Characterisation of potential iron transporters in the soybean nodule

Abstract

Iron is essential for symbiotic nitrogen fixation. It is required by the bacteroid for the production of nitrogenase and enzymes of the respiratory chain, and for the synthesis of leghemoglobin. Although the nodule has a high demand for iron the pathways for iron transport, particularly into the symbiosome are not clear. This study explores the role of putative iron transporters expressed in the nodule that have the potential to mediate iron transport into the symbiosome. GmVIT proteins are members of the vacuolar iron transporter family, which transport ferrous iron into the vacuole in Arabidopsis and yeast. Based on the orientation of the symbiosome membrane (SM) they are standout candidates for ferrous iron transporters on the symbiosome. GmVIT1 and GmVIT2 are predominantly expressed in infected cells. In the infected cell, GmVIT1 localises to the SM and its ability to complement the yeast vacuolar iron transport mutant Δccc1, suggests it transports ferrous iron across the SM. In addition, its ability to restore nitrogen fixation, when expressed in the L. japonicus mutant, sen1, highlights that it is essential for the symbiosis and that LjSEN1 may also transport ferrous iron. GmFWL1 and GmFWL3 were candidates based on their identification in the SM proteome and relationship to PLAC8 proteins, which transport divalent metals. GmFWL1 and GmFWL3 localised to the SM but their inability to restore growth of the Δccc1yeast mutant exposed to high iron concentrations, suggests that they do not transport ferrous iron into the symbiosome. To assess the direction of transport of previously characterised ferrous iron transporters, GmNRAMP5 and GmDMT1, their localisation in leek epidermal cells was assessed. The localisation of GmDMT1 to the vacuolar membrane supports a possible role in iron export from the symbiosome. The localisation of GmNRAMP5 suggests that it is unlikely to be an orthologue of the ferrous iron infected cell importer, M. truncatula NRAMP1 and this is supported by its phylogenetic relationship.