JPROT-02073; No of Pages 11 JOURNAL OF P ROTEOM IC S XX ( 2015) X XX–X XX

Available online at www.sciencedirect.com

ScienceDirect www.elsevier.com/locate/jprot

3Q3

Agnieszka Szubaa,b,⁎, Anna Kasprowicz-Maluśkic , Przemysław Wojtaszeka,c a

5

b

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznań, Poland Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, 62-035 Kórnik Poland c Department of Molecular and Cellular Biology, Adam Mickiewicz University, Umultowska 89, 61-613 Poznań, Poland

R O

6

O

4

F

2

Nitration of plant apoplastic proteins from cell suspension cultures

1Q2

7

AR TIC LE I NFO

ABSTR ACT

11 15

Article history:

Nitric oxide causes numerous protein modifications including nitration of tyrosine

12 16

Received 20 August 2014

residues. This modification, though one of the greatest biological importance, is poorly

13 17

Accepted 3 March 2015

recognized in plants and is usually associated with stress conditions. In this study we

D

14 18

analyzed nitrotyrosines from suspension cultures of Arabidopsis thaliana and Nicotiana

E

19 45 20

P

10 9

tabacum, treated with NO modulators and exposed to osmotic stress, as well as of BY2 cells long-term adapted to osmotic stress conditions.

46 21 47 22

Arabidopsis thaliana

Using confocal microscopy, we showed that the cell wall area is one of the compartments

Nicotina tabacum

most enriched in nitrotyrosines within a plant cell. Subsequently, we analyzed nitration of

48 23 49 24

Suspension cultures

ionically-bound cell-wall proteins and identified selected proteins with MALDI-TOF

Apoplast

spectrometry.

50 25 51 26

Proteomics

Proteomic analysis indicated that there was no significant increase in the amount of nitrated

Nitrotyrosines

proteins under the influence of NO modulators, among them 3-morpholinosydnonimine

E

C

T

Keywords:

52 27

R

(SIN-1), considered a donor of nitrating agent, peroxynitrite. Moreover, osmotic stress

28

conditions did not increase the level of nitration in cell wall proteins isolated from suspension

29

R

30

comparison with control samples. Among identified nitrotyrosine-containing proteins

31

34 35 36

N C O

33

dominated the ones associated with carbon circulation as well as the numerous proteins responding to stress conditions, mainly peroxidases. Biological significance High concentrations of nitric oxide found in the cell wall and the ability to produce large amounts of ROS make the apoplast a site highly enriched in nitrotyrosines, as presented in

U

32

Q4

cells, and in cultures long-term adapted to stress conditions; that level was even reduced in

Abbreviations: 1DE, one-dimensional electrophoresis; 2DE, two-dimensional electrophoresis; BSA, bovine serum albumin; BY2, tobacco cultivar Bright Yellow-2; CBB, Coomassie Brilliant Blue; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; cPTIO, 4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; GA, glutaraldehyde; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; MS, mass spectrometry; PFA, paraformaldehyde; PVDF, polyvinylidene fluoride; RNS, reactive nitrogen species; ROS, reactive oxygen species; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; SIN-1, 3-morpholinosydnonimine; SNAP, S-nitroso-N-acetyl-DL-penicillamine; TBST, Tris buffered saline with Tween; TRITC, tetramethylrhodamine isothiocyanate; Tyr-NO2, nitrotyrosine. ⁎ Corresponding author at: Laboratory of Proteomics, Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, 62-035 Kórnik, Poland. Tel.: +48 618170033; fax: +48 618170166. E-mail address: [email protected] (A. Szuba).

http://dx.doi.org/10.1016/j.jprot.2015.03.002 1874-3919/© 2015 Published by Elsevier B.V.

Please cite this article as: Szuba A, et al, Nitration of plant apoplastic proteins from cell suspension cultures, J Prot (2015), http:// dx.doi.org/10.1016/j.jprot.2015.03.002

Q5

2

JOUR NAL OF P ROTEOM ICS XX ( 2015) X XX–XX X

37

this paper. Analysis of ionically bound fraction of the cell wall proteins indicating generally

38

unchanged amounts of nitrotyrosines under influence of NO modulators and osmotic

39

stress, is noticeably different from literature data concerning, however, the total plant

40

proteins analysis. This observation is supplemented by further nitroproteome analysis, for

41

cells long-term adapted to stressful conditions, and results showing that such conditions

42

did not always cause an increase in nitrotyrosine content. These findings may be

43

interpreted as characteristic features of apoplastic protein nitration.

44 55 54 53 56

© 2015 Published by Elsevier B.V.

1. Introduction

2. Materials and methods

59

Nitric oxide, a radical molecule, modifies proteins by changing their structure and function [1,2]. One of the most biologically important chemical reactions of nitric oxide is the formation of 3-nitrotyrosines (Tyr-NO2). This modification involves addition of –NO2 groups to the aromatic ring of tyrosine residues. Despite the possibility of formation of Tyr-NO2 in many chemical reactions [3], it is commonly believed that in living organisms 3-nitrotyrosines are produced almost exclusively in the reaction of tyrosine with peroxynitrite (ONOO−), which is a product of reaction between the nitric oxide radical (NOU) and the superoxide anion (O2U−) [3,4]. Tyrosine nitration is usually associated with the activity of various ROS (reactive oxygen species) and RNS (reactive nitrogen species), and as such is regarded as a source of negative, pathological changes in the cell, often leading to apoptosis. Therefore, it is widely recognized as a marker of oxidative stress [5]. Studies on nitrotyrosines in plant tissues have been launched recently and nitration of plant proteins is known only from a few reports. In plants, nitration of tyrosine occurs under physiological conditions [6] and it may play a regulatory role in biological processes such as photosynthesis [7] or root development and senescence [8]. However, in most studies, also pertaining to animals, occurrence of nitrotyrosines is related to stress. For example, it has been shown that conditions of salt stress [9] or other abiotic [10] and biotic [11] stresses increased the amount of nitrotyrosines in plant tissues. This rise was associated with an increased production of nitric oxide [9], observed during stress, and is consistent with the assumption that NO acts via posttranslational protein modifications such as nitration of critical tyrosine residue [12,13]. Nitration of plant apoplastic proteins is still a completely unknown process. However, it was already reported that cell walls as part of an apoplastic space contain numerous sources of NO, the main factor responsible for nitration [14–16]. Moreover, it is known that cell walls are very important in plant signaling network [17] and are the first line of defense against stress conditions [18], especially against osmotic stress. To conclude, tyrosine nitration processing in the apoplast may be an important part of response to stress conditions. This paper presents a first attempt to investigate nitration occurring in the apoplast, based on localization of nitrated proteins in plant suspension cells and identification of nitrated ionically-bound cell wall proteins from intact suspension cells. Impact of NO modulators and osmotic stress conditions on this sub-proteome was analyzed as well.

2.1. Reagents

71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106

O

120

P

R O

2.2. Plant material

D

70

110

E

69

109

Rabbit polyclonal anti-nitrotyrosine antibody was from Molecular Probes. Immobilon-P membranes were from Millipore (Bedford, MA). Immobiline™ DryStrip gels were from GE Healthcare LS. Trypsin and α-cyano-4-hydroxycinnamic acid were from Promega. cPTIO (4-carboxyphenyl-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide) and SNAP (S-nitrosoN-acetyl-DL-penicillamine) were from Calbiochem. MemCode was from Thermo Fisher Scientific Inc. Other reagents, including SIN-1 (3-morpholinosydnonimine), were purchased from Sigma-Aldrich.

