ORIGINAL PAPER

Identification of changes in Triticum durum L. leaf proteomein response to salt stress by two-dimensional electrophoresisand MALDI-TOF mass spectrometry

Giuseppe Caruso & Chiara Cavaliere & Chiara Guarino &

Riccardo Gubbiotti & Patrizia Foglia & Aldo Laganà

Received: 20 December 2007 /Revised: 18 February 2008 /Accepted: 20 February 2008 /Published online: 27 March 2008# Springer-Verlag 2008

Abstract In order to understand the molecular basis ofsalt stress response, a proteomic approach, employing two-dimensional electrophoresis and matrix-assisted laserdesorption/ionization time of flight mass spectrometry(MALDI-TOF MS), was used to identify proteins affectedby salinity in wheat (Triticum durum ‘Ofanto’). Identifica-tion of proteins, whose levels were altered, was performedby comparing protein patterns of salt-treated and controlplants. A set of control plants was grown without NaCladdition under the same conditions as the salt-treatedplants. Proteins were extracted from the leaves of untreatedand NaCl-treated plants, and resolved using 24-cm immo-bilized pH gradient strips with a pH 4–7 linear gradient inthe first dimension and a 12.5% sodium dodecyl sulphatepolyacrylamide gel electrophoresis in the second dimen-sion; the gels were stained with Coomassie and imageanalysis was performed. Quantitative evaluation, statisticalanalyses and MALDI-TOF MS characterization of theresolved spots in treated and untreated samples enabled usto identify 38 proteins whose levels were altered inresponse to salt stress. In particular, ten proteins weredownregulated and 28 were upregulated. A possible role ofthese proteins in response to salinity is discussed.

Keywords Matrix-assisted laser desorption/ionization timeof flight . Proteomics . Salt stress . Triticum durum .Wheat .

Two-dimensional electrophoresis

Introduction

Salinity is one of the major abiotic stresses in plantagriculture worldwide, and an excess amount of salt in thesoil adversely affects plant growth and development. Nearly20% of the world’s cultivated area and about half of theworld’s irrigated lands are affected by salinity [1]. Salt stressis a limitation to agricultural production; in fact it is estimatedthat this environmental stress causes the loss of about 50% ofagricultural yield, compared with about 10–20% yield losscaused by pathogen agents [2]. Therefore, successfulstrategies to increase plant salt tolerance are in high demand.

Firstly, high salt concentration decreases the osmoticpotential of soil solution, creating a water stress in plantsthat cannot tolerate it (glycophytes) [3]. Secondly, high saltconcentration causes severe ion toxicity, because Na+ is notreadily sequestered into vacuoles as in halophytes [4].Finally, the interaction of salts with mineral nutrients mayresult in imbalances and deficiencies. Consequently mem-brane disorganization, photosynthesis inhibition, generationof toxic metabolites and reactive oxygen species (ROS),and attenuated nutrient acquisition could occur, followedeventually by cell and whole plant death [5]. Salt stressshows a high degree of similarity to dehydration stress withrespect to physiological, biochemical, molecular and genet-ical effects [6]. This is possibly due to the fact that thesublethal salt-stress condition is ultimately an osmoticeffect, which is apparently similar to that caused bywater deficit and to some extent by cold as well as heatstresses [7]. Plant response to salinity stress, and the

Anal Bioanal Chem (2008) 391:381–390DOI 10.1007/s00216-008-2008-x

G. Caruso : C. Cavaliere : C. Guarino : R. Gubbiotti : P. Foglia :A. LaganàDepartment of Chemistry, “Sapienza” University of Rome,Piazzale Aldo Moro 5,00185 Rome, Italy

A. Laganà (*)Dipartimento di Chimica, “Sapienza” Università di Roma,Box no. 34, Roma 62, Piazzale Aldo Moro 5,00185 Rome, Italye-mail: aldo.lagana@uniroma1.it

development of salt tolerance involve several extremelycomplex mechanisms [8], including molecular, biochem-ical and physiological processes as well as morphologicaland developmental changes [9].

The plant response to salt stress may be studied bycomparative proteomic research, because the proteome, incontrast to the genome, may markedly change as a result ofenvironmental factors [10]. Moreover, it is well known thatonly by direct protein analysis is it possible to study cellularand molecular mechanisms, because protein expressionlevels are not always correlated to messenger RNA levels[11], probably owing to large differences in proteinturnover and posttranslational modifications.

In salt resistance, specific changes in the profile ofproteins, whose biological functions are related to suchenvironmental stress tolerance, have been observed in manyplants [12–16], and several proteins have been character-ized to play prominent roles in response to salt stress.However, changes in protein profiles also differ dependingon plant organs and species [17].

Two-dimensional electrophoresis (2-DE) in conjunctionwith mass spectrometry (MS) analysis has been used toinvestigate root [18] and shoot [19] proteome-level differ-ences between varieties of wheat exhibiting differentialsalinity tolerance. The proteomic approach, based on 2-DEand MS analysis, may offer the possibility to identifyproteins associated with a particular environmental and/ordevelopmental signal [20].

