Anti-GLN2 | GS2, chloroplastic form of glutamine synthetase

Western blot analysis of selected candidate proteins (AtpB, 2Fe-2S, GLN2, Psb R, PRK, ALD) with their corresponding molecular weight in rice leaves during As stress under various S regimes (A); RuBisCo large subunit a band in coomassie blue staining (CBB) SDS gel served as a loading control (B), which used for normalization of detected proteins in densitometry studies (C) fold change indicated with respect of control samples.

Determination of GS2 oligomerization, activity, and stability under oxidative conditions. (A) GS2 protein complexes. Proteins from Wt and o/exTrxm chloroplasts were extracted under non-reducing conditions, separated by BN-PAGE, and analyzed by Western blot with anti-GS2 antibody. Photosynthetic thylakoid complexes are shown on the right and the molecular weight marker on the left. (B) Redox-sensitive GS activity. Protein extracts from Wt plants were incubated with or without 5 mM DTT or 100 µM CuCl2 for 30 min on ice, and the activity was then measured. Recovery of GS activity by reduction was performed by incubating with 5 mM DTT on ice after sample oxidation treatment (100 µM CuCl2). The activity of protein extracts from o/exTrxm plants was also measured. Results are expressed as means ±SE (n=3). (C) Quantification of GS2 in the o/exTrxm genotype. Total protein extracts (2.5 µg) were separated by SDS-PAGE and analyzed by western blot. Three different plants per line are shown in the blot. Immunoblots were analyzed with Gene Tools Analyzer software (SynGene). Data are given as means ±SE with the Wt relativized to 1. Statistical significance relative to the Wt is indicated by an asterisk (*P<0.05, Student’s t-test). (D) Degradation of GS2 protein in Wt and o/exTrxm plants. Leaves were incubated at 30 °C for up to 24 h in the presence of FeCl3, and the content of GS2 protein was analyzed by immunodetection. Similar results were obtained in eight independent biological replicates, and a representative blot is shown (upper panel). Immunoblots were analyzed with Gene Tools Analyzer software. Protein levels in plants treated for 4, 8, and 24 h are shown relative to the levels in plants treated for 0 h. Data are given as means ±SE. Statistical significance relative to Wt plants is indicated by asterisks (*P<0.05, Student’s t-test).