Identification and expression analysis of multiple FRO gene copies in Medicago truncatula

Iron (Fe) is an essential element for plant growth. Commonly, this element is found in an oxidized form in soil, which is poorly available for plants. Therefore, plants have evolved ferric-chelate reductase enzymes (FRO) to reduce iron into a more soluble ferrous form. Fe scarcity in plants induce the FRO enzyme activity. Although the legume Medicago truncatula has been employed as a model for FRO activity studies, only one copy of the M. truncatula MtFRO1 gene has been characterized so far. In this study, we identified multiple gene copies of the MtFRO gene in the genome of M. truncatula by an in silico search, using BLAST analysis in the database of the M. truncatula Genome Sequencing Project and the National Center for Biotechnology Information, and also determined whether they are functional. We identified five genes apart from MtFRO1, which had been already characterized. All of the MtFRO genes exhibited high identity with homologous FRO genes from Lycopersicon esculentum, Citrus junos and Arabidopsis thaliana. The gene copies also presented characteristic conserved FAD and NADPH motifs, transmembrane regions and oxidoreductase signature motifs. We also detected expression in five of the putative MtFRO sequences by semiquantitative RT-PCR analysis, performed with mRNA from root and shoot tissues. Iron scarcity might be a condition for an elevated expression of the MtFRO genes observed in different M. truncatula tissues.