ORIGINAL PAPER

High-throughput sequencing analysis of common fig (Ficus caricaL.) transcriptome during fruit ripening

Zohar E. Freiman & Adi Doron-Faigenboim &

Rajeswari Dasmohapatra & Zeev Yablovitz &

Moshe A. Flaishman

Received: 6 January 2014 /Revised: 18 March 2014 /Accepted: 28 March 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract The Ficus carica L. (Moraceae) tree belongs to oneof the largest genera of angiosperms and bears a unique closedinflorescence structure. Ripe fresh figs are highly perishableand require delicate postharvest handling. Studying the path-ways that lead to fig ripening may provide additional ways ofextending their storage life. We selected four developmentalstages of fig fruit for transcriptome sequencing and analysis toidentify the major activemetabolic pathways and transcriptionfactors during fig fruit ripening. We found 12,751 unigenes,93 % of which were homologous to at least one nonredundantdatabase sequence, and 46,927 singletons, 39 % with amatching sequence from the nonredundant database.Differential activity related to photosynthesis, anthocyaninand volatile metabolism, cell wall and wax metabolism, cellexpansion, transcription, DNA metabolism and organizationwas traced. In addition, ethylene-synthesis genes were identi-fied. Finally, 516 unigenes encoding transcription factors werefound which were active in the regulation of early andlate ripening processes. Focusing on eight FcMADS-box

transcription factors revealed three genes encoding membersof the AGL2 (SEP) subfamily, which is closely associatedwith ripening regulation. This study provides expressed-genedataset for multiple developmental stages of fig fruit(F. carica), and analysis directed to ripening metabolism,control, and regulation. It provides a potential platform forfurther studies of this unique plant family and contributes toripening process research in nonmodel systems.

Keywords Ethylene .Ficus carica . Fruit development .

MADS box . Ripening . Transcriptome

Introduction

The fig tree, Ficus carica L. (Moraceae), belongs to one of thelargest angiosperm genera. The genus is widely cultivated inthe tropics and subtropics and exhibits diverse growth habits,including trees, shrubs, hemi-epiphytes, climbers, andcreepers (Berg and Corner 2005). Among Ficus species, thefig is the only member suitable for fresh consumption byhumans and is believed to be one of the first domesticatedplants (Kislev et al. 2006). All Ficus species bear a uniqueinflorescence structure—the syconium. This closed inflores-cence produces an aggregate fruit, composed of small indi-vidual drupelets which develop from the ovaries followingpollination.

Female fig syconium development is characterized by adouble sigmoid growth curve comprised of three phases(Chessa 1997; Marei and Crane 1971). Phase I is character-ized by a rapid growth in size; during phase II, the fruitremains nearly the same size, color, and firmness. Phase IIIis the ripening phase and includes exceptionally rapid fruitgrowth, color change, softening, and alteration of the pulptexture to an edible state (Chessa 1997). The syconium de-velops into a succulent fruit by either pollination occurring at

Communicated by J.L. Wegrzyn

Electronic supplementary material The online version of this article(doi:10.1007/s11295-014-0732-2) contains supplementary material,which is available to authorized users.

Z. E. Freiman :A. Doron-Faigenboim :R. Dasmohapatra :Z. Yablovitz :M. A. FlaishmanInstitute of Plant Sciences, Agricultural Research Organization,P.O. Box 6, Bet-Dagan 50250, Israel

Z. E. FreimanThe Robert H. Smith Institute of Plant Sciences and Genetics inAgriculture, Faculty of Agriculture, Food and Environment, TheHebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel

M. A. Flaishman (*)Department of Fruit Tree Sciences, Agricultural ResearchOrganization, Volcani Center, Bet Dagan 50250, Israele-mail: vhmoshea@agri.gov.il

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the end of phase I or a parthenocarpic process, depending onthe female fig type—Smyrna, San Pedro, or common(Flaishman et al. 2008). The ripening process in the fig fruitis categorized as climacteric, showing a rise in respiration rateand ethylene production at the end of growth phase II.Surprisingly, ripening-related ethylene synthesis increases fol-lowing ethylene-perception blocking, in an unexpectedautoinhibitory behavior (Freiman et al. 2012; Marei andCrane 1971; Owino et al. 2006; Sozzi et al. 2005). Recently,a transcriptome analysis of caprifig (hermaphroditic fruitwhich functions as male) and common female fig fruits in latephase II was published. The study identified several unigenesencoding proteins involved in ethylene synthesis and signaltransduction, sugar metabolism and anthocyanins synthesis,all assembled from combined data from female cv.“Houraishi” and hermaphroditic ecotypes (Ikegami et al.2013).

Ripened fresh figs are highly perishable and require deli-cate postharvest handling (Chessa 1997). Fruit picked beforeoptimal maturity never reach the desirable parameters of size,color, flavor, or texture. On the other hand, fruit harvested toolate tend to perish due to over ripening and high susceptibilityto postharvest pathogens (Flaishman et al. 2008). Today,postharvest management of fresh figs includes compartmen-talized packaging and low-temperature storage conditions. Atthe center of research contributing to longer and higher-quality storage and shelf life, ripening regulation by climac-teric ethylene and transcription factors (TFs) has been inves-tigated in tomato and other fruits (Matas et al. 2009).

