Cu+ -chaperones are a diverse group of proteins that allocate Cu+ ions to specific copper proteins, creating different copper pools targeted to specific physiological processes. Symbiotic nitrogen fixation carried out in legume root nodules indirectly requires relatively large amounts of copper, for example for energy delivery via respiration, for which targeted copper deliver systems would be required. MtNCC1 is a nodule-specific Cu+ -chaperone encoded in the Medicago truncatula genome, with a N-terminus Atx1-like domain that can bind Cu+ with picomolar affinities. MtNCC1 is able to interact with nodule-specific Cu+ -importer MtCOPT1. MtNCC1 is expressed primarily from the late infection zone to the early fixation zone and is located in the cytosol, associated with plasma and symbiosome membranes, and within nuclei. Consistent with its key role in nitrogen fixation, ncc1 mutants have a severe reduction in nitrogenase activity and a 50% reduction in copper-dependent cytochrome c oxidase activity. A subset of the copper proteome is also affected in the ncc1 mutant nodules. Many of these proteins can be pulled down when using a Cu+ -loaded N-terminal MtNCC1 moiety as a bait, indicating a role in nodule copper homeostasis and in copper-dependent physiological processes. Overall, these data suggest a pleiotropic role of MtNCC1 in copper delivery for symbiotic nitrogen fixation.

Centro de Biotecnología y Genómica de Plantas Universidad Politécnica de Madrid Campus de Montegancedo UPM Pozuelo de Alarcón Madrid 28223 Spain

Department of Biotechnology Plant Biology Escuela Técnica Superior de Ingeniería Agraria Alimentaria y de Biosistemas Universidad Politécnica de Madrid Madrid 28040 Spain

Department of Experimental Plant Biology Faculty of Sciences University of South Bohemia Ceske Budejovice 37005 Czech Republic

Institute for Agricultural Biosciences Oklahoma State University Ardmore OK 73401 USA

Laboratory of Plant Biophysics and Biochemistry Institute of Plant Molecular Biology Biology Centre Czech Academy of Sciences Ceske Budejovice 37005 Czech Republic

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