Proteomic Signatures Uncover Hydrogen Peroxide and Nitric Oxide

Proteomic Signatures Uncover Hydrogen Peroxide and Nitric Oxide Cross-Talk Signaling Network in Citrus Plants Georgia Tanou,*,† Claudette Job,‡ Maya Belghazi,§ Athanassios Molassiotis,† Grigorios Diamantidis,† and Dominique Job‡ Aristotle University of Thessaloniki, School of Agriculture, University Campus, 54124 Thessaloniki, Greece, CNRS-Universite´ Claude Bernard Lyon-Institut National des Sciences Appliqueés-Bayer CropScience Joint Laboratory (UMR 5240), Bayer CropScience, F-69263 Lyon Cedex 9, France, and Centre d’Analyse Proteómique de Marseille, Institut Fe´de´ratif de Recherche Jean Roche, F-13916 Marseille Cedex 20, France Received July 28, 2010

Hydrogen peroxide (H2O2) and nitric oxide (•NO) elicit numerous processes in plants. However, our knowledge of H2O2 and •NO-responsive proteins is limited. The present study aimed to identify proteins whose accumulation levels were regulated by these signaling molecules in citrus leaves. To address this question, hydroponically grown citrus plants were treated by incubating their roots in the presence of H2O2 or the •NO donor, sodium nitroprusside (SNP). Both treatments induced H2O2 and •NO production in leaves, indicating occurrence of oxidative and nitrosative stress conditions. However, treated plants maintained their normal physiological status. The vascular system was shown to be involved in the H2O2 and •NO systemic signaling as evidenced by real-time labeling of the two molecules. Comparative proteomic analysis identified a number of proteins whose accumulation levels were altered by treatments. They were mainly involved in photosynthesis, defense and energy. More than half of them were commonly modulated by both treatments, indicating a strong overlap between H2O2 and •NO responses. Using a redox proteomic approach, several proteins were also identified as being carbonylation targets of H2O2 and SNP. The analysis reveals an interlinked H2O2 and •NO proteins network allowing a deeper understanding of oxidative and nitrosative signaling in plants. Keywords: carbonylation • citrus • hydrogen peroxide • nitric oxide • nitrosative stress • oxidative stress • proteomics • systemic signaling

Introduction Hydrogen peroxide (H2O2) and nitric oxide (•NO) have emerged as ubiquitous components of signal transduction in plants.1-7 Besides signaling regulation, both molecules can control physiological processes directly by modulating gene transcription.8 Genes regulated by H2O2,9-11 •NO,5,12 or both13 have been identified in plants. On the other hand, protein targets of H2O2 have been evidenced in Arabidopsis cell cultures14,15 and in rice leaves.16 Since •NO modulates protein homeostasis through S-nitrosylation and tyrosine nitration,17 recent proteomics studies have focused on the characterization of nitrosylated and nitrated proteins.6,18-22 However, the potential proteins targeted by the two molecules are yet unknown. We recently reported that root applied H2O2 or sodium nitroprusside (SNP) can induce tolerance against salinity in citrus.6 Also by conducting a proteome-wide analysis, we * To whom correspondence should be addressed. Georgia Tanou, Laboratory of Agricultural Chemistry, School of Agriculture, Aristotle University of Thessaloniki, University Campus, 54124 Thessaloniki, Greece. Tel/Fax: +30 2310 998882. E-mail: [email protected]. † Aristotle University of Thessaloniki. ‡ CNRS-Universite´ Claude Bernard Lyon-Institut National des Sciences Appliqueés-Bayer CropScience Joint Laboratory. § Institut Fe´de´ratif de Recherche Jean Roche.

5994 Journal of Proteome Research 2010, 9, 5994–6006 Published on Web 09/09/2010

observed that H2O2 and SNP-mediated tolerance responses are accompanied by interplay between the two molecules under salinity.6 This prompted us to use this experimental system to further unravel the molecular mechanisms underpinning H2O2 and •NO signaling pathways. The data uncovered the existence of a strong protein cross-talk network between the two molecules, which could serve as potent markers to monitor oxidative and nitrosative signaling in citrus.

Materials and Methods Plant Material and Chemical Treatments. According to the methodology previously described,6 5-month-old sour orange (Citrus aurantium L.) plants were hydroponically grown in Hoagland’s nutrient solution. Plants were treated with H2O2 or •NO by incubating their roots in the presence of 10 mM H2O2 (for 8 h) or 100 µM SNP (for 48 h, renewed every 12 h). Plants treated with distilled water served as control. At least 10 plants were used for each treatment. Following chemical treatments, plants were transferred to Hoagland’s nutrient solution for 16 d and fully expanded leaves were sampled. Growth and Physiological Measurements. Dry weight of leaves was obtained after drying at 60 °C for 72 h. Cellular membrane damage was assayed by measuring ion leakage as previously reported.23 Total chlorophyll content was deter10.1021/pr100782h

 2010 American Chemical Society

research articles

H2O2 and •NO Cross-Talk Signaling Network in Citrus Plants mined in ethanol extracts of leaf discs (1 cm in diameter) as described.24 The maximum photochemical efficiency of photosystem II (PSII) (Fv/Fm) was recorded using a pulsemodulated fluorimeter (FMS-2, Hansatech Instruments, Norfolk, U.K.). Determination of H2O2 and •NO Production. Content of H2O2 was measured using the fluorescent probe homovanillic acid (4-hydroxy-3-methoxy-phenylacetic acid; HVA) in a Shimadzu RF-500 spectrofluorimeter (Shimadzu Corporation, Japan) (excitation, 315 nm; emission, 425 nm).25 Nitric oxide production was determined based on Griess reaction.26 Imaging of Intracellular H2O2 and •NO Production. Realtime H2O2 and •NO imaging in cross sections of primary veins of petiole and in stems (approximately 0.5 cm from the top) was monitored using the fluorescent probes 2′,7′-dichlorofluorescin diacetate (DCF-DA; Molecular Probes, Eugene, OR) and 4,5-diaminofluorescin diacetate (DAF-2DA; Sigma-Aldrich, St. Louis, MO), respectively.27 Examination of DCF-DA and DAF2DA fluorescence was performed using laser scanning confocal microscopy (CLSM; Nikon D-Eclipse C1, Nikon Instruments, Melville, NY). All images shown represent typical results from observation of at least five biological replicates. Protein Extraction. Leaf samples were ground with mortar and pestle in liquid nitrogen and thawed in thiourea/urea buffer with the protease inhibitor cocktail ‘Complete Mini’ (Roche Diagnostics, Mannheim, Germany), 60 U/mL DNase, and 6 U/mL RNase as described.6 Following centrifugation (20 000g for 15 min at 4 °C) the supernatant, corresponding to the soluble proteins, was collected. Protein concentration was determined with a Bio-Rad assay kit following Bradford’s method.28 2-DE and Data Analysis. In 2-DE, PAGE gels were run as described previously.29 Protein (100 µg) was analyzed by isoelectric focusing on gel strips with an immobilized pH gradient from 3 to 10 (Immobiline DryStrip pH 3-10 NL, 24 cm, Amersham Biosciences, GE Healthcare, Munich, Germany). The second dimension was carried out as described30 using 10% polyacrylamide gels. Three gels were run in parallel for each treatment and stained with silver nitrate. Gels were scanned with a GS800 imaging densitometer (Bio-Rad) and analyzed with PDQuest Advanced 2-D Gel Analysis software (version 8.1, Bio-Rad) as described.6 Spots were detected, background subtracted, and matched, and quantitative determination of the spot volumes was performed (mode: total quantity of valid spots normalization). Statistics were performed by one-way analysis of variance significance (P < 0.05) and individual means were compared using Student’s t test (significance level 95%). In-Gel Digestion, Mass Spectrometry, and Database Searching. Spots were excised from 2-DE gels and digested with trypsin as described.30 Tryptic peptides were sequenced by nano-LC-MS/MS (QTOF-Ultima Global equipped with a nano-ESI source coupled with a Cap LC nanoHPLC, Waters Micromass) in the Data Dependent Acquisition mode allowing the selection of three precursor ions per survey scan. Only doubly and triply charged ions were selected for fragmentation over a mass range of m/z 400-1300. A spray voltage of 3.2 kV was applied. The peptides were loaded on a C18 column (AtlantisTM dC18, 3 µm, 75 µm × 150 mm Nano Ease, Waters) and eluted with a 5-60% linear gradient of water/acetonitrile 98/2 (v/v) containing 0.1% formic acid (buffer A) and water/acetonitrile 20/80 (v/v) containing 0.1% formic acid (buffer B) over 30 min at a flow rate of 200

