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The protein was further purified by size-exclusion chromatography on a Superdex 75 pg 26/60 column (Cytiva, Freiburg, Germany) pre-equilibrated with phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, 2 mM KHPO, pH 7.4) for polishing

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Add 8 ml 0.05 molL -1 sodium phosphate buffer (pH = 7.8) precooled in advance, centrifuge at 10000 rpm for 20 min, and store the supernatant at 4C for enzyme activity determination ( 2.4 Untargeted metabolomics of buckwheat leaf tissues Untargeted metabolomics, a useful approach for the simultaneous analysis of samples with significant phenotypic changes, has been used to detect metabolites after Cd treatment ( Non-targeted metabolic profiling analyses were profiled by Perkin Elmer 680 GC (Perkin Elmer Inc, Akron, OH, USA) and Q Exactive Focus Orbitrap LC-MS/MS (Thermo Scientific, Waltham, MA, USA)