**3.1. Endosperm development**

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

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

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

**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

and presented firm evidence that GMP quantity correlates to bread loaf volume.

to accurately measure size and conformation of wheat glutenins [57] (**Figure 5**).

bonds) [65] (**Figure 6**).

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elution time (dark line) (from Lemelin et al. [57]).

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

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 maximum dry weight from 40 DAA.

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 m2 . 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 plateau" phase) before decreasing at harvest time.

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

varies considerably, suggesting a phenomenon of differential regulation of this biosynthesis

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Thus, a certain accumulation asynchrony in the protein fractions of the grain can be highlighted. The albumins-globulins accumulate most rapidly in the grain, followed by the monomeric prolamins and finally the polymeric prolamins. As many researchers have shown [88–93], the accumulation of albumins-globulins is maintained only during the cell division phase, contrary to that of prolamins. This confirms the functional and/or structural role of

While the ratio between polymeric proteins and monomeric proteins is stable during the first stages of grain development (i.e. cell division and cell enlargement), this ratio increases significantly during the grain desiccation phase (i.e. after 35 DAA) (**Figure 8C**). A number of results in the literature are quite contradictory [90, 94, 95]. In our opinion, and in accordance with the

**Figure 8.** Evolution of the quantity of the different protein fractions (mg.kernel−1) for two common French wheat cultivars (A) Soissons and (B) Thésée, as a function of the days after anthesis. ( ) SDS-insoluble polymers; (O) SDS-soluble polymers; (■) albumins and globulins; ( ) monomers and (☐) total proteins. Evolution of (C) the polymer/monomer ratio (%) and (D) the quantity of monomers and total polymers (mg.kernel−1) as a function of days after anthesis. (O,●) total polymers of Thésée and Soissons, respectively; (☐,■) Monomers of Thésée and Soissons, respectively. Stages: (P1) cell division; (P2) cell enlargement and (P3) grain desiccation and maturation (from Carceller and Aussenac [67]).

(**Figure 8A** and **B**).

these specific proteins.

**Figure 7.** Grain filling period for a common wheat cultivar (*Soissons*). Evolution of (●) dry matter per kernel, (O) fresh matter per kernel, ( ) water quantity per kernel and (X) grain humidity. The vertical lines represent the standard deviation (*n* = 3) (from Carceller and Aussenac [67]).

filling phase) and the grain desiccation phase (the beginning of this phase corresponding to the acquisition of physiological maturity) [71] during which protein bodies disappear to form the protein matrix [72, 73].

Since 1970s, a great deal of work has been done to evaluate the effects of the environment on grain development. Thus, the effects of several environmental variables (i.e. light, temperature, water availability and nutrient availability), taken individually or in combination, have been studied [74–85]. In general, temperature and water availability strongly affect the filling rate (*V*) and the duration of grain filling (*D*), although some differences in behaviour may exist between wheat genotypes. Consequently, differences in thermal regimes and/or water regimes cause profound changes in the accumulation of the total dry matter (*P*) by affecting indifferently and without compensating the speed and duration of filling [86].

#### **3.2. Accumulation of Storage Proteins**

The accumulation of different protein fractions (albumins-globulins, gliadins and glutenins) is progressive from flowering until the acquisition of the physiological maturity of the grains (≈ 35–40 DAA). However, even if the time of initiation of the biosynthesis of the different proteins of the grain is not significantly different (5–7 DAA) [87], their rate of accumulation varies considerably, suggesting a phenomenon of differential regulation of this biosynthesis (**Figure 8A** and **B**).

Thus, a certain accumulation asynchrony in the protein fractions of the grain can be highlighted. The albumins-globulins accumulate most rapidly in the grain, followed by the monomeric prolamins and finally the polymeric prolamins. As many researchers have shown [88–93], the accumulation of albumins-globulins is maintained only during the cell division phase, contrary to that of prolamins. This confirms the functional and/or structural role of these specific proteins.

While the ratio between polymeric proteins and monomeric proteins is stable during the first stages of grain development (i.e. cell division and cell enlargement), this ratio increases significantly during the grain desiccation phase (i.e. after 35 DAA) (**Figure 8C**). A number of results in the literature are quite contradictory [90, 94, 95]. In our opinion, and in accordance with the

filling phase) and the grain desiccation phase (the beginning of this phase corresponding to the acquisition of physiological maturity) [71] during which protein bodies disappear to form

**Figure 7.** Grain filling period for a common wheat cultivar (*Soissons*). Evolution of (●) dry matter per kernel, (O) fresh matter per kernel, ( ) water quantity per kernel and (X) grain humidity. The vertical lines represent the standard

