Proteomic Identification of <em>S</em>-Nitrosylated Proteins in Arabidopsis<sup><a href="#fn1" rid="fn1" class=" fn">1</a>,</sup><sup><a href="#fn2" rid="fn2" class=" fn">[w]</a></sup>
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.
Overwhelming evidence suggests that nitric oxide (NO) is an integral part of normal physiological processes in animals (Nathan, 1995; Bogdan, 2001). By the late 1990s, NO was identified as an important messenger in plant defense signaling against microbial pathogens (Delledonne et al., 1998; Durner et al., 1998); it subsequently was shown to be a crucial player in the regulation of normal plant physiological processes including stomatal closure, growth, and development (Neill et al., 2002; Wendehenne et al., 2004), and recently, a hormone-activated NO producing enzyme was identified in Arabidopsis (Arabidopsis thaliana; Guo et al., 2003; Zeidler et al., 2004).
As a readily diffusible free radical, NO reacts with a variety of intracellular and extracellular targets and can act as activator or inhibitor of enzymes, ion channels, or transcription factors and in this way regulates specific processes during plant development and abiotic or biotic stress situations. The alteration of protein function/activity can be achieved by reaction of NO with sulfhydryl groups and transition metals (Stamler, 1994), and the resulting products, S-nitrosothiols and metal nitrosyls, respectively, have intrinsic reactivities that enable local action.
The majority of all NO-affected proteins seem to be regulated by S-nitrosylation of a single critical Cys residue, which occurs by oxygen-dependent chemical reactions or by the transfer of NO from a nitrosothiol to a protein sulfhydryl group (transnitrosylation). Because of their reactivity with intracellular reducing agents, e.g. ascorbic acid or glutathione (GSH), and with reduced metal ions, especially Cu, nitrosothiols are exceptionally labile. This lability results in tissue half-lives of seconds to a few minutes and therefore provides a very sensitive mechanism for regulating cellular processes. S-Nitrosylation is now regarded as posttranslational modification similar to phosphorylation.
In mammalians, especially S-nitrosoalbumin, S-nitrosohaemoglobin, and S-nitrosoglutathione (GSNO) are discussed as in vivo NO reservoirs and NO donors (Stamler et al., 1992a; Jia et al., 1996; Kluge et al., 1997; Tsikas et al., 1999). In plants, a strong GSNO reductase activity was demonstrated recently for GSH-dependent formaldehyde dehydrogenase (Diaz et al., 2003), an enzyme previously identified as formaldehyde detoxifying protein in maize (Zea mays; Fliegmann and Sandermann, 1997; Wippermann et al., 1999) indicating that GSNO and the formation of other nitrosothiols might play an important role in NO signaling in plants as well (Durner et al., 1999; Diaz et al., 2003). On the other side, it is also speculated whether metal nitrosyls are the dominant compounds for NO storage and NO transport in plants (Garcia-Mata and Lamattina, 2003).
Until now, little has been known about the dimension let alone the physiological function of S-nitrosylation in plants, and no endogenous S-nitrosylated plant protein has been described. To identify possible targets of S-nitrosylation, SwissProt database was searched for the consensus motif of S-nitrosylation sensitive Cys residues (Stamler et al., 1997). This search revealed 103 matches in 99 sequences from the deduced Arabidopsis proteome (Huber and Hardin, 2004). Except for this bioinformatics strategy, no approaches were undertaken to identify S-nitrosylated plant proteins.
The aim of this study was to identify possible candidates for S-nitrosylation in Arabidopsis cell suspension cultures and leaves to get insight into the regulatory function of NO on protein level in plants. Recently, Jaffrey et al. (2001) developed a highly specific biotin switch method for detection and purification of S-nitrosylated proteins in animals (Jaffrey et al., 2001; Kuncewicz et al., 2003; Foster and Stamler, 2004; Martinez-Ruiz and Lamas, 2004). A proteomics approach using this method in combination with nano liquid chromatography and tandem mass spectrometry (nanoLC/MS/MS) allowed us to identify 63 proteins from GSNO-treated cell culture extracts and 52 proteins from NO-treated Arabidopsis leaves, which represent targets for S-nitrosylation in plants. These proteins include stress-related proteins, signaling/regulating proteins, redox-related proteins, and cytoskeleton proteins as well as metabolic enzymes. Strikingly, about 60% of the identified proteins were already described in the animal system in context with S-nitrosylation or S-glutathionylation, underlining the specificity of the method and indicating that NO-regulated processes in plants and animals have common features.
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We thank Dr. Anna Sokolenko (LMU, Munich) for supplying antibodies against PSII oxygen-evolving complex 33, α-ATPase, and β-ATPase.
Notes
This work was supported by the Deutsche Forschungsgemeinschaft (SPP 1110 Innate Immunity) and by Bayerisches Staatsministerium für Umwelt, Gesundheit, und Verbraucherschutz.
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