Journal of Proteomics 211 (2020) 103530

Contents lists available at ScienceDirect

Journal of Proteomics

journal homepage: www.elsevier.com/locate/jprot

Qualitative proteomic comparison of metabolic and CM-like protein fractions in old and modern wheat Italian genotypes by a shotgun approach T ⁎ Antonella Di Francescoa, Rosaria Salettia, , Vincenzo Cunsoloa, Birte Svenssonb, Vera Muccillia, Pasquale De Vitac, Salvatore Fotia a Laboratory of Organic Mass Spectrometry, Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy b Department of Biotechnology and Bioengineering, Technical University of Denmark, Søltofts Plads, Building 224, Kgs. Lyngby DK-2800, Denmark c CREA Research Centre for Cereal and Industrial Crops (CREA-CI), S.S. 673 km 25.200, 71122 Foggia, Italy

ARTICLE INFO ABSTRACT

Keywords: The close relationship between diet and health is generally recognized and the growing wellness and con- Old and modern wheat genotypes sciousness, especially in developed countries, have led to increasing interest for old wheat genotypes, based on High resolution mass spectrometry perceived health benefits. Although nutritional comparison between old and modern wheat varieties is still Proteome analysis controversial, it is generally accepted that old wheat genotypes remained unchanged over the last hundred years. Proteins By contrast, modern wheat genotypes are derived by modification of old wheats during the so-called “Green- Allergens Revolution” in the second half of the 20th century focusing on obtaining properties in terms of higher grain Food and nutrition yield. The present work reports the first comprehensive proteomic profiling and qualitative comparison at the molecular level of metabolic and Chloroform-Methanol (CM)-like protein fractions extracted from mature ker- nels of two old Sicilian durum wheat landraces, Russello and Timilia Reste Bianche, and Simeto, an improved durum wheat variety widespread in Italy and other Mediterranean countries and chosen as representative of the most widely commercial cultivars. The results obtained reveal that metabolic and CM-like protein fractions of old and modern genotypes present remarkably high similarity with only minor differences. This leads to the conclusion that from a food and nutritional perspective there is a substantial equivalence of the protein com- position of the old and modern cultivars. Data are available via ProteomeXchange with identifier PXD014449. Biological significance: In recent years consumers have shown growing interest in the old wheat genotypes, which are generally perceived more “natural” and healthier than modern ones. However, comparison of nutritional value for modern and old wheat varieties is still controversial suggesting further studies. In particular proteome analysis of old and modern wheat genotypes is currently ongoing with particular focus on gluten proteins, whereas the metabolic protein fraction has not yet been investigated. In the present study, we conducted a comprehensive proteomic profile and qualitative comparison at the molecular level of metabolic and Chloroform-Methanol (CM)-like protein fractions of the old Sicilian landraces Russello and Timilia Reste Bianche and the modern cultivar Simeto by applying a shotgun approach. The results reveal that the metabolic and CM- like protein fractions of old and modern genotypes are remarkably similar with only minor differences, leading to the conclusion that from a food and nutritional perspective there is a substantial equivalence of these culti- vars. These results may contribute to improved understanding of the relationship between protein profiles of old wheat genotypes and their potential benefits for human consumption.

1. Introduction characterization of wheat proteins by MS-based methods [5–7]. The aim of these studies is not limited to characterization of the sequence of Wheat is one of the most important cereals for mankind. Wheat is kernel proteins or to proteomics investigation of cultivars [8–13], but not only a source of calories for the human diet but it also provides also include efforts to better understand the role of these components in essential amino acids, minerals, vitamins, and bioactive compounds the gluten matrix [14–17]. [1–4]. For this reason, wheat has been extensively studied over the In spite of its central role for human nutrition, wheat may also cause years. In particular, most of these studies focused on the several adverse reactions and disorders, such as immunoglobulin E

⁎ Corresponding author. E-mail address: [email protected] (R. Saletti). https://doi.org/10.1016/j.jprot.2019.103530 Received 3 May 2019; Received in revised form 3 September 2019; Accepted 17 September 2019 Available online 16 October 2019 1874-3919/ © 2019 Elsevier B.V. All rights reserved. A. Di Francesco, et al. Journal of Proteomics 211 (2020) 103530

(IgE) mediated allergies (celiac disease, CD), which requires a lifelong 2. Materials and methods gluten free diet (GFD) [18], and a less well-defined condition classified as non-celiac wheat sensitivity (NCWS) [19–23]. Therefore, the devel- 2.1. Chemicals opment and characterization of transgenic low-gliadin wheat lines suitable for CD is ongoing [24], even though this will be difficult to All chemicals were of the highest purity commercially available and achieve. The metabolic and CM-like protein fractions include other were used without further purification. KCl, K2HPO4, acetone, me- allergens, promoting IgE mediated reactions. A typical example of thanol, acetic acid and Tris-HCl were purchased from Carlo Erba wheat allergy is Baker's asthma [25,26]. The most commonly re- (Milan, Italy). Formic Acid (FA), Protease Inhibitor Cocktail, EDTA, cognized allergens are the α-amylase/trypsin inhibitor subunits [27], ammonium bicarbonate, ammonium acetate, dithiothreitol (DTT), io- but recently several other proteins have been linked to wheat allergy; in doacetamide (IAA) were obtained from Aldrich (St. Louis, Missouri, particular, lipid transfer proteins (LTPs) [28,29], peroxidase [26], USA). Modified porcine trypsin was purchased from Promega (Madison, thioredoxins [26], serine proteinase inhibitors (serpins) [26,30], thau- WI, USA). Water and acetonitrile (ACN) (OPTIMA® LC/MS grade) for matin-like protein [26], acyl-CoA oxidase [26], fructose-bisphosphate LC/MS analyses were purchased from Fisher Scientific (Milan, Italy). aldolase [26], triosephosphate isomerase [26], and glyceraldehyde-3- LDS sample buffer, Mark12™ Unstained Standards and SimplyBlue™ phosphate dehydrogenase [26]. Safe Stain were obtained from Invitrogen™ (Life Technologies™, Paisley, It has been suggested that old wheat species may have a healthier UK). and superior nutritional profile than modern wheat species, by being richer in vitamins, minerals and nutraceutical compounds [31–33]. 2.2. Samples collection and treatment Although there is no precise clarification, it is generally accepted that old wheat genotypes have remained unchanged over the last hundred Three biological replicates of Russello, Timilia RB and Simeto were years. By contrast, modern wheat cultivars were generated during the provided from CREA-CI. The genetic materials were sowed at Foggia, so-called “Green-Revolution” during the second half of the 20th cen- during the 2010–11 growing season, following a randomized block tury. The most common old wheat species commercially available are design with three replicates. Grain samples were harvested and the einkorn (T. monococcum L. ssp. monococcum), Emmer (T. turgidum L. ssp. flours were stored at 4 °C. Flours (200 mg) were suspended in 2 mL cold dicoccum), Khorasan (T. turgidum ssp. turanicum) and spelt (T. aestivum (4 °C) extraction solution (50 mM Tris-HCl, 100 mM KCl, 5 mM EDTA, L. ssp. spelta)[34]. In addition, there are several old genotypes of both Protease Inhibitor Cocktail, pH 7.8) in order to obtain the metabolic T. aestivum and T. durum, cultivated from the mid-1800s, to the be- and Chloroform-Methanol (CM)-like proteins. The solution was in- ginning of the 20th century (before the “Green Revolution) including cubated on ice (5 min) with intermittent mixing and centrifuged cultivars of durum wheat such as Russello, Senatore Cappelli, Timilia or (13,523g, 15 min, 4 °C). The supernatant was collected and added five Tumminia, and bread wheat such as Gentil Rosso, Maiorca, Sieve, Solina, volumes of 0.1 M ammonium acetate in methanol. Following incuba- and Verna [33]. In the literature, the nutritional comparison between tion overnight at −20 °C, the solution was centrifuged (30g, 15 min, old and modern wheat varieties is still debated, also due to the limited R.T.). The pellet (containing the metabolic proteins) was collected and number of genotypes investigated, and it has been suggested that fur- rinsed in 3 mL 0.1 M ammonium bicarbonate, pH 8.2. Proteins in the ther studies are urgently required [35]. Generally, it is reported that the supernatant (CM-like proteins) were precipitated by addition of four health benefit of old grains is not related to a single compound but to volumes of cold acetone, kept overnight at −20 °C and subsequently their general composition. In particular, information on the proteomic centrifuged (30g, 15 min, R.T.). Finally, the pellet containing CM-like comparison of these genotypes with modern wheat varieties is very proteins was rinsed by 1.5 mL 0.1 M ammonium bicarbonate, pH 8.2 scant [36]. Recently, a set of old and modern Italian tetraploid wheat [39]. The protein concentration for each extract was determined by a genotypes were compared in order to explore the effects of breeding fluorimetric assay using the Qubit Protein Assay kit with the Qubit 1.0 during the 20th century on gluten quality of durum wheat for proces- Fluorometer (ThermoFisher Scientific, Milan, Italy) [40]. Finally, 40 μL sing and health. The results obtained indicated that the breeding ap- (corresponding to about 60 μg) of each extract were reduced by adding parently improved the durum wheat gluten quality in relation to 40 μg of DTT dissolved in the same buffer (3 h, 20 °C), alkylated with technological performance, without aggravating the allergenic poten- 96 μg of IAA (1 h, in the dark at 20 °C) and digested by porcine trypsin tial and the content of potentially toxic immune-stimulating peptides (Sequencing Grade Modified Trypsin, Porcine, lyophilized, Promega) at [37]. Moreover, data reported in another recent investigation of gluten an enzyme-substrate ratio of 1:50 (overnight, 37 °C) [40]. proteins in whole-meal flours of old and modern genotypes suggested that beneficial features claimed for old genotypes such as a lower re- 2.3. Mass spectrometry analysis lease of CD-related peptides are not scientifically substantiated, whereas modern varieties do not seem worse in terms of characteristics Mass spectrometry data were acquired on a Thermo Fisher Scientific associated to health [38]. Orbitrap Fusion Tribrid® (Q-OT-qIT) mass spectrometer (Thermo Fisher Contrarily, no comparison of the metabolic protein fractions (salt- Scientific, Bremen, Germany). Liquid chromatography was carried out soluble proteins) in old and modern durum wheat genotypes has yet using a Thermo Scientific Dionex UltiMate 3000 RSLCnano system been reported. The present work aims at qualitative proteomic com- (Sunnyvale, CA). One microliter of peptide mixture was loaded onto an parison of the metabolic proteins as well as the CM-like proteins in old Acclaim ®Nano Trap C18 Column (100 μm i.d. × 2 cm, 5 μm particle and modern durum wheat genotypes. In particular, proteins were ex- size, 100 Å). After washing the trapping column with solvent A tracted from mature kernels of Russello and Timilia Reste Bianche (H2O + 0.1% FA) for 3 min at a flow rate of 7 μL/min, the peptides (hereafter called Timilia RB), two old Sicilian durum wheat landraces, were eluted from the trapping column onto a PepMap® RSLC C18 EASY- and Simeto, an improved modern durum wheat variety, released in Italy Spray column (75 μm i. d. × 50 cm, 2 μm particle size, 100 Å) and se- in 1988 and widely cultivated also in other European countries. All the parated by elution at a flow rate of 0.25 μL/min at 40 °C by a linear investigated genotypes were grown in the same agronomic conditions gradient of solvent B (ACN + 0.1% FA) in A, 5% for 3 min, followed by in a single field trial. Simeto was chosen because its protein profile can 5% to 20% in 32 min, 20% to 40% in 30 min, 40% to 60% in 20 min and be considered representative of commercial cultivars most commonly 60% to 98% in 15 min, finishing by holding 98% B 5 min, 98% to 5% in used in the current agriculture practice. 1 min. and re-equilibrating at 5% B for 20 min. The eluting peptide cations were converted to gas-phase ions by electrospray ionization using a source voltage of 1.75 kV and introduced into the mass spec- trometer through a heated ion transfer tube (275 °C). Survey scans of