-1

nL min . MSMS raw data were processed (smooth 3/2 Savitzky Golay and no deisotoping) using the ProteinLynx Global Server 2.05 software (Waters) and peak lists were exported in the micromass pkl format. Peak lists of precursor and fragment ions were matched automatically to proteins in the NCBI nonredundant database (version 2008.05.10 (6 512 701 sequences; 2 221 612 072 residues; taxonomy: Viridiplantae, 481 095 sequences)) and the GenBank viridiplantae (EST Viridiplantae version 2008.03.14 (91 083 810 sequences; 15 967 802 572 residues, taxonomy: Viridiplantae, 91 083 810 sequences)), using a local Mascot version 2.2 program (Matrix Science, London, http://www.matrixscience.com) with the following parameters: trypsin specificity, one missed cleavage, carbamidomethyl cysteine and oxidation of methionine, 0.2 Da mass tolerance on both precursor and fragment ions, and automatic error tolerant search. To validate protein identification, only matches with individual ion scores above 41 or 59 (protein or EST databanks, respectively), a threshold value calculated by the Mascot algorithm with our search parameters, were considered. All identified proteins have a MASCOT score greater than the significance level corresponding to P < 0.05. Moreover, among the positive matches, only protein identifications based on at least two different peptide sequences of more than 6 amino acids with an individual score above 20 were accepted. These additional validation criteria are a good compromise to limit the number of false positive matches without missing real proteins of interest. All peptide sequences from nano-LC-MS/ MS, accession number, database source, Mascot scores, and sequence coverage are provided in Supplementary Table S1. In some cases, protein sequences of matching plant ESTs were searched against the NCBI nonredundant protein database. Only the best BLASTP matches with E values < 10-30 (see Supplementary Table S1) were selected.31 Detection of Carbonylated Proteins by Two-Dimensional Immunoblotting. Proteins were separated by 2DE-PAGE as above and transferred to nitrocellulose sheets using standard procedures.31 Detection of carbonylated proteins was performed by derivatization of protein extracts with 2,4-dinitrophenylhydrazone (DNPH) and immunological detection of the DNP adducts with monoclonal anti-DNP antibody (OxyBlot Oxidized Protein Detection Kit; Chemicon, http://www. millipore.com) as described.32

Results and Discussion Rationale of the Experiments. In previous work, we set up an experimental protocol in which roots of citrus were treated with either H2O2 or SNP before applying a salt stress.6 This study revealed that a time point of 16 days after chemical treatments allowed to evidence at the leaf level (i) a priming effect against salinity stress and (ii) specific changes in accumulation of several proteins. The above data clearly demonstrate that exogenously applied H2O2 and SNP have a longterm profound impact on citrus’s proteome contributing to defense signaling under salt stress conditions and raise the question of whether the H2O2 and •NO-associated proteomic signatures vary between stress and unstress conditions. For these reasons, in the present work, we used exactly the same experimental model to analyze the direct impact of chemical treatments on H2O2 and •NO homeostasis, the physiology of plants and the leaf proteome responses. Such an experimental approach will help us not only to characterize H2O2 and •NOresponsive proteins, but also to understand how H2O2 and •NOJournal of Proteome Research • Vol. 9, No. 11, 2010 5995

research articles

Tanou et al.

Figure 1. Growth and physiological response of citrus plants upon treatments with H2O2 or •NO (applied as SNP). Roots of plants were treated with distilled water (control), 10 mM H2O2 (for 8 h), or SNP (100 µM for 48 h). After 16 days of treatments, dry weight (A), electrolyte leakage (B), chlorophyll content (C), and maximal photochemical efficiency of photosystem II (Fv/Fm) (D) were determined. Inset discs represent the sample leaf tissues used for the chlorophyll analysis. Means denoted by the same letter did not significantly differ at P < 0.05 (Duncan’s multiple range test). Data are means of 10 (A), 8 (B), or 5 (C and D) replications.

derived proteomic hallmarks are integrated to eventually regulate biological processes as priming phenomena. Physiological Characterization of Treated Citrus Plants. Plant growth, as expressed in terms of leaf dry weight, remained unchanged in treated plants (Figure 1A). Also, chemical treatments neither enhanced electrolyte leakage (Figure 1B) nor reduced chlorophyll content (Figure 1C) or the maximum photochemical efficiency of PSII (Fv/Fm) (Figure 1D). These data testify that treated citrus plants maintained their normal physiological status during treatments. H2O2 and •NO Production in Leaves after Root Chemical Treatments. To investigate the systemic impact of root treatments, the steady-state levels of H2O2 and •NO were quantified in citrus leaves. Compared with untreated control plants, an accumulation of both H2O2 and •NO was induced upon treatment with either H2O2 or SNP (Figure 2A,B). Time course analyses also revealed a sustained accumulation of these molecules in the leaves during the 16-day experimental period (data not shown). The above data suggest that citrus leaves faced oxidative and nitrosative microenvironment following H2O2 or SNP treatment. To get an enhanced resolution of the observed H2O2 and •NO systemic induction (Figure 2A,B), topological distribution of H2O2-depended DCF-DA and •NO-depended DAF-2DA signals was analyzed by CLSM. The specificity of DCF-DA and DAF2DA for H2O2 and •NO, respectively, was checked by pretreating samples with appropriate scavengers before fluorescent probe27 (data now shown). Both treatments caused a local induction of H2O2 and •NO in xylem and phloem of petiole (Figure 2C) and in stems (Figure 2D). This characteristic staining of citrus vessels is consistent with the reported production of both 5996

Journal of Proteome Research • Vol. 9, No. 11, 2010

H2O23,27 and •NO27,33-35 in vascular tissues during plant development or in defense reactions, further indicating that their spatial regulation is highly coordinated. Protein Abundance Changes in Response to H2O2 and SNP. Considering the substantial H2O2 and •NO accumulation seen in treated plants (Figure 2A,B), the absence of growth reduction and physiological damage in treated plants (Figure 1) suggested the possibility that citrus cells are able to develop specific adaptive response against both oxidative and nitrosative conditions. This question was addressed by proteomics. Significant differences in protein spot volumes were evidenced by 2DE-gel analysis between untreated control and H2O2/SNP-treated samples (Figure 3A,B). Varying spots were subjected to tandem mass spectrometry (MS/MS) analysis. The identified H2O2- and SNP-responsive protein spots are presented in Figure S1 (see Supporting Information) and listed in Table 1. This analysis revealed that the abundance of 46 proteins was changed in response to H2O2 while the abundance of 40 proteins was changed in response to SNP (Table 1; Figure 3A,B). It is interesting to note that a large portion of the H2O2- and SNP-regulated proteins detected under stress-free conditions in the current study (Table 1) was not previously characterized as protein targets for H2O2 and •NO in citrus plants submitted to salt stress,6 thus, indicating that H2O2 and •NO-associated proteomic signatures can be altered depending on specific environmental challenges. Identified proteins were then classified according to the functional categories proposed by Bevan et al.36 The Venn diagrams in Figure 4 summarize the functional classification and the number of proteins whose accumulation increased or decreased in response to chemical

H2O2 and •NO Cross-Talk Signaling Network in Citrus Plants

research articles

Figure 2. H2O2 (A) and •NO (B) leaf steady-state levels upon treatments with H2O2 or SNP. Treatments were conducted as described in Figure 1 and under Materials and Methods. Values are means ( SE (n ) 3). Means denoted by the same letter did not significantly differ at P < 0.05 (Duncan’s multiple range test). Representative CLMS analysis of intracellular H2O2 and •NO production (shown as DCF-DA and DAF-2DA fluorescence, respectively) in cross sections of primary veins of petiole (C) and stems (D) after treatment with H2O2 or SNP. Ph, phloem; X, xylem. Bar, 60 µm.

