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Old Apple (Malus domestica L. Borkh) Varieties with Hypoallergenic Properties: An Integrated Approach for Studying Apple Allergenicity Mara Vegro,† Giulia Eccher,† Francesca Populin,† Chiara Sorgato,† Federica Savazzini,§ Giulia Pagliarani,§ Stefano Tartarini,§ Gabriella Pasini,† Andrea Curioni,† Andrea Antico,⊥ and Alessandro Botton*,† †

Department of Agronomy, Food, Natural Resources, Animals and Environment − DAFNAE − Agripolis, University of Padova, Viale dell’università 16, 35020 Legnaro (Padova), Italy § Department of Agricultural Science, University of Bologna, Viale Fanin 46, 40127 Bologna, Italy ⊥ Allergy Unit, Ospedale Civile Srl, Via Guido Tonello 5, 46049 Volta Mantovana (Mantova), Italy S Supporting Information *

ABSTRACT: Freshly consumed apples (Malus domestica L. Borkh) can cause allergic reactions because of the presence of four classes of allergens. Knowledge of the genetic factors affecting the allergenic potential of apples would provide important information for the selection of hypoallergenic genotypes, which can be combined with the adoption of new agronomical practices to produce fruits with a reduced amount of allergens. In the present research, a multiple analytical approach was adopted to characterize the allergenic potential of 24 apple varieties released at different ages (pre- and post-green revolution). A specific workflow was set up including protein quantification by means of polyclonal antibodies, immunological analyses with sera of allergic subjects, enzymatic assays, clinical assessments on allergic patients, and gene expression assays on fruit samples. Taken as a whole, the results indicate that most of the less allergenic genotypes were found among those deriving from selection processes carried out prior to the so-called “green revolution”. KEYWORDS: Malus domestica, polyclonal antibodies, prick-by-prick tests, gene expression studies

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INTRODUCTION Studying the allergenicity of fruits has represented, in recent decades, a relevant challenge for scientists of diverse fields, such as allergologists, immunologists, biochemists, molecular biologists, agronomists, and food technologists. Differently from processed foods, the likely homogeneous features of which are typically achieved through a standardization of their transformation processes,1 fresh commodities, such as fruits and vegetables, are characterized by a wider variability, the latter often hindering a reliable definition of their allergenicity. Numerous studies have been specifically addressed to one particular species or another, often resulting into interesting achievements, although with no or few multidisciplinary approaches (i.e., only protein quantification or gene expression analyses or immunological assessment).2 Several studies were focused on apple allergenicity, as this widely consumed fruit not only is one of the most relevant for its nutritional value and nutraceutical properties3−5 but also because apples are among the most allergenic fruits.6 About 20 years ago, the major apple allergens were described and the corresponding genes identified and studied.7−14 Later, the genes encoding the four major allergens (i.e., Mal d 1, Mal d 2, Mal d 3, and Mal d 4) were also mapped onto the apple genome,15−18 and the effects of different factors on apple allergenicity were studied.10−12,14,19−21 Some investigations were also carried out on the interaction between the biochemical background of the fruit and its allergenic properties, pointing out an inverse correlation between polyphenol oxidase (PPO) activity and Mal d 1-related allergenicity.22,23 © XXXX American Chemical Society

A detailed characterization of the gene families coding for allergen isoforms is now more feasible thanks to the availability of the apple genome sequence.24 This information was recently used to identify all of the genes belonging to the Mal d 1 family and specifically quantify their expression in the fruit.25 In the meantime, several diagnostic tools were developed and/or improved, thus greatly refining the diagnosis of apple allergy, improving also the research on the plant’s side. Moreover, a hypoallergenic genetically modified apple was also obtained,26 and its allergenic properties were recently assessed,27 giving new food for thought to scientists dealing with this topic. Among the factors that most significantly affect the allergenic potential of apple, the genotype, here intended as the cultivar (i.e., the variability among different cultivars), is undoubtedly the most intriguing, as shown by previous studies dealing with it.12,21,28−30 Indeed, the availability of a hypoallergenic cultivar may have not only a short-term impact for allergic consumers but also long-term effects based on its adoption as starting material (i.e., as a source of hypoallergenic allelic variants) for the genetic improvement of apple, accelerated either by means of marker-assisted selection (MAS) or even by cisgenesis. Within this context, it is noteworthy that the “fruit allergenicity” (or “fruit allergenic potential”) trait was not only completely overlooked by breeders31 but most likely positively selected, taking into account that some basic defense-related traits (to Received: Revised: Accepted: Published: A

September 11, 2016 November 10, 2016 November 14, 2016 November 14, 2016 DOI: 10.1021/acs.jafc.6b03976 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry Table 1. Cultivars Assessed for Their Allergenicity Featuresa cultivar

acronym

susceptibilitiesb

approximate age of origin/release

Ambrosia Harmonie Delorina Forlady Gloster 69 Mairac La Flamboyante Modi CIV G198 Greenstar Nicogreen Pink Lady Cripps Pink

AM DE FL GL MA MO NI PL

“post-green revolution”

Annurcac Calvilla Bianca d’Inverno Golden Delicious Granny Smith Jonathan Magrè Mutsu Osnabruecker Renette Rosa Doppia Rosmarina rossa Sameling Tiroler Spitzlederer

AN CB GD GS JO MG MU OR RD RR SA SP

“pre-green revolution”

Braeburn Hillwell (bud mutation of Braeburn) Fuji Kiku8 (bud mutation of Fuji) Gala Schnitzer Schniga (bud mutation of Gala) Morgenduft (bud mutation of Rome Beauty)

BH FK GA MR

mutations

1980s 1990s 1980s 1960s 21st century 21st century 1980−1990s 1970s

S M S S S

16th century 16th century 19th century 19th century 19th century 19−20th century 1930s 19−20th century 18th century 19th century 19−20th century 19−20th century