2 A. Di Francesco, et al. Journal of Proteomics 211 (2020) 103530 peptide precursors from 200 to 1600 m/z were performed at 120 K re- uniprot.org/). It should be noted that, due to the limited annotation solution (@ 200 m/z). Tandem MS was performed by isolation at 1.6 Th of the wheat proteins, to obtain coding gene information an additional with the quadrupole, HCD fragmentation with a normalized collision step was required in many cases. Actually, when the gene symbol was energy of 35, and rapid scan MS analysis in the linear ion trap (low not available, the corresponding protein sequence was subjected to a resolution MS/MS analysis). Only those precursors with charge state sequence similarity search by BLAST (Basic Local Alignment Search 2÷4 and intensity above the threshold of 5·103 were sampled for MS2. Tool; http://blast.ncbi.nlm.nih.gov/Blast.cgi). By this strategy, many of The dynamic exclusion duration was set to 60 s with a 10 ppm tolerance the identified proteins whose gene code was not available were classi- around the selected precursor and its isotopes. Monoisotopic precursor fied by finding homologous proteins from the species closest related to selection was turned on. The instrument was run in top speed mode Triticum present in the databases and sharing at least more than 70% with 3 s cycles, meaning it would continuously perform MS2 events sequence similarity. Subsequently, to carry out the Gene Ontology (GO) until the list of non-excluded precursors diminished to zero or 3 s, analysis for each cultivar, the proteins were grouped, using the corre- whichever is shorter. MS/MS spectral quality was enhanced enabling sponding gene code, in unique gene products and subjected to GO the parallelizable time option (i.e. by using all parallelizable time analysis through the PANTHER (Protein ANalysis THrough during full scan detection for MS/MS precursor injection and detec- Evolutionary Relationship; version 14.1) system (http://www. tion). Mass spectrometer calibration was performed by using the pantherdb.org) by using the Triticum aestivum, Oryza sativa, Pierce® LTQ Velos ESI Positive Ion Calibration Solution (Thermo Fisher Brachypodium, Hordeum vulgare, Arabidopsis thaliana and Zea mays Scientific). MS data acquisition was carried out by utilizing the Xcalibur genome annotations as background. v. 3.0.63 software (Thermo Fisher Scientific). 3. Results 2.4. Gel electrophoresis Three biological replicates for each investigated genotype were One-dimensional polyacrylamide gel electrophoresis (1D-PAGE) analyzed. In order to increase the number of possible identification of was performed with 4–12% gradient, pre-cast polyacrylamide gels. proteins, the metabolic and CM-like protein fractions were isolated and Samples (metabolic 10 μg, CM-like 50 μg) were mixed with LDS sample analyzed separately. Firstly, a SDS-PAGE analysis of these fractions buffer under reducing conditions (50 mM DTT) and heated at 70 °C for extracted from two biological replicates for each genotype was carried 10 min. Following separation of samples and molecular weight markers out (Supplementary Fig. S1). Visual inspection of the electrophoretic (Mark12™ Unstained Standards), gels were fixed in a solution of 50% profiles indicated a substantial similarity at both qualitative and (v/v) methanol and 10% (v/v) acetic acid in water for 2 h, stained with quantitative level of the samples investigated. Then, a MS-based pro- SimplyBlue™ Safe Stain overnight, and destained in water for 2 h. All teomic approach was performed in order to give an additional insight gels, buffers, standards, and stains were obtained from Invitrogen™ into the samples investigated. To assess the reproducibility of the (Life Technologies™, Paisley, UK). available MS data, each extract was subjected to in-solution digestion followed by triplicate RP-nHPLC/nESI-MS/MS analyses and database 2.5. Database search, protein identification and gene ontology analysis search. As expected, separation of the proteins into metabolic and CM- like protein fractions was not completely selective and therefore partial MS data were processed using PEAKS de novo sequencing software cross-contamination between these two fractions resulted. To produce (v. 8.5, Bioinformatics Solutions Inc., Waterloo, ON Canada). Data were the final list of proteins detected in these two fractions of each geno- searched against a dedicated protein database (7612 protein se- type, the following method was adopted: firstly, the lists of the proteins quences), including only the reviewed entries of Triticum, Oryza, identified for each extract in the triplicate nLC-MS/MS analyses were Hordeum, Avena, Secale, Maize and Brachypodium species downloaded compared. Only those proteins identified at least twice were included. from the UniProt database (release July 2018). The common Repository Then, the lists of proteins identified in each biological replicate were of Adventitious Proteins (c-RAP) contaminant database was included in compared, and only those proteins identified at least in two replicates the database search. were considered for the compilation of the final list for each genotype Database search was carried out using the following parameters: i) (the raw lists of the proteins identified for each genotype are reported in full tryptic peptides with a maximum of 3 missed cleavage sites; ii) Supplementary Tables S2–S4). cysteine carbamidomethylation as a fixed modification; iii) oxidation of methionine, the transformation of N-terminal glutamine and N-terminal 3.1. Comparison of the metabolic protein fractions glutamic acid residue to pyroglutamic acid form as variable modifica- tions. The precursor mass tolerance threshold was set to 10 ppm and the Using the approach described above it was possible to identify 462 maximum fragment mass error was set to 0.6 Da. Peptide Spectral proteins for Russello, 471 for Timilia RB and 489 for Simeto, in their Matches (PSM) were validated using a Target Decoy PSM Validator metabolic fractions (Supplementary Table S1), which corresponded to a node based on q-values at a 0.1% False Discovery Rate (FDR). PEAKS gross-total of 603 different proteins. Of these identified proteins, 21.1% score thresholds for Peptide Spectral Matches (PSMs) were set in order were from Triticum genus, 57% from Oryza, 10.8% from Hordeum, 9.6% to achieve for each database search FDR values for PSMs, Peptide se- from Maize, 1% from Secale and 0.5% from Avena. The low percentage quences and Proteins identified below the 0.1% value. This resulted in a of identified proteins from Triticum can be explained by the low amount range for PEAKS score thresholds for peptide from 39 to 47 and for of reviewed entries from this species present in the UniProt database. protein from 20 to 90. A protein was considered identified if a On the other hand, the large presence of known homologous proteins minimum of two peptides were matched. Proteins containing the same from closely related cereals allowed cross-species identification for peptides and that could not be differentiated based on MS/MS analysis most of the detected proteins. The gene code was assigned to all 603 alone were grouped to satisfy the principles of parsimony (groups of identified proteins and they corresponded to 474 unique gene products. parsimony). In these cases, proteins from Triticum, when identified, For additional 42 proteins the gene code was not available (N/A). The were always chosen as the group's reference protein. When a group of list of these unique gene products is shown in Table 1. As displayed by parsimony did not contain a component from Triticum, the reference the Venn diagram (Fig. 1), a general qualitative comparison of the protein was selected from the species closest related to Triticum and metabolic fractions of Russello, Timilia RB and Simeto revealed that most represented in the group. of the unique gene products are common to the three genotypes (306 The gene code, when available, was assigned to the proteins here out of 516), whereas 51, 30 and 44 were exclusively detected in Simeto, identified by using the UniProt Knowledge database (http://www. Russello and Timilia RB, respectively. All the identified unique gene

3 A. Di Francesco, et al. Journal of Proteomics 211 (2020) 103530

TIMILIA RB are of primary importance in evaluating food quality [41]. (398) Taking into account that consumers have shown an increasing in- terest for the old wheat genotypes, which are generally perceived more “ ” 44 natural and healthier than modern ones, the present work reports the (8.5%) first in-depth qualitative comparison of the metabolic protein and CM- like protein fractions of two old Sicilian durum wheat landraces 20 28 (Russello and Timilia RB), and Simeto that represents one the most (3.9%) (5.4%) widely used commercial modern cultivars. Comparison of the protein 306 ff RUSSELLO (59.3%) SIMETO composition reveals remarkable similarity and only minor di erences (393) 30 51 (422) between old and modern cultivars. Particularly, the qualitative eva- 37 (5.8%) (9.9%) luation shows that the three genotypes contain the same CM-like pro- (7.2%) teins except for the alpha-amylase/trypsin inhibitor CM1 and CMd only identified in Timilia RB. The genotypes investigated share about 59% (306 out of 516) of the unique gene products identified in the respective Fig. 1. Venn diagrams giving the statistics of the identified proteins. metabolic fractions. By contrast, a limited number of components were fi Contribution to the total identi cations as obtained in Timilia RB, Simeto and exclusively found in a single cultivar: about 9.9% (corresponding to 51 Russello (the percentage values are also reported). unique gene products) for Simeto, 5.8% (i.e. 30 unique gene products) for Russello, and 8.5% (i.e. 44 unique gene products) for Timilia RB. products in the metabolic fraction of the three genotypes were classified However, the unique gene products detected in the three genotypes in the putative molecular function and biological process categories by show essentially the same distribution in the molecular function and Gene Ontology analysis. On the whole, a comparison of GO (Fig. 2) biological process categories and reflect the general protein composi- reveals a remarkable similarity between old and modern genotypes. In tion of the mature grains. The main molecular functions were “catalytic particular, the molecular function and biological process categories activity” and “binding”, followed by “structural molecule activity”, and show the same distributions in the three different genotypes indicating “transporter activity”, whereas the main categories of biological pro- that most of the proteins play a role in binding, catalytic activity, and cesses were “metabolic processes” and “cellular process”. Notably, a structural function (Fig. 2a) and are mainly involved in metabolic and pairwise comparison of the investigated genotypes revealed few, but cellular processes (Fig. 2b). Subsequently, a pairwise comparison of the interesting differences. In particular, Linoleate 9S-lipoxygenase 1 LOX1 three varieties was carried out. In detail, comparison of Russello and is exclusively present in the old genotypes. Lipoxygenase is a class of Simeto shows that these two genotypes share 343 unique gene products, non-heme iron-containing dioxygenases that catalyses the positional whereas 50 and 79 were exclusively identified in the metabolic frac- and specific dioxygenation of polyunsaturated fatty acids that contain tions of Russello and Simeto, respectively. 1,4-cis,cis pentadiene structures to produce the corresponding hydro- The lists of proteins identified in the metabolic fraction of Timilia RB peroxides. In plants, products of the LOX reaction have been shown to and Simeto share 334 proteins. In contrast, 64 proteins were unique for have roles in several processes, such as vegetative growth, wounding, Timilia RB and 88 for Simeto. response to herbivore and pathogen attack and also mobilisation of The comparison of the proteins identified in the metabolic fraction storage lipids during germination. In durum wheat semolina, radicals of the two old genotypes Timilia RB and Russello shows they share 326 produced during the intermediate states of linoleate hydroperoxidation unique gene products, while 72 proteins are unique for Timilia RB and can cause oxidation of carotenoid pigments, and consequently a loss of 67 for Russello. the yellow colour in pasta products [42–44]. In line with these data, it has been reported that, by contrast with Simeto that present a very low 3.2. Qualitative analysis of CM-like protein fractions of old and modern LOX activity, Russello and Timilia are characterized by a medium-high varieties LOX activity [44]. The L-ascorbate peroxidase 2 cytosolic, a protein associated with the Characterization of the CM-like fractions allowed the identification “response to stimulus”, was detected in Timilia RB and Simeto, but not in of 98 proteins for Russello, 108 for Timilia RB and 107 for Simeto. The Russello. This enzyme is a stress-responsive protein involved in the complete list of the detected proteins is reported in Supplementary metabolism of H2O2 in higher plants, and is up-regulated under fungal Table S5. As expected, a completely selective separation of metabolic (e.g. Aspergillus parasiticus or A. flavus) attack accompanied by drought and CM-like proteins was not achieved by the stepwise extraction stress. As a consequence, this protein might contribute to increase the method used, and contamination by metabolic components in this resistance of the plant to adverse biotic and abiotic stimuli [45]. An- fraction was observed. Comparison of the CM-like enriched fractions, other interesting class of identified proteins is represented by the tu- considering only the identified CM-like proteins, revealed, however, bulin (alpha and beta) family. While the alpha-type subunits are de- that all three investigated cultivars contain the same ten alpha-amylase tected in all three genotypes, the beta-type was found only in Russello and alpha-amylase/trypsin inhibitors. In addition, alpha-amylase/ and Simeto. Beta-tubulins show antifungal activity against fungal pa- trypsin inhibitor CM1 and CMd were identified exclusively in Timilia RB thogens, some of which may also produce mycotoxins that threaten (Table 2). human and animal health [46]. Moreover, Simeto shows one protein belonging to the glucose-1-phosphate adenyltransferase family (glu- 4. Discussion cose-1-phosphate adenyltransferase large subunit 1 chloroplastic/ amyloplastic) which is not detected in the old genotypes, whereas Ti- The advent of modern biotechnologies in the development of new milia RB contains glucose-1-phosphate adenyltransferase large subunit raw materials has generated concern and a wide request of transpar- 2 cytosolic, which is not present in Simeto. In particular, these enzymes ency about food origin and composition, because, in the extreme view, are associated with the plant reproduction processes and involved in any human intervention on what is considered the “natural” constitu- the starch biosynthetic pathway [47]. In this context, it has been de- tion of plant-based foods, is perceived potentially negative. Proteins are monstrated that the starch content influences wheat yield and also in- of central interest in food or feed safety assessment due to their in- fluences the processing quality of wheat flour [48]. volvement in metabolism and cellular development and since they can The finding therefore might be related to the genetic improvement potentially negatively impact human and animal health, behaving as carried out in Italy during the 20th century, which led to improvement toxins, anti-nutrients, or allergens. Therefore, proteomic investigations of wheat yield and of technological quality of durum wheat flour.

4 A. Di Francesco, et al. Journal of Proteomics 211 (2020) 103530

Fig. 2. Histograms of (a) molecular functions, and (b) biological processes pertaining to the 516 unique gene products detected in Russello, Timilia RB and Simeto genotypes.

