Published: November 16, 2011

r 2011 American Chemical Society 13295 dx.doi.org/10.1021/jf203583t | J. Agric. Food Chem. 2011, 59, 13295–13299

ARTICLE

pubs.acs.org/JAFC

Identification and Characterization of Genes Differentially Expressedin Cherimoya (Annona cherimolaMill) after Exposure to Chilling InjuryConditionsMauricio Gonz�alez-Ag€uero,*,†,‡ Nicol�as Cifuentes-Esquivel,§ Freddy Iba~nez-Carrasco,||

Orianne Gudenschwager,† Reinaldo Campos-Vargas,‡,^ and Bruno G. Defilippi†,‡

†Laboratorio de Postcosecha, Instituto de Investigaciones Agropecuarias (INIA-La Platina), Casilla 439/3, Santiago, Chile‡The Plant Cell Biotechnology Millennium Nucleus (PCB-MN), Santiago, Chile§Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Barcelona, Spain

)Department of Horticultural Sciences, Texas A&M University, Vegetable & Fruit Improvement Center, College Station,Texas 77843-2133, United States^Centro de Biotecnología Vegetal, Universidad Andres Bello, Rep�ublica 217, Santiago Chile

bS Supporting Information

ABSTRACT: Cherimoyas (Annona cherimola), like other subtropical/tropical fruits, are susceptible to damage from exposure totemperatures between 0 and 5 �C (chilling injury, CI), which may affect fruit quality. To increase our understanding of the molecularmechanisms involved in the CI response, a forward suppression subtractive hybridization (SSH) cDNA library was constructed. In thiswork, we obtained 75 genes that could potentially be involved in the CI response. The CI induced activation of genes that are involved ina range ofmetabolic pathways, such as primarymetabolism, transport, and endomembrane traffic, among others.We also characterized theexpression of 12 selected genes in different A. cherimola tissues by polymerase chain reaction (PCR), and we confirmed the differentialexpression of a subset in CI fruits by real-time quantitative PCR (qPCR). The expression of six A. cherimola genes: annexin (AcAnex),UDP-glucose pyrophosphorylase (AcUGP), syntaxin of plants 71 (AcSyp71), 1-aminocyclopropane-1-carboxylic-acid synthase (AcACS),ubiquitin carrier-like protein (AcUCP), and enolase (AcEnol), was up-regulated after cold storage for 12 days at 0 �C. These results implythat selected genes could be related to the development of internal browning observed in cherimoyas after exposure to CI conditions. Theinformation generated in this study provides new clues that may aid in understanding the cherimoya ripening process.

KEYWORDS: Annona cherimola, cold storage, suppression subtractive hybridization, gene expression

’ INTRODUCTION

Cherimoya (Annona cherimola Mill.) is a subtropical fruitbelonging to the Annonaceae family. This fruit requires lowtemperature storage (i.e., 10 �C) to delay postharvest softening.1Although cold storage of cherimoya and other fruits is consideredto be themost effectivemethod for preserving fruit quality, tropicaland subtropical fruits are susceptible to chilling injury (CI) byexposure to low temperatures. CI is a disorder of tropical andsubtropical crops and a major environmental factor that affectsplant growth and productivity because exposure to low, but notfreezing, temperatures eventually leads to cell death.2,3 Fruitresponses to CI are thought to be activated by cell membranedamage, which sets off a cascade of reactions that include alteredcellular structure, ethylene production, increased respiration,interference with energy production, reduced photosynthesis,oxidative events like enzymatic browning, and accumulation oftoxic compounds such as ethanol and acetaldehyde.3,4

Several genes have been implicated in the response to CI ingenetic and physiological studies. Such genes encode enzymesinvolved in cell wall modification, ethylene biosynthesis, andenzymatic reactions,5 such as the PPO gene that encodes apolyphenol oxidase that is responsible for fruit browning.1,6

However, in recent years, molecular approaches have contributed

to the characterization of genes induced during cold acclimation,to the characterization of mutations that affect freezing tolerance,and to QTL mapping to identify freezing-tolerance loci.4

The aim of this study was to identify genes involved in theresponse to CI during cold storage of cherimoya fruits. We useda suppression subtractive hybridization (SSH) strategy to isolate aset of cDNAs that correspond to mRNAs that are differentiallyexpressed in cherimoyas after storage for 12 days at 0 �C. Thisstrategy has been extensively used to identify differentially ex-pressed genes in many different organisms and is expected tofacilitate identification of genes with scarce transcripts because itcombines normalization and subtraction into a single procedure.2,7

