Cation selectivity of IRT1: Restricting cadmium uptake by plants

Iron plays a central role in key biological processes in both plants and animals. Lack of available iron commonly limits plant growth and nutritional value. Worldwide, dietary iron is primarily obtained from plant sources and, despite our best efforts, iron deficiency continues to be the most prevalent human nutritional disorder. In a global response to combating iron deficiency, efforts have been directed at iron fortification of crops prior to harvest, such as enhancing plant iron storage capacity through genetic engineering. However, increasing storage capacity actually depletes the pool of iron available for plant cellular demands, resulting in iron deficiency. As a result, plants increase expression of metal transporters in an attempt to acquire more iron from the soil. Unfortunately, the major transporter responsible for iron uptake from the soil, IRT1 (Iron Regulated Transporter1), also transports significant amounts of the toxic metal cadmium. Therefore, previous attempts to increase plant iron content resulted in the concomitant accumulation of cadmium.; In this work, we used the seemingly disadvantageous trait of IRT1 cadmium transport as a source of negative selection to isolate IRT1 variants that both retained the ability to transport iron and allowed both yeast and plants to survive in the presence of highly toxic levels of cadmium, thus demonstrating the specific elimination of cadmium transport via IRT1. We subsequently used the IRT1 variants identified as a source of functional information for transport specificity among the ZIP (Zrt Irt-like Protein) family of IRT1 homologs. We specifically targeted ZIPS, the only ZIP member identified in plants that lacks a DXXE metal binding motif in the loop region between transmembrane regions II and III. We substituted the conserved DXXE motif for the GGSK motif that normally occurs in ZIPS, resulting in a transporter now capable of cadmium transport and enhanced zinc and iron transport. These results parallel those seen previously with IRT1 and thus substantiate a role for the DXXE motif in ZIP transporter specificity. This work demonstrates that molecular breeding approaches can be used to enhance the iron content and reduce the cadmium content of plants.