Another additional protein detected exclusively in Simeto is a Modified (GM) crops compared to their conventional counterpart, can probable sucrose-phosphate synthase 1 (SPS1). This protein is a key be adopted. In light of this principle, the results obtained in the present regulatory enzyme in the pathway of Suc biosynthesis and have been work, all together indicate that there is a “substantial equivalence” in linked to quantitative trait loci controlling plant growth and yield [49]. the qualitative proteomic profile of the metabolic and CM-like fractions Finally, it is interesting to note that most of the main allergenic of the three investigated genotypes, despite some minor differences. proteins usually present in the metabolic fraction of wheat, such as lipid This conclusion is also corroborated by the consideration that qualita- transfer protein (LTP), peroxidase, thioredoxin, serine proteinase in- tive differences in the protein composition between old and modern hibitor (serpin), fructose-bisphosphate aldolase, triose-phosphate iso- varieties are similar to that observed by comparison of the old geno- merase cytosolic, and glyceraldehyde-3-phosphate dehydrogenase 1 types themselves, suggesting that the proteomic differences detected were identified in all the three genotypes investigated. among old and modern genotypes are very likely attributable to phy- Moreover,another putative allergen, a probable calcium-binding pro- siological variations rather than to genetic differences. tein CML7 [50] was detected in all genotypes investigated. Whereas other two putative allergens, glyceraldehyde-3-phosphate dehy- 5. Conclusions drogenase 2 and triose-phosphate isomerase chloroplastic were de- tected only in Timilia RB. In this work, a comparative proteomic analysis of the metabolic and It is reasonable to assume that, in the comparison of the proteomic CM-like protein fractions of three different durum wheat genotypes fi pro le of modern and old wheat genotypes under the aspect of the (Simeto, Russello and Timilia RB) was performed at a qualitative level. “ impact on health, an extension of the popular principle of substantial Simeto is representative of the modern cultivars used in the current ” equivalence , commonly employed for safety assessment of Genetically agronomic practice, while Russello and Timilia RB are old Sicilian

5 A. Di Francesco, et al. Journal of Proteomics 211 (2020) 103530

Table 1 List of unique gene products identified in the metabolic fractions of the three investigated genotypes in the present study: gene code, description of unique gene products, and genotypes in which they were identified. The description of unique gene products reported with an asterisk, was obtained by BLAST search (see the text).

N. Gene Description of unique gene product Russello TimiliaRB Simeto

1 ARD1 1 2-dihydroxy-3-keto-5-methylthiopentene dioxygenase 1 OS = Oryza sativa subsp. indica XX 2 GF14F 14-3-3-like protein A OS = Hordeum vulgare XX X 3 GF14E 14-3-3-like protein B OS = Hordeum vulgare* XX X 4 GRF1 14-3-3-like protein GF14–6OS=Zea mays XX X 5 GF14D 14-3-3-like protein GF14-D OS = Oryza sativa subsp. japonica XX X 6 hsp16.9A 16.9 kDa class I heat shock protein 1 OS = Triticum aestivum XX X 7 hsp16.9B 16.9 kDa class I heat shock protein 2 OS = Triticum aestivum X 8 HSP18 17.0 kDa class II heat shock protein OS = Zea mays X 9 HSP17.4 17.4 kDa class I heat shock protein OS = Oryza sativa subsp. japonica XX X 10 HSP17.9A 17.9 kDa class I heat shock protein OS = Oryza sativa subsp. japonica XX 11 HSP18.1 18.1 kDa class I heat shock protein OS = Oryza sativa subsp. japonica X 12 PER1 1-Cys peroxiredoxin PER1 OS = Triticum aestivum XX X 13 PGM1 2 3-bisphosphoglycerate-independent phosphoglycerate mutase OS = Zea mays XX X 14 HSP23.6 23.6 kDa heat shock protein mitochondrial OS = Oryza sativa subsp. japonica X 15 HSP24.1 24.1 kDa heat shock protein mitochondrial OS = Oryza sativa subsp. japonica XX X 16 RSCA 26 kDa endochitinase 2 OS = Hordeum vulgare XX X 17 RPN7 26S proteasome non-ATPase regulatory subunit 6 OS = Oryza sativa subsp. japonica XX X 18 TBP2 26S proteasome regulatory subunit 4 homolog OS = Oryza sativa subsp. japonica XX X 19 TBP1 26S proteasome regulatory subunit 6A homolog OS = Oryza sativa subsp. japonica XX X 20 RPT1A 26S proteasome regulatory subunit 7A OS = Oryza sativa subsp. japonica XX X 21 RPT1B 26S proteasome regulatory subunit 7B OS = Oryza sativa subsp. japonica XX X 22 BAS1 2-Cys peroxiredoxin BAS1 chloroplastic (Fragment) OS = Hordeum vulgare XX X 23 TSA 2-Cys peroxiredoxin BAS1 chloroplastic (Fragment) OS = Triticum aestivum XX X 24 Os01g0505400 2-hydroxyacyl-CoA lyase OS = Oryza sativa subsp. japonica X X 25 KAS12 3-oxoacyl-[acyl-carrier-protein] synthase I chloroplastic OS = Hordeum vulgare XX X 26 RPS10–1 40S ribosomal protein S10–1 OS = Oryza sativa subsp. japonica XX X 27 RPS10–2 40S ribosomal protein S10–2 OS = Oryza sativa subsp. japonica XX X 28 RPS11 40S ribosomal protein S11 OS = Zea mays X 29 RPS12 40S ribosomal protein S12 OS = Hordeum vulgare XX X 30 RPS13 40S ribosomal protein S13 OS = Zea mays XX X 31 RPS14C 40S ribosomal protein S14 OS = Zea mays XX X 32 RPS16A 40S ribosomal protein S16 OS = Oryza sativa subsp. indica X 33 RPS19A 40S ribosomal protein S19 OS = Oryza sativa subsp. japonica XX X 34 RPS20 40S ribosomal protein S20 OS = Oryza sativa subsp. japonica X 35 RPS21 40S ribosomal protein S21 OS = Oryza sativa subsp. japonica XX X 36 RPS26 40S ribosomal protein S26 OS = Oryza sativa subsp. japonica X 37 RPS27 40S ribosomal protein S27 OS = Hordeum vulgare XX 38 RPS3A 40S ribosomal protein S3a OS = Oryza sativa subsp. japonica XX X 39 RPS4 40S ribosomal protein S4 OS = Oryza sativa subsp. japonica XX X 40 RPS7 40S ribosomal protein S7 OS = Hordeum vulgare XX X 41 DPE1 4-alpha-glucanotransferase DPE1 chloroplastic/amyloplastic OS = Oryza sativa subsp. japonica XX X 42 DPE2 4-alpha-glucanotransferase DPE2 OS = Oryza sativa subsp. japonica XX X 43 Os12g0623900 5-methyltetrahydropteroyltriglutamate–homocysteine methyltransferase 1 OS = Oryza sativa subsp. japonica X X X 44 Os08g0130500 60S acidic ribosomal protein P0 OS = Oryza sativa subsp. japonica X X 45 RPP2B 60S acidic ribosomal protein P2 (Fragment) OS = Triticum aestivum XX X 46 RPP2A 60S acidic ribosomal protein P2A OS = Zea mays X 47 SC34 60S ribosomal protein L10–1 OS = Oryza sativa subsp. indica X 48 SG12 60S ribosomal protein L10–2 OS = Oryza sativa subsp. indica X 49 RPL10A 60S ribosomal protein L10a OS = Oryza sativa subsp. indica X X 50 RPL11 60S ribosomal protein L11 OS = Oryza sativa subsp. indica X X 51 N/A 60S ribosomal protein L17–1 OS = Hordeum vulgare XX 52 RPL17 60S ribosomal protein L17–2 OS = Hordeum vulgare* X X 53 RPL18A 60S ribosomal protein L18a OS = Oryza sativa subsp. japonica X X 54 RPL24 60S ribosomal protein L24 OS = Hordeum vulgare X 55 RPL3 60S ribosomal protein L3 OS = Oryza sativa subsp. japonica XX X 56 RPL30 60S ribosomal protein L30 OS = Triticum aestivum* XX X 57 Os01g0679700 60S ribosomal protein L37a-1 OS = Oryza sativa subsp. japonica X X 58 Os05g0557000 60S ribosomal protein L37a-2 OS = Oryza sativa subsp. japonica X X 59 RPL5A 60S ribosomal protein L5–1 OS = Oryza sativa subsp. indica X X 60 RPL7A-1 60S ribosomal protein L7a-1 OS = Oryza sativa subsp. japonica XX X 61 RPL7A-2 60S ribosomal protein L7a-2 OS = Oryza sativa subsp. japonica XX X 62 RPL9 60S ribosomal protein L9 OS = Oryza sativa subsp. japonica XX X 63 G6PGH1 6-phosphogluconate dehydrogenase decarboxylating 1 OS = Oryza sativa subsp. japonica XX X 64 G6PGH2 6-phosphogluconate dehydrogenase decarboxylating 2 chloroplastic OS = Oryza sativa subsp. japonica X X 65 At3g02360 6-phosphogluconate dehydrogenase decarboxylating 3 OS = Arabidopsis thaliana XX X 66 FKBP70 70 kDa peptidyl-prolyl isomerase OS = Triticum aestivum XX X 67 ACC2 Acetyl-CoA carboxylase 2 OS = Oryza sativa subsp. japonica XX X 68 ACT1 Actin-1 OS = Oryza sativa subsp. japonica XX X 69 ACT2 Actin-2 OS = Oryza sativa subsp. indica XX X 70 ACT3 Actin-3 OS = Oryza sativa subsp. indica XX X 71 ACT7 Actin-7 OS = Oryza sativa subsp. indica XX X 72 ACL1.3 Acyl carrier protein 3 chloroplastic OS = Hordeum vulgare XX X (continued on next page)

6 A. Di Francesco, et al. Journal of Proteomics 211 (2020) 103530

Table 1 (continued)