’MATERIALS AND METHODS

Plant Material, Treatments, and Internal Browning Quan-tification. Cherimoyas var. “Concha Lisa” were obtained from acommercial orchard located in Quillota Valley, Chile. Fruits were

Received: September 5, 2011Accepted: November 16, 2011Revised: November 15, 2011

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collected, considering ground color as a harvesting index, and transportedto the Postharvest Laboratory facility at INIA for ripening for 7 days at20 �C (mature fruit, MF). Then, cherimoyas were stored for up to 12 daysat 0 �C to induce CI and were sampled every 2 days. The occurrence ofinternal browning was determined by visual examination, with a hedonicscale from 1�5 with the following: 1, none; 2, slight; 3, moderate; 4,moderately severe; and 5, severe. At the end of each time point, four fruitswere halved, and one-quarter was immediately frozen in liquid nitrogenand stored at �80 �C. In addition, new leaves (L) from apical buds,flowers (F), and developing fruit (DF) were selected and stored at�80 �C until use.RNA Extraction, Isolation of Poly A+ mRNA, and cDNA

Synthesis. Total RNA from 0 day and 12 day samples was isolatedfollowing the hot borate method.8 Isolation of poly A+ mRNA fromtotal RNA was performed using the Oligotex mRNA Spin-Column kit(Qiagen, MD). The purified poly A+ mRNA was used for cDNA synthesisby the PCR-Select cDNA subtraction kit (Clontech, Palo Alto, CA). In addi-tion, total RNA and poly A+ mRNA from L, F, DF, and MF samples wereobtained and used as a template for reverse transcription reactions tosynthesize single strand cDNA, using MMVL-RT reverse transcriptase(Promega, Madison, WI) according to standard procedures.SSH cDNA Library Construction and EST Sequencing. SSH

was obtained using the PCR-Select cDNA subtraction kit (Clontech),and forward and reverse cDNA libraries were generated, according to thestandard manufacturer's procedure. Forward SSH was performed usingcDNA from samples at 0 �C for 12 days (with CI) as tester and cDNAfrom day 0 samples as driver. For the reverse SSH, 0 and 12 day sampleswere used as tester and driver, respectively. In this work, individualtransformants from the forward library were isolated and sequenced at acommercial DNA sequencing service (Macrogen Corp., Seoul, Korea).EST sequences were compared to the NCBI database using the BLASTTool (http://www.ncbi.nlm.nih.gov/BLAST/) and classified accordingto their metabolic function into 13 functional categories.Cloning of Selected Full Length A. cherimola Genes. To

obtain the full-length cDNA of selected genes, specific primers forpolymerase chain reaction (PCR) amplification were designed. RACE-PCRassays were conducted using the adaptors, primers, enzymes, andprocedures from the GeneRacer kit (Invitrogen, Breda, The Nether-lands). Amplified 30 and 50 RACE fragments were analyzed by agarosegel electrophoresis, and selected bands were purified using theQIAquickgel extraction kit (Qiagen). DNA fragments were cloned into pGEMT-Easy (Promega) according to the manufacturer's recommendations,and both strands were sequenced.Characterization of NewA. cherimolaGenes.We analyzed by

PCR the expression levels of the selected genes and other A. cherimolagenes in cDNAs from different tissues: L, F, DF, and MF. PCRconditions were as follows: denaturation at 94 �C for 30 s, annealingat 62�66 �C for 30 s, and primer extension at 68 �C for 2 min, all for30 cycles. All PCR reactions were performed in a MyCycler thermalcycler (Biorad, Hercules, CA).Analysis of Differential Expression of Genes by Real-Time

