Post-translational modification of N-terminal peptide of
MtERF75
In A. thaliana , RAP2.2/RAP2.12 degradation has been shown to be
dependent on the in vivo oxidation of its N-terminal cysteine,
after the removal of the N-terminal methionine by a MetAP, and that this
oxidation requires NO (Gibbs et al., 2014b; Hu et al., 2005). In
addition, NO is produced significantly in N2-fixing zone
of mature nodules (Baudouin et al., 2006; Shimoda et al., 2009). The
absence of proof of the S-nitrosylation of the Cys N-term of ERF-VII led
us to investigate the direct post-translational modification of the
N-terminal part of ERF75 by NO. The S-nitrosylation of Cys residue by NO
has been analysed by mass spectrometry on synthetic peptides
corresponding to the first 20 amino acids of MtERF75 protein (MR21) and
without the first methionine (CR20). By this direct MS approach, we
determined the level S-nitrosylation of these peptides after 5 min
treatment with 0, 5, 10, 50, 100 and 500 μM of the NO donor DEA-NONOate
(Fig. 9 ). Results showed a potential higher sensitivity of Cys
residue to S-nitrosylation in CR20 (10 fold) compared to MR21. Indeed,
at 50 µM of NO donor 25% of CR20 peptide were S-nitrosylated against
only 5% for MR21 peptide. The percentage of S-nitrosylation rises to
60% when the peptides are incubated with 500 μM, but at this dose the
same effect is observed on the 2 peptides. Dimerization, due to
formation of disulfide linkages between monomers, was also enhanced in
CR20 compared to MR21.