Figure 3. Differentially expressed leaf proteins upon treatments with H2O2 or SNP. Treatments were conducted as described in Figure 1 and under Materials and Methods. An equal amount (100 µg) of total protein extracts was loaded on each gel strip. (A) Representative 2DE silver-stained gel from control plants. (B) High magnification views of the same regions (1-4) of the 2DE-gels as shown in panel A for control plants and plants treated with H2O2 or SNP. Protein spot quantification was carried out as described in Materials and Methods, from at least three gels for each leaf sample. Proteins spots were labeled with the same numbers as in Table 1. Yellow and blue arrows indicate protein spots that were up-regulated or down-regulated, respectively, in leaves of treated plants compared with control plants.

treatments. Of these proteins, 21 had no MS/MS data or did not fit with the database and their identities need to be further confirmed. This analysis disclosed that the H2O2/SNP-responsive proteins were mainly associated with photosynthesis, energy

metabolism, and defense followed by secondary metabolism, amino acid metabolism, and transporters (Table 1; Figure 4). The H2O2/SNP-responsive proteins could further be grouped as follows: (i) a group of 16 H2O2-modulated proteins whose abundance was changed only by H2O2 Journal of Proteome Research • Vol. 9, No. 11, 2010 5997

Y

N N Y

N

Y

N N

N

N N

N

N

Y N N N N Y Y

Y

Y N

Y

Y

N

N

N N

Y

Y

N

101

102 201 202

203

205

301 302

303

305 402

406

601

611 802 815 816 817 1112 1201

1203

1207 1301

1302

1402

1607

1609

1613 1635

2104

2203

2206

C

D

D

C C

C

C

D

D

D C

D

U C C C C D D

C

C

C C

C

C C

D

C

C C D

D

Y

N

Y

N N

N

N

Y

N

Y N

Y

N N N N N N N

N

N

N N

N

N N

Y

N

N N N

Y

U

C

D

C C

C

C

D

C

D C

D

C C C C C C C

C

C

C C

C

C C

D

C

C C C

D

organism

Citrus sinensis

Citrus sinensis

Canavalia lineata

Citrus sinensis

Citrus sinensis

Citrus sinensis

Citrus sinensis

Brassica napus

Citrus limon

Pisum sativum

Citrus sinensis

Citrus sinensis

gi|63106891

gi|2213425

gi|57934502

gi|20559

Citrus x paradisi

Citrus x paradisi

Citrus sinensis

Petunia x hybrida

gi|114329663 Citrus sinensis

gi|2506277

gi|45448898

gi|56536510

Light-harvesting chlorophyll a/b-binding protein Chloroplast

Chloroplast

cellular localizationi

Ethylene-induced esterase

Glyoxalase I

Miraculin 2

HSP 70

Chaperonin 60 beta subunit ATP synthase F1 (subunit β)

Nucleoid DNA binding protein (aspartyl protease) ATP synthase F1 (subunit β) Rubisco activase

20S proteasome alpha subunit Pyruvate dehydrogenase (E1 beta subunit isoform 2)

Chaperonin 60 alpha subunit

Rubisco activase

Rubisco activase

Sedoheptulose-1,7bisphosphatase Rubisco activase

40-Non identified protein 06.13-Protein destination and storage/Proteolysis

40-Non identified protein 02.30-Energy/ Photosynthesis 02.30-Energy/ Photosynthesis 40-Non identified protein 02.30-Energy/ Photosynthesis 02.30-Energy/ Photosynthesis 06.01-Protein destination and storage/Folding and stability 40-Non identified protein 40-Non identified protein 40-Non identified protein 40-Non identified protein 40-Non identified protein 40-Non identified protein 06.13-Protein destination and storage/Proteolysis 02.01-Energy/Glycolysis

02.30-Energy/ Photosynthesis

40-Non identified protein 40-Non identified protein 06.01-Protein destination and storage/Folding and stability 02.30-Energy/ Photosynthesis

02.30-Energy/ Photosynthesis

functional categoryj

07.22-Transporters/ Transport ATPases Chloroplast 02.30-Energy/ Photosynthesis Chloroplast 02.30-Energy/ Photosynthesis Chloroplast 07.22-Transporters/ Transport ATPases 40-Non identified protein Cytosol 06.01-Protein destination and storage/Folding and stability Cytosol 11.05- Disease/Defense/ Stress responses Cytosol/ 11.06-Disease/Defense/ Mitochondria Detoxification Unclear 11.05- Disease/Defense/ Stress responses

Chloroplast

Chloroplast

Mitochondria

Cytosol

Chloroplast

Chloroplast

Chloroplast

Chloroplast

Chloroplast

Light-harvesting Chloroplast chlorophyll a/b-binding protein OEEp PsbO Chloroplast Oxygen-evolving protein 33 kDa of photosystem II

Citrus clementina HSP (ClpP) x Citrus tangerina

Hordeum vulgare

gi|110843127 Citrus clementina

gi|55931859

gi|28618410

gi|3790441

gi|57932636

gi|21652284

gi|57932636

gi|21652139

gi|5052366

gi|28630973

gi|63068047

gi|30081469

protein nameh

Journal of Proteome Research • Vol. 9, No. 11, 2010 g

spot H2O2 response SNP response no.a responsiveb to H2O2c responsived to SNPe

5998 acces. numberf

Table 1. List of the Soluble Proteins from Citrus Leaves Exposed to H2O2 or SNP Treatment

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

N

N

N

Y Y

Y

Y

Y

Y

Y Y

Y

N Y Y Y Y Y Y

Y

Y

Y Y

Y

Y Y

N

Y

Y Y Y

Y

C U

U

C

U

U

U U

U

U U U U C U

U

U

U U

U

U U

U

U U U

U

D D

U

C

C

C

C C

D

C U C C D C

D

D

D D

D

D D

D

D D D

D

1 1

1

1

1

1

1 1

1

1 1 1 1 1 1

1

1

1 1

1

1 1

1

1 1 1

1

carb. carb. 2-DE carb. status status oxyblot l m n kind status to H2O2 to SNP areao k

research articles Tanou et al.

N

N

N

N

N

N Y N

N

Y N Y

N

N

N Y

N

Y

N

Y

N

Y N

N Y

N

Y Y N

2322

2402

2403

2406 2506 2512

2626

2804 2808 3102

3110

3110

3210 3211

3305

3401

3502

3509

3514

3515 3518

3519 3702

3804

3817 4207 4301

N N N

2315 2318 2319

2320

N N

2207 2314

2321

N

2207

D D C

C

C U

U C

C

U

C

U

C

C D

C

C

D C D

C

C U C

C

C

C

C

C

C C C

C C

C

Y N N

N

N Y

N N

N

Y

N

Y

N

N N

Y

Y

Y N Y

N

N Y N

Y

N

N

N

N

N Y N

N N

N

D C C

C

C U

C C

C

U

C

U

C

C C

U

U

D C D

C

C U C

D

C

C

C

C

C U C

C C

C


Table 1. Continued

organism Poncirus trifoliata

g

Atalantia ceylanica

Citrus x paradisi x Poncirus trifoliata

Poncirus trifoliata

Oryza sativa

Oryza sativa

Citrus sinensis

Anemia phyllitidis Citrus sinensis

Citrus sinensis

Citrus sinensis

Citrus sinensis

Citrus sinensis Oryza sativa

Citrus sinensis

Hevea brasiliensis

Citrus x paradisi x Poncirus trifoliata Atalantia ceylanica

Nicotiana tabacum

gi|57932482

Citrus sinensis

Citrus sinensis Citrus x paradisi x Poncirus trifoliata gi|147842424 Vitis vinifera