The aim of the present research was to identify changesin protein profile, due to exposure to salt stress, in wheatplants (Triticum durum ‘Ofanto’) by comparative proteo-mics between control and salt-treated plants, in order toincrease the data set of proteins involved in plant toleranceto salinity. To our knowledge, this is the first work dealingwith salt-stress response in leaf proteome of durum wheatby 2-DE MS. This study may aid in improving agriculturalproduction by characterizing the salt-resistant varieties.

Materials and methods

Plant growth and stress treatment

Triticum durum ‘Ofanto’ plants (Experimental Institute ofCereal Research, Rome, Italy) were grown from seeds in agrowth cabinet under controlled conditions. The lightintensity was 300 μmol m−2 s−1 for a 12-h photoperiod,the temperature was 20 °C for the light period and 15 °Cfor the dark period, and the relative humidity was 70%.After two leaf stages (about 8 days) the experimental plantswere divided in two groups: a group of plants was irrigatedwith a 100 mmol L−1 NaCl solution for 2 days (stressedplants); the other group of plants was regularly irrigated

(control plants). All plants were harvested 2 days after thebeginning of the NaCl treatment.

Total soluble protein extraction and quantification

Proteins were extracted using the trichloroacetic acid(TCA)/acetone/2-mercaptoethanol precipitation method de-veloped by Damerval et al. [21], applying some modifica-tions. Wheat leaf tissue (5 g) was frozen in liquid N2 andground to a fine powder using a ceramic mortar and pestle.One gram of the resulting powder was suspended in 5 mL ofchilled (4 °C) extraction buffer containing 175 mmol L−1 tris(hydroxymethyl)aminomethane hydrochloride (pH 8.8),50 g L−1 sodium dodecyl sulphate (SDS), 150 mL L−1

glycerol, 0.70 mL L−1 2-mercaptoethanol and 10 mL L−1

plant protease inhibitor mix, and grinding was maintainedfor an additional 30 s. Homogenized cell debris wasremoved by filtering the homogenate through two layers ofmiracloth, and centrifuging at low speed (500 g) at 4 °C for15 min. The supernatant was collected and mixed byvortexing with 20 mL of cold (−20 °C) acetone containing100 g L−1 TCA and 0.70 mL L−1 2-mercaptoethanol, andwas maintained at −20 °C for at least 1 h to allow proteinprecipitation. Then, precipitated proteins were centrifuged at15,000 g at 4 °C for 45 min, and then the pellet was threetimes washed with a cold water/acetone solution (20:80, v/v)containing 0.70 mL L−1 2-mercaptoethanol and betweenrinses was centrifuged at 15,000 g for 15 min. Successively,the supernatant was removed and the pellet was slowly driedunder nitrogen and resuspended in the isoelectric focusing(IEF) extraction solution consisting of 9 mol L−1 urea,20 g L−1 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate, 30 g L−1 DTT and 20 mL L−1 pH 4–7ampholytes. In order to obtain a complete protein solubili-zation, the sample was incubated for 2 h at 33 °C. Finally,the sample was centrifuged at 15,000 g at 4 °C for 30 minand the supernatant was subjected to IEF. Protein concen-tration was determined using the 2D QUANT kit (GEHealthcare, Uppsala, Sweden) with bovine serum albumin asthe standard.

Two-dimensional electrophoresis

Two-dimensional electrophoresis (2-DE) of proteins wasperformed in accordance with the method of O’Farrel [22],with some modifications. Briefly, 450 μL of solutioncontaining 500 μg of proteins was applied in the strip holderand immobilized pH gradient strips (24 cm, pH 4–7) wereplaced and covered with paraffin oil. IEF was carried out withan Ettan IPGphor IEF system (GE Healthcare) applying thefollowing conditions. For the rehydration step the voltage wasmaintained for 12 h at 30 V, then the proteins were focused for1 h at 500 V, 1 h at 1000 Vand 8 h and 20 min at 8,000 V. The

382 Anal Bioanal Chem (2008) 391:381–390

temperature was maintained at 20 °C and the current was50 μA per strip. After IEF, the strips were equilibrated in DTTfollowed by iodoacetamide as described by Chivasa et al. [23]and then stored at −20 °C. The second-dimension separationof proteins was performed according to the method ofLaemmli [24] on a 12.5% SDS polyacrylamide gel using anEttan DALT Six electrophoresis unit (GE Healthcare). Theelectrophoresis was carried out at 25 °C and 2.5 W per gelfor 30 min and then 17 W per gel for 5 h and 40 min untilthe bromophenol blue dye front arrived at the bottom of thegels. Following SDS polyacrylamide gel electrophoresis(PAGE), gels were stained with Coomassie R 350 inaccordance with to the manufacture’s manual (GE Health-care). A total of six gels were analysed, three gels foruntreated plants and three gels for NaCl-treated plants.

