Differential transcript abundance and genotypic variation of ...

ORIGINAL PAPER

Differential transcript abundance and genotypic variationof four putative allergen-encoding gene familiesin melting peach

Zhaowei Yang & Yingtao Ma & Lin Chen & Rangjin Xie & Xianqi Zhang & Bo Zhang &

Meidan Lu & Shandong Wu & Luud J. W. J. Gilissen & Ronald van Ree &

Zhongshan Gao

Received: 8 June 2010 /Revised: 3 December 2010 /Accepted: 1 March 2011 /Published online: 19 March 2011# Springer-Verlag 2011

Abstract We analysed the temporal and spatial transcriptexpression of the panel of 18 putative isoallergens fromfour gene families (Pru p 1–4) in the peach fruit, anther andleaf of two melting cultivars, to gain insight into theirexpression profiles and to identify the key family members.Genotypic variation of abundantly expressed genes inmature fruit was further screened in nine additional melting

cultivars. In the Pru p 1 family, Pru p 1.01 and Pru p 1.06Bwere predominant and constitutively expressed in all thetissues, with large difference among cultivars observed inmature fruits. Pru p 1.02 was especially abundant only inthe leaf. A new member of the Pru p 1 family, Pru p 1.06D,was identified through peach genome mining. In the Pru p2 family, Pru p 2.01B was predominant in all tissues,whereas Pru p 2.01A was abundant in a few cultivars andundetectable in others. Pru p 3.01 was the most highlyexpressed member in all tissues except the mesocarp, whilethe other two members exhibited tissue specificity: Pru p3.02 was highly expressed in the leaf, and Pru p 3.03 in theanther. Both Pru p 4 isoallergen genes were equallyexpressed in all tissues of both cultivars. There was highexpression variability of Pru p 1 and Pru p 2 members inmature fruits among 11 cultivars, while relative lower forPru p 3 and Pru p 4. The location, arrangement andfeatures of peach isoallergen genes on the peach genomescaffolds were illustrated.

Keywords Gene expression . Peach allergen . Genotypicvariation . Real-time PCR

Background

Rosaceae species, particularly apple (Malus domesticaBorkh.) and peach (Prunus persica L. Batsch), are widelygrown and consumed, and are economically importantcrops. Despite being recommended for health benefits, forsensitized individuals they are, however, a potential triggerof allergic reactions. The increasing prevalence of fruitallergy has been reported in the Mediterranean countries ofEurope (Fernandez-Rivas et al. 1997; Kanny et al. 2001),

Communicated by A. Abbott

Electronic supplementary material The online version of this article(doi:10.1007/s11295-011-0383-5) contains supplementary material,which is available to authorized users.

Z. Yang :Y. Ma : L. Chen :R. Xie :B. Zhang :M. Lu :Z. Gao (*)Department of Horticulture,The StateAgricultureMinistry Laboratory ofHorticultural Plant Growth,Development and Quality Improvement, Zhejiang University,Hangzhou 310058, Chinae-mail: [email protected]

Z. Yang : S. Wu : Z. GaoAllergy Research Center, Zhejiang University,Hangzhou 310058, China

X. ZhangDepartment of Dermatology, The Second Affiliated Hospital,School of Medicine, Zhejiang University,Hangzhou 310009, China

L. J. W. J. GilissenPlant Research International,Wageningen University and Research Centre,PO Box 16, 6700AA Wageningen, The Netherlands

R. van ReeDepartment of Experimental Immunology,Academic Medical Center-University of Amsterdam,Room K0-130, Meibergdreef 9,Amsterdam 1105 AZ, The Netherlands

Z. W. Yang : Y. T. Ma : L. Chen : R. J. XieB. Zhang : M. D. Lu : Z. S. Gao (*)

Z. W. Yang : S. D. Wu : Z. S. Gao

X. Q. Zhang

Tree Genetics & Genomes (2011) 7:903–916DOI 10.1007/s11295-011-0383-5

http://dx.doi.org/10.1007/s11295-011-0383-5

China (Wen and Ye 2002) and Japan (Inomata et al. 2007).For peach, sensitization patterns are rather complex due toits geographic distribution (Cuesta-Herranz et al. 1998;Gamboa et al. 2007), leading to two prevalent symptoms:oral allergy syndrome (Inomata et al. 2007; Ortolani et al.1988) and systemic symptoms (Gamboa et al. 2007;Sanchez-Monge et al. 1999). Allergic syndromes relatedto cross-reactivity, based on IgE recognition of allergenssimilar in structure, have been well-documented amongRosaceae fruits (Rodriguez et al. 2000), vegetables(Fernandez-Rivas et al. 2008), pollen (Egger et al. 2006)and latex (Blanco et al. 1994). Allergy to pollen (Han et al.2008) and fruit skin hair (Li et al. 2003) in peach has alsobeen observed in China.

Molecular analysis of cross-reactivity has revealed thatmost Rosaceae fruit allergens can be assigned to a fewprotein families (Radauer et al. 2008). With regard topeach, four allergen families have been identified (Chen etal. 2008): the Bet v 1 homologues Pru p 1, the thaumatin-like protein (TLP) Pru p 2, the non-specific lipid transferprotein (nsLTP) Pru p 3 and the peach profilin Pru p 4. Theclinical relevance, biochemical and immunological proper-ties, and the recombinant forms of all these allergens havebeen extensively characterized, including the newly char-acterized peach TLP (Palacin et al. 2010). This has highamino acid sequence identity and 3D structure similarity(Botton et al. 2009) with known major allergens such asMal d 2 (apple) and Pru av 2 (cherry). Peach LTP(Pastorello et al. 1999) is the major peach allergen causingsevere symptoms in the Mediterranean area. It is the beststudied, regarding its IgE-binding epitopes (Garcia-Casadoet al. 2003) as well as its three-dimensional crystal structure(Pasquato et al. 2006). Isoform variation of the IgE-affinityspecificity has been shown (Pacios et al. 2008; Reuter et al.2005). In addition, recent genomic studies (Chen et al.2008) have revealed that the peach genome contains at least18 isoallergens (eight for Pru p 1, five for Pru p 2, three forPru p 3 and two for Pru p 4), which can be mapped on fivelinkage groups. Differential expression of these isoaller-gens, which can vary in IgE binding capacity among themembers of a single-allergen gene family, may result incomplex allergen variant (isoform) composition, leading tocomplex allergenicity.

