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, Tyr³⁶ of thaumatin-like protein E2 was identfied as a nitration site. The underlying mechanism and physiological relevance are discussed.

KEYWORDS:

• Apoplast
• defense/immunity-related proteins
• nitrogen dioxide
• pathogenesis-related proteins
• PR-1
• PR-3
• PR-5
• protein tyrosine nitration
• tobacco
• vacuole

Protein tyrosine nitration is a covalent post-translational protein modification that plays a critical role in cell physiological processes, including cellular signaling.^1-5 Plant proteomic studies have identified a variety of nitrated proteins in the leaves and seedlings of Arabidopsis [Arabidopsis thaliana (L.) Heynh.],^6-9 the hypocotyls of sunflower (Helianthus annuus L.),^10,11 and the roots of pea (Pisum sativum L.).¹² Each of these organs has shown nitration of a distinctly different set of proteins, and thus nitration is considered specific for organs analyzed.

Arabidopsis leaves following exposure to peroxynitrite,⁶ high light⁷ or nitrogen dioxide (NO₂)⁹ have identified nitration of 8, 14 and 6 different proteins, respectively. Interestingly, all these proteins but one cytosol protein⁶ are located in the chloroplast. Exposure to NO₂ induces nitration of PsbO and PsbP, extrinsic proteins of photosystem II (PSII), and 4 non-PSII proteins, including peroxiredoxin II E (PRX IIE).⁹ Furthermore, the proteomic analysis of Arabidopsis seedlings grown under unstressed condition also showed nitration of PsbP.⁸ Thus, chloroplasts may be a major target organelle of protein nitration in leaves. However, the precise regulatory mechanism controlling protein nitration remains unelucidated.

In order to gain insight into this aspect of protein nitration, we therefore performed proteomic identification of nitrated proteins in the leaves of tobacco, a fourth species, following exposure to NO₂. We found for the first time that nitration was exclusive to pathogenesis-related (PR)-1 (PR-1A and PR-1B), PR-3 (chitinases PR-P and PR-Q) and PR-5 (thaumatin-like proteins E22 and E2) proteins. All of these proteins are reportedly localized in the apoplast or vacuole. Furthermore, Tyr³⁶ of thaumatin-like protein E2 was identfied as a nitration site as shown in the followings.

Tobacco plants (Nicotiana tabacum cv. Xanthi XHFD8) were grown for 9 weeks and used in this study. They were exposed to NO₂ (4.0 ± 0.1 ppm) in an NO₂ chamber for 8 h in light (100 µmol m⁻² s⁻¹) and from them protein was extracted as described in Supplemental materials and methods.

One-dimensional polyacrylamide gel electrophoresis (1D PAGE) was carried out according to Görg et al. (1985).¹³ Immunoblotting was performed using a rabbit polyclonal anti-NT antibody (Upstate Biotechnology, Lake Placid, NY, USA), and a goat anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase(VECTOR Lab., Burlingame, CA, USA) as described in Supplemental materials and methods. Proteins extracted from tobacco leaves exposed or not exposed to NO₂ showed several NT-immunopositive bands or very faint bands, respectively, in 1D immunoblot gels (Fig. S1). Immunoblot analysis using nitrated bovine serum albumin (BSA) as a control confirmed the specificity of the anti-NT antibody (Fig. S1).

Two dimensional PAGE (2D PAGE) was carried out according to Görg et al. (1985)¹³ with some modifications as described elsewhere.¹⁴ For iodoacetamide treatment, after isoelectric focusing (IEF), the immobilized pH gradient (IPG) strips were equilibrated for 30 min in the dark with SDS buffer containing 2% (w/v) SDS, 50 mM Tris-HCl (pH6.8), 6 mM urea, 30% (w/v) glycerol, 25 mg/mL iodoacetamide and a trace of bromophenol blue, and applied to SDS gels for resolution in 2D. The 2D PAGE gels were stained with SYPRO Ruby stain (Bio-Rad), and protein spots were visualized using a fluorescence scanner (Bio-Rad Molecular Imager FX) and the PDQuest software (Bio-Rad), as described in Supplemental information. Immunoblotting was performed using the anti-NT antibodies, as described in Supplemental materials and methods.