Over the past few years, next generation sequencing (NGS)technologies have enabled high-throughput sequencing andbroad gene-expression studies in model and nonmodel plants,including fig (Feng et al. 2012; Ikegami et al. 2013; Li et al.2012; Sweetman et al. 2012; Yin et al. 2012). For tran-scriptome sequencing via 454 technology, we selected fourdevelopmental stages, in phase II and phase III describedabove (Chessa 1997), in the common fig fruit “BrownTurkey”, to identify the enriched metabolic pathways andactive TFs during fig fruit ripening. The current study extendsthe dataset of genes expressed during fruit ripening of aMoraceae member—the F. carica fruit tree—and is a potentialplatform for further studies on this unique plant family, whilecontributing to ripening process research in nonmodelsystems.

Materials and methods

Plant material

“Brown Turkey” common figs (F. carica) were collected froma commercial orchard located near Karmei Yosef in the Judeaplain, 137 m above sea level, in Israel. Fruits used for this

study were from the summer crop, July–August 2009, withday temperatures of 25–32 °C and night temperatures of 20–25 °C. Four developmental stages were sampled: fruit stage 1= early phase II; fruit stage 2 = late phase II—at both stagesthe fruit is dull green and stone hard; fruit stage 3 = transitionfrom phase II to phase III—the fruit is light green and minorfirmness loss is detectable; fruit stage 4 = phase III—the fruitis half purple, soft, and ready for commercial harvest (Fig. 1).Fruit stages 1 and 2 were sampled according to the fruits’position on the branch, exhibiting the differential fruit growthcharacteristic of the summer crop.

Fig RNA extraction

Total RNA was isolated from exocarp and mesocarp (peel andreceptacle) according to Jaakola et al. (2001). RNAconcentrationwas determined in a NanoDrop ND-1000 spectrophotometer.

Roche 454 cDNA library preparation

Equal amounts of total RNAwere pooled from the indicatedsamples and mRNA was purified from total RNA using thePolyATract mRNA Isolation Kit (Promega). ComplementaryDNA (cDNA) library construction and sequencing were per-formed at Dyn Diagnostics Ltd. (http://www.dyn.co.il). EachcDNA library was constructed using the cDNARapid LibraryPreparation Kit (454 Life Sciences, Roche). All steps—RNAfragmentation, cDNA synthesis, adaptor ligation, and productquantification—followed protocols provided by themanufacturer. The resulting cDNA libraries were run on theRoche 454 GS-FLX Titanium system.

454 GS-FLX Titanium pyrosequencing and bioinformaticsassembly

We used the 454 GS-FLX Titanium platform to sequence thetranscriptome of each fruit stage (Margulies et al. 2007). Eachlibrary was run on one-quarter of a PicoTiterPlate. Libraries2–4 were also subjected to additional runs: two additional 1/8lanes for sample 2, one additional 1/16 lane for sample 3, and

Fig. 1 Ficus carica fruit developmental stages used to prepare transcriptsfor 454 pyrosequencing: 1 nonripening stage—the fruit is green and stonehard, 2 preripening stage—the fruit is green and stone hard, 3 ripeningonset stage—minor loss in firmness detected, 4 ripening stage—the fruitis soft and ready for commercial harvest

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one additional 1/4 lane for sample 4. Removal of low-qualityregions and adaptors yielded ∼246Mb total data, equivalent toapproximately 809,000 raw reads, about 175,000–225,000reads per library, average length ∼320 bp (see SupplementalTable 1 and Fig. 2a). The four libraries were assembledseparately and as an “all samples” pool, using softwareversion 2.5 Newbler which has a cDNA option fortranscriptome assembly. This assembly results in theformation of contigs (which might be referred to asexons) and isotigs (which represent transcripts). Theisotigs are assigned into isogroups (unigenes representinggenes). Different isotigs within the same isogroup representalternative splice variants.

Annotation

The resulting contigs, isotigs, and singletons were annotatedusing the Basic Local Alignment Search Tool (BLASTX)(Altschul et al. 1990) against the nonredundant (nr) databasefrom the National Center for Biotechnology Information(NCBI) (http://www.ncb.nlm.nih.gov). The E value cutoffwas set at 10−3. Gene ontology (GO) term annotations wereintroduced based on the BLAST results using Blast2Go(Conesa et al. 2005). The sequences were distributed in threenon mutually exclusive GO categories: biological process,cellular component, and molecular function.

Digital expression analysis

The number of reads per unigene derived from each samplewas counted and normalized by total number of reads perlibrary multiplied by 106, resulting in transcripts per million(TPM) value.

GO-enrichment analysis

To obtain information on significantly enriched GO terms, GO-enrichment analyses were performed by singular enrichmentanalysis (SEA) in the agriGO web-based tool (http://bioinfo.cau.edu.cn/agriGO/index.php) (Du et al. 2010). The customizedannotation mode was used by providing a list of names andcorresponding GO accessions as both a query and a background.Different sets of lists were analyzed according to the fold-changeratio of i=2, 5, and 10: TPM2/TPM1 > i against TPM2/TPM1 <i as a background, TPM3/TPM2> i against TPM3/TPM2< i as abackground, TPM4/TPM3 > i against TPM4/TPM3 < i as abackground, TPM1/TPM2 > i against TPM1/TPM2 < i as abackground, TPM2/TPM3 > i against TPM2/TPM3 < i as abackground, and TPM3/TPM4 > i against TPM3/TPM4 < i as abackground (TPM1 is TPM at fruit stage 1, TPM2 at fruit stage2, etc.). Statistical significance was determined using the Chi-square test and the Yekutieli multitest adjustment (Benjamini andHochberg 1995).

