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Plant Physiology and Biochemistry 60 (2012) 35e45

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Plant Physiology and Biochemistry

journal homepage: www.elsevier .com/locate/plaphy

Research article

Heat treatment of peach fruit: Modifications in the extracellular compartmentand identification of novel extracellular proteins

Claudia A. Bustamante a, Claudio O. Budde b, Julia Borsani a, Verónica A. Lombardo a,Martin A. Lauxmann a,1, Carlos S. Andreo a, María V. Lara a, María F. Drincovich a,*

aCentro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, Rosario 2000,Argentinab Estación Experimental Agropecuaria San Pedro, Instituto Nacional de Tecnología Agropecuaria (INTA), Ruta Nacional n� 9, Km 170, San Pedro, Argentina

a r t i c l e i n f o

Article history:Received 18 May 2012Accepted 23 July 2012Available online 3 August 2012

Keywords:Cell wallDUF642GAPCHeat treatmentPeachPost-harvestPrunus persica

Abbreviations: 2D-DIGE, two-dimensional fluoresphoresis; ACO1, ACC (aminocyclopropane-1-carboxylinofuranosidase, a-L; CI, chilling injury; CW, cell walDUF, domain of unknown function; EG, endoglucanastosidase; GAPC, cytosolic glyceraldehyde-3-phosphaglyceraldehyde-3-phosphate dehydrogenase; Hsp, htreatment; IPG, immobilized pH gradient; NAD-MEphosphoenolpyruvate carboxylase; PEPCK, phosphoPG, polygalacturonase; PL, pectate lyase; PME, pectquantitative real-time reverse transcription-PCR; XYL* Corresponding author. Tel.: þ54 341 4371955; fax

E-mail address: drincovich@cefobi-conicet.gov.ar (1 Present address: Max-Planck-Institut für Moleku

Mühlenberg 1, 14476 Potsdam-Golm, Germany.

0981-9428/$ e see front matter � 2012 Elsevier Mashttp://dx.doi.org/10.1016/j.plaphy.2012.07.021

a b s t r a c t

Ripening of peach (Prunus persica L. Batsch) fruit is accompanied by dramatic cell wall changes that leadto softening. Post-harvest heat treatment is effective in delaying softening and preventing some chillinginjury symptoms that this fruit exhibits after storage at low temperatures. In the present work, the levelsof twelve transcripts encoding proteins involved in cell wall metabolism, as well as the differentialextracellular proteome, were examined after a post-harvest heat treatment (HT; 39 �C for 3 days) of“Dixiland” peach fruit. A typical softening behaviour, in correlation with an increase in 1-aminocyclopropane-1-carboxylic acid oxidase-1 (PpACO1), was observed for peach maintained at 20 �Cfor 3 days (R3). Six transcripts encoding proteins involved in cell wall metabolism significantly increasedin R3 with respect to peach at harvest, while six showed no modification or even decreased. In contrast,after HT, fruit maintained their firmness, exhibiting low PpACO1 level and significant lower levels of thetwelve cell wall-modifying genes than in R3. Differential proteomic analysis of apoplastic proteins duringsoftening and after HT revealed a significant decrease of DUF642 proteins after HT; as well as an increaseof glyceraldehyde-3-phosphate dehydrogenase (GAPC) after softening. The presence of GAPC in thepeach extracellular matrix was further confirmed by in situ immunolocalization and transient expressionin tomato fruit. Though further studies are required to establish the function of DUF642 and GAPC in theapoplast, this study contributes to a deeper understanding of the events during peach softening and afterHT with a focus on this key compartment.

� 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction

Peaches ripen and deteriorate quickly at ambient temperature.Cell wall changes associated with the softening process, including

cence difference gel electro-c acid) oxidase 1; ARF-arabi-l; dpi, days post-infiltration;e; Exp, expansin; Gal, galac-te dehydrogenase; GAPDH,eat shock protein; HT, heat, NAD-malic enzyme; PEPC,enolpyruvate carboxykinase;in methylesterase; qRT-PCR,, xylosidase.: þ54 341 4370044.M.F. Drincovich).lare Pflanzenphysiologie, Am

son SAS. All rights reserved.

modifications in the structure and composition of the constituentpolysaccharides, have been related to the expression of a number ofhydrolases and transglycosylases [1]. In this fruit, the activities ofseveral enzymes, such as exo- and endo-polygalacturonase (exo-PG,EC 3.2.1.67; endo-PG, EC 3.2.1.15), endo-b-1,4-mannanase (EC3.2.1.68), a-L-arabinofuranosidase (ARF, EC 3.2.1.55) and b-galacto-sidase (bGal, EC 3.2.1.23) are modified during ripening and areinvolved in the softening and texture modifications of this fruit [2].

Refrigeration is used to slow the ripening process as well as thedecay development during peach fruit storage and shipment [3].However, the storage of peaches at low temperatures for prolongedperiods induces chilling injury (CI). In peach, CI includes defectivecell wall disassembly and the development of a dry and woolly,rather than a soft and juicy, mesocarp [3]. The activities of severalcell wall-modifying enzymes are affected under cold storage, withimportant consequences for pectin metabolism [4]. In order toalleviate CI symptoms, exposure to sub-lethal high temperatureafter harvest has beenwidely used, as this heat treatment increases

C.A. Bustamante et al. / Plant Physiology and Biochemistry 60 (2012) 35e4536

the tolerance to subsequent chilling, delays ripening and softening,and reduces pathogen levels and disease development in severalfruits [5]. An approach to better understand and prevent CI is thestudy of the fruit changes that take place after a successful treat-ment that alleviates CI symptoms. In this regard, recent proteomicstudies on total peach mesocarp proteins after heat treatment (HT)identified several candidate proteins that may prevent some of theCI symptoms in peach fruit [6]. Besides, the analysis of the cell wallprotein composition of two peach cultivars with different suscep-tibility to CI and after cold storage have allowed the identification ofcandidate proteins involved in the protection against CI [7].However, to our knowledge, no studies regarding the impact of HTon the extracellular proteins of peach fruit have been performedyet.

