research article functions of defense-related proteins and

RESEARCH ARTICLE

Functions of defense-related proteins and

dehydrogenases in resistance response induced

by salicylic acid in sweet cherry fruits at

different maturity stages

Zhulong Chan1, 2*, Qing Wang1, 2*, Xiangbin Xu1, 2*, Xianghong Meng1,Guozheng Qin1, Boqiang Li1 and Shiping Tian1

1 Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany,Chinese Academy of Sciences, Beijing, China

2 Graduate School of the Chinese Academy of Sciences, Beijing, China

We report here a comparative analysis of sweet cherry (Prunus avium) fruits proteomeinduced by salicylic acid (SA) at different maturity stages. The results demonstrated that SAenhanced the resistance of sweet cherry fruits against Penicillium expansum, resulting inlower disease incidences and smaller lesion diameters, especially at earlier maturity stage.Based on proteomics analysis, 13 and 28 proteins were identified after SA treatment at ear-lier (A) and later (B) maturity stage, respectively. Seven antioxidant proteins and threepathogenesis related-proteins were identified at both A and B stages, while five heat shockproteins and four dehydrogenases were only detected at B stage. SA treatment also stimu-lated higher transcript levels of peroxidase, but repressed that of catalase. Moreover, someproteins regulated by SA at B maturity stage were identified as enzymes involved in glyco-lysis and tricarboxylic acid cycle. These findings indicated that younger sweet cherry fruitsshowed stronger resistance against pathogen invasion after SA treatment. It further indi-cated that antioxidant proteins were involved in the resistance response of fruits at everymaturity stage, while heat shock proteins and dehydrogenases might potentially act as fac-tors only at later maturity stages.

Received: December 16, 2007Revised: June 1, 2008

Accepted: June 18, 2008

Keywords:

Induced resistance / Maturity stage / Salicylic acid / Sweet cherry

Proteomics 2008, 8, 4791–4807 4791

1 Introduction

Fruits ripening are a unique physiological process of plantdevelopment and involve changes in color, texture, flavor,aroma, and nutritional quality [1]. With increasing maturityindex, fruits become more and more hypersensitive to infec-tion of pathogens [2]. To protect fruits against invasion ofpathogenic fungi, people rely heavily on the use of syntheticchemical fungicides for disease management. Because of the

Correspondence: Professor Shiping Tian, Key Laboratory ofPhotosynthesis and Environmental Molecular Physiology, Insti-tute of Botany, Chinese Academy of Sciences, Beijing 100093,ChinaE-mail: [email protected]: 186-10-8259-4675

Abbreviations: CAT, catalase; GPX, glutathione peroxidase; POD,peroxidase; PR-proteins, pathogenesis-related proteins; PRX,peroxiredoxin; ROS, reactive oxygen species; SA, salicylic acid;SOD, superoxide dismutase; SSC, soluble solids content; THX,thioredoxin * These authors contributed equally to this work.

DOI 10.1002/pmic.200701155

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

4792 Z. Chan et al. Proteomics 2008, 8, 4791–4807

development of resistance of fungal pathogens to fungicidesand growing public concern on the health and environmentalhazards, other potential alternatives to chemical fungicides forfruits disease control have been developed in recent years [3, 4].

It is well known that salicylic acid (SA) is important in theinduction of plant resistance [5]. Application of exogenous SAis sufficient to induce resistance to many normally virulentpathogens [6]. Some researches indicated that exogenous SAtreatment could enhance the immunity of fruits againstpathogen [7], and stimulate the expression of a set of defensegenes and the activation of systemic acquired resistance inmany plants [7–9]. A substantial body of evidence demon-strates that resistance to pathogen infection is accomplishedthrough a diverse array of antimicrobial chemicals and PR-proteins [10, 11], which serves to restrict the growth of invasivepathogens and the progression of diseases [12, 13].

Recently, some evidences show that SA has a potentialeffect on inducing resistance against post-harvest decay infruits [14, 15]. In the previous experiments, we found thatexogenous SA treatment could obviously reduce diseaseincidence caused by fungal pathogens, and significantlystimulate the activities of enzymes [14, 16], and increase totalprotein content with additional new proteins in harvestedsweet cherry fruits [17]. Chen et al. [18] found that post-har-vest application SA enhance the accumulation of antioxidantprotein mRNA in grape berry fruits. These results partiallyexplained the reasons that SA could enhance fruits resist-ance and inhibit the infection of fungal pathogens. However,those researches only focused on fruits at commercialmaturity and little information is available about the effect ofSA in activation of fruits immune response at differentmaturity stages in the orchard. Additionally, very few data areyet available on the molecular mechanism of ripening fruitsin response to pathogen, especially at the proteomic level.

As an effective strategy for protein analysis, 2-DE hasbeen used successfully to identify novel protein componentsin plant [19–22]. Faurobert et al. and Giribaldi et al. investi-gated dynamic changes which take place in the tomato andberry proteome during fruits development and ripening,respectively [20, 21]. We also identified some proteinsinvolved in the resistance of harvested peach fruits inducedby SA in the absence of pathogen [22]. In the present study,we mainly investigated the effect of SA on induced resistanceof sweet cherry fruit against blue mold rot caused by Peni-cillium expansum at different maturity stages, and analyzeddefense response proteins induced by SA using a proteomicsapproach. Particular attention was paid to the function of theproteins in fungal pathogen infection.

2 Materials and methods

2.1 Fungal culture

P. expansum Link (CGMCC3.3703) was initially isolatedfrom the naturally infected sweet cherry fruits, and routinely

cultured on potato dextrose agar (PDA) plates for 14 days at257C. Spores were obtained by flooding the surface of theculture with sterile distilled water containing 0.05% v/vTween-80 and gently scrubbing with a sterile spatula. Sporessuspension was filtered through four layers of sterile chee-secloth to remove any hyphal fragments. The concentrationof the suspension was adjusted to 16104 spores mL–1 withthe aid of a hemocytometer.

2.2 Plant material

Sweet cherry (Prunus avium L. cv. Hongdeng) fruits wereavailable in the experiment orchard of Institute of Botany,the Chinese Academy of Sciences, Beijing. Trees growing inthis orchard were 11 years old. Fruits were used for meas-urement at particular ripening stage defined by skin colorand development days of fruits after full blooming. Thematurity stages were as follows: A stage, fruits developing for38 d after full blooming with yellow skin; B stage, fruitsdeveloping for 42 days after full blooming with pale red skin;C stage, fruits developing for 46 days after full blooming withfull red skin. Treatments of SA were included in both pre-harvest (A and B) and after harvest (C) stages.

