Nitration is exclusive to defense-related PR-1, PR-3 and PR-5 proteins in tobacco leaves

Abstract

Protein tyrosine nitration is an important post-translational modification. A variety of nitrated proteins are reported in Arabidopsis leaves and seedlings, sunflower hypocotyls, and pea roots. The identities of nitrated proteins are species-/organ-specific, and chloroplast proteins are most nitratable in leaves. However, precise mechanism is unclear. Here, we investigated nitroproteome in tobacco leaves following exposure to nitrogen dioxide. Proteins were extracted, electrophoresed and immunoblotted using an anti-3-nitrotyrosine antibody. Mass spectrometry and FASTA search identified for the first time an exclusive nitration of pathogenesis-related proteins, PR-1, PR-3 and PR-5, which are reportedly located in the apoplast or the vacuole. Furthermore, Tyr(36) of thaumatin-like protein E2 was identfied as a nitration site. The underlying mechanism and physiological relevance are discussed.

Keywords: Apoplast; PR-1; PR-3; PR-5; defense/immunity-related proteins; nitrogen dioxide; pathogenesis-related proteins; protein tyrosine nitration; tobacco; vacuole.

Figure 1.

Two-dimensional PAGE of proteins from tobacco leaves exposed to NO₂. Nine-week-old tobacco plants (Nicotiana tabacum cv Xanthi XHFD8) were exposed to NO₂ at 4.0 ± 0.4 ppm for 8 h in light. Proteins were extracted from the exposed plants and subjected to 2D PAGE. Protein spots were visualized by staining with a polyclonal anti-3-NT antibody (A) and SYPRO Ruby (B). Nine-hundred micrograms of protein were loaded on each gel.

Figure 2.

Detection of 3-NT-containing peptides of thaumatin-like protein E2 by MALDI-TOF MS. Proteins were extracted from tobacco leaves exposed to 4.0 ± 0.4 ppm ¹⁵NO₂(51.6 atom% ¹⁵N), and subjected to 2D PAGE followed by SYPRO RUBY staining and immunoblotting using an anti-NT antibody. Spot 2 on the SYPRO-RUBY-stained gel corresponding to the signal obtained from immunoblot analysis was excised, in-gel digested with trypsin and V8 protease, and identified by MALDI-TOF/TOF MS. A Mascot search indicated that 2 singly charged [M + H]⁺ ion peaks at m/z 2023.3 and 2069.3 correspond to residues 30–48 (³⁰IVNQCTYTVWAAASPGGGR⁴⁸) of E2, without or with one nitrated tyrosine, respectively. Nitration (replacement of H with an ¹⁵NO₂ group) resulted in a mass shift of +46 Da.