N. Gene Description of unique gene product Russello TimiliaRB Simeto

73 AT9 Acyl transferase 9 OS = Oryza sativa subsp. japonica X 74 APT1 Adenine phosphoribosyltransferase 1 OS = Triticum aestivum X 75 SAHH Adenosylhomocysteinase OS = Triticum aestivum XX X 76 ADK-A Adenylate kinase 3 OS = Oryza sativa subsp. japonica XX X 77 ADK-B Adenylate kinase 4 OS = Oryza sativa subsp. japonica XX X 78 PURA1 Adenylosuccinate synthetase chloroplastic (Fragment) OS = Triticum aestivum XX X 79 ANT-G1 ADP ATP carrier protein 1 mitochondrial OS = Triticum aestivum X X 80 Os01g0813400 ADP-ribosylation factor 1 OS = Oryza sativa subsp. japonica XX 81 ARF ADP-ribosylation factor 2 OS = Oryza sativa subsp. japonica XX X 82 ARF1 ADP-ribosylation factor OS = Zea mays XX 83 ALAAT2 Alanine aminotransferase 2 OS = Hordeum vulgare XX X 84 ADH1 Alcohol dehydrogenase 1 OS = Hordeum vulgare XX X 85 ADH2 Alcohol dehydrogenase 2 OS = Hordeum vulgare XX 86 ADHIII Alcohol dehydrogenase class-3 OS = Oryza sativa subsp. indica XX X 87 Os02g0815500 Alcohol dehydrogenase class-3 OS = Oryza sativa subsp. japonica X X 88 FDH Alcohol dehydrogenase class-3 OS = Zea mays XX X 89 N/A Aldose reductase OS = Hordeum vulgare XX X 90 N/A Alpha/beta-gliadin A-IV OS = Triticum aestivum X 91 N/A Alpha/beta-gliadin A-V OS = Triticum aestivum XX X 92 N/A Alpha/beta-gliadin clone PW8142 OS = Triticum aestivum XX X 93 N/A Alpha/beta-gliadin OS = Triticum aestivum XX 94 THI1.1 Alpha-1-purothionin (Fragment) OS = Triticum aestivum XX X 95 N/A Alpha-amylase inhibitor 0.19 OS = Triticum aestivum XX 96 IMA1 Alpha-amylase inhibitor 0.28 OS = Triticum aestivum X 97 N/A Alpha-amylase inhibitor 0.53 OS = Triticum aestivum XX X 98 IHA-B1–2 Alpha-amylase inhibitor WDAI-3 (Fragment) OS = Triticum aestivum XX X 99 IAT3 Alpha-amylase/trypsin inhibitor CM16 OS = Triticum aestivum X 100 IAT2 Alpha-amylase/trypsin inhibitor CM2 OS = Triticum aestivum XX X 101 IAT3 Alpha-amylase/trypsin inhibitor CM3 OS = Triticum aestivum XX X 102 Os10g0493600 Alpha-galactosidase OS = Oryza sativa subsp. japonica XX X 103 PHS2 Alpha-glucan phosphorylase H isozyme OS = Triticum aestivum X X 104 Os02g0218200 Aminopeptidase M1-A OS = Oryza sativa subsp. japonica XX X 105 Os08g0398700 Aminopeptidase M1-B OS = Oryza sativa subsp. japonica XX X 106 N/A Antifungal protein R (Fragment) OS = Hordeum vulgare XX X 107 PIP2–3 Aquaporin PIP2–3 OS = Zea mays X 108 B0616E02-H0507E05.7 Arginase 1 mitochondrial OS = Oryza sativa subsp. indica X X 109 ARG1 Arginase 1 mitochondrial OS = Oryza sativa subsp. japonica XX X 110 Os01g0760600 Aspartate aminotransferase cytoplasmic OS = Oryza sativa subsp. japonica XX X 111 PYRB Aspartate carbamoyltransferase chloroplastic OS = Oryza sativa subsp. japonica X X 112 RAP Aspartic proteinase OS = Oryza sativa subsp. japonica XX X 113 ATP9 ATP synthase subunit 9 mitochondrial OS = Triticum aestivum X 114 ATPA ATP synthase subunit alpha mitochondrial OS = Triticum aestivum X X 115 ATPB ATP synthase subunit beta mitochondrial OS = Oryza sativa subsp. japonica X 116 ACLA-3 ATP-citrate synthase alpha chain protein 3 OS = Oryza sativa subsp. japonica X X 117 ACLB-1 ATP-citrate synthase beta chain protein 1 OS = Oryza sativa subsp. japonica XX X 118 AVNLA Avenin-like a4 OS = Triticum aestivum XX X 119 AVNLB Avenin-like b1 OS = Triticum aestivum XX X 120 N/A B3-hordein (Fragment) OS = Hordeum vulgare XX 121 rscc Basic endochitinase C OS = Secale cereale XX X 122 BMY1 Beta-amylase OS = Triticum aestivum XX X 123 BGLU26 Beta-glucosidase 26 OS = Oryza sativa subsp. japonica XX X 124 BGLU8 Beta-glucosidase 8 OS = Oryza sativa subsp. japonica X 125 BADH1 Betaine aldehyde dehydrogenase OS = Hordeum vulgare* X 126 BADH2 Betaine aldehyde dehydrogenase OS = Hordeum vulgare* XX X 127 NIT4 Bifunctional nitrilase/nitrile hydratase NIT4 OS = Oryza sativa subsp. japonica X X 128 CPK15 Calcium-dependent protein kinase 15 OS = Oryza sativa subsp. japonica X 129 CAL1 Calmodulin OS = Triticum aestivum XX X 130 CAM3 Calmodulin-3 OS = Oryza sativa subsp. indica XX X 131 CML1 Calmodulin-like protein 1 OS = Oryza sativa subsp. indica X X 132 Os07g0246200 Calreticulin OS = Oryza sativa subsp. japonica XX X 133 CARB Carbamoyl-phosphate synthase large chain chloroplastic OS = Oryza sativa subsp. japonica XX X 134 N/A Carbonic anhydrase chloroplastic OS = Hordeum vulgare XX X 135 CAT1 Catalase isozyme 1 OS = Hordeum vulgare XX X 136 CAT2 Catalase isozyme 2 OS = Hordeum vulgare* XX X 137 CATA Catalase isozyme A OS = Oryza sativa subsp. japonica X 138 CLPB1 Chaperone protein ClpB1 OS = Oryza sativa subsp. japonica XX X 139 CLPB2 Chaperone protein ClpB2 chloroplastic OS = Oryza sativa subsp. japonica XX X 140 CLPB3 Chaperone protein ClpB3 mitochondrial OS = Oryza sativa subsp. japonica XX X 141 CLPC1 Chaperone protein ClpC1 chloroplastic OS = Oryza sativa subsp. japonica XX X 142 CPN60I Chaperonin CPN60–1 mitochondrial OS = Zea mays XX X 143 CPN60II Chaperonin CPN60–2 mitochondrial OS = Zea mays X 144 Os11g0104900 Clathrin heavy chain 1 OS = Oryza sativa subsp. japonica X X 145 Os03g0711400 Coatomer subunit alpha-1 OS = Oryza sativa subsp. japonica XX X 146 Os06g0143900 Coatomer subunit beta’-1 OS = Oryza sativa subsp. japonica XX X 147 Os11g0174000 Coatomer subunit beta-1 OS = Oryza sativa subsp. japonica X X (continued on next page)

7 A. Di Francesco, et al. Journal of Proteomics 211 (2020) 103530

Table 1 (continued)

N. Gene Description of unique gene product Russello TimiliaRB Simeto

148 Os02g0209100 Coatomer subunit beta’-2 OS = Oryza sativa subsp. japonica X X 149 Os01g0281400 Coatomer subunit beta-2 OS = Oryza sativa subsp. japonica XX X 150 Os05g0310800 Coatomer subunit delta-1 OS = Oryza sativa subsp. japonica X 151 Os05g0311000 Coatomer subunit delta-2 OS = Oryza sativa subsp. japonica XX X 152 Os01g0833700 Coatomer subunit delta-3 OS = Oryza sativa subsp. japonica X 153 Os03g0227000 Coatomer subunit gamma-1 OS = Oryza sativa subsp. japonica XX X 154 Os07g0201100 Coatomer subunit gamma-2 OS = Oryza sativa subsp. japonica X X 155 OsI_13867 Cupincin OS = Oryza sativa subsp. indica XX X 156 Os01g0270100 Cysteine proteinase inhibitor 12 OS = Oryza sativa subsp. japonica XX X 157 RCS1 Cysteine synthase OS = Oryza sativa subsp. japonica X 158 CYS1 Cysteine synthase OS = Triticum aestivum XX X 159 Os05g0108800 Cytochrome b5 OS = Oryza sativa subsp. japonica XX 160 CYTC Cytochrome c OS = Triticum aestivum XX X 161 RH15 DEAD-box ATP-dependent RNA helicase 15 OS = Arabidopsis thaliana XX X 162 AIP2 DEAD-box ATP-dependent RNA helicase 15 OS = Oryza sativa subsp. japonica X 163 RH37 DEAD-box ATP-dependent RNA helicase 37 OS = Arabidopsis thaliana X 164 PL10A DEAD-box ATP-dependent RNA helicase 37 OS = Oryza sativa subsp. japonica XX X 165 Os03g0158200 DEAD-box ATP-dependent RNA helicase 38 OS = Oryza sativa subsp. japonica XX X 166 PL10B DEAD-box ATP-dependent RNA helicase 52B OS = Oryza sativa subsp. japonica X X 167 Os11g0599500 DEAD-box ATP-dependent RNA helicase 52C OS = Oryza sativa subsp. japonica XX X 168 RH56 DEAD-box ATP-dependent RNA helicase 56 OS = Arabidopsis thaliana XX X 169 N/A Defensin Tk-AMP-D1 OS = Triticum kiharae X 170 N/A Defensin Tk-AMP-D1.1 OS = Triticum kiharae XX 171 N/A Defensin Tk-AMP-D4 OS = Triticum kiharae X X 172 N/A Defensin Tm-AMP-D1.2 OS = Triticum monococcum XX 173 N/A Defensin-like protein 1 OS = Triticum aestivum XX X 174 N/A Defensin-like protein 2 OS = Triticum aestivum XX X 175 RAB15 Dehydrin Rab15 OS = Triticum aestivum XX 176 P5CS1 Delta-1-pyrroline-5-carboxylate synthase 1 OS = Oryza sativa subsp. japonica X X 177 HEMB Delta-aminolevulinic acid dehydratase chloroplastic OS = Hordeum vulgare XX 178 DBB1 DNA damage-binding protein 1 OS = Oryza sativa subsp. japonica XX X 179 MT21A EC protein III OS = Triticum aestivum* XX X 180 REFA1 Elongation factor 1-alpha OS = Oryza sativa subsp. japonica X X 181 TEF1 Elongation factor 1-alpha OS = Triticum aestivum XX X 182 EF1A Elongation factor 1-alpha OS = Zea mays X 183 Os07g0662500 Elongation factor 1-beta OS = Triticum aestivum XX X 184 Os03g0406200 Elongation factor 1-delta 2 OS = Oryza sativa subsp. japonica X 185 Os06g0571400 Elongation factor 1-gamma 3 OS = Oryza sativa subsp. japonica XX X 186 EMH2 Em protein H2 OS = Triticum aestivum X 187 EMH5 Em protein H5 OS = Triticum aestivum XX X 188 EM Em protein OS = Triticum aestivum XX X 189 RASI Endogenous alpha-amylase/subtilisin inhibitor OS = Triticum aestivum XX X 190 GLU4 Endoglucanase 11 OS = Oryza sativa subsp. japonica XX X 191 N/A Endoplasmin homolog OS = Hordeum vulgare XX X 192 ENO2 Enolase 2 OS = Zea mays XX X 193 ENO1 Enolase OS = Oryza sativa subsp. japonica XX X 194 Os08g0327400 Enoyl-[acyl-carrier-protein] reductase [NADH] 1 chloroplastic OS = Oryza sativa subsp. japonica XX X 195 Os09g0277800 Enoyl-[acyl-carrier-protein] reductase [NADH] 2 chloroplastic OS = Oryza sativa subsp. japonica XX X 196 Os02g0146600 Eukaryotic initiation factor 4A OS = Triticum aestivum X 197 Os06g0701100 Eukaryotic initiation factor 4A OS = Triticum aestivum XX X 198 TIF4A-2 Eukaryotic initiation factor 4A-2 OS = Arabidopsis thaliana X 199 EIF4A3A Eukaryotic initiation factor 4A-III homolog A OS = Oryza sativa subsp. japonica X 200 EIF4A3B Eukaryotic initiation factor 4A-III homolog B OS = Oryza sativa subsp. japonica X X 201 N/A Eukaryotic translation initiation factor 2 subunit beta OS = Triticum aestivum XX X 202 TIF3K1 Eukaryotic translation initiation factor 3 subunit K OS = Oryza sativa subsp. japonica XX X 203 EIF4B Eukaryotic translation initiation factor 4B1 OS = Triticum aestivum XX 204 N/A Eukaryotic translation initiation factor 4E-1 OS = Triticum aestivum X 205 N/A Eukaryotic translation initiation factor 4G OS = Triticum aestivum XX 206 TIF5A Eukaryotic translation initiation factor 5A OS = Zea mays X 207 Os04g0499300 Eukaryotic translation initiation factor isoform 4G-1 OS = Triticum aestivum XX X 208 GLU Ferredoxin-dependent glutamate synthase chloroplastic OS = Oryza sativa subsp. Japonica* XX X 209 GLSF Ferredoxin-dependent glutamate synthase chloroplastic OS = Zea mays X 210 N/A Ferredoxin-dependent glutamate synthase (Fragment) OS = Hordeum vulgare X 211 Os03g0784700 Ferredoxin–NADP reductase root isozyme chloroplastic OS = Oryza sativa subsp. japonica X 212 Os06g0486800 Formate dehydrogenase mitochondrial OS = Hordeum vulgare XX X 213 Os06g0486800 Formate dehydrogenase 1 mitochondrial OS = Oryza sativa subsp. japonica X 214 FRK1 Fructokinase-1 OS = Oryza sativa subsp. japonica XX X 215 OsI_04558 Fructose-1 6-bisphosphatase cytosolic OS = Oryza sativa subsp. indica X X 216 CFBP1 Fructose-1 6-bisphosphatase cytosolic OS = Oryza sativa subsp. japonica X 217 FBA1 Fructose-bisphosphate aldolase 1 cytoplasmic OS = Oryza sativa subsp. japonica XX X 218 FBA2 Fructose-bisphosphate aldolase 2 cytoplasmic OS = Oryza sativa subsp. japonica XX X 219 FAH Fumarylacetoacetase OS = Oryza sativa subsp. japonica XX X 220 OsI_17385 Gamma-aminobutyrate transaminase 1 mitochondrial OS = Oryza sativa subsp. indica X X 221 OSL2 Gamma-aminobutyrate transaminase 1 mitochondrial OS = Oryza sativa subsp. japonica X 222 N/A Gamma-gliadin (Fragment) OS = Triticum aestivum XX X (continued on next page)

8 A. Di Francesco, et al. Journal of Proteomics 211 (2020) 103530

Table 1 (continued)

