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. 2022 Sep 10;191(1):515–527. doi: 10.1093/plphys/kiac423

Figure 3.

Figure 3

DEF8 mediates Cd efflux. A and B, Metal binding assay. Recombinant proteins TF-fDEF8 or TF-tDEF8 were purified from E. coli cells and incubated with 100 μM CdCl2 at pH 5.5 and 7.5 for 1 h before ICP-MS analysis to determine the stoichiometry of DEF8:Cd (A), and binding affinity of the mature tDEF8 to Cd was determined by ITC at pH 7.5 (B). TF represents the E. coli trigger factor protein fused to the N-terminus of target proteins. C and D, Cd efflux in protoplasts. Rice seedlings of the wild-type (ZH11) and def8 mutants (def8-1, def8-2, def8-3) were grown for 14 days in hydroponics supplemented with 5 µM Cd, and leaf sheath was sampled to isolate protoplasts, which were then cultivated in W5 buffer for 6 h before determining the contents of Cd (C) or other metals as indicated (D). Numbers above the bars represent Cd decrease rate at 6 h compared with 0 h. Values are mean ± sd, and significance was determined by Student’s t test in (A) and ANOVA test (P < 0.05) in (C and D), n = 6. Asterisks indicate difference at P < 0.01 (**).