expression of defense genes in strawberry fruits treated with different resistance inducers

Expression of Defense Genes in Strawberry Fruits Treated withDifferent Resistance InducersLucia Landi, Erica Feliziani, and Gianfranco Romanazzi*

Department of Agricultural, Food, and Environmental Sciences via Brecce Bianche, Marche Polytechnic University, Ancona 60131,Italy

ABSTRACT: The expression of 18 defense genes in strawberry fruit treated with elicitors: chitosan, BTH, and COA, at 0.5, 6,24, and 48 h post-treatment was analyzed. The genes were up-regulated differentially, according to the elicitor. Chitosan andCOA treatments promoted the expression of key phenylpropanoid pathway genes, for synthesis of lignin and flavonoids; onlythose associated with flavonoid metabolism were up-regulated by BTH. The calcium-dependent protein kinase, endo-β 1,4-glucanase, ascorbate peroxidase, and glutathione-S-transferase genes were up-regulated by BTH. The K+ channel,polygalacturonase, polygalacturonase-inhibiting protein, and β-1,3-glucanase, increased in response to all tested elicitors. Theenzyme activities of phenylalanine ammonia lyase, β-1,3-glucanase, Chitinase, and guaiacol peroxidase supported the geneexpression results. Similarity of gene expression was >72% between chitosan and COA treatments, while BTH showed lowersimilarity (38%) with the other elicitors. This study suggests the relationship between the composition of the elicitors and aspecific pattern of induced defense genes.

KEYWORDS: benzothiadiazole, chitosan, elicitors, Fragaria × ananassa, gene expression

■ INTRODUCTION

Strawberry (Fragaria × ananassa) is one of the most widelyconsumed berries, and it is a good source of naturalantioxidants.1 However, strawberry fruits are highly perishableand very susceptible to fungal decay in the field, and even moreso during postharvest storage. This can result in severe croplosses. Application of natural compounds known as resistanceinducers or elicitors is an innovative approach to prolong theshelf life of fresh fruit, through the reduction of diseaseincidence and with increased ecological security and safety forconsumers. To reduce the postharvest decay of strawberries,the application of these natural compounds has beeninvestigated as an alternative to the use of syntheticfungicides.2−4 These compounds act as elicitors, as theyactivate the natural phenomenon known as induced resistance,with effects that are localized or, more often, systemic and thatpromote nonspecific resistance to pathogens.5,6

In the present study, we investigated three differentresistance inducers that are based on natural compounds totest their activation of the resistance mechanism. Thebiopolymer chitosan is an N-deacetylated form of thepolysaccharide chitin that is found in the cell wall of manyfungi. Chitosan has been shown to have a double action in plantprotection: it inhibits the development of decay-causing fungithrough the production of a film on treated surfaces,7,8 and itinduces resistance responses in plant tissues. Benzothiadiazole(BTH), which is the functional analogue of the plantendogenous hormone-like compound salicylic acid, protectsdifferent plant species against diseases caused by viral, bacterial,and fungal pathogens.9 The third product is a commercialformulation that is based on a mixture of calcium and organicacids (COA), according to the well-known effects of calcium invegetal tissues for the binding of pectins and for strengtheningthe plant cell wall.10,11

The signaling pathways that control systemic resistance aremultiple component networks with characteristic schemes thatlead to plant resistance.12 However, the transcription factorsproduced as a result of signal transduction can trigger theexpression of a large number of genes, with the consequentphysiological events usually involving changes in cell-wallcomposition, ion fluxes, de novo production of pathogenesis-related (PR) proteins, synthesis of phytoalexins, and reactiveoxygen species (ROS) production. 13

Several studies have shown the involvement of phenoliccompounds14,15 and cell-wall degradation enzyme activities3,16

in the responses of strawberry fruit exposed to postharvesttreatments with elicitors. However, the relationships betweenresistance inducers and the genes used as potential markers forresistance induction in harvested strawberries have not beeninvestigated.The aim of the present study was to setup a method based on

reverse transcription−quantitative real-time polymerase chainreaction (RT-qPCR) to analyze changes in expression ofselected defense genes induced in strawberry fruit at 0.5, 6, 24,and 48 h following short (30 s) treatments with the elicitorschitosan, BTH, and COA. The 18 genes analyzed wereassociated with Ca2+ and K+ ion fluxes; calcium-dependentprotein kinase (CDPK); K+ channels; ROS cell responses,including glutathione S-transferase (GST) and ascorbateperoxidase (APX); secondary metabolites of the phenyl-propanoid pathway, including phenylalanine ammonia-lyase(PAL); biosynthesis of different flavonoid branches, includingchalcone synthase (CHS), chalcone isomerase (CHI), flavanon

Received: October 1, 2013Revised: March 14, 2014Accepted: March 16, 2014Published: March 16, 2014

