Ital. J. Food Sci., vol. 30, 2018 - 497 PAPER COMPARATIVE 1D- AND 2D-ELECTROPHORETIC PROTEIN PROFILES OF ANCESTRAL AND MODERN BUCKWHEAT SEEDS GROWN IN THE ITALIAN ALPINE REGION J. CAPRARO*1, C. MAGNI1, A. GIORGI2, M. DURANTI1 and A. SCARAFONI1 1Department of Food, Environmental and Nutritional Sciences (DeFENS), Faculty of Food and Agriculture, Università degli Studi di Milano, via Celoria 2, Milan, Italy 2Department of Agricultural and Environmental Sciences, UNIMONT, Via Morino 8, 25048 Edolo, Brescia, Italy *Corresponding author: Tel.: +39 0250316823; Fax: +39 0250316800 E-mail address: jessica.capraro@unimi.it ABSTRACT Buckwheat is an old crop whose seeds are under-utilized. The protein composition of these seeds, however, makes them suitable as much needed ingredients for the production of gluten-free products. Several buckwheat species and local cultivars are known worldwide. In this work, 1D and 2D electrophoresis were used to characterize and compare the seed protein profiles of two buckwheat species (Fagopyrum esculentum and Fagopyrum tataricum). The two analyzed cultivars of F. esculentum represent authentic landraces of an Italian Alpine valley, named Valtellina. The protein profiles of F. tataricum and the two F. esculentum cultivars did not show major differences. However, narrow but significant differences were present between these two landraces, allowing their discrimination at protein level. This work represents a molecular-based approach to the designation of origin and authenticity of local buckwheat varieties and their tracing in flours for human food. Keywords: buckwheat, electrophoresis, Fagopyrum spp., historical landraces, proteome, traditional cultivation Ital. J. Food Sci., vol. 30, 2018 - 498 1. INTRODUCTION Buckwheat is a pseudo-cereal seed of the class Dicotyledoneae, genus Fagopyrum, and smartweed family. It originated from and was domesticated in Eastern Himalaya regions and can be cultivated in flat and mountainous regions, as long as the climate is cold. Buckwheat came to Europe in the late Middle Age (OHNISHI, 1993) and, early traces of its cultivation in Italy were found in property documents of a family in Teglio, Valtellina, dating back to the middle of the sixteenth century (FERRANTI et al., 2002). After a long history of growth and food use and a remarkable decline in the last decades, there has been a renewed interest in the crop. Among the reasons for this recent reappraisal are the growing worldwide need for sustainable nutrient sources (DURANTI and SCARAFONI, 2015) and the claimed health benefits of various buckwheat components (LI et al., 2008; IZYDORCZYK et al., 2014; ZHOU et al., 2015), as it occurs for many other seeds (SCARAFONI et al., 2007). Buckwheat seeds (Fagopyrum spp.) display a high fiber content, ranging from 12 to 18% with very low lipid levels (about 3%) and relatively low carbohydrate levels, around 50% (EGGUM et al., 1980). The presence of biologically-relevant compounds, such as flavonoids, flavones, phytosterols, thiamin-binding proteins (LI and ZHANG, 2001) and rutin, with antioxidant properties (KREFT et al., 2002), all add nutritional value to this seed. The protein content is similar to or greater than that of wheat, from 12% d.w. upward. The peculiar composition of the protein fraction makes these seeds and their proteins suitable as main ingredients for many food applications, including foods for coeliac patients. Phylogenetic relationships and polymorphisms of buckwheat have been studied at both genetic and protein levels (LI et al., 2008; OHNISHI and MATSUOKA, 1996; YASUI and OHNISHI, 1998; DU et al., 2004; ZELLER et al., 2004; ROUT and CHRUNGOO; 2007). Wide margins for implementing tailored and finalized applications exist, because not all molecular and compositional features of these seeds have been thoroughly investigated for their optimal exploitation. Modern analytical approaches allow the use of molecular-based strategies for the comparison, selection and improvement of crops and these activities are becoming crucial for mankind in the near future. Electrophoretic techniques applied to the protein fraction may represent a complementary and effective approach to the genetic/genomic analysis of plants (GORINSTEIN et al., 2005; CAPRARO et al., 2008). In this