111 112 113 114 115 116 117 118 119

Nicotiana tabacum BY-2 (Bright Yellow-2) suspension-cultured cells were obtained from the Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland while Arabidopsis thaliana cell suspension was kindly provided by Rafael Pont-Lezica from CNRS–Université Paul Sabatier, Toulouse, France. Cells were cultured in a modified Murashige and Skoog medium [19] enriched with 3% (w/v) sucrose, myo-inositol (100 mg/1), and respective additional compounds: thiamine hydrochloride (1 mg/l), KH2PO4 (370 mg/1) and 2,4-dichlorophenoxyacetic acid (0.2 mg/l) for BY2 cells and 1-naphthaleneacetic acid (1 mg/l), kinetin (1 mg/l) and thiamine hydrochloride (1 mg/l) for A. thaliana suspension cells. N. tabacum suspension-cultured cells were adapted to osmotic stress conditions in the Department of Molecular and Cellular Biology, Adam Mickiewicz University, Poznan, Poland. Cells were gradually [20] adapted to 190 mM NaCl, 180 mM KCl, 450 mM mannitol, and 450 mM sorbitol solutions. All cultures were maintained at 22 °C with constant shaking (120 rpm, 2 in.) in the dark and were subcultured every 10 days.

121

2.3. Suspension cell treatments

141

NO modulators were used in final concentrations: 250 μM SNAP, 250 μM SIN-1 and 200 μM cPTIO. SNAP and SIN-1 were first dissolved in 20 μl DMSO (dimethyl sulfoxide; with appropriate compensation in the control probe), and next, as cPTIO, were dissolved in deionized water, all done to obtain 25 mM stocks, just before application to the suspension culture medium. Cells were incubated in the dark with constant shaking (120 rpm) for either: 30 min, 1 h, 2 h, 5 h or 24 h. After incubation, the cell wall proteins were immediately isolated or cells were used for microscopic analyses.

142

T

68

C

67

E

66

R

65

R

64

O

63

C

62

N

61

U

60

108 107

F

Q6 58 57

Please cite this article as: Szuba A, et al, Nitration of plant apoplastic proteins from cell suspension cultures, J Prot (2015), http:// dx.doi.org/10.1016/j.jprot.2015.03.002

122 123 124 Q7 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140

143 144 145 146 147 148 149 150 151

3

JOURNAL OF P ROTEOM IC S XX ( 2015) X XX–X XX

169

To extract ionically-bound cell wall proteins, a calcium chloride wash method [21] was used. Briefly, intact 4-day-old suspension-cultured cells were filtered on a funnel through Miracloth (culture media were collected and treated as described below) and re-suspended in an equal volume of 0.2 M CaCl2 and incubated with constant shaking (120 rpm) for 40 min. Supernatants were collected and reduced by ultrafiltration utilizing a Y10 filter (10 kDa cut-off; Amicon, Milipore, Milford, MA, USA) and equilibrated with TBS (Tris buffered saline), pH 7.5. For two-dimensional electrophoresis (2DE), proteins were additionally cleared with phenol extraction [22].

170

2.4.2. Protein preparation and electrophoresis methods

171

191

For one-dimensional electrophoresis (1DE), 40 μg protein fractions per well were re-suspended in sample buffer (10% glycerol, 2% SDS (sodium dodecyl sulfate), 5% β-mercaptoethanol, 0.01% bromophenol blue, 150 mM Tris–HCl pH 6.8) and SDS-PAGE (SDS-polyacrylamide gel electrophoresis) was carried out according to Laemmli [23] in 10% acrylamide gels. Gels were stained with Coomassie Brilliant Blue G-250 or were used for Western blot analysis. For 2DE, 150 μg of protein per strip was rehydrated overnight in a buffer containing 5 M urea, 2 M thiourea, 2% CHAPS, 2% carrier ampholyte (Pharmalyte 3–10, GE Healthcare LS), and a trace amount of bromophenol blue. Final volumes of 125 μl were loaded on 7-cm pH 3–10 linear IPG strips (GE Healthcare LS). IPG strips were focused on Multiphor II (Amersham Biosciences) with voltage program as follows: 300 V for 90 min; 1500 V for 1 h; then up to 3500 V for 6.5 h. Following isoelectric focusing, strips were equilibrated (15 min) in equilibration buffer (6 M urea, 0.75 M Tris, pH 8.8, 2% SDS, 30% glycerol) containing 1% DL-dithiothreitol, and then for 15 min in 2.5% iodoacetamide. After equilibration, proteins were electrophoresed on 12% SDS-PAGE as described above.

192

2.4.3. Immunodetection of nitrated proteins

193

Following standard wet transfer, PVDF (polyvinylidene fluoride) membranes were blocked for minimum 1 h in 1% BSA (bovine serum albumin) solution in TBST (Tris-buffered saline with Tween;10 mM Tris–HCl pH 8.0; 150 mM NaCl; 0.05% Tween 20). Membranes were incubated overnight, at room temperature, with anti-nitrotyrosine antibody at 1/2000 dilution in 1% BSA/TBST, prior to incubation for 2 h, at room temperature, with goat anti-rabbit alkaline phosphatase-conjugated antibody at 1/30,000 dilution in TBST. After several washes with TBST, membranes were developed in SigmaFast solution. 5 μg of nitrated BSA (Sigma) was used as a positive control. In some experiments, before immunodetection, proteins were visualized with MemCode (Thermo Fisher Scientific Inc, 24585), according to the manufacturer's protocol, washed and then immunodetected as described above.

165 166 167 168

172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190

194 195 196 197 198 199 200 201 202 203 204 205 206 207

C

164

E

163

R

162

R

161

N C O

160

U

159

F

158

O

2.4.1. Protein extraction

R O

157

P

2.4. Proteomic procedures

Protein spots of interest were excised from CBB-stained gels using a clean Pasteur pipette and digested according to the standard “in-gel digestion” protocol. Briefly, CBB was removed by double washing with 50 mM NH4HCO3:acetonitrile (1:1; v/v) solution. Reduction and alkylation were achieved by, respectively, 45-min incubation of the gel fragments in 10 mM DL-dithiothreitol in 50 mM NH4HCO3 at 56 °C and 30-min incubation in a solution of 55 mM iodoacetamide in 50 mM NH4HCO3 in the dark, at room temperature. Digestion of proteins, performed by incubation of gel fragments in 10 μl of 50 mM NH4HCO3 containing trypsin in a concentration of 20 ng/ml, was carried out overnight at 37 °C. After digestion, 1 μl of 1:1 (v/ v) mixture of the digested protein and freshly prepared matrix solution (saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile, 0.1% trifluoroacetic acid) was spotted onto a MALDI AnchorChip target plate and dried at room temperature. The spectra were recorded in the Autoflex spectrometer (Bruker Daltonics GmbH) in positive-ion reflector mode. Minimum one hundred laser shots were taken per spectrum. All spectra were externally calibrated using a standard peptide solution (Bruker Daltonics GmbH). Peptide mass fingerprints were compared with the Mass Spectrometry Database or the Swiss-Prot Database. The search was performed using a peptide mass tolerance of 200 ppm with carboxymethylation as a fixed modification and oxidation of methionine as variable modification. One missed cleavage was allowed.

D

156

154

2.4.4. Spot excision, tryptic digestion and MALDI-TOF analysis 208

T

155

Influence of osmotic stress condition: A. thaliana suspension cells were incubated for 30 min, 5 h or 24 h in 100 mM NaCl, 100 mM KCl or 200 mM mannitol solutions, respectively, and subsequently treated as above.