N. Gene Description of unique gene product Russello TimiliaRB Simeto

223 N/A Gamma-gliadin B OS = Triticum aestivum XX X 224 N/A Gamma-gliadin B-I OS = Triticum aestivum XX X 225 N/A Gamma-gliadin OS = Triticum aestivum XX X 226 GME-2 GDP-mannose 3 5-epimerase 2 OS = Oryza sativa subsp. japonica X X 227 N/A Glucan endo-1 3-beta-glucosidase GI OS = Hordeum vulgare X 228 N/A Glucan endo-1 3-beta-glucosidase GIV OS = Hordeum vulgare XX X 229 Os05g0140800 Glucose and ribitol dehydrogenase homolog OS = Oryza sativa subsp. japonica XX X 230 AGP-L Glucose-1-phosphate adenylyltransferase large subunit chloroplastic/amyloplastic OS = Triticum aestivum X X X 231 N/A Glucose-1-phosphate adenylyltransferase large subunit 1 chloroplastic/amyloplastic OS = Hordeum vulgare X 232 AGPL2 Glucose-1-phosphate adenylyltransferase large subunit 2 cytosolic OS = Oryza sativa subsp. japonica X 233 AGP-S Glucose-1-phosphate adenylyltransferase small subunit chloroplastic/amyloplastic OS = Triticum aestivum X X X 234 AGPS1 Glucose-1-phosphate adenylyltransferase small subunit 1 chloroplastic/amyloplastic OS = Oryza sativa subsp. XX X japonica 235 AGPS2 Glucose-1-phosphate adenylyltransferase small subunit 2 chloroplastic/amyloplastic/cytosolic OS = Oryza sativa XX subsp. japonica 236 Os03g0776000 Glucose-6-phosphate isomerase cytosolic A OS = Oryza sativa subsp. japonica XX X 237 Os06g0256500 Glucose-6-phosphate isomerase cytosolic B OS = Oryza sativa subsp. japonica XX X 238 PHI1 Glucose-6-phosphate isomerase cytosolic OS = Zea mays XX X 239 GDH1 Glutamate dehydrogenase 1 mitochondrial OS = Oryza sativa subsp. indica X 240 GDH2 Glutamate dehydrogenase 2 mitochondrial OS = Oryza sativa subsp. indica XX X 241 GDH3 Glutamate dehydrogenase 3 mitochondrial OS = Oryza sativa subsp. japonica X 242 GSA Glutamate-1-semialdehyde 2 1-aminomutase chloroplastic OS = Hordeum vulgare XX X 243 GSH1–2 Glutamate–cysteine ligase B chloroplastic OS = Oryza sativa subsp. japonica XX X 244 GLN4 Glutamine synthetase root isozyme 3 OS = Zea mays XX 245 GLN5 Glutamine synthetase root isozyme 4 OS = Zea mays XX X 246 GRC2 Glutathione reductase cytosolic OS = Oryza sativa subsp. japonica X 247 GSTA1 Glutathione S-transferase 1 OS = Triticum aestivum XX X 248 GSTA2 Glutathione S-transferase 2 OS = Triticum aestivum XX X 249 N/A Glutenin high molecular weight subunit 12 OS = Triticum aestivum XX X 250 GLU-D1–2B Glutenin high molecular weight subunit DY10 OS = Triticum aestivum XX X 251 N/A Glutenin low molecular weight subunit 1D1 OS = Triticum aestivum XX X 252 N/A Glutenin low molecular weight subunit OS = Triticum aestivum XX X 253 N/A Glutenin low molecular weight subunit PTDUCD1 OS = Triticum aestivum XX X 254 GAPC Glyceraldehyde-3-phosphate dehydrogenase 1 cytosolic OS = Hordeum vulgare XX X 255 GAPC1 Glyceraldehyde-3-phosphate dehydrogenase 1 cytosolic OS = Oryza sativa subsp. japonica XX X 256 GAPC2 Glyceraldehyde-3-phosphate dehydrogenase 2 cytosolic OS = Oryza sativa subsp. japonica X 257 blt801 Glycine-rich RNA-binding protein blt801 OS = Hordeum vulgare XX X 258 WAXY Granule-bound starch synthase 1 chloroplastic/amyloplastic OS = Triticum aestivum XX X 259 RAN2 GTP-binding nuclear protein Ran-2 OS = Oryza sativa subsp. indica XX X 260 RACK1A Guanine nucleotide-binding protein subunit beta-like protein A OS = Oryza sativa subsp. japonica XX X 261 BIP1 Heat shock 70 kDa protein BIP1 OS = Oryza sativa subsp. japonica XX X 262 BIP2 Heat shock 70 kDa protein BIP2 OS = Oryza sativa subsp. japonica XX X 263 BIP5 Heat shock 70 kDa protein BIP5 OS = Oryza sativa subsp. japonica X 264 HSP70 Heat shock 70 kDa protein OS = Zea mays XX X 265 HSP81–1 Heat shock protein 81–1 OS = Oryza sativa subsp. indica XX X 266 HSP81–3 Heat shock protein 81–3 OS = Oryza sativa subsp. japonica XX X 267 HSP82 Heat shock protein 82 OS = Zea mays XX X 268 Os05g0150900 Histidine–tRNA ligase cytoplasmic OS = Oryza sativa subsp. japonica X 269 N/A Histone H1 OS = Triticum aestivum XX X 270 H2A-9 Histone H2A.1 OS = Triticum aestivum XX 271 Os01g0502700 Histone H2A.2.1 OS = Triticum aestivum* XX X 272 H2B.10 Histone H2B.1 OS = Triticum aestivum X 273 H2B.2 Histone H2B.2 OS = Oryza sativa subsp. indica X 274 H2B.3 Histone H2B.3 OS = Triticum aestivum* X 275 H2B.4 Histone H2B.4 OS = Oryza sativa subsp. indica X 276 N/A Histone H2B.5 OS = Triticum aestivum X 277 H2B.6 Histone H2B.6 OS = Oryza sativa subsp. indica X 278 H2B.7 Histone H2B.7 OS = Oryza sativa subsp. indica X 279 H2B.8 Histone H2B.8 OS = Oryza sativa subsp. indica X 280 H3.1–1 Histone H3.2 OS = Triticum aestivum XX X 281 OsI_011536 Histone H3.3 OS = Oryza sativa subsp. indica XX X 282 H3 Histone H3.3 OS = Oryza sativa subsp. japonica XX X 283 H4C7 Histone H4 OS = Zea mays XX X 284 SIH4 Histone H4 variant TH011 OS = Triticum aestivum XX X 285 H4 Histone H4.3 OS = Zea mays X 286 MNB1B HMG1/2-like protein OS = Triticum aestivum X X 287 HOP Hsp70-Hsp90 organizing protein OS = Triticum aestivum XX X 288 Os01g0253300 Importin subunit alpha-1a OS = Oryza sativa subsp. japonica XX X 289 Os05g0155601 Importin subunit alpha-1b OS = Oryza sativa subsp. japonica XX X 290 AO2 Indole-3-acetaldehyde oxidase OS = Zea mays X X 291 Os05g0125500 Isovaleryl-CoA dehydrogenase mitochondrial OS = Oryza sativa subsp. japonica XX X 292 Os05g0573700 Ketol-acid reductoisomerase chloroplastic OS = Oryza sativa subsp. japonica XX X 293 GLYI-11 Lactoylglutathione lyase OS = Oryza sativa subsp. japonica XX X 294 APX2 L-ascorbate peroxidase 2 cytosolic OS = Oryza sativa subsp. japonica X X 295 HVA1 Late embryogenesis abundant protein group 3 OS = Triticum aestivum XX X (continued on next page)

9 A. Di Francesco, et al. Journal of Proteomics 211 (2020) 103530

Table 1 (continued)

N. Gene Description of unique gene product Russello TimiliaRB Simeto

296 LEA1 Late embryogenesis abundant protein 1 OS = Oryza sativa subsp. indica X 297 LEA17 Late embryogenesis abundant protein 17 OS = Oryza sativa subsp. japonica XX X 298 B19.1A Late embryogenesis abundant protein B19.1A OS = Hordeum vulgare XX X 299 B19.1B Late embryogenesis abundant protein B19.1B OS = Hordeum vulgare X 300 B19.3 Late embryogenesis abundant protein B19.3 OS = Hordeum vulgare XX X 301 Os02g0794700 Leucine aminopeptidase 2 chloroplastic OS = Oryza sativa subsp. japonica XX X 302 LOX1.1 Linoleate 9S-lipoxygenase 1 OS = Hordeum vulgare XX 303 Os03g0699700 Linoleate 9S-lipoxygenase 1 OS = Oryza sativa subsp. japonica X 304 BIPE2 Luminal-binding protein 2 OS = Zea mays XX X 305 Os03g0586800 Lysine–tRNA ligase OS = Oryza sativa subsp. japonica XX X 306 Os10g0478200 Malate dehydrogenase cytoplasmic OS = Oryza sativa subsp. japonica XX X 307 Os12g0632700 Malate dehydrogenase glyoxysomal OS = Oryza sativa subsp. japonica XX X 308 LIP Malate synthase glyoxysomal OS = Zea mays X 309 MS Malate synthase OS = Oryza sativa subsp. japonica X 310 MSBP1 Membrane steroid-binding protein 1 OS = Oryza sativa subsp. japonica X 311 IDI2 Methylthioribose-1-phosphate isomerase OS = Hordeum vulgare XX X 312 OsI_35550 Methylthioribose-1-phosphate isomerase OS = Oryza sativa subsp. indica X 313 MDAR3 Monodehydroascorbate reductase 3 cytosolic OS = Oryza sativa subsp. japonica XX X 314 MDAR4 Monodehydroascorbate reductase 4 cytosolic OS = Oryza sativa subsp. japonica X X 315 MDAR5 Monodehydroascorbate reductase 5 chlorplastic OS = Oryza sativa subsp. japonica X X 316 NAD9 NADH dehydrogenase [ubiquinone] iron‑sulfur protein 3 OS = Oryza sativa subsp. japonica X 317 ME6 NADP-dependent malic enzyme chloroplastic OS = Oryza sativa subsp. japonica XX X 318 ME1 NADP-dependent malic enzyme OS = Phaseolus vulgaris X X 319 OsI_07785 NAP1-related protein 2 OS = Oryza sativa subsp. indica XX X 320 Os02g0576700 NAP1-related protein 2 OS = Oryza sativa subsp. japonica XX X 321 CPA N-carbamoylputrescine amidase OS = Oryza sativa subsp. japonica XX X 322 LTP1500 Non-specific lipid-transfer protein (Fragment) OS = Triticum aestivum XX X 323 N/A Non-specific lipid-transfer protein 2G OS = Triticum aestivum XX X 324 LTP2 Non-specific lipid-transfer protein 2P OS = Triticum aestivum XX X 325 NDKR Nucleoside diphosphate kinase 1 OS = Oryza sativa subsp. indica XX X 326 NAP1;2 Nucleosome assembly protein 1 X 327 YchF1 Obg-like ATPase 1 OS = Oryza sativa subsp. japonica XX X 328 OBAP1A Oil body-associated protein 1A OS = Zea mays XX X 329 OBAP2A Oil body-associated protein 2A OS = Zea mays XX X 330 OBAP2B Oil body-associated protein 2B OS = Zea mays XX X 331 CYP Peptidyl-prolyl cis-trans isomerase OS = Zea mays XX X 332 PRX112 Peroxidase 1 OS = Hordeum vulgare XX 333 PRXIIE-1 Peroxiredoxin-2E-1 chloroplastic OS = Oryza sativa subsp. japonica X 334 PRXIIE-2 Peroxiredoxin-2E-2 chloroplastic OS = Oryza sativa subsp. japonica XX X 335 PRXIIF Peroxiredoxin-2F mitochondrial OS = Oryza sativa subsp. japonica X 336 MFP Peroxisomal fatty acid beta-oxidation multifunctional protein OS = Oryza sativa subsp. japonica XX X 337 N/A Phosphoenolpyruvate carboxykinase (ATP) OS = Zea mays X 338 PEP1 Phosphoenolpyruvate carboxylase 1 OS = Zea mays X X 339 PEP4 Phosphoenolpyruvate carboxylase 2 OS = Zea mays XX X 340 PCKA Phosphoenolpyruvate carboxylase 3 OS = Sorghum bicolor XX 341 PGM2 Phosphoglycerate kinase chloroplastic OS = Triticum aestivum X X 342 PGK3 Phosphoglycerate kinase cytosolic OS = Triticum aestivum XX 343 PGM3 Phosphoglycerate kinase cytosolic OS = Triticum aestivum X 344 PLD1 Phospholipase D alpha 1 OS = Oryza sativa subsp. japonica XX X 345 Os05g0567100 Phytepsin OS = Hordeum vulgare XX X 346 PARP3 Poly [ADP-ribose] polymerase 3 OS = Oryza sativa subsp. japonica XX X 347 OsI_031067 Probable 6-phosphogluconolactonase 4 chloroplastic OS = Oryza sativa subsp. indica X 348 Os10g0138100 Probable aldehyde oxidase 1 OS = Oryza sativa subsp. japonica XX X 349 PIP2–6 Probable aquaporin PIP2–6 OS = Oryza sativa subsp. japonica X 350 TIP3–1 Probable aquaporin TIP3–1 OS = Oryza sativa subsp. japonica XX 351 CML7 Probable calcium-binding protein CML7 OS = Oryza sativa subsp. japonica XX X 352 CAD1 Probable cinnamyl alcohol dehydrogenase 1 OS = Oryza sativa subsp. japonica X 353 DHAR1 Probable glutathione S-transferase DHAR1 cytosolic OS = Oryza sativa subsp. japonica XX X 354 Os03g0162200 Probable histone H2A variant 1 OS = Oryza sativa subsp. japonica X X 355 Os03g0743400 Probable histone H2A variant 3 OS = Oryza sativa subsp. japonica XX X 356 OsI_26380 Probable histone H2A.1 OS = Oryza sativa subsp. indica X 357 Os07g0545300 Probable histone H2A.1 OS = Oryza sativa subsp. japonica X 358 UAM2 Probable inactive UDP-arabinopyranose mutase 2 OS = Oryza sativa subsp. japonica XX X 359 APX6 Probable L-ascorbate peroxidase 6 chloroplastic/mitochondrial OS = Oryza sativa subsp. japonica XX X 360 AGD2 Probable LL-diaminopimelate aminotransferase chloroplastic OS = Oryza sativa subsp. japonica XX X 361 Os06g0508700 Probable methionine–tRNA ligase OS = Oryza sativa subsp. japonica X 362 Os03g0617900 Probable N-acetyl-gamma-glutamyl-phosphate reductase chloroplastic OS = Oryza sativa subsp. japonica X X X 363 VIP3 Probable prefoldin subunit 4 OS = Avena fatua X 364 Os02g0149800 Probable protein phosphatase 2C 10 OS = Oryza sativa subsp. japonica XX X 365 Os06g0698300 Probable protein phosphatase 2C 59 OS = Oryza sativa subsp. japonica XX X 366 PDX11 Probable pyridoxal 5′-phosphate synthase subunit PDX1.1 OS = Oryza sativa subsp. japonica XX X 367 SPS1 Probable sucrose-phosphate synthase 1 OS = Oryza sativa subsp. indica X 368 SPS4 Probable sucrose-phosphate synthase 4 OS = Oryza sativa subsp. japonica XX X 369 VTE1 Probable tocopherol cyclase chloroplastic OS = Oryza sativa subsp. japonica X 370 UPTG Probable UDP-arabinopyranose mutase 1 OS = Zea mays XX X (continued on next page)

