Nor. Afr. J. Food Nutr. Res. 2020; 04(07):280-284 //www. najfnr.org DOI 10.5281/zenodo.3974052 Proteome consistency of the aleurone layer in grain of major wheat species grown over different years Samira Meziani ’ * ® Isabelle Nadaud ^ ^ Laboratory of Biotoxicology, Djillali Liabes University, Faculty of Life and Natural Sciences, Department of Biology, Algeria ^ UMR. INRA GDEC-UCA 1095, 5 Chemin de Beaulieu, 63000 Clermont-Ferrand, France ABSTRACT Background: Aleurone layer (AL), being a living cell layer among the peripheral layers of the grain structure obtained after milling wheat, is rich in, vitamins, minerais, and antioxidants potentially nutritional value of the flour. Objectives: To isolate AL in the mature grain of the three major species; Common wheat (CW), Durum wheat (DW), and Einkorn wheat (EW) that were grown at two different years as well as to analyze and compare their proteomes revealed through two-dimensional electrophoresis (2DE) and image analysis. Methods: The AL was hand dissected and unicellular purity verified using scanning électron microscopy. AL proteins were separated using lEE pH3- 10 X SDS-PAGE then Coomassie-stained. The gels were scanned and the images were compared using Samespot (Nonlinear Dynamics) and were proteins identified using mass spectrometry and database interrogation. Results: Eor CW and DW samples, no significant quantitative or qualitative différences were observed between the AL proteome in the two years. However, a few quantitative différences were revealed between EW for AL of 2006 and 2007. The identified proteins were classified in the carbohydrate pathway and stress defense response. Conclusion: This remarkable stability over environmental growing conditions strengthens the need to pay greater attention to this unicellular living cell of the wheat grain. Keywords: Wheat, durum, einkorn, aleurone layer, metabolic pathways Received: May 12, 2020 Accepted: August 04, 2020 Published: August 11, 2020 1 Introduction Wheat represents one of the main cereals consumed by humans and animais. Knowledge of wheat grain structure and the composition of storage proteins, starch, lipids, vitamins, and micronutrients is essential for producing high-quality grain Wheat species, such as durum and einkorn wheat are high nutritive value of their grain Durum wheat is an extremely important species in Mediterranean régions, where is used in the production of semolina and pasta Einkorn wheat is one of the ancient wheats and is often grown in environmentally friendly organic farming. This wheat is also known for its high protein and carotenoid contents Einkorn wheat is a potential source of food with high nutritional properties The peripheral layers (PL) of wheat, including the aleurone layer (AL), are a considérable source of micronutrients. The AL is used as a food ingrédient for animal feed, the presence of its nutritional and phytochemical éléments increases the quality of food Lor several years, studies on AL hâve mainly focused on analyzing its nutritional potential for health. AL contains vitamins more particularly vitamin B and E, and minerais and phenolic antioxidants, mainly ferulic acid compounds, which exhibit a potential nutritional value. However, few studies hâve been carried out to study the potential health effects of isolated aleurone on biomarkers for cancer Numerous studies focusing on the proteome variations of the wheat grain were reported since the last decade. Based on the two- dimensional electrophoresis of the proteins extracted either from grain peripheral layers or from the hand isolated AL using binocular a large set of enzymes and proteins, spécifie of this unicellular layer, were revealed. In the aim to compare AL of the major wheat species, three représentatives of common wheat (T, âestivum) and three varieties of T. durum were analyzed The same proteomic approach was also developed in the comparison of AL from the mature grain of three représentative cultivars of T, aesdvum and T. monococcum The influence of different growing environmental conditions on the AL proteome was also partly reported in a previous study Our study was focused on possible proteomic variations of the AL isolated in mature grain from one variety of common (T. aestivum), durum (T. durum) and Einkorn (T. monococcum) wheat grown at two consecutive years in different conditions. Each of these cultivars has been produced in nurseries, under conventional growing conditions, with sufficient minerai fertilization to achieve high grain yields. The production of these cultivars was carried out with complété phytosanitary protection on the plant and the ear. * Corresponding author: Samira Meziani Laboratory of Biotoxicology, Djillali Liabes University, Faculty of Life and Natural Sciences, Department of Biology, Algeria. Téléphoné No: 00213 560 337 210. E-mail: meziani samira@yahoo.fr Meziani & Nadaud Figure 1 : Transversal scanning electro-microscopy of aleurone layer fully separated from the other layers. Scale 100 |im. (a) AL CW, (b) Al DW, (c) AL EW AL CW: Aleurone layer in common wheat ALDW: Aleurone layer in durum wheat AL EW: Aleurone layer in Einkinon wheat 2 Material and Methods 2.1 Plant material One cultivât représentative of each specie was used to characterize the AL. The three cultivars such as the common wheat Chinese Spring (CS); durum wheat Bidi 17 (Bidil7) and the Einkorn wheat DV92 (DV92), were collected from different experimental CS. Bidi 17 was grown in 2004 and 2007, not at two consecutive years like the CS and DV92. The dissection of the ALs was performed under the binocular microscope The isolated AL was kept at -80 °C before protein extraction and characterization. 