153

E

152

209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235

2.4.5. Microscopic procedures — localization of nitrated tyrosine 236 residues with confocal laser scanning microscopy 237 Location of nitrated proteins in fixed BY-2 cells was investigated according to the whole-mount protocol, originally developed for the analysis of Arabidopsis seedlings [24], with some modifications. After treatment with NO modulators, initial fixation of the cells was performed by mixing 1:1 (v/v) the cell suspension with a solution of 4% paraformaldehyde (PFA) and 1% glutaraldehyde (GA) in a microtubule-stabilizing buffer MTSB [25]; final concentrations amounted to 2% PFA and 0.5% GA. Cells were then incubated with constant shaking (120 rpm) for 25 min at 22 °C. Subsequently, the cells were fixed in 4% PFA and 1% GA in MTSB solutions for 35 min. The fixed cells were washed in MTSB containing 0.1% Triton X-100, in water. Each time the cells were sedimented by centrifugation (500 rpm, 1 min). Cell walls were predigested during 20-min incubation in a solution of 1% cellulose (1 U/mg; Serva, Cat. No.16413) and 0.35% Macerozyme (0.104 U/mg, Serva, Cat. No.28302) in MTSB with Triton X-100, and rinsed. Afterwards, cells were incubated in 10% DMSO, 3% NP-40 in MTSB for 50 min and washed. The final fixation was performed during a 15-min incubation in 4% PFA and 1% GA in ½MTSB, followed by another washing of cells. Blocking of free aldehydes, leading to reduced auto-fluorescence of the preparation, was obtained by a 10-min incubation in a solution of NaBH4 (1 mg ml−1) in MTSB. Nonspecific attachment of antibodies was blocked during a 30-min incubation in 2% BSA in MTSB at room temperature. The cells were incubated in a solution of the primary anti-nitrotyrosine antibody, diluted 1:200, overnight at 4 °C. After repeated washing, cells were incubated for 2 h in the solution of the secondary anti-rabbit antibody

Please cite this article as: Szuba A, et al, Nitration of plant apoplastic proteins from cell suspension cultures, J Prot (2015), http:// dx.doi.org/10.1016/j.jprot.2015.03.002

238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266

4

282 283 284 285 286 287 288 289 290 291 292 293

A

B

F

Cell walls form part of the cell wall–plasma membrane– cytoskeleton continuum, an important structural and signaling network [16–18,20,26]. Therefore, the chosen, final concentrations of NO modulators as well as times of cell treatments were selected very carefully and were similar to conditions applied previously for studies on NO-induced cytoskeleton rearrangements [26,27]. Immunolocalization of nitrotyrosines in fixed tobacco BY2 cells indicated their greatest accumulation within the area of cell walls and around the nucleus (Fig. 1). In order to confirm the presence of modified residues in the cell wall, the procedure of in vivo immunolocalization was used. Indeed, the obtained data confirmed the apoplastic occurrence of nitrotyrosines (Fig. 2). Furthermore, the results evidence that in

90 80

C

D

70 60 50 40 30 20 10 0

H

E

Control

296 297 298 299 300

303

O

281

3.1. Distribution of nitrotyrosines

Max grey value

280

302 301

R O

279

3. Results

P

278

D

277

E

276

T

275

C

274

295

E

273

The maximum gray values of the representative cell wall area's cross sections (n = 30) were estimated using ImageJ 1.48v software. The obtained data were analyzed using STATISTICA 5.1 (StatSoft, USA) and means were compared by using the Fisher's least significant difference test. Differences were considered significant at p ≤ 0.05.

R

272

294

R

271

O

270

2.5. Statistics

C

269

linked to TRITC (tetramethylrhodamine isothiocyanate) at a dilution of 1:400. After washing away the secondary antibody, cells were stained with 100 μM DAPI (4′,6-diamidino-2phenylindole) for 10 min and with 0.01% toluidine blue in PBS for further 10 min. After final washing, cells were mounted on a microscope slide for examination with a confocal laser scanning microscope (Zeiss LSM 510) using argon laser (488 nm) and 540DF30 filter. In all cases, the images were processed and analyzed using Zeiss LSM Image Browser and Adobe PhotoShop7.0. Analysis of nitrotyrosine location in cell walls was performed on suspension cells, applying the in vivo detection procedure. In consequence, all available epitopes were contained in or exposed to the extracellular space. All stages of analysis were conducted in a fresh medium (see above) and all incubations were carried out with gentle shaking (30 rpm). The suspension (0.5 ml) was incubated in 1% BSA for 60 min to block nonspecific attachment of antibodies. Then intact cells were repeatedly washed with a fresh culture medium, each time being left on the table top for 5 min to sediment. Primary anti-nitrotyrosine antibody, dissolved at a ratio of 1:200, was used in the next 1-hour incubation, followed by another washing and adding the secondary anti-rabbit antibody linked to TRITC, at a dilution of 1:400, in which the cells were incubated for one more hour. After washing, nitrotyrosines were detected using a confocal microscope as described above.

N

268

U

267

JOUR NAL OF P ROTEOM ICS XX ( 2015) X XX–XX X

cPTIO

SNAP

F

Fig. 1 – Nitrotyrosine (Tyr-NO2) distribution in tobacco BY-2 cell suspension cultures. Images were taken under confocal laser scanning microscope; cross sections (A, B and C) and 3D images respectively (D, E and F), both obtained by overlapping multiple optical sections. Tyr-NO2 was detected in fixed suspension BY2 cells treated with NO modulators. Images A and D — cells incubated for 2 h in 250 μM SNAP; B and E — cells grown in normal conditions; images C and F — cells incubated for 2 h in 200 μM cPTIO. Scale — 50 μm. Maximal of gray values obtained during analysis of cell wall cross sections (G) of the above images. Values followed by different letters are significantly different from each other at p ≤ 0.05 according to the Fisher least significant difference test. Please cite this article as: Szuba A, et al, Nitration of plant apoplastic proteins from cell suspension cultures, J Prot (2015), http:// dx.doi.org/10.1016/j.jprot.2015.03.002

304 305 306 307 308 309 310 311 312 313 314 315 316

5

B

R O

A

O

F

JOURNAL OF P ROTEOM IC S XX ( 2015) X XX–X XX

3.2. Proteomic analysis

328

339

Analyses of the nitroproteome were carried out using a well-described anti-nitrotyrosine antibody [6,8–10], which specificity was additionally tested (data not shown). Studies were performed mainly on proteins ionically-bound to cell walls, isolated from suspension-cultured cells of A. thaliana and tobacco BY2 using the procedure that does not disturb the integrity of plasma membrane [21]. In some cases, proteins released from cell suspensions to the culture medium were also investigated. Analyses showed a high repeatability of nitrated protein profiles in biological repetitions (Fig. 3) and supported the previous reports [28] on nitration as a selective process, also in the extracellular space.

340

3.3. Nitroproteome and NO modulators

341

NO modulators did not show a significant effect either on the 1DE profiles of the cell wall proteins or on the nitroproteome (as indicated by the example of 250 μM SNAP; Fig. 4), even after long-term treatments. The effect of SIN-1 was analyzed with the use of 2DE (Fig. 5). Cell suspensions were incubated for 2 h in control conditions and in a 250 μM SIN-1, a nitric oxide donor additionally generating a superoxide anion. After isolation of ionically-bound cell wall proteins, their 2DE and transfer onto the Immobilon-P membrane, proteins were stained with

329 330 331 332 333 334 335 336 337 338

342 Q8 343 344 345 346 347 348 349 350

C

325

E

324

R

323

R

322

N C O

321

U

320

reversible MemCode dye to visualize the protein profile. No significant changes were detected in the cell wall proteome. After rinsing the dye, nitrotyrosines were immunodetected. Most cell wall proteins of both suspension cultures were strongly alkaline (Fig. 5) and included high amounts of nitrotyrosines. Samples treated with SIN-1 displayed only a slight increase in the nitrotyrosine content as compared with the control.