gi|46211630 gi|57923466

gi|46211630 gi|56784991

gi|56590455

gi|231586

gi|4206588

gi|57923828

gi|19875

gi|110847344 Citrus clementina gi|57932482 Citrus sinensis

gi|57931319

gi|28615709

gi|57934502

gi|115452237 Oryza sativa

gi|4206588

gi|57922400

gi|31670123

gi|29665484

gi|29665484

gi|45448898

gi|3746940 gi|57932636

gi|55292234 Poncirus trifoliata gi|116787631 Picea sitchensis

gi|31670830

acces. numberf Chloroplast



02.30-Energy/ Photosynthesis NADPH:quinone reductase Chloroplast 02.20 Energy/E-transport Phosphoribulokinase Chloroplast 02.30-Energy/ Photosynthesis 40-Non identified protein Actin 3 Cytoskeleton 09.04-Cytoskeleton Rubisco activase Chloroplast 02.30-Energy/ Photosynthesis Rubisco activase Chloroplast 02.30-Energy/ Photosynthesis Rubisco activase Chloroplast 02.30-Energy/ Photosynthesis Rubisco activase Chloroplast 02.30-Energy/ Photosynthesis Rubisco activase Chloroplast 02.30-Energy/ Photosynthesis Succinyl-CoA ligase beta Mitochondria 02.10-Energy/TCA pathway subunit 40-Non identified protein 40-Non identified protein ATP synthase F1 (subunit Chloroplast 07.22-Transporters/ β) Transport ATPases HSP 70 Cytosol 06.01-Protein destination and storage/Folding and stability 40-Non identified protein 40-Non identified protein Miraculin 2 Cytosol 11.05-Disease/Defense/ Stress responses Fe-superoxide dismutase Chloroplast 11.06-Disease/Defense/ Detoxification Carbonic anhydrase Chloroplast 02.30-Energy/ Photosynthesis NADPH:quinone reductase Chloroplast 02.20-Energy/E-transport Fructose 1,6 bisphosphate Chloroplast 02.30-Energy/ aldolase Photosynthesis Glutamate-1-semialdehyde Chloroplast 02.30-Energy/ 2,1-aminomutase Photosynthesis Phosphoglycerate kinase Chloroplast 02.30-Energy/ Photosynthesis ATP synthase F1 (subunit Chloroplast 07.22-Transporters/ β) Transport ATPases ATP synthase F1 (subunit Mitochondria 07.22-Transporters/ β) Transport ATPases Mitochondrial processing Mitochondria 06.04-Protein destination peptidase subunit alpha and storage/Targeting Enolase Cytosol 02.01-Energy/Glycolysis ATP synthase F1 (subunit Mitochondria 07.22-Transporters/ β) Transport ATPases Enolase Cytosol 02.01-Energy/Glycolysis Transketolase Chloroplast 02.30-Energy/ Photosynthesis HSP (clpB) Chloroplast 06.01-Protein destination and storage/Folding and stability 40-Non identified protein 40-Non identified protein Fructose 1,6 bisphosphate Chloroplast 02.30-Energy/ aldolase Photosynthesis

Rubisco activase

protein nameh

S

S

S S

S S

S

S

S

S

S

S S

Y N Y

Y

Y Y

Y Y

Y

Y

Y

Y

Y

Y N

N

M M

N

S

Y

Y N Y

Y

Y

Y

Y

Y

Y Y Y

Y Y

Y

Y Y N

S

S

S

S

S

S

S

S S

M S

M

U

D

D

C D

D C

D

U

D

U

C

U

D D

U

U

U

U

U

U

U

U

U U U

U U

U

U

D

D

C D

C C

C

C

D

D

C

U

D D

D

C

C

D

C

D

C

C

C C C

C C

C

1

1

1

1 2

1 1

1

1

1

1

1

1

1 1

1

1

1

1

1

1

1

1

1 1 1

1 1

1



research articles

Journal of Proteome Research • Vol. 9, No. 11, 2010 5999

6000

N

N Y N N

N N N

N

N

N

N

Y

N

Y

Y

N

N

N

N N

N N N

N

N

N

N

N

N

N

N

4311

4312 4315 4318 4402

4402 4410 4411

4604

4701

4702

4703

4704

4705


5205

5206

5211

5212

5306

5311 5315

5316 5320 5402

5406

5406

5407

5412

5412

U5501

5601

5602

C

C

C

C

C

C

C

C

C C C

C C

C

C

C

D

D

C

U

C

C

C

C

C C C

C U C C

C

N

N

Y

N

N

N

N

N

N N N

N N

Y

Y

N

Y

Y

N

N

N

N

N

N

N N N

N Y N N

N

C

C

D

C

C

C

C

C

C C C

C C

U

U

C

D

D

C

C

C

C

C

C

C C C

C U C C

C


Table 1. Continued

organism

protein nameh

Transketolase

Phosphoglycerate kinase Enolase S-adenosylhomocysteine hydrolase Aldehyde dehydrogenase

Citrus x paradisi x Transketolase Poncirus trifoliata Citrus x paradisi x Transketolase Poncirus trifoliata Citrus jambhiri Transketolase

Citrus jambhiri

Citrus sinensis Citrus sinensis Petroselinum crispum Citrus sinensis

Nicotiana tabacum GAPDH

Citrus x paradisi x Phosphoglycerate kinase Poncirus trifoliata

g

Citrus sinensis

Poncirus trifoliata

gi|57932630

gi|57932559

gi|42478020

gi|45452721

gi|4206550

gi|21652369

gi|90464008

gi|80973462

gi|120665

Fructose 1,6 bisphosphate aldolase Fructose 1,6 bisphosphate aldolase Fructose 1,6 bisphosphate aldolase

Isoflavone reductase

Citrus sinensis

Citrus sinensis

Citrus sinensis

Pleiospermium alatum Citrus sinensis

Aldehyde dehydrogenase

Alanine aminotransferase

Pyruvate dehydrogenease (dihydrolipoamide acetyltransferase) Aldehyde dehydrogenase

Rubisco large subunit

GDP-D-mannose-3′,5′epimerase Helianthus annuus ATP synthase F1 (subunit R) Citrus sinensis Aldehyde dehydrogenase

Malpighia glabra

Nicotiana tabacum GAPDH

gi|118487575 Populus trichocarpa gi|21311559 Hevea brasiliensis

gi|57932482

gi|57875834

Citrus x paradisi x Transketolase Poncirus trifoliata gi|110851670 Citrus clementina Cinnamoyl-CoA reductase

gi|57922485

gi|51303009

gi|57922485

gi|57922485

gi|51303009

gi|57932630

gi|56533131 gi|46211630 gi|417744

gi|120665

gi|57923828

acces. numberf 02.30-Energy/ Photosynthesis 40-Non identified protein 40-Non identified protein 40-Non identified protein 02.30-Energy/ Photosynthesis 02.01-Energy/Glycolysis 02.01-Energy/Glycolysis 01.01-Metabolism/Amino acid 11.06-Disease/Defense/ Detoxification 02.30-Energy/ Photosynthesis 02.30-Energy/ Photosynthesis 02.30-Energy/ Photosynthesis 02.30-Energy/ Photosynthesis 02.30-Energy/ Photosynthesis 20.01-Secondary metabolism/ Phenylpropanoids/ Phenolics 20.01-Secondary metabolism/ Phenylpropanoids/ Phenolics 02.30-Energy/ Photosynthesis 02.30-Energy/ Photosynthesis 02.30-Energy/ Photosynthesis 40-Non identified protein 02.30-Energy/ Photosynthesis 40-Non identified protein 40-Non identified protein 11.06-Disease/Defense/ Detoxification 07.22-Transporters/ Transport ATPases 11.06-Disease/Defense/ Detoxification 02.30-Energy/ Photosynthesis 02.01-Energy/Glycolysis


Mitochondria 11.06-Disease/Defense/ Detoxification Unclear 01.01-Metabolism/Amino acid Mitochondria 11.06-Disease/Defense/ Detoxification 40-Non identified protein

Mitochondria

Chloroplast

Mitochondria

Mitochondria

Unclear

Chloroplast

Chloroplast

Chloroplast

Chloroplast

Unclear

Unclear

Chloroplast

Chloroplast

Chloroplast

Chloroplast

Chloroplast

Mitochondria

Cytosol Cytosol Cytosol

Chloroplast

Chloroplast


S

S

M

M

S

M

M

S

S

S

S

S

S

S

S

S

S

S

S

S

M S S

M

S

Y

Y

N

Y

Y

Y

Y

Y

Y Y Y

Y Y

N

Y

Y

N

N

Y

Y

Y

Y

Y

Y

Y Y Y

Y N Y Y

Y

D

D

C

C

D

D

D

C D U

D D

C

C

D

D

D

D

D

D

D

D

D

D

C

D

D

D D D

D D

D

D

D

D

D

D

D

D

D D D

U D

U D D C C

D

D

C

C

2

2

2

2

2

2

2 2 2

2 2

2

2

2

2

2

2

2

2

2 2 2

1 2

1

2



N

Y

Y

Y

Y

N

Y N

N Y

Y

Y

N

N

N

Y

Y N

Y

N

Y

Y

N

N

N

N

N

N

5603

6105

6109

6201

6203

6204

6302 6304

6401 6402

6414

6521

6601

6603

7111

7201

7202 7218

7302

7318

7401

7404

7410

7415

7415

7415

7431

7432

C

C

C

C

C

C

D

D

C

D

D C

D

C

C

C

U

U

C D

D C

C

D

D

D

D

C

N

N

N

N

N

Y

Y

Y

N

Y

Y N

Y

N

N

N

N

N

N Y

Y N

N

Y

Y

Y

Y

N

C

C

C

C

C

D

D

D

C

D

D C

D

C

C

C

C

C

C D

D C

C

D

D

D

D

C


Table 1. Continued

organism

g

Citrus sinensis

Poncirus trifoliata

Citrus jambhiri

Citrus sinensis

Citrus sinensis

Cucumis sativus

Citrus sinensis

Hevea brasiliensis

Capsicum annuum

Poncirus trifoliata

Poncirus trifoliata

Severinia buxifolia



gi|57874007

gi|4206520

gi|118723752 Handeliodendron bodinieri gi|19070130 Vitis vinifera

gi|46212454

gi|118564

gi|57931635

gi|21311559

gi|18072795

gi|57570529

Citrus x paradisi x Poncirus trifoliata gi|118484540 Populus trichocarpa

gi|57923318



Alcohol dehydrogenase

Ferredoxin-NADP+ oxidoreductase Fructose 1,6 bisphosphate aldolase

Cinnamoyl-CoA reductase

Miraculin

Glycine decarboxylase L subunit Glutathione S-transferase

protein nameh


Chloroplast

Cytosol

Chloroplast

Chloroplast

01.01-Metabolism/Amino acid Rubisco large subunit Chloroplast 02.30-Energy/ Photosynthesis Catalase Peroxisome 11.06-Disease/Defense/ Detoxification Rubisco large subunit Chloroplast 02.30-Energy/ Photosynthesis Serine Mitochondria 01.01-Metabolism/Amino hydroxymethyltransferase acid Rubisco large subunit Chloroplast 02.30-Energy/ Photosynthesis Pyruvate dehydrogenease Chloroplast 02.30-Energy/ (dihydrolipoamide Photosynthesis acetyltransferase)