2.3 Pre- and post-harvest treatment with SA

SA solution was prepared with distilled water and neu-tralized to pH 7.0 with 1 N NaOH. To eliminate the possibleeffect of pH value of the neutralized buffer (1 N NaOH), dis-tilled water was used as the control treatment for all of thefollowing treatments. According to the results of preliminaryexperiment, 2 mM SA was used as the treatment concentra-tion in this experiment because the fruits did not appear anyphyto-toxicity and SA did the best control effect against P.expansum when sweet cherry fruits were treated with thisconcentration (data not shown).

The treatments at three different maturity stages weredescribed as the following: W-N, fruits sprayed with water,non-wounded, and non-inoculated with P. expansum; SA-N,fruits sprayed with 2 mM SA solution, non-wounded, andnon-inoculated with the pathogen; W-P, fruits sprayed withwater, then wounded (a uniform wound of 4-mm deep63-mm wide was made at the equator of fruits using a sterilenail) and inoculated with P. expansum (10 mL of the pathogensuspension at 16104 spores mL–1 was inoculated into eachwound site); SA-P, fruits sprayed with 2 mM SA solution,and then wounded and inoculated with the pathogen asdescribed above (Table 1).

At A and B maturity stages, sweet cherry fruits were trea-ted on trees in the orchard, and the fruits with approximatesize and no physical injuries or infections were selected forthe experiment. After SA application, all fruits from fourtreatments were harvested, then transported to the laboratory,and analyzed immediately. At C maturity stage, sweet cherryfruits were harvested and immediately transported to thelaboratory, where the fruits were sorted on size and the


Proteomics 2008, 8, 4791–4807 Plant Proteomics 4793

Table 1. Detailed treatments on sweet cherry fruits and possible comparisons used for proteomic analysis

Maturity stages Treatment time and storage temperature Treatment with SA Comparisons

Stage A(38 d after full blooming

with yellow skin)

Pre-harvest treatment in the orchard W-N: no wound, no pathogen, treatment with water W-N_v_SA-NSA-N: no wound, no pathogen, treatment with SA W-P_v_SA-PW-P: wound, pathogen inoculation, treatment with waterSA-P: wound, pathogen inoculation, treatment with SA

Stage B(42 d after full blooming

with pale red skin)

Pre-harvest treatment in the orchard W-N: same as above W-N_v_SA-NSA-N: same as above W-P_v_SA-PW-P: same as aboveSA-P: same as above

Stage C-R(46 d after full blooming

with full red skin)

Post-harvest treatmentStorage at 25 7C

W-N: same as above W-N_v_SA-NSA-N: same as above W-P_v_SA-PW-P: same as aboveSA-P: same as above

Stage C-L(46 d after full blooming

with full red skin)

Post-harvest treatmentStorage at 0 7C

W-N: same as above W-N_v_SA-NSA-N: same as above W-P_v_SA-PW-P: same as aboveSA-P: same as above

absence of physical injuries or infections. The fruits weredivided into four groups and treated as described above. Allfruits were put into 2006130650 mm plastic boxes withplastic film to maintain a high relative humidity (95%).Half of them were stored at 257C (C-R) and the rest at 07C(C-L). Pathogen-inoculated fruits first showed watery spots,and then the typical symptom of blue mold developedgradually from the inoculation site. Sampling was per-formed when all WP-treated fruits showed symptom ofblue mold rot and lesion diameters were about 1 cm.Therefore, fruits were harvested 4 days after SA treatmentat A stage, 3 d at B and C-R stages, and 18 days at C-Lstage according to the corresponding lesion diameter. Toavoid the contamination of proteins from P. expansum, thepulp around the wound was removed, and the remainingpulp was sampled for analysis.

Disease incidence and lesion diameter were measuredevery day according to the following formulas. There werethree replications with 15 fruits in each treatment, and theexperiment was repeated twice.

Disease incidence (%) =S Number of decayed sweet cherry fruit

Total number of treated fruit� 100

Lesion diameter (cm) =S Lesion diameter of decayed sweet cherry fruit

Total number of treated fruit

2.4 Assay of fruits quality parameters

SSC of sweet cherry fruits was determined using an Abberefractometer (10481 S/N, Reichert, Buffalo, NY). Flesh pHvalue was measured by a pH analyzer (pHS-3B, Shanghai).There were three replications in each analysis per treatmentand the experiment was conducted twice.

2.5 Preparation of total protein from fruits

Total protein extracts were prepared from sweet cherry fruitsat different maturity stages according to the method of Sar-avanan and Rose with some modifications [23]. All proceduresdescribed below were carried out at 47C. Briefly, 4 g of fleshfrom ten fruits was ground in liquid nitrogen using mortarand pestle, and then homogenized in 4 mL of a extractionbuffer containing 20 mM Tris-HCl pH 7.5, 250 mM sucrose,10 mM EGTA, 1 mM PMSF, 1 mM DTT, and 1% Triton X-100. The mixture was extensively homogenized on ice, andsubsequently extracted with an equal volume of Tris-HCl pH7.8 buffered phenol for 30 min. After centrifugation(10 0006g, 40 min, 47C), the phenol phase was re-extractedtwo or three times with extraction buffer as above. Proteinswere precipitated from the final phenol phase with fivevolumes of ice-cold saturated ammonium acetate in methanolovernight at 2207C. The proteins were collected by cen-trifugation (10 0006g, 30 min; 47C) and washed twice withcold saturated ammonium acetate in methanol and acetoneeach. The precipitate was air-dried for 1 h at 47C and thensolubilized in 250 mL of thiourea/urea lysis buffer containing2 M thiourea, 7 M urea, 4% w/v CHAPS, 1% w/v DTTand 2%v/v of a mixture of carrier ampholytes of pH 5–8 and pH 3.5–10 in a ratio of 1:1. Protein samples were kept at 2807C untilused. The protein concentration was determined according toBradford’s method using BSA as standard [24]. There werethree technical replicates in each treatment for proteinextraction, and the whole experiment was repeated twice.