In the present study, the level of transcripts encoding proteinsinvolved in the cell wall metabolism and the differential proteomeof extracellular proteins of the fruit, obtained by an infiltrationmethod, were examined during post-harvest ripening and after HTof “Dixiland” peach fruit. The analysis of peach fruit extracellularproteins is of great relevance as these proteins are involved in the

Fig. 1. Relationship between fruit firmness and levels of PpACO1 and cell wall-modifyingimmediately after harvest (R0), after 3 days at 20 �C (R3) or after heat treatment (HT). Valushown). Level of PpACO1 was determined by qRT-PCR. (BeF) Level of transcripts encodingripening or after HT. The means of the results obtained, using 3 independent mRNAs as tto P. persica DNA-repair enzyme mRNA. Y axis refers to the fold difference in a particular trPpARF/XYL values are expressed relative to those found in peach kept at 20 �C for 3 daysdifferent (P < 0.05). nd: not detected.

perception and transduction of environmental signals, cell wallmodification and reconstruction, and defence responses, althoughcomprise only 5e10% of the wall dry weight [8]. Considering thesoftening delay and prevention of CI symptoms induced by HT, theobjectivewas to search for proteins that are differentially expressedduring fruit softening and after HT, with a focus on the extracellularcompartment, in order to identify novel protein targets to beselected when trying tomodulate the peach fruit softening process.

2. Results

2.1. Fruit firmness and levels of cell wall-modifying genes

“Dixiland” peaches were harvested at an average firmness of52.1 � 6.5 N. Fruit kept at ambient temperature showed a signifi-cant decrease in firmness (from R0 to R3), while fruit exposed to39 �C for 3 days (HT) exhibited practically the same firmness ofthose fruit recently harvested (Fig. 1A). In a previous work, it wasshown that levels of the transcript encoding 1-aminocyclopropane-1-carboxylic acid oxidase 1 (PpACO1), involved in ethylene

transcripts in fruit kept at 20 �C or after HT. (A) Firmness was measured in peacheses represent the mean of 15 independent determinations using different fruit (SDs areproteins involved in cell wall metabolism, assessed by qRT-PCR, during post-harvest

emplate, are shown. Each reaction was normalized using the Ct values correspondinganscript level relative to its amount found in peaches after harvest (R0). In the case of(R3). Standard deviations are shown. Bars with the same letters are not significantly

C.A. Bustamante et al. / Plant Physiology and Biochemistry 60 (2012) 35e45 37

synthesis, were positively correlated to ethylene production [9].Fig. 1A shows that HT prevents the PpACO1 increase that usuallyoccurs during fruit ripening at 20 �C, as fruit exposed to HT dis-played a much lower level than fruit of the same post-harvest agemaintained at ambient temperature.

The levels of twelve transcripts encoding proteins involved incell wall metabolism were investigated by quantitative real-timePCR (qRT-PCR). The genes evaluated were the following: an a-L-arabinofuranosidase/b-xylosidase (PpARF/XYL) [10], a b-galactosi-dase (Pp-bGal) [10], an endo-b-1,4-glucanase (PpEG4) [11], threeexpansins (PpExp1ePpExp3) [10], two polygalacturonases (PpPG1and PpPG2) [12], two pectate lyases (PpPL1 and PpPL2) [12] and twopectin methylesterases (PpPME1 and PpPME2) [12]. During “Dixi-land” peach post-harvest softening, transcripts of all these geneswere detected, but accumulated differentially (Fig. 1BeF). Levels ofPpARF/XYLwere not detected at R0, but accumulated after 3 days at20 �C (Fig. 1B). While Ppb-Gal, PpExp1, PpExp3, PpPG1 and PpPG2increased (30-, 10-, 3-, 5- and 300-times, respectively) from R0 toR3 (Fig. 1BeD); PpEG4 and PpPME2 decreased nearly 2- and 5-fold,respectively (Fig. 1B and F). PpExp2, PpPL1, PpPL2 and PpPME1 didnot significantly change in fruit kept at 20 �C for 3 days with respectto R0 (Fig. 1C, E and F). On the contrary, after heat treatment of“Dixiland” peach fruit (HT), with the exception of Ppb-Gal, PpExp1,PpPG1 and PpPG2 whose levels remained practically unchangedwith respect to R0, the levels of the other transcripts analysed wereextremely low or even undetectable, as the case of PpARF/XYL,PpPME1 and PpPME2 (Fig. 1). On the other hand, when compared toR3, lower levels of all the transcripts analysed were found in HT(Fig. 1).

2.2. Extraction of extracellular proteins, 2D-DIGE analysis and MS/MS identification

By using a non-destructive procedure, extracellular (apoplastic)proteins were extracted from peach mesocarp. The infiltrationmethod used in this work was modified from the one employed inthe extraction of weakly bound cell wall proteins and proteins fromthe intercellular spaces from rice calli [13]. The proteins obtainedwere first analysed using pH 3e10 immobilized pH gradient (IPG)strips; however, as the majority of the proteins were detected overthe range from pH 7 to 10 (not shown), pH 7e10 IPG strips and

Table 1Sixteen most abundant spots from 2D gels (pH 7e10 and 12.5% SDS-PAGE) of extracellul

Spot Protein identification Organism Accession number (GenBank ID) Sc

1 Aspartyl protease Vitis vinifera XP_002264426 72 Aspartyl protease Vitis vinifera XP_002264426 53 Aspartyl protease Vitis vinifera XP_002264426 84 Aspartyl protease Vitis vinifera XP_002264426 145 DUF642 Arabidopsis thaliana NP_566328 206 DUF642 Arabidopsis thaliana NP_566328 57 Apr protease precursor Bacillus licheniformis CAH036708 Apr protease precursor Bacillus licheniformis CAH03670 69 Apr protease precursor Bacillus licheniformis CAH0367010 Peroxidase Artemisia annua AA045182 2911 Peroxidase Artemisia annua AA045182 2912 Small Hsp Prunus salicina ACV9324813 Germin-like protein Vitis vinifera XP_002278736 1814 Aspartyl protease Vitis vinifera XP_002264426 715 Small Hsp Prunus salicina ACV93248 1016 Porin Solanum tuberosum CAA56601 21

a Mascot MOWSE store.b Number of peptides matched in databases.c Experimental data.d Theoretical data.e N-terminal protein signal was predicted by iPSORT software (http://www.hypothesi

signal peptide.