02.30-Energy/ Photosynthesis 02.13-Energy/Respiration

02.30-Energy/ Photosynthesis 40-Non identified protein 02.30-Energy/ Photosynthesis 02.01-Energy/Glycolysis

Mitochondria 11.06-Disease/Defense/ Detoxification Cytosol 06.13-Protein destination and storage/Proteolysis

11.06-Disease/Defense/ Detoxification Cytosol 11.05-Disease/Defense/ Stress responses Unclear 20.01-Secondary metabolism/ Phenylpropanoids/ Phenolics Chloroplast 02.30-Energy/ Photosynthesis Chloroplast 02.30-Energy/ Photosynthesis 40-Non identified protein Unclear 11.06-Disease/Defense/ Detoxification 40-Non identified protein Chloroplast 02.30-Energy/ Photosynthesis Chloroplast 02.30-Energy/ Photosynthesis Chloroplast 02.30-Energy/ Photosynthesis Mitochondria 02.13-Energy/Respiration

Cytosol

Mitochondria 02.13-Energy/Respiration


Glycerate dehydrogenase Peroxisome (NADH-dependent hydroxypyruvate reductase) Aspartate aminotransferase Unclear

Fructose 1,6 bisphosphate aldolase GAPDH

GAPDH

Ubiquitin-conjugating enzyme E2, catalytic (UBCc) domain Fructose 1,6 bisphosphate aldolase

Glycine decarboxylase L subunit Aldehyde dehydrogenase

Heloniopsis orientalis Rubisco large subunit


gi|47604670


gi|51302785

gi|56534483


gi|147791392 Vitis vinifera


gi|38035194

gi|31670267


acces. numberf

S

S

M

M

M

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

Y

Y

Y

Y

Y

N

N

Y

Y

Y

N Y

N

Y

Y

Y

Y

Y

Y Y

N Y

Y

N

N

N

N

Y

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D D

D

D

D

D

D

C

C

C

D

U

D

D

D

D

D

D

D

D D

D

D

D

2

2

2

2

2

2

2

2

2

3

2

2

2

2

2 2

2

2

2



research articles


6002


Y

N Y Y

N

N N

Y

8113

8127 8128 8202

8208

8303 8606

9401

D

C C

C

C D D

D

D

D D C

N

N N

N

N N N

Y

N

Y Y Y

C

C C

C

C C C

D

C

D D U

Citrus junos

organismg

Citrus aurantium

Glycine decarboxylase L subunit Miraculin 2

Citrate synthase

protein nameh


b

Cytosol

Mitochondria 02.10-Energy/TCA pathway 40-Non identified protein Mitochondria 02.13-Energy/Respiration


11.05-Disease/Defense/ Stress responses gi|71598377 Citrus sinensis Miraculin Cytosol 11.05-Disease/Defense/ Stress responses 40-Non identified protein 40-Non identified protein gi|12240092 Citrus reticulata Rubisco small subunit Chloroplast 02.30-Energy/ Photosynthesis gi|118487575 Populus trichocarpa Fructose 1,6 bisphosphate Chloroplast 02.30-Energy/ aldolase Photosynthesis gi|21650412 Citrus sinensis GAPDH Cytosol 02.01-Energy/Glycolysis gi|57931430 Citrus sinensis Serine Mitochondria 01.01-Metabolism/Amino hydroxymethyltransferase acid 40-Non identified protein

gi|61691363


gi|40646744

acces. numberf

S S

S

S

S

S

S

S

N

Y Y

Y

Y N N

N

N

N N Y

C D

D

D

D

U D

D

D

D

2 2

2

3

3

carb. carb. 2-DE carb. status status oxyblot kindk statusl to H2O2m to SNPn areao

Spot no., spot label on the reference map presented in Supporting Information Figure S1. H2O2 responsive protein, protein whose accumulation level varied under H2O2 treatment; Y, yes; N, no. c Response to H2O2, pattern of accumulation; C, protein whose accumulation level was constant; U, up-regulated protein; D, down-regulated protein. d SNP responsive protein, protein whose accumulation level varied under SNP treatment; Y, yes; N, no. e Response to SNP, pattern of accumulation; C, protein whose accumulation level was constant; U, up-regulated protein; D, down-regulated protein. f Accession number, accession number in NCBI database. g Organism, organism in which the protein has been identified. h Protein name, identified peptide names. i Cellular localization, subcellular localization was assigned based on database searches. j Function category, functional category defined according to the ontological classification of Bevan et al.36 k Kind, single-protein protein spot (S) or multiprotein spot (M). l Carbonylation status, Y, yes; N, no. m Carbonylation status to H2O2, C, protein whose carbonylation level was constant; U, protein whose carbonylation level was up-regulated; D, protein whose carbonylation level was down-regulated. n Carbonylation status to SNP, C, protein whose carbonylation level was constant; U, protein whose carbonylation level was up-regulated; D, protein whose carbonylation level was down-regulated. o 2-DE oxyblot area, location of cabonylated proteins on 2-DE (oxyblots) gels as shown in Figure 5.

Y

8109

a

Y Y N

7502 7504 7601


Table 1. Continued



research articles

Figure 4. Venn diagram analysis of the differentially expressed protein in citrus plants treated with H2O2 or SNP. Treatments were conducted as described in Figure 1 and under Materials and Methods. Number of all leaf citrus proteins whose abundance levels were modulated by H2O2 (A) or SNP (B) treatment and the overlap among the treatments (C). The total number of proteins that were up-regulated (+) or down-regulated (-) were also given for each category. The percentage of proteins modulated (up-regulated or down-regulated) by signaling molecule treatments were assigned to functional categories according to Bevan et al.36

Figure 5. Representative 2-DE immunoblots of carbonylated citrus leaf proteins in control (A) and plants treated with H2O2 (B) or SNP (C). Treatments were conducted as described in Figure 1 and under Materials and Methods. Specific areas of 2-DE immunoanalysis undergoing varying carbonylation spot patterns in response to H2O2 or SNP treatments (areas 1,-3) are marked. (D) Carbonylated proteins are labeled with blue arrows on a reference 2DE-gel of total soluble citrus leaf proteins stained with silver nitrate. Numbers correspond to the carbonylated proteins that have been identified by mass spectrometry are shown in Figure S2 and listed in Table 1. (E) Carbonylated citrus proteins are classified according to Bevan et al.36 Journal of Proteome Research • Vol. 9, No. 11, 2010 6003