work, we applied a fast and reliable methodology based on electrophoretic analyses of seed proteins to identify candidate quality marker to be used to test and guarantee the designation of origin and authenticity of local buckwheat varieties. We focused our attention on two remnant cultivars of Fagopyrum esculentum, L.; one which is locally named ‘Nustran’ and the other called ‘Furest’ or ‘Francese’ or else ‘Curunin’ which has recently been identified as an authentic Valtellina landrace by genetic analyses (BARCACCIA et al., 2016). In this work we will refer to this latter cultivar as ‘Furest’. It is still cultivated in Teglio (Valtellina), an Italian Central Alpine valley village. The first landrace, ‘Nustran’, is an original Teglio ecotype, whereas ‘Furest’ has been cultivated in Teglio only since the beginning of the twentieth century. In appearance, the two cultivar seeds show only slight morphological differences (BARCACCIA et al., 2016). In addition, a modern variety of Fagopyrum tataricum has also been included in our comparative work. Ital. J. Food Sci., vol. 30, 2018 - 499 2. MATERIALS AND METHODS 2.1. Materials Buckwheat seeds were kindly supplied by Raetia Biodiversità Alpine, Teglio, Valtellina, Italy. Fagopyrum esculentum, L. was of the varieties Nustran and Furest; Fagopyrum tataricumwas of variety n’Zibaria. 2.2. Methods 2.2.1 Protein extraction Dry seed kernels were manually ground to a meal in a mortar. The protein fractions of the resulting flours were extracted under stirring at room temperature for two hours following two procedures: a non-denaturing one, consisting of a solution containing 50 mMTris-HCl and 0.5 M NaCl at pH 7.5, and a denaturing one (redry solution), consisting of a solution containing 8 M urea, 2 M thiourea, 20 mg/mL3-[(3- Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate(CHAPS) and 65 mM 1,4-dithiothreitol (DTT) at pH 8.5. The ratios, 1/20 (w/v) in non-denaturing conditions and 1/40 (w/v) in the denaturing buffer, were used. The slurries were centrifuged at 12,000g at room temperature for 20 min, and the extracted proteins were conserved at -20°C until used. Two experimental replicas for each condition were carried out. 2.2.2 Electrophoretic techniques Isoelectric focusing (IEF) was performed on 7 cm pH 3–10 linear IPG strips (Amersham Biosciences) as described by CAPRARO et al. (2008). SDS-PAGE for either 1D and 2D analyses was carried out according to LAEMMLI (1970) on 12% polyacrylamide gel using a mini-Protean II cell (Bio-Rad). The gels were stained with Coomassie blue. Protein extracts were analyzed in triplicate. 3. RESULTS AND DISCUSSION Buckwheat seed proteins include either albumins and globulins, typical of the legume grains, and prolamins at high and low molecular weight (HMW and LMW) (NAIŁĘCZ et al., 2009), typical of cereals seeds. For this reason, the performances of two extraction protocols were assessed. The extraction of proteins under non-denaturing conditions using a saline alkaline buffer (Fig. 1, lanes marked with ND, namely TND, NND and FND) resulted in the prevalent solubilization of buckwheat globulins, as shown by 1D SDS- PAGE analysis. The presence of two main bands at about 40 and 25 kDa (LA and LB, respectively in Fig. 1) is typical of the reduced 13S globulin family of most species (CASEY et al., 1985) and the greater Mrminor band components around 60 kDa likely correspond to the 7S globulin subunits (V in Fig. 1). Under these conditions, a lower number of high Mr bands with respect to the samples extracted under denaturing conditions was visible. According to NAIŁĘCZ et al. (2009), these new bands likely correspond to the prolamin family, which are insoluble, unless denatured and reduced. Ital. J. Food Sci., vol. 30, 2018 - 500 Figure 1. SDS-PAGE under reducing conditions of proteins from seeds of Fagopyrum species, namely tataricum (T), and esculentum landraces, ‘Nustran’ (N) and ‘Furest’ (F) extracted under non-denaturing (ND) and denaturing extraction conditions (D). LA and LB stand for 13S globulin acidic and basic subunits, respectively; V stands for 7S globulin family. See text for further details. In the 1D separation of Fig. 1, a clearly distinct pattern of F. tataricum protein profile from those of the two F. esculentum cultivars was visible in the range 