12.5% SDS-PAGEwere used for electrophoretic separation. About 60spots were detected on the two-dimensional electrophoresis mapobtained with apoplastic protein from peach fruit at harvest (datanot shown). From these spots, the sixteen most abundant proteinswere chosen and identified by fingerprinting mass analysis,showing matches with proteases, proteins of unknown functionDUF642, a germin-like protein, peroxidases, a porin and small heatshock proteins (Table 1). To assess the contamination with intra-cellular proteins, Western blot analysis using antibodies againstheat shock protein 70 (Hsp70) and phosphoenolpyruvate carbox-ykinase (PEPCK), typical cytosolic proteins, and against NAD-malicenzyme (NAD-ME), a mitochondrial protein, were performed.These proteins were detected in total peach fruit extracts; however,no signal was detected in the apoplastic fractions (Fig. 2).

The differential proteome of the extracellular proteins from R0,R3 and HT peach samples was analysed by Two-DimensionalFluorescence Difference Gel Electrophoresis (2D-DIGE). Thecomparison of the gels obtained, allowed the identification of fourspots with significant variation by a factor �1.5 (t-test withsignificance P < 0.05, Fig. 3) among the different apoplastic peachprotein extracts. One spot increased in R3 with respect to R0 (spot17, Fig. 3), while the other three polypeptides decreased in HT inrelation to R3 (spots 18e20, Fig. 3). Three of these differentiallyexpressed proteins were identified by fingerprinting mass analysis,with spot 17 matching with a cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) from Plantago major (NCBIaccession number gi106879588 and Uniprot accession numberQ1EMQ8) and spots 19 and 20 showing matches with a protein ofunknown function (DUF642) from Prunus persica (NCBI accessionnumber gi22486324). Identifications gave the following parame-ters for spots 17, 19 and 20: (i) scores: 66, 281 and 270 (MASCOT,MOWSE score); (ii) number of peptides identified: 4, 8 and 7; and(iii) coverage: 20%, 38% and 38%; respectively. For spot 18 (Fig. 3) noprotein homology could be assigned.

2.3. Extracellular localization of GAPC

The presence of GAPC, a classical cytosolic enzyme, in the apo-plast of peach fruit was examined using affinity-purified antibodiesagainst recombinant GAPC of Triticum aestivum [14]. A GAPCimmunoreactive band of 37 kDa was detected by Western blot in

ar proteins extracted from R0 peach fruit and bioinformatics analysis.

orea Queries matchedb MWc (kDa) pIc MWd (kDa) pId Protein sorting signale

3 1 50 7.8 47 9.2 SP0 1 50 7.9 47 9.2 SP1 1 50 8.0 47 9.2 SP8 2 50 8.1 47 9.2 SP7 6 48 7.2 39 7.5 SP3 1 45 7.2 39 7.5 SP

28 8.5 63 10.7 mTP4 5 28 8.6 63 10.7 mTP

28 7.4 63 10.7 mTP4 3 40 8.5 35 8.6 SP1 3 40 8.7 35 8.6 SP

20 8.0 18 5.6 e

5 3 32 9.0 23 8.4 SP1 1 50 7.7 47 9.2 SP2 5 20 8.4 18 5.6 e

3 4 34 7.6 29 8.0 e

screator.net/iPSORT/); mTP, mitochondrial targeting peptide; SP, secretory pathway

Fig. 2. Western blot analysis of intracellular proteins. The presence of Hsp70, PEPCKand NAD-ME in total (R3t) and apoplastic (R3ap) protein extracts obtained from R3fruit is shown by immunoblotting using antibodies against Hsp70 of N. tabacum, PEPCKof cucumber and aNAD-ME of A. hypochondriacus. Twenty mg of protein extracted frompeach fruit were added per lane. Molecular masses of the immunoreactive bands areshown on the left (kDa). Coomassie brilliant blue-stained gels were loaded withequivalent amounts of the samples used in the western blots and served as a loadingcontrol (not shown).

Fig. 4. Western blot analysis of GAPC. (A) The presence of GAPC in total (R3t) andapoplastic (R3ap) protein extracts obtained from R3 fruit is shown by immunoblottingusing antibodies against GAPC of T. aestivum. (B) Western blot analysis of theexpression of GAPC in apoplastic protein extracts obtained from R0 (R0ap), R3 (R3ap)and HT (HTap) fruit. Twenty mg of protein extracted from peach fruit were added perlane. Molecular masses of the immunoreactive bands are shown on the left (kDa).Coomassie brilliant blue-stained gels were loaded with equivalent amounts of thesamples used in the western blots and served as a loading control (not shown).

C.A. Bustamante et al. / Plant Physiology and Biochemistry 60 (2012) 35e4538

both total and apoplastic protein extracts from R3 fruit (Fig. 4A), incontrast to Hsp70, PEPCK and NAD-ME, which were only detectedin total protein extracts (Fig. 2). On the other hand, whencomparing apoplastic extracts from R0, R3 and HT, no significantchanges in the immunoreactive GAPC bands, estimated by densi-tometric analysis (not shown), were detected (Fig. 4B).

To confirm the occurrence of GAPC in the apoplast of peach fruit,in situ immunolocalization of GAPC was conducted and examinedby confocal microscopy. Sections were counterstained withcellulose-binding Calcofluor white fluorochrome in order to visu-alize the cell wall of mesocarp cells. As shown in Fig. 5AeD, it isclear that GAPC is present in the cytoplasm, cell wall and inter-cellular spaces of R3 peach fruit. On the other hand, fluorescencefrom the immunolocalizations of b-xylosidase (b-XYL) and phos-phoenolpyruvate carboxylase (PEPC) indicated only cell wall orcytosolic localization of the mentioned proteins, respectively(Fig. 5EeL).

2.4. Analysis of transcripts encoding differential apoplastic proteinsamong R0, R3 and HT samples

The levels of the transcripts encoding GAPC and DUF642proteins from peach were further investigated by qRT-PCR in R0, R3and HT peach samples.

