Figure 4.

GmNTL1 is oxidized under H₂O₂ treatment. A) Schematic diagram of GmNTL1, GmNTL1ΔTM (TM), and GmNTL1ΔC (ΔC, lacking the C-terminal region). The highly conserved NAC domain is in the N-terminal region (black boxes). Red box, α-helical TM. The numbers indicate the position of cysteine residues. B) In vitro analysis of the oxidative modification of GmNTL1 by the biotin switch assay. Recombinant GmNTL1-His was pretreated with different H₂O₂ concentrations. Biotin-conjugated iodoacetamide (BIAM)-tagged proteins in the samples were captured with streptavidin beads and detected by immunoblot using an anti-His antibody. Data shown are the results of 3 biological experiments. C) In vitro analysis of the oxidative modification of GmNTL1ΔC by the biotin switch assay. Data shown are the results of 2 biological experiments. D, E) In vitro analysis of the oxidation of GmNTL1ΔTM and GmNTL1ΔC variants after the recombinant proteins were treated with water (0 µM) or 2 mm H₂O₂ for 30 min. Data shown are the results of 3 biological experiments. F, G) Immunodetection of oxidized GmNTL1 in roots from 5-d-old wild-type seedlings treated with 150 mm NaCl for 6 h, 0, 10, 100, or 1000 µM H₂O₂ for 12 h using the anti-GmNTL1 antibody. Error bars denote SD (n = 3 from 3 biological experiments). The intensity of each band was measured using ImageJ, and relative protein levels were normalized against those in untreated controls, which were set to 1. Lowercase letters indicate significant differences between labeled samples as determined by 1-way ANOVA, P < 0.05. H–K) In vivo analysis of the oxidative modification of GmNTL1, GmNTL1^8CS (with all 8 Cys residues mutated to Ser), and GmNTL1^C247S (with the Cys-247 residue mutated to Ser) proteins in plants by the biotin-switch assay. Total proteins from MYC-GmNTL1, MYC-GmNTL1^8CS, and MYC-GmNTL1^C247S transgenic soybean hairy roots treated with or without 150 mm NaCl (H) for 6 and 1 mm H₂O₂ (J) for 12 h were sequentially treated with N-Ethylmaleimide (NEM), dithiothreitol (DTT), and BIAM and then analyzed for biotin label. The intensity of each band was measured using ImageJ, and relative protein levels were normalized against those in untreated controls, which were set to 1. Error bars denote SD (n = 3 from 3 biological experiments). Lowercase letters indicate significant differences between labeled samples determined by 2-way ANOVA, P < 0.05 in (I) and (K). Please see Supplemental Data Set 4 for detailed statistical analyses.