Article

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© 2014 American Chemical Society 3047 dx.doi.org/10.1021/jf404423x | J. Agric. Food Chem. 2014, 62, 3047−3056

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3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR),flavonol synthase (FLS), anthocyanidin synthase (ANS), andflavonoid 3-O-glucosyltransferase (UFGT); cell-wall degrada-tion enzymes, including endo-β-1,4-glucanase (endoβGlu),polygalacturonase (PG), polygalacturonase-inhibiting protein(PGIP); and encoding of PR proteins, including β-1,3-glucanase (βGlu), class III Chitinase (Chi3), and PR-1. Inthe same way, the enzyme activities related to PAL, βGlu, totalChitinase (Chi), and guaiacol peroxidase (GPX) were tested.The results of this study provide new information on the effectof resistance inducers on strawberry fruit.

■ MATERIALS AND METHODSFruit Material. Experiments were carried out on fruits of

strawberries cv. Camarosa grown according to organic agriculturepractices, from commercial orchards located in the Marche region,central-eastern Italy. The fruits were selected for the absence ofdefects, uniformity in size, and degree of ripening (2/3 of surface red),and they were used for the experiments on the day of their harvest.Treatments. The physiological changes in the strawberry fruit were

analyzed following treatments with commercial elicitor formulations:

1% (w/v) chitosan (Chito Plant, ChiPro GmbH, Bremen, Germany),1% (v/v) COA (Fitocalcio, Agrisystem, Lamezia Terme, CZ, Italy),and 1% (w/v) BTH (Bion, Syngenta, Milano, MI, Italy). Thecommercial products were prepared by dissolving or diluting them indistilled water. Distilled water was used as the control.

Strawberry fruits (800 g) were pooled together and randomized,and then they were immersed for 30 s in 1 L of one of the threeresistance inducers or water. After the treatments, the strawberrieswere dried in air for 30 min and then subdivided into 4 groups of 200g each. After air drying, one group was stored at −80 °C (0.5 h post-treatment), and the other groups were arranged in small plastic boxesthat were placed in bigger covered plastic boxes. These groups werestored up to a total of 6, 24, and 48 h post-treatment, at 20 °C and95%−98% relative humidity. At each time, fruits corresponding to 20 gwere randomly selected and stored in two separated tubes at −80 °C,until RNA extraction. The experiments were repeated at least twice.

Gene Expression Analysis. Gene expression analysis on thestrawberry fruit after the treatments with the different resistanceinducers was performed by RT-qPCR, using a SYBR-green dye system,according to Minimum Information for Publication of QuantitativeReal-Time PCR Experiments (MIQE) guidelines.17 The comparative−ΔΔCt method

18 was used to evaluate the relative quantities of eachof the amplified products in the samples.

Table 1. Primers Selected for the Gene Expression Analyses of Strawberry Fruits Treated with Chitosan, COA, and BTHCommercial Formulationsa

aPCR amplification efficiencies and regression coefficients for the standard curves are reported for each primer pair. *, reference genes.

Journal of Agricultural and Food Chemistry Article

dx.doi.org/10.1021/jf404423x | J. Agric. Food Chem. 2014, 62, 3047−30563048

RNA Extraction. High quality total RNA was obtained from thefruit according to the protocol of Landi and Romanazzi.19 Briefly, 10 gof strawberry tissue samples including both achenes and receptacle wasground in liquid nitrogen, and 400 mg of the resulting fruit powderswas randomly collected for RNA extraction. Extraction buffer wasadded (1 mL; 100 mM Tris-HCl, pH 8.0, 25 mM EDTA, pH 8.0, 2%[w/v] CTAB [Sigma], 2% [v/v] β-mercaptoethanol, 2.5 M NaCl, and2% [w/v] soluble PVP-40), and the samples were incubated at 65 °Cfor 30 min. The supernatants were transferred to new tubes with anequal volume of chloroform/isoamyl alcohol (24:1) and centrifuged at10,000g for 5 min at 4 °C. This last step was repeated two more times.The total RNA were precipitated in 0.25 vol 10 M LiCl, with thereaction left to proceed overnight at 4 °C. The samples were thencentrifuged at 10,000g for 30 min at 4 °C, washed in 70% ethanol,dried, and resuspended in 50 μL of double-distilled diethylpyrocarbonate water. RNA integrity was verified by agarose gels thatwere stained using SYBRSafe (Invitrogen, Carlsbad, CA, USA). RNApurity was assessed based on an absorbance ratio of 1.80 to 1.90 at260/280 nm, using BioPhotometer plus (Eppendorf Inc., Westbury,NY, USA) and 1.8 to 2.0 at 230/260 nm.Reverse Transcription. A total of 40 ng to 50 ng RNA was used