10 A. Di Francesco, et al. Journal of Proteomics 211 (2020) 103530

Table 1 (continued)

N. Gene Description of unique gene product Russello TimiliaRB Simeto

371 UAH Probable ureidoglycolate hydrolase OS = Oryza sativa subsp. japonica XX 372 KOB1 Probable voltage-gated potassium channel subunit beta OS = Oryza sativa subsp. japonica XX X 373 PRO1 Profilin-1 OS = Triticum aestivum XX X 374 PRO2 Profilin-2 OS = Triticum aestivum XX X 375 Os02g0805200 Proliferating cell nuclear antigen OS = Oryza sativa subsp. japonica XX X 376 PAF1 Proteasome subunit alpha type-1 OS = Oryza sativa subsp. japonica XX X 377 PAB1 Proteasome subunit alpha type-2 OS = Oryza sativa subsp. indica XX X 378 OsI_021120 Proteasome subunit alpha type-4-1 OS = Oryza sativa subsp. indica X X 379 PAC1A Proteasome subunit alpha type-4-1 OS = Oryza sativa subsp. japonica X X 380 OsI_021067 Proteasome subunit alpha type-4-2 OS = Oryza sativa subsp. indica XX 381 Os06g0167600 Proteasome subunit alpha type-4-2 OS = Oryza sativa subsp. japonica XX 382 Os06g0177100 Proteasome subunit alpha type-4-3 OS = Oryza sativa subsp. japonica X X 383 PAE1 Proteasome subunit alpha type-5 OS = Oryza sativa subsp. japonica XX X 384 PAA1 Proteasome subunit alpha type-6 OS = Oryza sativa subsp. japonica XX X 385 OsI_029135 Proteasome subunit alpha type-7-A OS = Oryza sativa subsp. indica XX X 386 Os08g0548900 Proteasome subunit alpha type-7-A OS = Oryza sativa subsp. japonica XX X 387 PAD1 Proteasome subunit alpha type-7-B OS = Oryza sativa subsp. indica XX X 388 PBF1 Proteasome subunit beta type-1 OS = Oryza sativa subsp. japonica XX X 389 PBD1 Proteasome subunit beta type-2 OS = Oryza sativa subsp. japonica XX X 390 PDIL1–1 Protein disulfide isomerase-like 1–1 OS = Oryza sativa subsp. japonica X 391 PDIL2–1 Protein disulfide isomerase-like 2–1 OS = Oryza sativa subsp. japonica XX X 392 PDIL2–2 Protein disulfide isomerase-like 2–2 OS = Oryza sativa subsp. japonica XX X 393 PDIL2–3 Protein disulfide isomerase-like 2–3 OS = Oryza sativa subsp. japonica XX X 394 PDI Protein disulfide-isomerase OS = Triticum aestivum XX X 395 H2A-4 Protein H2A.7 OS = Triticum aestivum XX 396 RIP30A Protein synthesis inhibitor II OS = Hordeum vulgare XX X 397 TPR1 Protein TPR1 OS = Oryza sativa subsp. japonica X 398 PCM Protein-L-isoaspartate O-methyltransferase OS = Triticum aestivum XX X 399 Os08g0562700 Puromycin-sensitive aminopeptidase OS = Oryza sativa subsp. japonica XX 400 THI1.3 Purothionin A-1 OS = Triticum aestivum XX X 401 Os08g0191100 Putative aconitate hydratase cytoplasmic OS = Oryza sativa subsp. japonica XX X 402 Os02g0209000 Putative coatomer subunit beta’-3 OS = Oryza sativa subsp. japonica X X 403 Os02g0773300 Putative D-cysteine desulfhydrase 1 mitochondrial OS = Oryza sativa subsp. japonica X 404 PPDK1 Pyruvate phosphate dikinase 1 chloroplastic OS = Oryza sativa subsp. japonica XX X 405 PPDK2 Pyruvate phosphate dikinase 2 OS = Oryza sativa subsp. japonica XX X 406 PDC2 Pyruvate decarboxylase 2 OS = Oryza sativa subsp. indica XX X 407 PDC3 Pyruvate decarboxylase 3 OS = Oryza sativa subsp. indica X 408 Os02g0739600 Pyruvate dehydrogenase E1 component subunit alpha-1 mitochondrial OS = Oryza sativa subsp. japonica X 409 Os06g0246500 Pyruvate dehydrogenase E1 component subunit alpha-2 mitochondrial OS = Oryza sativa subsp. japonica X X X 410 Os08g0536000 Pyruvate dehydrogenase E1 component subunit beta-1 mitochondrial OS = Oryza sativa subsp. japonica X X X 411 Os09g0509200 Pyruvate dehydrogenase E1 component subunit beta-2 mitochondrial OS = Oryza sativa subsp. japonica X X X 412 Os03g0645100 Pyruvate dehydrogenase E1 component subunit beta-4 chloroplastic OS = Oryza sativa subsp. japonica X 413 OsI_35105 Pyruvate kinase 1 cytosolic OS = Oryza sativa subsp. indica X 414 Os11g0148500 Pyruvate kinase 1 cytosolic OS = Oryza sativa subsp. japonica X 415 RIC1 Ras-related protein RIC1 OS = Oryza sativa subsp. japonica XX 416 RIC2 Ras-related protein RIC2 OS = Oryza sativa subsp. japonica X 417 RIDA Reactive Intermediate Deaminase A chloroplastic OS = Arabidopsis thaliana X 418 Os02g0714600 Ribose-phosphate pyrophosphokinase 4 OS = Oryza sativa subsp. japonica XX X 419 R40C1 Ricin B-like lectin R40C1 OS = Oryza sativa subsp. japonica XX X 420 R40G2 Ricin B-like lectin R40G2 OS = Oryza sativa subsp. japonica XX X 421 R40G3 Ricin B-like lectin R40G3 OS = Oryza sativa subsp. japonica XX X 422 N/A RuBisCO large subunit-binding protein subunit alpha chloroplastic (Fragment) OS = Triticum aestivum XX X 423 CPN60 RuBisCO large subunit-binding protein subunit beta chloroplastic (Fragment) OS = Secale cereale XX X 424 SAMS1 S-adenosylmethionine synthase 1 OS = Triticum monococcum X X 425 SAMS S-adenosylmethionine synthase OS = Triticum aestivum XX X 426 CBP2 Serine carboxypeptidase 2 OS = Triticum aestivum XX X 427 CBP3 Serine carboxypeptidase 3 OS = Triticum aestivum XX X 428 CXP;2–1 Serine carboxypeptidase II-1 (Fragment) OS = Hordeum vulgare X 429 CBP31 Serine carboxypeptidase-like OS = Oryza sativa subsp. japonica XX X 430 SAPK7 Serine/threonine-protein kinase SAPK7 OS = Oryza sativa subsp. japonica X X 431 BSL2 Serine/threonine-protein phosphatase BSL2 homolog OS = Oryza sativa subsp. japonica X 432 Os03g0268000 Serine/threonine-protein phosphatase PP1 OS = Oryza sativa subsp. japonica X 433 PP2A1 Serine/threonine-protein phosphatase PP2A-1 catalytic subunit OS = Oryza sativa subsp. indica X 434 PP2A3 Serine/threonine-protein phosphatase PP2A-3 catalytic subunit OS = Oryza sativa subsp. indica X X 435 PP2A4 Serine/threonine-protein phosphatase PP2A-4 catalytic subunit OS = Oryza sativa subsp. japonica X X 436 WZCI Serpin-Z1A OS = Triticum aestivum XX X 437 PAZX Serpin-ZX OS = Hordeum vulgare XX X 438 Os03g0610650 Serpin-ZXA OS = Oryza sativa subsp. japonica XX X 439 SKP1 SKP1-like protein 1 OS = Oryza sativa subsp. japonica XX X 440 HSP21 Small heat shock protein chloroplastic OS = Triticum aestivum XX X 441 SUMO1 Small ubiquitin-related modifier 1 OS = Oryza sativa subsp. japonica X 442 IPP Soluble inorganic pyrophosphatase OS = Hordeum vulgare subsp. vulgare XX X 443 SPDSYN1 Spermidine synthase 1 OS = Oryza sativa subsp. japonica XX X 444 U2AF65A Splicing factor U2af large subunit A OS = Oryza sativa subsp. japonica X 445 U2AF65B Splicing factor U2af large subunit B OS = Triticum aestivum X (continued on next page)

11 A. Di Francesco, et al. Journal of Proteomics 211 (2020) 103530

Table 1 (continued)

