Soybean (Glycine max (L.) Merr.) plants were exposed to various Cd concentrations from background and low non-toxic (0.5-50 nM) via sublethally toxic (< 550 nM) to highly, ultimately lethally toxic (3 µM) concentrations. Plants were cultivated hydroponically for 10 weeks until pod development stage of the control plants. The threshold and mechanism of sublethal Cd toxicity was investigated by metabolomics and metalloproteomics (HPLC-ICP-MS) measuring metal binding to proteins in the harvested roots. Spatial distribution of Cd was revealed by µXRF-CT. Specific binding of Cd to proteins already at 50 nM Cd revealed the likely high-affinity protein binding targets in roots, identified by protein purification from natural abundance. This revealed allantoinase, aquaporins, peroxidases and protein disulfide isomerase as the most likely high-affinity targets of Cd binding. Cd was deposited in cortex cell vacuoles at sublethal and bound to the cell walls of the outer cortex and the vascular bundle at lethal Cd. Cd binding to proteins likely inhibits them, and possibly induces detoxification mechanisms, as verified by metabolomics: allantoic acid and allantoate increased due to sublethal Cd toxicity. Changes of the Cd binding pattern indicated a detoxification strategy at lower Cd, but saturated binding sites at higher Cd concentrations.

Czech Academy of Sciences Biology Centre Institute of Plant Molecular Biology Department Plant Biophysics and Biochemistry Branišovská 31 1160 CZ 37005 České Budějovice Czech Republic

Czech Academy of Sciences Biology Centre Institute of Plant Molecular Biology Department Plant Biophysics and Biochemistry Branišovská 31 1160 CZ 37005 České Budějovice Czech Republic; University of South Bohemia Faculty of Science Department of Experimental Plant Biology Branišovská 31 1160 CZ 37005 České Budějovice Czech Republic

Deutsches Elektronen Synchrotron DESY Notkestr 85 22607 Hamburg Germany

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