D

327

319

T

326

both live and fixed cells nitrotyrosines were distributed unevenly and locally formed distinct clusters (Fig. 2; arrowheads). The effect of NO modulators on the distribution and level of nitration in cell suspensions (Fig. 1) was also studied. Differences (p = 0,0001; Fig. 1G) in fluorescence intensity were observed, as it increased in cells incubated in a NO donor, 250 μM SNAP (Fig. 1; images A and D), and was reduced in samples incubated with NO scavenger, 200 μM cPTIO (Fig. 1; images C and F), compared to control groups (Fig. 1; images B and E).

318

E

317

P

Fig. 2 – In vivo immunolocalization of nitrotyrosines. Transmitted light image of BY2 plant cells (A) and distribution of epitopes of anti-nitrotyrosine antibodies in the cell wall area (B). Images were taken under confocal laser scanning microscope and show cross-sections. Arrowheads — immune-positive spots forming distinct clusters. Scale — 50 μm.

CW M

CW1 CW2 M1

PROTEOME

NITROPROTEOME

M2

C

205 kDa 116 kDa 97 kDa 66 kDa 55 kDa

29 kDa 20 kDa

Fig. 3 – Profiles of proteins containing nitrotyrosines. Proteins of interest were separated with 10% 1DE; nitrotyrosines were detected after transfer onto Immobilon-P. (PROTEOME): representative profiles of all apoplastic proteins stained with CBB G-250. (NITROPROTEOME): results of immunodetection. CW — ionically bound cell wall proteins of A. thaliana suspension cells; M — proteins released into culture medium by cell suspensions of A. thaliana; C — nitrated BSA (5 μg, Sigma). 1,2 — independent biological repetitions of experiment.

Please cite this article as: Szuba A, et al, Nitration of plant apoplastic proteins from cell suspension cultures, J Prot (2015), http:// dx.doi.org/10.1016/j.jprot.2015.03.002

351 352 353 354 355 356 357 358

6

JOUR NAL OF P ROTEOM ICS XX ( 2015) X XX–XX X

1

34 kDa

26 kDa

PROTEOME

NITROPROTEOME

Fig. 4 – Results of incubation of BY2 cells in 250 μM solution of SNAP. PROTEOME — 12% 1DE SDS-PAGE gel stained with CBB-G250; NITROPROTEOME — results of immunodetection. 1–5: cell wall proteins isolated from the control sample (t0, 30 min, 1 h, 2 h, 5 h, respectively). Tracks 6–10: proteins isolated from suspension incubated in SNAP, taken at the same time points. M — molecular weight marker. Arrow — protein identified in MS. “1” — One of the most nitrated proteins from the BY2 cell wall proteome (results of MS identifications are presented in Table 1).

3.4. Apoplastic nitrotyrosines and osmotic stress conditions

360

Experiments involved the use of 100 mM NaCl or KCl as well as 200 mM mannitol or sorbitol solutions. Concentrations of osmotically active agents were selected in such a way as not to induce increased mortality of cells as detected with Evans blue staining (data not shown). Conditions of osmotic stress resulted in significant changes in the profiles of ionically-bound proteins (Fig. 6) as well as in profiles of proteins released to the culture medium (Fig. 7). However, in both groups, no increase in nitrotyrosines was recorded under stressful conditions. Results of immunodetection provided evidence for massive changes in protein profiles. All heavily nitrated proteins were identified using MS

368 369 370 371

C

E

367

R

366

R

365

373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399

4 . Discussion

401 400

Apoplastic proteome seems to be particularly interesting when considering the distribution of NO in plant cells. Available literature data [29,30] and our analyses [12,26] indicate that the cell wall area is the compartment most enriched in NO within a plant cell. This may result from the activity of membrane enzymes, nitrite:NO reductase and plasma membrane-bound nitrate reductase, which are the

402

O

364

C

363

Control

N

362

U

361

372

T

359

F

43 kDa

O

56 kDa

(mass spectrometry) methods, results of which are presented in Table 1. Cell wall proteins were also isolated from tobacco BY2 cell suspensions long-term adapted to 190 mM NaCl, 180 mM KCl, 450 mM mannitol or 450 mM sorbitol. Protein profiles of adapted cells differed significantly from the control and were characteristic for each variant analyzed (Fig. 8). The level of protein nitration was significantly lower in proteins isolated from the adapted cell suspensions compared to the control sample. In samples isolated from cells adapted to stressful conditions, only a few heavily nitrated proteins were detected (Fig. 8; proteins no. 20–22). Moreover, level of their nitration depended on the osmotic agent used and was lower following salt treatments (Fig. 8; NaCl and KCl lines). In the analysis of nitrated proteins, considering all osmotic stress conditions, redox state-regulating enzymes, stressrelated enzymes and numerous peroxidases (Table 1) were the main ones identified. Nitrated peroxidases occurred in the pool of apoplastic proteins isolated from cell suspensions incubated in solutions of NO modulators (Fig. 4; protein nr 1) and under stress conditions (Fig. 7; protein nos. 18 and 19), and were the most heavily nitrated cell wall proteins (Fig. 8; protein nos. 21 and 22). They were the most nitrated proteins of the apoplastic proteome also in control conditions, especially in the pool of proteins released into the culture medium (Fig. 7). Furthermore, osmotic stress or incubation with NO donors did not cause a significant increase in the amount of nitrotyrosine in these proteins.

R O

6 7 8 9 10 M

P

1 2 3 4 5

D

6 7 8 9 10

E

M K 1 2 3 4 5

95 kDa 72 kDa

SIN-1

PROTEOME

A

NITROPROTEOME

PROTEOME

NITROPROTEOME

B

Fig. 5 – Effects of SIN-1 on cell wall proteome and nitroproteome of suspension cultures from BY2 (A) and A. thaliana (B). Cell wall proteins were separated in 2DE in a linear pH 3–10 gradient in the first dimension and 12% SDS-PAGE in the second dimension. After transfer onto Immobilon-P, proteins were stained with MemCode (PROTEOME panels) and after dye rinsing the nitrotyrosines were detected (NITROPROTEOME panels). Proteins were extracted from the control cells (Control line) and from suspension incubated in 250 μM solution of NO donors (SIN — 1 line). Please cite this article as: Szuba A, et al, Nitration of plant apoplastic proteins from cell suspension cultures, J Prot (2015), http:// dx.doi.org/10.1016/j.jprot.2015.03.002

403 404 405 406 407 408

7

JOURNAL OF P ROTEOM IC S XX ( 2015) X XX–X XX

M 12 22 32 15 25 35 124 224 324 M

95 kDa

2

72 kDa

5

56 kDa

6

9

C 12 22 32 15 25 35 124 224 324 2

3 4

7 8

6

9

10

43 kDa

3 4

5

7 8 10

11

11

34 kDa

12

12

13

13

NITROPROTEOME

O

PROTEOME

F

26 kDa

415 416 417

M

1

2

3

4

M

72 kDa 14

56 kDa 43 kDa

34 kDa

26 kDa

15 16

1

2

4

14 15 16

17

17

18

18

19

19

PROTEOME

3

NITROPROTEOME

Fig. 7 – Effect of 2-hour incubation of A. thaliana suspension in osmotic stress conditions on the nitration of proteins secreted into the culture medium: M — molecular weight marker; 1 — control: proteins isolated from suspension incubated in normal conditions; 2 — suspension incubated in 100 mM KCl; 3 — suspension incubated in 100 mM NaCl; 4 — suspension incubated in 200 mM mannitol. PROTEOME — CBB G-250 stained gel obtained with 10% 1DE SDS PAGE; NITROPROTEOME — results of nitrotyrosine immunodetection. Arrows — nitrated proteins of interest (14–19) identified in MS (see Table 1).