2.2 Protein extraction and quantification Total protein contents were quantified using the method reported in previous studies 12,15,16 sample, from a given génotype, the samples were ground in liquid nitrogen introduced into Eppendorf tubes and separated into two biological extracts, allowing two répétitions per extract. These two extracts were then stored at - 80 °C for analysis 2.3 Proteins identification In this part, the spots, which are qualitatively and quantitatively different, hâve been selected and which the p-value is greater than or equal 0.05 to eut and hydrolyze by an enzyme and then identified by MALDI-TOE mass spectrometry. According to some authors proteins are identified if they match a single reference in the databases: NCBI Viridiplantae, Poacea, and Wheat EST database. 3 Results As reported in previous publications the protein maps of the AL showed a wide distribution of spots in the pl range from 3 to 10 and a mass range between 10 and 110 kDa for CW and DW, respectively Each 2DE revealed more than one thousand AL spots for each variety, while others were found common (identical position for pl and apparent MW) between CW and DW as well as for CW and EW (Eigure 2). Thanks to the linear relation of the Coomassie staining, between protein contents and optical density, the proteome comparison was carried out for DW grown in 2004 and 2007. Neither qualitative nor quantitative différences between the grains harvested during the two different years were observed. Proteins grouped in spécifie zones Most of AL grown in the two years, had spots localized in three main zones (Eigure 2). In addition, the results of the image analysis of AL proteome comparison did not reveal any qualitative différences between samples of EW grown in 2006 and 2007. Among eleven hundred AL spots, only 15 spots hâve significantly different in abundance between the two consecutive growing years of EW. Zone I was composed of two proteins. Aldose reductase (spot 1302) involved in metabolism pathway; involved in environmental functions, metabolism and defense, and including polysaccharide, glycolysis, Iperoxyredoxin (spot 1717) both involved was involved in protection against oxidative stress (Table 1: supplementary data) This protein was more abundant in EW grown in 2006. Zone II and III out of these 15 spots were more abundant in EW grown in 2007. Ten spots were identified as Glo-3 involved in storage proteins; this globulin is light molecular weight. Among the nine spots, seven were globulins (spot number 2184, 2369, 2370, 3387, 2313, 3390 and 2249) of low molecular weight (19 kDa to 37 kDa). Those spots were obtained in the acid and basic région of the gel, when, one was a glyoxalase family protein (spot 2605) that was more abundant in EW grown in 2006. 4 Discussion The proteomic approach used in our study was based on manual dissected AL under binocular. At least 30 grains per biological replicate were needed to isolate AL with sufficient protein amount to perform 2DEs for a given génotype. 281 Nor. Afr. J. Food Nutr. Res. Volume 04, Issue 7, 2020 Meziani & Nadaud Figure 2: 2DE gel immobiline pH 3-10 x SDS-PAGE of the proteins extracted from AL of EW. The normalized volume of the surrounded and numbered spots differed significantly between the two growing years The laborious hand isolation of AL was verified for each species using scanning électron microscopy (SEM) to guarantee the purity of the AL isolated (Figure 1). The proteome analysis revealed that the number of AL protein spots was not additive like the cumulated genomes in wheat grain: Le.: common and durum wheat’s genome ABD and AB did not show three tintes and two tintes the number of spots of Einkorn cultivât (genome A). Instead, most of the spots were revealed as common in interspecies proteomic comparisons For each of these species, a comparison of AL proteome grown in different environmental and weather conditions did not reveal any différences in terms of presence or absence between the AL proteome of the grains harvested the two years. In addition, no quantitative différences, in terms of spot abundance, were detected between AL proteome for both common wheat CW and DW. Only 15 spots (1 % of the stained AL spots) displayed différences between the two growing years for the EW. Why did we obtain such a consistency in the proteomic composition of the AL of wheat grain? - First, we may recognize that ail wheat was grown under complété fungicide protection. Spike diseases, may attack grains and consequently induce different protein expressions, such as protease inhibitors, in the AL. - Secondly, field growing conditions, which differed mainly in température and rainfall amount, had an impact on the grain size and grain yield. The proteome of the AL in hexaploid and tetraploid wheat cultivars was however not quantitatively affected, whereas only EW had few proteins whose abundance was affected. This finding showed that the AL proteome is highly genetically controlled. The fact that most of the