Gel image and data analysis

The 2-DE image and statistical analysis was performed byusing the ImageScanner™ II (GE Healthcare) and theImageMaster 2D Platinum software (GE Healthcare). Inorder to evaluate if the observed qualitative or quantitativechanges arise from analytical variability or from biologicalconditions, three biologically independent samples wereprepared for each group of plants, stressed plants andcontrol plants. The stained gels were divided into two setscorresponding to the two different group of plants; prior toperforming spot matching between gel images, one gelimage was selected as a reference. Image analysis wascarried out by spot detection, spot measurement, back-ground subtraction and spot matching. After automatic spotdetection, gel images were manually edited, such asartificial spot deletion, spot splitting and merging. Theamount of a protein spot was expressed as a volume,corresponding to the pixel density. For protein expressionanalyses, the volume of each spot was expressed as relativevalue, calculated as ratio between the individual spotvolume and the sum of all spot volumes (percentagevolume). This procedure was corrected for experimentalvariations due to protein loading and staining. Gel patternsfrom each independent analysis were matched together andthe relative abundances of each spot (percentage volume) inthe two gel sets (control and stressed) were compared. Dataanalysis was carried out at two different levels: proteinexpression changes within a set of gels, and significantprotein expression changes between sets of gels. Forintraset data analysis the following were used: (1) scatterplots, to analyse gel similarities or experimental variationssuch as disparities in stain intensity or sample loading; (2)descriptive statistics of central tendency and dispersion ofthe spot volume, which can be calculated and displayed inintraset reports and histograms; (3) factor analysis, enablingthe identification of similar gels, and of spots that are

characteristic for a particular population of gels; (4)heuristic clustering, an artificial intelligence technique toautomatically classify sets of gel and highlight significantproteins spots. For interset data analysis the following wereused: (1) overlapping measures, which summarize each setof gels as an interval and measure the overlap betweenthese intervals; (2) statistical tests, which asses thedifference between populations and give an estimate forthe significance of the difference. These statistics tools wereused in accordance with the guidelines of the ImageMaster2D Platinum software. The differences in expressionbetween control and treated samples were analysed byStudent’s t test, and the protein spots showing a significantdifference (p<0.05) of abundance change (up or down)were considered as “proteins of interest”.

Spot excision and protein in-gel digestion

The “protein spots of interest” were excised from two-dimensional gels with razor-shortened plastic pipette tipsand the gel plugs were dislodged with an intact pipette tipinto wells of a 96-well polypropylene ZipPlate micro-SPEplate (Millipore Corporation, Billerica, MA, USA). Trypticin-gel digestion was based, with some modifications, on theprotocol of Parker et al. [25]. The procedure was optimizedfor polyacrylamide gels of 1.5-mm thickness. Briefly, each gelpiece was rinsed once for 30 min with 100 μL of 25 mmol L−1

ammonium bicarbonate/acetonitrile (95:5, v/v), twice for30 min with 100 μL of 25 mmol L−1 ammoniumbicarbonate/acetonitrile (50:50, v/v), and then the gel pieceswere dehydrated for 10 min with 200 μL of acetonitrile. Theenzymatic digestion was therefore performed at 37 °C for3 h with 15 μL of trypsin (11 ng μL−1); then 130 μL of a20 mL L−1 trifluoroacetic acid (TFA) solution was added toeach well containing a gel piece, and the mixture wasincubated at room temperature for 30 min. The gel pieceswere dried under vacuum and twice washed with 100 μL ofthe 20 mL L−1 TFA solution. Finally, the resulting trypticfragments were recovered with 20 μL of acetonitrile/water(50:50, v/v) containing 0.50 mL L−1 TFA.

Matrix-assisted laser desorption/ionization time of flightMS analysis and database searching

For matrix-assisted laser desorption/ionization (MALDI) timeof flight (TOF) MS, the peptide sample was mixed with anequal volume of the matrix solution, consisting of 5 g L−1

recrystallized α-cyano-4-hydroxycinnamic acid, dissolved inacetonitrile/water (50:50, v/v), and 0.50 mL L−1 TFA, andthen 1 μL of the mixture was spotted on a MALDI plate.Mass spectra were acquired using a Voyager-DE STR™mass spectrometer (Applied Biosystems, Framingham, MA,USA), and peptides were detected in the reflectron-delayed

Anal Bioanal Chem (2008) 391:381–390 383

extraction mode, in the m/z range 500–4,500. A closeexternal calibration was employed using calibration mixture1 (Applied Biosystems) followed by internal mass correctionwith the accurate mass of peptide ions generated by trypsinautolysis (at m/z 842.51 and 2,211.10). Spectra from 100shots at ten different positions were combined to generate apeptide mass fingerprinting (PMF) for each protein sample.Peptide spectra were automatically processed applyingbaseline correction, noise removal and peak deisotoping. InFig. 1 is displayed an example of a MALDI-TOF massspectrum of the tryptic digest (protein spot 6); a correctisotopic distribution and a signal-to-noise of more than 2 areminimal requirements for the definition of mass peaks. Thedetermination of the first isotope with accuracy is sometimesdifficult for masses with a mass in excess of 2,500 Da,depending on the resolution of the peak. The mass accuracyof the measurements strongly depends on the mass spec-trometer. Improving the mass accuracy by internal calibra-tion is one way to reduce false-positive protein matches. Theprocessed spectra were used for searching in MSDB andNCBInr databases, using Mascot (http://www.matrixscience.com). Since only partial sequence information is availablefor the Triticum durum genome and many genes forabundant proteins are highly conserved in plants, availableproteins were searched from all higher plants (Streptophyta).This allowed us to identify proteins using homology tohighly conserved proteins from other plants, especiallyTriticum aestivum, for which the most extensive informationabout genome sequence is currently available.