heavily enriched in nitrotyrosines (Figs. 1 and 2), certainly present in the apoplast (Fig. 2). Stress signaling pathways involve numerous ROS and RNS, including NO [28,35,36]. Therefore, we examined how both NO donors and osmotic stress would affect nitration of cell wall proteins. Studies of the effect of NO modulators on the cell wall sub-proteome revealed an interesting feature of suspension cells. Experiments showed no significant influence of both NO donor and NO scavenger on the nitration of ionically-bound cell wall proteins, even during long-term incubation (Fig. 4). Because direct nitration agent is considered to be ONOO−, we checked if peroxynitrite itself will result also in unaffected nitrotyrosine level in analyzed proteome. Indeed, even the SIN-1, considered a source of ONOO−, treatment caused only a slight increase, compared to control, in the level of tyrosine residue nitration (Fig. 5). Following literature data [11], application of SIN-1 should result in a significant increase in nitration. However, the mentioned analyses were carried out for total proteins. Simultaneously, our microscopic studies indicated that NO modulators significantly changed the level of nitration in the cell wall area of plant cells (Fig. 1G). These results are consistent with literature data showing increased nitration of the plant whole proteome [11]. They do not, however, contradict our proteomic analysis. We examined ionically-bound cell wall proteins — one of the apoplast proteome fraction [37,38], isolated under the procedure that minimizes sample contamination with membrane or cytoplasmic proteins [21]. It is also possible that altered nitration levels observed under microscope pertain e.g. to membrane proteins as well as cortical microtubules, already known for their specific nitrotyrosine enrichment from earlier studies [39,40] Because NO is over-produced by the plant cells during stress response [9], we proceeded with verifying whether osmotic stress would cause an increase in the nitration of cell wall proteins. The presented study analyzes the influence of

E

T

C

414

E

413

R

412

R

411

main source of NO in plant cells apart from nitrate reductase [15,16]. Moreover, it was also shown that NO can be produced in the walls via non-enzymatic pathway [14]. To complement this picture, cell walls are the site of release of superoxide anion by apoplastic peroxidases [31]. Accumulation of these two substrates, necessary for biosynthesis of ONOO−, leads to the generation of main nitrating agent [32–34] and may cause nitration of apoplastic proteins. Indeed, our microscopic results indicate that in cell suspensions cell wall areas are

N C O

410

U

409

D

P

R O

Fig. 6 – Effect of osmotic stress conditions on cell wall nitroproteome of A. thaliana suspension cells. M — molecular weight marker; C — nitrated BSA (Sigma, 5 mg) 1 — control: cell wall protein isolated from suspension incubated in normal conditions; 2 — proteins isolated from suspension incubated in 100 mM NaCl; 3 — cell wall proteins isolated from suspension incubated in 200 mM mannitol. Samples were taken at various time points: X2 were incubated for 2 h, X5 — for 5 h, and X24 — for 24 h. PROTEOME — CBB G-250 stained gel obtained in 12% 1DE SDS PAGE; NITROPROTEOME — results of nitrotyrosine immunodetection. Arrows — nitrated proteins of interest (2–13) identified in MS (see Table 1).

Please cite this article as: Szuba A, et al, Nitration of plant apoplastic proteins from cell suspension cultures, J Prot (2015), http:// dx.doi.org/10.1016/j.jprot.2015.03.002

418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453

8

t1:5

Table 1 – Identification of selected nitrated proteins isolated from cell walls of various suspension-cultured cells (in the order in which they appear in the main text). All proteins were cut out from comparative gels stained with CBB, digested according to a standard “in-gel digestion” protocol and analyzed in the MALDI-TOF spectrometer. Results were compared with the Mass Spectrometry Database or the Swiss-Prot Database using the MASCOT platform. Signal peptide column: S — secreted.

t1:6

No.

t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15

Nitrated cell wall protein isolated from BY2 suspension cells treated with NO modulators (see. Fig. 4) 1 Q9XIV8_TOBAC Peroxidase (Nicotiana tabacum) 135 45% Nitrated cell wall proteins 2 T44928 3 T44928 4 Q8RWM8_ARATH 5 Q8LGC3_ARATH 6 F86163

t1:16

7

Q8LF70_ARATH

t1:17

8

T47545

t1:18 t1:19 t1:20

9 10 11

H96826 Q9C6U3_ARATH Q8LAN4_ARATH

t1:21 t1:22 t1:23 t1:24 t1:25 t1:26 t1:27

12 13

T47537 T50646

t1:28 t1:29 t1:30 t1:31 t1:32 t1:33 t1:34 t1:35

17 18 19

NCBI no.

MASCOT identity

33

S

63.6 63.7 48.0 52.3 46.4

86 84 57 56 50

S S – S S

46.5

46

S

46.6

42

–

42.2 34.9 36.1

40 39 35

– – S

29.3 27.4

31 28

S –

69 59 51

S S S

49 32 28

S S S

86 49 39

– S S

D

P

R O

O

isolated from A. thaliana suspension cells incubated in osmotic stress conditions (see Fig. 6) L-ascorbate oxidase (A.thaliana) 92 20% 9 L-ascorbate oxidase (A.thaliana) 99 28% 14 Enolase (A.thaliana) 145 44% 14 Putative disease resistance protein (A.thaliana) 153 50% 19 Extracellular dermal glycoprotein, putative 76 31% 13 (A.thaliana) Extracellular dermal glycoprotein, putative 69 27% 12 (A.thaliana) Monodehydroascorbate reductase (NADH)-like 83 31% 12 protein (A.thaliana) Phosphoglycerate kinase (A.thaliana) 95 54% 17 Hypothetical protein (A.thaliana) 82 35% 9 Possible apospory-associated like protein 89 41% 9 (A.thaliana) Expansin-like 1 precursor (A.thaliana) 84 40% 10 Triose-phosphate isomerase (A.thaliana) 101 48% 11

36.2

E

from culture medium of A. thaliana suspension cells incubated in osmotic stress conditions (see Fig. 7) Subtilisin-like serine proteinase (A.thaliana) 140 25% 12 80.0 Pectinesterase, putative (A.thaliana) 114 35% 15 60.2 FAD-binding domain-containing protein 116 24% 11 60.7 (A.thaliana) Extracellular dermal glycoprotein (A.thaliana) 110 28% 9 46.4 Probable peroxidase (A.thaliana) 203 58% 15 37.6 Peroxidase (A.thaliana) 198 51% 10 35.4

R

isolated from BY2 suspension cells long-term adapted to osmotic stress conditions (see Fig. 8) Heat shock protein 70 (Nicotiana tabacum) 78 18% 7 Peroxidase (Nicotiana tabacum) 92 25% 8 Peroxidase (Nicotiana tabacum) 65 25% 6