2.6 2-DE

The 2-DE was carried out according to the method ofKomatsu et al. [25] with minor modification. The IEF gel so-lution contained 10% NP-40, 30% w/v acrylamide, 9.5 M



urea, 10% ammonium persulfate, and an equal mixture of2% carrier ampholytes pH 3.5–10 and 5–8. The gels werepolymerized in glass tubes (Daiichi pure Chemicals, Tokyo,Japan) to obtain gels of 13-cm length and 3-mm diameter.Approximately 600-mg of proteins was applied to the gel. IEFwas performed at 200 V for 30 min, 400 V for 10 h, and then800 V for 6 h. After the first dimensional run, IEF gels weretransferred to equilibration buffer containing 10% v/v glyc-erol, 2.5% w/v SDS, 125 mM Tris-HCl pH 6.8, and 5% v/v b-mercaptoethanol and incubated for 15 min at room temper-ature twice. SDS-PAGE in the second dimension was per-formed with homemade 15% separation gels and 5% stack-ing gels (175620061 mm) at a constant current of 30 mA.Equilibrated IEF gel was placed onto the stacking gel andsealed with 0.5% agarose in equilibration buffer. After elec-trophoresis, 2-D gels were fixed with the buffer containing50% v/v ethanol and 10% v/v acetic acid for 1 h, and thenstained for 2 h with CBB R-250 solution containing 50% v/vmethanol, 15% v/v acetic acid and 0.1% w/v CBB R-250. Thegels were destained with the buffer containing 30% v/vethanol and 8% v/v acetic acid to desired intensity.

2.7 Gel image analysis and in-gel tryptic digestion

The colloidal CBB-stained gels were scanned using a flatbedscanner (Amersham Bioscience) with 300 dpi resolution andsaved in TIF format. Comparison of protein expression in 2-D gel images was performed using Image Master 2D Elitesoftware (Amersham Pharmacia Biotech, Uppsala, Sweden).To account for experimental variation, at least triplicate gels,resulting from protein extracts obtained from independentexperiments, were analyzed for each treatment. Spot detec-tion was carried out automatically and those spots showingfaint intensity near the detection limit of colloidal CBB werenot included in the comparisons. Prior to automatic match-ing of spots between gel images, one gel was selected as thereference gel of each treatment. The amount of a protein spotwas calculated based on the volume of that spot. To reflect thequantitative variations in intensity of protein spots betweencontrol and treated samples, the spot volume was normal-ized as a percentage of the total volume of all spots on thecorresponding gel. Statistical analysis of the data was per-formed using SPSS software (SPSS, Chicago, IL). The nor-malized intensity of spots on nine replicates 2-D gels wasaveraged and the SD was calculated for each treatment.

All data were analyzed as a one-variable general linearmodel procedure (analysis of variance) with SPSS (SPSS).Mean separations were performed using the least significantdifference test. Differences at p ,0.05 were considered signif-icant. Results presented were pooled across repeated experi-ments. Only spots that changed significantly in averaged nor-malized spot volume were excised for protein identification.

In-gel digestion was performed as described by Shen et al.[26]. Protein spots were excised from the gels and cut in about1 mm2 pieces, then destained with a 1:1 v/v solution of meth-anol and 50 mM NH4HCO3 while changing solutions every

1 h until the blue color of Coomassie was removed. Proteinswere reduced with 10 mM DTTand 10 mM EDTA in 100 mMNH4HCO3 for 1 h at 607C. After drying in a vacuum cen-trifuge, the gels were incubated with 40 mM iodoacetamide in100 mM NH4HCO3 for 30 min at room temperature in thedark, then washed several times with water and completelydried in a vacuum centrifuge. Gel pieces were re-hydratedwith a digestion buffer of 5 ngmL–1 trypsin (sequencing grade,modified; Promega, USA) in 100 mM NH4HCO3 and digest-ed for 14 to 16 h at 377C After digestion, the gel slices werewashed with 0.1% TFA in 50% v/v ACN three times to extractthe peptides. The collected solutions were concentrated to10 mL, and then desalted with ZipTipC18 (Millipore, Bedford,MA). Peptides were eluted from the column in 2 mL of 0.1%TFA in 50% ACN.

2.8 Protein spot identification by MALDI-TOF-MS and

ESI-Q-TOF-MS/MS

MALDI-TOF-MS analysis was performed on an AXIMA-CFRplus MALDI-TOF MS (Shimadzu Biotech, Kyoto, Japan)equipped with a flight tube (reflectron mode, 225-cm long),laser (N2, 337 nm), and scout 384 target system. Before eachanalysis, the instrument was externally calibrated by threepeptide (ACTH 18 - 39, angiotensin a and bradykinin) spot-ted as near as possible to the sample. PMF were matched tothe National Center for Biotechnology Information non-redundant (NCBInr) protein databases using the searchengine Matrix Science at http://www.matrixscience.com.

When a spot failed to be identified with the MALDI-TOF,ESI-MS/MS analysis was performed using an ESI Q-TOFhybrid mass spectrometer (ESI Q-TOF; micro, Micromass,Altrincham, UK) with a z-spray source. Before loading thedigested peptide, the instrument was externally calibratedwith the fragmentation spectrum of the doubly charged1571.68 Da (785.84 m/z) ion of fibrinopeptide B. The peptideswere introduced by nanoelectrospray needle (gold-coatedborosilicate glass capillaries, Micromass). The applied sprayvoltage was 800 V, with a sample cone working on 30 V. Thecollision energy was varied from 14 to 40 V dependent on themass and charge state of the peptides. MS/MS data were pro-cessed using the MaxEnt3 (Micromass), and searched inNCBInr protein databases with the MASCOT MS/MS IonsSearch program on the Matrix Science public web site. Apeptide tolerance of 62.0 Da for the precursor ions, an MS/MS tolerance of 60.3 Da for the fragment ions was set. Pep-tide charges of 12 and 13, monoisotopic mass was chosen,and the instrument type was set to ESI-QUAD-TOF.

Identification of proteins was performed using the MAS-COTsoftware (http://www.matrixscience.com/) by searchingMSDB 20060831 (3 239 079 sequences; 1 079 594 700 resi-dues) and NCBInr 20080328 (6 348 806 sequences;2 166 943 470 residues) databases. Both “Viridiplantae” and“Fungi” were selected as the taxonomic category for all MSresults. “Viridiplantae” was chosen because the sweet cherrygenome is not completely sequenced and only 405 sequences



are available now. To make sure that pathogen protein wasremoved completely during sampling and protein isolation,we chose “Fungi” as the second taxonomic category for Mascotsearch so that we could eliminate the possibility of pathogenprotein contamination. MASCOT uses a probability-based“MOWSE Score” to evaluate data obtained from MS or MS/MS. More strict criteria were used to identify proteins withMALDI-TOF. Only proteins that met the following criteriawere accepted as unambiguously identified: (i) MASCOTscore�90/46 for MALDI-TOF/Q-TOF, respectively (MOWSEScores were reported as 2106log10 (p) where p is the prob-ability that the observed match between the experimental dataand the database sequence is a random event. Scores .66/46for MALDI-TOF/Q-TOF indicate identity or extensive homol-ogy; p ,0.05); (ii) number of matched peptides �4/2 forMALDI-TOF/Q-TOF, respectively; (iii) sequence coverage�10/4% for MALDI-TOF/Q-TOF, respectively; (iv) allowedmissed cleavage: 1; (v) allowed modifications: Carbamido-methylation of cysteine (157.02 Da), oxidation of methionine(1 16 Da), and N-terminal pyroglutamine (217.02 Da); and(vi) maximum allowed molecular mass deviation: 0.3 Da.