research articles treatment (Table 1; Figure 4A); (ii) a group of 10 SNPmodulated proteins whose abundance was changed only by SNP treatment (Table 1; Figure 4B); and (iii) a group of 30 H2O2 and SNP-modulated proteins whose abundance was changed by both treatments (Table 1; Figure 4C). Among the 16 proteins exclusively regulated by H2O2, 5 were up-regulated and 11 were down-regulated (Table 1). Several of them are involved in energy/photosynthesis (Table 1, Figure 4A), as is the case for transketolase (spot 4704; Table 1), Rubisco large subunit (spots 6414 and 6521; Table 1), and Rubisco small subunit (spot 8202; Table 1). Changes in the abundance of HSP (ClpP) (spot 202; Table 1), 20S proteasome alpha subunit (spot 1201; Table 1), ATP synthase F1 (subunit β) (spot 1302; Table 1), and enolase (spot 3515; Table 1) confirmed previous observations that these genes or gene products are targets of oxidative stress in plants,9-11,14,16,37-39 thereby lending support to the current results. Ten citrus proteins were exclusively affected by SNP with seven up-regulated and three down-regulated (Table 1). As observed in H2O2-treated plants, several enzymes involved in photosynthesis were identified with altered abundance in response to SNP (Table 1). Among SNP-responsive proteins were also alanine aminotransferase (spot 5501; Table 1) and glycine decarboxylase L subunit (spot 7601; Table 1), both being involved in photorespiration. This observation is worth noting as photorespiration is a major source of H2O2 in photosynthetic cells and, furthermore, makes a key contribution to •NO production through nitrogen homeostasis in plants. In support of the present results, Palmieri et al.40 recently showed that • NO can modify the glycine decarboxylase complex and the photorespiratory system in Arabidopsis cells. Finally, the observation that actin 3 (spot 2318; Table 1) was up-regulated in the presence of SNP is consistent with a recent report showing that •NO modulates actin cytoskeleton assembly and organization in maize cells.41 Transcriptomic analysis has previously revealed considerable overlap between H2O2- and •NO-inducible genes in catalasedeficient tobacco plants.13 The present comparative proteomic analysis further strengthens the parallels between the mode of action of these two molecules since a high portion of the H2O2- and •NO-affected proteome (comprised of 30 proteins) was similarly altered by treatments (Figure 4C; Table 1). This strong protein cross-talk between the two signaling molecules may represent the major etiologic factor for the relationship between H2O2 and •NO observed during various physiological processes in plants.2,4,6,7,13,42,43 Particularly relevant is also the fact that a majority of the proteins affected by both treatments were reduced in abundance relative to control (Table 1, Figure 4C), disclosing a negative correlation between signaling with these two molecules and proteome activity. The largest categories of the commonly H2O2- and SNPregulated proteins belong to photosynthesis and especially to the Calvin-Benson cycle (Table 1, Figure 4C), again indicating that photosynthetic factors are preferential targets for the two signaling compounds. Also, it is interesting that only glutathione S-transferase (GST, spot 6105; Table 1) was classified as ROS-related enzyme in the group of the H2O2 and SNP overlapping proteins. These results are consistent with previous observations2 showing association between GST transcript and H2O2/•NO signaling. In addition, four forms of miraculin (spots 2104, 3102, 6109, and 8113; Table 1) were decreased in abundance in treated plants, suggesting that this protein could play a role in mediating cross-talk between H2O2 and •NO 6004


Tanou et al. signaling pathways. The currently proposed role of miraculins as defense proteins in plants, including citrus exposed to abiotic/biotic stress conditions,6,44-46 could explain their sensitivity to H2O2 and SNP. Several proteins related to the energy metabolic category were identified exhibiting a decreased abundance upon H2O2 or SNP exposure (Table 1). In particular, the down-regulation of the cytosolic form of fructose 1,6 bisphosphate aldolase (spot 7302; Table 1) by both treatments suggests that H2O2 and •NO signaling may be accompanied by a decrease in the glycolytic production of ATP. Also, the depression of the tricarboxylic acid cycle (TCA) as indicated by the down-regulation of pyruvate dehydrogenase (spot 1203; Table 1) and citrate synthase (spot 7502; Table 1) is likely to impose significant flux restrictions on the TCA cycle and electron transport in treated citrus plants. Impact of H2O2 and SNP on Protein Carbonylation Patterns. The fact that both H2O2- and •NO accumulated in leaves of treated plants (Figure 2A,B) together with the observation that most of the H2O2- and SNP-responsive proteins were down-regulated (Table 1, Figure 4C) led us to hypothesize that a specific set of citrus proteins can undergo oxidative modifications. To address this question, 2DE-gel analysis coupled with immunoblotting experiments using anti-dinitrophenyl-group antibodies was employed to characterize oxidatively modified proteins in citrus leaves (Figure 5A-C). Protein carbonylation was readily observed in untreated plants (Figure 5A), verifying that some plant proteins are highly susceptible to oxidation.31,47 Exposure to H2O2 (Figure 5B) or SNP (Figure 5C) specifically altered carbonyl signals compared to control (Figure 5A). For example, the spot pattern observed in the area 1 by 2DE-DNPH immunoanalysis was induced by SNP and H2O2. By contrast, carbonylated signals in the areas 2 and 3 of oxyblots showed lower intensity in treated plants compared to control. Carbonylated proteins were then tentatively identified by spot matched anti-DNP blots to corresponding protein spots on 2DE-silver stained gels (Figure 5D). The carbonylation status and the position area in 2DE-immunoblots of specific proteins are indicated in Table 1 and Figure S2 (see Supporting Information). Among the 107 carbonylated proteins presently characterized in citrus leaves, we can define a group of 91 proteins whose carbonylation status changed (47 decreased and 44 increased in abundance) upon H2O2 treatment and a group of 76 proteins whose carbonylation status changed (69 decreased and 7 increased in abundance) upon SNP treatment (Table 1). Proteins classified to photosynthesis corresponded to the largest category of carbonylated proteins whose levels were changed by H2O2 (38.5%, 35 proteins) and SNP (38.2%, 29 proteins) exposure. Also, we found that proteins involved in energy, defense, protein/amino acid metabolism, transport and cytoskeleton were oxidative targets in citrus leaves (Table 1; Figure 5E). Among the carbonylated citrus proteins affected by H2O2 treatment, it is worth noting the behavior of Rubisco activase and Rubisco large subunit. Thus, H2O2 treatment entailed oxidative carbonylation of all detectable forms of Rubisco activase (spots 303, 402, 406, 1402, 2207, 2319, 2320, 2321, 2322, and 2402; Table 1; Figure 5), whereas it depressed the accumulation of all carbonylated forms of Rubisco large subunit (spots 5407, 6402, 6414, 6521, 7415, and 7431; Table 1; Figure 5). These completely distinct features suggest that the RuBisCO complex is in a dynamic balance of synthesis and catabolism in response to exogenous H2O2. Apart from Rubisco activase and Rubisco large subunit, various carbonylated proteins were differentially accumulated upon H2O2 or SNP treatments.

research articles

H2O2 and •NO Cross-Talk Signaling Network in Citrus Plants Indeed, molecular chaperones such as HSP (ClpP) (spot 202; Table 1; Figure 5), chaperonin 60 alpha subunit (spot 601; Table 1; Figure 5), and HSP 70 (spots 1635 and 2626; Table 1; Figure 5) were identified as strong oxidative targets after H2O2 but not after SNP treatment. The specific oxidation of these proteins is interesting because it was suggested that, under oxidative stress, chaperones act as shields protecting proteins or interact with damaged proteins to assist their refolding48 and supports earlier proposals that chaperones are sensitive to carbonylation in plants.29,32 Similarly, protein metabolism was also oxidatively targeted by H2O2, since 20S proteasome alpha subunit (spot 1201; Table 1; Figure 5) and nucleoid DNA binding protein (spot 1301; Table 1; Figure 5) exhibited strong responsiveness to carbonylation under H2O2 treatment. Given that the chaperone/ proteolytic mechanism is highly dependent on ATP availability49 and that many forms of ATP synthase F1 (subunit β) (spots 1302, 1609, 2512, and 3509; Table 1; Figure 5) were carbonylated by H2O2, a link between the status of carbonylation and ATP synthesis of the H2O2-treated citrus cells and its ability to control protein folding/stability is likely. Besides the above results showing distinct regulation of protein carbonylation by H2O2 and SNP, we found that several proteins were similarly modified upon chemical treatments. Of the 47 proteins whose carbonylation was depressed by H2O2, 40 proteins also showed decreased levels of carbonylation in response to SNP treatment. Notably, carbonylated forms of transketolases (spots 3702, 4701, 4702, 4703, 4704, and 4705; Table 1; Figure 5) were identified among the proteins whose carbonylation was depressed by both molecules. Transketolase has been shown previously to be highly sensitive to H2O2derived oxidation in yeast and plants.1,50 Also, recent proteomic studies disclosed that transketolase is •NO inducible via Snitrosylation and tyrosine nitration.20,22 Transketolase catalyzes two independent reactions of the pentose phosphate pathway (PPP), which generates NADPH and ribose-5-phosphate. An activation of the PPP can be useful to generate reductive power (NADPH) needed to maintain redox (disulfide) balance in protein through the thioredoxin system.32 In addition, decreased carbonylation levels of aldehyde dehydrogenase (spots 4604, 5406, 5601, and 6603; Table 1; Figure 5), which is involved in the metabolic control of potentially toxic aldehydes, were also observed in response to both treatments, contributing to cellular defense in the detoxification of these oxidative stressgenerated reactive compounds in treated plants. It is important to note that 15 mitochondrial proteins, such as pyruvate dehydrogenase (spots 1203, 5412; Table 1; Figure 5), succinyl-CoA ligase beta subunit (spot 2403; Table 1; Figure 5), mitochondrial processing peptidase subunit alpha (spot 3514; Table 1; Figure 5), and serine hydroxymethyltransferase (spot 7415; Table 1; Figure 5) were strongly carbonyl-tagged in control plants showing that mitochondrial components are susceptible to oxidation. Previous proteomics studies pointed extensive carbonylation of mitochondrial proteins by H2O214,49,51,52 and it is reported to be associated to the metal-catalyzed oxidation of mitochondrial matrix enzymes.53 However, the carbonylation status in a very large portion of the carbonylated citrus mitochondrial proteins remains constant or depressed upon external H2O2 and SNP application (Table 1), whereas the abundance of most of these mitochondrial proteins was unaffected by both treatments (Table 1). These data suggest that the network of mitochondrial proteins is able to perform their functions in treated citrus plants and add an important role for mitochondrial proteins in oxidative and nitrosative signal transduction mechanisms. In