Since cultivars may differ in allergenicity, strategies forselection and breeding of hypoallergenic cultivars havebeen developed for apple under a joint EU-project(Hoffmann-Sommergruber and Consortium 2005). A sim-ilar approach was initiated in peach recently. The startingpoint is the assumption that the degree of (hypo-)allergenicity depends on qualitative and quantitativecultivar-specific factors (Chen et al. 2008). Immunologicalstudies, such as skin prick tests (SPT) and double-blind,placebo-controlled food challenges in vivo, and immuno-

blotting and ELISA in vitro, have been used for thedetection and quantification of cultivar-specific allerge-nicity or total allergen content. These have demonstratedvariations in allergenicity among different apple (Sanchoet al. 2008) and peach (Brenna et al. 2004) cultivarstested. Allele diversity analysis of intron-containingmembers of Mal d 1 indicates that protein variantcomposition and allele doses are both associated withallergenicity (Gao et al. 2008).

Considering the varying IgE binding capacity ofallergen variants and the small number of allergenicmolecules needed to trigger an allergic reaction, infor-mation is required on relative expression abundance andIgE-binding properties of individual isoallergens orvariants. Association analysis combining clinical data,expression profiles at both mRNA and protein level, andthe allergenic property of the individual allergen variantsis needed to further unravel the allergenicity of acultivar.

Previous studies have shown markedly different allergenaccumulation in diverse peach and nectarine cultivars(Brenna et al. 2004), in agreement with results found inapple (Sancho et al. 2008). Differential tissue localizationof major apple allergens has been identified by immuno-histochemical methods (Marzban et al. 2005), such as theimmuno-tissue-print assay, showing that Mal d 1 and Mal d2 are distributed throughout the apple peel and pulp whileMal d 3 is restricted to the peel. Similar distribution andbiological functions of the allergens of apple and peachcould be considered in view of the conserved structures ofthese proteins. Northern blotting (Botton et al. 2002, 2006)and MALDI-mass spectrometry imaging (Cavatorta et al.2009) have, in fact, confirmed that peach LTP (Pru p 3)essentially concentrates in peel, with less in pulp. Pru p 3has also been detected in fruit surface fuzz (Asero et al.2006), leaves (Garcia et al. 2004) and pollen (Marzban etal. 2006). Less Pru p 1 and Pru p 4 have been found in thefruit compared with Pru p 3 (Gaier et al. 2008; Rodriguez-Perez et al. 2003).

Information on transcript expression and differentialtissue accumulation of peach allergen isoforms is limited,although other members have recently been identified(Chen et al. 2008). Two members of peach LTP (Pru p3.01 and Pru p 3.02) have been identified, with majordifferences in mRNA and protein accumulation, and inpatterns of transcript regulation (Botton et al. 2002;Marzban et al. 2005). Similar expression patterns of twoisoforms (Pru p 1.01 and Pru p 1.06D) of Pru p 1 havebeen found at the mRNA level, during fruit development inpeach skin (Zubini et al. 2009). It has also been found thatPru p 2.02 (AF362987, namely PpAz8) does not accumu-late in peach fruit, and is only detected (by northern blotanalysis) in leaf and flower, while a Pru p 2.01 homologue

904 Tree Genetics & Genomes (2011) 7:903–916

(AF362988, namely PpAz44) accumulates abundantly inripening peach fruit and the senescing flower, but isabsent in the leaf (Ruperti et al. 2002). Recent studieswith a nectarine cultivar, on transcript expression andregulation of previously identified isoallergen genes, haveshown distinct expression patterns of different allergenvariants (Botton et al. 2009).

The multiple isoallergens of the four peach allergenfamilies and their encoded allergen variants make it difficultto characterize and identify putative allergens and variantsthrough traditional immunological approaches. Real-timeRT-PCR (also known as quantitative RT-PCR (qPCR)) is apowerful tool with high sensitivity and specificity formonitoring and quantifying gene expression. This can beuseful to discriminate closely related genes and weaklyexpressed genes at the mRNA level, and to narrow downthe candidate list of peach allergy-related variants forfurther investigation, at the individual protein level, bymass spectrometry-based proteomics methods (Helsper etal. 2002; Monaci and Visconti 2009; Napoli et al. 2008;Schenk et al. 2009), for breeding hypoallergenic cultivars.

A preliminary study on the peach allergen encodinggenes (Botton et al. 2009) did not cover all the membersidentified by genomic research. In the present study,transcript accumulation of all known putative allergengenes from the four gene families was investigated inepicarp and mesocarp from two melting peach cultivarswith regard to regulation patterns during fruit development.It was also investigated in anther and young leaf to gaininsight into the general expression profiles and distinguishbetween the potential key members specifically involved infruit allergy of the two peach cultivars, with an additionalnine varieties screened for expression level of key allergengene members.

Materials and methods

Plant materials and tissue sampling

Two melting flesh peach cultivars (cv. ‘Yulu’ and cv.‘Hujingmilu’) were obtained from Fenghua Honey PeachInstitute (Zhejiang, China). Young leaves and anthers werecollected at full bloom. Twenty-four fruits (without visibledefects) from the two cultivars were harvested during thefour physiological phases of fruit development (S1–S4) anddivided into three replicates. Ripe fruits (S4) were sampledfrom two consecutive harvest seasons (2008 and 2009) inorder to confirm the results. In 2010, fully ripe fruits fromeleven diverse peach cultivars, ‘Yulupantao’, ‘Hujingmilu’,‘Hongburuan’, ‘Baifeng’, ‘Yuhualu’, ‘Yulu’, ‘Bailu’,‘Akatsuki’, ‘Jinxiuhuangtao’, ‘Okubo’ and ‘Zhonghuashoutao’(in the order of fruit firmness from 4.2 to 43.1 N, the

first six cultivars are soft melting type, the latter five ishard melting type) collected from four provinces, wereused to identify expression diversity of the abundantlyexpressed isoallergen gene members. The epicarp(including the outer epidermis and a few layers ofhypodermal cells) and mesocarp (a 1-cm thick regionof the mesocarp closest to the epicarp) were excised andtreated separately. The samples were immediately frozenin liquid nitrogen and stored at −40°C.