71.2 39.3 39.3

457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474

C

456

osmotic stress on apoplastic proteome and nitroproteome in cell suspension cultures (Figs. 6–8). As already mentioned, due to participation of superoxide anion in the generation of nitrotyrosine [41], this modification is considered a marker of the oxidative stress [5]. Although it is known that not all stress conditions result in an increase in plant proteins nitration [42], it is commonly believed that nitration serves as a fingerprint of RNS [43], production of which rises under stress conditions [9]. Indeed, several published examples of analyses of tyrosine nitration in plants showed an increase in the nitration level under stress [11,42,44] including the herein studied salt stress [9]. However, our results indicate that cell wall sub-proteome does not respond to osmotic stress with increased nitration at least when considering the analyzed suspensions (Figs. 4 and 5). Taking advantage of a unique set of BY2 suspensions, undergoing long-term, over 18-month-long, adaptation to various osmotic stress conditions, we were able to analyze the impact of chronic stress. Analyses of adapted suspension lines are represented by only sparse literature [45]. Nevertheless, our results confirmed that adaptation should result in a change in

N

455

U

454

O

R

Nitrated cell wall proteins 20 Q84QJ3_TOBAC 21 Q50LG5_TOBAC 22 Q50LG5_TOBAC

E

C

F86163 H84560 CAA67551

10

T

Nitrated proteins isolated 14 JC7519 15 Q8L790_ARATH 16 T10628

MASCOT Coverage Peptides Expected Observed Signal score (%) matched MW MW peptide (kDa) (kDa)

F

t1:1 t1:2 t1:3 t1:4 Q1

JOUR NAL OF P ROTEOM ICS XX ( 2015) X XX–XX X

the structure and proteome of the adapted cell walls [45] (Fig. 8). Most interesting, however, was the significant decrease in the level of protein nitration in the walls of cells adapted to osmotic stress. The lowered level of nitration was comparable in all variants of stress. These results were recently supported by research on the acclimation of citrus plants to salinity stress [36], in which roots subjected to long-term salt treatment showed a lower number of nitrotyrosines than the control. This outcome suggests that a decreasing nitration level may by a common reaction observed during acclimatization to osmotic stress. Such decreasing nitrotyrosine level (Fig. 8) raises also a general question about the nature of this posttranslational modification. Nitration was previously considered as a non-reversible protein modification [46]. However, according to the growing number of reports, nitration occurs in plants under physiological conditions [6–8]. This suggests that the effect of nitration of plant proteins is dose-dependent [40,47] and at physiological level tyrosine nitration may play a role in the signaling pathways [48]. In this context nitration of plant proteins should be considered reversible [28]. Such thesis was recently supported by the analysis of plant tubulin cytoskeleton [40,47], in which it

Please cite this article as: Szuba A, et al, Nitration of plant apoplastic proteins from cell suspension cultures, J Prot (2015), http:// dx.doi.org/10.1016/j.jprot.2015.03.002

475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495

9

JOURNAL OF P ROTEOM IC S XX ( 2015) X XX–X XX

95 kDa

M

C NaCl KCl Man Sor

M

C NaCl KCl Man Sor

20

20 72 kDa

56 kDa

43 kDa

22

21

21

22

34 kDa

520

Conflict of interest

567 Q11 566

The authors have no conflicts of interest to declare.

568

Acknowledgments

570 569

The research was supported by grants of the Ministry of Science and Higher Education (PBZ-KBN-110/P04/2004 and 1644/B/P01/2008/35) and the “Mobilitas.pl” Research Network. We would also like to thank Maciej Stobiecki, Magdalena

571

R O

O

F

26 kDa

of large amounts of highly nitrated HSP70 in the walls of BY2 cells long-term adapted to osmotic stress. These molecular ER chaperones were previously described as present in the walls [56]. Our results, supported by former studies of aquatic plants [57], suggest that this protein may serve as a plant biomarker of chronic hyper-osmotic stress and that its nitration may play an important role in stress responses. It shall be noted that nitro-proteomes isolated from suspension cells were highly enriched mostly in peroxidases (Figs. 4, and 6–8). Although links between tyrosine nitration and peroxidases are known from numerous examples, as the enzymes are involved in nitrotyrosine production in the so-called enzymatic pathway [58,59], nitration of plant peroxidases has been reported only once, for the whole proteome of A. thaliana [60], while nitration of extracellular peroxidases has still not been observed. However, it was recorded that peroxidase activity is inhibited by nitric oxide [49]. Application of various NO donors results only in temporary inhibition of peroxidases activity, but SIN-1 causes an irreversible loss of enzymatic activity of peroxidases [49], suggesting, in our opinion, the role of nitration in this process. Consequently, the massive nitration of apoplastic peroxidases, observed here, should be likely to affect extracellular peroxidase activity as nitration of critical tyrosines inhibits the activity of plant enzymes [6,8,61], and alter stress response pathways associated with peroxidases. Nevertheless, the reason why peroxidases are the most nitrated apoplastic proteins in cell suspension cultures remains to be identified. There is evidence for extracellular peroxidases being involved not only in the removal of H2O2 but also in the wound-induced production of ROS [62], including O2U−, in the apoplastic area [31]. In combination with high concentration of NO in apoplast, an environment suitable for increased nitration may be formed in the surrounding of extracellular peroxidases. In plant suspension, peroxidases were heavily nitrated also in control conditions. However, our analysis of endogenous nitration under physiological conditions showed that in maize roots these proteins, although being the best represented ones in apoplastic proteome, did not contain nitrotyrosines [26]. This result may be explained by the fact that during mechanical stress activated by shaking of cultured plant cells [63–65], an increased production of ROS (which induce nitration), does not occur in roots. Considering all the above-mentioned data, extracellular peroxidases appear to be a very interesting object for further studies in the context of nitration processes occurring in the apoplastic space.

17 kDa

499 500 501 502 503 504 505 506 507 Q9 508 509 510 511 512 513 514 515 516 517 518 519

R

498

N C O

497

was found that low concentration of nitrotyrosines caused partly reversible cytoskeleton changes. However, high concentration of nitrating agents caused irreversible, negative protein changes [6,40,42,46,47,49]. Plants under chronic stress cannot change their location and have to cope with extremely negative environmental conditions. To avoid the harmful effects of long-term ROS/RNS actions and to maintain the activity of the enzymatic pathways it is considered that those plants developed some unknown, specific protecting/adaptive systems (e.g. “repair” of proteins via denitrification [50–53]); the consequence of adaptation is decrease of nitration level observed under chronic stress conditions [36]. However, such speculative hypothesis requires further detailed analysis. This may be the topic of particular interest because of the unusual nature of observed changes (e.g. in the animal systems chronic stress leads usually to an increase in TyrNO2 levels [54]). However, plants and animals differ significantly in many aspects, and e.g. the role of peroxynitrite is among those differences [55]. Therefore, such differences in the regulation of nitration level are possible. Analysis of proteins nitrated under various osmotic stress conditions resulted in the identification of several proteins, mainly redox state-regulating enzymes and stress-related enzymes (Table 1). Particularly interesting seems to be the presence

D

522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565

U

496

R

E

C

T

Fig. 8 – Analysis of nitroproteome of cell wall proteins isolated from BY2 suspension cells adapted to different conditions of osmotic stress. M — molecular weight marker; C — cell wall proteins isolated from control samples; NaCl — proteins isolated from suspension adapted to 190 mM NaCl; KCl — proteins isolated from suspension adapted to 180 mM KCl; Man — proteins isolated from suspension adapted to 450 mM mannitol; Sor — protein isolated from suspension adapted to 450 mM sorbitol. PROTEOME — CBB G-250 stained gel obtained with 12% 1DE SDS PAGE; NITROPROTEOME — results of nitrotyrosine immunodetection. Arrows — nitrated proteins of interest (20–22) identified in MS (see Table 1).

