**2. Molecular weight distribution (MWD) of wheat storage proteins**

#### **2.1. Classification and polymorphism of wheat grain proteins**

As early as the 1950s, thanks to very good recombination experiments with flour constituents, Finney [1] confirms that the baking capacity is essentially conferred by gluten. Gluten, which can be defined as a viscoelastic protein complex formed after hydration and the addition of flour, consists of a heterogeneous mixture of prolamins (i.e. gliadins and glutenins) associated with covalent (S-S) and non-covalent bonds (hydrogen, hydrophobic and ionic). The specific role of certain protein fractions (monomer to polymeric proteins ratio) in the different proper-

During the period 1970–1990, it became clear that the variation of the baking capacity of a flour is based on the ability of its storage proteins (i.e. prolamins) to form, during mixing, a three-dimensional plastic structure (**Figure 1A** and **B**). This remarkable structure creates a cohesive and viscoelastic network, insoluble in water, ensuring the retention of carbon dioxide, ethanol and aromas, during the fermentation of the dough and unlike other cereals for which these properties are non-existent (**Figure 2**). Thus, a common wheat is all the more breadmaking that its storage proteins have a strong tendency to aggregate into a threedimensional viscoelastic network during mixing. Thus, gluten is considered a transient network whose mechanical properties depend on the density of the junction zones between the

Since the 1990s, thanks to the integration of many complementary scientific approaches (i.e. molecular biology, biochemistry, analytical chemistry, rheology, etc.), a clearer vision of the transformation processes and the role of the main protein constituents within them have begun to take shape [3]. Thus, attention has been focused on the (polymeric) glutenin fraction because a strong relationship has been established between breadmaking properties, such as mixing time, extensibility and loaf volume and the molecular weight distribution (MWD) of

**Figure 1.** Scanning electron micrographs of durum wheat (A) flour and (B) dough particles (From Hoseney and Rogers

ties of wheat doughs was also highlighted.

elements that compose it [2].

134 Global Wheat Production

[3]).

the polymeric protein components [4].

Like all grain seeds, wheat grain contains a large number of proteins classified as structural proteins, functional proteins and reserve proteins. They are unequally distributed within the different cell of the grain. A natural gradient of distribution can be highlighted. As a result, the starch to protein ratio significantly increases from the peripheral to the central regions of the grain. Given the relative weight of these different cells, 70–80% (w/w) of the proteins are in the albumen.

The classification system for cereal proteins is mainly based on Osborne's historical work, in 1907 [5], based on their differences in solubility later used in sequential extractions (**Table 1**). As a result, four major protein fractions have been defined: albumins (soluble in water), globulins (soluble in dilute salt solutions), gliadins (soluble in diluted alcohols, 70% ethanol) and finally, glutenins (residual proteins, partially soluble in diluted acids and bases). Other authors have enriched these classifications based on structural and/or functional properties [6, 7].Within the large family of the storage proteins (prolamins), two main classes can be


Gliadins have a large genetic polymorphism, it has been possible to detect between 20 and 40 different constituents for a wheat variety [9]. Within a class of gliadins, it is possible to find several sub-families depending on the composition and richness of certain amino acids (the ω1 and ω5 gliadins differ in basic amino acids, glutamine and proline, than that γ1, γ2 and γ3 differ in their richness in tyrosine, lysine and methionine). Thus, the polymorphism of gliadins is very important that it serves as a basis for the varietal identification of wheat [10]. The polymorphism of low molecular weight glutenic subunits (LMW-GS) is less important than that of gliadins. For a given variety, there are 7–6 LMW-GS. But 40 different LMW-GS were found in 222 varieties of soft wheat [11]. Finally, high molecular weight glutenic subunits (HMW-GS) are the prolamins that have the lowest polymorphism. The association of two genes at each Glu-A1, Glu-B1 and Glu-D1 locus was noted. The *x*-type genes express subunits of masses greater than those encoded by *y*-type genes in SDS-PAGE [12]. However, some *y*-type HMW-GS (notably subunit 12) have been shown to have important immunochemical similarities with α/β and γ-gliadins [13]. In all cases, recombination between these genes is very rare. The different wheat varieties contain between 3 and 5 HMW-GS. Indeed, 1Ay genes

Storage Proteins Accumulation and Aggregation in Developing Wheat Grains

http://dx.doi.org/10.5772/intechopen.75182

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are never expressed, and 1By and 1Ax genes are only expressed in some varieties [14].

for the repetitive domain (**Figure 3**).