S S R, S PD, S, M S

1950s (original clone); 1980s (mutation) 1930s (original clone); 1990s (mutation) 1930s (original clone); 1970s (mutation) 19th century (original clone and mutation)

BP, BW, FL, S S S S

resistancesb S

S S S

S

a

An acronym is indicated along with an approximate age in which the genotypes were constituted and/or released into the market for cultivation, subdivided in pre- and post-green revolution, with a separate category including the cultivars obtained from mutations. bBP, bitter pit; BW, browning; FL, firmness loss during storage; M, mildew; PD, preharvest fruit drop; R, rust; S, scab. cProbably cited with the name “Mala Orcula” by Pliny the Elder in manuscripts of the 1st century. the Streif index suitable for commercial harvest, as previously described.12 Cubes of cortex (pulp) and squares of epidermis (peel) of equal dimensions were separately excised from the same area of each fruit (i.e., the equatorial belt), immediately frozen in liquid nitrogen, and stored at −80 °C. Three biological replicates were obtained in total, each including a pool of samples collected from 12 fruits. Polyclonal Antibodies Construction. To obtain high-coverage antibodies specific for each of the four major apple allergen classes (Mal d 1−4), a survey was performed on the available apple genome sequence24 to identify new allergen-encoding sequences. This search was performed using the available sequences of the four apple allergens to generate the patterns to be used in a Hidden Markov Model (HMM)-based search performed with HMMER v3.0 software (http:// hmmer.janelia.org/), as described by Eccher et al.35 Sequences were filtered according to their scores and manually checked. The final selection was then aligned with the ClustalW algorithm implemented within CLC Sequence Viewer 7 (CLC Bio, Aarhus, Denmark) using default parameters, and the consensus sequence was used to identify the most conserved portions on which the immunogenic peptides could have been designed, taking also into account the information available regarding the known IgE epitopes (see the Supporting Information for further details and literature, Figure S1 and Table S1). Candidate synthetic peptides were located on the deduced amino acid sequences of Mal d 1−4 isoallergens and selected according to their putative hydrophobic profiles (excluding the most hydrophobic ones or, when possible, by replacing the hydrophobic residues) and antigenic properties. The chosen peptides were synthesized and used for antiserum production in rabbits and polyclonal antibodies purification (Primm S.r.l., Milano, Italy) (see the Supporting Information, Table S2). Protein Extraction. Apple samples were manually ground with a mortar and pestle in liquid nitrogen. Five grams of powdered tissue

both biotic and abiotic stresses) may be ascribed, at least in part, to pathogenesis-related (PR) proteins, among which three of four of the apple allergens are included.32 Indeed, a likely relationship seems to exist between the so-called “green revolution”, during which plant’s genetic improvement was greatly accelerated, and the increasing prevalence of food allergies.33,34 Although many studies were already performed on the relationships between genotype and fruit allergenicity, the present research was specifically focused on the evaluation of the allergenic potential of 24 apple cultivars released in different eras, including (i) the pre-green revolution, (ii) the early postgreen revolution, and (iii) the current century. Apple allergenicity was assessed throughout a multidisciplinary approach, thus providing ready-to-use results and relevant guidelines to inspire the future improvement of apple varieties.

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MATERIALS AND METHODS

Plant Material. Apples belonging to a total of 24 cultivars (Table 1) were chosen according to the following parameters, sorted by priority: (i) their age of origin (or release to the market), (ii) diffusion at national/international level, (iii) diffusion at local level, and (iv) sample availability. Fruits were harvested from trees grown at the experimental orchard of Maso Part (Val d’Adige, Trento, Italy, 210 m asl) of the Edmund Mach Foundation (San Michele all’Adige, Trento, Italy) for two subsequent growing seasons (2011 and 2012). All of the trees underwent exactly the same agronomical practices, following the standard integrated pest management (IPM) allowed in European apple orchards. Thirty-six apples were picked from six homogeneous trees of each variety at comparable ripening stages assessed through B