for cDNA synthesis with reverse-transcription PCR, using iScript TMcDNA synthesis kits (Bio-Rad, Hercules, CA, USA), according to themanufacturer’s instructions. From each RNA extraction, cDNAsynthesis was performed twice, and the products were mixed beforethe gene expression studies.Primers and Reference Gene Selection. Specific primer sets

were designed using the Primer3 software (http://biotools.umassmededu/bioapps/primer3_www.cgi) from the specific sequenceof Fragaria × ananassa deposited in NCBI GenBank (Table 1). Theprimer pairs were chosen and validated in silico using primer BLAST-specific analysis (http://www.ncbi.nlm.nih.gov/Blast.cgi) and thenaccording to the melting profiles obtained from the quantitative real-time PCR conditions (qPCR), as described later.The relative expression stabilities of the candidate reference genes

of 18S-rRNA, actin, histone H4, and GAPDH2 were validated usingthe geNorm method, using the 3.5 version.20

Quantitative Real-Time PCR. qPCR reactions were performed in96-well clear multiplate PCR plates (Bio-Rad, Hercules, CA, USA)using the iQ SYBR Green Supermix (Bio-Rad) on an iCycler iQ Real-Time PCR Detection System (Bio-Rad) under the followingconditions: an initial denaturating cycle (5 min at 95 °C), followedby 40 cycles of three steps of denaturation, annealing, andpolymerization (30 s at 95 °C, 20 s at 55 °C, and 30 s 72 °C).PCR amplification was carried out in a total volume of 22 μL,containing 9 μL of diluted (1:10) cDNA (duplicates), 0.25 μM of eachprimer, and 11 μL of iQ SYBR Green Supermix. All of the assaysincluded no-RT and no-template controls, to determine thenonspecific amplification. To determine the specificity of theamplicons, melting curve analysis was performed over the range of55 to 98 °C. The qPCR efficiency (E) of each primer pair wasdetermined using standard curves generated according to the equationE = 10−1/slope of five triplicate cDNA pool dilutions (undiluted, 0.25,0.0625, 0.015, and 0.003).Enzyme Activities. Spectrophotometric assays were used to

determine the βGlu EC 3.2.1.6, Chi EC 3.2.1.14, PAL EC 4.3.1.24,and GPX EC 1.11.1.7 activities of the strawberry fruits at 0.5, 6, 24,and 48 h post-treatment with chitosan, COA, and BTH. Frozen fruits(1 g fresh weight) were ground, and βGlu and Chi were extractedusing 1% (w/v) polyvinylpolypyrrolidone in 50 mM sodium acetatebuffer (pH 5.0) containing 1 mM dithiothreitol and 0.2% (w/v)phenylmethylsulfonyl fluoride. The PAL was obtained using 100 mMpotassium phosphate buffer (pH 8.0) containing 1% (w/v)polyvinylpolypyrrolidone and 1.4 mM β-mercaptoethanol. The GPXwas obtained with 100 mM potassium phosphate buffer (pH 7.3)containing 1 mM EDTA. The homogenates were centrifuged at15000g for 15 min at 4 °C, and the resulting supernatants were used asthe crude enzyme extracts. The protein content in the enzyme extractswas determined according to the Bradford assay21 (Sigma-Aldrich),using bovine serum albumin as the standard. βglu activity was assayed

according to Derckel et al.22 The Chi and PAL activities weremeasured according to Trotel-Aziz, et al.23 and the GPX according toAmako et al.24 All of the enzyme activities were analyzed using a UV1800 spectrophotometer (Shimadzu Corp., Tokyo, Japan). For all ofthe assays, the absorbance was measured against a blank (crude proteinextract in incubation mixture). The specific activities of the enzymesare expressed as Units (U) mg−1 protein.

Data Analysis. The gene expression study was performed usingthe −ΔΔCt method.

18 According to this method, the expression oftarget genes was given as the mean fold-changes in gene expressionnormalized to an endogenous reference gene and compared to theuntreated controls. For each individual sample, three replications wereanalyzed. The gene expression calculations were performed using theExcel program Gene Expression Analysis for iCycler iQ Real TimePCR Detection System (Bio-Rad). The calculations in this spreadsheetwere derived from the algorithms outlined by Vandesompele et al.20

All of the analyses were performed twice.For the analysis of the enzyme activities, for each individual sample

three replications were analyzed. The experiments were performedtwice. The data from each sampling point are shown as the mean ±SD and were statistically evaluated by ANOVA, followed by individualcomparisons using Duncan’s Multiple Range Test, at p ≤ 0.05.

