Protein tyrosine nitration in pea roots during development and senescence

Abstract

Protein tyrosine nitration is a post-translational modification mediated by reactive nitrogen species (RNS) that is associated with nitro-oxidative damage. No information about this process is available in relation to higher plants during development and senescence. Using pea plants at different developmental stages (ranging from 8 to 71 days), tyrosine nitration in the main organs (roots, stems, leaves, flowers, and fruits) was analysed using immunological and proteomic approaches. In the roots of 71-day-old senescent plants, nitroproteome analysis enabled the identification a total of 16 nitrotyrosine-immunopositive proteins. Among the proteins identified, NADP-isocitrate dehydrogenase (ICDH), an enzyme involved in the carbon and nitrogen metabolism, redox regulation, and responses to oxidative stress, was selected to evaluate the effect of nitration. NADP-ICDH activity fell by 75% during senescence. Analysis showed that peroxynitrite inhibits recombinant cytosolic NADP-ICDH activity through a process of nitration. Of the 12 tyrosines present in this enzyme, mass spectrometric analysis of nitrated recombinant cytosolic NADP-ICDH enabled this study to identify the Tyr392 as exclusively nitrated by peroxynitrite. The data as a whole reveal that protein tyrosine nitration is a nitric oxide-derived PTM prevalent throughout root development and intensifies during senescence.

Fig. 1.

Protein and tyrosine nitration pattern in organs of pea plants during natural development (8–16-days-old) and senescence (71-days-old). Silver-stained SDS gels (10%) and Western blots probed with a rabbit anti-nitrotyrosine polyclonal antibody at a 1:8000 dilution. (A) Root samples of pea plants aged 8–71 days (5 µg per lane); (B) stem samples of pea plants aged 8–71 days (10 µg per lane); (C) leaf samples of pea plants aged 8–71 days (20 µg per lane); (D) flowers (Fw) and fruits (Fr) of pea plants aged 71 days (10 µg per lane). Arrows indicate bands that have changed their intensity, disappeared, or become visible.

Fig. 2.

Analysis of superoxide dismutase (SOD) isozymes present in roots of pea plants during natural development (8–16 days) and senescence (71 days). (A) Activity of SOD isozymes; SODs were separated by native PAGE on 10% (w/v) polyacrylamide gels, and gels were stained by the photochemical nitroblue tetrazolium method. (B and C) Western blot of pea root samples probed with antibodies against pea MnSOD (1:2000 dilution) and Equisetum CuZnSOD (1:3000 dilution), respectively. Proteins (8 µg per lane) were separated by 12% SDS-PAGE and transferred onto a PVDF membrane.

Fig. 3.

Representative images illustrating confocal laser scanning microscopic detection of nitric oxide (NO), peroxynitrite (ONOO^–), and protein 3-nitrotyrosine (NO₂-Tyr) in root cross-sections of pea plants aged 8 and 71 days. (A–F) Cross-sections of pea roots (100 µm thick) were incubated with 10 µM 4,5-diaminoflorescein diacetate to detect NO (A and B), with 10 µM 3’-(p-aminophenyl) fluorescein to detect ONOO^– (C and D), and with a specific antibody against NO₂-Tyr (E and F). (G and H) show representative bright-field image of the corresponding samples. The bright-green fluorescence corresponds to the detection of ^·NO, ONOO^–, or NO₂-Tyr in the corresponding panels. En, Endodermis; Ep, epidermis; Pc, parenchyma cells of the cortex; Rh, root hairs; V, vascular tissues. Bar = 300 µm (this figure is available in colour at JXB online).

Fig. 4.

Detection of nitrated proteins in roots of senescent pea plants by 2D electrophoresis and immunoblot. (A) Representative 2D electrophoresis (pH 5–8 for the first dimension) of pea root samples stained with Sypro Ruby and its corresponding immunoblot probed with a polyclonal antibody against nitrotyrosine (1:8000 dilution). Molecular-mass standards are indicated on the left in kDa. Approximately 150 µg protein was loaded per gel. Arrows indicate all the immunoreactive spots, and the numbers refer to the proteins listed in Table 1. (B) Zoom boxes illustrate details of immunoreactive spots.

Fig. 5.

Purification of recombinant cytosolic NADP-isocitrate dehydrogenase (ICDH) and effect of SIN-1 (peroxynitrite donor) on its activity. (A) SDS-PAGE analysis of the purification of the recombinant pea NADP-ICDH; Coomassie blue staining; E1–E7, elution fractions; FT, flow-through; I, total protein in induced culture; IF, insoluble fraction; M, molecular markers; NI, total protein in no-induced culture; SF, soluble fraction; W, wash. (B) Effect of SIN-1; purified NADP-ICDH was incubated with different concentrations of SIN-1 at 37 °C for 60min. The specific activity of the recombinant peroxisomal NADP-ICDH was 10.1±0.3 µmol NADPH min^–1 mg^–1 protein. Data are mean ± SEM of at least three replicates. Asterisks indicate significant differences from control values (P < 0.05). (C) Representative immunoblot showing the grade of tyrosine nitration of the cytosolic NADP-ICDH treated with different concentrations of SIN-1 and detected with an antibody against 3-nitrotyrosine (1:2500 dilution).

Fig. 6.

Comparison of the nitrated (top) and unmodified (bottom) MS/MS spectra of the identified peptide (EHYLNTEEFIDAVAAELK) from the cytosolic pea NADP-isocitrate dehydrogenase.

Fig. 7.

Three-dimensional model of cytosolic NADP-isocitrate dehydrogenase showing Tyr392 (red) and its potential interactions. Conserved residues implicated in the binding of Mg²⁺ (green), isocitrate (blue), and NADP (yellow); catalytic residues are shown as purple or orange. The NADP-binding site includes Lys376 and His317 and is at 8.6 Å of the nitrated Tyr392 (this figure is available in colour at JXB online).

Fig. 8.

NADP-isocitrate dehydrogenase and catalase activities in roots of young (8-day-old) and senescent (71-day-old) pea plants. Asterisks indicate significant differences from control values (P < 0.05)