40-70 kDa. In particular, in samples TD, three distinct bands were visible in the range 50 - 66 kDa while only two polypetides were detectable in the other two samples. Overall, the 1D protein profiles of F. esculentum ‘Nustran’ and ‘Forest’ were more similar to each other than that of F. tataricum. The greater resolution of 2D electrophoretic analysis was used to get a more detailed comparison of the three protein patterns (Fig. 2). Based on the results described above, the 2D IEF/SDS-PAGE analyses were carried out under denaturing and reducing extraction conditions to get the most complete picture of the respective proteomes. Indeed, the 2D maps of the analyzed Fagopyrum spp. allowed the identification of some of the seed’s main protein components. The spot groups marked with LA and LB display intensities and positions which definitely identifies these spots as acidic and basic subunits of the 13S globulin, respectively. The ‘train spot’ group is typical of the high Mr 7S globulin chains with their peculiar pI isoforms (RADOVIĆ et al., 1996; MAGNI et al., 2007). In this latter case, however, closely migrating HMW prolamins with variable pI and Mr around 50 kDa were likely present too, as detailed by NAIŁĘCZ et al. (2009). Prolamins of intermediate and low Mr, though barely recognizable, seemingly spread in the map at more acidic pIs and with greater migration coefficients, according to NAIŁĘCZ et al. (2009). The 2D electrophoretic maps confirm and detail the 1D profiles by showing a greater similarity between the two F. esculentum cultivars and their difference with F. tataricum spot pattern. Indeed, the region of the 13S globulin acidic subunits appears quite different and some major spots, which are circled in the panel T of Fig. 2, contributed to significantly diversify the map of F. tataricum with respect to those of F. esculentum. However, differences between the two F. esculentum landraces were also detectable. Quantitative differences among common spots, could be noted too. Most of these differences were found in the low Mr, neutral pH region of the 2D maps. The observed intervarietal differences cannot be attributed to climatic, pedological or edaphological causes, since the seeds arise from same cultivation area and grower. Likely, these Ital. J. Food Sci., vol. 30, 2018 - 501 differences represent authentic biodiversity source. In an extension of this work, it would be interesting to identify those differing spots, which are unlikely related to classical storage proteins because of their low Mr and clear-cut shape of the spots, and to associate them with peculiar phenotypic or nutritional features of the two cultivars. Figure 2. Two-dimension electrophoretic maps of proteins extracted from seeds of Fagopyrum species, namely tataricum (T) and esculentum landraces: ‘Nustran’ (N) and ‘Furest’ (F), all extracted under denaturing conditions (see details under Methods). V: 7S globulin chains; P: prolamins chains; LA: 13S globulin acidic subunits; LB: 13S globulin basic subunits. 4. CONCLUSIONS In the perspective of future uses of landraces and their derivatives in foods suitable for people with coeliac disease or in nutraceutical formulations, the development of an agile Ital. J. Food Sci., vol. 30, 2018 - 502 methodology to identify landraces components is needed. This work represents a first step in this direction, making available specific 2D electrophoretic maps for given landraces and thus helping in their identification and tracing in flours for human food. It is worthy of note that the two cultivar seeds are very similar in appearance, with ‘Furest’ being smaller in size and of lighter grey color (BARCACCIA et al., 2016). These minimal differences make the need for molecular fingerprinting more compelling. This work, by revealing even minor interspecific and intervarietal differences in the protein expression patterns of buckwheat seeds, may open the gateway to the identification of hidden useful properties, such as resistance to adverse environmental conditions and seed quality characteristics, and to the valorization of these crop populations. REFERENCES Barcaccia G., Volpato M., Gentili R., Abeli T., Galla G., Orsenigo S., Citterio S., Sgorbati S. and Rossi G. 2016. 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