2.9 Cloning of CAT and GPX and Northern blotting

Total RNA was extracted from sweet cherry fruits by hot-phe-nol isolation protocol as described by Chan et al. [22]. First-strand cDNA was synthesized with first-strand cDNA synthe-sis kit (TRANS). Degenerate primers of CAT2 were designedbased on the full-length cDNA sequences of peach (AJ496419)and pumpkin (D55646): 50 - ATG GAT CCW TAC ARG YAC -30 (forward), 50 - TCA AAT GCT KGG CCT CAC - 30 (reverse).PCR conditions were 947C for 5 min, 33 cycles of 947C for30 s, 507C for 30 s, 727C for 100 s; and finally 727C for10 min.The degenerate primers of GPX were designed based on thefull-length cDNA sequences of peach (DW353011) and apple(AF403707): 50 - ATG GCT RGC CAK TCY GRG - 30 (forward),50 - TCA GTT TCT TMW YAY AGA - 30 (reverse). The follow-ing thermal profile was used for PCR: 947C for 5 min, 33cycles of 94 7C for 30 s, 527C for 30 s, 727C for 45 s; and finally727C for10 min. The PCR products were cloned in the pGEM-T Easy vector (Tiangen, China) and sequenced.

For Northern blot, aliquots of 10 mg of total RNA per lanewere loaded in a 1.2% denaturing formaldehyde agarose gel,and hybridization with [32P] dCTP-labeled PCR probe wascarried out as described by Sambrook et al. [27].

3 Results

3.1 SA induced the resistance of sweet cherry fruits

at early maturity stage and did not affect fruit

quality

P. expansum, a main fungal pathogen, can infect sweetcherry fruits both before and post harvest. Figs. 1a and 1bshowed the symptoms of blue mold caused by P. expansum at

B maturity stage with or without SA treatment, respectively.Application of SA for 72 and 96 h could delay pathogeninfection and resulted in significant lower disease incidencethan those of water-treated fruits at A stage (Fig. 1c). Thedisease incidences were also declined at 24 and 48 h after SAtreatments at B, and C-R stages (Figs. 1d and e). The resultsshowed that SA treatment could inhibit the pathogengrowth, resulting in smaller lesion diameter in SA-treatedfruits than that of controls at A, B, and C-R stages (Figs.1g, h,and i). Low temperature (07C), as a commercial storageapproach for harvested sweet cherry fruit, did inhibit thedevelopment and infection of blue mold pathogen, resultingin much lower disease incidence and lesion diameter. How-ever, no significant difference was found between water- andSA-treated fruits stored at 07C (Figs. 1f and j). Moreover, bothdisease incidence and lesion diameter at later maturitystages were higher than those at earlier stages. The lesiondiameters of water-treated fruits were about 1 cm at 4, 3, 3,and 18 days post inoculation (dpi) at A, B, C-R, and C-Lstages. It indicates that fruits at later stages were more sus-ceptible to fungal infection than those at earlier stages.

3.2 Fruit flavor and quality were not affected by SA

treatment

Soluble solids content and pH value in flesh, as importantflavor and quality parameters, showed no significant differ-ences between SA- and water-treated fruits at all maturitystages (Figs. 2a and b). These results indicated that SA solu-tion did not change fruit quality.

3.3 Proteins were induced by SA in sweet cherry

fruits at different maturity stages

Comparative analysis of the proteome was performed toinvestigate the profiles of differentially expressed proteins atA and B maturity stage. A 2-D gel of fruit proteins revealedmore than 600 protein spots after ignoring very faint spots andspots with undefined shapes and areas using Image Master2D Elite software (Smooth ,4, Min Area ,20, and Saliency,300 000) (Fig. 3). At A stage and in the absence of pathogen,the abundances of 33 protein spots in sweet cherry fruits werealtered by SA treatment. Among them, eight were identifiedby MALDI-TOF-MS and ESI-Q-TOF-MS/MS (labeled as spotN-), with three of them enhanced and five repressed by SAtreatment (Table 2). While in the presence of P. expansum, 19proteins showed significant changes in relative abundancesafter SA treatment at A stage. Five proteins were identified(labeled as spot P-). Among them, abundance of four proteinswas increased and other two was decreased (Table 2). At Bstage, 22 proteins were differentially expressed after SAtreatment in the absence of P. expansum. We identified 13 ofthem with significant MOWSE scores (p ,0.05) (Fig. 3;Table 2), in which six proteins were up-regulated and sevenproteins were down-regulated. In the presence of pathogen atB stage, the abundances of 25 proteins were regulated by SA,



Figure 1. Effects of SA on disease development in sweet cherry fruits and fruits quality parameters. (a) And (b) Symptom of blue mold insweet cherry fruits at B stage after water and SA treatment, respectively. Representative fruits were shown in the orchard. (c)-(f) Diseaseincidence of blue mould in sweet cherry fruits after water and SA treatment at A, B, C-R, and C-L stages, respectively. (g)-(j) Lesion diameterof blue mould in sweet cherry fruits after treatment with water or SA solution at A, B, C-R, and C-L stages, respectively. A, B, C representdifferent maturity stages of sweet cherry fruits as described in Section 2. C-R and C-L represent fruits at C maturity stage stored at 25 and07C after treatment, respectively. W-P and SA-P represent fruits treated with water and 2 mM SA in the presence of P. expansum, respec-tively. Data are the means 6 SE of three replications. Bars represent standard deviation of the treatment means of pooled data.

and 15 of them were identified (Fig. 3; Table 2). Only theabundance of one protein was down-regulated after SAtreatment.

Functions of all these identified proteins were involved inmetabolism and energy pathway (16 proteins), defense andstress response (15 proteins), iron homeostasis (3 proteins),signal transduction/transcription (5 proteins), cell structure(1 protein), and others (1 protein) according to Bevan et al.[28] and Schiltz et al. [29]. Of all identified proteins, we wereparticularly interested in those involved in defense and stressresponse, including antioxidant proteins, heat shock pro-teins and pathogenesis-related proteins, since they play adirect role in defense responses. Dehydrogenases, kinases

and some other proteins involved in iron homeostasis werealso found to be induced by SA treatment (Table 2).