RNA extraction and cDNA synthesis

Total RNA was isolated from pooled samples according toChang et al. (Chang et al. 1993). For each sample, 2 g ofdissociative fruit tissues or 1 g of leaves and flowers wereextracted in 15 ml of extraction buffer. Contaminatinggenomic DNA was removed by RNase-free DNase I(TaKaRa, Japan) treatment. The concentration of isolatedtotal RNA was determined by the absorbance at 260 nm(A260) using the NanoDrop® ND-3300 Fluorospectrometerwith Quant-iT™ RiboGreen® RNA Reagent (Invitrogen,USA) following the manufacturer’s instructions. Theintegrity was evaluated by electrophoresis on 1.2% agarosegels. First-strand cDNA was synthesized using 1.0 μg oftreated RNA for each sample with AMV Reverse Tran-scriptase XL (TaKaRa, Japan) and oligo(dT) as primers.The cDNA was tested by PCR, using specific primersflanking an intron sequence to confirm the absence ofgenomic DNA contamination, and adjusted to thresholdcycle (Ct) values within the mean range ±1 of the referencegene to ensure similar cDNA yield for each qPCR reaction.All cDNAs were stored at −20°C.

Gene-specific PCR primers

Primer pairs used for quantitative PCR were designedusing the software program PRIMER DESIGNERv. 2.0 (Scientific and Educational Software, Cary, NC)described previously (Chen et al. 2008), with meltingtemperatures (Tm) of 60±2°C to facilitate multi-parallelqPCR using a standard PCR program. Primer pairsflanking an intron were compared by standard PCR usinggenomic DNA and cDNA as templates. The specificity ofeach primer pair was analyzed by a melting curve at theend of the PCR run, with one single peak revealing thatthe fluorescence signal was derived from the intendedamplicon with no dimer formation. PCR products werecloned into the pGEM T-easy vector (Promega, Madison,Wis.) and sequenced (Invitrogen, Shanghai, China).Their size was checked on 1.5% agarose gels stainedwith ethidium bromide. The primer sequences, ampliconsizes, and Tm of all PCR products are indicated inTable 1.

Tree Genetics & Genomes (2011) 7:903–916 905

Real-time RT-PCR

Real-time RT-PCR reactions were performed in a totalvolume of 12.5 μl, containing 0.625 μl (10 μmol/L) of eachprimer, 0.5 μl diluted cDNA, and 6.25 μl SYBR® PremixEx Taq™ (TaKaRa, Japan) on a LightCycler 1.5 (Roche,Germany). Initiation was by a preliminary step of 10 s at95°C, followed by 45–50 cycles of 10 s at 95°C fortemplate denaturation, 20 s at 60°C for annealing and 20 sat 72°C for extension and fluorescence measurement. No-template controls for each primer pair were included ineach run. The specificity of amplification was confirmed bymelting curve analyses and the correct size of theamplification products checked by the presence of a singleband of expected size for each primer pair in electropho-resis gels. For each gene, qPCR was carried out induplicate, using RNA from three independent extractions.

Data analysis

CT values were determined using the LightCycler® soft-ware 3.5 (Roche, Germany), using the second derivativemaximum method, and exported to Microsoft Excel. Real-time PCR data were presented according to the comparative

method (2�ΔCt ) as elaborated by Thomas (Schmittgen andLivak 2008) for relatively complicated treatments (Jin et al.2009; Zhang et al. 2006), where ΔCt is the difference inthreshold cycles for the target (Ct sample) and reference (Ct

TEF2). The gene expression value was initially normalizedto peach Actin (peach EST database, TC12104) thenchanged to TEF2 (TC3544), which appeared to be themost stable reference gene between the different cultivars,tissues and fruit development stages (Tong et al. 2009),giving a logarithmic scale ratio. The GraphPad Prismsoftware (version 5, Mac OS X 10.4) was used for allstatistical analyses and for plotting the figures.

Genomic data mining and promoter analysis

The peach (v1.0) and apple (v1.0) genome sequences weresearched for homology of putative allergen sequences usingthe GDR NCBI BLAST Server. The phylogenetic tree ofderived sequences was built using the Neighbor-Joiningmethod after translation to amino acid sequences, with theGeneious program (version 5, Mac os X 10.6). The2,000 bp upstream of the translation start (ATG) of eachindividual gene was scanned to analyze the transcriptionfactor binding sites (TFBS), tandem repeat and CpNpG

Table 1 Gene-specific primers used for real-time RT-PCR

Gene Forward primer sequence [5′-3′] Reverse primer sequence [5′-3′] Accessionnumber

Ampliconsize (bp)

