Increasing seed iron content by gene manipulation in Arabidopsis
Authors: Ghalamkari Zahra , Buckhout Thomas J. 
Published: June 2021

Iron‐deficiency anemia is the most widespread human micronutrient deficiency in the world. According WHO more than two billion people suffer from Fe‐deficiency anemia. Children and women in particular are at higher risk. Food‐based methods may increase iron intake by incorporating iron‐rich foods into the diet. However, due to difficulties with changing dietary habits and the cost of post‐harvest supplementations, these methods have met with only partially success.Here we report on our results on attempts to increase Fe in plants by genetic manipulation. The non‐proteinogenic amino acid nicotianamine (NA) chelates and transports essential metal ions such as Fe(II) and Zn(II) in plants. NA has been detected in roots and leaves and in phloem sap. It is critical for seed development. Recent investigations have already shown an important role of NAS genes in increasing the Fe content in plants. Alternatively, a small Vacuolar Iron Transporter‐Like (VTL) gene family is found in both mono‐ and dicotyledon plants. VTL1, 2 and 5 are vacuolar Fe importers, and our investigations reveal that over‐expression of the VTLs in plants can lead to moderate increases in seed Fe content. In my research I have attempted to combine an increase in the Fe transport capacity by over‐expression of AtNAS3 with an increased Fe storage capacity through overexpression of AtVTL5. To this end plants that over‐expressed the NA synthesis gene, AtNAS3, in Arabidopsis were crossed with AtVTL5 over‐expressing lines. The results showed that the Fe content in the wild‐type (WT) was 166 μg/g fresh weight, where as in the AtVTL5 over‐expressing line it was 170 μg/g and in the AtNAS3 over‐expressing line 182 μg/g. In the AtVTL5‐AtNAS3 double overexpressing line the Fe content was 207 μg/g.The expression level of transcription regulators MYB72, bHLH38, PYE, ILR3 as well as the Fe transporter IRT1, which are all known to be involved in Fe homeostasis, was determined in WT, VTL5‐OE, NAS3‐OE and VTL5‐NAS3 double overexpressing plants that were grown 14 days under sufficient Fe concentrations and then seedlings were transferred for 72h in 0 or 40 μM Fe. Results from quantitative RT‐PCR analyses indicate that MYB72, bHLH38 and PYE in VTL5‐OE, NAS3‐OE and VTL5‐NAS3 double overexpressing were up‐regulated compared with the WT, whereas ILR3 as a repressor of Fe hemostasis genes was down‐regulated. Morphological analyzes of 7 day‐old seedlings, which were grown in 0 and 40 μM Fe, revealed that the ratio of root length in Fe deficient to Fe sufficient NAS3‐OE was 0.9 and VTL5‐NAS3 double overexpressing was 0.8, whereas in the WT it was 0.6. A visualization of ferric reduction in +Fe and ‐Fe plants showed a stronger pattern of reduction activity in NAS3‐OE and VTL5‐NAS3 double overexpressing plants compared to the WT. In conclusion, we have shown that the double over‐expression of AtNAS3 and AtVTL5 successfully increased the Fe content in Arabidopsis seeds. Furthermore, based on growth of these plants the increased Fe appears to be biologically available for growth and seed germination.Support or Funding InformationYousef Jameel FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.