Quantitative PCR (qPCR). To confirm the expression of up-regulated genes in cherimoyas with CI, a qPCR approach was used.Quantitative PCR was performed with the LightCycler real-time PCRsystem (Roche Diagnostics, Mannheim, Germany), using SYBR andgene-specific primers (IDT, Coralville, San Diego, CA) that weredesigned using the Primer Premier 5.0 software (Premier BiosoftInternational). The primer sequences are as follows: forAcAnex, forward50-AAAAGTTGCTGTTGGCTTATGTGA-30 and reverse 50-TGCTA-GATGGGCCCTGCTTC-30; for AcUGP, forward 50-GTGGGTGAA-CTTGAGTGCTATTA-30 and reverse 50-ATGGTATAAAGATCC-GACTGGAC-30; for AcSyp71, forward 50-TCAGGCTCAAGGAGA-CAGTTAAT-30 and reverse 50-TCACGCTAGGAGGAATACAGCA-30;for AcACS, forward 50-CACCGGAAGGAGCATCAGAATT-30 and

reverse 50-TAGACGGGTTGGTTATGAGCAG-30; forAcUbiq, forward50-TCCTGCAGAATCAGTGGAGTC-30 and reverse 50-AGGA-ACCAAATCCGCAAACAGC-30; and for AcEnol, forward 50-AAG-GAGGTAATGAAGATGGGTGT-30 and reverse 50-TAGAAACAAT-CGGGTATTCAGACA-30. Conditions, procedures, and analyses forqPCR were performed as previously described.9,10 Quadruplicate qPCRexperiments were performed for each sample, and the expression valueswere normalized against the 18S rRNA gene (GenBank AY819054).Statistical Analyses. For internal browning quantification and

qPCR assays, there were at least six and four replicates for evaluation,respectively. Data were subjected to analyses of variance, and meanswere separated by LSD test at the 5% level of significance usingStatgraphics Plus 5 (Manugistics, Inc., Rockville, MD).

’RESULTS

Internal Browning as a CI Indicator during Cold Storage ofCherimoya. As shown in Figure 1, we found that cherimoyafruits stored at 0 �C increased internal browning after 2 days,which indicates that these fruits are under CI. However, the PPOactivity in the cherimoya fruit extracts did not show significantdifferences during cold storage (data not shown), similar toresults observed in a previous study.1,11 These parameters weremeasured to ensure proper induction of CI in cherimoya fruits.Isolation of Cold-Induced Genes and Functional Classifi-

cation. A forward SSH cDNA library from A. cherimola fruitexposed to 0 �C for 12 days was obtained, consisting of genesactivated during cold storage and possibly related to CI. Weisolated 115 differentially expressed clones with a mean length of449 bp, ranging from 200 to 1400 bp. The library redundancy wasnear 35%, containing 75 unique sequences. The full list of clonesis available in Table S1 in the Supporting Information. Theidentified genes were grouped into 13 functional categoriesaccording to the putative function of their encoded proteins(Figure 2).Annotation and Characterization of Novel A. cherimola

Genes. To identify and characterize genes with a putative role inresponse toCI, we selected 12 genes based on their high representa-tion (three or more clones) in the forward SSH library. From these,we isolated and annotated in the GenBank database the sequenceof 10 novel A. cherimola genes: pectinesterase (AcPE, accessionno. FJ664260); AP2 complex subunit (AcAP2, FJ664261);

Figure 1. Internal browning determined in cherimoya fruits aftertime�course analysis at 0 �C (0�12 days). Browning was determinedby visual examination using a hedonic scale (1�5): 1, none; 2, slight; 3,moderate; 4, moderately severe; and 5, severe. Different letters betweeneach treatment represent significant differences at Pe 0.05 by the LSDtest. A.U. means arbitrary units for internal browning quantification.

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4-α-glucanotransferase (AcDPE2, FJ664262); ubiquitin carrier-like protein (AcUCP, FJ664263); enolase (AcEnol, FJ664264);β-ketoacyl�acyl carrier protein synthase (AcBKAC, FJ664265);annexin (AcAnex, FJ664266); UDP-glucose pyrophosphorylase(AcUGP, FJ664267); clathrin-coat assembly protein (AcCCAP,FJ664268); and syntaxin of plants 71 (AcSyp71, FJ664269). Inaddition, we have analyzed the expression levels of these selectedgenes and other cDNAs from different cherimoya tissues,including leaves, flowers, developing fruits, and mature andripened fruit (Figure 3). It was found that most of the genesselected as “novel” from our SSH library are present in differentcherimoya tissues, with the exception of annexin, 4-α-glucano-transferase, and putative rhamnogalacturonate lyase, which arenot expressed in developing fruit tissue.Expression Analysis of Up-Regulated Genes during Cold

Storage of Cherimoyas. The expression patterns of five novelA. cherimola genes: AcAnex, AcUGP, AcSyp71, AcUCP, AcEnol,and one control gene,AcACS (GenBank access AF443280), weredetermined by qPCR in fruits that were maintained at 0 �C for 0or 12 days (Figure 4). For each gene, the relative abundance ofmRNA was normalized to Ac18sRib (18S rRNA gene, GenBankaccess AY819054) abundance in the corresponding samples. Theresults are presented in Figure 4, showing genes that were up-regulated in samples obtained from fruits with CI. Next, wechecked the quality of the clones obtained from the forwardlibrary as possible genes involved in response to CI.