■ RESULTSSelection of Appropriate References Genes for RT-

qPCR in the Treated Strawberry Fruits. For this study, theRT-qPCR was set up for analysis of the strawberry fruits treatedfor 30 s with the three elicitors. The specific primer setsidentified for all of the genes analyzed showed specific single-peak melting curves (data not shown), which confirmed thehomogeneity and specificity of the amplicons produced in theRT-qPCR for each of the 18 target genes. No amplification wasobserved in any of the control assays, which confirmed that thesamples were free of contamination with genomic DNA orRNA, or the cDNA template (data not shown). Theamplification efficiency (E) ranged from 96.2% to 106.2%,according to the standard curve analysis in RT-qPCR of eachgene-specific primer pair (Table 1).The validation of four putative candidate reference genes,

18S-RNA, actin, glyceraldehyde 3-phosphate dehydrogenase(GAPDH2), and histone H4, according the geNorm method,showed differences within the treatments. However, thesereference genes had M values <1.1, which is below the defaultlimit of M < 1.5 (Figure 1). For the analysis, the two moststable genes were selected. In particular, on strawberry fruitstreated with COA, among these reference genes the most stablewere the 18S-RNA and GAPDH2 genes (M = 0.49) (Figure1A). According to the analysis of reference genes on thestrawberry fruits treated with BTH, the genes for actin andhistone H4 were the most stable (M = 0.43) (Figure 1B). Usingchitosan and the water control, the best expression stability wasusing the 18S-RNA and actin genes. In particular, the M valuewas 0.46 in samples treated with chitosan (Figure 1C), and 0.51in the control samples (Figure 1D).

Effects of Three Resistance Inducers on the Ex-pressions of Defense Genes. The biochemical reactions inthe cell that are catalyzed by enzymes and genes that were up-regulated in strawberry fruits after the chitosan, COA, and BTHtreatments are illustrated schematically in Figure 2.The expression level of the 18 genes was compared to water

control treatment, and it was defined as fold-stimulation overthe control according to these analysis times, as shown inFigures 3 and 4. In general, the shortest time from thetreatment of 0.5 h had no significant effects across these data(except where specified below).



http://biotools.umassmededu/bioapps/primer3_www.cgi

http://biotools.umassmededu/bioapps/primer3_www.cgi

http://www.ncbi.nlm.nih.gov/Blast.cgi

The expression levels of CDPK were increased in thestrawberry fruit treated with BTH, by 2.8-fold (6 h post-treatment), 2.5-fold (24 h post-treatment), and 3.6-fold (48 hpost-treatment). This gene was affected by chitosan only at 24h post-treatment, with a 2.7-fold increase seen. There were nochanges in CDPK gene expression with COA (Figure 3). Forthe gene encoding a K+ channel protein, the transcript levelsincreased with all of the resistance inducers tested. In particular,after chitosan and COA, the K+ channel expression increased at6, 24, and 48 h post-treatment by 8.3-fold, 15.4-fold, and 9.7-fold, respectively, with chitosan, and 13.3-fold, 8.8-fold, and13.4-fold, respectively, with COA. After the BTH treatment, asignificant increase of the transcript was observed only after 48h post-treatment, with an 11.4-fold increase (Figure 3).The genes encoding for enzymes involved in the scavenging

of oxygen species that were analyzed here were those of APX

and GST. These showed increased gene expression with BTHat 6, 24, and 48 h post-treatment of 7.4-fold, 3.8-fold, and 1.8-fold, respectively, for APX, and of 3.2-fold, 4.9-fold, and 1.7-fold, respectively, for GST (Figure 3).For the expression patterns of the flavonoid upstream

pathway gene PAL and for the genes that are more related tobiosynthesis across the different flavonoids branches, as CHS,CHI, F3H, DFR, FLS, ANS, and UFGT, different data wereobtained according to the resistance inducers tested (Figure 3).The PAL gene was up-regulated at 24 and 48 h post-treatmentby 4.0-fold and 4.5-fold, respectively, after chitosan treatment,and 6.1-fold and 5.0-fold, respectively, after COA treatment,with no significant changes in transcription with BTH.Conversely, CHS gene expression was enhanced only afterBTH treatment, by 3.2-fold (6 h post-treatment), 2.9-fold (24 hpost-treatment), and 5.6 fold (48 h post-treatment). Among theCHS downstream pathway genes, the CHI gene showedincreased expression after the chitosan and BTH treatments. Inparticular, it increased by 7.5-fold (24 h post-treatment) and4.9-fold (48 h post-treatment) with chitosan, and 3.5-fold (6 hpost-treatment), 2.9-fold (24 h post-treatment), and 4.8-fold(48 h post-treatment) with BTH. The DFR and F3H geneswere up-regulated after the BTH treatment. For DFR, thetranscript levels were increased by 9.6-fold at 48 h post-treatment, while for the F3H gene, the increase was 3.2-fold atboth 6 and 48 h post-treatment. For the flavonoid biosyntheticpathway, the gene showing the highest expression levels wasFLS. The expression of the FLS gene increased following thetreatments with each of the elicitors tested, although at differenttimes of treatment. In particular, at 6, 24, and 48 h post-treatment, the increases were 5.2-fold, 6.7-fold, and 11.4-fold,respectively, for chitosan, and 12.8-fold, 20.4-fold, and 14.1-fold, respectively, for COA; BTH only produced a significantincrease in FLS gene expression at 48 h post-treatment of 4.3-fold (Figure 3). For the ANS gene, which leads to anthocyaninand proanthocyanidin synthesis, the only significant increases ingene expression were seen for the chitosan treatment: 8.9-fold(6 h post-treatment), 7.4-fold (24 h post-treatment), and 12.6-fold (48 h post-treatment). UFGT gene expression wasincreased after BTH treatment, by 2.6-fold (6 h post-treatment), 3.2-fold (24 h post-treatment), and 2.0-fold (48 hpost-treatment), while with COA it increased by 2.5-fold at 6 hpost-treatment and 3.0-fold at 24 h post-treatment. Nosignificant increase in UFGT transcript induction was seenafter the chitosan treatment (Figure 3).According to the cell wall degrading enzymes (Figure 4), for

