Understanding the Effects of Genotype, Growing Year, and Breeding

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Understanding the Effects of Genotype, Growing Year and Breeding on Tunisian Durum Wheat Allergenicity (Part 1): The Baker’s asthma case Fatma Boukid, Barbara Prandi, Stefano Sforza, Rhouma Sayar, Yong Weon Seo, Mondher Mejri, and Ines Yacoubi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02040 • Publication Date (Web): 23 Jun 2017 Downloaded from http://pubs.acs.org on June 26, 2017

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Journal of Agricultural and Food Chemistry

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Understanding the Effects of Genotype, Growing Year and Breeding on Tunisian

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Durum Wheat Allergenicity (Part 1): The Baker’s asthma case

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Fatma Boukid1, 2, Barbara Prandi2*, Stefano Sforza2, Rhouma Sayar3, Yong Weon Seo4, Mondher

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Mejri1*, Ines Yacoubi1

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1

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Tunisia

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2

Food and Drug Department, University of Parma, Parco Area delle Scienze 27/A, 43124 Parma, Italy

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3

Tunisian Higher School of Agriculture of Kef Boulifa, 7119 Kef, Tunisia

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Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Anam-

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Dong, Seongbuk-Gu, Seoul 136-713, Republic of Korea.

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*Corresponding authors:

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Mondher Mejri, Center of biotechnology of Sfax, University of Sfax, B.P 1177 Sfax 3018, Tunisia; e-

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mail: [email protected]; tel: +21674871816

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Barbara Prandi, Food and Drug Department, University of Parma, Parco Area delle Scienze 27/A,

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43124 Parma, Italy; e-mail: [email protected]; tel: +390521906079

Plant protection and improvement laboratory, Center of biotechnology of Sfax, University of Sfax,

1 ACS Paragon Plus Environment


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Abstract:

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Baker’s asthma is a serious airway disease triggered by wheat proteins CM3 α-

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amylase/trypsin inhibitor. The purpose of the present study was to investigate the impact of

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genotype and crop year on allergen CM3 α-amylase/trypsin inhibitor associated to baker’s

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asthma. A historical series of Tunisian durum wheat (100 accessions), derived from three crop

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years, was used to compare the amount of CM3 from landraces to advanced cultivars. CM3

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protein quantification was assessed after an enzymatic cleavage of the soluble protein extracts

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on an UPLC/ESI-MS system, using a marker peptide for its quantification. Combined data

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analysis of variance revealed an important effect of genotype, crop year and their interaction.

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The CM3 allergenic proteins were found to significantly vary among studied genotypes, as

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confirmed by genetic variability, coefficient of variance, heritability and genetic advance.

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Keywords: CM3 α-amylase/trypsin inhibitor, Baker’s asthma, Allergenicity, Durum wheat


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Introduction

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Cereal researchers and breeders focused on achieving food security, via improving yield rate,

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overlooking food safety. In the past, the safety of food did not emerge as a problem because

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the crop varieties had a long history of safe consumption 1. Food allergies are one of the most

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serious food safety issue2, and in recent decades, there is a mounting prevalence of allergies

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and certain intolerances, which are probably associated with the changes in consumers’

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lifestyle and food habits as well as the intervention of modern breeding 3.

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Allergic sensitization to wheat flour components is one of the most common triggers of

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occupational asthma 4. Baker’s asthma is a serious work-related obstructive respiratory

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disease, which affect from 4% to 25% of bakery workers 5. However, the recent development

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and progress achieved in the field of allergy exposure and response might provide further

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clarifications to better understand baker's asthma 6. Indeed, it affects several professions

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dealing with the wheat grains cultivation, transportation, production and transformation.

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Although wheat is assumed to be the major trigger of baker’s asthma, other grains, such as

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barley and rice, might be also involved 7.

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Several allergens as well as some additives, have been involved in baker's asthma, but it

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remains great focus was attributed to the water or salt soluble fraction of wheat proteins

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(albumins and globulins) as probably the major cause, indeed, the activity of specific IgE-

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binding accounts for about 70% to 80% 8. A wide spectrum of wheat grain proteins have been

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also identified to be involved with bakers' asthma, such as serine proteinase inhibitors,

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thioredoxin, agglutinin, α- and β-amylases, peroxidase, acyl CoA oxidase, glyceraldehyde-3-

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phosphate dehydrogenase and triose phosphate isomerase

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amylase/trypsin inhibitor family is considered the major allergen of baker’s asthma

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Indeed, they are from 12 to 16 kDa polypeptides with 4 to 5 intrachain disulphide bonds

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responsible of their inhibitory activity

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soluble in solution prepared by chloroform and methanol

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activity, diagnostic tools are mainly skin prick tests, radioallergosorbent test and

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immunoblotting assays 12, 13, 17.

