Transition metal (TM) distribution through the phloem is an essential part of plant metabolism and is required for systemic signaling and balancing source-to-sink relationships. Due to their reactivity, TMs are expected to occur in complexes within the phloem sap; however, metal speciation in the phloem sap remains largely unexplored. Here, we isolated phloem sap from Brassica napus and analyzed it via size exclusion chromatography coupled online to sector-field ICP-MS. Our data identified known TM-binding proteins and molecules including metallothioneins (MT), glutathione, and nicotianamine. While the main peak of all metals was low MW (∼1.5 kD), additional peaks ∼10 to 15 kD containing Cu, Fe, S, and Zn were also found. Further physicochemical analyses of MTs with and without affinity tags corroborated that MTs can form complexes of diverse molecular weights. We also identified and characterized potential artifacts in the TM-biding ability of B. napus MTs between tagged and non-tagged MTs. That is, the native BnMT2 binds Zn, Cu, and Fe, while MT3a and MT3b only bind Cu and Zn. In contrast, his-tagged MTs bind less Cu and were found to bind Co and Mn and aggregated to oligomeric forms to a greater extent compared to the phloem sap. Our data indicates that TM chemistry in the phloem sap is more complex than previously anticipated and that more systematic analyses are needed to establish the precise speciation of TM and TM-ligand complexes within the phloem sap.

Czech Academy of Sciences Biology Centre Institute of Plant Molecular Biology Laboratory of Plant Biophysics and Biochemistry České Budějovice Czech Republic

Czech Academy of Sciences Biology Centre Institute of Plant Molecular Biology Laboratory of Plant Biophysics and Biochemistry České Budějovice Czech Republic; Department of Experimental Plant Biology University of South Bohemia České Budějovice Czech Republic

Department of Plant Sciences Qwaqwa Campus University of the Free State Phuthaditjhaba South Africa

Division of Biological Sciences University of Missouri Columbia Columbia Missouri USA

Division of Plant Sciences University of Missouri Columbia Columbia Missouri USA

Division of Plant Sciences University of Missouri Columbia Columbia Missouri USA; Environmental Biotechnology Laboratory Department of Biotechnology University of the Western Cape Bellville South Africa

Environmental Biotechnology Laboratory Department of Biotechnology University of the Western Cape Bellville South Africa

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