Fig. 3. Differential proteome of the extracellular proteins of peach fruit ripening at 20 �C orapoplastic proteins from R3 peach fruit. Samples were analysed at harvest (R0) and after 3 din intensity) among the different samples analysed are marked on the gel and numbered. (B)filled circle indicates the spot that is more abundant in R3 than in R0 samples, while open cirbelow shows the pI and molecular mass (MW) of each differential polypeptide as well as thecomparison. For spot N� 18 no protein homology could be assigned.

In higher plants, three distinct isoforms of phosphorylatingglyceraldehyde-3-phosphate dehydrogenase (GAPDH) are found,which exhibit specific cell compartmentalization: the cytosolicGAPDH (GAPC) that catalyses the oxidative phosphorylation ofglyceraldehyde-3-phosphate into 1,3-bisphosphoglycerate by con-verting NAD into NADH; the phosphorylating NADP-specific chlo-roplastic GAPDH (GAPA/B) involved in photosynthetic CO2 fixation;and the NAD-dependent GAPDH (GAPCp) that is closely related toGAPC and involved in the carbohydrate catabolism of non-greenplastids [14]. Two putative transcripts, named PpGAPDH1 andPpGAPDH2, encoding proteins with high sequence identity toGAPCs from higher plants (more than 85% sequence identity), werefound in peach EST databases (Table S1). The level of these tran-scripts was further analysed by qRT-PCR. As shown in Fig. 6A,PpGAPDH1 and PpGAPDH2 did not significantly change during fruitripening and after heat treatment.

On the other hand, DUF642 genes encode for a plant-specificdomain, DUF642, whose function is still unknown [15]. A tran-script, named PpDUF642, which encodes the protein identified asdifferentially expressed when comparing R3 and HT extracellularsamples (Fig. 3) was found in peach EST databases (Table S2).Deduced amino acid sequence of PpDUF642 showed high identitywith sequences of DUF642 proteins from Ricinus communis (77.8%),

subjected to HT. (A) A typical 2D-DIGE gel is presented showing the spot distribution ofays at 20 (R3) or 39 �C (HT). The protein spots with significant changes (by a factor �1.5The panels show typical partial sections of the 2D-gels from the indicated samples. Thecles show spots that are less abundant in samples from HT than from R3 fruit. The tableratio of decrease in the first condition with respect to the second of the corresponding

Fig. 5. Immunolocalization of GAPC in peach mesocarp visualized by confocal microscopy. (A, E and I) Calcofluor staining of cellulose in R3 fruit. (B) Mesocarp cells after incubationof R3 fruit section with affinity-purified antibodies against GAPC of T. aestivum. The green fluorescence obtained with the anti-GAPC/Alexa 488 stain is indicative of the presence ofGAPC in the cell wall (cw), cytoplasm (c) and intercellular spaces (i). (C) Section immunolabelled with GAPC and counterstained with Calcofluor showing the presence of GAPC in thecell wall (Pearson’s colocalization coefficient ¼ 0.4). (D) Superposition of bright field and fluorescent images of GAPC. (F and J) Sections immunolabelled with affinity-purifiedantibodies against b-XYL of F. x ananassa and PEPC of A. viridis were used as cell wall and cytosolic controls, respectively. (G and K) Immunofluorescence detection of b-XYLand PEPC in mesocarp cells counterstained with Calcofluor. (H and L) Superposition of bright field and fluorescent images of b-XYL and PEPC. Scale bars represent 20 mm. (Forinterpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

C.A. Bustamante et al. / Plant Physiology and Biochemistry 60 (2012) 35e45 39

Populus trichocarpa (76.4%), Vitis vinifera (76.0%) and Gylcine max(72.2%), among others (Table S2). The level of PpDUF642 was ana-lysed by qRT-PCR in peach samples. As shown in Fig. 6B, PpDUF642markedly decreased after HT, according to the results obtained atprotein level by 2D-DIGE analysis (Fig. 3).

2.5. Transient expression of GAPC and DUF642 fusion proteins intomato fruit

Localization of GAPC and DUF642 was also analysed by GFPfluorescence. PpGAPDH1, PpGAPDH2 and PpDUF642 cDNAs were

fused to GFP, expressed in tomato fruits at mature green stage andvisualized by confocal microscopy (Fig. 7). In tomato fruits agro-injected with p35STPpGAPDH1:GFP and p35STPpGAPDH2:GFP,which expressed PpGAPDH1-GFP and PpGAPDH2-GFP fusionproteins, the green fluorescence was associated with cytosol, cellwall and intercellular spaces (Fig. 7DeI). In the case of PpDUF642-GFP, the green fluorescence was detected in the cell wall and also inthe intercellular spaces (Fig. 7JeL). In contrast, in tomato fruitsagroinjected with the control construct p35STGFP (expressing theGFP coding sequence alone) the green fluorescence was distributedin the cytoplasm (Fig. 7AeC).

Fig. 6. Level of transcripts encoding proteins identified in 2D-DIGE studies. (A) Level ofPpGAPDH1-2, assessed by qRT-PCR, during post-harvest ripening and after HT. (B) qRT-PCR study of PpDUF642 in R0, R3 and HT fruits. The means of the results obtained,using 3 independent mRNAs as template, are shown. Each reaction was normalizedusing the Ct values corresponding to P. persica DNA-repair enzyme mRNA. Y axis refersto the fold difference in a particular transcript level relative to its amount found inpeaches after harvest (R0). Standard deviations are shown. Bars with the same lettersare not significantly different (P < 0.05). nd: not detected.