Results

In order to investigate the changes of leaf proteome inresponse to salt stress, 2-DE analysis of the total proteins inTriticum durum leaves was carried out. Figure 2 shows thereference 2-DE electrophoretic maps obtained from controlplants (Fig. 2a) and NaCl-treated plants (Fig. 2b), and Fig. 3shows examples of protein spots with a significant difference(p<0.05) in abundance (up or down) between control andNaCl-treated plants. The protein spots showed a broaddistribution in the pI range from 4.0 to 7.0 and the massrange from 10 to 120 kDa. Approximately 850 protein spotswere detected on Coomassie R 350 stained gels and about600 protein spots were matched between three control gelsand three treated gels. Spot intensity variations betweenuntreated and treated samples were quantified by softwareimage analysis, and the protein spots showing significantdifferences (p<0.05) were selected and excised for trypticdigestion and MALDI-TOF MS PMF characterization. SomeNaCl-responsive protein spots were not excised owing totheir low abundance. Indeed, these protein spots were belowthe threshold of Coomassie R 350 staining, and thereforewere not well visible for picking up. In Fig. 2 and Table 1are shown and listed the identified protein spots whoseexpression level was observed to change in response to saltstress. It was found that some proteins were identified inmore than one spot, although they were excised from thesame gel (Fig. 2, Table 1). For instance, ATP synthase wasidentified in two spots (spots 2 and 4), phosphopyruvate

%INTENSITY

MASS (m/z)

1.0 E+ 6

Fig. 1 Matrix-assisted laser desorption/ionization time of flight mass spectrum of the tryptic digest (protein spot 6), obtained in the positive iondelayed extraction reflector mode for highest resolution and mass accuracy

384 Anal Bioanal Chem (2008) 391:381–390

hydratase was identified in two spots (spots 7 and 8), S-adenosylmethionine synthetase was identified in two spots(spots 10 and 11), plastid glutamine synthetase wasidentified in three spots (spots 14–16), ferredoxin NADP(H) oxidoreductase was identified in two spots (spots 19 and20), ribulose 1,5-bisphosphate carboxylase–oxygenase(RuBisCO) large subunit (fragment) was identified in twospots (spots 22 and 23), carbonic anhydrase was identified intwo spots (spots 25 and 27), ascorbate peroxidase wasidentified in two spots (spots 26 and 31), triosephoshate

isomerase was identified in three spots (spots 28–30), thiol-specific antioxidant protein was identified in two spots (spots33 and 34) and RuBisCO small subunit was identified in twospots (spots 37 and 38). Proteins present in more than onespot could be isoforms, perhaps due to post-translationalmodification, or degradation. Posttranslational modificationssuch glycosylation, phosphorylation, etc. can change themolecular weight and/or the pI of proteins. Among 38identified protein spots, 28 proteins were upregulated, andten proteins were downregulated (Fig. 2, Table 1).

pH7 IEF pH4

S

D

S

P

A

G

E

2

1

3 4

11

7

5

8

12

9

13 14

6

15 16

17

10

2018

2223

21

2826

27

29

24

30 31

25

36

33 34

32

37 38

35

19

pH7 IEF pH4

S

D

S

P

A

G

E

1

2 3 45 6

7 89

10 11 1213

17

15 16

18

1920 21

2223 24

2526

27

2829 30 31 32

33 34

35

36

37 38

14

a

b

Fig. 2 Reference two-dimensional electrophoreticpatterns of soluble leaf proteinsobtained from a control andb NaCl-treated plants. Proteinswere resolved using a lineargradient pH 4–7 in the firstdimension and 12.5% sodiumdodecyl sulphate (SDS) poly-acrylamide gel electrophoresis(PAGE) in the second dimen-sion. Gels were stained withCoomassie R 350. Protein spotsidentified are indicated with acircle and a number. IEF iso-electric focusing

Anal Bioanal Chem (2008) 391:381–390 385

Discussion

Our research allowed the identification of 38 protein spotswhose levels were altered by salinity exposure; however,other proteins could be differentially expressed but notdetected in our two-dimensional gels owing to their lowconcentration. Some of the identified proteins have beenwell characterized in terms of their response to salinity andother abiotic stress; on the other hand, the role of otherproteins is not clear yet in abiotic stress. All these proteinsare involved in regulation of carbohydrate, amino acid,nitrogen and energy metabolism, ROS scavenging andnucleic acid and protein processing. Under NaCl stress,plants decrease energy metabolism rates to conserve energyand limit further generation of ROS [26]. Therefore, it isnot surprising to observe that in the present study theabundance of proteins, involved in carbohydrate and energymetabolism, decreased after NaCl treatment (Table 1),whereas the abundance of some proteins involved in ROSscavenging increased (Table 1).