PG gene expression, there was a similar pattern across all ofthese resistance inducers, with a significant increase at 6 h post-treatment, which was lost at 24 h post-treatment but returnedagain at 48 h post-treatment. In particular, at 6 and 48 h post-treatment there were 3.8-fold and 5.6-fold increases,respectively, with chitosan, 7.2-fold and 7.4-fold, respectively,with COA, and 4.7-fold and 14.1-fold, respectively, with BTH;COA also showed significant up-regulation of the PG transcriptat 24 h post-treatment of 2.5-fold. However, the PGIPtranscripts showed an inverted pattern of expression comparedto that of the PG gene, with maximum gene expression at 24 hpost-treatment using all of the elicitors. Here, significant geneup-regulation was seen at 0.5, 6, 24, and 48 h post-treatmentwith the chitosan and COA treatments, as 2.9-fold, 3.7-fold,3.9-fold, and 3.4-fold, and as 3.1-fold, 5.9-fold, 8.7-fold, and 3.7-fold, respectively. With BTH, PGIP gene expression was onlysignificantly increased at 6 and 24 h post-treatment, by 4.4-fold

Figure 1. Stability of the reference genes in strawberry fruit. Stabilityvalue (M) for a set of reference genes was analyzed with the geNormalgorithm. Strawberry fruits were treated with COA (A), BTH (B),chitosan (C), and water, as the control (D). The reference genes onthe X-axis are ranked from left to right based on their mean geneexpression stability. In each panel, the genes on the right show thehighest expression stability among the reference genes. See Table 1 forthe names of the reference genes.



and 3.3-fold, respectively. The βGlu gene expression wassignificantly increased with all of the resistance inducers tested.Starting from 6 h post-treatment, with chitosan there was aconsistent increase through 24 and 48 h post-treatment, of16.7-fold, 16.2-fold, and 21.3-fold, respectively. For BTH andCOA, while remaining significantly increased, at 24 h post-treatment the effects were reduced, with increased βGlu geneexpression at 6, 24, and 48 h post-treatment of 10.3-fold, 2.9-fold, and 16.6-fold, respectively, for BTH, and 14.4-fold, 4.5-fold, and 10.3-fold, respectively, for COA. The other two genesinvolved on plant-cell degradation, as those for endoβGlu andChi3, showed different results again. endoβGlu gene expressionwas only significantly increased with BTH treatment, by 5.4-fold (6 h post-treatment), 5.8-fold (24 h post-treatment), and12.9-fold (48 h post-treatment). Conversely, Chi3 geneexpression was significantly increased only after chitosan andCOA, showing increases at 0.5, 6, 24, and 48 h post-treatmentof 2.7-fold, 13.6-fold, 13.6-fold, and 22.0-fold, respectively, forchitosan, and 1.4-fold, 9.4-fold, 8.6-fold, and 5.5-fold,respectively, for COA.As for Chi3, expression of the gene coding for PR protein-1

(PR-1) was only significantly increased with chitosan and COA(Figure 4). After chitosan treatment, the PR-1 transcript levelswere significantly increased at 6, 24, and 48 h post-treatment,by 7.6-fold, 13.0-fold, and 23.1-fold, respectively. The differenteffects with COA showed the increased PR-1 transcript level at

6, 24, and 48 h post-treatment of 17.8-fold, 9.7-fold, and 5.1-fold, respectively.The relationships between the paired resistance inducers and

these gene responses were then calculated with respect to thenumbers of genes showing the same response (unvaried, up-regulated, or down-regulated) of the total set of 18 genesanalyzed. The highest similitude level was seen at 0.5 h post-treatment, as at this time, a few of the genes showed somechanges with the elicitors: chitosan/COA, 100%; BTH/chitosan, 88%; and BTH/COA, 88%. The similitude levelsacross these elicitors then differed substantially at 6, 24, and 48h post-treatment. In particular, the paired chitosan/COA geneexpression similarity was the greatest, at 88%, 72%, and 88%,respectively, with BTH/chitosan at 33%, 38%, and 28%,respectively, and BTH/COA, at 38%, 33%, and 27%,respectively (Figure 5).

