Cadmium (Cd) is dispersed in natural and agricultural environments through human activities and natural occurrence. Japanese arable lands, especially paddy fields, are contaminated to some extent by Cd through irrigation with river water originating from mines or through emission from metal-smelters. Rice is a staple crop in Japan and is a major source for dietary intake of Cd. In addition, soybean or wheat is often used as a rotation crop or a secondary crop, respectively, in paddy fields; therefore Cd concentrations of soybean and wheat should also be taken care of as well as rice. The rice Nramp5 is well known to mediate Cd and manganese (Mn) uptake in the root and the knockout line greatly reduces Cd concentration in the grains. In a recent study, the Cd transport activity in TaNramp5A and 5D from hexaploid wheat was investigated. In this study, we examined if the Nramp5 orthologs in soybean and two types of wheat, i.e., tetraploid and hexaploid wheat, have ability for Cd and Mn transport.

To examine the effects of Cd on Mn uptake in the plants, the seedlings of rice, soybean, and wheat were treated for 3 days with different levels of Cd (0, 0.05, 0.1, and 0.5, 1 mg Cd/L) in nutrient solution and the Cd and Mn concentrations of plants were determined by ICP-MS or ICP-OES after acid-digestion. The full-length ORF of Nramp5 in rice (OsNramp5), soybean (GmNramp5), tetraploid wheat (TdNramp5A and TdNramp5B), and hexaploid wheat (TaNramp5A, TaNramp5B, and TaNramp5D) was cloned by PCR and sequenced. For subcellular localization analysis, confocal microscopy of rice protoplasts transiently producing the Nramp5 orthologs fused with green fluorescence protein (GFP) was performed. The two yeast mutants, Cd-sensitive Δycf1 and Mn uptake-defective Δsmf1, were used to analyze Cd and Mn transport activities of Nramp5 orthologs.

Metal analysis showed that the Cd concentrations of shoots and roots in all plant species were increased in dose-depending manner, but the Mn concentrations remarkably decreased with increasing Cd levels, indicating antagonistic interaction between Cd and Mn uptake by roots in plant species tested. The cDNA sequences between TdNramp5B and TaNramp5B matched perfectively. High identity of more than 80% was found between rice and wheat in amino acid sequence of Nramp5. Amino acid sequence of OsNramp5 and GmNramp5 shared 69% identity. Confocal microscopy of rice protoplasts expressing GFP-fused proteins demonstrated that the all Nramp5 orthologs are localized at the plasma membrane. The growth of Δycf1 cells expressing OsNramp5 or GmNramp5 was strongly impaired by Cd, whereas all Nramp5 homologs in wheat did not affect the growth. The growth of Δsmf1 was improved by the expression of OsNramp5 or GmNramp5, whereas was not by all Nramp5 homologs in wheat. These results suggest high or low ability for Cd and Mn transport through GmNramp5 or Nramp5 homologs in wheat, respectively.