Flankingregionb

Pru p 1.01 GAGCGAGTTCACCTCTGAGA TCCTTCAAGGATTTCAGAATG EU424240 121 1

Pru p 1.02 CCAACAGCAGTGAAAGATACC TCAATCAAAGTGTAACTGTATGTAA EU424242 158 1/2

Pru p 1.03 TGGAACCATCAAGAAAATTACG TCAAAGTGTAGCTGTAAGAAAAC EU424244 107 1/2

Pru p 1.04 TGGAACCATCAAGAAAATTAA TCACACTGTACTTGTACACG EU424246 107 1/2

Pru p 1.05 CTGCAGATGGAGGTTCAGTA CAATAATCTTGAACAAAGCATGG EU424248 123 2

Pru p 1.06A TCAGACAAGGTTGAGAAAATCAG CCTAGAGCATGCAGTATATTTCA EU424252 239 2/3′UTR

Pru p 1.06B AAAGCCCTTGTTCTTGAAGCG CATCTCCTTCAACCAAAGTGTAGT EU424250 211 1/2

Pru p 1.06C TCAGACAAGGTTGAGAAAATCAG AGACTCTCTTCAACATGCTACA EU424253 230 2/3′UTR

Pru p 2.01A AACAAAGTGTGCCCGGCTGA GTCTCCGGCTTGTCGTTAGGG EU424257 127 2

Pru p 2.01B AACAAAGTGTGCCCGGCTCC TCTCCGGCTTGTCGTTAGGT EU424259 127 2

Pru p 2.02 CATGTGCAACGGTAAGACTG CGCTCCCATCGGACCCTATC EU424255 227 2

Pru p 2.03 AGTGAACAAGTGTCGGTACG GTGACGCAGGAGAAGTTGGT EU424261 192 2

Pru p 2.04 GGTGAACAAGTGCGAGTACA GAGGTTGGAGCAGAGATGGT EU424263 123 2

Pru p 3.01 CTTTGGTGGTGGCCTTGT CTCACGTAGGGTATGCATGG EU424265 103 1

Pru p 3.02 CTCCTCAAGCTCGTTTGCCT CCCACCGTTTGCCACGTAGT EU424267 135 1

Pru p 3.03 CTCTTCATGTGCATGGTGGT GAGCCGACCAGTCTTTTGAT EU424269 158 1

Pru p 4.01 AGCAGTACGTCGATGACCAC ACCGGCTTCACCTTGGATCA EU424271 230 1/2

Pru p 4.02 GTAGACGACCATCTGATGTG TGTGCCTCCAAGATACAACC EU424273 192 1/2

ACTIN AATGGAACTGGAATGGTGAAGGC TGCCAGATCTTCTCCATGTCATCCCA TC12104a 227 –

TEF2 GGTGTGACGATGAAGAGTGATG TGAAGGAGAGGGAAGGTGAAAG TC3544a 129 –

a Peach EST database (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=peach) accession numberb Numbers refer to position of exons which primers belong to


http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=peach

islands of the promoter region by the PlantPAN online tool(Chang et al. 2008) and Phobos plugin (version 3.3.12) inthe Geneious program.

Results

Tissue expression of peach isoallergens

In both melting peach cultivars (cv. ‘Yulu’ and cv.‘Hujingmilu’), transcript accumulation of a total of 18isoallergens from the four gene families was quite distinctin the epicarp and mesocarp of ripening fruit and in leaf andanther (Fig. 1). Most isoallergens were expressed andlargely accumulated in epicarp and anther, but only lowlevels (one or two orders of magnitude lower) weredetected in the mesocarp and leaf. The transcript levels ofa few isoallergens exhibited tissue specificity. Allergengene expression in ripe fruit (epicarp and mesocarp) of the

two cultivars was measured in two subsequent seasons.Because similar expression patterns for both cultivars werefound, only the data from the second season are given(Fig. 1a, b). In both cultivars, most of the individualisoallergens gave similar expression patterns but at differentlevels. Our results also clearly demonstrate that the overallexpression level in the epicarp was ten to 50 times higherthan in the mesocarp.

With regard to Pru p 1, Pru p 1.01 and Pru p 1.06B weremost abundant in both cultivars and predominant in the fourtissues, independent of their expression level. The amountsof the other five members of Pru p 1, except for Pru p 1.02,were also 50 to 100 times higher in the epicarp than in themesocarp, although differences existed between the culti-vars. Pru p 1.02 was predominant in the young leaf, butthere was little accumulation in other tissues (Fig. 1d).

Transcript expression of Pru p 2 was generally lowerthan that of the other allergens. Pru p 2.01B was the mostabundant member in all tissues. A significant difference

Fig. 1 Spatial transcript expression of peach isoallergens. Differentialexpression levels of peach isoallergens analyzed from four tissues:epicarp (a) and mesocarp (b) at S4 stage and anther (c) and young leaf(d). The black bar indicates cv. ‘Yulu’ and the white bar cv.

‘Hujingmilu’. Each value represents the mean of six replicates (threeseparated cDNAs with each run in duplicate). The line bar shows thestandard error. All expression levels are normalized to peach TEF2and presented as a ratio in logarithmic scale


was observed for Pru p 2.01A in the two cultivars, with lowlevels being accumulated in the epicarp and anthers ofHuijingmilu, and almost undetectable in the mesocarp andleaf of Yulu. Pru p 2.02 was accumulated in the epicarp at asimilar level to Pru p 2.01B in leaf and anther. Accumu-lation of Pru p 2.03 was low in all tissues of both cultivars.Pru p 2.04 was similarly expressed, at a low level, in alltissues.

The patterns of the three isoallergens of Pru p 3 weredivergent. Pru p 3.01 was predominant in all tissues,accumulating mainly in epicarp, anther and leaf and to alesser extent in mesocarp. Pru p 3.02 was abundant in theleaf and Pru p 3.03 in the anther, but both were almostundetectable in fruit tissues.

With regard to Pru p 4, the two isoallergens, Pru p 4.01and Pru p 4.02, showed similar transcript abundance in alltissues except anther, where the expression level of Pru p4.02 was about ten times lower in cv. ‘Hujingmilu’(Fig. 1c).

Temporal expression pattern of isoallergens during fruitdevelopment

To analyze the expression of the various allergensduring fruit development in peach, the expressionpattern of the isoallergens was measured at fourdevelopmental stages:

& S1: first exponential growth phase& S2: phase of pit hardening and temporary halt in growth& S3: second exponential growth phase and& S4: fruit ripening phase

Overall (Fig. 2), most isoallergens had similar expressionpatterns in the two cultivars, independent of the accumu-lation levels.