C.A. Bustamante et al. / Plant Physiology and Biochemistry 60 (2012) 35e4540

3. Discussion

3.1. Levels of cell wall-modifying transcripts during “Dixiland”peach softening and upon heat treatment

Softening of “Dixiland” peach fruit at 20 �C was accompanied byan increase in the levels of six cell wall-related genes (PpARF/XYL,Ppb-Gal, PpExp1, PpExp3, PpPG1 and PpPG2) and the decrease of two(PpEG4 and PpPME2) (Fig. 1). After heat treatment of peach fruit,which inhibits fruit softening (Fig. 1A), similar or lower levels of allthe cell wall-related genes analysed were detected when comparedto fruit at harvest (R0), while significant lower levels were found inall cases when compared to R3 (Fig.1). Thus, taking into account theresponse of the cell wall-related genes to HT, they can be classifiedinto three groups: 1) genes induced after peach softening for 3 daysbut repressed by HT (in comparison to R3): PpARF/XYL, Ppb-Gal,PpExp1, PpExp3, PpPG1 and PpPG2; 2) genes that are not modifiedafter peach softening for 3 days but resulted decreased by HT:PpExp2, PpPL1, PpPL2 and PpPME1; 3) genes that are decreased afterpeach softening for 3 days and that resulted further decreased byHT: PpEG4 and PpPME2. Genes belonging to group 1 are highlyprobably induced by ethylene and involved in peach softening.Moreover, the lack of induction of these genes after HT is correlatedto the softening inhibition produced by this treatment. On the other

hand, genes belonging to groups 2 and 3 are not correlated ornegatively correlated to softening, being insensitive or repressed byethylene, respectively. However, these transcripts decrease afterHT, indicating that the alteration of the cell wall metabolismproduced by HT is not only due to the repression of the ethylenesynthesis, as previously suggested [16]. Thus, additional factors thatinducemodifications in proteins involved in cell wall metabolism inpeach fruit, apart from the decrease in ethylene, are taking placewhen peach fruit is exposed to HT [17].

3.2. Identification of novel proteins from peach apoplast withdifferential expression during fruit softening or upon heat treatment

Recently, proteomic analysis was performed in order to examinechanges in total peach mesocarp proteins during the post-harvestand after heat treatment [6,9]. However, such a proteomicapproach to identify apoplastic proteins that are differentiallyexpressed during peach softening or after HT has not been per-formed before. In the present work, 2D-DIGE analyses performedwith apoplastic peach proteins identified GAPC and DUF642proteins as differentially expressed in this compartment duringripening and after heat treatment, respectively (Fig. 3).

Cytosolic GAPDH (GAPC) is a highly conserved enzyme which,besides its participation in the glycolysis, is thought to be involvedin additional cellular functions [18]. In this work, proteomic andWestern blot studies showed the presence of GAPC in the apoplastof peach fruit (Figs. 3 and 4), which was confirmed by in situimmunolocalization in peach (Fig. 5) and by transient expression intomato fruits (Fig. 7). Thus, these results indicate that, besides thealready known plastidic and cytosolic localization of the enzyme,GAPC would also be targeted to the extracellular matrix in peachfruit. In relation, previous proteomic approaches have also revealedthe presence of GAPC in the cell wall of grape berry [19], Arabi-dopsisthaliana[20],Medicago truncatula [21] and maize [22], and onthe surface of yeast and bacteria [23,24]. However, only in the caseof microorganisms it has been unequivocally demonstrated thatGAPC is a secreted protein [25]. It remains an open questionwhether GAPC fulfils a discrete function in the apoplast and if thereis a mechanism involved in the export of this protein from thecytosol. Moreover, the results obtained in this work, suggest thatGAPC in the extracellular matrix may be linked to the ripening ofpeach fruit.

Although an increase in the intensity of the spot identified asGAPC was detected in apoplast extract from R3 compared to R0(spot 17, Fig. 3), no significant changes in the total amount ofimmunoreactive GAPC were detected by Western blot (Fig. 4B).Several explanations may account for these results. First, it may bepossible that, from the total GAPC detected by Western blot in theextracellular matrix, just a fraction, or alternatively a particularGAPC isoform, may change its isoelectro-focussing characteristicsby post-translational modification during peach post-harvestripening (Fig. 3). In this regard, in silico analysis of PpGAPDH1and PpGAPDH2 (PROSITE; http://www.expasy.org/prosite/)suggests that these enzymes could be subjected to several types ofpost-translational modifications, including N-glycosilation, phos-phorylation and N-myristoylation. This hypothesis is also linked tothe practically constant levels of PpGAPDH1 and PpGAPDH2 duringfruit ripening (Fig. 6). Secondly, the results may also be explainedby considering that the antibodies used in the present work do notdetect the GAPC isoform that is up-regulated and/or translocated tothe apoplast during peach ripening. In this sense, in a previousproteomic analysis using total peach mesocarp protein, GAPC wasalso up-regulated during post-harvest ripening [26].

DUF642 family proteins contain a conserved region found ina number of uncharacterised plant proteins [14]. Proteins of

Fig. 7. Transient expression of GFP fusion proteins in tomato fruits. (A, D, G and J) Calcofluor staining of cellulose in tomato fruit at 7 dpi. (B, E, H, K) GFP fluorescence in tomato fruittransiently transformed with: Untargeted GFP (B); PpGAPDH1-GFP (E); PpGAPDH2-GFP (H); PpDUF642-GFP (K). (C, F, I, L) Superposition of fluorescent images between A and B (C);D and E (F); G and H (I) and J and K (L). For both PpGAPDH1-GFP and PpGAPDH2-GFP, in which the fusions are detected in the cytosol, cell wall and intercellular spaces, a Pearson’scolocalization coefficient of 0.2 was obtained for Calcofluor and GFP fluorescence. In the case of PpDUF642-GFP, a Pearson’s colocalization coefficient of 0.3 was obtained. In tomatofruits agroinjected with the control construct p35STGFP the green fluorescence was distributed in the cytoplasm (Pearson’s colocalization coefficient ¼ 0.1). c: cytoplasm, cw: cellwall, i: intercellular spaces. Scale bars represent 20 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

C.A. Bustamante et al. / Plant Physiology and Biochemistry 60 (2012) 35e45 41

unknown function DUF642, which contain putative galactose-binding domains, have been identified in A. thaliana [27] andMedicago sativa [28] cell wall. In the present work, two spots fromthe apoplastic proteome of peach fruit matching with DUF642proteins were repressed in heated fruit with respect to R3 samples(Fig. 3). This differential expressionwas validated at transcript levelby qRT-PCR (Fig. 6). Analysis of PpDUF642 sequence (PROSITE;http://www.expasy.org/prosite/) indicates possible post-translational modifications, such as N-glycosylation, phosphoryla-tion and N-myristoylation that may explain the presence of twospots with different pI. The presence of putative galactose-binding

domains in its sequence and the extracellular localization of theprotein suggest that PpDUF642 could be playing a role in the cellwall metabolism of peach fruit. However, additional work isrequired to establish the participation of this protein in theresponse to HT and the softening process.