N. Gene Description of unique gene product Russello TimiliaRB Simeto

446 WSSI-2 Starch synthase 1 chloroplastic/amyloplastic OS = Triticum aestivum X 447 N/A Subtilisin-chymotrypsin inhibitor WSCI OS = Triticum aestivum XX X 448 SDH1 Succinate dehydrogenase [ubiquinone] flavoprotein subunit mitochondrial OS = Oryza sativa subsp. japonica X X X 449 Os07g0577700 Succinate–CoA ligase [ADP-forming] subunit alpha mitochondrial OS = Oryza sativa subsp. japonica XX X 450 Os02g0621700 Succinate–CoA ligase [ADP-forming] subunit beta mitochondrial OS = Oryza sativa subsp. japonica XX X 451 ALDH5F1 Succinate-semialdehyde dehydrogenase mitochondrial OS = Oryza sativa subsp. japonica XX X 452 SS1 Sucrose synthase 1 OS = Hordeum vulgare XX X 453 SS2 Sucrose synthase 2 OS = Hordeum vulgare XX X 454 SUS3 Sucrose synthase 3 OS = Oryza sativa subsp. japonica XX X 455 SUS4 Sucrose synthase 4 OS = Oryza sativa subsp. japonica XX X 456 SPP2 Sucrose-phosphatase 2 OS = Oryza sativa subsp. japonica XX X 457 SODA.4 Superoxide dismutase [Mn] 3.1 mitochondrial OS = Zea mays XX X 458 CCT5 T-complex protein 1 subunit epsilon OS = Avena sativa XX X 459 N/A Thioredoxin H-type OS = Triticum aestivum XX X 460 TKL-2 Transketolase chloroplastic OS = Zea mays XX X 461 TCTP Translationally-controlled tumor protein homolog OS = Triticum aestivum XX X 462 OsI_18044 Transportin-1 OS = Oryza sativa subsp. indica XX X 463 TRN1 Transportin-1 OS = Oryza sativa subsp. japonica XX X 464 ROMT-17 Tricin synthase 2 OS = Oryza sativa subsp. japonica XX 465 TPIP1 Triosephosphate isomerase chloroplastic OS = Secale cereale X 466 TPI Triosephosphate isomerase cytosolic OS = Hordeum vulgare XX X 467 TPP2 Tripeptidyl-peptidase 2 OS = Oryza sativa subsp. japonica XX X 468 N/A Trypsin/alpha-amylase inhibitor CMX1/CMX3 OS = Triticum aestivum XX X 469 N/A Trypsin/alpha-amylase inhibitor CMX2 OS = Triticum aestivum XX X 470 TUBA Tubulin alpha chain OS = Triticum aestivum X 471 TUBA2 Tubulin alpha-2 chain OS = Hordeum vulgare XX 472 TUBA3 Tubulin alpha-3 chain OS = Hordeum vulgare X 473 TUBA5 Tubulin alpha-5 chain OS = Zea mays XX X 474 TUBA6 Tubulin alpha-6 chain OS = Zea mays XX X 475 TUBB Tubulin beta chain OS = Hordeum vulgare X X 476 TUBB2 Tubulin beta-2 chain OS = Triticum aestivum X 477 TUBB3 Tubulin beta-3 chain OS = Triticum aestivum X 478 TUBB5 Tubulin beta-5 chain OS = Triticum aestivum X 479 MUB1 Ubiquitin-40S ribosomal protein S27a OS = Hordeum vulgare XX X 480 MUB2 Ubiquitin-40S ribosomal protein S27a OS = Hordeum vulgare XX X 481 RPS27AA Ubiquitin-40S ribosomal protein S27a-1 OS = Oryza sativa subsp. japonica X 482 Ub-CEP52–1 Ubiquitin-60S ribosomal protein L40–1 OS = Oryza sativa subsp. japonica XX X 483 Ub-CEP52–2 Ubiquitin-60S ribosomal protein L40–2 OS = Oryza sativa subsp. japonica XX X 484 UBA1 Ubiquitin-activating enzyme E1 1 OS = Triticum aestivum X 485 UBA2 Ubiquitin-activating enzyme E1 2 OS = Triticum aestivum X 486 UBA3 Ubiquitin-activating enzyme E1 3 OS = Triticum aestivum XX X 487 UBC5B Ubiquitin-conjugating enzyme E2 5B OS = Oryza sativa subsp. japonica XX X 488 UBC7 Ubiquitin-conjugating enzyme E2 7 OS = Triticum aestivum X 489 Os01g0962400 Ubiquitin-fold modifier 1 OS = Oryza sativa subsp. japonica XX X 490 OsI_33008 Ubiquitin-fold modifier-conjugating enzyme 1 OS = Oryza sativa subsp. indica X 491 Os10g0205200 Ubiquitin-fold modifier-conjugating enzyme 1 OS = Oryza sativa subsp. japonica X 492 Os02g0506500 Ubiquitin-like modifier-activating enzyme 5 OS = Oryza sativa subsp. japonica X X 493 RUB1 Ubiquitin-NEDD8-like protein RUB1 OS = Oryza sativa subsp. japonica XX X 494 RUB2 Ubiquitin-NEDD8-like protein RUB2 OS = Oryza sativa subsp. japonica XX X 495 UAM1 UDP-arabinopyranose mutase 1 OS = Oryza sativa subsp. japonica XX X 496 Os01g0969100 UDP-D-apiose/UDP-D-xylose synthase OS = Oryza sativa subsp. japonica XX X 497 UGD1 UDP-glucose 6-dehydrogenase 1 OS = Oryza sativa subsp. japonica X 498 UGD4 UDP-glucose 6-dehydrogenase 4 OS = Oryza sativa subsp. japonica X X 499 UGD5 UDP-glucose 6-dehydrogenase 5 OS = Oryza sativa subsp. japonica X 500 USP UDP-sugar pyrophosphorylase OS = Oryza sativa subsp. indica X 501 UREG Urease accessory protein G OS = Oryza sativa subsp. indica XX X 502 UMPS1 Uridine 5′-monophosphate synthase OS = Oryza sativa subsp. japonica XX X 503 UMPS2 Uridine 5′-monophosphate synthase OS = Oryza sativa subsp. japonica X 504 N/A UTP–glucose-1-phosphate uridylyltransferase OS = Hordeum vulgare XX X 505 CVA69.24 V-type proton ATPase catalytic subunit A (Fragment) OS = Hordeum vulgare X X 506 VHA-A V-type proton ATPase catalytic subunit A (Fragment) OS = Hordeum vulgare X 507 CVA69.25 V-type proton ATPase catalytic subunit A (Fragment) OS = Zea mays X 508 VHA-B1 V-type proton ATPase catalytic subunit A (Fragment) OS = Zea mays X 509 CVA69.26 V-type proton ATPase subunit B 1 OS = Hordeum vulgare X 510 VHA-B2 V-type proton ATPase subunit B 1 OS = Hordeum vulgare X 511 VATC V-type proton ATPase subunit C OS = Hordeum vulgare XX X 512 PR4A Wheatwin-1 OS = Triticum aestivum XX X 513 PR4B Wheatwin-2 OS = Triticum aestivum XX X 514 XIPI Xylanase inhibitor protein 1 OS = Triticum aestivum XX X 515 XYLA Xylose isomerase OS = Hordeum vulgare XX X 516 Os03g0733400 Zinc finger BED domain-containing protein RICESLEEPER 2 OS = Oryza sativa subsp. japonica X

12 A. Di Francesco, et al. Journal of Proteomics 211 (2020) 103530

Table 2 List of proteins identified in the CM-like fractions of the three investigated genotypes in the present study: accession number and description.

Accession Description Russello TimiliaRB Simeto

P01085 Alpha-amylase inhibitor 0.19 OS = Triticum aestivum X X X P01083 Alpha-amylase inhibitor 0.28 OS = Triticum aestivum X X X P01084 Alpha-amylase inhibitor 0.53 OS = Triticum aestivum X X X P10846 Alpha-amylase inhibitor WDAI-3 (Fragment) OS = Triticum aestivum X X X P16850 Alpha-amylase/trypsin inhibitor CM1 OS = Triticum aestivum X P16159 Alpha-amylase/trypsin inhibitor CM16 OS = Triticum aestivum X X X P16851 Alpha-amylase/trypsin inhibitor CM2 OS = Triticum aestivum X X X P17314 Alpha-amylase/trypsin inhibitor CM3 OS = Triticum aestivum X X X P11643 Alpha-amylase/trypsin inhibitor CMd OS=Hordeum vulgare X P16347 Endogenous alpha-amylase/subtilisin inhibitor OS = Triticum aestivum X X X Q43723 Trypsin/alpha-amylase inhibitor CMX1/CMX3 OS = Triticum aestivum X X X Q43691 Trypsin/alpha-amylase inhibitor CMX2 OS = Triticum aestivum X X X

durum wheat landraces traditionally cultivated in some areas of Sicily analysis of mature cereal kernels, Mass Spectrom. Rev. 31 (2012) 448–465, https:// and used in the production of bread and pasta. The qualitative pro- doi.org/10.1002/mas.20347. [7] V. Cunsolo, S. Foti, R. Saletti, Mass spectrometry in the characterisation of cereal teomic analysis of these durum wheat varieties revealed a remarkable seed proteins, Eur. J. Mass Spectrom. 10 (2004) 359–370, https://doi.org/10.1255/ similarity in the protein composition between old and modern geno- ejms.609. types. These results lead to the conclusion that there is a “substantial [8] S. Foti, G. Maccarrone, R. Saletti, P. Roepstorff, S. Gilbert, A. Tatham, P. Shewry, ” Verification of the cDNA deduced sequence of Glutenin subunit 1Dx5 and an Mr58 equivalence between old and modern varieties, despite some minor 000 repetitive peptide by matrix-assisted laser desorption/ionisation mass spec- differences. trometry (MALDI-MS), J. Cereal Sci. 3 (2000) 173–183, https://doi.org/10.1006/ This study represents the first comprehensive proteomic profile and jcrs.1999.0292. qualitative comparison at the molecular level of the metabolic and CM- [9] V. Cunsolo, S. Foti, R. Saletti, S. Gilbert, A.S. Tatham, P.R. Shewry, Structural studies of glutenin subunits 1Dy10 and 1Dy12 by matrix-assisted laser desorption/ like protein fractions of the old Sicilian landraces Russello and Timilia ionisation mass spectrometry and high-performance liquid chromatography/elec- RB with respect to a modern cultivar. The outcome of this comparison trospray ionisation mass spectrometry, Rapid Commun. Mass Spectrom. 17 (2003) – helps to understand the relationship between protein profiles of old 442 454, https://doi.org/10.1002/rcm.938. fi [10] V. Cunsolo, S. Foti, R. Saletti, S. Gilbert, A.S. Tatham, P.R. Shewry, Investigation wheat genotypes and modern ones and illustrates the potential bene ts and correction of the gene-derived sequence of glutenin subunit 1Dx2 by matrix- for human consumption. Further investigation by in vivo or in vitro assisted laser desorption/ionisation mass spectrometry, Rapid Commun. Mass tests of fractions of known protein components to evaluate their specific Spectrom. 16 (2002) 1911–1918, https://doi.org/10.1002/rcm.807. ff [11] V. Cunsolo, S. Foti, R. Saletti, S. Gilbert, A.S. Tatham, P.R. Shewry, Structural immune stimulatory e ects may provide more detailed insight on this studies of the allelic wheat glutenin subunits 1Bx7 and 1Bx20 by matrix-assisted aspect. laser desorption/ionization mass spectrometry and high-performance liquid chro- Supplementary data to this article can be found online at https:// matography/electrospray ionization mass spectrometry, J. Mass Spectrom. 39 (2004) 66–78, https://doi.org/10.1002/jms.558. doi.org/10.1016/j.jprot.2019.103530. [12] V. Muccilli, V. Cunsolo, R. Saletti, S. Foti, S. Masci, D. Lafiandra, Characterization of B- and C-type low molecular weight glutenin subunits by electrospray ionization Declaration of Competing Interest mass spectrometry and matrix-assisted laser desorption/ionization mass spectro- metry, Proteomics 5 (2005) 719–728, https://doi.org/10.1002/pmic.200401029. [13] G. Mamone, S. De Caro, A. Di Luccia, F. Addeo, P. Ferranti, Proteomic-based ana- The authors declare that they have no conflicts of interest with the lytical approach for the characterization of glutenin subunits in durum wheat, J. contents of this article. Mass Spectrom. 44 (2009) 1709–1723, https://doi.org/10.1002/jms.1680. [14] F. Scossa, D. Laudencia-Chingcuanco, O.D. Anderson, W.H. Vensel, D. Lafiandra, R. D'Ovidio, S. Masci, Comparative proteomic and transcriptional profiling of a Acknowledgments bread wheat cultivar and its derived transgenic line overexpressing a low molecular weight glutenin subunit gene in the endosperm, Proteomics 8 (2008) 2948–2966, The mass spectrometry proteomics data have been deposited to the https://doi.org/10.1002/pmic.200700861. [15] V. Muccilli, V. Cunsolo, R. Saletti, S. Foti, B. Margiotta, F. Scossa, S. Masci, ProteomeXchange Consortium via the PRIDE [51] partner repository D. Lafiandra, Characterisation of a specific class of typical low molecular weight with the dataset identifier PXD014449. The authors gratefully ac- glutenin subunits of durum wheat by a proteomic approach, J. Cereal Sci. 51 (2010) 134–139, https://doi.org/10.1016/j.jcs.2009.11.003. knowledge the Bio-Nanotech Research and Innovation Tower (BRIT; fi fi [16] F. Sestili, F. Paoletti, E. Botticella, S. Masci, R. Saletti, V. Muccilli, D. La andra, PON project nanced by the Italian Ministry for Education, University Comparative proteomic analysis of kernel proteins of two high amylose transgenic and Research MIUR). This research was partly supported by “Piano durum wheat lines obtained by biolistic and Agrobacterium-mediated transforma- della Ricerca di Ateneo 2016–2018” of the University of Catania, Italy. tions, J. Cereal Sci. 58 (2013) 15–22, https://doi.org/10.1016/j.jcs.2013.05.001. [17] F. Sestili, R. Pagliarello, A. Zega, R. Saletti, A. Pucci, E. Botticella, S. Masci, S. Tundo, I. Moscetti, S. Foti, D. Lafiandra, Enhancing grain size in durum wheat References using RNAi to knockdown GW2 genes, Theor. Appl. Genet. 132 (2019) 419–429, https://doi.org/10.1007/s00122-018-3229-9. [18] G. Vici, L. Belli, M. Biondi, V. Polzonetti, Gluten free diet and nutrient deficiencies: [1] J. Mattei, V. Malik, N.M. Wedick, F.B. Hu, D. Spiegelman, W.C. Willett, H. Campos, a review, Clin. Nutr. 35 (2016) 1236–1241, https://doi.org/10.1016/j.clnu.2016. reducing the global burden of type 2 diabetes by improving the quality of staple 05.002. foods: the global nutrition and epidemiologic transition initiative, globalization and [19] A. Sapone, J.C. Bai, C. Ciacci, J. Dolinsek, P.H.R. Green, M. Hadjivassiliou, health 11T, Global Nutr. Epidemiol. 23 (2015), https://doi.org/10.1186/s12992- K. Kaukinen, K. Rostami, D.S. Sanders, M. Schumann, R. Ullrich, D. Villalta, 015-0109-9. U. Volta, C. Catassi, A. Fasano, Spectrum of gluten-related disorders: consensus on [2] P.R. Shewry, S.J. Hey, The contribution of wheat to human diet and health, Food. new nomenclature and classification, BMC Med. 10 (2012), https://doi.org/10. Energy Sec. 4 (2015) 178–202, https://doi.org/10.1002/fes3.64. 1186/1741-7015-10-13. [3] Z. Šramkováa, E. Gregováb, E. Šturdík, Chemical composition and nutritional [20] B.P. McAllister, K. Clarke, E. Williams, A comprehensive review of celiac disease/ quality of wheat grain, Acta Chim. Slov. 2 (2009) 115–138. gluten-sensitive enteropathies, Clin. Rev. Allergy Immunol. (2018) 1–18, https:// [4] Public Health England, Results of the National Diet and Nutrition Survey (NDNS) doi.org/10.1007/s12016-018-8691-2. Rolling Programme for 2008 and 2009 to 2011 and 2012, https://www.gov.uk/ [21] M.D.E. Potter, M.M. Walker, N.J. Talley, Non-coeliac gluten or wheat sensitivity: government/statistics/national-diet-andnutrition- survey-results-from-years-1-to-4- emerging disease or misdiagnosis? Med. J. Aust. 207 (2017) 211–215, https://doi. combined-of-the-rollingprogramme-for-2008-and-2009-to-2011-and-2012 (11/04/ org/10.5694/mja17.00332. 2019). [22] A. Sapone, K.M. Lammers, V. Casolaro, M. Cammarota, M.T. Giuliano, M. De Rosa, [5] V. Cunsolo, V. Muccilli, R. Saletti, S. Foti, Mass spectrometry in food proteomics: a R. Stefanile, G. Mazzarella, C. Tolone, M.I. Russo, P. Esposito, F. Ferraraccio, tutorial, J. Mass Spectrom. 49 (2014) 768–784, https://doi.org/10.1002/jms.3374. M. Carteni, G. Riegler, L. de Magistris, A. Fasano, Divergence of gut permeability [6] V. Cunsolo, V. Muccilli, R. Saletti, S. Foti, Mass spectrometry in the proteome