Moreover, several proteins induced by SA treatment wereidentified in both pathogen infected and non-pathogeninfected fruits, indicating they were induced by both SAtreatment and pathogen infection (or wounding). For exam-ple, P-6 and N-12 were peroxidase; P-11 and N-23 were cata-lase; N-40 and P-20 were enolase; and N-39 and P-23 wereNAD-dependent malate dehydrogenase. In addition, twoprotein spots (P-11 and P-12) were identified as the sameprotein, catalase (Table 2), and their location in the gels dif-fered slightly in Mr and pI (Fig. 4). This indicates that theymight be isoforms of catalase because of PTM.



Figure 2. Changes in soluble solids content and pH value ofsweet cherry fruits after SA treatment. W-N and SA-N representfruits treated with water and SA in the absence of pathogeninoculation, respectively. A, B, C-R, C-L, W-P and SA-P representthe same meaning as described in Fig. 1.

3.4 Antioxidant and pathogenesis related proteins

were induced by SA at all maturity stages

SA treatment enhanced the abundance of POD (N-12 and P-6) both in the presence and absence of P. expansum, espe-cially at A and C-R stages. The expression of CAT (N-23) wasinhibited by SA in the absence of pathogen at A and C-Rstages, while CAT (P-11) and CAT (P-12) were induced in thepresence of pathogen (Fig. 4) at A, B, and C-L stages. More-over, the abundance of THX (N-35) and PRX (P-15) wasenhanced at B stage, and that of and SOD (P-26) wasincreased at B and C-R stages after SA treatment (Table 2,Fig. 4).

The expression of major allergen Pru av 1 (Pru a 1) (N-18and N-31) was repressed after SA treatment in the absence ofpathogen at A and B stages. Abundance of major allergenPru av 1 (N-18) was also decreased slightly at C stage after SAtreatment (Fig. 4).

3.5 HSP and dehydrogenases were induced by SA at

later maturity stages

Figure 5 showed the abundance variations of HSP anddehydrogenases in sweet cherry fruits after SA treatment. SAtreatment enhanced the abundance of HSP 70 kDa (P-19) atB and C stages, and Cytosolic class II low molecular weightheat shock protein (N-30) at C stage, but inhibited others,

including Heat shock protein 60 (P-18), Hsp20 (N-27), andlow-molecular weight HSP (N-42). No differences wereobserved at early (A) maturity stage after SA treatment. Theabundances of dehydrogenases (P-22, P-23, N-37, and N-39)were enhanced by SA treatment. Additionally, those changeshappened mainly at later maturity stages B and C. No sig-nificant differences were observed at early (A) maturity stage.

3.6 SA treatment induced GPX expression but

repressed CAT2 in sweet cherry fruits

POD (N-12 and P-6), CAT (N-23, P-11, and P-12), SOD (P-26),THX (N-35), and PRX (P-15) are directly involved in reactiveoxygen species (ROS) metabolism. We tried to clone thosegenes in order to verify their expression levels by Northernblot. Unfortunately, only one glutathione peroxidase gene(GPX) and one catalase gene (CAT2) were successfullycloned from sweet cherry fruit. However, both genes are di-rectly involved in reactive oxygen species metabolism, cata-lyzing the reactions, which convert hydrogen peroxide(H2O2) to water (H2O). Full-length cDNA sequences of CAT2and GPX genes were obtained and designated as CAT2 andGPX (GeneBank accession nos. EF165590 and EF165591,respectively). The CAT2 gene contains an open readingframe of 1479 bp encoding 492 amino acid residues (Sup-porting Information Fig. 1). The GPX gene has an openreading frame of 513 bp encoding 170 amino acid residues(Supporting Information Fig. 1).

The expression level of CAT2 and GPX genes in sweetcherry fruits was analyzed using Northern blotting (Fig. 6).SA treatments significantly repressed the expression of CAT2at B and C stage, but not at A stage. For GPX, the transcriptlevel was elevated by SA treatment at B and C-R stages, butno significance was observed at A stage.

4 Discussion

Fruit ripening is a complex, well-documented, but not fullyelucidated phenomenon [2]. SA is well known as an impor-tant component in response to systemic acquired resistance[7, 9]. Our previous study showed that SA treatment stimu-lated the activities of polyphenoloxidase, phenylalanineammonia-lyase, and b-1,3-glucanase in sweet cherry fruits,resulting in lower disease incidence and smaller lesion di-ameter relative to control [14, 17]. In this study, as indicatedin Fig. 1, we further found that SA treatment showed bettercontrol efficacy in sweet cherry fruits at earlier maturitystage, as was illustrated by much lower disease incidence andsmaller lesion diameter. The relationship between plant ageand disease resistance has been investigated in many plant-pathogen systems [30, 31]. Some plants become more sus-ceptible [32], but others display increased resistance to cer-tain pathogens as they develop [33]. Gevens et al. [34] con-sidered that susceptibility of cucumber fruit to Phytophthorainfection was related to fruit maturation. Our results indi-



Figure 3. Two-dimensional patterns of proteins from sweet cherry fruits after SA treatment. Sweet cherry fruits were treated with 2 mM SAsolution or distilled water. Protein extraction and 2-D PAGE protocol were described in Section 2. W-N and SA-N represent fruits treatedwith water and 2 mM SA in the absence of P. expansum, respectively. W-P and SA-P represent fruits treated with water and 2 mM SA in thepresence of P. expansum, respectively. A, B, C represent different maturity stages of sweet cherry fruits as described in Section 2. C-R and C-L represent fruits at C maturity stage stored at 25 and 07C after treatment, respectively.

cated that younger sweet cherry fruits would more easilyintensify their defense response system when induced bySA, and then could show stronger resistance against Peni-cillium invasion (Fig. 1a), suggesting fruit susceptibility toSA treatment and pathogen infection changed at differentmaturity stages. In addition, the differences in disease inci-dence and lesion diameter between SA treated- and the watercontrol-fruits at 24 and 48 h were not very evident (Fig. 1). Itmight be due to the time limit of induced resistance, andindicates that induced resistance individually by abioticagents (such as SA) was not powerful enough to control post-

harvest disease. Our recent researches indicated that appli-cation of antagonistic yeast in combination with somechemicals could inhibit the infection of pre- and post-harvestpathogens more effectively [4, 14, 35].

The actual mechanisms responsible for the maturity-related resistance were studied in a preliminary manner inonly a few cases [36]. Using a proteomic approach, someinsights were given at protein level about the mechanisms ofinduced resistance related to fruits maturity in this paper. Atotal of 41 proteins regulated by SA were identified in sweetcherry fruits across different maturity stages.