4. Concluding remarks

Data presented in this study indicate that after HT ripening isimpaired with maintenance of fruit firmness and decrease inthe levels of genes involved in cell wall metabolism. During

C.A. Bustamante et al. / Plant Physiology and Biochemistry 60 (2012) 35e4542

ripening at ambient temperature, softening of peach fruit isaccompanied by increase in the levels of PpARF/XYL, Ppb-Gal,PpExp1, PpExp3, PpPG1 and PpPG2 genes. Proteomic analysis ofthe fruit apoplast showed a significant decrease in DUF642protein levels after HT, while a spot identified as GAPC wasinduced during ripening. The results obtained in this workreveal that both classical and non-classical extracellularproteins could be involved in the dramatic cell wall changesthat accompany the onset of ripening in peach fruit, identifyingnovel proteins targets to be selected when trying to modulatethe fruit softening process.

5. Experimental

5.1. Plant material and heat treatment

Assayswere conducted with peach fruit (P. persica [L.] Batsch, cv.Dixiland) grown in the Estación Experimental Agropecuaria INTA,San Pedro, Argentina [16], during 2007 and harvested in 2008 andrepeated with fruits grown during 2009 and harvested in 2010. Theflesh firmness of the fruit at harvest was typically 52.1 � 6.5 N,which corresponded to approximately 93 days after bloom.Immediately after harvest, fruit were manually selected foruniformity of colour, size and firmness and divided into twogroups: one was kept in a chamber at 20 �C and 90% relativehumidity for 3 days and the other was held in a chamber at39� 1 �C and 90% relative humidity for 3 days. Samples were takenimmediately after harvest (R0), after 3 days in the chamber at 20 �C(R3) and after 3 days at 39� 1 �C (HT). About 20e30 fruit from eachgroup were used for firmness measurements. Representativemesocarp tissue was also collected from the different sample fruit,immediately frozen in liquid nitrogen and stored at �80 �C forfurther experiments.

Tomato fruits (Solanum lycopersicum cv Micro Tom) at maturegreen stage (22e25 days after anthesis) were used for transientexpression of fusion proteins. About 10 fruits were infiltrated foreach construction.

5.2. Firmness determination

Flesh firmness was evaluated with a penetrometer (Effegi 327,Italy) with a 7.9 mm tip and expressed in Newtons (N). Measure-ments were carried out on two opposite sides of each individualfruit after removal of the peel [16].

5.3. Extracellular protein extraction

The extraction of extracellular (apoplastic) proteins from peachfruit was carried out according to Chen et al. [13] with somemodifications. Mesocarp tissue was cut in pieces and extractedwith one volume of extraction buffer (0.1 M TriseHCl, pH 8.0;0.1 M KCl; 10% (v/v) glycerol; 10 mM EDTA; 0.3 M mannitol; 0.2 MCaCl2 and 1 mM PMSF) for 1 h at room temperature after infil-tration with a vacuum pump for 1 min. Proteins extracted werecollected by centrifugation for 15 min at 20,000 g. One volume ofTriseHCl, pH 8.8-saturated phenol was mixed thoroughly with theextract, followed by centrifugation at 5000 g for 15 min. Aftercentrifugation, protein samples in the phenolic phase werecollected, and the remaining phenolic layer was back extractedfour times with 100 mM TriseHCl, pH 8.4; 20 mM KCl; 10 mMEDTA and 0.4% (v/v) b-mercaptoethanol. The phenolic phase wasprecipitated by the addition of five volumes of 100 mM ammo-nium acetate in methanol at �20 �C overnight. The precipitatedproteins were then recovered by centrifugation for 20 min at

20,000 g at 4 �C and washed twice with 100 mM ammoniumacetate in methanol, followed by a final wash in 80% (v/v) acetone.The pellet was resuspended in 2D-DIGE buffer (30 mM TriseHCl,pH 8.5; 7 M urea; 2 M thiourea; 4% (w/v) CHAPS) for 2D-DIGE ordiluted in 0.25 M TriseHCl, pH 7.5; 2% (w/v) SDS; 0.5% (v/v) b-mercaptoethanol and 0.1% (v/v) bromophenol blue and boiled for2 min for SDS-PAGE.

5.4. Total protein extraction

Approximately 2 g of mesocarp material was ground in liquidnitrogen using a ceramic mortar and pestle, sand and polyvinylpolypirrolidone (PVPP), transferred to an SS34 tube containing10 ml of extraction buffer (100 mM TriseHCl, pH 8.8; 2% (w/v)SDS; 0.4% (v/v) b-mercaptoethanol; 10 mM EDTA; 1 mM PMSF;0.9 M sucrose) and 10 ml of ice-cold TriseHCl, pH 8.8-saturedphenol, and then agitated at 4 �C for 30 min. The aqueous pha-ses were back-extracted with extraction media and phenol byvortexing. Tubes were centrifuged at 5000 g for 15 min at 4 �Cand the phenolic phases were transferred to a new tube leavingthe interface intact. Proteins were precipitated with 5 volumes ofcold 0.1 M ammonium acetate in methanol at �20 �C overnight.The samples were collected by centrifugation at 20,000 g at 4 �Cfor 20 min. Next, the pellet was washed with 1.5 ml of coldammonium acetate/methanol and two times with cold 80% (v/v)acetone. A final wash used 1.5 ml of cold 70% (v/v) ethanol. Thepellet was resuspended in 2D-DIGE buffer and diluted in 0.25 MTriseHCl, pH 7.5; 2% (w/v) SDS; 0.5% (v/v) b-mercaptoethanoland 0.1% (v/v) bromophenol blue and boiled for 2 min for SDS-PAGE.

5.5. Protein quantification

Protein concentration was determined by the method of Brad-ford [29] using the Bio-Rad protein assay reagent (Bio-Rad,Hercules, CA, USA) and bovine serum albumin as standard.

