**2.2. Gliadin to glutenin ratio**

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

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

**Osborne [5] classification Shewry et al. [6] classification Singh and Shepherd [7]** 

(prolamins) - ω Poor in S *Gli-1*(1A,1B,1D)S

Reach in S Polymers



Solubility MW (kDa) Composition Structure Gene localization\* Function

5–90 Structural and

25–75 Monomers Storage proteins

**classification**

functional proteins

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, repre-

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 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

more complex material than the gliadins.

Protein fraction

136 Global Wheat Production

Albumins and globulins

Gliadins Diluted

Glutenins Acids, bases,

short arm, (L) long arm.

Water neutral salts

alcohols

reductants, detergents

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

sent 44–60%, 30–46% and 6–20% of total gliadins) [8].

100 to several millions

(LMW-GS) and high molecular weight subunits (HMW-GS).

fall into two groups: HMW-GS*y* (67–74 kDa) and HMW-GS*x* (83–88 kDa).

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

**Figure 3.** Schematic structures of typical primary structures of (A) ω-giadin, (B) α-gliadin, (C) γ-gliadin, (D) LMW-GS, (E) HMW-GS*y* and (F) HMW-GS*x* [15, 16]. Repetitive sequences are shaded and disulphide bonds between conserved cysteine residues (1–8) in the γ-gliadin are shown as lines. SH denotes the positions of cysteine residues in the HMW prolamins. Single letter abbreviations for amino acids: F = phenylalanine; G = glycine; L = leucine; P = proline; Q = glutamine; S = serine; V = valine and Y = tyrosine.

The molecular weight distribution (MWD) of prolamins is becoming recognized as the main determinant of physical dough properties [30, 31]. However, in theory, the MWD can be altered from one sample of wheat (or one cultivar) to another by changes in the relative proportions of monomeric proteins and polymeric proteins (gliadins to glutenins ratio) but also

**Figure 4.** A structural model for wheat gluten in which the HMW subunits provide a disulphide-bonded backbone which interacts with other gluten proteins through disulphide bonds (LMW subunits) and non-covalent interactions

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Chen and Bushuk [33] revealed that part of the glutenin is soluble in acetic acid thus making the distinction between an insoluble and a soluble fraction. The importance of this distinction became clear when Orth and Bushuk [34] demonstrated a positive correlation between the amount of acetic acid insoluble glutenin and bread loaf volume. From then on, insoluble glutenin became widely recognized as the key protein fraction that can explain differences in dough strength and breadmaking quality [35]. The use of detergents (SDS) and organic solvents (propanol) [36] allowed an even better separation and led to the conclusion that insolubility was due to size and a very high degree of polymerization. Other groups developed methodology with propanol to further separate soluble protein parts from the insoluble glutenin. Currently, two main methods are in use to quantify and characterize this fraction. The first corresponds to the so-called unextractable polymeric protein (UPP) method using propanol and during which unextractable polymeric protein (UPP) fraction is obtained. Upon sonication, this fraction becomes soluble in SDS [28, 29] and can be analysed using size exclusion chromatography [27, 37]. The other method is the SDS method as advanced by Graveland et al. [38] resulting in the SDS-insoluble gel protein fraction. This fraction was renamed glutenin macro polymer (GMP) to reflect its highly aggregated nature [39, 40]. Moonen et al. [41]

by changes in the size distribution of polymeric proteins [32].

**2.3. Size distribution of polymeric proteins**

(gliadins) (From Shewry et al. [19]).

(hydrogen and hydrophobic). The viscoelasticity of gluten depends on its state of polymerization and the interactions between polymers [2].

A large number of conventional fractionation and reconstitution tests have been conducted based on the differential physical properties observed in purified gliadins and glutenins. The aim of these studies was to link variations in molecular weight distribution (i.e. monomer to polymer ratio) with the rheological characteristics of the glutens obtained. In the majority of cases, the results obtained during these different reconstitution studies have demonstrated that the rheological properties of the restructured flours generated are strongly influenced by the ratio of these two protein fractions [20, 21]. With a constant amount of prolamins, the strength of the reconstituted flour, measured at the time of the dough making with a mixograph (i.e. peak time value mix (MPT)), is related to the proportion of polymeric proteins.

