Proteomic identification of S-nitrosylated proteins in Arabidopsis

Abstract

Although nitric oxide (NO) has grown into a key signaling molecule in plants during the last few years, less is known about how NO regulates different events in plants. Analyses of NO-dependent processes in animal systems have demonstrated protein S-nitrosylation of cysteine (Cys) residues to be one of the dominant regulation mechanisms for many animal proteins. For plants, the principle of S-nitrosylation remained to be elucidated. We generated S-nitrosothiols by treating extracts from Arabidopsis (Arabidopsis thaliana) cell suspension cultures with the NO-donor S-nitrosoglutathione. Furthermore, Arabidopsis plants were treated with gaseous NO to analyze whether S-nitrosylation can occur in the specific redox environment of a plant cell in vivo. S-Nitrosylated proteins were detected by a biotin switch method, converting S-nitrosylated Cys to biotinylated Cys. Biotin-labeled proteins were purified and analyzed using nano liquid chromatography in combination with mass spectrometry. We identified 63 proteins from cell cultures and 52 proteins from leaves that represent candidates for S-nitrosylation, including stress-related, redox-related, signaling/regulating, cytoskeleton, and metabolic proteins. Strikingly, many of these proteins have been identified previously as targets of S-nitrosylation in animals. At the enzymatic level, a case study demonstrated NO-dependent reversible inhibition of plant glyceraldehyde-3-phosphate dehydrogenase, suggesting that this enzyme could be affected by S-nitrosylation. The results of this work are the starting point for further investigation to get insight into signaling pathways and other cellular processes regulated by protein S-nitrosylation in plants.

Figure 1.

Nitrosothiol content in Arabidopsis cell suspension culture extracts after treatment with NO donors and in leaves of NO-treated Arabidopsis plants. After treatment of cell culture extracts with either 250 μm GSNO, 250 μm SNP, or water, remaining NO donors were removed by chromatography on a Sephadex G-25M column. Nitrosothiol contents were determined according to Saville (1958). Additionally, nitrosothiol content from leaves of Arabidopsis plants treated with NO gas and from leaves of untreated plants was measured. Values represent mean of at least two independent determinations.

Figure 2.

Detection of S-nitrosylated proteins of Arabidopsis cell culture extracts. Extracts containing 100 μg protein were treated with different concentrations of GSNO or GSH and labeled with biotin using the biotin switch method. Additionally, proteins were S-nitrosylated with 250 μm GSNO and reduced with 100 mm DTT after biotinylation. The sample on the right side was treated with 250 μm GSNO and underwent biotin switch method without MMTS treatment (blocking step). Proteins were separated by SDS-PAGE and blotted onto polyvinylidene difluoride-membrane. Detection of biotinylated proteins was achieved using anti-biotin antibody. The relative masses of protein standards are shown on the right.

Figure 3.

S-Nitrosylated proteins of Arabidopsis cell cultures. A total of 10 mg of cell culture proteins were treated with 250 μm GSH or GSNO, subjected to the biotin switch method, and biotinylated proteins were purified by affinity-chromatography using neutravidin-agarose. Eluates (E) were separated by SDS-PAGE and visualized by Coomassie Blue staining. Protein bands corresponding to predominant bands of the immunoblot analysis (IB) were identified by nanoLC/MS/MS. The percentage of protein covered by the matched peptides is given in brackets. The relative masses of protein standards are shown on the left.

Figure 4.

Immunoblot analysis of in vitro S-nitrosylated proteins. Leaf extracts were treated with 250 μm GSNO or GSH and analyzed with the biotin switch method. After biotinylation, proteins were purified with neutravidin-agarose, separated by SDS-PAGE, and immunoblotted with anti-PSII oxygen-evolving complex 33, anti-α-ATPase, and anti-β-ATPase antibodies. Additionally, cell culture extracts were treated and prepared in the same way and analyzed with anti-α-tubulin antibody.

Figure 5.

Effect of GSNO and SNP on GAPDH activity. Crude extracts of Arabidopsis cell cultures were treated with different concentrations of GSNO (diagonal lines) or SNP (black) and enzyme activity was determined according to Mohr et al. (1996). The GAPDH activity in untreated control extract was set at 100%. For restoring GAPDH-activity, 10 mm DTT was added to extracts with inhibited enzymes. For each concentration, measurements were done at least in triplicates.