For most members of Pru p 1 there was an increase inthe transcript during fruit ripening, especially in the epicarp,from the S2 phase onwards, except for Pru p 1.02, and Prup 1.03 in the mesocarp (Fig. 2a). Significantly highertranscript levels were found in the epicarp as comparedwith mesocarp. The expression level of Pru p 1.01gradually increased during fruit development, with aparallel trend in epicarp and mesocarp of cv. ‘Hujingmilu’.For Pru p 1.02, the level of transcript in epicarp andmesocarp from the two cultivars was totally reversed.Transcript levels of most isoallergens were roughly tentimes higher in cv. ‘Hujingmilu’ than in cv. ‘Yulu’.

The transcript levels of the isoallergens of Pru p 2 werelow throughout the four growth stages, compared withmembers of other allergens (Fig. 2b). A large differencewas observed for Pru p 2.01A, with a 100-fold lowerexpression in the cultivar Yulu. Disparate patterns werefound for other isoallergens between the two cultivars. The

transcript levels of Pru p 2.04 were similar in mesocarp andepicarp.

For the three isoallergens of Pru p 3, only the expressionof Pru p 3.01 was abundant and remained constant duringthe four phases in the epicarp, but this decreased threeorders of magnitude in the mesocarp during further fruitdevelopment (Fig. 2c). Expression of Pru p 3.02 in bothcultivars was only detectable at S1 in the epicarp (data notshown), and there was no accumulation of Pru p 3.03during the four phases.

Similar patterns of expression and amounts of transcriptwere found for Pru p 4.01 and Pru p 4.02 (Fig. 2d). Inaddition, the initial expression levels in the cultivar Yuluwere ten times lower than in Hujingmilu, but this differencegradually decreased during fruit development.

Differential expression abundance of key isoallergens in 11varieties

Transcript analysis of seven key isoallergen genes wassubsequently conducted on eleven diverse melting varieties(including Yulu and Hujingmilu). A similar transcriptpattern of the four allergen family was confirmed betweencv. ‘Hujingmilu’ and ‘Yulu’, and large differences for somespecific members were observed (Fig. 3).

For two Pru p 1 genes, Pru p 1.01 expression levels inepicap and mesocarp were 13 and 11 times differentbetween the highest and lowest in the 11 cultivars,respectively. The lowest level of expression in epicarpwas found in ‘Bailu’, and in mesocarp, ‘Yuhualu’. For Prup 1.06B, the difference is much larger, 67 and 126 times inepicarp and mesocarp, respectively. For each cultivar, adiverse pattern was observed in the relative abundance ofthe two genes. The ratio in transcript abundance of Pru p1.01 and Pru p 1.06B was 44 in cv. ‘Hongburuan’ but only0.1 in cv. ‘Bailu’ in the epicarp. In mesocarp, there was 103times difference in cv. ‘Hujingmilu’ but the same abun-dance in cv. ‘Okubo’. In the case of Pru p 2, a significantdiversity of transcript level was observed for both genes,Pru p 2.01A and Pru p 2.01B. The ratio between the geneswas 17–1,446 in the epicarp. In mesocarp, Pru p 2.01Awasalmost undetectable in most cultivars, and very low levelsof Pru p 2.01B was found. The diffrence between the twogenes of Pru p 2 was as high as 887 times in a few cultivarsbut equally abundant in other cultivars. With regard to Prup 3.01, the difference seems small, as only 4 times variationwas observed in epicarp between the highest cv. ‘Zhong-huashoutao’ and the lowest ‘Bailu’. A small difference wasalso observed for Pru p 4.01 (four and six times in epicarpand mesocarp, respectively) and Pru p 4.02 (five and sixtimes, respectively). No general correlation was foundbetween relative abundance of allergen genes and fruitflesh types (soft melting or hard melting).


Genomic data mining of candidate allergen genesfrom peach and apple

Location and arrangement of peach isoallergen genes wereidentified, including eight paralogs (Online Resource 1)retrieved by homology search on the whole peach genomesequence (peach genome V1.0). The four peach allergengene families were located in five scaffolds, Pru p 1, somePru p 2 and Pru p 3 members as gene clusters (Fig. 4). Thebin mapping positions were also identified using closely

linked markers on peach genome scaffolds (see OnlineResource 1). Paralogs of the Pru p 1 family included onecopy of Pru p 1.05 and Pru p 1.6A, respectively, the lattercorresponding to be a pseduogene shortened by a prematurestop codon, and three more Pru p 1.06 homologous (Pru p1.06D/E/F) were also found. All Pru p 1 genes weregrouped in a single cluster on scaffold 1 of the peachgenome or between two bins (1:14 and 1:15) of the TxElinkage map excluding the pseduogene version of Pru p1.06A, which was located on scaffold 6 or bin 6:25.

Fig. 2 Temporal transcript expression of peach isoallergens duringpeach development. Expression profiles of peach isoallergens duringfruit development are listed where detected. Expression values are

normalized to peach TEF2. Error bars indicate SE from six replicates.All data were presented in logarithmic scale


Members of Pru p 2 and Pru p 4 distributed over severalscaffolds. Two copies of Pru p 2.01A and Pru p 2.01B eachwere grouped on scaffold 3 or bin 3:37, with a copy of Prup 2.02 nearby. Pru p 2.03 was located on scaffold 8 or bin8:41. Peach scaffold 1 contained Pru p 2.04 (1:50) and Prup 4.01 (1:73) whereas scaffold 7 contained Pru p 4.02(between bin 7:56 and 7:71) and another copy of Pru p 2.02(7:25). The four members of Pru p 3 were grouped on

scaffold 6 or bin 6:25. The peach EST databases (ESTreeand GDR database) were also mined towards newfoundisoallergens, however, only one entry was obtained frompeach mesocarp (BU043492) corresponding to the Pru p1.06D sequence. Due to the clinical importance of peachPru p 3, the 2,000 bp promoter regions of the fourisoallergen genes (Pru p 3.01–04) from the ATG werederived from the genome sequences. A list of TFBS was