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Until now, few studies

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amylase/trypsin inhibitor CM3 allergenicity of wheat. In this research paper, we aimed to

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study the allergenic potential relative to the salt soluble albumins and globulins throughout the

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9,10,11,12

. More particularly, the α13, 14

.

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. They are known as CM proteins because they are 16

. Furthermore, to assess their

were performed on the immunochemical assessment of α-



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progress of breeding from landraces to high yielding genotypes of Tunisian durum wheat

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using many accessions grown in three growing seasons. For the furtherance of these aims, we

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sought firstly to identify asthma marker peptide for CM3 protein using the UPLC-MS

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technique, secondly to investigate allergenic variation during a century of breeding program

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in Tunisia and ultimately to compare CM3 proteins in different wheat accessions grown in

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three different crops years.

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Material and methods

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Description of sampling

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To study genotype and crop year influence on the content of the CM3 proteins associated to

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baker asthma, representative durum wheat sets, released during the 20th century in Tunisia,

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were used. Three sets were grown during three seasons (2011-2012, 2013-2014 and 2014-

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2015) as mentioned below in details.

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Set 1 (season 2014-2015) comprises 70 accessions of durum wheat consisting in landraces,

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old cultivars, advanced genotypes made by international breeding programs (CYMMIT and

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ICARDA) and recent lines selected by national breeding program (INRAT). The set was

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subdivided into five groups according to the year in which each cultivar was released:

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Group 1 (from 1900 to 1940) comprises 51 indigenous and exotic landraces: Hmira (4

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accessions), Jenah Khottifa (3 accessions), Azizi (3 accessions), Aouij (1 accession), Sbei (2

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accessions), Derbassi (2 accessions), Wared Lebled (2 accessions), Biadha (2 accesions),

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Swabaa Elgia (2 accessions), Echatla (2 accessions), Roumani (2 accessions), Aoudy (2

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accessions), Richi (2 accessions), Biskri (3 accessions), Agili (3 accessions), Arbi (2

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accessions), Mahmoudi (3 accessions), Bidi (3 accessions), Bidi 17 (1 accession) and LS (7

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accessions).

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Group 2 (from 1940 to 1970) comprises 7 old cultivars that were adopted from international

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programs (Chili (3 accessions) and Kyperounda (1 accession)) and varieties selected from

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crosses involving landraces (INRAT 69 (2 accessions) and Badre (1 accession)).

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Group 3 (from 1970 to 1980) comprises 1 CIMMYT genotype Maghrebi (1 accession)


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Group 4 (from1980-1990) comprises 3 CIMMYT genotypes (Karim (1 accession) and Khiar

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(2 accessions) and 6 ICARDA genotypes (Waha (2 accessions), Om Rabia (2 accessions) and

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Nasr (2 accessions)).

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Group 5 comprises 2 modern lines selected after the 1990s (Maali (2 accessions)).

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Set 2 (season 2013-2014) comprises 15 accessions (6 landraces (Hamira, Jenah Khottifa,

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Richi, Beskri, Mahmoudi and Bidi), 3 old cultivars (Chili, Kyperounda and INRAT 69) and 6

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high yielding varieties results of crosses made in CIMMYT (Karim and Khiar), in ICARDA

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(Om Rabia and Nasr) and in INRAT (Maali and Salim)).

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Set 3 (season 2011-2012) comprises 15 accessions (6 landraces (Hamira, Jenah Khottifa,

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Richi, Beskri, Mahmoudi and Bidi), 3 old cultivars (Chili, Kyperounda and INRAT 69) and 6

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high yielding varieties results of crosses made in CIMMYT (Karim and Khiar), in ICARDA

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(Om Rabia and Nasr) and in INRAT (Maali and Salim)).

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Field set up

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Field experiments were carried out in the trial field of the Graduate School of Agriculture of

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Kef during three growing seasons (2011–2012, 2013–2014 and 2014–2015) on a clay-loam

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soil. Each experimental trial was arranged on randomized complete block design comprising

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plots (6m rows, spaced 0.20 m apart), with three replicated checks for each variety.