Phosphoglycerate kinase (spot 12) utilizes ATP tophosphorylate 3-phosphoglycerate to form 1,3 bisphospho-glycerate. This reaction represents the first reaction in thereduction step of the Calvin cycle. Decreased expression ofthis enzyme could indicate a decrease in photosyntheticcarbon assimilation owing to a direct reduction in stomatalconductance and subsequent low CO2 levels followingexposure to a long-term salt stress. Phosphoribulokinase(spot 17) catalyses the ATP-dependent phosphorylation ofribulose-5-phosphate to ribulose-1,5-phosphate, a key stepin the pentose phosphate pathway where CO2is assimilatedby autotrophic organisms. Our results show that under thesalt-stress condition phosphoribulokinase was downregu-lated. Fructose 1,6-bisphosphate aldolase (spot 21) is a keymetabolic enzyme catalysing the cleavage of β-fructose-1,6-phosphate to D-glyceraldehyde-3-phosphate and dihy-droxyacetone phosphate in glycolysis and the reversereaction in gluconeogenesis. Two different forms ofaldolase are present in higher plants: cytoplasmic and

plastidic. The physiological meaning of cytoplasmatic forminduction must be different from that of plastidic forminduction, because the two forms are involved in differentmetabolic pathways and catalyse the same reactionin opposite directions. The abundance of fructose 1,6-biphosphate aldolase identified from wheat in this investi-gation was decreased in salt-treated plant in comparisonwith the controls. Two protein spots (spots 37 and 38) wereidentified as RuBisCO small subunit. RuBisCO is the keyenzyme of the Calvin cycle and catalyses the reaction of D-ribulose 1,5-bisphosphate and CO2 to two 3-phospho-D-glycerate molecules. It was noted that the abundance ofRuBisCO small subunit decreased in response to salinity.We found that photosystem II (PII) oxygen-evolvingcomplex protein 1 (spot 24) precursor was downregulatedin response to salt stress. In contrast, Abbasi and Komatsu[27] showed that this protein was upregulated in rice leafsheath under salt stress. It is important to note that in theirresearch the increased expression of protein was lesspronounced in the 50 mmol L−1 NaCl treatment group thanin the groups receiving 100 or 150 mmol L−1 NaCl for24 h. Our plants were exposed to 100 mmol L−1 NaCl for48 h, and this more severe treatment could have led todownregulation of the PSII oxygen-evolving complexprotein 1 precursor. β-Glucosidase (spot 3) catalyses thehydrolysis of 1,3-β-D-glucosidic linkages in 1,3-β-D-glucans and it is implicated in three processes. First, it isinvolved in the alteration of specific β-linked polysacchar-ides during cell expansion in development [28]. Moreover,it is involved in pathogen defence reactions by cyanogen-esis, because the enzyme catalyses the hydrolysis ofglucosides after pathogen attack [29]. Furthermore, β-glucosidase could release active cytokinins, gibberellinsand auxins from biologically inactive hormone–glucosideconjugates [30]. In our study, β-glucosidase was down-regulated in salt-treated plants, confirming its involvement inthe response to salt stress, although its function(s) in salt stressis not known. Another energy-related protein, ATP synthaseCF1α subunit (spots 2 and 4), was found to be downregulated

3636

Control NaCl-treated

1111

Control NaCl-treated

Control NaCl-treated Control NaCl-treated

3838

2121

Fig. 3 Examples of proteinspots showing a significant dif-ference (p<0.05) in abundance(up or down) between controland NaCl-treated plants; thenumbers refer to those in Fig. 2

386 Anal Bioanal Chem (2008) 391:381–390

Tab

le1

Identificationof

saltrespon

sive

proteins

inTriticum

durum

L.leaf

bypeptidemassfing

erprintin

g

Spo

tnu

mbera

Increasing

ordecreasing

compo

nent

Theoretical

pI/m

olecular

mass(kDa)

Exp

erim

ental

pI/m

olecular

mass(kDa)

Protein

identificationandspecies

Accession

number

MOWSE

score

Num

berof

peptides

matched

Cov

erage

(%)

1↑

6.32/112

6.29

/111

Glycine

dehydrog

enase(decarboxy

latin

g)Hordeum

sp.×Triticum

sp.

T46

636

397

4958

2↓

6.11/55

6.41

/65

ATPsynthase

CF1αsubu

nitTriticum

aestivum

gi|14017

569

186

2348

3↓

6.35/65

6.20

/69

β-G

lucosidase

Triticum

aestivum

Q1X

IR9

178

2853

4↓

5.70/56

6.00

/63

ATP1Triticum

aestivum

gi|81176

509

193

2445

5↑

5.43/30

5.58

/78

Phospho

glyceratemutase,

(fragm

ent)Triticum

aestivum

Q7X

YD2

158

2062

6↑

4.88/54

5.27

/74

RuB

isCO

subu

nitbind

ingproteinβsubu

nitSecale

cereale

gi|24936

5029

632

737

↑5.70/48

5.65

/59

Phospho

pyruvate

hydrataseZea

mays

T02

221

116

1950

8↑

5.70/48

5.55

/60

Phospho

pyruvate

hydrataseZea

Mays

T02

221

7612

239

↑5.92/59

5.41/62

Glucose-6-phosphate

dehydrogenaseTriticum

aestivum

Q9L

RJ0

6813

3710

↑5.61/43

5.88

/49

S-Adenosylm

ethioninesynthetase

1Triticum

mon

ococcum

gi|115

5897

4417

821

5611

↑5.61/43

5.73

/50

S-Adenosylm

ethioninesynthetase

1Triticum

mon

ococcum

gi|115

5897

4419

521

5812

↓6.58/50

5.41

/50

Phospho

glyceratekinase

Triticum

aestivum

gi|12991

517

621

6113

↑8.62/47

5.36/48

RuB

isCO

activ

aseisoform

1Hordeum

vulgaresubsp.