view of the above observations and the fact that the treated plants displayed high vigor (Figure 1A-C) the current data support the proposal of Job et al.32 that protein carbonylation represent an adaptive mechanism of cellular metabolism to environmental conditions.

Concluding Remarks The present experiments delineate a signaling pathway in which root treatments with H2O2 or SNP lead to both H2O2 and • NO accumulation in citrus leaves. Using a global proteomic strategy, we identified H2O2 and •NO-responsive proteins and metabolic pathways and highlighted an extensive overlap in protein expression regulated by these molecules. The current study also uncovered that protein carbonylation serves as a mechanism to couple H2O2 and •NO signals to proteins modifications signposts. Because roots are exposed to many (a)biotic rhizosphere factors leading to ROS and RNS accumulation,54 the H2O2/•NO systemic loading system along with its corresponding protein network described here may be physiologically relevant from an ecological point of view during whole plant responses to a broad spectrum stressful phenomena in nature.

Acknowledgment. G.T. is grateful to the State Scholarship’s Foundation of Greece for a fellowship. Supporting Information Available: Supplementary Table S1, sequence data for citrus leaf proteins. Supplementary Table S2, quantitative data for citrus leaf protein spot volumes. Supplementary Figure S1, reference map for citrus leaf proteins. Supplementary Figure S2, reference map for citrus leaf carbonylated proteins. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Juhnke, H.; Krems, B.; Kftter, P.; Entian, K. D. Mutants that show increased sensitivity to hydrogen peroxide reveal an important role for the pentose phosphate pathway in protection of yeast against oxidative stress. Mol. Genet. Genomics 1996, 252, 456–464. (2) Delledonne, M.; Zeier, J.; Marocco, A.; Lamb, C. Signal interactions between nitric oxide and reactive oxygen intermediates in the plant hypersensitive disease resistance response. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 13454–13459. (3) Ros Barcelo´, A. Xylem parenchyma cells deliver the H2O2 necessary for lignification in differentiating xylem vessels. Planta 2005, 220, 747–756. (4) Bright, J.; Desikan, R.; Hancock, J. T.; Weir, I. S.; Neill, S. J. ABA induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis. Plant J. 2006, 45, 113–122. (5) Ahlfors, R.; Brosche´, M.; Kollist, H.; Kangasja¨rvi, J. Nitric oxide modulates ozone-induced cell death, hormone biosynthesis and gene expression in Arabidopsis thaliana. Plant J. 2009, 58, 1–12. (6) Tanou, G.; Job, C.; Rajjou, L.; Arc, E.; Belghazi, M.; Diamantidis, G.; Molassiotis, A.; Job, D. Proteomics reveals the overlapping roles of hydrogen peroxide and nitric oxide in the acclimation of citrus plants to salinity. Plant J. 2009a, 60, 795–804. (7) Tanou, G.; Molassiotis, A. Diamantidis, Gr. Hydrogen peroxideand nitric oxide-induced systemic antioxidant prime-like activity under NaCl-stress and stress free conditions in citrus plants. J. Plant Physiol. 2009b, 166, 1904–1913. (8) Palmieri, M. C.; Sell, S.; Huang, X.; Scherf, M.; Werner, T.; Durner, J.; Lindermayr, C. Nitric oxide-responsive genes and promoters in Arabidopsis thaliana: a bioinformatics approach. J. Exp. Bot. 2008, 59, 177–186. (9) Desikan, R.; Mackerness, A. H. S.; Hancock, J. T.; Neill, S. J. Regulation of the Arabidopsis transcriptome by oxidative stress. Plant Physiol. 2001, 127, 159–172. (10) Vandenabeele, S.; Van Der Kelen, K.; Dat, J.; Gadjev, I.; Boonefaes, T.; Morsa, S.; Rottiers, P.; Slooten, L.; Van Montagu, M.; Zabeau, M.; Inze´, D.; Van Breusegem, F. A comprehensive analysis of hydrogen peroxide-induced gene expression in tobacco. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 16113–16118.


research articles (11) Vanderauwera, S.; Zimmermann, P.; Rombauts, S.; Vandenabeele, S.; Langebartels, C.; Gruissem, W.; Inze, D.; Van Breusegem, F. Genome-wide analysis of hydrogen peroxide-regulated gene expression in Arabidopsis reveals a high light-induced transcriptional cluster involved in anthocyanin biosynthesis. Plant Physiol. 2005, 139, 806–821. (12) Parani, M.; Rudrabhatla, S.; Myers, R.; Weirich, H.; Smith, B.; Leaman, D. W.; Goldman, S. L. Microarray analysis of nitric oxide responsive transcripts in Arabidopsis. Plant Biotechnol. J. 2004, 2, 359–366. (13) Zago, E.; Morsa, S.; Dat, J. F.; Alard, P.; Ferrarini, A.; Inze, D.; Delledonne, M.; Van Breusegem, F. Nitric oxide- and hydrogen peroxide-responsive gene regulation during cell death induction in tobacco. Plant Physiol. 2006, 141, 404–411. (14) Sweetlove, L. J.; Heazlewood, J. L.; Herald, V.; Holtzapffel, R.; Day, D. A.; Leaver, C. J.; Millar, A. H. The impact of oxidative stress on Arabidopsis mitochondria. Plant J. 2002, 32, 891–904. (15) Hancock, J. T.; Henson, D.; Nyirenda, M.; Desikan, R.; Harrison, J.; Lewis, L.; Hughes, J.; Neill, S. J. Proteomic identification of glyceraldehyde 3-phosphate dehydrogenase as an inhibitory target of hydrogen peroxide in Arabidopsis. Plant Physiol. Biochem. 2005, 43, 828–835. (16) Wan, X. Y.; Liu, J. Y. Comparative proteomics analysis reveals an intimate protein network provoked by hydrogen peroxide stress in rice seedling leaves. Mol. Cell. Proteomics 2008, 7, 1469–1488. (17) Corpas, F. J.; Del Rio, L. A.; Barroso, J. B. Post-translational modifications mediated by reactive nitrogen species: Nitrosative stress responses or components of signal transduction pathways? Plant Signal. Behav. 2008, 3, 301–303. (18) Lindermayr, C.; Saalbach, G.; Durner, J. Proteomic identification of S-nitrosylated proteins in Arabidopsis. Plant Physiol. 2005, 137, 921–930. (19) Romero-Puertas, M. C.; Campostrini, N.; Matte`, A.; Righetti, P. G.; Perazzolli, M.; Zolla, L.; Roepstorff, P.; Delledonne, M. Proteomic analysis of S-nitrosylated proteins in Arabidopsis thaliana undergoing hypersensitive response. Proteomics 2008, 8, 1459–1469. (20) Abat, J. K.; Deswal, R. Differential modulation of S-nitrosoproteome of Brassica juncea by low temperature: Change in S-nitrosylation of Rubisco is responsible for the inactivation of its carboxylase activity. Proteomics 2009, 9, 4368–4380. (21) Cecconi, D.; Orzetti, S.; Vandelle, E.; Rinalducci, S.; Zolla, L.; Delledonne, M. Protein nitration during defense response in Arabidopsis thaliana. Electrophoresis 2009, 30, 2460–2468. (22) Chaki, M.; Valderrama, R.; Fernańdez-Ocan ˜ a, A. M.; Carreras, A.; Lo´pez-Jaramillo, J.; Luque, F.; Palma, J. M.; Pedrajas, J. R.; BegaraMorales, J. C.; Sańchez-Calvo, B.; et al. Protein targets of tyrosine nitration in sunflower (Helianthus annuus L.) hypocotyls. J. Exp. Bot. 2009, 60, 4221–4234. (23) Rizhsky, L.; Hallak-Herr, E.; Van Breusegem, F.; Rachmilevitch, S.; Barr, J. E.; Rodermel, S.; Inze´, D.; Mittler, R. Double antisense plants lacking ascorbate peroxidase and catalase are less sensitive to oxidative stress than single antisense plants lacking ascorbate peroxidase or catalase. Plant J. 2002, 32, 329–342. (24) Wintermans, J. F.; de Mots, A. Spectrophotometric characteristics of chlorophylls a and b and their pheophytins in ethanol. Biochim. Biophys. Acta 1965, 109, 448–453. (25) Pazdzioch-Czochra, M.; Widenska, A. Spectrofluorimetric determination of hydrogen peroxide scavenging activity. Anal. Chim. Acta 2002, 452, 177–184. (26) Ding, A. H.; Nathan, C. F.; Stuehr, D. J. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. J. Immunol. 1998, 141, 2407–2412. (27) Rodrı´guez-Serrano, M.; Romero-Puertas, M. C.; Zabalza, A.; Corpas, F. J.; Go´mez, M.; Del Rıó, L. A.; Sandalio, L. M. Cadmium effect on oxidative metabolism of pea (Pisum sativum L.) roots. Imaging of reactive oxygen species and nitric oxide accumulation in vivo. Plant Cell Environ. 2006, 29, 1532–1544. (28) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Chem. 1976, 72, 248–254. (29) Rajjou, L.; Lovigny, Y.; Groot, S. P. C.; Belghazi, M.; Job, C.; Job, D. Proteome-wide characterization of seed aging in Arabidopsis: a comparison between artificial and natural aging protocols. Plant Physiol. 2008, 148, 620–641. (30) Catusse, J.; Strub, J. M.; Job, C.; Van Dorsselaer, A.; Job, D. Proteome-wide characterization of sugarbeet seed vigor and its tissue specific expression. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 10262–10267. (31) Imin, N.; Kerim, T.; Weinman, J. J.; Rolfe, B. G. Characterization of rice anther proteins expressed at the young microspore stage. Proteomics 2001, 1, 1149–1161.