’DISCUSSION

To assess changes in gene expression associated with coldstorage inA. cherimola, we inducedCI by storing fruits for 12 daysat 0 �C and observed a higher occurrence of browning under thistreatment. In this study, we used a forward SSH to identify genesdifferentially expressed between cold storage and noncold stor-age fruit tissues. In general, the results are similar to other studieson cold acclimation in terms of the distribution of functional

classes in our EST libraries.12�14 There was a relatively highabundance of differentially expressed genes involved in primarymetabolism, endomembrane traffic, cell signaling, protein syn-thesis, and stress response. Our SSH library of A. cherimola fruits

Figure 2. Construction and characterization of a cDNA library of A. cherimola under cold storage. A forward-subtracted cDNA library was made usingthe PCR-select cDNA subtraction kit (Clontech). Individual clones carrying cDNA fragments (400�2000 bp) were manually selected and sequenced.A classification by putative function of almost 80 differentially expressed genes was performed (a full listing of clones is available in Table S1 in theSupporting Information), and the results are shown grouped into 13 functional categories (A�M).

Figure 3. Characterization of tissue-specific expression of selectedgenes from the cherimoya library. We have identified and analyzed theexpression levels of 12 selected genes and four control genes in cDNAsfrom different cherimoya tissues by RT-PCR, including leaves (L),flowers (F), developing fruits (DF), and mature and ripened fruit (MF).The genes analyzed were as follows: 1, clone 027ssh, annexin (GenBankFJ664266); 2, clone 032ssh, AP-2 complex subunit (GenBank FJ664261);3, clone 004ssh, syntaxin of plants 71 (GenBank FJ664269); 4, clone041ssh, 4-α-glucanotransferase (GenBank FJ664262); 5, clone 015ssh,β-ketoacyl�acyl carrier protein synthase (GenBank FJ664265); 6, clone019ssh, putative rhamnogalacturonate lyase; 7, clone 072ssh, enolase(GenBank FJ664264); 8, ACC synthase (GenBank AF443280); 9, clone030ssh, putative actin-related protein; 10, clone 020ssh, putative metal-lothionein-like protein; 11, clone 003ssh, UDP-glucose pyrophosphor-ylase (GenBank FJ664267); 12, clone 006ssh, clathrin-coat assemblyprotein (GenBank FJ664268); 13, polyphenol oxidase (GenBankDQ990911); 14, 18S rRNA gene (GenBank AY819054); 15, putativeactin; and 16, clone 101ssh, ubiquitin carrier-like protein (GenBankFJ664263).

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contained 75 genes that were up-regulated in cold storage, andeach of these genes was isolated and sequenced. For several ofthese genes, orthologous sequences were found in different plantspecies such Ricinus communis, Arabidopsis thaliana, Zea mays,and Populus trichocarpa, among others, suggesting that thesegenes are conserved within the plant kingdom. The diversefunctions of the 75 cold-induced genes found in this study(Figure 2) indicate the complexity and the number of differentpathways involved in the stress response in cherimoya, which hasalso been shown for other plants.15,16

From the 75 cold-induced genes found in our study, weselected and obtained the full-length cDNAs using RACE-PCRassay. With the full-length cDNAs, we were able to annotate thenovel A. cherimola genes and analyze the expression of thesegenes in different cherimoya tissues (Figure 3). To evaluate theexpression of novel genes, including AcAnnex, AcUGP, AcSyp71,AcACS, AcUCP, and AcEnol, the abundance of mRNAs wasquantified by qPCR. Our analysis indicated that in fruit undercold storage, levels of these genes increased in terms of mRNAexpression and putative gene product (Figure 4).