TF analysis

TFs from five species (Vitis vinifera, Solanum lycopersicum,Arabidopsis lyrata, Malus × domestica, and Prunus persica)were downloaded from the Plant Transcription FactorsDatabase (TFDB) (http://planttfdb.cbi.edu.cn/) (Zhang et al.2011) and were used as the database for a BLASTX searchagainst the fig transcripts (cutoff E value of 0.05). The iden-tified fig TFs were clustered based on their expression pro-files; clustering analysis was performed through the Expandertool using the CLICK algorithm (Shamir et al. 2005).

Phylogenetic analysis of MADS-box homologs

FcMADS-box geneswere isolated from a total cDNAmixture ofall fruit stages. Total RNA from each fig stage sample wasdigested with RQ1-DNase (Promega). First-strand cDNA syn-thesis was performed using oligo-dT primer with the Verso™RT-PCR Kit (Thermo Fisher Scientific). Eight MADS-box tran-scripts were successfully isolated using primers designed accord-ing to the 454 sequences (Supplemental Table 8). The amino acidsequences of MADS-box TFs from Arabidopsis were obtainedfrom the NCBI database (http://www.ncbi.nlm.nih.gov/); MIPScodes of the translated genes are as follows: AtAGL5(At2g42830), AtAGL1 (At3g58780), AtAGL11 (At4g09960),AtAG (At4g18960), AtAGL9 (At1g24260), AtAGL4(At3g02310), AtAGL2 (At5g15800), AtAGL3 (At2g03710),AtAGL13 (At3g61120), AtAGL6 (At2g45650), AtCAL(At1g26310), AtAP1 (At1g69120), AtAGL8 (At5g60910),AtAGL42 (At5g62165), AtAGL72 (At5g51860), AtAGL71(At5g51870), AtSOC1 (At2g45660), AtAGL19 (At4g22950),AtAGL14 (At4g118800), AtAGL21 (At4g37940), AtAGL17(At2g22630), AtAGL16 (At3g57230), AtANR1 (At2g14210),AtAGL18 (At3g47390), AtAGL15 (At5g13790), AtSVP(At2g22540), AtAGL24 (At4g24540), AtABS (At5g23260),AtPI (At5g20240), AtAP3 (At3g54340), AtFLC (At5g10140),AtAGL69 (At5g65070), AtAGL68 (At5g65080), AtAGL27(At1g77080), AtAGL70 (At5g65060), and AtAGL31(At5g65050). To compare them with putative fig MADS-boxgenes, we used predicted amino acid sequences originated fromthe longest transcripts, all including the specific C-terminal do-main characteristic of MADS-box TFs. Alignment withAtMADS-box TFs was performed by MUSCLE program usingdefault parameters (http://www.ebi.ac.uk/Tools/msa/muscle)(Edgar 2004). A phylogenetic tree was reconstructed based onthe maximum likelihood (ML) framework using PhyML soft-ware (Guindon and Gascuel 2003) based on the JTT matrix-based model (Jones et al. 1992). A bootstrap consensus tree wasinferred from 1,000 replicates and the number of replicate trees inwhich the associated clade clustered together in the bootstrap testis designated next to the branches (Felsenstein 1985). The treewas graphically designed with the use of Fig Tree version 1.4(http://tree.bio.ed.ac.uk/software/figtree/).

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Validation of digital expression values

To validate the digital expression evaluation, expression levelsof eight FcMADS-box genes were assessed using quantitativereal-time RT-PCR. Total RNA from each fig-stage sample wasdigested with RQ1-DNase (Promega). First-strand cDNAsynthesis was performed, using oligo-dT primer with theVerso™ RT-PCR Kit (Thermo Fisher Scientific).Quantitative real-time PCR was carried out in a 12-μl reactionvolume using Absolute QPCR SYBR Green mix (ABgene).Actin was used as an internal control, to normalize variationsin the amount of cDNA template. Primers used for the real-time PCRs are detailed in Supplemental Table 8. The real-timePCR analysis was performed in a Rotor-Gene 6000™(Corbett Life Science) under the following conditions: 95 °Cfor 15 min followed by 40 cycles of 95 °C for 5 s, annealing at60 °C for 15 s, 72 °C for 20 s, and a melting step up to 99 °C.Analysis was performed on three biological replicates and twotechnical replicates. Expression is presented as percentage ofthe gene’s highest expression, to compare digital values andreal-time PCR relative expression values.