DOI: 10.1021/acs.jafc.6b03976 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry was extracted by stirring for 1 h at room temperature using a buffer solution containing 25 mM Tris, 192 mM glycine (pH 9.0), 6 M urea, 10% (w/v) glycerol, 2.5% SDS (w/v), and 2.5% β-mercaptoethanol (v/v) in a ratio 1:2 (w/v). The homogenates were then heated at 100 °C for 10 min and centrifuged at 10000g (4 °C) for 30 min. Supernatants were used for the SDS-PAGE and immunoblotting analyses. SDS-PAGE and Immunoblotting. Protein extracts were analyzed by SDS-PAGE36 in a Mini Protean II cell apparatus (Bio-Rad, Milan, Italy). Gels were prepared with a total polyacrylamide concentration of 15%. Electrophoresis was carried out at 50 mA until the tracking dye bromophenol blue ran off the gel. Standard broad range proteins (BioRad) were myosin (205.7 kDa), β-galactosidase (113.9 kDa), bovine albumin (79.7 kDa), ovalbumin (47 kDa), carbonic anhydrase (34.1 kDa), soy trypsin inhibitor (27 kDa), lysozyme (17.5 kDa), and aprotinin (6.0 kDa). Gels were stained with Coomassie Brillant Blue37 or used for immunoblotting. To this aim, SDS-PAGE separated proteins were transferred onto a PVDF membrane 0.2 μm (GE Healthcare, Little Chalfont, UK) using a Trans-Blot Turbo semidry cell (Bio-Rad) at 25 V for 30 min. Then, the membrane was treated with Tris buffer saline (TBS) containing 0.05% Tween 20 (TBS-T) and 5% (w/v) skim milk powder (M-TBS-T) for 3 h. Loadings were checked by incubating the membranes with an antiactin monoclonal antibody (A0480; Sigma-Aldrich, St. Louis, MO, USA) diluted 1:6000 (v/v) in M-TBS-T for 20 min. After three washes (15 min each) with M-TBS-T, IgG binding was detected by incubation with polyclonal anti-mouse IgG peroxidase conjugate (A9044; SigmaAldrich) diluted 1:10000 (v/v) in M-TBS-T for 90 min. All steps were carried out at room temperature with gentle agitation. Finally, the membrane was developed with Western Lightning Ultra Substrate (PerkinElmer, Waltham, MA, USA), and the chemioluminescent reaction was detected by ChemiDoc XRS+ (Bio-Rad). Densitometric analyses were performed with ImageJ (https://imagej.nih.gov/ij/), and loading volumes of all samples were corrected accordingly to achieve similar amounts of total protein loaded into the gel. Membranes obtained after SDS-PAGE with the same protein quantity, as obtained by the previous volume correction, were used for detection of the protein bands corresponding to Mal d 1, Mal d 2, and Mal d 3 antigen detection. Mal d 4 was not assessed, because its concentration in the protein extracts was under the detection limit in the preliminary analyses performed with the four antibodies. Incubation with the primary antibody (anti-Mal d 1, -Mal d 2, -Mal d 3) at a ratio 1:2000 (v/v) was followed by 2 h of immunoreaction with anti-rabbit IgG peroxidase conjugate diluted 1:2000 (v/v) in MTBS-T. For detection of serum IgE binding to apple proteins, membranes were incubated overnight with the sera of selected allergic patients and, after five washings with M-TBS-T, blots were incubated for 1 h with an anti-human IgE peroxidase conjugate antibody (Sigma-Aldrich) diluted 1:10000 in M-TBS-T. The membranes were developed and the signals acquired and measured as described above. Patient Profiling, Serum Collection, and Skin Prick Tests (SPTs). Patients were selected from a number of consecutive adult subjects (76 people >18 years old, living in the Lombardia region of northern Italy) with clinical history of apple allergy with or without adverse reactions to other fruits or vegetables. Sensitization profiles were obtained by skin prick testing all patients with a large panel of commercial extracts of pollens and vegetable foods (Lofarma, Milan, Italy), including, but not limited to, birch, grass, mugwort, olive, latex, and Rosaceae fruits. All patients were also tested with commercial extracts of LTP and profilin (ALK-Abellò, Madrid, Spain). SPTs were carried out using the standard method and device (prick lancet with 1 mm tip, Stallergen Italia, Milan, Italy). Reading and scoring were performed according to EAACI guidelines,38 and the size of skin reactions was recorded as the mean of the longest and midpoint orthogonal diameters. To search for primary sensitization from different allergen sources, available recombinant allergens of birch (Bet v 1, Bet v 2, Bet v 4), peach (Pru p 1, Pru p 3, Pru p 4), and apple (Mal d 1, Mal d 3) were

used to quantify specific IgE on all patients by means of the ImmunoCAP system (Phadia, Uppsala, Sweden). A cutoff of 0.35 kUA/l was chosen for positivity in IgE dosage, according to both the manufacturer’s instructions and the clinical practice. On the basis of patients’ history and skin test results, when appropriate, recombinant allergens of grass (Phl p 1, Phl p 2, Phl p 5, Phl p 12), mugworth, olive, latex, and other suspected plant foods (e.g., nuts) were also tested. After this first selection, a total of 27 patients were retained for serum collection and further investigations. Sera were tested by immunoblot (as described above) on apple peel and pulp extracts to detect IgE binding to the different bands. Selected apple cultivars were used for prick-by-prick tests on a selection of patients who were considered representative of the two main apple allergies: Mal d 1-mediated, for which the main clinical manifestation is the oral allergy syndrome; and Mal d 3-mediated, characterized by more or less serious systemic symptoms. A 1 cm3 cube of fruit of the selected varieties, including the peel on one side, was used for these tests. Written informed consent was obtained from all patients. Because the study was based on routine procedures in compliance with Lombardia Regional Authorities regulations (Regional Decree No. 11960/13.07.2004) and classified as “observational study”, no further notification to the Local Ethical Committee was required. Polyphenol Oxidase Activity Measurements. PPO activity was assayed on apple pulp as previously described.22 RNA Extraction, cDNA Synthesis, and Quantitative RealTime PCR. Total RNA was extracted as previously described,12 starting from 4 g of cortex (i.e., the pulp) and 1 g of epidermis (i.e., the peel). Twenty micrograms of total RNA was treated with 10 units of RQ1 RNase-free DNase (Promega) and 1 unit of RNase inhibitor (GE Healthcare) for 30 min at 37 °C, then purified by phenol−chloroform extraction, and precipitated in isopropyl alcohol according to standard methods. Complementary DNA was synthesized with the SuperScript VILO cDNA Synthesis Kit (Life Technologies) from 1 μg of DNA-free total RNA in a final volume of 40 μL, according to the instructions provided by the manufacturer. The reaction was performed in a Gene Amp PCR System 9700 thermocycler (Applied Biosystems). Quantitative real-time PCR was carried out as described by Pagliarani et al.25 A preliminary screening of the Mal d 1 genes with highest expression levels in the fruit tissues was performed, allowing to focus the following assessments on 10 genes (Mal d 1.01, 1.02, 1.03A, 1.03D, 1.03E, 1.06A, 1.06B, 1.07, 1.11A, 1.13A). Statistical Analyses. Descriptive statistics, principal component analysis (PCA), hierarchical clustering, and all general statistical analyses (i.e., Pearson’s correlation coefficients and significance) were performed with R software version 3.2.2 (www.r-project.org/) using its standard packages. All multiple comparison statistics were calculated using the same software. In detail, normality was verified with the Shapiro−Wilk test, homoscedasticity was verified with Bartlett’s and/ or nonparametric Levene’s test, and differences among samples were verified with either ANOVA (normality and homogeneous variances) or Welch’s one-way ANOVA (normality and nonhomogeneous variances) followed by post hoc LSD or Waller−Duncan test, respectively, and with Kruskal−Wallis (non-normality and homogeneous variances) or Friedman test (non-normality and nonhomogeneous variances). For all statistics a P value threshold of 0.05 was adopted.