vulgare

gi|16709

613

423

5114

↑6.04/47

5.09

/47

Plastid

glutam

inesynthetase

isoform

GS2b

Triticum

aestivum

Q45

NB3

144

1431

15↑

5.42/47

4.99

/48

Plastid

glutam

inesynthetase

isoform

GS2a

Triticum

aestivum

gi|71362

455

6611

2616

↑5.75/47

4.91

/48

Plastid

glutam

inesynthetase

isoform

GS2c

Triticum

aestivum

Q45

NB2

127

1640

17↓

5.72/45

5.05

/45

Phospho

ribulokinase

Triticum

aestivum

S1658

515

522

6418

↓7.21/41

6.83/43

GADPH

(fragm

ent)Zea

mays

Q6L

BU9

7910

2819

↑8.29/39

6.35

/41

Ferredo

xinNADP(H

)ox

idoreductase

Triticum

aestivum

Q8R

VZ9

144

2253

20↑

8.29/39

6.08

/42

Ferredo

xinNADP(H

)ox

idoreductase

Triticum

aestivum

Q8R

VZ9

114

1843

21↓

9.01/42

5.61

/41

Fructose1,6-bisphosphate

aldolase

Avena

sativa

Q9L

LD7

8013

3322

↑7.07/50

6.83

/35

RuB

isCO

largesubu

nit,(fragm

ent)Triticum

aestivum

RKWTLC

124

2636

23↑

6.28/46

6.57

/33

RuB

isCO

largesubu

nit,(fragm

ent)Triticum

aestivum

Q37

335

8316

3724

↓5.89/35

5.27/34

Photosystem

IIoxygen-evolvingcomplex

protein1Com

mon

toba

cco

T02

066

737

1825

↑8.93/36

6.59

/28

CarbonicanhydraseHordeum

vulgare

gi|72900

317

616

4926

↑5.85/28

6.31

/29

Ascorbate

peroxidase

Hordeum

vulgare

O23

983

112

1143

27↑

8.93/36

6.18

/25

CarbonicanhydraseHordeum

vulgare

gi|72900

313

614

4328

↑5.38/27

5.89/27

Triosephosphat-isom

eraseTriticum

aestivum

Q9F

S79

139

1454

29↑

6.00/32

5.42/28

Triosephosphat-isom

eraseSecale

cereale

S5376

117

018

6830

↑6.00/32

5.31/27

Triosephosphat-isom

eraseSecale

cereale

S5376

122

417

6131

↑5.10/28

5.25

/27

Ascorbate

peroxidase

Hordeum

vulgare

Q94

5R5

145

1253

32↑

4.76/31

4.35

/27

Nucleic

acid

bind

ingproteinHordeum

vulgare

T05

727

119

1449

33↑

5.48/23

4.97

/24

Thiol-specificantio

xidant

proteinHordeum

vulgare

gi|24994

7796

747

34↑

5.48/23

4.85

724

Thiol-specificantio

xidant

proteinHordeum

vulgare

gi|24994

7714

311

7135

↑4.84/17

4.58/20

Cold-responsive

LEA/RAB-related

CORproteinTriticum

aestivum

gi77

1695

683

953

36↑

5.35/20

5.31

/19

Cu/Znsuperoxide

dism

utaseTriticum

aestivum

gi|15686

3910

57

5337

↓8.80/20

6.15

/15

RuB

isCO

smallsubunitTriticum

aestivum

Q9F

RZ4

206

1871

38↓

8.80/20

5.73

/15

RuB

isCO

smallsubunitTriticum

aestivum

Q9F

RZ4

207

1253

RuB

isCO

ribu

lose

1,5-bispho

sphate

carbox

ylase–ox

ygenase

aFor

spot

numbers

seeFig.2.

Anal Bioanal Chem (2008) 391:381–390 387

in response to salinity stress. The decrease in ATP synthaseCF1 α subunit could be associated with transiently decreasedphotosynthesis rates and the downregulation of the Calvincycle enzyme phosphoglycerate kinase.