Approximately 600 protein spots were detected on a SYPRO Ruby-stained 2D gel (Fig. 1B) of proteins extracted from exposed tobacco leaves. Among them, 8 spots (spots 1–8) were detected by an anti-NT antibody (Fig. 1A). These proteins were in the pI range of 4.7–6.2 and the molecular mass range of 18–37 kDa. Six spots (1–4, 7, and 8) were further investigated, while spots 5 and 6 showed no detectable peptides by mass spectrometry.

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.

These spots were individually excised and in-gel digested by trypsin (Promega) according to Shevschenko et al. (1996).¹⁵ The tryptic digests were analyzed using a hybrid quadrupole time-of-flight mass spectrometer (Q-TOF MS) (Micromass, Manchester, UK) in the MS and MS/MS mode according to Kristensen et al. (2000).¹⁶ The Q-TOF mass spectra (parent ions) of the tryptic peptides were first obtained in the MS mode. The parent ions of interest were then subjected to sequence analysis in the MS/MS mode to determine amino acid sequences (designated internal sequences). The MS and MS/MS data were subjected to a FASTA search against the SwissProt and GenBank databases using the following parameters: one missed cleavage was allowed; oxidation of methionine (+15.99 Da) and pyroglutamination of the amino-terminal glutamine (−17.06 Da) were set as variable modifications, and carbamidomethylation of cysteine (+57.02 Da) was set as a fixed modification. Additionally, some peptides were analyzed using a matrix- assisted laser desorption ionization-time of flight mass spectrometer (MALDI-TOF MS) according to Matsuo et al. (2006)¹⁷ as described in Supplemental materials and methods. In this study, unless noted otherwise, all MS analyses were performed using Q-TOF MS. The results are summarized in Table 1. Detailed analyses of the data obtained by MS analyses are available in Supplemental results.

Table 1. Nitrated proteins identified by Q-TOF MS analysis of tryptic peptides of proteins extracted from tobacco leaves following exposure to NO₂.

Spot no.	Charge^1)status	m/z^1)measured	m/z^1)calculated	Sequence^2)	Identity	Protein name	pI (Mw^3)) measured	pI (Mw^3)) calculated	UniProtKB accession number
1	2	1015.51	1015.01	^50LDSGQSWSINVNPGTVQAR^68	19/19	E22	5.6 (25)	4.94 (21.7)	P13046
2	2	1079.61	1078.55	^49QLNSGQSWSINVNPGTVQAR^68	20/20	E2	5.7 (25)	4.87 (21.6)	P07052
3	2	871.89	872.40	^31QNSQQDYLDAHNTAR^45	15/15	PR-1A, B, C			P08299, P07053, P09042
	2	1483.42	1483.60	^143VQCNNGGYVVSCNYDPPGNYRGESPY^168	26/26	PR-1A	4.7 (19)	4.57 (15.2)	P08299
4	2	871.93	872.40	^31QNSQQDYLDAHNTAR^45	15/15	PR-1A, B, C			P08299, P07053, P09042
	2	1429.	1429.65	^143VKCNNGGYVVSCNYDPPGNVIGQSPY^168	26/26	PR-1B	5.3 (18)	4.89 (15.1)	P07053
7	2	1065.99	1066.53	^25QGIGSIVTNDLFNEMLKNR^43	19/19	PR-P	5.4 (27)	5.00 (24.8)	P17513
	2	1073.94	1075.59	^25QGIGSIVTNDLFNEMLKNR^43	19/19	PR-P			P17513
	2	1137.90	1138.47	^230YCGMLNVAPGDNLDCYNQR^248	19/19	PR-P			P17513
8	2	714.35	714.82	^65SFPGFGTTGDDTAR^78	14/14	PR-Q	5.7 (27)	5.22 (25.0)	P17514

¹⁾ Shown are charge status and the measured and calculated m/z values of parent ions detected in the Q-TOF mass spectra of trypsin digests of NT-positive protein spots from tobacco leaves following exposure to NO₂.