Fig. 3 Variability of keyisoallergens transcript patternsin epicarp (a) and mesocarp (b)of diversified melting peachcultivars at maturation stage.The 11 melting varietiesanalyzed are, listed in order ofthe horizontal axis, ‘Yulupantao’(a), ‘Hujingmilu’ (b),‘Hongburuan’ (c), ‘Baifeng’ (d),‘Yuhualu’ (e), ‘Yulu’ (f), ‘Bailu’(g), ‘Akatsuki’ (h),‘Jinxiuhuangtao’ (i), ‘Okubo’ (j)and ‘Zhonghuashoutao’ (k). Allexpression levels are normalizedto peach TEF2 and given on alogarithmic scale. Error barindicates SE from six replicates

scaffold 1

Pp1.04 Pp1.05

Pp1.06C

Pp1.03

Pp1.06B

Pp1.06A

Pp1.05

Pp1.02 Pp1.01

Pp1.06F Pp1.06D Pp1.06E Pp2.04 Pp4.01

Pp2.01B

Pp2.01A

Pp2.01B

Pp2.01Ascaffold 3

Pp2.02

Pp3.03 Pp3.04

Pp3.01 Pp3.02

scaffold 6

Pp2.03scaffold 8 scaffold 7

Pp2.02 Pp4.02

Fig. 4 Location andarrangement of candidate peachisoallergens on the assembledpeach genome scaffolds


found for all the four promoters covering hormone (ABA),cold, drought and defense-responsive elements (data notshown). A transcription factor, AINTEGUMENTA (ANT),was found only in the region of Pru p 3.03 implyingpossible regulation during floral organ development. Asignificant difference was observed in tandem repeatswithin promoters (see Online Resource 3) which wasassumed to also be involved in gene expression regulation.No CpNpG island was observed in the promoter regions ofthe four members.

The genome of domesticated apple (M. domesticaBrokh.; apple genome V1.0) was searched for homologoussequences of all known apple allergen families. Aftercorrection for complete ORF and the calculated molecularweight of the predicted protein, fifty sequences wereretained (Online Resource 2). The phylogeny of homolo-gous sequences of the four allergen families from apple andpeach genome is also given (Online Resources 4, 5, 6 and7). The Mal d 1 family comprised 26 genes of 30 identifiedsequences, in several clusters on chromosomes 1, 6, 13 and16: nine genes of Mal d 2 were scattered on chromosome 1,9, 10 and 17, and five members of Mal d 3 were locatedmainly on chromosome 4, 12 and 13. Only two genes ofMal d 4 were identified, including three copies of Mal d4.01B clustering on chromosome 8 and a single gene ofMal d 4.01A on chromosome 9.

Discussion

Peach allergy is an intricate problem involving distinctsymptoms in individuals from different geographic areas,e.g. northern and southern Europe. Many factors can beresponsible for this, of which, along with individual or localhuman sensitivity, the genetic basis of the allergens in thevarious cultivars might be of great importance. Analysis oftranscript expression of peach isoallergen genes by RT-PCRgives insight into the involvement, allergenicity and differ-ences in tissues and cultivars of specific members of thegene families.

Predominant isoallergens involved in peach allergy

Previous studies have shown that fruit peel is moreallergenic than the pulp, with peach allergenicity mainlyascribed to Pru p 3 (nsLTP) and Pru p 1 (Bet v 1homologues) (Gaier et al. 2008). In addition, Pru p 4(profilin) has been identified as a food allergen of clinicalrelevance (Rodriguez-Perez et al. 2003) as well as thenewly reported Pru p 2 (Palacin et al. 2010). Theseallergens occur in peach as gene families, of which severalallergen isoforms may be expressed in different tissues,resulting in complex allergen mixtures. Investigating the

expression profiles of these predominant isoallergens canbe helpful to identify the pivotal allergen involved. Ourresults unambiguously showed a large variation in tran-script levels between different allergens and their isoaller-gens in the different tissues.

In recent years, two-dimensional gel electrophoresis (2-DE) as well as MS-based proteomic tools have been usedfor allergen characterization. An increasing number ofallergen isoforms of Rosaceae species have been identifiedat the protein level (Cavatorta et al. 2009; Chan et al. 2007;Nilo et al. 2010), with their abundance at transcript levelintimately connected. For PR-10 (Bet v 1 homologues),protein analyses confirm the existence of two cherryisoallergens designated as Pru av 1.01 and Pru av 1.02(Reuter et al. 2005), whose amino acid sequences matchthose of peach Pru p 1.01 and Pru p 1.06, respectively.Furthermore, three variants of Pru av 1.02 were identified,which, on the basis of their mRNA expression (Fig. 1),implies that several Pru p 1.06 variants are present in peachfruit. This is similar to the case in apple, where Mal d 1.02(Mal d 1b) is the most predominant isoallergen member atboth transcript and protein level (Beuning et al. 2004;Botton et al. 2008; Puehringer et al. 2003). Subsequently,Mal d 1.06A (Mal d 1e) (Beuning et al. 2004) and Mal d1.03A (Zheng et al. 2007) were verified at the protein level,although multiple spots were detected in the 2-DE map(Herndl et al. 2007).

The peach Pru p 1.01 and Pru p 1.06 most closely matchMal d 1.02 and Mal d 1.06, respectively. Therefore, thegenomic synteny of PR-10 genes in peach and apple alsohas impact on their expression. Evidence of Pru p 2.01B,Pru p 2.01A and Pru p 3.01 in fruit is supported by theliterature (Cavatorta et al. 2009, Nilo et al. 2010; seeTable 2). In apple fruit, the amino sequence of Mal d 2protein (Krebitz et al 2003) is matched to Mal d 2.01although several putative isoallergens have been suggested(Gao et al. 2005). In a recent 2-DE map, two spots of TLP(Mal d 2) have been found, but the corresponding memberscould not be matched (Smole et al. 2008). Apple Mal d 3has been confirmed as Mal d 3.01 (Sancho et al. 2006;Herndl et al 2007) and is highly similar to Pru p 3.01.Direct protein identification and sequence confirmation ofpeach profilin is still lacking, although two apparent spotsof almond (very closely related to peach) profilin have beendetected by 2-DE immunoblot (Tawde et al. 2006),suggesting the expression of multiple isoforms. From this,it can be predicted that a limited number of isoallergenshave the major contribution to specific allergenicity.

