Structural and functional characteristics of cGMP-dependent methionine oxidation in Arabidopsis thaliana proteins

Here we use tandem mass spectrometry-based proteomics to show that treatment of Arabidopsis thaliana cells with a non-oxidative signaling molecule, the cell-permeant second messenger analogue, 8-bromo-3,5-cyclic guanosine monophosphate (8-Br-cGMP), results in a time-dependent increase in the content of oxidised methionine residues. Interestingly, the group of proteins affected by cGMP-dependent methionine oxidation is functionally enriched for stress response proteins. Furthermore, we also noted distinct signatures in the frequency of amino acids flanking oxidised and un-oxidised methionine residues on both the C- and N-terminus.Given both a structural and functional bias in methionine oxidation events in response to a signaling molecule, we propose that these are indicative of a specific role of such post-translational modifications in the direct or indirect regulation of cellular responses. The mechanisms that determine the specificity of the modifications remain to be elucidated.The debate of whether methionine (Met) oxidation of proteins is a purely chemical consequence of cellular oxidative damage or a protective mechanism against oxidative damage, or indeed a post-translational modification that can act as a specific cellular signal and/or response, is ongoing [1-3]. To shed light on this question we treated Arabidopsis suspension culture cells with the cell permeant second messenger analogue 8-bromo 3,5-cyclic guanosine monophosphate (8-Br-cGMP). Cyclic GMP has a signaling role in many plant responses, including responses to light [4], hormones [5-8], signaling peptides [9], salt and drought stress [10,11], ozone, and defence responses [12-14]. Given that cGMP is not an oxidising agent and does not induce protein Met oxidation in vitro (Additional file 1), we tested if cGMP causes protein oxidation in vivo. To this end we used an OxiSelect？ Intracellular ROS Assay Kit (Cell Biolabs, Inc.) and show that cGMP can cause protein oxidation (Figure 1). We therefore