Results and discussion

Generation of transcriptome dataset for ripening fig fruit(F. carica)

To characterize the fig fruit transcriptome during ripening,pyrosequencing was conducted on four poly-A cDNA librar-ies originating from exocarp and mesocarp of the dark com-mon fig cv. Brown Turkey, at several developmental stages:(1) nonripening fruit stage (early growth phase II of fig devel-opment), (2) preripening fruit stage (late growth phase II)—atboth of these stages the fruit is green and stone hard, (3)ripening onset fruit stage (transition from growth phase II togrowth phase III)—a minor loss in firmness is detectablealong with fading of the green hue, (4) ripening fruit stage(growth phase III)—the fruit is soft and ready for commercialharvest (Fig. 1). We chose 454 pyrosequencing since it pro-duces relatively long reads (250–400 bp), allowing de novoassembly when no reference sequence is publicly available(Margulies et al. 2007). Transcriptome sequencing providedus with expression-level data during fig development andripening. The F. carica fruit transcript dataset was establishedthrough de novo assembly of raw data from all four librariescombined and on each library separately (SupplementalTable 1). Assembly of the reads from all libraries togethergenerated 16,634 transcripts (isotigs on Fig. 2a, constructedfrom 602,297 reads) with an average transcript size of1,254 bp and 46,927 singletons representing genes with lowexpression. The transcripts were assigned to 12,751 unigenes(isogroups on Fig. 2a). These results resemble the assembly

outcome from a single developmental stage of the common“Houraishi” fig (Ikegami et al. 2013). Summing up individualassembly results of the four libraries generated 28,168 tran-scripts with an average size of less than 1,000 bp and 157,771singletons. Assigning those transcripts to unigenes for eachlibrary separately created a total of 24,459 unigenes, suggest-ing that a large proportion of the genes are expressed atdifferent developmental stages (Fig. 2b and SupplementalTable 1). To annotate the gene dataset, transcripts resultingfrom the combined library assembly were compared to theNCBI nr protein database by BLASTX program (Altschulet al. 1990). More than 93.5 % of the transcripts were matchedwith homologous sequences, corresponding to 93 % of theunigenes. In addition, 39.7 % of the singletons were matchedwith homologous sequences from the nr database. Furtheranalysis showed that most blast hits originated from Vitisvinifera, followed by Arabidopsis thaliana, Oryza sativa,and Populus trichocarpa—the main plant organisms withavailable and well-defined genomic resources (SupplementalFig. 1). A flow chart of the construction of the fig fruittranscriptome dataset is presented in Fig. 2a.

Main processes involved in fig fruit developmentand ripening

After assigning annotations to the gene set according to theBLASTX results by Blast2GO tool (Conesa et al. 2005), anenrichment analysis was performed by agriGO tool (Du et al.2010) to identify prominent changes during fig fruit ripening.Represented by more than one read, we assume unigenesrepresent the main fig ripening events. To focus on theseevents, further analysis was performed on the unigenes as-sembled from all four fruit stages. Lists of genes that were up-and downregulated between stages were analyzed to findenriched GO terms in the differentially expressed genes.Three cutoff values—2-, 5- and 10-fold change—were ana-lyzed to trace processes exhibiting different fold-change ra-tios. Full lists of significantly enriched terms are detailed inSupplemental Tables 2–6; no significantly enriched termswere found in the analysis of genes that were downregulatedtoward fruit stage 2.

Enrichment analysis revealed changes in photosynthesis-related genes. The rise in these genes toward the preripeningfruit stage (Fig. 3a) is in agreement with the continuousincrease in fig carbohydrate content during growth phase II(Crosby 1954; Marei and Crane 1971). A recent study hasshown delayed ripening of tomato (Solanum lycopersicum)mutants exhibiting chlorophyll-deficient phenotypes as a re-sult of reduced chlorophyll synthesis (Barry et al. 2012). Theopposite phenomenon was observed in a transgenic system oftomato overexpressing a SnRK1 (sucrose non-fermenting-1-related protein kinase 1) gene from pingyitiancha (Malushupehensis) (Wang et al. 2012). The protein SnRK1 regulates

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carbon metabolism by inactivating several key enzymes in thecell carbon cycle and thus plays a role in carbon assimilation(Halford 2006). The rise in photosynthesis-related gene ex-pression toward the preripening stage in fig fruit can thereforebe a function of sugar accumulation; it could serve as aninternal signal for ripening onset, as has been shown in straw-berry (Fragaria x ananassa) in which silencing of FaSUT1, asucrose transporter gene, led to fruit ripening arrest, whileoverexpression of FaSUT1 accelerated fruit ripening (Jiaet al. 2013). As implied by the fading green color fromripening onset on photosynthesis-related elements are contin-uously reduced toward the fruit ripening stage (Fig. 4a, b),similar to the decline reported in tomato and grapevine berry(Vitis vinifera) (Blanke and Lenz 1989; Lijavetzky et al. 2012;Piechulla et al. 1986).

One of the unique characteristics of fig fruit is its rapidripening accompanied by rapid growth in size—both weightand diameter. The transformation to an edible state (fromripening onset) can be completed within only 2 days in thesummer crop in Israel, the main crop of most fig cultivars.Upregulation of cell-fate specification toward fruit phase III,ripening onset, serves as an indication of the tissue’s commit-ment to the ripening plan (Fig. 3b). Rapid transformation maywell require an increase in RNA transcript toward thepreripening fruit stage (Fig. 3a). This expected trend in tran-scription processes has been documented in transcriptomestudies of date palm (Phoenix dactylifera) and bayberry(Myrica rubra) fruits (Feng et al. 2012; Yin et al. 2012). Acontinuous decrease following this peak (Fig. 4a, b) has beendocumented in preripening figs after ethylene treatment,which triggered ripening accompanied by peaks in sRNA,rRNA, ribosome, and protein synthesis (Marei and Romani1971). A similar trend is evident in date palm and grapevine

berry transcriptome studies (Sweetman et al. 2012; Yin et al.2012).

