Fig. 5: Copper and cadmium toxicity correlate with accumulation of diphthamide-unmodified eEF2 protein.

a Shoot fresh biomass of 18-day-old seedlings. Wild-type (WT; n = 5), iron uptake-defective irt1 (n = 3), sulfate uptake-defective sultr1;1 sultr1;2 (sultr)(n = 3), dph1-1 (n = 5), and dph1-2 (n = 7) mutants were cultivated in modified Hoagland medium. Additionally, WT was cultivated in modified Hoagland medium supplemented with 5 µM Cu (n = 3), 80 µM Zn (n = 4), 2 µM Cd (n = 3), 5 nM methyl viologen (MV; n = 4), 25 µM Pb (n = 4), and a specific Pb-free control medium (C_Pb; n = 3). Shown are mean ± s.d., n biologically independent pools of 17 seedlings, with each pool sampled from a replicate petri plate, from one experiment. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA with Games-Howell test, or two-tailed Student’s t-test compared with the respective control). b Immunoblot detection of unmodified and global eEF2 protein in total protein extracts from shoot tissues of the seedlings shown in a. c, d Photographs of 18-day-old WT seedlings cultivated on series of CuSO₄ (c) or CdCl₂ (d) concentrations in modified Hoagland medium (Controls: 0.5 μM Cu, 0 µM Cd). Scale bars, 10 mm. e, f Immunoblots (as in b for shoots of Cu-exposed (e) and Cd-exposed seedlings (f), respectively. Numbers indicate the fold increase in unmodified eEF2 protein amount relative to controls, after normalization to global eEF2 protein levels. Protein extracts from dph1-1 and dph1-2 seedlings served as controls. g, h Relationships between the amount of unmodified eEF2 protein and shoot growth inhibition in Cu-exposed (g, y = 1.80 x − 1.15, R² = 0.9995) and Cd-exposed (h, y = 3.68 x + 5.00, R² = 0.9384) WT seedlings. A.U. arbitrary units. i Immunoblot detection of unmodified and global eEF2 protein in total protein extracts from DPH1^−/− mutant and WT of human MCF-7 cell line cultivated in liquid medium without or with addition of 125 μM CuSO₄ for 3 days.