Medicago truncatulaMOT1.3 is a plasma membrane molybdenum transporter required for nitrogenase activity in root nodules

Abstract

SummaryMolybdenum, as a component of the iron-molybdenum cofactor of nitrogenase, is essential for symbiotic nitrogen fixation. This nutrient has to be provided by the host plant through molybdate transporters.Members of the molybdate transporters family MOT1 were identified in the model legume Medicago truncatula and their expression in nodules determined. Yeast toxicity assays, confocal microscopy, and phenotypical characterization of a Tnt1 insertional mutant line were carried out in the one M. truncatula MOT1 family member expressed specifically in nodules.Among the five MOT1 members present in M. truncatula genome, MtMOT1.3 is the only one uniquely expressed in nodules. MtMOT1.3 shows molybdate transport capabilities when expressed in yeast. Immunolocalization studies revealed that MtMOT1.3 is located in the plasma membrane of nodule cells. A mot1.3-1 knockout mutant showed an impaired growth concomitant with a reduction in nitrogenase activity. This phenotype was rescued by increasing molybdate concentrations in the nutritive solution, or upon addition of an assimilable nitrogen source. Furthermore, mot1.3-1 plants transformed with a functional copy of MtMOT1.3 showed a wild type-like phenotype.These data are consistent with a model in which MtMOT1.3 would be responsible for introducing molybdate into nodule cells, which will be later used to synthesize functional nitrogenase.

MtMOT1.2 is responsible for molybdate supply to Medicago truncatula nodules

“…Nitrate reductase activity was analysed as described by Tejada‐Jimenez et al (). Briefly, a crude extract was obtained from approximately 100 mg of fresh material in 100 mM potassium phosphate, pH 7.5, 5 mM magnesium acetate, 10% glycerol ( v /v), 10% polyvinylpolypyrrolidone ( w /v), 0.1% Triton X‐100, 1 mM ethylenediaminetetraacetic acid, 0.05% ß‐mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride (PMSF).…”

“…For instance, Glycine max has seven members; Phaseolus vulgaris has four; and Medicago truncatula has five (MtMOT [ Medicago truncatula Molybdate Transporter] 1.1 to MtMOT1.5). Two legume MOT1 proteins have been characterized to date (Duan et al, ; Gao et al, ; Tejada‐Jimenez et al, ). LjMOT1 would be responsible for molybdate uptake from soil and its distribution to plant sink organs (Duan et al, ; Gao et al, ), in a role similar to that played by AtMOT1 (Tomatsu et al, ).…”

“…However, LjMOT1 would not primarily be involved in molybdate delivery to the nodules, since lack of this transporter has no significant effect on nitrogen fixation capabilities. In contrast, mutation of the other characterized legume MOT1 protein, nodule‐specific MtMOT1.3, results in nearly a total loss of nitrogenase activity (Tejada‐Jimenez et al, ). Immunolocalization of this transporter in the plasma membrane and characterization of its transport capabilities indicate that its main function would be introducing molybdate into nodule cells.…”

“…Immunolocalization of this transporter in the plasma membrane and characterization of its transport capabilities indicate that its main function would be introducing molybdate into nodule cells. However, mot1.3‐1 mutant nodules still accumulate more Mo than wild‐type ones, suggesting that the delivery of this metal to the nodules is not impaired by MtMOT1.3 mutation (Tejada‐Jimenez et al, ). Consequently, we still need to identify the transporters responsible for molybdate release from the vasculature into the nodules.…”

“…Among the four remaining MOT1 transporters in M. truncatula , MtMOT1.1 is the most closely related to LjMOT1 and would likely play a similar role (Tejada‐Jimenez et al, ). MtMOT1.4 and MtMOT1.5 are expressed all over the plant, while MtMOT1.2 is the only one of these four genes that is expressed exclusively in roots and nodules, with a maximum of expression in the latter (Tejada‐Jimenez et al, ). Here, we characterize the function of MtMOT1.2 as a likely candidate for molybdate uptake from the vasculature by endodermal cells.…”