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. 2014 Sep;65(17):5049-62.
doi: 10.1093/jxb/eru270. Epub 2014 Jun 24.

MSRB7 reverses oxidation of GSTF2/3 to confer tolerance of Arabidopsis thaliana to oxidative stress

Shu-Hong Lee  1 , Chia-Wen Li  2 , Kah Wee Koh  3 , Hsin-Yu Chuang  3 , Yet-Ran Chen  4 , Choun-Sea Lin  4 , Ming-Tsair Chan  5
Affiliations

MSRB7 reverses oxidation of GSTF2/3 to confer tolerance of Arabidopsis thaliana to oxidative stress

Shu-Hong Lee et al. J Exp Bot. 2014 Sep.

Abstract

Methionine sulfoxide reductases (MSRs) catalyse the reduction of oxidized methionine residues, thereby protecting proteins against oxidative stress. Accordingly, MSRs have been associated with stress responses, disease, and senescence in a taxonomically diverse array of organisms. However, the cytosolic substrates of MSRs in plants remain largely unknown. Here, we used a proteomic analysis strategy to identify MSRB7 substrates. We showed that two glutathione transferases (GSTs), GSTF2 and GSTF3, had fewer oxidized methionine (MetO) residues in MSRB7-overexpressing Arabidopsis thaliana plants than in wild-type plants. Conversely, GSTF2 and GSTF3 were highly oxidized and unstable in MSRB7-knockdown plants. MSRB7 was able to restore the MetO-GSTF2M100/104 and MetO-GSTF3M100 residues produced during oxidative stress. Furthermore, both GSTs were specifically induced by the oxidative stress inducer, methyl viologen. Our results indicate that specific GSTs are substrates of MSRs, which together provide a major line of defence against oxidative stress in A. thaliana.

Keywords: A. thaliana; LC-MS/MS; glutathione transferase; methionine sulfoxide reductase B (MSRB).; methyl viologen; oxidative stress.

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Figures

Fig. 1.
Fig. 1.
Flowchart of the steps used for comparative proteomic analysis using the CNBr digestion approach.
Fig. 2.
Fig. 2.
Induction of MSRB7, GSTF2, and GSTF3 by oxidative stress. (A) Expression patterns of MSRB7. Real-time PCR analysis of transcripts of 10-d-old A. thaliana plants treated with 10 μM MV for 15min to 24h. The data represent the means±SD (n=10) of three independent experiments. (B, C) Histochemical GUS staining and GUS activity. A. thaliana seedlings harbouring the MSRB7 promoter (B7pro)-driven GUS were untreated (C) or treated (M) with 10 μM MV for 8h and GUS expression (B) and activity (C) were determined. Wild-type (WT) and pCAMBIA1301 transgenic plants (CaMV35Spro-GUS; 1301) served as negative and positive controls, respectively. (D) Immunoblotting of MSRB7. Ten-d-old wild-type seedlings were treated with 10 μM MV for 0–24h and expression of the MSRB7 protein was detected with a specific anti-MSRB7 antibody. Protein stained with Coomassie Brilliant Blue (CBB) was used as a loading control.
Fig. 3.
Fig. 3.
Induction of GSTF2 and GSTF3 by oxidative stress. (A, B) Expression patterns of GSTF2 and GSTF3. Real-time PCR analysis of transcripts in 10-d-old A. thaliana plants treated with 10 μM MV for 15min to 24h. The data represent the means±SD (n=10) of three independent experiments. (C) Immunoblotting of GSTF2/3. Ten-d-old wild-type seedlings were treated with 10 μM MV for 0–24h. GSTF2/3 expression was detected using an anti-GSTF2/3 antibody. Protein stained with Coomassie Brilliant Blue (CBB) was used as a loading control.
Fig. 4.
Fig. 4.
Interaction of MSRB7 with GSTs. (A) BiFC assay for interaction of MSRB7 with GSTs. Yellow indicates CY-B7 [MSRB7 fused with the C-terminal fragment of yellow fluorescence protein (YFP)] and NY-GSTF2/3/8 (GSTF2/3/8 fused with the N-terminal fragment of YFP) dimerization, as determined by BiFC. Red denotes chloroplast autofluorescence and blue denotes nuclear localizing marker (bZIP63–CFP). (B) Co-immunoprecipitation of MSRB7, GSTF2, GSTF3, and GSTF8. Recombinant GSTs proteins were co-precipitated with flag–MSRB7 using an anti-Flag antibody and detected with an anti-His antibody. WB, Western blot. (C) Yeast two-hybrid assay verifying the interactions of GSTF2, GSTF3, and GSTF8 with MSRB7. Controls were performed by co-transforming pEXP32/Krev1 with pEXP22/ RalGDs-WT (strong interaction), pEXP22/RalGDs-m1 (weak interaction), and pEXP22/RalGDs-m2 (no interaction). L, Leu; W, Trp; H, His.
Fig. 5.
Fig. 5.
Restoration of oxidized-GST enzymatic activity by MSRB7 in vitro. The enzymatic activities of GSTF2 (A), GSTF3 (B), and GSTF8 (C). HOCl-treated recombinant GSTF2, GSTF3, and GSTF8 proteins were co-incubated with MSRB7 for 1h at 25 °C. Enzymatic activities were determined. Recombinant GFP protein was used as a negative control. Data are means±SD (n=3) of three independent experiments. Data were analysed statistically using Duncan’s test and different letters indicate significant differences at P<0.05.
Fig. 6.
Fig. 6.
Maintenance of GST stability by MSRB7 in vivo. (A, B) Expression patterns of GSTF2 and GSTF3. Real-time PCR analysis of GSTF2 and GSTF3 transcripts in 10-d-old B7Ox, B7i, and 1301 plants treated with 10 μM MV for 8h. (C) Immunoblotting of GSTF2/3 in aerial parts and roots. Ten-day-old 1301, B7Ox, and B7i seedlings were pre-treated with 10 μM MV for 8h, followed by treatment with 0.5mM CHX for 0–36h. GSTF2/3 was detected using an anti-GSTF2/3 antibody. Protein stained with Coomassie Brilliant Blue (CBB) was used as a protein loading control. (D, E) Relative expression of GSTF2/3. The relative amounts of GSTF2/3 in the aerial parts and roots were determined using immunoblot analysis, and quantified using G:Box iChemi XL (Syngene). Data were analysed statistically using Duncan’s test and different letters indicate significant differences at P<0.05. (F) Total GST activity of MSRB7 transgenic plants. Ten-day-old 1301, B7Ox, and B7i seedlings were pre-treated with 10 μM MV for 8h followed by treatment with 0.5mM CHX for 0–48h. GST activity was measured. Data represent the means±SD (n=10) of three independent experiments.
Fig. 7.
Fig. 7.
Reduction of Met residues of GSTF2 and GSTF3 by MSRB7. The activities of wild-type and mutated GSTs are shown. HOCl-oxidized recombinant GSTF2, GSTF2M100L (Met replaced with Leu), GSTF2M104L, and GSTF2M100/104L (A), and GSTF3 and GSTF3M100L (B) proteins were co-incubated with MSRB7 for 1h at 25 °C and assayed for GST activity. Data denote means±SD (n=3) of three independent experiments. Data were analysed statistically using Duncan’s test and different letters indicate significant differences at P<0.05.
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