Aside from the expected terms of ripening andanthocyanin-precursor metabolism (phenylpropanoid and fla-vonoid), another prominent up-regulated process at ripeningonset was amino acid catabolism (Fig. 3b). Detailed inspec-tion revealed genes associated with flavonoid and thiamin(vitamin B1) synthesis, one of the fig’s beneficial nutritionalcomponents (Flaishman et al. 2008). Another fate for fig fruitamino acids is volatiles. As the fig’s volatile profile is rich inacetaldehyde, ethyl acetate, methanol, ethanol, and others,amino acids can serve as a pool for the rapid synthesis ofaroma and flavor compounds (Gonda et al. 2010; Oliveiraet al. 2010a, b; Schwab et al. 2008).

Fig fruit softening occurs in parallel to changes in color,flavor, and aroma. As fig fruit has a short shelf life and storageperiod, fig softening has been studied mainly from a postharvestpoint of view and is amain target for improving storage and shelflife of the fresh commodity (Flaishman et al. 2008; Owino et al.2006, 2004a, b; Sozzi et al. 2005). Genes for polygalacturonase,glucanase, galactosidase, endotransglycosylase, andarabinofuranosidase functions have been shown active in ripen-ing “Masui Dauphine” fig fruit at different stages (Owino et al.2004b). In addition, in caprifig and “Houraishi” common fig—pectin-modifying genes, encoding glycosyl hydrolases, beta glu-cosidase, and glycosyltransferases, constitute several of the 20most common transcripts toward ripening phase (Ikegami et al.2013). The expression of some of these gene families has beencorrelated with softening patterns in several fruits, includingapple (Malus × domestica), date palm (Phoenix dactylifera),ber (jujube, Ziziphus mauritiana), kiwi (Actinidia chinensis),and apricot (Prunus armeniaca) (Atkinson et al. 2012; Harbet al. 2012; Leida et al. 2011; Mworia et al. 2012; Rastegar

Fig. 2 Assembly and analysis of fig transcriptome reads dataset. a Flow chart of fig fruit transcriptome dataset construction. bVenn diagram of numberof unigenes expressed at different fruit stages

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et al. 2012; Yadav et al. 2012). Concurring with these findings,fig fruit cell wall-modifying enzymesweremarkedly upregulatedtoward ripening onset (Fig. 3b) and could contribute to figsoftening during ripening and postharvest life (Brummell 2006;Rugkong et al. 2011). However, fruit softening is not influencedby cell wall degradation alone; it is also a function of water lossand maintenance of cell turgor. In the “Delayed FruitDeterioration” (DFD) tomato mutant, the cuticle was clearlyshown to have an influence on intact tomato fruit firmness andripening physiology. DFD fruits had a 36 % higher wax contentthan the related Ailsa Craig (AC) tomatoes when ripe, allowingthe fruits to stay intact even after 7 months at room temperature(Saladie et al. 2007). The importance of wax as a key player infruit softening was also concluded from rin, nor, and Alcobaçafruits, whose cuticle lipid compositions differed from that of ACtomatoes (Kosma et al. 2010). Wax metabolism was found to bea major downregulated process toward the fig ripening stage(Fig. 4b), in agreement with the decrease in wax content

documented in three other fig cultivars and the transcriptomeprofile documented in grapevine berry (Chessa et al. 1992;Lijavetzky et al. 2012). This decrease in wax synthesis duringrapid size growth might be partially responsible for fruit soften-ing, decay, and shriveling problems, the elucidation of which iscentral to improving fig storage (Flaishman et al. 2008).

The enrichment in ascorbate oxidase function in the genesthat were upregulated toward ripening onset (Fig. 3b) mightrelate to the rapid cell expansion observed in figs duringripening. In tomato, ascorbate oxidase is only expressed indeveloping fruit at the early growth stages and is reduced atthe mature green stage, when growth stops (Ioannidi et al.2009). Since fig fruit growth arrests before ripening andresumes at a high speed in parallel to ripening processes,ascorbate oxidase activity may well contribute to the cellexpansion that occurs toward full ripening. The high abun-dance of genes related to DNA metabolism and chromatin atthe ripening fruit stage (Fig. 3c) may also relate to rapid cellexpansion coupled with endoreduplication events, which havebeen documented in fig and in other fleshy fruits (Bourdonet al. 2010). The relationship between cell size and ploidylevels has been studied during fruit development in tomato(Bourdon et al. 2012). Though the role of endoreduplication isunknown and, in addition, fruit growth has been successfullyuncoupled from endoreduplication progress in transgenic to-mato, in natural systems, endoreduplication is a feature ofspecies exhibiting rapid fruit development, whereas it is ab-sent in fruits with longer developmental periods (Bourdonet al. 2010). This said, the rapid growth of fig fruit can be anadvantage for studies of endoreduplication events by servingas a snapshot of the processes involved.