Effects of Three Resistance Inducers on EnzymeActivity. For strawberry fruits, the PAL, βGlu, Chi, and GPXenzymatic activities were also assayed following the 30-schitosan, COA, and BTH treatments, at 0.5, 6, 24, and 48 hpost-treatment (Figure 6).The control PAL activity (12.4−15.58 U mg−1 protein)

increased after chitosan and COA treatments only. Inparticular, for chitosan treatment, PAL activity was significantlyincreased at 24 and 48 h post-treatment, by 11.8 units mg−1

protein (1.9-fold) and 17.9 U mg−1 protein (2.1-fold),

Figure 2. Gene expression and enzyme activity involved in resistance induced by chitosan, COA, and BTH (see legend). The metabolic roles of boththe genes and enzymes analyzed are shown. Genes associated with ion fluxes, CDPK, the K+ channel; genes associated with oxidative stress, GST,APX; genes associated with secondary metabolism, such as phenylpropanoid, PAL, CHS, CHI, F3H, FLS, DFR, ANS, and UFGT; genes associatedwith cell-wall degradation enzymes, endoβgluc, PG, and PGPI, some of which are known as PR proteins, as βglu, Chi3, and with the addition of thePR-1 protein. The up-regulation of genes by the resistance inducers and post-treatment times are shown with circular lines surrounding theabbreviated gene names, with different colors and typologies (see legend). The metabolic reactions of the analyzed enzyme activities are also shown:GPX, PAL, Chi, and βglu, surrounded by square lines (see legend).



respectively. Similarly, for COA treatment, the increases were9.1 U mg−1 protein (1.7-fold) and 12.9 U mg−1 protein (1.8-fold), respectively.The control βGlu activity (2.2−3.2 U mg−1 protein)

increased with all of the inducers. For both chitosan andCOA, this was significant at 24 and 48 h post-treatment, with1.9 U mg−1 protein (1.7-fold) and 1.8 U mg−1 protein (1.6-fold) increases, respectively, for chitosan and 4.8 U mg−1

protein (2.7-fold) and 4.5 U mg−1 protein (2.4-fold) increases,respectively, for COA. With BTH, the βGlu activity wasincreased only at 48 h post-treatment by 5.6 U mg−1 protein(2.8-fold).The control total Chi activity (0.9−1.4 U mg−1 protein) also

increased with all of the elicitors tested. At 24 and 48 h post-treatment, these increases in Chi activity were of 0.8 U mg−1

protein (1.8-fold) and 1.8 U mg−1 protein (2.9-fold), afterchitosan treatment, 1.4 U mg−1 protein (2.4-fold) and 1.6 Umg−1 protein (2.7-fold), with COA, and only at 48 h post-treatment of 2.7 U mg−1 protein (3.0-fold), with BTH.For the control GPX activity (10.1−16.0 U mg−1 protein),

this was significantly increased only after BTH treatment at 6

and 24 h post-treatment by 5.4 U mg−1 protein (1.4-fold) and14.2 U mg−1 protein (2.4-fold), respectively.

■ DISCUSSION

The ability to quickly induce defense mechanisms is acharacteristic of incompatible (resistant) plant−pathogeninteractions, whereby the plant can efficiently block pathogenpenetration. The application of resistance inducers promotesincreases in the potential immunity of susceptible plants andallows the plants to combat possible future pathogen infections.In the present study, we investigated the gene expression ofselected markers associated with defense mechanisms ofstrawberry fruits exposed to resistance inducers.This study determined the relationships between the

different resistance inducers of BTH, chitosan, and COA thathave been shown to be effective in the control of post-harvestdiseases of strawberry4 and 18 defense-related genes that areassociated with different biological processes. With this aim, weset up an analytical protocol based on RT-qPCR, and wedefined the appropriate reference genes for the strawberry fruitsunder elicitor stress conditions. It is known that the expression

Figure 3. Relative gene expression of defense genes in strawberry fruit after treatment with resistance inducers. Strawberry fruits were treated withchitosan, BTH, and COA, and with water as the control. The relative gene expression of the defense genes (as indicated) is referred to as 0.5, 6, 24,and 48 h post-treatment. Data are the means + SD, and values with the same letter are not statistically different, according to Duncan’s multiplerange test, at P ≤ 0.05. See Table 1 for the names of the genes.



