Identification of dimedone-trapped sulfenylated proteins in plants under stress

Abstract

In stressed plants, the reactive oxygen species (ROS) levels rise. Key to ROS signaling research are detection and identification of the protein cysteine sulfenylation (-SOH), the ROS-mediated oxidative product of a thiol (-SH). Arabidopsis thaliana seedlings were stressed with hydrogen peroxide (H₂O₂) and the sulfenylated proteins were tagged with dimedone. Dimedone-tagged sulfenic acid proteins were visualized on a two-dimensional electrophoresis (2DE) immunoblot with an anticysteine sulfenic acid antibody and were subsequently detected by mass spectrometry. We optimized the detection method for protein sulfenylation in Arabidopsis. We conclude that dimedone can penetrate the cell wall, does not stress plants, and can "read" the changes in the protein sulfenylation pattern under oxidative stress. We observed that the number of sulfenylated proteins in plants treated with 10 mM H₂O₂ was higher than that in untreated plants. A total of 39 sulfenylated protein spots were found on 2DE immunoblots. By means of mass spectrometry, 11 sulfenylated proteins were discovered involved in primary metabolism, redox regulation, translation and signaling pathways. Hence, by combining an immunochemical 2DE strategy with mass spectrometry, we were able to identify sulfenylated proteins in H₂O₂-stressed Arabidopsis seedlings. The sulfenylated proteins can be considered for further validation as redox regulators in plants.

Keywords: Arabidopsis; Dimedone; Oxidative stress; Sulfenylation.

Fig. 1

Schematic representation of the method to identify dimedone-tagged sulfenic acid proteins. Upon H₂O₂ stress, sulfenic acids are formed on specific cysteine thiols of plant proteins. (A) Penetration of dimedone into the plant cells and reaction with the sulfenic acid proteins. (B) Extraction of the proteins in the presence of iodoacetamide (IAM) and N-ethylmaleimide (NEM) to block all free thiols and to prevent aspecific oxidation during the extraction procedure. (C) Formation by the dimedone-tagged sulfenylated proteins of a unique epitope for recognition by anti-cysteine sulfenic acid antibodies. (D) Detection of spots of sulfenylated proteins by combining the information of the two-dimensional immunoblots with the SDS-PAGE, and identification with mass spectrometry.

Fig. 2

Detection by dimedone of dose-dependent changes in H₂O₂-induced protein sulfenylation in Arabidopsis seedlings. Ten-day-old Arabidopsis seedlings were treated with 0, 5, and 10 mM H₂O₂ for 1 h following 5 mM dimedone incubation for 15 and 60 min. The H₂O₂ dose-dependent sulfenylation was visualized on an anti-cysteine sulfenic acid immunoblot and equal protein loading was confirmed on a CBB-stained SDS-PAGE gel.

Fig. 3

Oxidative stress induction by H₂O₂ treatment. Both H₂O₂ and dimedone treatment influenced the efficiency of PSII. Fv′/Fm′ levels were measured in 10-day-old Arabidopsis seedlings grown in a 6-well plate before and after dimedone treatment under both non-treated (A, B) and H₂O₂-treated conditions (C, D). Fv′/Fm′ levels are depicted by color codes, with blue and yellow-green representing high and low values, respectively. Fv′/Fm′ levels were high in non-stressed and dimedone-treated plants (A and B), indicating that dimedone did not interrupt the PSII efficiency in the control plants, but were lower under 10 mM H₂O₂ stress, indicating that dimedone blocked sulfenylated proteins with an effect on the Fv′/Fm′ readout.

Fig. 4

Protein pattern visualized on 2DE gels and the corresponding sulfenylation pattern on the respective immunoblots. Ten-day-old Arabidopsis seedlings were treated with 0 and 10 mM H₂O₂ for 1 h and subsequently treated with 5 mM dimedone for 15 min. After extraction, the proteins were separated on 2DE SDS-PAGE and the gels were stained with G250 CBB or immunoblotted with anti-cysteine sulfenic acid antibodies, followed by visualization with anti-rabbit-HRP. The number of sulfenylated proteins was higher in 10-mM-stressed plants than in non-stressed plants.

Fig. 5

Analysis of the sulfenylated proteins in Arabidopsis seedlings identified by dimedone under 10 mM H₂O₂ stress. (A) A total of 39 sulfenylated protein spots detected in 10-mM H₂O₂-stressed plants after 2DE SDS-PAGE and immunoblot analysis with Image Master 2D platinum software. These dimedone-labeled sulfenylated proteins were identified by mass spectrometry. (B) The 11 identified sulfenylated proteins, of which 11 had previously been reported for reactive cysteine containing sulfenic acid (SOH), disulfide (S-S), S-glutathionylation (S-SG), S-nitrosylated (SNO), and Trx/Grx target cysteine proteins. (C) Seven cytoplasmic sulfenylated proteins identified by dimedone are common proteins with either cytoplasmic YAP1C (1) or cytoplasmic DYn-2 (4) or both YAP1C and DYn-2 (2).