The development of the original analytical approaches (i.e. high performance liquid chromatography of size exclusion, SEC-HPLC) during the 1980s confirmed the vast majority of these hypothesis, which were essentially based on results obtained from differential solubility protocols (i.e. gliadins vs. glutenins). Thus, many authors [22–29] have confirmed the existence of a significant relationship between the relative amount of glutenin aggregates and the baking quality of many everyday wheat genotypes.

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**Figure 4.** A structural model for wheat gluten in which the HMW subunits provide a disulphide-bonded backbone which interacts with other gluten proteins through disulphide bonds (LMW subunits) and non-covalent interactions (gliadins) (From Shewry et al. [19]).

The molecular weight distribution (MWD) of prolamins is becoming recognized as the main determinant of physical dough properties [30, 31]. However, in theory, the MWD can be altered from one sample of wheat (or one cultivar) to another by changes in the relative proportions of monomeric proteins and polymeric proteins (gliadins to glutenins ratio) but also by changes in the size distribution of polymeric proteins [32].

#### **2.3. Size distribution of polymeric proteins**

(hydrogen and hydrophobic). The viscoelasticity of gluten depends on its state of polymeriza-

**Figure 3.** Schematic structures of typical primary structures of (A) ω-giadin, (B) α-gliadin, (C) γ-gliadin, (D) LMW-GS, (E) HMW-GS*y* and (F) HMW-GS*x* [15, 16]. Repetitive sequences are shaded and disulphide bonds between conserved cysteine residues (1–8) in the γ-gliadin are shown as lines. SH denotes the positions of cysteine residues in the HMW prolamins. Single letter abbreviations for amino acids: F = phenylalanine; G = glycine; L = leucine; P = proline;

A large number of conventional fractionation and reconstitution tests have been conducted based on the differential physical properties observed in purified gliadins and glutenins. The aim of these studies was to link variations in molecular weight distribution (i.e. monomer to polymer ratio) with the rheological characteristics of the glutens obtained. In the majority of cases, the results obtained during these different reconstitution studies have demonstrated that the rheological properties of the restructured flours generated are strongly influenced by the ratio of these two protein fractions [20, 21]. With a constant amount of prolamins, the strength of the reconstituted flour, measured at the time of the dough making with a mixograph (i.e. peak time value mix (MPT)), is related to the proportion of polymeric proteins.

The development of the original analytical approaches (i.e. high performance liquid chromatography of size exclusion, SEC-HPLC) during the 1980s confirmed the vast majority of these hypothesis, which were essentially based on results obtained from differential solubility protocols (i.e. gliadins vs. glutenins). Thus, many authors [22–29] have confirmed the existence of a significant relationship between the relative amount of glutenin aggregates and the baking

tion and the interactions between polymers [2].

Q = glutamine; S = serine; V = valine and Y = tyrosine.

138 Global Wheat Production

quality of many everyday wheat genotypes.

Chen and Bushuk [33] revealed that part of the glutenin is soluble in acetic acid thus making the distinction between an insoluble and a soluble fraction. The importance of this distinction became clear when Orth and Bushuk [34] demonstrated a positive correlation between the amount of acetic acid insoluble glutenin and bread loaf volume. From then on, insoluble glutenin became widely recognized as the key protein fraction that can explain differences in dough strength and breadmaking quality [35]. The use of detergents (SDS) and organic solvents (propanol) [36] allowed an even better separation and led to the conclusion that insolubility was due to size and a very high degree of polymerization. Other groups developed methodology with propanol to further separate soluble protein parts from the insoluble glutenin. Currently, two main methods are in use to quantify and characterize this fraction. The first corresponds to the so-called unextractable polymeric protein (UPP) method using propanol and during which unextractable polymeric protein (UPP) fraction is obtained. Upon sonication, this fraction becomes soluble in SDS [28, 29] and can be analysed using size exclusion chromatography [27, 37]. The other method is the SDS method as advanced by Graveland et al. [38] resulting in the SDS-insoluble gel protein fraction. This fraction was renamed glutenin macro polymer (GMP) to reflect its highly aggregated nature [39, 40]. Moonen et al. [41] found that the SDS-insoluble glutenin-gel protein fraction highly correlated with SDS sedimentation values and loaf volume. Weegels et al. [40, 42] studied this fraction in great detail and presented firm evidence that GMP quantity correlates to bread loaf volume.