5.6. Gel electrophoresis

SDS-PAGE was performed in 10% (w/v) polyacrylamide gelsaccording to Laemmli [30]. Proteins were visualized with Coo-massie blue or electroblotted onto a nitrocellulose membrane forimmunoblotting. Bound antibodies were located by linking toalkaline phosphatase-conjugated goat anti-rabbit IgG according tothe manufacturer’s instructions (Bio-Rad, Hercules, CA, USA). Theantibodies used for detection were the following: 1:200 anti-T.aestivum GAPC [14]; serum against the a subunit of NAD-ME(diluted 1:1000) from Amaranthus hypochondriacus [9]; 1:200anti cucumber PEPCK [9] and 1:200 anti Nicotiana tabacumHSP70 [5].

5.7. Protein labelling with dyes

To perform Two-Dimensional Fluorescence Difference GelElectrophoresis (2D-DIGE) experiments, proteins were labelledwith Alexa 532 (excitation, 532 nm; emission peak, 554 nm) orAlexa 594 (excitation, 590; emission, 617 nm) after adjustingthe pH to 8.5 using the supplier’s instructions (MolecularProbes Inc., Invitrogen Ltd). Proteins dissolved in 2D-DIGEbuffer were labelled at the ratio of 100 mg of protein to 20 nmolof Alexa protein minimal labelling dye in dimethylformamide.After vortexing, samples were incubated for at least 2 h on ice.The reaction was quenched by addition of 1 ml of 1 mM lysineand 20 mM DTT, and 4% (v/v) isoelectrofocusing (IEF) buffer pH7e9 was added (Amersham Biosciences).

C.A. Bustamante et al. / Plant Physiology and Biochemistry 60 (2012) 35e45 43

5.8. Two-dimensional gel electrophoresis

A 75 mg aliquot of Alexa 532-labelled sample was mixed with75 mg of Alexa 594-labelled protein prior to 2D gel electrophoresis.Protein samples at harvest (R0), from fruit kept at 20 �C for 3 days(R3) or exposed to HT were analysed, mixing differentially labelledR0 and R3 proteins and R3 and HT proteins. Each mixture was runat least in triplicate using proteins extracted from different bio-logical samples. A Protean IEF Cell instrument (Bio-Rad) was usedfor IEF with pre-cast IPG strips (pH 7e10, linear gradient, 17 cm;Bio-Rad). Samples of 300 ml containing the labelled proteins wereloaded by in-gel rehydration according to the manufacturer’sprotocol. The strips were subjected to IEF using the followingprogram: 12 h at 50 V; 1 h at 500 V; 1 h at 1000 V and 8000 V untila final voltage of 50,000 V was reached. Focused gel strips wereequilibrated in SDS equilibration buffer (350 mM TriseHCl, pH 8.0;20% (v/v) glycerol; 2% (w/v) SDS and 6 M urea), first with buffercontaining 130 mM DTT for 15 min and afterwards with buffercontaining 135 mM iodoacetamide for 15 min. The strips werewashed briefly with running buffer, loaded on top of a preparedSDS-PAGE Laemmli gel cast with 12.5% (w/v) acrylamide, andcovered with 1.5% (w/v) agarose. Proteins were separated at 15 mAper gel for 12e15 h at 15 �C using a Hoefer TMSE 600, 18 � 16 cm(Amersham, Uppsala, Sweden), and the gels were scanned usinga BioChemi System UVP BioImaging System. Data were saved in tiffformat. To excise samples for mass spectrometric analysis,a preparative gel loaded with 0.5 mg of protein was run.

5.9. Gel image analysis

Images were analysed using Image Master 2D-Platinum (GEHealthcare) using the protocol described in Casati et al. [31]. Anormalization procedure was used to allow for variation in totalprotein loading onto the gel(s). Total spot volume was calculated,and each spot was assigned a normalized spot volume asa proportion of this total value. Normalized spot volumes werecompared between Alexa 532- and Alexa 594-labelled samples oneach gel. Difference thresholds were then applied to identify theproteins with a statistically significant 1.5-fold difference innormalized spot volume (P < 0.05) [31].

5.10. In-gel digestion, mass spectrometry, and database searching

Before the spots were removed, the gel was stained usingcolloidal Coomassie blue stain. Gel spots of interest were manuallyexcised from the gels and sent to theMS facility CEQUIBIEM (Centrode Estudios Químicos y Biológicos por Espectrometría de Masa;Facultad de Ciencias Exactas y Naturales, Universidad de BuenosAires, Argentina) for further analyses. Spots were subjected to in-gel digestion (http://donatello.ucsf.edu/ingel.html) with trypsinaccording to [31]. Themass spectrometric datawere obtained usinga MALDI-TOF-TOF spectrometer, Ultraflex II (Bruker). The spectraobtained were submitted for National Center for BiotechnologyInformation (NCBI) database searching using MASCOT (www.matrixscience.com) [32] and analysed as previously described[31]. Protein functional classification was carried out according toliterature data. The presence of N-terminal signal peptides andpost-translational modifications were predicted by iPSORT (http://www.hypothesiscreator.net/iPSORT/) and PROSITE (http://www.expasy.org/prosite/) software, respectively.

5.11. In situ immunolocalization by confocal microscopy

Peach fruit at R3 stage were cut into pieces approximately3e4 mm diameter and fixed in FAA solution (50% (v/v) ethanol, 10%

(v/v) formaldehyde and 5% (v/v) acetic acid) at room temperaturefor 2 days. After dehydration of the samples in a graded series ofethanol, the material was embedded in paraffin. Sections of 8 mmwere collected on gelatine-coated slides and immunostained. Forimmunolabelling, slides were deparaffinised in xylol and rehy-drated. Sections were permeabilized with 0.5% (v/v) Triton X-100,blocked with 3% (w/v) BSA in 1 � PBS for 1 h and then incubatedwith 1:20 anti-T. aestivum GAPC [13], 1:5 anti-Amaranthus viridisPEPC [8], 1:5 anti-Fragaria x ananassa XYL [33] or 1 � PBS (negativecontrol) overnight at 4 �C. After washing the sections with 1 � PBScontaining 0.05% (v/v) Tween 20, slides were incubated with 1:500Alexa Fluor 488 goat anti-rabbit IgG for 1 h in the dark. Sampleswere washed and counterstained for 5 min with 17.5 mg ml�1

Calcofluor white (Sigma) to detect the presence of cellulose. Afterwashing with 1 � PBS, the sections were mounted in anti-fadesolution (0.1% (w/v) p-phenylenediamine and 50% (v/v) glycerolin 1 � PBS) and observed on a confocal microscope (ModelTE-2000-E2; Nikon). Pearson’s correlation coefficient was calcu-lated for each image using the program “Image J” (http://imagej.nih.gov/ij).