In particular, we identified a cDNA homologous to annexin(AcAnnex), which is in a group of multifunctional proteins ineukaryotic cells that bind membranes in a calcium-dependentmanner.17 These proteins have the capacity to interact with lipidsand protein moieties at the membranes, contributing to stressadaptation via regulation of various signaling pathways.18 In plant

cells, annexins help confer tolerance to stress responses.18,19

Thus, the increase in relative expression of AcAnnex in plants incold storage may suggest that its gene product is involved inregulating stress-activated signaling pathways.

We found an increase in a cDNA homologous to UDP-glucosepyrophosphorylase (AcUGP), and the associated protein is in-volved in the synthesis and pyrophosphorolysis of UDP-glucose,the principal precursor for sucrose formation and cellulose.20

Furthermore, it has a crucial role in quality control of proteinstransported to the endoplasmic reticulum, where glycosylationprocesses occur.21UGP gene expression is up-regulated by sucroseand low temperature stress, as seen in potato tubers22 andArabidopsis leaves.23 Thus, the increase in relative expression ofAcUGP may suggest that its gene product is involved in cellwall remodeling and protein folding pathways in cold storageA. cherimola fruits.

Another novel gene that increased during cold storage was aputative syntaxin 71 (AcSyp71). This protein is within theSNARE (soluble N-ethyl-maleimide sensitive factor attachmentprotein receptors) family of proteins, which play a critical role inthe membrane fusion step of the vesicular transport system ineukaryotes.24 The Arabidopsis SNARE SYP71 is predominantlyexpressed in all vegetative tissues and is mainly localized to theplasma membrane, but it is also localized to the endoplasmicreticulum in the dividing cells of various types of tissues.25 Wethink that the possible function of AcSyp71 in the cold storageA. Cherimola fruits is to maintain adequate vesicular transport.

Another gene up-regulated at low temperature was Enolase(AcEnol), also known as phosphopyruvate dehydratase, which isinvolved in glycolysis26 and is responsive to many environmentalstresses, including salt stress, drought, cold, and anaerobic stressin different plant species.27 Thus, the increase in relative expres-sion of AcEnol in cold storage may suggest that its gene product isinvolved in maintaining primary metabolism through properglycolysis.

Other genes up-regulated in cold storage A. cherimola fruitswere AcUCP and AcACS. Ubiquitin carrier-like protein (AcUCP)is part of the proteasome system that is a crucial regulatorymechanism for protein degradation in all eukaryotic cells. Thissystem degrades a wide range of proteins in the nucleus andcytoplasm and plays a key role in the control of cellular functionsas diverse as cell cycle progression, endocytosis, protein sorting,embryogenesis, hormone responses, defense against pathogens,and senescence.28�30 ACC synthase (AcACS) is a key enzymethat controls biosynthesis of the plant hormone ethylene.Ethylene is necessary in the normal response to cold stressbecause increased ethylene synthesis enhances the cold responseduring postharvest of several fruits.31�33 Thus, a change inethylene biosynthesis decreases the cold response of A. cherimolafruits.

In conclusion, the results of this investigation provide aninitial characterization of a set of genes with diverse functions thatare up-regulated in response to cold storage. Although the resultsof this study are preliminary, it provides a group of candidategenes for in depth and detailed studies on CI of A. cherimolafruits.

’ASSOCIATED CONTENT

bS Supporting Information. Table of a full listing of clones.This material is available free of charge via the Internet at http://pubs.acs.org.

Figure 4. Gene expression analysis for six transcripts that were up-regulated during cold storage of cherimoya. The relative abundance ofthe transcripts of the following genes was determined by qPCR in threefruits from two time points at 0 �C (0 and 12 days): (A) AcAnex,(B) AcUGP, (C) AcSyp71, (D) AcACS, (E) AcUCP, and (F) AcEnol.The relative abundance of each mRNAwas normalized to the 18S rRNAgene (Ac18sRib). The results are presented as relative expressionas compared to the transcript amount of the corresponding gene at0 days at 0 �C, which is given a nominal value of 1. Different lettersbetween each treatment represent significant differences at Pe 0.05 bythe LSD test.

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’AUTHOR INFORMATION

Corresponding Author*Tel: +56-2-7575161. Fax: +56-2-7575104. E-mail: maugonzalez@inia.cl.

Funding SourcesM.G.-A. gratefully acknowledges the PBCT-Conicyt (PSD03)project for financial support.

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