Figure 4. Abundance varianceof antioxidant and pangenesis-related proteins in sweet cherryfruits at different maturity stageafter treatments with 2 mM SA.Protein extraction and 2-D PAGEprotocol were described in Sec-tion 2. The number of each pro-tein spot corresponds to its list-ing in Table 2. The spot volumewas normalized as a percentageof the total volume of all spotson the corresponding gel. Therelative abundance ratio insweet cherry fruits treated withSA was divided by that in fruitstreated with water, from threedifferent gels and independentextractions. The protein spotswith significant changes (p,0.05) were considered to bedifferent. The graph representsan average of three biologicalreplicates. Bars represent stand-ard errors of the mean. A, B, C-R,C-L, W-P, SA-P, W-N and SA-Nrepresent the same meaning asdescribed in Fig. 3.



Table 2. Proteins induced by SA treatment on sweet cherry fruits and their relative abundance in the presence (P) or absence (N) ofpathogen

Spotno.a)

Protein name StageA

StageB

Accessionno.

Mr (kDa) pI Species MASCOTscored)

NP SC(%)

N P N P Theoretic Experimental Theoretic Experimental /Threshold

Metabolism and energy pathway

N-8b) Lactuca sativa 9-cis-epoxycarotenoid 4dioxygenase

D gi)84579412 64.0 47.2 7.65 6.43 Lactuca sativa 101/66 8 19

N-13b) Acyl-ACP thioesterase I gi)12004041 47.3 35.8 5.67 6.84 Iris tectorum 91/66 6 20N-33c) Putative aminoacylase I gi)42408797 49.79 47.8 5.90 5.42 Oryza sativa 104/46 2 5N-34c) prunasin hydrolase

isoform PH I precursorD gi)15778638 58.27 69.3 5.47 5.21 Prunus serotina 133/48 3 7

N-37c) Glucose-6-phosphate1-dehydrogenase

I gi)585165 58.43 66.3 6.00 6.87 Solanumtuberosum

90/67 8 18

N-38b) V-ATPase catalyticsubunit A

I gi)15982954 68.52 70.3 5.30 5.43 Prunus persica 122/67 9 24

N-39b) NAD-dependent malatedehydrogenase


N-40b) Enolase I gi)33415263 47.7 48.3 6.16 5.80 Gossypiumbarbadense

112/67 8 26

N-43c) ATP synthase subunitalpha, mitochondrial

D gi)231585 55.29 48.2 6.23 6.14 Glycine max 533/46 10 21

P-1b) Temperature-inducedlipocalin

D gi)77744893 21.5 22.3 5.60 5.59 Prunus arme-niaca

105/66 7 34

P-20b) Enolase I gi)33415263 47.7 56.7 6.16 6.25 Gossypiumbarbadense

112/67 8 26

P-22c) Phosphoglyceratedehydrogenase

I gi)18394525 66.41 61.2 5.81 5.78 Arabidopsisthaliana

82/46 2 4

P-23b) NAD-dependent malatedehydrogenase


P-27c) UTP–glucose-1-phos-phate uridylyltrans-ferase

I gi)6136112 51.81 52.5 5.99 6.29 Pyrus pyrifolia 544/46 12 28

P-28c) S-adenosylmethioninesynthetase

I gi)1346523 42.74 46.6 5.47 6.86 Petunia xhybrida

86/46 2 4

P-32c) Fumarate hydratase I gi)1769568 52.79 51.4 7.98 7.37 Arabidopsisthaliana

281/46 3 9

Defense and stress response

N-12b) Peroxidase I gi)167531 32.3 34.5 6.00 6.78 Cucumis sativus 95/66 7 28N-18b) Major Cherry Allergen

Pru Av 1D gi)159162232 17.5 16.2 5.87 6.72 Prunus avium 118/66 7 55

N-23b) Catalase D gi)32526568 57.4 58.2 6.95 7.46 Prunus persica 187/66 16 34N-27c) Heat shock protein

Hsp20D gi)122199396 18.18 19.3 5.80 6.25 Medicago

truncatulaa126/46 3 19

N-30c) Cytosolic class II lowmolecular weightheat shock protein

D gi)5257560 17.49 17.5 5.60 5.48 Prunus dulcis 85/46 2 16

N-31c) Major allergen Pru av 1(Pru a 1)

D gi)7388028 17.64 18.2 5.90 5.79 Prunus avium 193/46 3 23

N-35c) Thioredoxin H I gi)16588843 14.48 14.3 5.60 5.26 Prunus persica 155/46 3 14N-42c) Low molecular weight

heat shock proteinD gi)6969974 18.21 16.4 5.39 5.17 Malus x

domestica207/46 8 25

P-6b) Peroxidase I gi)167531 32.7 34.5 6.00 6.78 Cucumis sativus 95/66 7 28P-11b) Catalase I gi)32526568 57.0 58.2 6.95 7.46 Prunus persica 187/66 16 34P-12b) Catalase I gi)121078773 57.4 58.9 6.95 7.82 Prunus avium 168/66 12 27P-15c) Chain A, Prx D (Type Ii) I gi)66360171 17.42 16.8 5.56 5.61 Populus

trichocarpa114/46 2 6



Table 2. Continued

Spotno.a)

Protein name StageA

StageB

Accessionno.

Mr (kDa) pI Species MASCOTscored)

NP SC(%)

N P N P Theoretic Experimental Theoretic Experimental /Threshold

P-18c) Heat shock protein 60D

gi)24637539 57.72 59.3 5.26 5.39 Prunus dulcis 367/46 6 14P-19c) Heat shock 70 kDa

proteinI gi)399940 72.49 71.6 5.95 5.57 Phaseolus

vulgaris475/46 8 17

P-26c) Superoxide dismutase[Mn]

I gi)464775 25.82 24.8 7.10 6.74 Heveabrasiliensis

232/67 4 12

Iron homeostasisN-36c) K1 channel protein D gi)1063415 36.36 35.8 8.22 7.53 Arabidopsis

thaliana213/46 6 4

P-2b) Putative iron-sulfurcluster-bindingprotein

D gi)50919237 60.0 60.3 7.56 6.35 Arabidopsisthaliana

114/66 12 32

P-34c) Ferritin I gi)50787937 28.24 29.5 5.55 5.58 Conyzacanadensis

75/46 2 6

Signal transduction/transcriptionN-17b) Putative glycine-rich

RNA-binding proteinD gi)34851124 17.3 16.5 7.82 6.17 Prunus avium 175/66 10 49

N-24b) Transmembranereceptor

D gi)42566973 31.7 32.4 9.59 7.12 Arabidopsisthaliana

133/66 9 31

P-13c) Phosphoglyceratekinase

I gi)2499498 42.33 44.5 5.69 6.25 Nicotianatabacum

247/46 4 13

P-14c) Phosphoenolpyruvatecarboxykinase

I gi)13785467 73.12 63.9 7.61 7.83 Flaveria trinervia 167/46 5 10

P-16c) 26S proteasomesubunit 11

I gi)94442896 25.99 31.5 8.04 7.72 Platanus xacerifolia

77/46 2 9

Cell structureP-30b) Actin isoform B I gi)6683504 41.7 42.4 5.31 5.35 Mimosa pudica 151/67 10 4