Fig. 4 Significantly enrichedbiological processes, cellcomponents, and molecularfunctions of the genesdownregulated during figdevelopment. a Significantlyenriched GO termsdownregulated toward fruitripening onset. Enrichmentanalysis is described in the legendto Fig. 3, query list TPM2/TPM3> i against background listTPM2/TPM3 < i. b Significantlyenriched GO termsdownregulated toward theripening stage, query listTPM3/TPM4 > i againstbackground list TPM3/TPM4 < i

�Fig. 3 Significantly enriched biological processes, cell components, andmolecular functions of the genes upregulated during fig development. aSignificantly enriched GO terms upregulated toward the preripening stage.Enriched GO terms of the gene list satisfy the criteria TPM2/TPM1 > iagainst the background list containing genes in which TPM2/TPM1 < i.TPM transcripts per million, i.e., total number of reads mapped to a geneper library multiplied by 106; i=2, 5, and 10. Percentage of eachsignificantly enriched term of the examined list (query or background) ispresented. Statistical significance was determined using the Chi-square testand the Yekutieli multitest adjustment in the agriGO tool. b Significantlyenriched GO terms upregulated toward fruit ripening onset, query listTPM3/TPM2 > i against background list TPM3/TPM2 < i. cSignificantly enriched GO terms upregulated toward the ripening stage,query list TPM4/TPM3 > i against background list TPM4/TPM3 < i

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TFs expressed during fig fruit ripening

Finding genes of TFs that might regulate fig fruit ripeningprocesses required further investigation of the assembled tran-scripts. This time, isotigs were compared to the Plant TFDB,resulting in 516 unigenes encoding TFs from 40 different TFfamilies (Supplemental Table 7). The three most abundant TFfamilies in the fig fruit transcriptome were the basic/helix-loop-helix (bHLH) family, the Cys3His zinc finger domain-containing proteins (C3H), and the ethylene response factors(ERF) (presented in Fig. 5a are TF families that constitute atleast 1 % of the total 516 genes identified as TFs). The bHLHfamily, which constituted 10% of the total TF genes comparedwith 8.7 % of the TFDB collection, was found to be related toanthocyanin biosynthesis and fruit dehiscence, among otherprocesses (Buck and Atchley 2003; Su et al. 2012). The C3Hfamily, which constituted close to 10 % of the total TF genescompared with 3.3 % of the TFDB collection, was first asso-ciated with embryogenesis (Grabowska et al. 2009; Li andThomas 1998). The ERF family, which constituted around7 % of the total TF genes and the same percentage of the

TFDB collection, include TFs induced by the hormone andinvolved in the hormone signal’s transduction (Ohme-Takagiand Shinshi 1995). TFs preceding ethylene synthesis in cli-macteric fruits, triggering ripening onset in nonclimactericfruits, and regulating advanced ripening processes have beenisolated from several species. In tomato, members of the genefamilies NAC,MADS-box, and SPB-box have been thorough-ly examined as regulators of ripening onset (Klee andGiovannoni 2011). In grapevine berry, candidate genes forripening onset and regulation belong to the families AP2/ERF,bHLH, MYB, Trihelix, WRKY, Homeobox, TCP, and GATA(Lijavetzky et al. 2012; Sweetman et al. 2012). A blueberry(Vaccinium corymbosum) transcriptome survey revealed TFsexpressed during fruit ripening—members of the familiesnoted above with the addition of GRAS, bZIP, C2H2, ARF,COL, and C3H (Li et al. 2012). In our search for TFs that areactive in the fig fruit, the above gene families constituted closeto 80 % of the TFs expressed during fig development andripening.

Based on the TPM values of the 516 TFs, clustering analysiswas performed. Six clusters of expression patterns of the TF

Fig. 5 Fig fruit transcriptionfactor (TF) analysis. a TF familiesthat are active during fig fruitdevelopment. Active TFs in figfruit were targeted throughBLASTX of the all stages datasetagainst Plant TFDB. TF familiesconstituting over 1 % of all TFgenes are presented. b Expressionpatterns of 516 fig fruittranscription factors. Clusteringwas performed with the Expandertool using the CLICK clusteringalgorithm. TPM1 normalizedvalue of transcripts per million atfruit stage 1, TPM2 normalizedvalue of transcripts per million atfruit stage 2, etc.

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genes were obtained (Fig. 5b and Supplemental Table 7). Thelargest cluster, cluster 1 comprising 132 genes, presented expres-sion patterns which decrease from the fruit stage 1 to the fruitstage 4 andmay therefore include ripening inhibitors. The secondlargest cluster, cluster 2 comprising 129 genes and cluster 4comprising 79 genes, presented expression patterns which in-crease gradually toward ripening onset and ripening stages andtherefore may include genes involved in ripening processes.Cluster 3, comprising 123 genes, and cluster 6, comprising 21genes, presented expression patterns that peak at the preripeningstage 2 and decrease toward ripening onset and therefore mayinclude specific ripening onset regulators. Particularly interestingwas the fact that 46% of the TF family of ERFswere assigned toclusters 3 and 6, and 45 % were assigned to clusters 2 and 4 (seeSupplemental Table 7), corresponding to ripening onset regulat-ing TF’s and advanced ripening processes regulating TF’s, re-spectively. Only 9% of the ERFswere assigned to other clusters,implying a close relation of the ERF family to ripening eventsregulation.