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RESULTS Characterization of Allergic Patients and Serum Selection. This step of the study was not aimed to provide a complete and exhaustive profiling of apple-allergic patients but rather to select sera showing specific profiles (i.e., possibly with monosensitization) suitable to be used as a “quantification tool” for the different apple allergens. The use of patient’s sera for this aim, even through ELISA, was shown to be reliable and C


Article

Journal of Agricultural and Food Chemistry Table 2. Characteristics of Allergic Patientsa IgE (kUA/L) apple patient ID

a b

F49b

immunoblot positivity birch

Mal d 1 Mal d 3 Bet v 1 Bet v 2 Bet v 4

Mal d 1 (∼18 kDa)

Mal d 2 (∼23 kDa)

Mal d 3 (∼9 kDa)

peel

pulp

peel

pulp

peel

pulp

other bands

xx

xx

x

xx

xxx

xx

∼66 kDa in both peel and pulp

1G

12.00

21.60

8.42

91.7

19.7

na

2B

6.63

25.90

na

37

na

na

3T 4A

na 1.81

na 1.41

na na

na 1.44

na 5.14

na 0.18

5M

2.46

NA

NA

NA

NA

NA

6P 7M 8R

4.95 3.88 na

na 2.21 na

5.73 3.71 na

na 33.4 na

na na na

na na na

9A

1.39

2.27

0.50

16.5

na

na

10C

11.60

22.50

na

100

na

na

11M

0.31

0.91

na

3.11

na

na

12C

1.29

3.72

na

23.7

na

na

13S

1.01

3.43

na

7.74

na

na

14B

2.86

3.41

0.12

15.7

NA

NA

15A

1.23

5.73

na

23

na

na

16B 17B 18S

2.81 0.20 2.86

11.60 0.55 7.87

na na na

24.3 17.7 29.3

na na na

na na na

x

xx

19M

12.00

21.60

8.42

45.8

na

na

xx

x

21P

NA

0.27

NA

1.18

na

na

22C

17.90

32.30

0.63

46

na

na

xxx

xx

23T

3.43

6.71

na

9.69

na

na

xx

x

24B

1.70

0.26

na

20.3

na

na

25G 26T 27E 28A

2.84 0.1 0.56 na

na 0.10 1.10 na

3.88 0.33 na na

na na 29.4 na

na na na na

na na na na

∼31to ∼66 kDa in both peel and pulp ∼66 kDa in peel xx x

∼31 to ∼66 kDa in both peel and pulp

∼40 kDa in both peel and pulp xxx

x

main symptoms upon apple consumption rhinitis with secondary asthma rhinitis with secondary asthma rhinitis rhinitis, OAS rhinitis with secondary asthma urticaria rhinitis rhinitis with secondary asthma rhinitis with secondary asthma rhinitis with secondary asthma rhinitis with secondary asthma rhinitis with secondary asthma rhinitis, OAS rhinitis with secondary asthma rhinitis rhinitis rhinitis asthma with secondary rhinitis rhinitis with secondary asthma asthma with secondary rhinitis asthma with secondary rhinitis rhinitis with secondary asthma rhinitis with secondary asthma OAS asthma rhinitis rhinitis

The intensity of the immunoblot bands is expressed as follows: x, weak, xx, strong; xxx, very strong. na, not detectable; OAS, oral allergy syndrome. Apple extract.

effective in several cases, especially dealing with peanut allergens.39−41 As usually seen in clinical practice, a significant number of apple-allergic patients showed cross-reactivity to other fruits, mainly peach, and vegetables, as well as skin test positivity and specific IgE for a number of different pollens and related/ unrelated foods, some of them not clinically relevant. A summary of the most relevant results of this screening is shown in Table 2. Twenty of 27 patients (74%) were positive to apple extract. Among these, 11 had specific IgE only for Mal d 1, 2 only for Mal d 3, and 5 to both allergens. All patients positive for Mal d 1 had also specific IgE for Bet v 1. When tested by

immunoblotting (see Figure S2 in the Supporting Information) on apple peel and pulp (cv. ‘Golden Delicious’ was chosen because of its wide diffusion and worldwide importance, both as a commercial variety in its own right and as breeding stock for many other varieties), the sera were able to bind Mal d 1 in only five cases (all with a high dosage of specific IgE for this allergen), four of which were monosensitized (i.e., showing only the Mal d 1 band). The same was observed for Mal d 3, with four sera showing specificity for this allergen and one with double sensitization (i.e., showing IgE binding to both Mal d 1 and Mal d 3 bands). It is worth noting that four patients’ sera were unable to bind either Mal d 1 or Mal d 3, but displayed clearly visible bands ranging from 30 to 66 kDa. On the basis of D


Article

Journal of Agricultural and Food Chemistry

Figure 1. Immunoblot quantification of Mal d 1 (a, peel; b, pulp), Mal d 2 (c, peel; d, pulp), and Mal d 3 (e, peel only) allergens in the 24 apple varieties listed in Table 1. The charts represent the intensity (as arbitrary units) of the bands measured in three sample replicates. Bars represent standard deviation. Letters indicate statistically different values (P ≤ 0.05; n = 3) resulting from different post hoc tests (see Materials and Methods). A representative image of the blots is shown below the chart.