²⁾ Shown is the amino acid sequence of the tryptic fragment. Internal sequences determined by the Q-TOF MS analysis are shown in bold letters. Q; Pyroglutamine, C; carbamidomethylated cysteine, M; oxidized methionine. Sequences were subjected to a FASTA search against the Swiss-Prot database to identify proteins.

³⁾ Mw values are shown in kDa.

The MS mode analysis of the trypsin digest of spot 1 detected a doubly charged [M + 2H]²⁺parent ion at m/z 1015.51, and the subsequent MS/MS mode analysis determined an internal sequence consisting of 17 residues (Table 1). A detailed analysis of these data (see Supplemental results) identified spot 1 as thaumatin-like protein E22 (Table 1).

A doubly charged parent ion at m/z 1079.61 was detected in the MS spectra of the trypsin digests of spot 2, and from this parent ion, a 17-residue internal sequence identical to that of spot 1 was identified (Table 1). A detailed analysis of these data (see Supplemental results) identified spot 2 was as thaumatin-like protein E2 (Table 1).

E22 and E2 are isoforms of the group 5 PR proteins in tobacco.^18,19 Both the PR-5 proteins share significant amino acid sequence homology with the sweet-tasting protein, thaumatin, in the fruits of the tropical plant Thaumatococcus daniellii and are thus referred to as thaumatin-like proteins.²⁰

In the MS spectra of the trypsin digests of spot 3, 2 doubly charged parent ions at m/z 871.89 and 1483.42 were detected, from which 13- and 7-residue internal sequences, respectively, were determined (Table 1). A detailed analysis of these data (see Supplemental results) identified spot 3 as PR-1A.

In the MS spectra of the trypsin digests of spot 4, 2 doubly charged parent ions at m/z 871.93 and 1429.37 were detected, which 13- and 12-residue internal sequences, respectively, were determined (Table 1). A detailed analysis of these data (see Supplemental results) identified spot 4 as PR-1B. PR-1A and PR-1B are localized in the apoplast (extracellular space) and/or vacuole, depending on the cell type, in tobacco.^21,22 PR-1 proteins are the most abundant PR protein.^18,19

In the MS spectra of the trypsin digests of spot 7, 3 doubly charged parent ions were detected at m/z 1065.99, 1073.94 and 1137.90 (Table 1). The internal sequences determined from the former 2 ions consisted of 16 identical residues. A detailed analysis of these data together with peptide finger printing (PMF) analysis using MALDI-TOF MS (see Supplemental results) identified spot 7 as PR-P protein.

In the MS spectra of tryptic digests of spot 8, a parent ion at m/z 714.35 was detected, from which a 12-residue internal sequence was determined. A detailed analysis of these data together with PMF analysis (see Supplemental results) identified spot 8 as PR-Q protein (Table 1). PR-P and PR-Q are acidic chitinases of the group 3 PR proteins in tobacco (Table 1).^18,19 Both PR-3 proteins reportedly display antifungal activities and are located in the apoplast.¹⁸

A basic endochitinase and a Populus trichocarpa chitinase family protein are nitrated in pea roots.¹² Although these chitinase species are distinct from tobacco acidic PR-3 chitinases (PR-P and PR-Q) (see above), it is noteworthy that chitinase species are a common target for nitration in various organs of different species.

The PR proteins identified in this study have 5 to 11 tyrosine residues per molecule (UniProt). However, no nitrated peptides were detected in the tryptic digests of tobacco leaf proteins by the present Q-TOF analyses. We therefore analyzed the nitrated peptides using MALDI-TOF MS according to Matsuo et al. (2006).¹⁷ Tobacco leaves were exposed to 4.0 ± 0.4 ppm ¹⁵N-labeled NO₂(51.6 atom% ¹⁵N) for 8 h in light.¹⁴ Leaf proteins were extracted from exposed plants and subjected to 2D PAGE and immunoblotting using the anti-NT antibody as described above, except that iodoacetamide was omitted from the SDS buffer so that cysteine residues were not carbamidomethylated. Formation of a cysteine acrylamide adduct (+71.08 Da) was set as a variable modification. Since nitration results in a mass shift of +45 or +46 Da (replacement of H with a NO₂ or ¹⁵NO₂ group, respectively), we searched pairs of ions having such a mass shift in the spectra of the in-gel digests of spot 2 digested by trypsin and V8 protease (Sigma).