Allergen levels in some peach and nectarine cultivars(Ahrazem et al. 2007), and in a few apple cultivars (Bolhaaret al. 2005; Sancho et al. 2008), have been assessed byimmuno-detection methods, but details of the relativeabundance of the various allergens and their isoallergens


or variants is lacking. Only mean levels have been reported forthe distribution of peach allergens in peel and pulp for Pru p 3(132.86 and 0.61 μg/g, respectively) and Pru p 1 (0.62 and0.26 μg/g, respectively) (Ahrazem et al. 2007), and no definiteresults have previously been reported for quantification of Prup 2 and Pru p 4. Our results demonstrate that, for Pru p 1 andPru p 3, the transcript levels in mesocarp were significantlylower than in epicarp. Moreover, Pru p 1.01, Pru p 1.06B andPru p 3.01 were predominant in epicarp (Fig. 1a) whereas inmesocarp, although at relatively low levels, Pru p 1.01, Pru p1.06B, Pru p 2.01B, Pru p 3.01, Pru p 4.01 and Pru p 4.02were predominant (Fig. 1b). Equal expression of Pru p 4members was detected in all tissues, whereas expression ofPru p 4.01 has been shown to be 105 times higher than that ofPru p 4.02 in fruit tissues of nectarine (Botton et al. 2009).Nevertheless, these allergens may be involved in allergenicitycaused by peeled peach fruit.

There is also evidence for the presence of Pru p 1 and Pru p3 in pollen (Marzban et al. 2006) and leaf (GarcÌa et al.2004). A correlation of expressed isoforms between tran-script and protein level has been shown in apple (Mal d 1)(Beuning et al. 2004; Marzban et al. 2006) and Birch (Bet v1) (Schenk et al. 2009) pollen. The transcript expression

profile of the four allergens in the anther (Fig. 1c) wassimilar to that in the epicarp. In contrast to fruit tissues, Pru p1.02 was the predominant Pru p 1 in peach leaf. Interestinglywe also found similar levels of expression for Pru p 3.03 andPru p3.01, indicating that both might be involved in peachpollen allergy. A similar amount of LTP (Mal d 3) in peachleaf and fruit skin has been previously reported (GarcÌa et al.2004), and our results show that two isoallergens Pru p 3.01and Pru p 3.02 might be present.

Differential expression of peach isoallergens

Peach allergens are mainly restricted to four proteinfamilies. It has been suggested that their biological functionis structurally related (Table 3), based on experimentalevidence and sequence similarity, with the number ofprotein isoforms possibly accounting for the observeddiversity in function. The differential temporal and spatialexpression of peach isoallergens reported here reinforcesthis hypothesis, pointing to a specific function of eachisoform. Two isoforms of PR-10, Pru p 1.01 and Pru p1.06D, have been found to have different ribonucleaseactivity, ligand binding and enzymatic activity (Zubini et al.

Table 2 Protein evidence of allergen isoforms in peach fruit based on literature

Peach isoform Matched spota Peptide sequenceb Reference

Pru p 1.01 C19 TYESEFTSELPPPR Chan et al. (2007)

N021 VFTYESEFTSEIPPPR AFVLDADNLVPK HSEILEGDGGPGTIKITFGEGSQYGYVK LVASPSGGSIIK GNVEIKEEHVK

Nilo et al. (2010)

N029 AFVLDADNLVPK HSEILEGDGGPGTIKKITFGEGSQYGYVKHIDSIDKENHSYSYTLIEGDALGDNLEK LVASPSGGSIIK

Nilo et al. (2010)

Pru p 1.06c C1 SDESTSVIPPPRIF Chan et al. (2007)

Pru p 2.01A AKITFTNK SVDAPSPWSGRF Palacin et al. (2010)

Pru p 2.01B N310 SVDAPSPWSGR SACLAFNQPK Nilo et al. (2010)

Pru p 3.01 – ITCGQVSSSLAPCIPY CGVSIPYK Cavatorta et al. (2009)

a Spot matched in corresponding 2-DE mapb Sequence of peptides identified by MS-based methodcMatching amino acid consensus sequences of Pru p 1.06 A, 1.06 B and 1.06 C, not assignable to the specific variants

Table 3 Classification and properties of peach allergens

Peach allergen (protein family) Putative isoallergennumber

Function Stability Allergenicitya Syndrome

Pru p 1 (Bet v 1 homologue) 8 PR-10 Heat labile, unstableon pepsin digestion

+ OAS, cross-reactive withapple, birch pollen

Pru p 2 (TLP) 5 PR-5 Stable + Urticaria/angioedema oranaphylactic symptoms

Pru p 3 (nsLTP) 3 PR-14 Heat-stable, resistant togastric digestion

++ Severe, systemic reactions

Pru p 4 (Profilin) 2 Actin-binding Heat labile, vulnerable togastric digestion

+ OAS, cross-reactive withgrass pollen

a Degree of importance and clinical relevance of the peach allergy: mild (+) and severe (++)


2009). Antifungal activity has been demonstrated by Mal d2 (TLP; PR-5) in vitro (Krebitz et al. 2003), but not by twonatural TLPs from apple and cherry (Menu-Bouaouiche etal. 2003), with moderate endo-b1,3-glucanase activity,possibly due to the occurrence of different isoforms ofTLP. The three isoforms of LTP (PR-14) in peach may alsohave different biological roles, as they have low sequencehomology (Chen et al. 2008) and differential spatialexpression. Moreover, for most isoallergens of Pru p 1,Pru p 2 and Pru p 3 we found a strong correlation betweenthe amount of transcripts and the ripening process,consistent with previously reported data for apple (Goulaoand Oliveira 2007), peach (Botton et al. 2009) and pear(Fonseca et al. 2004) trees, suggesting a role in plantdefense and ripening. This also gives us the possibility ofinvestigating the expression abundance of peach isoaller-gens in different cultivars, through transcriptional analysis.