the immunoblotting results obtained with individual sera, two pools were obtained: one from sera of patients 18S, 19M, 22C, and 23T for Mal d 1 specificity and the other from sera 6P, 7M, 16B, and 25G for Mal d 3. These two pools were thereafter used in immunoblotting experiments for allergen quantification in the different apple varieties. Quantification of the Main Allergens in the Different Varieties by Specific Antibodies. Four polyclonal antibodies were raised in rabbit against four different sets of synthetic peptides specific for the major apple allergens (as described under Materials and Methods). The obtained antibodies were tested by immunoblotting on the protein extracts of both apple peel and pulp. Three of them displayed a satisfactory

performance in terms of binding, sensitivity, and specificity (data not shown) and were thus used for Mal d 1, Mal d 2, and Mal d 3 quantification. The unsatisfactory performance of the anti-Mal d 4 antibody was likely due to either the very low amount of this allergen in the fruit and/or its low extractability with the protocol herein adopted. When tested on the protein extracts of the 24 apple varieties, the 3 antibodies displayed large differences in terms of binding intensity for the allergens present in the peels and pulps of the different varieties (Figure 1), and this result was not due to differences in the loaded protein quantity, which was the same for all of the samples. Moreover, the low variability obtained for E


Article

Journal of Agricultural and Food Chemistry Table 3. Correlations among the Different Parameters Quantified with either the Antibodies or the Patients’ Seraa Mal d 1 (antibody, peel) Mal d 1 (antibody, peel) Mal d 1 (sera, peel) Mal d 1 (antibody, pulp) Mal d 1 (sera, pulp) Mal d 2 (antibody, peel) Mal d 2 (antibody, pulp) Mal d 3 (antibody, peel) Mal d 3 (sera, peel) a

Mal d 1 (sera, peel)

Mal d 1 (antibody, pulp)

Mal d 1 (sera, pulp)

Mal d 2 (antibody, peel)

Mal d 2 (antibody, pulp)

Mal d 3 (antibody, peel)

Mal d 3 (sera, peel)

0.04287

0.00004

0.00119

0.01032

0.16133

0.01682

0.19726

0.04782

0.09172

0.04827

0.16506

0.05691

0.02475

0.00001

0.00781

0.03233

0.14465

0.15866

0.00025

0.00054

0.01932

0.14853

0.00019

0.02490

0.29339

0.00071

0.00359

0.41658 0.73949

0.40795

0.62141

0.35191

0.76675

0.51324

0.40722

0.52935

0.68031

0.29524

0.29275

0.43792

0.65302

0.68947

0.48299

0.39380

0.30690

0.47388

0.45661

0.64257

0.27274

0.45705

0.29705

0.30410

0.22368

0.57066

0.00001 0.76295

Pearson’s coefficients (ρ) are reported (below the diagonal), along with their significance (P value; above the diagonal).

Figure 2. Immunoblot quantification of Mal d 1-related (a, peel; b, pulp) and Mal d 3-related (c, peel only) immunoreactivity in the 24 apple varieties listed in Table 1, as performed with patients’ sera. The charts represent the intensity (as arbitrary units) of the bands measured in three sample replicates. Bars represent standard deviation. Letters indicate statistically different values (P ≤ 0.05; n = 3) resulting from different post hoc tests (see Materials and Methods). A representative image of the blots is shown below the chart.

different replicates (not shown) indicated a very satisfactory reliability of this quantification assay. Specifically, concerning Mal d 1, highly correlated (i.e., statistically significant Pearson’s correlation ρ = 0.73949, with P = 0.00004) quantifications were pointed out in pulp and peel (Table 3), with the highest average levels of these allergenic proteins observed in cvs. ‘Ambrosia’, ‘Gala’, ‘Forlady’, ‘Mairac’, ‘Delorina’, ‘Gloster’, and ‘Fuji’, and the lowest in ‘Tiroler Spitzlederer’, ‘Calvilla Bianca d’Inverno’, ‘Rosa Doppia’, ‘Pink Lady’, ‘Jonathan’, and ‘Morgenduft’. The remaining genotypes showed intermediate levels.

For Mal d 2, a statistically significant correlation (ρ = 0.68947; P = 0.00019) was found again between the allergen amounts in the two tissues considered. The highest amounts of this allergen class were measured in cvs. ‘Modi’,̀ ‘Ambrosia’, ‘Gloster’, ‘Mairac’, ‘Nicogreen’, ‘Forlady’, and ‘Golden Delicious’, whereas the lowest were assessed in ‘Rosa Doppia’, ‘Tiroler Spitzlederer’, ‘Morgenduft’, and ‘Calvilla Bianca d’Inverno’. The Mal d 3 allergenic fraction was immunologically detected only in the peel, as in the pulp the level of this protein is known to be extremely low.42 In this case, the highest values were measured in cvs. ‘Gloster’, ‘Modi’,̀ and ‘Forlady’ and F


Article


Figure 3. Polyphenol oxidase (PPO) activity of the pulp of the 24 apple varieties listed in Table 1. Bars represent standard deviation. Letters indicate statistically different values (P ≤ 0.05; n = 3) resulting from Friedman’s post hoc test (see Materials and Methods).

