Identification and characterization of several new members of the ZIP family of metal ion transporters in Medicago truncatula

To broaden our understanding of micronutrient metal transport in plants, we have identified cDNAs for six new metal transporters in the model legume Medicago truncatula. All of the predicted proteins have high similarity to the ZIP protein family, and have been designated MtZIP1, MtZIP3, MtZIP4, MtZIP5, MtZIP6, and MtZIP7. The six predicted proteins ranged from 350 to 372 amino acids in length; sequence analysis revealed that all proteins contained eight transmembrane domains and the highly conserved ZIP signature motif. Most of the proteins also exhibited a histidine-rich region in the variable sequence between transmembrane domains III and IV. When MtZIPs were transformed into appropriate metal-uptake defective yeast mutants and grown on metal-limited media, MtZIP1, MtZIP5, and MtZIP6 proteins restored yeast growth on Zn-limited media, MtZIP4 and MtZIP7 proteins restored yeast growth on Mn-limited media, and MtZIP3, MtZIP5, and MtZIP6 proteins restored yeast growth on Fe-limited media. Therefore, we conclude that these proteins function as metal transporters in Medicago truncatula. The expression pattern for each gene was studied by semi-quantitative RT-PCR in roots and leaves from plants grown under various metal supplies. MtZIP1 transcripts were only detected in Zn-deficient roots and leaves. MtZIP3 and MtZIP4 expression was down regulated in leaves from Mn- and Fe-deficient plants and appeared to be upregulated under Zn-deficient conditions in both roots and leaves. MtZIP5 was upregulated in leaves under Zn and Mn deficiency. The expression of MtZIP6 and MtZIP7 was unaffected by the metal supply, at least in root and leaf tissues. Characterizing these proteins in a single organism will allow us to understand the interplay between various ZIP genes, and the role they play in the regulation/execution of plant metal homeostasis.