In contrast, three proteins, phosphopyruvate hydratase,triose phosphate isomerase and glucose-6-phosphate dehy-drogenase, involved in regulation of carbohydrate metabo-lism, increased in salt-treated plants. Differential regulationof these proteins has previously been reported in NaCl-treated roots, and it may reflect subfunctionalization ofrelated enzymes for optimal activity in different cells orcellular microenvironments [31]. It has been noted thatphosphopyruvate hydratase (spots 7 and 8), which catalysesthe formation of high-energy phosphoenol pyruvate from 2-phosphoglycerate in the glycolytic pathway, increased aftersalt-stress treatment. It is notable that phosphopyruvatehydratase is also involved in the response to otherenvironmental stresses, such as anaerobic stress of maizeroots [32], heat shock in yeast [33], heat shock, salt stress,abscisic acid (ABA) treatment, and water stress in thecommon ice plant [34]. Triosephosphate isomerase (spots28–30) catalyses the reversible interconversion of the triosephosphate isomers dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate. It plays an important role inglycolysis and it is essential for efficient energy production.Previously 2-DE analysis showed that drought induces inmaize expression of triosephosphate isomerase [35]. Ourresults show that it was also induced by salt stress in wheat.Glucose-6-phosphate dehydrogenase (spot 9) is a keyenzyme that catalyses a nonequilibrium reaction, and thusregulates the flux of carbon through the pentose phosphatepathway. It is also a key enzyme that catalyses the first stepof the oxidative pentose phosphate pathway. The mainfunction of the pentose phosphate pathway is to provideNADPH and other intermediates, such as pentose anderythrose 4-phosphate [36]. A preceding study [37]reported that transcriptional activation of the gene ofglucose-6-phosphate dehydrogenase in wheat specificallyresponds to NaCl stress treatment. This is agreement withour results, in which the glucose-6-phosphate dehydroge-nase was upregulated.

The amount of proline and other amino acids is reportedto increase following NaCl treatment [38, 39]. In studiedplants most there is an increased accumulation of aminoacids and amines (e.g. proline, β-alanine, glycine betaine)in their tissues in response to salt stress. The way thesecompounds are accumulated differs among species, andranges from only one to several different compounds beingaccumulated. The synthesis and the storage of lowmolecular weight metabolites, known as compatible sol-utes, is a ubiquitous mechanism for osmotic adjustment inplants. Their main role is to increase the ability of cells toretain water without affecting the normal metabolism [40].

It was observed that the abundance of three enzymes relatedto amino acid biosynthesis was influenced by NaCl(Table 1). Glycine is a precursor for glycine betaine; thereare at least two mechanisms by which the two-carbonmoiety of glycine betaine is derived from glycine. Glycinedehydrogenase (decarboxylating), an enzyme involved inthe C-2 glycolate pathway in higher plants, can decarbox-ylate glycine to form serine, which can generate ethanol-amine after decarboxylation again. Moreover, ethanolaminecan undergo a series of methylations to form choline andthen glycine betaine is formed as a result of the oxidation ofcholine. Glycine dehydrogenase (decarboxylating) (spot 1)was found to be lightly upregulated in response to salinity.S-Adenosylmethionine synthetase (spots 10 and 11) wasfound to be upregulated, it catalyses the biosynthesis of S-adenosyl-L-methionine, which serves as a methyl groupdonor in highly specific transmethylation reactions involv-ing all kinds of acceptor molecules such as proteins, nucleicacids, phenylpropanoid derivatives, polysaccharides, cyclicfatty acids, etc [41]. In plants, this enzyme also plays a rolein amino acid biosynthesis and polyamine biosynthesis.Polyamines are aliphatic amines of low molecular weightcharged positively at physiological pH. The positive chargepermits their interaction with proteins, membrane lipids andDNA. It is well known that the activity of the plantenzymes of polyamine biosynthesis is induced underabiotic stress, including salinity. In addition, induction ofS-adenosylmethionine synthetase by salt stress might benecessary to cope with a higher demand for S-adenosyl-L-methionine for the increased lignification, because ligninmonomers were methylated before polymerization. Gluta-mine synthetase catalyses the ATP-dependent condensationof ammonium with glutamate to yield glutamine, whichthen provides nitrogen groups for the biosynthesis of allnitrogenous compounds in the plant. Two distinct isozymes,plastidic and cytosolic, have been identified in higher plants[42]. Proline is an important component of salt-stressresponses of plants; it is an osmoprotector and anosmorregulator. Recent data suggest that glutamine is themajor amino acid involved in proline synthesis. In thiswork, we identified three different glutamine synthetaseisoforms (spots 14–16) that were upregulated. A reportshowed that proline accumulation was at least in part due tothe increased glutamine synthetase activity under salt stressin cashew [43].

In order to maintain homeostasis under stress conditions,plants need to fortify the resistance mechanisms, such asROS scavenging and cell defence. ROS can cause damageto cellular components and can act as signalling moleculesfor stress responses [44]. Plants can regulate the ROS levelthrough complex mechanisms of several enzymes whosefunctions are implicated in cell detoxification and celldefence. In this study, the abundance of proteins involved

388 Anal Bioanal Chem (2008) 391:381–390

in these mechanisms was increased following NaCltreatment. One of the major antioxidant enzymes and free-radical scavengers in plants is ascorbate peroxidase (spots26 and 31), which can detoxify H2O2 by oxidizing specificsubstrates such as ascorbate. Ascorbate peroxidase has beenfound to be upregulated by salt stress. It was reported, forexample, that the transcripts of ascorbate peroxidase werestrongly induced by salt stress in pea [45]. The chloroplast[Cu–Zn] superoxide dismutase (spot 36) was shown toincrease its level of expression after salinity was increased.Superoxide dismutase forms part of an enzymatic detoxi-fication system for the scavenging of ROS [46]. The O��

2

radical is the main source of oxidative injury in plants andthe dismutation reaction catalysed by superoxide dismutasemaintains intracellular O��

2 within normal levels, andvarious authors have implicated this enzyme in theprotection of cells from hydric-oxidative stress [47].Thiol-specific antioxidant protein (spots 33 and 34) wasthe first peroxiredoxin to be identified. The peroxiredoxinsare a family of multiple isozymes that catalyse thereduction of H2O2 and protect the cells against oxidativedamage in plants. The upregulation of the thiol-specificantioxidant protein, identified in this study, indicates that itmight play an important role in ROS scavenging under saltstress. Ferredoxin NADP+ oxidoreductase (spots 19 and 20)is a flavoenzyme that catalyses the reversible electrontransfer between NADP(H) and [2Fe–2S] ferredoxins orflavodoxins [48]. In plants ferredoxin NADP+ oxidoreduc-tase is implicated in photosynthesis and nitrogen fixation inplastids; recently a new role was proposed for it in cellprotection against ROS, but at present, the mechanism bywhich ferredoxin NADP+ oxidoreductase protects cellsagainst the presence of ROS in plants remains unknown[49]. In wheat, salt stress induced an upregulation offerredoxin NADP+ oxidoreductase.

Carbonic anhydrase (spots 25 and 27) was upregulatedin salt-treated plants. It is a zinc-containing metalloenzyme,and widely distributed in animals, plants, archaebacteriaand eubacteria, it catalyses the reversible interconversion ofCO2 and HCO�

3 CO2 þ H2O $ HCO�3 þ Hþ� �

[50, 51].Increased expression of carbonic anhydrase may influencethe rate of the reversible reaction HCO�

3 þ Hþ $ CO2þH2O, and thus it may play a role in the buffering capacityof plant cells suffering from high concentrations of HCO�

3

and CO2�3 and facilitating CO2 diffusion. Under salt stress

the enzyme activities are affected and the basic metabo-lisms are disturbed.

Some proteins may be degraded during abiotic stress,and ROS may contribute to the degradation of proteinsunder stress conditions. It was found that two differentfragments (spots 22 and 23) of the RuBisCO large subunitas well as one fragment of phosphoglycerate mutase (spot5) were upregulated in salt treated plants. Hajduch et al.

[52] reported an increase in the number of RuBisCO largesubunit breakdown products in metal-stressed rice leaves.In contrast, one fragment of GADPH (spot 18) was foundto be downregulated in treated plants.

ABA is one of the key plant hormones responding toenvironmental stresses. Salt, drought and, to some extent,cold stress cause increased biosynthesis and accumulationof ABA, which can be rapidly catabolized following therelief of stress. Increased ABA levels under drought andsalt stress are mainly achieved by the induction of genescoding for enzymes that catalyse ABA biosyntheticreactions. Cold-responsive LEA/RAB-related COR protein(spot 35) is responsive to the ABA pathway [53]. Thisprotein, which in this study was upregulated, in both plantsand animals is associated with tolerance to abiotic stress.However, although various functions of cold-responsiveLEA/RAB-related COR protein have been proposed, itsprecise role has not yet been defined. RuBisCO largesubunit binding protein subunit β (spot 6) was induced bysalt stress. The RuBisCO-binding protein is the predomi-nant molecular chaperone of the chloroplasts; it consists oftwo seven-subunit rings which contain two homologous,but distinct, nuclear-encoded subunit polypeptides, desig-nated as α and β forms of the functional complex [54].Molecular chaperones bind and stabilize proteins atintermediate stages of folding, assembly, translocationacross membranes and degradation, and thus they arerepresentative of potential stress proteins that might play arole in protecting proteins against denaturation under stressconditions. It is possible that salt stress may directlyinterfere with some protein–protein interactions, which arecrucial for protein processing. RuBisCO activase (spot 13)belongs to the AAA+ family of proteins, which playfundamental roles in all three kingdoms of life. Theyprobably act as molecular chaperones in aiding theassembly or disassembly of proteins or protein complexes.The main role of activase is the maintenance of the catalyticactivity of RuBisCO by removal of inhibitory sugars fromthe active site of uncarbamylated and carbamylatedRuBisCO [55]. RuBisCO activase was upregulated inresponse to salt stress. This result is in accordance withthat of Parker et al. [56], who reported that upregulation ofactivase activity in rice leaf lamina may be required in orderto tolerate salt stress, owing to a direct reduction in stomatalconductance and subsequent low CO2 levels. Nucleic acidbinding protein (spot 32) belongs to a family of nuclear-encoded chloroplast proteins which share a commondomain structure and may be involved in posttranscription-al regulation of chloroplast gene expression. Posttranscrip-tional regulation is a process that could be a target of saltstress in plants. Protein–RNA interactions are crucial forRNA processing; thus, upregulation of nucleic acid bindingprotein in salt-treated plants may confer salt tolerance.

Anal Bioanal Chem (2008) 391:381–390 389

This study gives new insights into the salt-stressresponse in wheat and demonstrates the power of theproteomic approach in plant biology studies. This studyalso indicates that the identified proteins in wheat leaf areimplicated in diverse physiological and defence processes,and while some are probably part of a general stressresponse to help plants survive in suboptimal conditions,others may contribute to the reduction of the negativephysiological effects of the salt treatment.

Acknowledgement This work was supported by the Italian Depart-ment of Agriculture Food and Forestry, in the framework of Triticumdurum quality programme (FRUMISIS project).

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