MADS-box gene candidates for regulation of fig fruitripening

In plants, MADS-box genes encode transcription factors thatregulate floral development, as well as fruit development and

ripening. The study of caprifig and “Houraishi” common figintroduced nine FcMADS-box genes, in order to find genesinfluencing the differences between the hermaphroditic fruitand the female fruit (Ikegami et al. 2013). Gene expressionwas detected to some level in both fruit types in phase I and II,prior to ripening. Regarding fruit development and ripening,MADS-box genes have been found to regulate these processesin several species: RIN (Ripening Inhibitor) and TAGL1(Tomato AGAMOUS Like) from the climacteric tomato(Solanaceae), PLENA from the climacteric peach (Prunuspersica; Rosaceae), MADS9 from the nonclimacteric straw-berry (Fragaria x ananassa; Rosaceae), and MaMADS1–5from the monocotyledonous climacteric banana (Musaacuminata) (Choudhury et al. 2012; Elitzur et al. 2010; Itkinet al. 2009; Seymour et al. 2011; Tadiello et al. 2009; Vrebalovet al. 2009). In light of these discoveries, MADS-box proteinsmay act as key players in the regulation of fig fruit ripening,similar to their role in other fruits. In an attempt to relateFcMADS-box genes to ripening events, we isolated elevengenes homologous to theMADS-box family from common figin phase II and III and examined their expression during thesestages. Eight out of eleven MADS-box transcripts were suc-cessfully isolated using primers designed according to our 454sequences. Quantitative PCR was performed to validate thepatterns of the digital expression values of the different

Fig. 6 Real-time PCR (qPCR) analysis of the expression of eightMADS-box genes, compared to digital expression values. Expression is presented aspercentage of the gene’s highest expression, to compare digital values and real-time PCR relative expression values

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FcMADS-box transcripts (Fig. 6). Correlation analysis be-tween relative expression values and digital expression TPMvalues show high correlation coefficients for all eightFcMADS-box genes tested (Supplemental Fig. 2).

Phylogenetic analysis of deduced amino acids (Fig. 7)showed that one FcMADS gene belongs to the AGL6 subfamily(Isogroup 1917), two belong to the SQUA (SQUAMOSA)subfamily (isogroups 4355 and 7031), one gene is part of theSTMADS11 subfamily (Isogroup 10006), and one is related tothe FLC (Flowering Locus C) subfamily (Isogroup 9067).Subfamilies are defined by the genes from Arabidopsis (Beckerand Theissen 2003). Of particular interest, three FcMADS genesencoding AGL2 (SEP) subfamily members were identified(isogroups 1334, 5572 and 139). This subfamily includes the

proteins LeRIN, FaSEP9, MaMADS1, 2, and 4, andMdMADS8 and 9, all related to fruit ripening (Ireland et al.2013; Itkin et al. 2009; Seymour et al. 2011; Tadiello et al. 2009;Vrebalov et al. 2002). The deduced amino acid sequence ofisogroup 1334 and isogroup 5572 isolates showed the highestsimilarity among tomato MADS proteins to LeMADS1 andLeMADS5, respectively (through BLASTX against theSolanum lycopersicum nr collection in NCBI, data not shown).Transcripts of LeMADS1 and LeMADS5 were located inflowers and exhibited a continuous decrease during tomato fruitdevelopment and ripening (Gaffe et al. 2011). Similar to theirtomato homologous, neither isogroup’s expression profile couldbe related to fruit ripening induction, since their transcription islow at ripening onset, and increases only slightly toward the

Fig. 7 Phylogenetic analysis of FcMADS-box proteins. A maximumlikelihood tree with 1,000 bootstrap replicates was constructed based onalignment of the predicted amino acid sequences of eight fig fruit MADS-box homologous and Arabidopsis MADS-box proteins. Subfamiliesaccording to (Becker and Theissen 2003) are noted on the right.AtAGL12 was used as the root. Deduced fig proteins are namedisogroups 5572, 1334, 139, 1917, 4355, 7031, 10006, and 9067.ArabidopsisMIPS codes of the translated genes are as follows: AtAGL5(At2g42830), AtAGL1 (At3g58780), AtAGL11 (At4g09960), AtAG(At4g18960), AtAGL9 (At1g24260), AtAGL4 (At3g02310), AtAGL2(At5g15800), AtAGL3 (At2g03710), AtAGL13 (At3g61120), AtAGL6

(At2g45650), AtCAL (At1g26310), AtAP1(At1g69120), AtAGL8(At5g60910), AtAGL42 (At5g62165), AtAGL72 (At5g51860),AtAGL71 (At5g51870), AtSOC1 (At2g45660), AtAGL19(At4g22950), AtAGL14 (At4g118800), AtAGL21 (At4g37940),AtAGL17 (At2g22630), AtAGL16 (At3g57230), AtANR1(At2g14210), AtAGL18 (At3g47390), AtAGL15 (At5g13790), AtSVP(At2g22540), AtAGL24 (At4g24540), AtABS (At5g23260), AtPI(At5g20240), AtAP3 (At3g54340), AtFLC (At5g10140), AtAGL69(At5g65070), AtAGL68 (At5g65080), AtAGL27 (At1g77080),AtAGL70 (At5g65060), and AtAGL31 (At5g65050)

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ripening stage (Fig. 6). One MADS-box gene, Isogroup 139,showed expression levels similar to those of LeRIN duringtomato ripening, increasing toward and during ripening(Vrebalov et al. 2002) (Fig. 6). This isogroup showed the highestsimilarity to LeRIN among tomato MADS proteins (throughBLASTX against the S. lycopersicum nr collection in NCBI,data not shown), and we therefore intend to further investigate itsfunction in fig fruit ripening.