Genomic information for characterization of peachisoallergens

The recent public release of the peach (published by theInternational Peach Genome Initiative) and apple (Velascoet al 2010) genome sequence has facilitated genomicidentification of allergy-related genes and their geneticorganization. All the 18 isoallergens previously reportedwere identified in peach as several clusters. Furthermore,five paralogous sequences were obtained, including threecopy genes, one pseudogene shortened by a premature stopcodon, three homologs of Pru p 1.06 and one homolog ofPru p 3.01. However, only Pru p 1.06D was identified, inthe EST database for peach (ESTree DB) (Lazzari et al.2008), as matching BU043492 from mesocarp, which hasnot been studied before. A preliminary analysis of promoterregions of Pru p 3 genes confirmed the involvement ofdisease response regulation, common in the LTP superfam-ily. The ANT TFBS found in Pru p 3.03 may account forits expression in anther tissue. Due to the important role oftandem repeats in gene expression regulation, the diver-gence observed should be further verified by experimentalanalysis. Genomic sequence data also provided interestinginformation such as genome position, direction and closelylinked markers, and can be further compared with pub-lished genetic maps. The Prunus bin map (TxE map)anchored with peach isoallergens (Chen et al. 2008) wasmostly confirmed using closely linked markers, thusdemonstrating bin mapping to be effective and useful.Moreover, ass the Pru p 1 culster falls into a gap betweenbin 1:14 and 1:15, new bins need to be discovered toincrease accuracy, with the published genome sequenceinformation. With the apple genome sequence, we werealso able to identify homologous sequences of the fourRosaceae allergen families (Online Resource 2). The

synteny conservation of allergen genes between the peachand apple linkage maps (Chen et al. 2008) was partlyidentified between isoallergen clusters, with the direction ofapple linkage groups in reverse order.

Genotypic variation and strategies for hypoallergenic peachvarieties

Recently, strategies for producing hypoallergenic plants as amethod of allergy prevention have been described (Gilissenet al. 2004). These strategies may involve selection,breeding and genetic modification, and there have beenremarkable achievements in apple. The selected low-allergenic apple cultivar from The Netherlands, ‘Santana’,can now be marketed as suitable for individuals with mildapple allergy (Bolhaar et al. 2005). Using a differentapproach, the apple Mal d 1 gene has been successfullysilenced through RNA interference (Gilissen et al. 2005),suggesting this type of genetic modification may be a goodprospect and one likely to be accepted by the general public(Schenk et al. 2008). Genetic and genomic analysis of appleallergen genes for breeding purposes has also been carriedout (Gao et al. 2008).

The gene expression of 11 diverse melting peachcultivars showed complex differences between individualisoallergen genes and also their relative abundance orcomposition. This knowledge is important for variabilityin phenotype, as allergenicity is the result of both contentand IgE-reactivity of individual allergen isoforms. Aprevious study also indicated that different compositionand IgE-reactivity of Betula allergen isoforms (Bet v 1)affected their allergenic potency (Schenk et al 2009). Thegenetic basis provides useful breeding information forselection of hypoallergenic peach germplasm and cultivarranking. For Pru p 1 and Pru p 2, it is essential toinvestigate the relative abundance of the whole panel of keyisoallergens, while combining data from IgE-reactivity ofeach isoform, best examined in its recombinant form.Genotypic variation of Pru p 3 and Pru p 4 seems smallin melting varieties, in which case further screening ofPru p 3.01, 4.01 and 4.02 is sufficient. In addition, nosignificant correlation was found between transcriptabundance and fruit firmness of the test cultivars. Thus,a wider range of cultivars, including different types ofpeach, should be screened for a hypoallergenic cultivarsuch as the reported ‘Rita Star’ (nectarine) with unde-tectable LTP (Botton et al. 2006). It is also possible todesign hypoallergenic cultivars by replacing highlyexpressed, high-allergenic with low-allergenic allelesthrough cross-breeding.

Further studies will focus on: (a) identification of trueallergenic variants and isoforms expressed in peach fruit atprotein level; (b) characterization of the allergenicity of


individual isoallergens in recombinant forms by immuno-logical methods; (c) genotyping and investigation of theallele diversity of expressed isoallergens in a wide range ofvarieties; and (d) association analysis using the allergenicityof individual cultivars, based on immunological (SPT andfood challenge) as well as pedigree relationships andlinkage disequilibrium analysis, to detect allele-specificmarkers for low allergenicity.

Conclusions

This research provides information on the temporal andspatial transcript expression of four putative peach allergen-encoding gene families and genotypic variation of keyisoallergens in 11 melting cultivars. A thorough analysiswas conducted on the transcript expression of all peachisoallergens known to date. Our results on their differentialexpression indicate the key allergen isoforms that may beinvolved in peach allergy. The genomic characterization ofthe whole panel of isoallergens and genetic diversity of keyisoallergens in peach germplasm is an essential step toelaborate phenotype (allergenicity) variation. This informa-tion facilitates genome-based breeding of hypoallergeniccultivars for related Rosaceae species and for investigatingexpression and regulation mechanisms and functions offruit isoallergens.

Acknowledgements This study was carried out with financialsupport from the Natural Science Foundation of China (30971970)and the Special Research Fund for Public Welfare in ChineseAgriculture (contract no. 200903044). We specially acknowledge theInternational Peach Genome Initiative (IPGI) for providing the wholegenome sequences of peach (peach genome v1.0). The plant materialswere provided by Fenghua Honey Peach Institute. ZJU students TangQin, Dai Gaofeng and Hu Zhangjian did some lab work as part of anSRTP training program.

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