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Climatic data

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Rainfall distribution and temperature trends in the three years are presented in figure 1. High

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significance differences in the trends of accumulated precipitation were shown during the

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grain filling period (April). Accumulated precipitation during April was high in season 2011-

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2012 (40.2mm), moderate in season 2013-2014 (9.4mm) and an almost complete absence in

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season 2014-2015 (0.8mm).

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In the first year (2011–2012; Fig. 1a) accumulated precipitation average value was the highest

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(570 mm). Important precipitation was marked mainly during autumn and winter. The second

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year (2013–2014; Fig. 1b) was characterized by 389 mm (average accumulated precipitation),

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9.3 °C (average minimum temperature) and 22.1 °C (average maximum temperature).

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Moderate rainfall was recorded during winter followed by a raise during Mars, while the last



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year (2014–2015; Fig. 1a) was the driest. Also, considerable difference in the trends of

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temperature was shown in the three crop seasons, more specifically during the spring.

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Salt protein extraction

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Soluble proteins were extracted from wheat kernels (0.5 g) using 0.5 M NaCl aqueous

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solution (10 ml) for 2 h and 30 min at room temperature. The supernatant was recuperated by

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centrifugation (3,220g, 15 min, 4°C), and then stocked at 20 °C 18.

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In vitro enzymatic digestion

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The enzymatic in vitro digestion was described by

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under nitrogen flux, and then redissolved with 10 mM HCl aqueous solution (500 ml, pH 2).

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For the in vitro digestion, 1 mg/ml pepsin solution (20 µl) was added and the mixture was

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incubated for 3 h at 37 °C. Then, 100 mM NaH2PO4 solution (300 µl, pH=7.2), 1 mg/ml

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chymotrypsin solution (20 µl) and 1 mg/ml trypsin solution (20 µl) were added and the

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mixture was incubated for 4 h at 37°C. The samples were boiled in a water-bath (5min, 95°C),

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dried under nitrogen flux, reconstituted with 0.1% formic acid solution (300 µl), and

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centrifuged (15,093g, 10 min, 4°C). For the UPLC/ESI-MS, 250 µl of supernatant were

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injected with 5 µl of internal standard (FIA (d3) LPVPSQPVDPR solution (0.214 mM)) 18.

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Ultra-performance liquid chromatography (UPLC/ESI-MS) analysis

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The obtained mixture was separated by a RP column (Aeris peptide C18, 1.7 µm, 150*2.10

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mm) in a UHPLC/ESI-MS system (Dionex ultimate 3000 with a triple quadrupole Thermo

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TSQ vantage) using a gradient elution. Eluent A was bidistilled water with 0.1% formic acid

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and 0.2% acetonitrile, eluent B was acetonitrile with 0.1% formic acid; gradient: 0–7 min

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100% A, 7–50 min from 100% A to 50% A, 50–52.6 min 50% A, 52.6–53 min from 50% A

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to 0% A, 53–58.2 min 0% A, 58.2–59 min from 0% A to 100% A, 59–72 min 100% A. The

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digested sample extracts were analysed by the mass analyser running in the Full Scan mode.

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Flow is 0.2 ml/min; analysis time 72 min; column temperature 35 °C; sample temperature 18

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°C; injection volume 2 µl; acquisition time 7–58.2 min; ionization type positive ions; scan

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range 100–2000 m/z; spray voltage 3500 V, vaporizer temperature 250°C; sheath gas pressure

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22; auxiliary gas pressure 10; capillary temperature 250°C.

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Data processing

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. In brief, the extract (1 ml) was dried


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The characteristic ions (695.6m/z, 768.9m/z and 770.5 m/z) were extracted, obtaining eXtract

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Ion Chromatograms (XICs), in which the areas of the identified peptides and internal standard

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FIA(d3)LPVPSQPVDPR were integrated with the Thermo Scientific™ Xcalibur™ software.

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The result was expressed as the ratio peptide area/internal standard area multiplied by the

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moles of internal standard.

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Data analysis

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Statistical analysis

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The Statistical Package for the Social Sciences (SPSS for windows version 11.0; SPSS Inc,

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Chicago) was used for the statistical analyses. One-way analysis of variance (ANOVA) and

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combined analysis of variance were performed using SPSS software (SPSS, Inc). Genotype

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was regarded as a fixed effect, while environment was regarded as a random effect. The level

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of 5significance was expressed as significant at p

Understanding the Effects of Genotype, Growing Year, and Breeding

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