Figure 4. Principal component analysis of all the data concerning the quantification of the allergen fractions, their immunoreactivity, and the PPO activity in the 24 apple varieties listed in Table 1. Arrows indicating the loadings are labeled as follows: aM1-pulp/peel, antibody quantification of Mal d 1 in pulp or peel; aM2-pulp/peel, antibody quantification of Mal d 2 in pulp or peel; aM3-peel, antibody quantification of Mal d 3 in peel; sM1-pulp/peel, Mal d 1 immunoreactivity in pulp or peel; sM3-peel, Mal d 3 immunoreactivity in peel; PPO, PPO activity. Pre- and post-green revolution genotypes are indicated with white and black squares and grouped with dashed and dotted ellipses at 0.60 confidence level, respectively. Genotypes derived from mutations were included in one of the two categories according to the age of release of the original clone, taking the 1950s as a breaking point for the green revolution. The percentage of variance explained by each principal component is also indicated.

the lowest in ‘Rosa Doppia’, ‘Granny Smith’, ‘Mairac’, ‘Sameling’, ‘Gala’, ‘Mutsu’, ‘Jonathan’, ‘Rosmarina Rossa’, ‘Osnabruecker Renette’, and ‘Tiroler Spitzlederer’. IgE Immunoreactivity of the Different Varieties. The two pools of sera from apple-allergic patients obtained as described above were used to quantify both the Mal d 1- and Mal d 3-related IgE binding of the same samples previously assessed with the polyclonal antibodies (Figure 2). With regard to Mal d 1 in the peel, the highest immunoreactivity was found in cvs. ‘Gloster’, ‘Gala’, and ‘Forlady’ and the lowest in ‘Mutsu’, ‘Nicogreen’, ‘Morgenduft’, ‘Braeburn Hillwell’, ‘Calvilla Bianca d’Inverno’, and ‘Granny Smith’. In the pulp, the highest signal was detected in cv. ‘Ambrosia’, whereas the lowest was detected in ‘Braeburn Hillwell’, ‘Pink Lady’, ‘Jonathan’, ‘Annurca’, ‘Rosa Doppia’, ‘Calvilla Bianca d’Inverno’, ‘Tiroler Spitzlederer’, and ‘Morgenduft’. It is noteworthy that, whereas the IgE reactivity of the pulp samples was significantly and highly correlated (ρ = 0.76675; P = 0.00001) with the total amount of the protein

corresponding to Mal d 1 protein as assessed in the same tissue with the polyclonal antibody, the values detected in the peel by using the Mal d 1-specific serum pool were related neither to the antibody quantification of Mal d 1 in the same tissue (ρ = 0.41658; P = 0.04287) nor to its levels in the pulp (ρ = 0.40795; P = 0.04782). The highest Mal d 3-related immunoreactivity was found in cv. ‘Gloster’ and the lowest in ‘Rosmarina Rossa’, ‘Sameling’, ‘Osnabruecker Renette’, and ‘Tiroler Spitzlederer’. Intermediate levels were measured in the other samples. The correlation between these values and the specific protein amount as measured with the rabbit antibody was very significant (ρ = 0.76295; P = 0.00001). Polyphenol Oxidase Activity. Considering the importance of the PPO activity in determining the Mal d 1-related allergenicity of the apple pulp,22,23 this parameter was assayed in all pulp samples of the 24 varieties (Figure 3). Taken as a whole and based upon the statistical analysis, the PPO activity levels of the varieties could be divided into three G


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a

32.30 6.71 11.40 0.00 2.21 0.00 22C 23T 19M 6P 7M 25G

The results of the tests normalized according to the positive control are shown in parentheses. ImmunoCAP and immunoblot results (already shown in Table 2) are also reported. bRecombinant nonspecific lipid transfer protein.

(0) (3.3) (3.8) (7.7) (2.9) (5.1) (3.8) (2.5) 0 3 3 8 3 6 3.8 2.8 (2.9) (6.6) (3.8) (3.8) (7.7) (2.6) (4.6) (2.1)

GL

3 6 3 4 8 3 4.5 2.1 (4.8) (4.4) (0) (7.7) (0) (5.1) (3.7) (3.1)

GD

5 4 0 8 0 6 3.8 3.3 (0) (0) (0) (6.7) (5.8) (6) (3.1) (3.4)

CB

0 0 0 7 6 7 3.3 3.7 (3.8) (6.6) (3.8) (7.7) (2.9) (2.6) (4.6) (2.1) 4 6 3 8 3 3 4.5 2.1

AM nsLTP

3 (2.9) 0 (0) 0 (0) 10 (9.6) 13 (12.5) 13 (11.1) mean (mm) SD 8 7 6 8 8 9

positive control (histamine) negative control

0 0 0 0 0 0 N N N Y Y Y

Mal d 3 Mal d 1 Mal d 1 patient ID

Mal d 3

Y Y Y N N N

prick-by-prick tests

b

immunoblotb immunoCAP

Table 4. Results of the Prick-by-Prick Tests Carried out on Six Selected Patients with Six Selected Apple Varietiesa H