OthersN-10b) Os01g0942200 I gi)115442165 48.0 41.6 6.52 6.49 Oryza sativa 102/66 7 26

Proteins were identified with MALDI-TOF MS or ESI-Q-TOF-MS/MS. Fruits at early maturity stage (A) and middle maturity stage (B) weretreated with 2 mM SA in the orchard and then harvested 4 and 3 days after SA treatment for protein isolation. The assigned protein of thebest matched was given with the organism in which it has been identified and its GenBank accession number. Spot no.: spot number;Accession no.: accession number from NCBI database of matched protein; Mr (kDa)/pI: theoretical molecular mass and isoelectric pointbased on amino acid sequence of the identified protein; NP: the number of matched peptides; SC: amino-acid sequence coverage for theidentified proteins. D: protein intensity was decreased after SA treatment. I: protein intensity was increased after SA treatment.a) N means without pathogen inoculation. P means with pathogen inoculation.b) Proteins identified with MALDI-TOF.c) Proteins identified with ESI-Q-TOF-MS/MS.d) MASCOT scores greater than the thresholds are statistically significant (p ,0.05).

Without pathogen inoculation, three and five defenseand stress-response proteins were induced by SA in sweetcherry fruits at A and B stage, respectively. Functions of heatshock proteins [Hsp20 (N-27), cytosolic class II low-molecu-lar weight heat shock protein (N-30), and low-molecularweight heat shock protein (N-42)] and major allergen Pruproteins [major allergen Pru av 1 (Pru a 1) (N-18) and majorallergen Pru av 1 (Pru a 1) (N-31)] had been well character-ized [37, 38]. Other proteins including peroxidase (PR-pro-tein 9) (N-12), catalase (N-23), and thioredoxin H (N-35) are

part of the main enzymatic systems for protecting cellsagainst oxidative damage [39, 40, 41]. They are directly relat-ed to the defense response and exhibit inhibitory activityagainst a variety of pathogens. Moreover, two proteinsbelonging to signal transduction/transcription at A stagewere responsible to SA treatment in sweet cherry fruits,including RNA-binding proteins (N-17) and transmembranereceptor (N-24) [42-44]. One protein, K1 channel protein(N-36), was involved in iron homeostasis and identified at Bmaturity stage after SA treatment [45].



Figure 5. Abundance varianceof heat shock proteins anddehydrogenases in sweet cherryfruits at different maturity stageafter treatments with 2 mM SA.Protein extraction and 2-D PAGEprotocol were described in Sec-tion 2. The number of each pro-tein spot corresponds to its list-ing in Table 2. The spot volumewas normalized as a percentageof the total volume of all spotson the corresponding gel. Therelative abundance ratio insweet cherry fruits treated withSA was divided by that in fruitstreated with water, from threedifferent gels and independentextractions. The protein spotswith significant changes (p,0.05) were considered to bedifferent. The graph representsan average of three biologicalreplicates. Bars represent stand-ard errors of the mean. A, B, C-R,C-L, W-P, SA-P, W-N and SA-Nrepresent the same meaning asdescribed in Fig. 3.



After inoculation with P. expansum, the activities of anti-oxidant proteins, including peroxidase (P-6) and catalase (P-11 and P-12) at maturity stage A, and superoxide dismutase[Mn] (P-26) at maturity stage B were enhanced in SA-treatedfruits (Fig. 2; Table 2). These results were slightly divergentfrom the results of gene expression (Fig. 6). Some reasonscould be attributed to the discrepancy. It is known that theprotein level integrates post-transcriptional and post-transla-tional processing that modulates the quantity, temporalexpression, localization, and efficiency of the final product inthe cell when compared with the RNA level. We could,indeed, identify some protein modifications in the form ofmultiple spots. Part of them showed slightly different pat-terns of expression (e.g. P-11 and P-12 were identified as cat-alase; and N-18 and N-31 were identified as Major allergenPru av 1) (Table 2), suggesting regulation mechanisms andpossibly different effects in sweet cherry fruits.

Treatment with SA increased the abundances of fiveantioxidant proteins [Peroxidase (N-12), Catalase (N-23), Per-oxidase (P-6), Catalase (P-11), and Catalase (P-12)] at A stageand two antioxidant proteins [Thioredoxin H (N-35), Super-oxide dismutase [Mn] (P-26)] at B stage. It is well known thatan early response to pathogen attack is oxidative burst, whichleads to the production of ROS. As key enzymes for detox-ifying ROS in plant cells, POD, CAT, SOD directly play a rolein conferring resistance to a wide range of pathogens [46, 47].THXs, as electron donors to peroxidases, are also small redoxproteins [41] (Fig. 7a). One possible model of SA effect onROS is that SA inhibits the H2O2-degrading activity of cata-lase, thereby leading to an increase in the endogenous levelof H2O2. The H2O2, and other ROS derived from it, may thenserve as second messengers to activate the expression ofplant defense-related genes and strengthen mechanical bar-riers [8]. A large body of evidence has shown that exogenousSA treatment can induce the accumulation of H2O2 levels inplant tissues [48-50]. However, ROS, when produced athigher concentration during pathogenesis, may initiatedegradative reactions [51]. Therefore, efficient antioxidantactivity is essential in order to maintain the concentration ofROS at relatively low levels. Our previous results indicatedexogenous SA application enhanced the activity of catalaseand CAT expression in peach fruit [22]. Ananieva et al. alsoreported that SA treatment enhanced CAT activity in barley[52]. The results in this study further demonstrated that SAtreatment stimulated POD transcript level, but inhibited thatof CAT (Fig. 6), as well as changed the activities of anti-oxidant proteins (Fig. 4). These enzymes modulate the con-centration of H2O2 in plant cells. In fact, the balance betweenSOD, POD, CAT and/or THX activities in cells is crucial fordetermining the steady-state level of O2

.2 and H2O2 [53]. Ourrecent study indicates that antioxidant proteins are alsoinvolved in pathogenicity of P. expansum [54]. Therefore,efficient antioxidant activity might play crucial role in theinteraction between host and pathogens.