5.12. RNA isolation and reverse transcription

Total RNA from different samples of peaches was isolated from4 g of tissue using the method described by Meisel et al. [34]. Theintegrity of the RNA was verified by agarose electrophoresis. Thequantity and purity of RNA were determined spectrophotometri-cally [8]. First-strand cDNA was synthesized with MoMLV-reversetranscriptase following the manufacturer’s instructions (Promega,Madison, WI, USA) and using 3 mg of RNA and oligo(dT).

5.13. Quantitative real-time PCR

Relative transcript level was determined by performing quan-titative real-time PCR (qRT-PCR) in an iCycler iQ detection systemwith the Optical System Software version 3.0a (Bio-Rad), using theintercalation dye SYBRGreen I (Invitrogen) as a fluorescent reporter,with 2.5 mMMgCl2; 0.5 mMof each primer and 0.04 U ml�1 of GoTaqPolymerase (Promega). PCR primers were designed based on peachfruit cDNA sequences published in GenBank and P. persicaexpressed sequence tag (EST) databases (TIGR Plant TranscriptAssemblies; http://plantta.tigr.org) [35], with the aid of the web-based program “primer3” (http://www.frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) in order to produce amplicons of141e246 bp in size (Table S3). The sequences of the primers andamplicons were analysed further using peach EST databases(ESTree Database; http://www.itb.cnr.it/estree/; and GDR GenomeDatabase for Rosaceae, http://www.bioinfo.wsu.edu/gdr/) [36,37].A 10-fold dilution of cDNA obtained as described above was used astemplate. PCR controls were performed in the absence of addedreverse transcriptase to ensure RNA samples were free of DNAcontamination. Cycling parameters were as follows: initial dena-turation at 94 �C for 2 min; 40 cycles of 96 �C for 10 s, 58 �C for 15 sand 72 �C for 1 min; and 72 �C for 10 min. Melting curves for eachPCR were determined by measuring the decrease of fluorescencewith increasing temperature (from 65 �C to 98 �C). The specificity ofthe PCRs was confirmed by melting curve analysis using theappropriate software as well as by agarose gel electrophoresis ofthe products. Relative t was calculated using the “Comparative2�DDCT” method [38] and DNA-repair enzyme (PpDNArep) asreference gene. Each RNA sample was run in triplicate and repeatedin at least two independent sets of treatments generating a total of6 replicates per gene per sample. To test whether PpDNArepbehaves as housekeeping gene in the analysed samples, the gene

C.A. Bustamante et al. / Plant Physiology and Biochemistry 60 (2012) 35e4544

expression index was plotted against the sample and linearity andlow slope were verified.

5.14. Construction of gene fusions and transient transformation

Full-length cDNAs encoding PpGAPDH1, PpGAPDH2 andPpDUF642 were amplified by RT-PCR using RNA extracted from R3peach fruit. In the case of PpGAPDH1, the oligonucleotide pairGAPDH1F (50-GGTACCATGGGATCTGACAAG-30) and GAPDH1R (50-GGATCCGAGAGTGGATGCTAC-30) was used. PpGAPDH2 was ampli-fied with the primer pair GAPDH2F (50-GGTACCATGGGGAA-GATCAAGA-30) and GAPDH2R (50-GGATCCAGCATGATCAACAGAT-30), and PpDUF642 with DUFF (50-GGGGTACCATTCTTCTTGTTTT-30)and DUFR (50-GGATCCAGCAACAGGGTAAAC-30). The PCR productswere cloned into pGEM-T Easy Vector (Promega) and completelysequenced. The primers were designed to introduce unique KpnIand BamHI sites at the 50 and 30 ends, respectively, to facilitate thesubcloning into the pGREEN II vector. In each pGREEN II vectorcontaining the inserts of PpGAPDH1, PpGAPDH2 and PpDUF642,cDNAs were fused in-frame to GFP to obtain the fusion proteins.Binary vectors carrying the fusion genes or GFP alone were intro-duced into A. tumefaciens strain GV3101. The resulting bacteriawerethen used for transient transformation according to Orzaez et al.[39]. Sections of fruits were counterstained with Calcofluor andimages were takenwith a confocal microscope (Model TE-2000-E2;Nikon). Agroinfiltrated tomatoes showed the highest levels of GFPfluorescence at 7 dpi (days post-infiltration). Negative controls,consisting in non-agroinjected and PGREEN II-agroinjected toma-toes, did not render significant green fluorescence. Pearson’scorrelation coefficient was calculated for each image using theprogram “Image J” (http://imagej.nih.gov/ij).

5.15. Statistical analysis

Data from the quantitative real time experiments and firmnessvalues were tested using one-way analysis of variance (ANOVA).Minimum significant differences were calculated by the Bonferroni,Holm-Sidak, Dunett and Duncan tests (a ¼ 0.05) using the SigmaStat Package.

Acknowledgements

This work was supported by Argentine National ResearchCouncil (CONICET, PIP 0679). CB, MFD, MVL and CSA are membersof the Researcher Career of CONICET and JB and ML are fellows ofthe same Institution. COB is member of INTA. The authors thank Dr.Diego Gómez-Casati (CEFOBI) and Drs. GustavoMartínez and PedroCivello (IIB-INTECH) for kind gift of the anti-GAPC and anti-b-XYLantibodies, respectively.

Appendix A. Supplementary material

Supplementary material associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.plaphy.2012.07.021.

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