stability of reference genes varies with the environment, plantgrowth, plant tissue, and different biotic and abiotic stress.17

Therefore, normalization of the relative RT-qPCR data usingsuitable reference genes is a crucial prerequisite for obtainingreliable data. Our results showed differences between theelicitor treatments and suggested the appropriate referencegene selection for RT-qPCR analysis for new materials.Our data show that the target genes analyzed associated with

plant natural defense mechanisms are involved in different waysaccording to the elicitors. The earliest responses of plant cellsto elicitors involve changes in membrane potential, Ca2+

signaling, and the production of ROS. 25 Our study showsthe more constant involvement of the CDPK transcript, whichis known in plants as a Ca2+-binding sensor protein26 in thestrawberry fruit BTH response, with chitosan effects only seenat 24 h post-treatment in these strawberry fruits. However,although the expression of the K+ channel was enhancedfollowing the use of all of these elicitors, its early activation was

only seen in the strawberry fruits treated with chitosan andCOA; with BTH, there was only an enhancement of this geneat 48-h post-treatment. CDPKs have roles in early signalprocesses for multifaceted functions in the complex immuneand stress signaling networks, including for oxidative burst,stomatal movements, hormonal signaling, and gene regula-tion.27 In the same way, K+ channel activation during stress isaccompanied by an oxidative burst.28 Our data suggestdifferences between these chitosan, COA, and BTH treatmentsin the activation of strawberry fruit plasma-membrane ionfluxes. This might be associated with different innate immunitysignal transduction in these fruit, which might involvedownstream signaling events, such as oxidative stress.29 Inthis regard, the genes associated with the nonenzymaticcomponents of the antioxidative defense system that wereanalyzed in this study, such as APX and GST, which canremove H2O2 from the cell,30 were up-regulated in thesestrawberry fruits treated with BTH; likewise, the enzymeactivity of GPX increased only after the BTH treatment. Ourdata suggest the involvement of oxidative stress in strawberryfruits treated with BTH, although we do not exclude theinvolvement of oxygen scavenger enzymes belonging to otherbiosynthetic pathways using the other elicitors, as previouslyhypothesized.3,4

Furthermore, as some of the major secondary metabolites inplants, phenolic compounds are well-known to have antioxidantproperties31 and to lead to the synthesis of phytoalexins, whichare known to have antimicrobial activity.32 In the present study,differences in the expression of the genes involved in thephenylpropanoid pathway were observed according to theelicitor used and the time from the treatment. In particular, theBTH treatment enhanced the expression of most of thesegenes. Some increases were apparent from 6 h post-treatment(CHS, F3H, CHI, and UFGT), while others were more delayedto 48 h post-treatment (FLS, DFR). Chitosan up-regulated

Figure 4. Relative gene expression of defense genes in strawberry fruit after treatment with resistance inducers. The strawberry fruits were treatedwith chitosan, BTH, and COA, and with water as the control. The relative gene expression of the defense genes (as indicated) are referred to as 0.5,6, 24, and 48 h post-treatment. Data are the means + SD, and values with the same letter are not statistically different, according to Duncan’smultiple range test, at P ≤ 0.05. See Table 1 for the names of the genes.

Figure 5. Relationships between the resistance inducers used and thecorrespondent gene responses in treated strawberry fruit. Therelationships between the resistance inducers and gene responsescalculated according to the numbers of genes showing the sameinduction response of the total genes analyzed.



PAL, CHI, FLS, and ANS; COA only up-regulated PAL andFLS, and usually from 6 h post-treatment. Between these genes,there was the remarkable involvement of the FLS and ANSgenes, which encode the main enzymes that lead to the flux ofanthocyanin and proanthocyanidin synthesis.33 Interestingly,the PAL gene, which is pivotal for the phenylpropanoidpathway that includes, among others, the lignin branchpathway34 was enhanced only in strawberry fruit treated withthe chitosan and COA elicitors, while the downstream geneCHS, which is the key enzyme of flavonoid synthesis35,36 wasup-regulated in the strawberry fruit treated with the BTHelicitor.Although our data suggest the prevalent involvement of

flavonoid branches relative to all of the elicitors, theinvolvement of other pathway branches for lignin synthesiscan be proposed as among the effects of the chitosan and COAtreatments. The analysis of the PAL enzyme activity confirmedthese PAL gene expression data.Genes involved in cell-wall-modifying processes, such as PG,