13 A. Di Francesco, et al. Journal of Proteomics 211 (2020) 103530

and mucosal immune gene expression in two gluten-associated conditions: celiac peptides and potential prebiotic carbohydrates in old and modern Triticum turgidum disease and gluten sensitivity, BMC Med. 9 (2011) 23, https://doi.org/10.1186/ ssp. genotypes, Food Res. Int. 120 (2019) 568–576, https://doi.org/10.1016/j. 1741-7015-9-23. foodres.2018.11.007. [23] A. Fardet, Wheat-based foods and non celiac gluten/wheat sensitivity: is drastic [39] W.J. Hurkman, C.K. Tanaka, Extraction of wheat endosperm proteins for proteome processing the main key issue? Med. Hypotheses 85 (2015) 934–939, https://doi. analysis, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 849 (2007) 344–350, org/10.1016/j.mehy.2015.09.007. https://doi.org/10.1016/j.jchromb.2006.11.047. [24] M.D. Garcia-Molina, V. Muccilli, R. Saletti, S. Foti, S. Masci, F. Barro, Comparative [40] R. Saletti, S. Reina, M.G.G. Pittalà, A. Magrì, V. Cunsolo, S. Foti, V. De Pinto, Post- proteomic analysis of two transgenic low-gliadin wheat lines and non-transgenic translational modifications of VDAC1 and VDAC2 cysteines from rat liver mi- wheat control, J. Proteome 165 (2017) 102–112, https://doi.org/10.1016/j.jprot. tochondria, BBA-Bioenergetics 1859 (2018) 806–816, https://doi.org/10.1016/j. 2017.06.010. bbabio.2018.06.007. [25] G. Salcedo, S. Quirce, A. Diaz-Perales, Wheat allergens associated with Baker’s [41] I. Ortea, G. O’Connor, A. Maquet, Review on proteomics for food authentication, J. asthma, J Investig. Allergol. Clin. Immunol. 21 (2011) 81–92. Proteome 147 (2016) 212–225, https://doi.org/10.1016/j.jprot.2016.06.033. [26] A.S. Tatham, P.R. Shewry, Allergens in wheat and related cereals, Clin. Exp. Allergy [42] H.H. Chen, P. Chu, Y.L. Zhou, Y. Ding, Y. Li, J. Liu, L.W. Jiang, S.Z. Huang, Ectopic 38 (2008) 1712–1726, https://doi.org/10.1111/j.1365-2222.2008.03101.x. expression of NnPER1, a Nelumbo nucifera 1-cysteine peroxiredoxin antioxidant, [27] A. Capocchi, V. Muccilli, V. Cunsolo, R. Saletti, S. Foti, D. Fontanini, A hetero- enhances seed longevity and stress tolerance in Arabidopsis, Plant J. 88 (2016) tetrameric alpha-amylase inhibitor from emmer (Triticum dicoccon Schrank) seeds, 608–619, https://doi.org/10.1111/tpj.13286. Phytochemistry 88 (2013) 6–14, https://doi.org/10.1016/j.phytochem.2012.12. [43] C. Haslekas, M.K. Viken, P.E. Grini, V. Nygaard, S.H. Nordgard, T.J. Meza, 010. R.B. Aalen, Seed 1-cysteine peroxiredoxin antioxidants are not involved in dor- [28] A. Palacin, S. Quirce, A. Armentia, M. Fernandez-Nieto, L.F. Pacios, T. Asensio, mancy, but contribute to inhibition of germination during stress, Plant Physiol. 133 J. Sastre, A. Diaz-Perales, G. Salcedo, Wheat lipid transfer protein is a major al- (2003) 1148–1157, https://doi.org/10.1104/pp.103.025916. lergen associated with baker’s asthma, J. Allergy Clin. Immunol. 120 (2007) [44] A. Verlotta, V. De Simone, A.M. Mastrangelo, L. Cattivelli, R. Papa, D. Trono, 1132–1138, https://doi.org/10.1016/j.jaci.2007.07.008. Insight into durum wheat Lpx-B1: a small gene family coding for the lipoxygenase [29] A. Capocchi, D. Fontanini, V. Muccilli, V. Cunsolo, F. Saviozzi, R. Saletti, R. Lorenzi, responsible for carotenoid bleaching in mature grains, BMC Plant Biol. 10 (2010) S. Foti, L. Galleschi, NsLTP1 and NsLTP2 isoforms in soft wheat (Triticum aestivum 263–280, https://doi.org/10.1186/1471-2229-10-263. cv. centauro) and farro (Triticum dicoccon Schrank) bran, J. Agric. Food Chem. 53 [45] T. Wang, E.H. Zhang, X.P. Chen, L. Li, X.Q. Liang, Identification of seed proteins (2005) 7976–7984, https://doi.org/10.1021/jf0580465. associated with resistance to pre-harvested aflatoxin contamination in peanut [30] C. Constantin, S. Quirce, M. Grote, A. Touraev, I. Swoboda, A. Stoecklinger, A. Mari, (Arachis hypogaea L), BMC Plant Biol. 10 (2010) 267, https://doi.org/10.1186/ J. Thalhamer, E. Heberle-Bors, R. Valenta, Molecular and immunological char- 1471-2229-10-267. acterization of a wheat serine proteinase inhibitor as a novel allergen in baker’s [46] K.-X. Gu, X.-S. Song, X.X.-X.D. Xue-Mei, W.Y.-B.D. Jian-Xin, Y.-P. Hou, M.-G. Zhou,

asthma, J. Immunol. 180 (2008) 7451–7460, https://doi.org/10.4049/jimmunol. A β 2 -tubulin dsRNA derived from Fusarium asiaticum confers plant resistance to 180.11.7451. multiple phytopathogens and reduces fungicide resistance, Pestic. Biochem. [31] Z. Kohajdová, J. Karovičová, Nutrional value and baking applications of spelt Physiol. 153 (2019) 36–46, https://doi.org/10.1016/j.pestbp.2018.10.005. wheat, Acta Sci. Pol. Technol. Aliment. 7 (2008) 5–14. [47] E.S. Xu, M.M. Chen, H. He, C.F. Zhan, Y.H. Cheng, H.S. Zhang, Z.F. Wang, [32] A. Hidalgo, A. Brandolini, Lipoxygenase activity in wholemeal flours from Triticum Proteomic analysis reveals proteins involved in seed imbibition under salt stress in monococcum, Triticum turgidum and Triticum aestivum, Food Chem. 131 (2012) Rice, Front. Plant Sci. 7 (2017), https://doi.org/10.3389/fpls.2016.02006. 1499–1503, https://doi.org/10.1016/j.foodchem.2011.09.132. [48] X.W. Zhang, S.Y. Li, L.L. Zhang, Q. Yang, Q.T. Jiang, J. Ma, P.F. Qi, W. Li, [33] M. Dinu, A. Whittaker, G. Pagliai, S. Benedettelli, F. Sofi, Ancient wheat species and G.Y. Chen, X.J. Lan, M. Deng, Z.X. Lu, C.J. Liu, Y.M. Wei, Y.L. Zheng, Structure and human health: biochemical and clinical implications, J. Nutr. Biochem. 52 (2018) expression analysis of genes encoding ADP-glucose pyrophosphorylase large sub- 1–9, https://doi.org/10.1016/j.jnutbio.2017.09.001. unit in wheat and its relatives, Genome 59 (2016) 501–507, https://doi.org/10. [34] P.R. Shewry, S. Hey, Do “ancient” wheat species differ from modern bread wheat in 1139/gen-2016-0007. their contents of bioacive components? J. Cereal Sci. 65 (2015) 236–243, https:// [49] C.K. Castleden, N.A. Aoki, V.J. Gillespie, E.A. MacRae, W.P. Quick, P. Buchner, doi.org/10.1016/j.jcs.2015.07.0104. C.H. Foyer, R.T. Furbank, J.E. Lunn, Evolution and function of the sucrose-phos- [35] P.R. Shewry, Do ancient types of wheat have health benefits compared with modern phate synthase gene families in wheat and other grasses, Plant Physiol. 135 (2004) bread wheat? J. Cereal Sci. 79 (2018) 469–476, https://doi.org/10.1016/j.jcs. 1753–1764. 2017.11.010. [50] R. Valenta, B. Hayek, S. Seiberler, A. Bugajska-Schretter, V. Niederberger, [36] V. Muccilli, M. Lo Bianco, V. Cunsolo, R. Saletti, G. Gallo, S. Foti, High molecular A. Twardosz, S. Natter, L. Vangelista, A. Pastore, S. Spitzauer, D. Kraft, Calcium- weight gluten in subunits in some durum wheat cultivars investigated by means of binding allergens: from plants to man, Int. Arch. Allergy Immunol. 117 (1998) mass spectrometric techniques, J. Agric. Food Chem. 59 (2011) 12226–12237, 160–166, https://doi.org/10.1159/000024005. https://doi.org/10.1021/jf203139s. [51] Y. Perez-Riverol, A. Csordas, J. Bai, M. Bernal-Llinares, S. Hewapathirana, [37] M.A. De Santis, M.M. Giuliani, L. Giuzio, P. De Vita, A. Lovegrove, P.R. Shewry, D.J. Kundu, A. Inuganti, J. Griss, G. Mayer, M. Eisenacher, E. Pérez, J. Uszkoreit, Z. Flagella, Differences in gluten protein composition between old and modern J. Pfeuffer, T. Sachsenberg, S. Yilmaz, S. Tiwary, J. Cox, E. Audain, M. Walzer, durum wheat genotypes in relation to 20th century breeding in Italy, Eur. J. Agron. A.F. Jarnuczak, T. Ternent, A. Brazma, J.A. Vizcaíno, The PRIDE database and re- 87 (2017) 19–29, https://doi.org/10.1016/j.eja.2017.04.003. lated tools and resources in 2019: improving support for quantification data, [38] D.B.M. Ficco, B. Prandi, A. Amaretti, I. Anfelli, A. Leonardi, S. Raimondi, Nucleic Acids Res. 47 (D1) (2019) D442–D450, https://doi.org/10.1093/nar/ N. Pecchioni, P.D. Vita, A. Faccini, S. Sforza, M. Rossi, Comparison of gluten gky1106.

14