Ethylene synthesis during fig fruit ripening

The fig is categorized as a climacteric fruit with changingethylene evolution as growth progresses. Ethylene productionrate is relatively high at the beginning of growth phase II(Fig. 1, fruit stage 1); it then declines and rises again in parallelwith the onset of ripening in growth phase III (Fig. 1, fruit stage3) (Marei and Crane 1971). Unlike the autocatalytic ethylenesynthesis typical of ethylene system II in climacteric fruits, thefig fruit response to ethylene-perception blocking agent 1-MCP(1-methylcyclopropene) application at ripening onset is actual-ly a sharp rise in ethylene production (Freiman et al. 2012;Owino et al. 2006; Sozzi et al. 2005). In the “Brown Turkey”fig fruit transcriptome, four gene members of the 1-aminocyclopropane-1-carboxylate synthase (ACS) family weredetected. Two unigenes (Fig. 8) and 2 singletons, this comparedto 3 contigs and 11 singletons found in the joined assembly ofcaprifig and common fig (Ikegami et al. 2013). In the “BrownTurkey” fig fruit transcriptome, five gene members of the 1-aminocyclopropane-1-carboxylate oxidase (ACO) family weredetected, all unigenes, this compared to 26 contigs and over 89singletons found in the joined assembly of caprifig and com-mon fig (Ikegami et al. 2013). The difference between thesegene sets can be explained by the caprifig contribution of genesthat are absent in the common fig. According to our findings,one ACS (Isogroup 413) was 99 % homologous to the

nucleotide sequence of ACS1 previously documented in thevariety Masui Dauphine. One ACO (Isogroup 41) was foundto be 100 % homologous to the nucleotide sequence of asingle gene documented in the same study (Owino et al.2006, data not shown). Expression of both of these genesincreased toward ripening, similar to the pattern observed inthe harvested “Masui Dauphine” fruit, except that in ourstudy, the fruit were allowed to reach the specific develop-mental stage on the tree. Two ACS genes expressed in“Masui Dauphine” fruit 6 days postharvest, were not de-tected in our experiment. As presented in Fig. 8, expressionof all unigenes involved in ethylene synthesis increasedtoward ripening, except two ACO genes (isogroups 4960and 6420) which showed a minor decrease from ripeningonset to the ripening stage. The latter two ACO genesshowed relatively high expression at the nonripening stage.As for the traditional definition, these genes may be consideredethylene system I synthesis genes though, as mentioned, in figfruit, the behavior of ethylene is not restricted to climactericcharacteristics. To define the ethylene synthesis systems in figfruit, further examination is necessary based on the recenttranscriptome studies.

Conclusions

This study provides the first expressed gene dataset for mul-tiple developmental stages of fig fruit (F. carica) and analysisdirected to ripening metabolism, control, and regulation. Aforward bioinformatics approach was taken to locate the mainchanges during fig fruit ripening. We chose an enrichmentanalysis that was based on differential expression and notbiased by our knowledge of ripening processes. In addition,a complementary reverse approach was taken to present atranscriptomic view of ethylene synthesis and TF genes thatare active during fig fruit ripening. A comparison to thecaprifig and common fig transcriptome sequencing data willprovide extensive knowledge on the processes differentiatingthe development of caprifig from that of common fig. Furtherstudies of fig will contribute to understanding the complexethylene mechanism, which shows both climacteric andnonclimacteric features. Since all spoilage phenomena aretypically associated with overripe fig fruit, studying the path-ways that lead to fig ripening can provide additional ways ofextending the fruit’s storage life. Fig fruit storage and dietaryvalues have yet to be directly targeted in genetically basedbreeding; further mapping of these traits on the basis of thisstudy will improve fig breeding programs and may serve as amodel for other heterozygous fruit trees. The data presented inthis work, stemming from large-scale sequencing of aMoraceae member, can serve for both evolutionary and eco-logical studies of other Ficus species, in addition to ripeningprocess research in nonmodel systems.

Fig. 8 Expression of ethylene-synthesis genes during fig fruit develop-ment and ripening. Heatmap of the expression levels of ACS and ACOgenes generated based on the TPM value of the unigene at each devel-opmental stage, using the heatmap.2 package and default complete link-age method in R. The color in the heatmap represents Z-score-normalizedTPM for each unigene

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Acknowledgments The authors wish to thank Dr. Vered Caspi, InbarPlaschkes, and Guy Rapaport of the Bioinformatics Core Facility, TheNational Institute for Biotechnology in the Negev, Ben-Gurion Universi-ty, Beer-Sheva, Israel, for providing us with computer services andsupport for the annotation analysis. We also thank Dr. Amir Sherman,Dr. Ron Ophir, Dr. Nurit Katzir, and Dr. Shiri Freilich for their helpfuladvice. This work was supported by a grant from The Ministry ofAgriculture, Israel.

Data archiving statement The sequence data generated in this studyhave been deposited at NCBI in the Sequence Read Archive (SRA) underthe accession numbers SRR1174869- SRR1174872 (sample 1–4, respec-tively). The Transcriptome Shotgun Assembly project has been depositedat DDBJ/EMBL/GenBank under the accession GAYT00000000. Theversion described in this paper is the first version, GAYT01000000.Sequences of cDNA isolates of Isogroups 1917, 4355, 7031, 10006,9067, 1334, 5572, and 139 have been deposited at NCBI under theaccessions KJ506152- KJ506159 (FcMADS1-8, respectively).

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