A pair of 2 singly charged ions at m/z 2023.3 and 2069.3 was found (Fig. 2). The Mascot database search indicated that these ions correspond to residues 30–48 (³⁰IVNQCTYTVWAAASPGGGR⁴⁸) of E2, without or with one nitrated tyrosine, respectively. Formation of an acrylamide adduct at Cys³⁴ was postulated in both ions. The nitration or replacement of H with a ¹⁵NO₂ group resulted in a mass shift of +46 Da from m/z 2023.3 to 2069.3 (Fig. 2), identifying Tyr³⁶ of E2 as the site of nitration. However, all attempts to determine nitration site identification in nitrated proteins other than E2 have been in vain. Reason(s) for this is unclear.

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.

It is possible that exogenously applied NO₂ enters cells and nitrate proteins. It is also possible that exogenously applied NO₂ elevate nitrosative stress in cells to stimulate production²⁴ of endogenous NO₂, which nitrate proteins. Thus, whether the nitro groups of the nitrated proteins following NO₂ exposure are derived from the exogenously supplied NO₂ or from endogenous²⁴ NO₂ has so far been unclear. Our present finding of a mass shift of +46 Da, in turn, indicates that the nitro group in nitrated proteins produced by NO₂-induced nitration treatment is derived from exogenously supplied NO₂.

Our present study showed that of ∼600 protein spots 6 NT-positive proteins were all identified as PR proteins, PR-1A, PR-1B, PR-P, PR-Q, thaumatin-like protein E2 and thaumatin-like protein E22. Protein tyrosine nitration is not a random chemical process, but is instead a selective process in which only specific proteins are nitrated,^1,2 and neither the abundance of a protein nor the abundance of tyrosine residues in a given protein can predict whether it is a target for protein tyrosine nitration.^1,2 However, this exclusive nitration of PR proteins in tobacco leaves was a totally unexpected observation. Nonetheless, we thought our present finding important in light of the reported fact that PR proteins play a vital role in plant defense/immune systems.^17,18 However, in order to clarify whether apoplastic / vacuolar proteins are nitratale, immuno-electron microscopic evidence²⁵ to show localization of nitrated proteins in the apoplast / the vacuole in tobacco leaves following exposure to NO₂ is absolutely required.

PR proteins are defined as inducible defense-related proteins.^17,18 They are inducible upon pathogen infection and biotic/abiotic stress and play a role in the surveillance mechanism in the plant immune system.¹⁸ They are currently classified into 17 families.^17,18 Those PR proteins whose nitration was identified in the present study are inducible by abiotic stresses such as nitric oxide (NO),²⁶ salicylic acid²⁷ and ozone²⁸ in tobacco leaves. They may also have been induced upon exposure to NO₂.

It should be noted, however, that the concentration of NO₂ used in the present study is ∼100-fold higher than those (10–200 ppb) used in NO₂-mediated regulation of plant growth.^9,29,30 Therefore, further investigations are required to establish the physiological relevance of the present observations on nitration of these PR proteins. Future studies will focus on site-directed mutagenesis of Tyr³⁶ of E2, a nitration site identified in this study, followed by introduction of a mutated E2 gene in tobacco plants. The engineered and non-engineered control plants will be grown in the presence or absence of physiological concentrations of NO₂ to explore the relationship between nitration of defense-related PR proteins and NO₂-mediated growth regulation.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

Financial support from the Nippon Life Insurance Foundation (to MT) is acknowledged. This research was supported in part by the Research for the Future Program, Japanese Society for the Promotion of Science (JSPS-RFTF96L00604) and by a Grant-in-Aid for Creative Scientific Research (no. 13GS0023) from the Japan Society for the Promotion of Science (to HM).