0.63 0.00 0.15 5.73 3.71 3.88

MO

0 3 4 6 0 5 3.0 2.5

SP

classes, that is, high, medium, and low. The highest PPO activity was found in cv. ‘Nicogreen’, although several other genotypes showed comparable levels not statistically differing from the former. Intermediate levels were measured in the samples of ‘Forlady’, ‘Granny Smith’, ‘Osnabruecker Renette’, and ‘Ambrosia’. The lowest activity was assessed in ‘Pink Lady’, with seven other genotypes not statistically differing from this cultivar. No statistically significant correlation was found between PPO activity and either of the other parameters described above (data not shown). Data Integration through Principal Component Analysis. All of the data concerning the quantification of the allergen fractions, their IgE reactivity, and the PPO activity were integrated by means of a PCA (the mean values were used; n = 3), to give an overview of how the different varieties behave with respect to all of the measured parameters. The first two principal components explained 63.4% of the total variance and are summarized in Figure 4. The first principal component (PC1) was able to explain 48.9% of the total variance observed for the parameters considered in the analysis and was able to discriminate between “old” and “new” varieties. The second component explained 14.5% of the total variance and was shown to separate the samples with high Mal d 1-related measurements (i.e., antibody quantification in both tissues and IgE binding in the pulp) from those regarding Mal d 3. With regard to the loadings, most of the variables, with the exception of only PPO activity, were negatively correlated with PC1. This would mean that, because the old varieties are mainly on the “right side” of the chart and the new ones on the opposite side, the latter are characterized by higher levels of all the allergenicity parameters measured. For the PPO activity, a negative correlation was reported only with PC2, thus being unable to divide the samples according to their age of origin. It is worth noting that the newer genotypes displayed also a higher variability, as they appear to be “spread” in the PCA chart, oppositely to the old ones, which clustered more closely with one another. A summary of the PCA results is also provided in the Supporting Information (Tables S3−S5). Prick-by-Prick with Candidate Varieties. On the basis of the results of the PCA, six varieties were chosen as representative of the different positions within the chart (Figure 4) and, thus, with a putatively different allergenicity. These varieties, that is, ‘Ambrosia’ (putatively high Mal d 1related allergenicity), ‘Gloster’ (putatively high Mal d 3-related allergenicity), ‘Modı ̀’ (putatively high allergenicity), ‘Golden Delicious’ (putative intermediate allergenicity), and ‘Calvilla Bianca d’Inverno’ and ‘Tiroler Spitzlederer’ (both with a putatively low allergenicity), were used for prick-by-prick tests carried out on six patients selected among those listed in Table 2, according to their different profiles (Table 4). A nonsymptomatic patient (with negative ImmunoCAP and no IgE reactivity in immunoblotting) was also assessed during these tests, showing skin reaction only to the positive control (i.e., histamine; data not shown). Taken as a whole, the results of the skin tests mostly reflected the findings previously pointed out by the PCA. ‘Calvilla Bianca d’Inverno’ and ‘Tiroler Spitzlederer’ displayed the lowest skin reactivity, followed by ‘Golden Delicious’ and ‘Modı ̀’, the latter scoring intermediately in the PCA. The highest reactions in the skin tests were provoked by ‘Ambrosia’ and ‘Gloster’. Worthy of note is that ‘Calvilla Bianca d’Inverno’ did not induce any wheal

(0) (3.3) (5.1) (5.8) (0) (4.3) (3.1) (2.5)



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Figure 5. Quantitative real-time PCR expression analyses performed on samples of pulp (white) and peel (gray) of six selected varieties (AM, ‘Ambrosia’; CB, ‘Calvilla Bianca d’Inverno’; GD, ‘Golden Delicious’; GL, ‘Gloster’; MO, ‘Modı ̀’; SP, ‘Tiroler Spitzlederer’). Ten Mal d 1-encoding genes were assayed (see Pagliarani et al.25 for details) and expressed as arbitrary units of mean normalized expression. Bars represent standard error. Letters indicate, when present, statistically different values (P ≤ 0.05; n = 3) resultimg from different post hoc tests (see Materials and Methods) performed separately on the two tissues for each gene.

previously tested on patients (Figure 5), to highlight possible links between allergenicity and the relative expression of the different isoforms. Among the 10 Mal d 1 genes expressed in the fruit, Mal d 1.01 and Mal d 1.02 showed the highest expression levels, and their highest amount of transcripts was always found in the peel. Oppositely, the lowest expression was detected for Mal d 1.11A, regardless of tissue.

development in the patients characterized by a Mal d 1-related allergy. Expression of Mal d 1 Genes in Candidate Varieties and Hierarchical Clustering of Key Data. Because of the findings pointed out by the skin tests, especially concerning the absence of reactivity to ‘Calvilla Bianca d’Inverno’ in Mal d 1 monosensitized patients, the expression levels of the genes coding for this allergen were measured in the same six varieties I


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Figure 6. Heatmaps (a) and hierarchical clustering (a, b) of data resulting from the prick-by-prick tests (SPT), from gene expression assays on Mal d 1 genes, from antibody quantification of Mal d 1 allergen (aM1), and from the immunoreactivity as tested with the patients’ sera (sM1). Data are shown for both peel and pulp of six selected varieties (AM, ‘Ambrosia’; CB, ‘Calvilla Bianca d’Inverno’; GD, ‘Golden Delicious’; GL, ‘Gloster’; MO, ‘Modı ̀’; SP, ‘Tiroler Spitzlederer’), except for the skin tests, which were carried out concurrently on both tissues. Clustering was performed with the Ward agglomerative method and by calculating the Euclidean distance. The clustering was tested by calculating the AU (approximately unbiased) P value and BP (bootstrap probability) value (b).

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DISCUSSION Genetic improvement of apple varieties in the past decades has been focused on traits mainly related to four aspects: (i) the aesthetics of the fruit (i.e., skin color and sparkle, fruit shape and size); (ii) fruit quality as related to taste, texture, and storability; (iii) tree architecture; and, last but not least, (iv) resistance traits to biotic and abiotic stresses.31 However, because consumers’ attention on new traits related to the health and safety properties of the fruits has been growing in recent years along with the demand for environmentally sustainable production practices, breeders are now facing new challenges and are called to answer questions dealing with the nutraceutical content of fruits and vegetables, the allergenic potential of these commodities, and the impact of the production chain on the environment.43,44 Within this context, apple represents an interesting model for several reasons. First, apple fruit was proved to provide significant health benefits;3−5 second, apple is a model fruit tree with a sequenced genome24 and numerous tools available to researchers; and, last but not least, apple allergens are known and well-characterized, and the clinical manifestation of apple allergy has been studied for a long time.6,8−14,18−21 In the present study, 24 apple genotypes released in different eras, that is, prior to, during, or after the socalled “green revolution” (Table 1), were assessed for their allergenicity throughout an integrated approach. Recovery, profiling, and selection of patients’ sera represented not only the most difficult step of this study but also the most important tool, as it added very relevant and reliable information to the