Interestingly, several spots induced by SA treatment wereidentified as proteins involved in iron homeostasis, includ-

Figure 6. Effect of SA on gene transcripts of GPX and CAT2 insweet cherry fruits. Twenty micrograms of total RNA from fruitswere fractionated by gel electrophoresis, blotted and hybridizedwith probes. The intensity of the bands was quantified by scan-ning densitometry of the autoradiographs using Scion ImageSoftware (Scion Corporation, Frederick, MD). A, B, C-R, C-L, W-P,SA-P, W-N and SA-N represent the same meaning as described inFig. 3.

ing K1 channel protein (N-36), putative iron-sulfur cluster-binding protein (P-2), and ferritin (P-34). Ferritin, one of themajor proteins of iron metabolism, may constitute a missinglink in the regulatory loop between iron and ROS [55, 56]. K1

channel protein and iron-sulfur cluster-binding protein werealso involved in redox reaction [44, 57, 58]. Microarray dataexhibited that iron deficiency directly affected expressionlevel of ROS-related genes [59]. These results indicated thatthe relationships among SA, ROS pathway, and iron home-ostasis were complex and would be worthy of further re-search.

Our comparative proteomic analysis also showed that theabundances of five HSPs, including Hsp20 (N-27), cytosolicclass II low-molecular weight HSP (N-30), low-molecularweight HSP (N-42), Hsp 60 (P-18), and 70-kDa HSP (P-19),and four dehydrogenases, including glucose-6-phosphate 1-dehydrogenase (N-37), NAD-dependent malate dehy-drogenase (N-39), phosphoglycerate dehydrogenase (P-22),and NAD-dependent malate dehydrogenase (P-23), had beenchanged after SA treatment at latter maturity stage (B)



Figure 7. SA treatment affectedantioxidant and metabolicalpathway on sweet cherry fruits.(A) Proteins, whose abundanceswere regulated by SA in sweetcherry fruits, were identified asenzymes involved in antioxidantpathway. Step 1: catalyzed bySOD (P-26); Step 2: catalyzed byCAT (N-23, P-11, and P-12); Step3: catalyzed by POD (N-12, andP-6); Step 4: catalyzed by THX(N-35). (B) Proteins, whoseabundances were regulated bySA in sweet cherry fruits, wereidentified as enzymes involvedin glycolysis and tricarboxylicacid cycle. Step 1: catalyzed byUTP–glucose-1-phosphate uri-dylyltransferase (P-27); Step 2:catalyzed by Phosphoenolpyr-uvate carboxy kinase (P-14);Step 3: catalyzed by Glucose-6-phosphate 1-dehydrogenase (N-37); Step 4: catalyzed by Phos-phoglycerate kinase (P-13); Step5: catalyzed by Phosphoglyce-rate dehydrogenase (P-22); Step6: catalyzed by Enolase (N-40);Step 7: catalyzed by Fumaratehydratase (P-32); Step 8: cata-lyzed by NAD-dependent malatedehydrogenase (N-39 and P-23).



(Table 2). HSP are one of the major classes of chaperonemolecules and play many roles in eukaryotic cells. Theseproteins may act as a primary defense during oxidativestresses caused by pathogens, thus preventing damage ofROS to cellular membranes. Dehydrogenase oxidizes a sub-strate by transferring one or more protons and a pair ofelectrons to an acceptor, usually NAD/NADP or a flavincoenzyme such as FAD or FMN. They are directly involved inenergy pathway. It is postulated that heat shock proteins anddehydrogenases may act in the resistance response of fruitsat latter maturity stage.

Moreover, 18 identified proteins were related to metabo-lisms and energy pathway in the present study. Amongthem, 7 proteins [glucose-6-phosphate 1-dehydrogenase (N-37), NAD-dependent malate dehydrogenase (N-39), enolase(N-40), phosphoglycerate dehydrogenase (P-22), NAD-de-pendent malate dehydrogenase (P-23), UTP–glucose-1-phosphate uridylyltransferase (P-27), and fumarate hydra-tase (P-32)], which catalyze the reactions during sugar me-tabolism and energy pathway, were found to be altered insweet cherry fruits at maturity stage B after SA treatments.Those proteins together with 2 kinases [phosphoglyceratekinase (P-13), and phosphoenolpyruvate carboxykinase (P-14)] are directly involved in glycolysis and tricarboxylic acidcycle (Fig. 7b). It indicated that SA treatment might affectsugar metabolism and change energy pathway in sweetcherry fruits. Further detailed researches are needed to char-acterize the potential roles of sugar metabolism and energypathway in the resistance induced by SA.

Finally, it should be mentioned that sampling time is alsoimportant to reflect fruit response to SA application andpathogen attack. Younger fruits, as they had stronger resist-ance to pathogen attack, usually showed symptom moreslowly than older fruits. In addition, when pathogen-inocu-lated fruits at the same maturity stage were stored at lowtemperature, the symptom appeared much later than thatkept at room temperature, as low temperature inhibited thegrowth of P. expansium in sweet cherry fruits (Fig. 1).Therefore, sampling in this experiment was performed whenall WP-treated fruits showed the symptom of blue mold rotwith about 1 cm lesion diameter (Fig. 1), i.e. at 4, 3, 3, and18 days after SA application for fruits at A, B, C-R, and C-Lstages, respectively. Such a strategy might affect the directrelationship between pathogen attack response and fruitmaturity at the gene expression level.

Taken together, the novel findings of this study includ-ed: (i) younger sweet cherry fruits treated by SA showedstronger resistance against pathogen invasion; (ii) anti-oxidant proteins were involved in fruit resistance responseat every maturity stage, while HSPs and dehydrogenasesmay potentially act as factors at latter maturity stage; and(iii) the resistance induced by SA in sweet cherry fruits maybe related to metabolism and energy pathway. In addition,our data also revealed that exogenous SA treatment mightaffect endogenous level of H2O2 in sweet cherry fruits. Thefindings provide insight into the mechanisms of SA-

induced resistance that counts for the inhibitory effect ofpathogenic P. expansum in sweet cherry fruits at differentmaturity stages.

We thank Dr. Shihua Shen for his advice in the proteomicsexperiment. This study was supported by the National NaturalScience Foundation of China (30430480), by the Ministry ofScience and Technology of China (2006CB1019007), and by theKnowledge Innovation Program of the Chinese Academy of Sci-ences (KSCX2-YW-G-010).

The authors have declared no conflict of interest.

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6 Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL databases under the following accession numbers: CAT2,EF165590; and GPX, EF165591.


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