P

NITROPROTEOME

E

PROTEOME

521

Please cite this article as: Szuba A, et al, Nitration of plant apoplastic proteins from cell suspension cultures, J Prot (2015), http:// dx.doi.org/10.1016/j.jprot.2015.03.002

572 573 Q10 574

10

57 7

REFERENCES

578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639

[1] Schöneich Ch, Sharov VS. Mass spectrometry of protein modifications by reactive oxygen and nitrogen species. Free Radic Biol Med 2006;41:1507–20. [2] Tuteja N, Chandra M, Tuteja R, Misra MK. Nitric oxide as a unique bioactive signaling messenger in physiology and pathophysiology. J Biomed Biotechnol 2004;4:227–37. [3] Halliwell B, Gutteridge JM. Free radicals in biology and medicine. 3rd ed. Oxford: Oxford University Press; 1998. [4] Pietraforte D, Salzano AM, Marino G, Minetti M. Peroxynitrite-dependent modifications of tyrosine residues in hemoglobin. Formation of tyrosyl radical(s) and 3-nitrotyrosine. Amino Acids 2003;25:341–50. [5] Turko IV, Murad F. Protein nitration in cardiovascular diseases. Pharmacol Rev 2002;54:619–34. [6] Chaki M, Valderrama R, Fernández-Ocaña AM, Carreras A, López-Jaramillo J, Luque F, et al. Protein targets of tyrosine nitration in sunflower (Helianthus annuus L.) hypocotyls. J Exp Bot 2009;60:4221–34. [7] Galetskiy D, Lohscheider J, Kononikhin A, Popov I, Nikolaev E, Adamska I. Mass spectrometric characterization of photooxidative protein modifications in Arabidopsis thaliana thylakoid membranes. Rapid Commun Mass Spectrom 2011; 25:184–90. [8] Begara-Morales JC, Chaki M, Sánchez-Calvo B, Mata-Pérez C, Leterrier M, Palma JM, et al. Protein tyrosine nitration in pea roots during development and senescence. J Exp Bot 2013;64: 1121–34. [9] Valderrama R, Corpas FJ, Carreras A, Fernandez-Ocana A, Chaki M, Luque L, et al. Nitrosative stress in plants. FEBS Lett 2007;581:453–61. [10] Corpas FJ, Chaki M, Fernández-Ocaña A, Valderrama R, Palma JM, Carreras A, et al. Metabolism of reactive nitrogen species in pea plants under abiotic stress conditions. Plant Cell Physiol 2008;49:1711–22. [11] Saito S, Yamamoto-Katou A, Yoshioka H, Doke N, Kawakita K. Peroxynitrite generation and tyrosine nitration in defense responses in tobacco BY-2 cells. Plant Cell Physiol 2006;47:689–97. [12] Szuba A, Wojtaszek P. Structural modifications of proteins induced by nitric oxide. Postepy Biochem 2010;56:107–14. [13] Chaki M, Carreras A, López-Jaramillo J, Begara-Morales JC, Sánchez-Calvo B, Valderrama RJ, et al. Tyrosine nitration provokes inhibition of sunflower carbonic anhydrase (b-CA) activity under high temperature stress. Nitric Oxide 2013;29:30–3. [14] Bethke PC, Badger MR, Jones RL. Apoplastic synthesis of nitric oxide by plant tissues. Plant Cell 2004;16:332–41. [15] Wendehenne D, Pugin A, Klessig D, Durner J. Nitric oxide: comparative synthesis and signaling in animal and plant cells. Trends Plant Sci 2001;6:177–83. [16] Wojtaszek P. Nitric oxide in plants. To NO or not to NO. Phytochemistry 2000;54:1–4. [17] Rodakowska E, Derba-Maceluch M, Kasprowicz A, Zawadzki P, Szuba A, Kierzkowski D, et al. Signaling and cell walls. In: Baluska F, Vivanco JM, editors. Signaling and communication in plants. Springer; 2009. p. 173–93. [18] Wojtaszek P. Organismal view of a plant and a plant cell. Acta Biochim Pol 2001;48:43–451. [19] Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 1962;15: 473–97. [20] Kasprowicz A. The role of cell wall-plasma membrane-cytoskeleton continuum in the maintenance of

[22]

[23]

[24]

[25]

[26]

E

D

[27]

[28]

T

C

E

R

R

O

C

N

U

F

[21]

structural and functional integrity of plant cells adapted to increased osmotic conditions. (PhD Thesis) Poznan, Poland: Adam Mickiewicz University; 2009. Wojtaszek P, Trethowan J, Bolwell GP. Specificity in the immobilization of cell wall proteins in response to different elicitor molecules in suspension-cultured cells of French bean (Phaseolus vulgaris L.). Plant Mol Biol 1995;28:1075–87. Hurkman WJ, Tanaka CK. Solubilization of plant membrane proteins for analysis by two- dimensional gel electrophoresis. Plant Physiol 1996;81:802–6. Laemmli UK. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 1970; 227:680–5. Friml J, Benkova E, Mayer U, Palme K, Muster G. Automated whole mount localization techniques for plant seedlings. Plant J 2003;3:115–24. Baluška F, Parker JS, Barlow PW. Specific patterns of cortical and endoplasmic microtubules associated with cell growth. Rearrangements of F-actin arrays in growing cells of intact maize root apex tissues, a major developmental switch occurs in the postmitotic transition region. Eur J Cell Biol 1992;72:113–21. Szuba A. Analysis of modifications of proteins within plant cell wall–plasma membrane–cytoskeleton continuum evoked by nitric oxide. (PhD Thesis) Poznan, Poland: Institute of Bioorganic Chemistry, Polish Academy of Sciences; 2010. Kasprowicz A, Szuba A, Volkmann D, Baluska F, Wojtaszek P. Nitric oxide modulates dynamic actin cytoskeleton and vesicle trafficking in a cell type-specific manner in root apices. J Exp Bot 2009;60:1605–17. Ischiropoulos H. Biological selectivity and functional aspects of protein tyrosine nitration. Biochem Biophys Res Commun 2003;305:776–83. Corpas FJ, Barroso JB, Carreas A, Quriós M, León AM, Romero-Puertas MC, et al. Cellular and subcellular localization of endogenous nitric oxide in young and senescent pea plants. Plant Physiol 2004;136:2722–33. Floryszak-Wieczorek J, Milczarek G, Arasimowicz M, Ciszewski A. Do nitric oxide donors mimic endogenous NO-related response in plants? Planta 2006;224:1363–72. Minibayeva F, Kolesnikov O, Chasov A, Beckett RP, Lüthje S, Vylegzhanina N, et al. Wound-induced apoplastic peroxidase activities: their roles in the production and detoxification of reactive oxygen species. Plant Cell Environ 2009;32:497–508. del Río LA, Corpas FJ, Sandalio LM, Palma JM, Barroso JB. Plant peroxisomes, reactive oxygen metabolism and nitric oxide. IUBMB Life 2003;55:71–81. Heijnen H, van Donselaar E, Slot J, Fries D, Blachard-Fillion B, Hodara R, et al. Subcellular localization of tyrosine-nitrated proteins is dictated by reactive oxygen species generating enzymes and by proximity to nitric oxide synthase. Free Radic Biol Med 2006;40:1903–13. Meany D, Poe B, Navratil M, Moraes C, Arriaga E. Superoxide released into the mitochondrial matrix. Free Radic Biol Med 2006;41:950–9. Bernstein N, Shoresh M, Xu Y, Huang B. Involvement of the plant antioxidative response in the differential growth sensitivity to salinity of leaves vs roots during cell development. Free Radic Biol Med 2010;49:1161–71. Tanou G, Filippou P, Belghazi M, Job D, Diamantidis G, Fotopoulos V, et al. Oxidative and nitrosative-based signaling and associated post-translational modifications orchestrate the acclimation of citrus plants to salinity stress. Plant J 2012;72:585–99. Jamet E, Canut H, Boudart G, Pont-Lezica RF. Cell wall proteins: a new insight through proteomics. Trends Plant Sci 2006;11:33–9. Jamet E, Albenne C, Boudart G, Irshad M, Canut H, Pont-Lezica R. Recent advances in plant cell wall proteomics. Proteomics 2008;8:893–908.