Although not always with statistical significance, 8 genes of 10 (i.e., Mal d 1.01, 1.02, 1.03A,D,E, 1.06B, 1.07, and 1.13A) were expressed at their highest level in the peel of the cv. ‘Golden Delicious’. In nine cases, most of which involve the cv. ‘Ambrosia’, a higher transcript amount was observed in the pulp than in peel. However, the statistical significance of these data (i.e., pulp vs peel) was not tested. To achieve a complete overview of these data and improve their readability, they were clustered together with the results of skin tests, antibody quantification of Mal d 1 allergen, and Mal d 1-related immunoreactivity as tested with the patients’ sera (Figure 6). Two main clusters were pointed out, both supported by significant statistical scores. The most numerous one included a subgroup with the gene expression patterns of seven genes in the pulp correlating with Mal d 1 quantification in the peel, and a second subgroup, the most interesting one, including one gene (Mal d 1.02 in the pulp) correlating with immunoreactivity in both tissues, Mal d 1 quantification in the pulp, and, worthy of note, the results of the prick-by-prick tests. The second main cluster, including all of the remaining parameters mostly regarding the peel, could be further subdivided into three subgroups, the first characterized by high values in cvs. ‘Ambrosia’ and ‘Golden Delicious’, the second by high levels only in the latter, and the third one including only Mal d 1.11A peaking in both tissues in the cv. ‘Calvilla Bianca d’Inverno’. J



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quantification of allergen fractions performed with the specific rabbit antibodies. Worthy of note is the absence of correlation between Mal d 1 quantification in the peel and its immunoreactivity (Figures 1 and 2 and Table 3). This result would indicate that the allelic composition and/or dosages in the peel of the different varieties are different, in agreement with previous results.29 The most relevant findings, however, concern the difference in terms of global allergenicity observed between the pre- and post-green revolution genotypes, with the former showing generally lower allergenicity along with a reduced variability of all the considered parameters (Figure 4). It is noteworthy that these results are substantially paralleled by those pointed out by the skin tests (Table 4), with the two old local varieties ‘Calvilla Bianca d’Inverno’ and ‘Tiroler Spitzlederer’ confirmed to be the less allergenic. In particular, it is noteworthy that the former genotype did not trigger any immunological reaction, at least at the skin level, in the Mal d 1-monosensitized patients. A low allergenic potential has also been reported for ‘Durello di Forli’̀ 45 and ‘Szara Reneta’,46 two old apple cultivars from the Italian and Polish germplasms, respectively, thus further strengthening the findings herein described. On the other hand, whereas also the results concerning the low allergenicity of ‘Pink Lady’ were consistent with previous findings, this was not the case of ‘Modı ̀’.30 The inconsistency sometimes found among the results achieved in the different studies, carried out in diverse years and locations by several research groups and with different patients, can be mainly ascribed to the genotype × environment interactions, both on the plant’s side and on the patient’s side, as previously shown.12−47 Gene expression profiling attempted to give an explanation to the different behaviors observed in the six varieties that were chosen to represent the diverse allergenicity levels. Hierarchical clustering (Figure 6) pointed out that the allergenicity of the six genotypes, measured in terms of Mal d 1 allergen content, IgE, and skin reactivity, is correlated with expression of Mal d 1.02 in the pulp, in agreement with previous findings.26 In the cv. ‘Calvilla Bianca d’Inverno’, the low expression level of this gene is strongly compensated by a very high (the highest) transcript accumulation of Mal d 1.11A (Figure 5). It may be speculated that the latter gene may functionally counteract the low expression levels of the prevalent isoform (i.e., Mal d 1.02). In conclusion, the present study pointed out that “allergenicity” as a fruit trait was not only completely overlooked by breeders31 but most likely positively selected due to its possible link with other traits, such as those conferred by the PR proteins (i.e., pathogen response). It is noteworthy that the cvs. ‘Santana’ and ‘Elise’, both shown to be hypoallergenic,30 carry also a scab resistance trait conferred by genes (i.e., the Vf genes) that do not encode PR proteins.48 However, the likely relationship existing between the so-called “green revolution”, during which the plant’s genetic improvement was greatly accelerated, and the increasing prevalence of food allergies33,34 has been substantially confirmed. Because several other desirable and important features, such as the nutraceutical properties of the apple,49 were almost completely “forgotten” by the apple breeders in the past decades, a deep revision of the currently ongoing breeding programs regarding this important fruit species must be taken into account to provide future consumers with a healthy and safe apple, possibly produced with more environmentally friendly agronomical practices.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b03976. Table S1, additional references. Table S2, sequences of synthetic peptides used to obtain polyclonal antibodies against apple allergens. Table S3, standard deviation, proportion of variance, and cumulative proportion of the variance explained by the first nine principal components. Table S4, scores of 24 varieties as related to the first two principal components. Table S5, loadings (weights) of different variables on the first two principal components (PDF) Figure S1, multiple amino acid alignments of apple sequences belonging to four allergen classes (PDF) Figure S2, immunoblottings performed with sera of single patients on extracts of apple pulp and peel (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(A.B.) Phone: +39 049 8272849. Fax: +39 049 8272850. Email: [email protected]. ORCID

Alessandro Botton: 0000-0001-5054-1479 Funding

This research was carried out with financial support from the Progetto Agroalimentare e Ricerca - AGER (Grant 2010-2119) and by Project ex60% 2014 (Grant 60A08-4842/14) and 2015 (Grant 60A08-0582/15) funded by the University of Padova. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge Paolo Lezzer and Edmund Mach Foundation (San Michele all’Adige, Trento, Italy) for providing the apples used in experiments.

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REFERENCES

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