6006


Tanou et al. (32) Job, C.; Rajjou, L.; Lovigny, Y.; Belghazi, M.; Job, D. Patterns of protein oxidation in Arabidopsis seeds and during germination. Plant Physiol. 2005, 138, 790–802. (33) Corpas, F. J.; Barroso, J. B.; Carreras, A.; Quiros, M.; Leon, A. M.; Romero-Puertas, M. C.; Esteban, F. J.; Valderrama, R.; Palma, J. M.; Sandalio, L. M.; et al. Cellular and subcellular localization of endogenous nitric oxide in young and senescent pea plants. Plant Physiol. 2004, 136, 2722–2733. (34) Gaupels, F.; Furch, A. C. U.; Will, T.; Mur, L. A. J.; Kogel, K.-H.; van Bel, A. J. E. Nitric oxide generation in Vicia faba phloem cells reveals them to be sensitive detectors as well as possible systemic transducers of stress signals. New Phytol. 2008, 178, 634–646. (35) Molassiotis, A.; Tanou, G.; Diamantidis, G. NO says more than ‘YES’ to salt tolerance: Salt priming and systemic nitric oxide signaling in plants. Plant Signal. Behav. 2010, 5, 1–4. (36) Bevan, M.; Bancroft, I.; Bent, E.; Love, K.; Goodman, H.; Dean, C.; Bergkamp, R.; Dirkse, W.; Van Staveren, M.; StiekemaW; et al. Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana. Nature 1998, 391, 485–488. (37) Komatsu, S.; Wada, T.; Abal, Y.; Nouri, M.-Z.; Nanjo, Y.; Nakayama, N.; Shimamura, S.; Yamamoto, R.; Nakamura, T.; Furukawa, K. Analysis of plasma membrane proteome in soybean and application to flooding stress response. J. Proteome Res. 2009, 8, 4487–4499. (38) Komatsu, S.; Yamamoto, R.; Nanjo, Y.; Mikami, Y.; Yunokawa, H.; Sakata, K. A comprehensive analysis of the soybean genes and proteins expressed under flooding stress using transcriptome and proteome techniques. J. Proteome Res. 2009, 8, 4766–4778. (39) Sobhanian, H.; Razavizadeh, R.; Nanjo, Y.; Ehsanpour, A. A.; Jazii, F. R.; Motamed, N.; Komatsu, S. Proteome analysis of soybean leaves, hypocotyls and roots under salt stress. Proteome Sci. 2010, 8, 19. (40) Palmieri, M. C.; Lindermayr, C.; Bauwe, H.; Steinhauser, C.; Durner, J. Regulation of plant glycine decarboxylase by S-nitrosylation and glutathionylation. Plant Physiol. 2010, 152, 1514–1528. (41) 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, 1605–1617. (42) Neill, S. J.; Desikan, R.; Clarke, A.; Hurst, R. D.; Hancock, J. T. Hydrogen peroxide and nitric oxide as signalling molecules in plants. J. Exp. Bot. 2002, 53, 1237–1247. (43) Neill, S. Interactions between abscisic acid, hydrogen peroxide and nitric oxide mediate survival responses during water stress. New Phytol. 2007, 175, 4–6. (44) Nouar, E. H.; Vereecke, D.; Goethals, K.; Jaziri, M.; Baucher, M. Screening for differential gene expression in Atropa belladonna leafy gall induced following Rhodococcus fascians infection. Eur. J. Plant Pathol. 2003, 109, 327–330. (45) Tsukuda, S.; Gomi, K.; Yamamoto, H.; Akimitsu, K. Characterization of cDNAs encoding two distinct miraculin-like proteins and stressrelated modulation of the corresponding mRNAs in Citrus jambhiri lush. Plant Mol. Biol. 2006, 60, 125–136. (46) Gimeno, J.; Gadea, J.; Forment, J.; Perez-Valle, J.; Santiago, J.; Martinez-Godoy, M.; Yenush, L.; Belles, J. M.; Brumos, J.; Colmenero-Flores, J. M. Shared and novel molecular responses of mandarin to drought. Plant Mol. Biol. 2009, 70, 403–420. (47) Johansson, E.; Olsson, O.; Nystrom, T. Progression and specificity of protein oxidation in the life cycle of Arabidopsis thaliana. J. Biol. Chem. 2004, 279, 22204–22208. (48) Nystro¨m, T. Role of oxidative carbonylation in protein quality control and senescence. EMBO J. 2005, 24, 1311–1317. (49) Møller, I. M.; Kristensen, B. K. Protein oxidation in plant mitochondria as a stress indicator. Photochem. Photobiol. Sci. 2004, 3, 730–735. (50) Zhang, J. H.; Sun, L.; Liu, L. L.; Lian, J.; An, S. L.; Wang, X.; Zhang, J.; Jin, J. L.; Li, S. Y.; Xi, J. H. Proteomic analysis of interactions between the generalist herbivore Spodoptera exigua (Lepidoptera: Noctuidae) and Arabidopsis thaliana. Plant Mol. Biol. Rep. 2010, 28, 324–333. (51) Qin, G.; Wang, Q.; Liu, J.; Li, B.; Tian, S. Proteomic analysis of changes in mitochondrial protein expression during fruit senescence. Proteomics 2009, 9, 4241–4253. (52) Qin, G.; Meng, X.; Wang, Q.; Tian, S. Oxidative damage of mitochondrial proteins contributes to fruit senescence: A redox proteomics analysis. J. Proteome Res. 2009, 8, 2449–2462. (53) Tan, Y.-F.; O’Toole, N.; Taylor, N. L.; Harvey Millar, A. H. Divalent metal ions in plant mitochondria and their role in interactions with proteins and oxidative stress-induced damage to respiratory function. Plant Physiol. 2010, 152, 747–761. (54) Stohr, C.; Stremlau, S. Formation and possible roles of nitric oxide in plant roots. J. Exp. Bot. 2006, 57, 463–470.

PR100782H

Proteomic Signatures Uncover Hydrogen Peroxide and Nitric Oxide

Recommend Documents