In addition to these classical approaches (UPP and/or GMP), new analytical protocols have been developed since the early 2000s to separate and more accurately characterize the molecular size distribution of the polymeric proteins. Flow field-flow fractionation (FFFF) [43–46], which is a new separation technique without any stationary phase, and which is therefore not hampered by a steric exclusion limit [47–49], has been used successfully to separate a number HMW fractions [50–52]. Furthermore, the MALLS technique which is one of the most effective means of determining molecular weight, size and conformation of glutenin polymers without reference to standards [48, 53–56] has been applied in combination with the A-FFFF method to accurately measure size and conformation of wheat glutenins [57] (**Figure 5**).

The glutenin association level (i.e. the size distribution) is strongly correlated with the HMW-GS/LMW-GS [58–60] ratio and the nature of the HMW-GS present (especially HMW-GS pair 5 + 10 vs. HMW-GS pair 2 + 12 coded by Glu-*D1*). As demonstrated by the different experimental approaches carried out in recent years [61–64], the different glutenin subunits (i.e. HMW-GS, LMW-GS and HMW-GS *x* and *y* type) are unequally distributed within polymers. These results demonstrate the existence of a highly ordered structures in which some subunits play a predominant role, notably because of their difference in functionality (i.e. number and especially position of cysteine residues capable of forming intermolecular bonds) [65] (**Figure 6**).

**3. Accumulation of prolamins in developing wheat grains**

The development of the wheat endosperm which has been well described at the microscopic level, as reviewed by Bechtel et al. [66], can be quite easily characterized by the study of the temporal variations of several quantitative components of it such as the accumulation of the total dry matter, the water content of the grain and the accumulation of total protein and

**Figure 6.** Hierarchical arrangement of HMW-GS and LMW-GS in relation to their intrinsic contribution to polymer

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The accumulation of total dry matter in the grain provides a good insight into the functioning of different accumulation metabolisms (i.e. nitrogen translocation and post-flowering photosynthesis) [68]. Thus, after an initial lag phase (up to 10–15 days after anthesis (DAA)), it is easy to observe a phase of linear accumulation of this dry matter; wheat grains reaching a

During this linear phase, the observed phenomena depend on two main variables: the duration (*D*) and the speed or flux of assimilates towards the grain (*V*), so that the weight of a grain (*P*) is given by the relation *P* = *V* × *D* [69]. *D* can be expressed in days or in the sum of average daily temperatures (i.e. degree-days (*DD*)). The filling speed is the limiting factor in the development of the weight of a grain. This speed is mainly by the number of grains per

. Finally, under natural conditions, the duration *D* cannot compensate for the weight loss produced by any reduction in the rate of accumulation. The amount of water per grain that gradually increases to about 20 DAA remains relatively constant up to ≈ 35 DAA (i.e. "water

The higher the rate of water accumulation in the grain, the greater the height of the "water plateau" and the higher the weight of the grain at maturity [70]. Based on changes in the amount of water and total dry matter per grain after anthesis, three particular phases of grain development can be estimated: the cell division phase, the cell enlargement phase (i.e. grain

**3.1. Endosperm development**

formation (from Kasarda [65]).

maximum dry weight from 40 DAA.

plateau" phase) before decreasing at harvest time.

starch [67] (**Figure 7**).

m2

**Figure 5.** Asymmetrical flow field-flow fractionation (A4F) profiles of total solubilized storage proteins of a common French wheat cultivar (Soissons). UV (blue line), light scattering at 90° (red line) and molecular weight in relation to elution time (dark line) (from Lemelin et al. [57]).


**Figure 6.** Hierarchical arrangement of HMW-GS and LMW-GS in relation to their intrinsic contribution to polymer formation (from Kasarda [65]).