**2.2. Gliadin to glutenin ratio**

The primary structure of the storage proteins is well understood. They comprise three distinct domains (**Figure 3**): a central domain made up of repeated sequences and two domains formed of non-repeated sequences at the ends (i.e. C- and N-terminal). The understanding of these sequences has made it possible to locate particularly important cysteine residues because of their ability to form disulphide bonds (intra and/or intermolecular). α-, β- and γ-gliadins are provided with cysteines at their C-terminal domains; these all being involved in the formation of intramolecular disulphide bridges. HMW-GS have unpaired cysteine in their C-terminal domain and several others in their N-terminal domain; LMW-GS carrying seven C-terminal cysteines and one N-terminal cysteine. Thanks to these unpaired cysteines, unlike gliadins, HMW-GS and LMW-GS are able to form intermolecular disulphide bridges. Some authors report a globular type structure for the N- and C-terminal and a spiral structure

Generally, it is accepted that the functional properties of gluten proteins are related to their ability to form a network during technological processes [17, 18]. However, gliadins and glutenins do not have the same effect on the rheological properties of doughs or glutens. Consequently, gliadins explain the viscous nature, while glutenins determine elasticity. In fact, the small quantity of cysteine residues in these storage proteins makes it possible to establish an important structural and functional distribution between gliadins and glutenins (**Figure 4**).For the former, all cysteine residues are involved in the establishment of intramolecular disulphide bridges while for both high and low molecular weight glutenins, a number of cysteines not involved in intramolecular bonds are therefore available to establish intermolecular links with other subunits. Glutenins are therefore likely to constitute polymers with a real consistency, thanks to the formation of intermolecular disulphide bridges, while gliadins remain in the monomeric state. The latter may, however, be aggregated by weak bonds

\**Allelic blocks*, wheat homologous chromosomes (noted 1–6), wheat genomes (A, B and D) and chromosome position: (S) short arm, (L) long arm.

**Table 1.** Classification of wheat grain proteins.

differentiated due, to their degree of aggregation/polymerization. Thus, on the one hand, gliadins (soluble monomeric proteins in aqueous alcohols), which represent approximately 30–40% (w/w) of flour proteins and on the other hand, glutenins representing, 40–45% (w/w) of the total flour proteins. The latter are polymeric and aggregated proteins, forming a much more complex material than the gliadins.

Gliadins correspond to a mixture of monomeric proteins of molecular weight between 25 and 75 kDa and are characterized by their richness in glutamine and proline. They represent 45% (w/w) of the total prolamins. There are four classes based on their electrophoretic behaviour (i.e. increasing mobility in acid medium): α/β, γ and ω-gliadins (which, respectively, represent 44–60%, 30–46% and 6–20% of total gliadins) [8].

Glutenins, for their part, represent 40–50% (w/w) of total proteins; they are rich in proline and glutamic acid and their content in basic amino acids is higher than that of gliadins. They constitute a much more complex material formed of an assembly of polypeptide chains, commonly called subunits, linked together mainly by intermolecular disulphide bridges. These subunits have been grouped into two different subgroups: low molecular weight subunits (LMW-GS) and high molecular weight subunits (HMW-GS).

LMW-GS account for an average of two-thirds of total glutenins. They are very polymorphic and have molar masses between 30 and 50 kDa. Given their similarity to some gliadins, these have sometimes been difficult to quantify. HMW-GS, as their name indicates, have higher molecular weights ranging from 95 to 130 kDa. According to their SDS-PAGE migration, they fall into two groups: HMW-GS*y* (67–74 kDa) and HMW-GS*x* (83–88 kDa).

Gliadins have a large genetic polymorphism, it has been possible to detect between 20 and 40 different constituents for a wheat variety [9]. Within a class of gliadins, it is possible to find several sub-families depending on the composition and richness of certain amino acids (the ω1 and ω5 gliadins differ in basic amino acids, glutamine and proline, than that γ1, γ2 and γ3 differ in their richness in tyrosine, lysine and methionine). Thus, the polymorphism of gliadins is very important that it serves as a basis for the varietal identification of wheat [10].

The polymorphism of low molecular weight glutenic subunits (LMW-GS) is less important than that of gliadins. For a given variety, there are 7–6 LMW-GS. But 40 different LMW-GS were found in 222 varieties of soft wheat [11]. Finally, high molecular weight glutenic subunits (HMW-GS) are the prolamins that have the lowest polymorphism. The association of two genes at each Glu-A1, Glu-B1 and Glu-D1 locus was noted. The *x*-type genes express subunits of masses greater than those encoded by *y*-type genes in SDS-PAGE [12]. However, some *y*-type HMW-GS (notably subunit 12) have been shown to have important immunochemical similarities with α/β and γ-gliadins [13]. In all cases, recombination between these genes is very rare. The different wheat varieties contain between 3 and 5 HMW-GS. Indeed, 1Ay genes are never expressed, and 1By and 1Ax genes are only expressed in some varieties [14].

The primary structure of the storage proteins is well understood. They comprise three distinct domains (**Figure 3**): a central domain made up of repeated sequences and two domains formed of non-repeated sequences at the ends (i.e. C- and N-terminal). The understanding of these sequences has made it possible to locate particularly important cysteine residues because of their ability to form disulphide bonds (intra and/or intermolecular). α-, β- and γ-gliadins are provided with cysteines at their C-terminal domains; these all being involved in the formation of intramolecular disulphide bridges. HMW-GS have unpaired cysteine in their C-terminal domain and several others in their N-terminal domain; LMW-GS carrying seven C-terminal cysteines and one N-terminal cysteine. Thanks to these unpaired cysteines, unlike gliadins, HMW-GS and LMW-GS are able to form intermolecular disulphide bridges. Some authors report a globular type structure for the N- and C-terminal and a spiral structure for the repetitive domain (**Figure 3**).