PGIP, and endoβGlu were also analyzed. Here, their expressionpatterns provided evidence for the synchronized expression ofPG and PGIP according to all of the elicitors analyzed. Inparticular, the complementary gene expressions of the PG andPGPI transcripts with time could suggest a feedbackmechanism, where PGPI gene expression is modulated in

response to plant PG gene expression. PG is involved in pectinmetabolism in the production of oligogalacturonides, which canelicit different cellular responses during plant−pathogeninteractions in strawberry fruit.37 The PGIPs that have rolesin strawberry defense38 are extracellular plant proteins that caninhibit fungal endopolygalacturonases, while endoβGlu acts onhemicellulose degradation.39 Differences between the resistanceinducers were seen for endoβGlu gene expression, which wasonly affected by BTH treatment. The role of this enzyme inplant defense is still unknown, although it is clear that also inthis case, the elicitor typology affected the gene expression.However, the βGlu and Chi enzymes that are involved in

cell-wall degradation are those most frequently analyzed interms of these plant−elicitor interactions.3 βGlu, which is also aPR-2 protein,13 showed both early gene expression and enzymeactivity enhancement with all of the tested elicitors. Thisemphasizes the wide involvement of some structural genes inimmune responses. However, related to the Chi group, ourstudy shows Chi3 (PR-8 family) up-regulation by the chitosanand COA elicitors but not by BTH, while the total Chi enzymeactivity was up-regulated with all of the elicitors tested. Ourdata thus indicate that there are roles for other classes of Chifollowing BTH treatment. The differences observed associatedwith Chi3 as well as in the PR-1 gene expression might berelated to the elicitor-specific induction of these PR proteins instrawberry fruit and to the relationships between elicitors dosesand the plant species.13 Our study shows different levels ofinvolvement of these genes that are associated with plantdefense that depends on the resistance inducer applied. Usually,gene activation was apparent from 6 h post-treatment. Only thePGPI and Chi3 genes were up-regulated at the first time pointof 0.5 h post-treatment and only when using the chitosan andCOA elicitors. In this regard, starting from 6 h post-treatment,the highest similarities in the changes in gene expression wereseen between the commercial formulations of chitosan andCOA (>70%), compared to the similarities between BTH andboth chitosan and COA (both <40%). Microarray analysis ofArabidopsis thaliana in response to treatment with chitinshowed 43% similarity with the genes that are up-regulated bysalicylic acid treatment,40 and BTH is a light-resistant form ofthis SAR signaling compound. Thus, a similar level of similarityis seen in the present study in terms of the relationship betweenthe gene expression responses of strawberry fruit treated withchitosan, which is the N-deacetylated form of chitin, and BTH,which is, as indicated, an analogue of salicylic acid. Thissuggests that the expression of these defense genes occursaccording to the nature of the elicitor.In investigating the possible similarities between the actions

of these resistance inducers, both chitosan and COA provide aprotective physical barrier to pathogen invasion, although indifferent ways. Chitosan forms a surface coating on the fruit,while COA uses calcium to reinforce the structural compositionof the plant cell wall, through the binding of pectins withsalts.4,11 Instead, as an analogue of salicylic acid, BTH is knownto primarily induce the expression of SAR without interactionswith the plant cell wall.In conclusion, in this study we set up the relevant RT-qPCR

method to test for different gene expressions in strawberryfruits treated with different resistance inducers. Our data showdifferent suitable combinations for reference genes according tothe resistance inducer, also indicating the importance of thevalidation of the reference genes before the experiments. In thisstudy, all of the resistance inducers analyzed had previously

Figure 6. Enzyme activities of defense enzymes in strawberry fruitsafter treatment with resistance inducers. The strawberry fruits weretreated with chitosan, BTH, and COA, and with water as the control.The enzyme activities (as indicated) are referred to as 0.5, 6, 24, and48 h post-treatment. Data are the means + SD, and values with thesame letter are not statistically different, according to Duncan’smultiple range test, at P ≤ 0.05.



been shown to reduce both gray mold and Rhizopus rot diseasein postharvest strawberry fruit,4 and here, they triggered theexpression of a large number of genes that lead to thephysiological events involved in plant defense. This supportsthe effectiveness of these compounds for the induction ofresistance in strawberry fruit. However, there were differencesbetween the elicitor resistance at both the transcriptional levelof the genes analyzed and the enzyme activities of some of theirproducts that were observed at different times from treatments.This suggests that there is a similarity between chitosan andCOA in their target defense gene activation, while their generesponses differ from those due to BTH. This study thusindicates that the composition of the resistance inducers andtheir interaction with the host plant determine the pattern ofinduction of the specific defense-marker genes. Informationgained with this study can be useful to choose proper resistanceinducers for gray mold management.

■ AUTHOR INFORMATIONCorresponding Author*Tel: +39-071-2204336. Fax: +39-071-2204856. E-mail: [email protected] work was supported by EUBerry Project [EU FP7 KBBE2010-4, Grant Agreement No. 265942].NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThanks are expressed to Mark Davey for critical revision of themanuscript.

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mailto:[email protected]

mailto:[email protected]

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expression of defense genes in strawberry fruits treated with different resistance inducers

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