O

Łuczak and Łukasz Marczak from the Institute of Bioorganic Chemistry, PAS for help and fruitful discussions.

R O

576

P

575

JOUR NAL OF P ROTEOM ICS XX ( 2015) X XX–XX X

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

Please cite this article as: Szuba A, et al, Nitration of plant apoplastic proteins from cell suspension cultures, J Prot (2015), http:// dx.doi.org/10.1016/j.jprot.2015.03.002

640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708

11

JOURNAL OF P ROTEOM IC S XX ( 2015) X XX–X XX

N C O

R

R

E

C

D

P

R O

O

F

[52] Kuo WN, Kocis JM, Mewar M. Protein denitration/ modification by glutathione-S-transferase and glutathione peroxidase. J Biochem Mol Biol Biophys 2002;6:143–6. [53] Kuo WN, Kocis JM, Webb JK. Protein denitration/modification by Escherichia coli nitrate reductase and mammalian cytochrome P-450 reductase. Front Biosci 2002;7:9–14. [54] Leung TM, Lu Y, Yan W, Morón-Concepción JA, Ward SC, Ge X, et al. Argininosuccinate synthase conditions the response to acute and chronic ethanol-induced liver injury in mice. Hepatology 2012;55:1596–609. [55] Arasimowicz-Jelonek M, Floryszak-Wieczorek J. Understanding the fate of peroxynitrite in plant cells — from physiology to pathophysiology. Phytochemistry 2011;72:681–8. [56] Łuczak M, Bugajewska A, Wojtaszek P. Inhibitors of protein glycosylation or secretion change the pattern of extracellular proteins in suspension-cultured cells of Arabidopsis thaliana. Plant Physiol Biochem 2008;46:962–9. [57] Elyse Ireland H, Harding SJ, Bonwick GA, Jones M, Smith CJ, Williams JH. Evaluation of heat shock protein 70 as a biomarker of environmental stress in Fucus serratus and Lemna minor. Biomarkers 2004;9:139–55. [58] Bian K, Ke Y, Kamisaki Y, Murad F. Proteomic modification by nitric oxide. J Pharmacol Sci 2006;101:271–9. [59] Duguet A, Iijima H, Eum SY, Hamid Q, Eidelman DH. Eosinophil peroxidase mediates protein nitration in allergic airway inflammation in mice. Am J Respir Crit Care Med 2001; 164:1119–26. [60] Lozano-Juste J, Colom-Moreno R, León J. In vivo protein tyrosine nitration in Arabidopsis thaliana. J Exp Bot 2011;62: 3501–17. [61] Chaki M, Valderrama R, Fernández-Ocaña AM, Carreras A, Gómez-Rodríguez MV, Pedrajas JR, et al. Mechanical wounding induces a nitrosative stress by down-regulation of GSNO reductase and an increase in S-nitrosothiols in sunflower (Helianthus annuus) seedlings. J Exp Bot 2011;62: 1803–13. [62] Huang X, Stettmaier K, Hutzler Ch, Mueller M, Durner J. Nitric oxide is induced by wounding and influences jasmonic acid signaling in Arabidopsis thaliana. Planta 2004;218:938–46. [63] Cazalé AC, Rouet-Mayer MA, Barbier-Brygoo H, Mathieu Y, Laurière C. Oxidative burst and hypoosmotic stress in tobacco cell suspensions. Plant Physiol 1998;16:659–69. [64] Legendre L, Rueter S, Heistein PF, Low PS. Characterization of the oligogalacturonide-induced oxidative burst in cultured soybean (Glycine max) cell. Plant Physiol 1993;102:233–40. [65] Yahraus T, Chandra Ch, Legendre L, Low P. Evidence for a mechanically induced oxidative burst. Plant Physiol 1995;109: 1259–66.

E

T

[39] Wilhelmova N, Fuksova H, Srbova M, Mikova D, Mytinova Z, Prochazkova D, et al. The effect of plant cytokinin hormones on the production of ethylene, nitric oxide, and protein nitrotyrosine in ageing tobacco leaves. Biofactors 2006;27: 203–11. [40] Blume YB, Krasylenko YA, Demchuk OM, Yemets AI. Tubulin tyrosine nitration regulates microtubule organization in plant cells. Front Plant Sci 2013;4:530. [41] Larrainzar E, Urarte E, Auzmendi I, Ariz I, Arrese-Igor C, González EM, et al. Use of recombinant iron-superoxide dismutase as a marker of nitrative stress. Methods Enzymol 2008;437:605–18. [42] Chaki M, Valderrama R, Fernández-Ocaña AM, Carreras A, Gómez-Rodríguez MV, López-Jaramillo J, et al. High temperature triggers the metabolism of S-nitrosothiols in sunflower mediating a process of nitrosative stress which provokes the inhibition of ferredoxin-NADP reductase by tyrosine nitration. Plant Cell Environ 2011;34:1803–18. [43] Kharitonov SA, Barnes PJ. Nitric oxide, nitrotyrosine, and nitric oxide modulators in asthma and chronic obstructive pulmonary disease. Curr Allergy Asthma Rep 2003;3:121–9. [44] Morot-Gaudry-Talarmain Y, Rocke P, Moureaux T, Quillere I, Leydecker T, Kaiser WM, et al. Nitrite accumulation and nitric oxide emission in relation to cellular signaling in nitrite reductase antisense tobacco. Planta 2002;215:708–15. [45] Iraki NM, Bressan RA, Carpita NC. Extracellular polysaccharides and proteins of tobacco cell cultures and changes in composition associated with growth-limiting adaptation to water and saline stress. Plant Physiol 1989;91: 54–61. [46] Ischiropoulos H. Biological tyrosine nitration: a pathophysiological function of nitric oxide and reactive oxygen species. Arch Biochem Biophys 1998;365:1–11. [47] Lipka E, Müller S. Nitrosative stress triggers microtubule reorganization in Arabidopsis thaliana. J Exp Bot 2014;65:4177–89. [48] Monteiro HP. Signal transduction by protein tyrosine nitration: competition or cooperation with tyrosine phosphorylation-dependent signaling events? Free Radic Biol Med 2002;33:765–73. [49] Clark D, Durner J, Navarre DA, Klessig DF. Nitric oxide inhibition of tobacco catalase and ascorbate peroxidase. Mol Plant Microbe Interact 2000;13:1380–4. [50] Kamisaki Y, Wada K, Bian K, Balabanli B, Davis K, Martin E, et al. An activity in rat tissues that modifies nitrotyrosine-containing proteins. Biochemistry 1998;95:11584–9. [51] Kuo WN, Kocis JM. Nitration/S-nitrosation of proteins by peroxynitrite-treatment and subsequent modification by glutathione S-transferase and glutathione peroxidase. Mol Cell Biochem 2002;233:57–63.

U

709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 806

Please cite this article as: Szuba A, et al, Nitration of plant apoplastic proteins from cell suspension cultures, J Prot (2015), http:// dx.doi.org/10.1016/j.jprot.2015.03.002

758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805