AL protein spots were common between the three species, and revealed through high-resolution 2DE gels, suggests that many genes encoding spécifie AL proteins in the Einkorn (genome A) are also présent and probably also duplicated in the genome B and D of durum wheat and common wheat. Together with the gene duplication, the passage from the genome (A) to the durum wheat (genome AB) and common wheat (genome ABD) has undoubtedly led to a specialization of genes making it possible to respond better to climatic and environmental variations. The polyploid wheat possessing a larger area of production has progressively acquired more genes related to environmental response as compared to Einkorn with a narrower area of production. Thirdly, the possible major reason of this remarkable AL proteome consistency was due to the fact that AL is a living cell layer. The grain peripheral layers, composed of emptied cells together with endosperm, are ail dead tissues. The AL and embryo are the living tissues of mature grain. Hence, to protect the endosperm sac against fungi attacks and possible conséquences of microfliora from hydrolytic influence, the AL has undoubtedly acquired during wheat évolution the needed numerous genes associated with proteome consistency and better response to environmental variation. Nadaud et ai., analyzed the AL proteome évolution at fifteen stages during grain development. Previous proteomic analyses showed that globulins were the major AL proteins (representing 65% of CW spots) of mature seed In our study, seven globulins were significantly more abundant in EW grown in 2007 than in 2006. These globulins correspond to 7S-globulins, which were identified in wheat species Globulins are involved in response to biotic stresses 282 Nor. Afr. J. Food Nutr. Res. Volume 04, Issue 7, 2020 Meziani & Nadaud DVfl2(!0é) DV52(!07) Zonfiin 13 » Figure 3: The three major zones where qualitative and quantitative différences for the AL proteins of the EW in mature wheat significantly between the two growing years (synthesis of defense enzymes and inhibitors to face fungi attack, and bacteria) during grain formation, in response to abiotic stresses (mainly thermal and water) during the grain storage period Globulins are also involved in the synthesis of enzymes such as proteases and amylases in response to the stimulation of gibberellic acid causing germination. Other proteins differently expressed in EW were identified: one belonging to stress/defense family proteins: the 1-Cys peroxiredoxin (1-Cys Prx), this protein possesses thioredoxin peroxidase activity, and plays a rôle in the réduction of hydroperoxides, being induced during oxidative stress. This protein présent in cereals, which identified in the wheat endosperm (2), and AL (14), the 1-Cys peroxiredoxin genes are présent in seeds, i.e. the embryo and the AL. The protein is expressed during the maturation phase of the grain, a sensitive phase in grain development but also during germination, where this protein is involved in protection against oxidative stress One aldose reductase and one glyoxalase were differently expressed in 2007 and 2006 in DV92. These two enzymes participate in the conversion of glucose to fructose, the détoxification of methylglyoxal, and the aldéhydes of cellular metabolism (Table 1: supplementary data). 5 Conclusion The aleurone layer constitutes an important source of micronutrients and its rôle in the mobilization of kernel reserves for germination has been widely studied. Our results clearly showed the proteome consistency (or proteome stability) of the AL from mature grains collected in the three major wheat species, when grown at different years with fungicide protection. This finding needs to be confirmed with wheat harvested in different controlled environments. Consequently, the genetic improvement of the nutritional value of wheat should make significant advances particularly through the knowledge acquired on the AL for human health benefit. Acknowledgment: The authors are thankful to the laboratory of Biotoxicology, Sidi-Bel-Abbes University, and INRA for providing laboratory facilities to carry out the research work. Author contribution: S.M., and I.N. conceived, designed the study and undertook the literature research. S.M., and I.N. participated in the experiment I.N. performed the data acquisition. Data and statistical analysis were achieved by both authors. Ail authors prepared, drafted the manuscript. 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Plant Physiology, 152(3), 1459- 1470. https://doi.org/ 10.1104/pp. 109.149864 21. Shewry, P. R., & Halford, N. G. (2002). Cereal seed storage proteins: Structures, properties and rôle in grain utilization. Journal of Experimental Botany, 53(370), 947- 958. https://d 0 i. 0 rg/l 0.1093/jexbot/53.370.947 Cite this article as: Meziani, S. & Nadaud, I. (2020). Proteome consistency of the aleurone layer in grain of major wheat species grown over different years. North African Journal of Food and Nutrition Research, January - June, 04(07), 280-284. https://doi.org/10.5281/zenodo.3974052 © 2020 The Author(s). This is an open-access article distributed under the terms ofthe Creative Gommons Attribution License (http://creativecommons.Org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 284 Nor. Air. J. Food Nmr. Res. Volume 04, Issue 7, 2020