Abstract
Heavy metal contamination poses significant threats to global food security worldwide, necessitating the development of crops with enhanced metal tolerance. Natural resistance-associated macrophage proteins (NRAMPs) constitute a conserved family of divalent metal transporters crucial for maintaining cellular metal homeostasis and conferring heavy metal tolerance in plants. Despite extensive characterization in model species, comprehensive genomic analysis of NRAMP gene families in forage legumes remains limited. Here, we report the first genome-wide identification and characterization of the NRAMP transporter family in sainfoin (Onobrychis viciifolia), a high-value forage legume with exceptional nutritional properties. Phylogenetic analysis, gene structure examination, conserved motif identification, chromosomal distribution mapping, and cis-regulatory element analysis were performed. Expression profiling was conducted under lead, cadmium, and arsenic stress conditions using quantitative real-time PCR in both soil-based long-term and hydroponic short-term experimental systems. Functional validation was accomplished through heterologous expression in Saccharomyces cerevisiae. A total of 32 OvNRAMP genes were identified and mapped across 20 chromosomes, exhibiting uneven distribution with evidence of tandem duplications and segmental duplications. Phylogenetic analysis revealed three distinct subgroups (Groups I-III) with conserved structural features but functional diversification. All genes showed plasma membrane localization and contained characteristic NRAMP domains with 7 or 10 conserved motifs. Comprehensive promoter analysis identified 34 types of cis-regulatory elements, including hormone-responsive (ABA, MeJA) and stress-responsive elements (drought, low-temperature, anaerobic conditions). Expression profiling revealed concentration-dependent and metal-specific responses: low metal concentrations generally induced gene upregulation, while high concentrations showed varied responses. Heterologous yeast expression confirmed functional diversity among OvNRAMP proteins, with differential metal tolerance capabilities for lead, cadmium, and arsenic transport. This study provides the first comprehensive genomic characterization of the NRAMP gene family in sainfoin, revealing complex regulatory networks governing metal homeostasis and stress responses. The identification of functionally diverse OvNRAMP genes with distinct expression patterns and metal transport capabilities establishes a foundation for molecular breeding programs aimed at developing sainfoin cultivars with enhanced heavy metal tolerance and reduced metal accumulation. These findings significantly advance our understanding of metal transport mechanisms in forage crops and provide valuable genetic resources for sustainable agriculture in metal-contaminated environments.