental for plant manipulation approaches to modify tolerance to Fe deficiency and Fe accumulation through alterations of bHLH104 gene expression.