Since 1970s, a great deal of work has been done to evaluate the effects of the environment on grain development. Thus, the effects of several environmental variables (i.e. light, temperature, water availability and nutrient availability), taken individually or in combination, have been studied [74–85]. In general, temperature and water availability strongly affect the filling rate (*V*) and the duration of grain filling (*D*), although some differences in behaviour may exist between wheat genotypes. Consequently, differences in thermal regimes and/or water regimes cause profound changes in the accumulation of the total dry matter (*P*) by affecting

The accumulation of different protein fractions (albumins-globulins, gliadins and glutenins) is progressive from flowering until the acquisition of the physiological maturity of the grains (≈ 35–40 DAA). However, even if the time of initiation of the biosynthesis of the different proteins of the grain is not significantly different (5–7 DAA) [87], their rate of accumulation

indifferently and without compensating the speed and duration of filling [86].

the protein matrix [72, 73].

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deviation (*n* = 3) (from Carceller and Aussenac [67]).

**3.2. Accumulation of Storage Proteins**

**Figure 8.** Evolution of the quantity of the different protein fractions (mg.kernel−1) for two common French wheat cultivars (A) Soissons and (B) Thésée, as a function of the days after anthesis. ( ) SDS-insoluble polymers; (O) SDS-soluble polymers; (■) albumins and globulins; ( ) monomers and (☐) total proteins. Evolution of (C) the polymer/monomer ratio (%) and (D) the quantity of monomers and total polymers (mg.kernel−1) as a function of days after anthesis. (O,●) total polymers of Thésée and Soissons, respectively; (☐,■) Monomers of Thésée and Soissons, respectively. Stages: (P1) cell division; (P2) cell enlargement and (P3) grain desiccation and maturation (from Carceller and Aussenac [67]).

remarks of Stone and Nicolas [92], most of these differences can be explained by the fact that the methods of extraction and analysis of the polymeric proteins retained are extremely varied from one research group to another; it is therefore certain that all the researchers did not take into account the same protein entities in the calculation of the polymers/monomers ratio.

of attention during the last 15 years because these fractions became widely recognized as the key protein fraction that can explain differences in dough strength and breadmaking quality. According to the various physiological observations carried out since the early 2000s [67, 93, 100–102], it appears that the UPP accumulation phase coincides very strongly with the grain desiccation phase (**Figure 9B**), whatever the culture conditions applied (i.e. light, temperature, water availability and nutrient availability). Thus, 95–100% (w/w) of the UPPs present in the grain at harvesting accumulates during the grain desiccation phase. Finally, several experiments of artificial dehydration of wheat grains have confirmed the strong relationship

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Although today the mechanisms responsible for the formation of UPPs are still the subject of discussions and/or hypotheses, many observations seem to confirm that the strengthening of the aggregation character in these polymeric proteins during grain desiccation results from the reinforcement of intermolecular interactions (mainly covalent interactions) between the different glutenin subunits (HMW-GS and LMW-GS) [103, 104]. This phenomenon has led to a very significant increase in the different molecular dimensions (Mw and Rg) of the glutenin

Studying the function of free glutenin sulfhydryl (SH) and disulphide (SS) groups in glutenins of developing wheat for UPP formation, we showed that the major wheat glutenin subunits residing in the protein bodies undergo redox change during the development and the maturation of the grain [103] (**Figure 10**). Indeed, during the cell division and grain filling, glutenin

**Figure 10.** Change in sulfhydryl status of wheat proteins during grain development and maturation. MBBr-derivatized (fluorescence photography) storage proteins of a common French wheat cultivar (Soissons). Days after anthesis (DAA)

between grain water loss and UPP accumulation [93, 102].

polymers [103].

(from Rhazi et al. [103]).

The accumulation of SDS-soluble polymers that starts very early in the grain (from 7 DAA), is very slow and continues up to the beginning of the drying phase of the grain. The accumulation of SDS-insoluble polymers (i.e. UPP) is, in turn, really visible only when the grain begins to lose its water balance (i.e. end of the "water plateau") [67, 92, 96] (**Figure 9B**).

These elements must be compared with the observations of researchers such as Woodman and Engledow who, as early as the 1920s, noted the increase in the ability of proteins to form a coherent mass, gluten, in relation to the beginning of the grain desiccation [97]. The accumulation of the protein polymers in the broad sense coincides perfectly with the accumulation of the different glutenin subunits (LMW-GS and HMW-GS) in the grain [91, 98]; the HMW-GS/LMW-GS ratio being an important parameter for differentiating wheat genotypes from each other. For example, in the framework of our own research [67, 99], we have been able to demonstrate that at harvest time, the association state of polymeric proteins (i.e. SDSinsoluble polymers/total polymers ratio) is strongly correlated with the HMW-GS/LMW-GS ratio. Thus, at maturity, with the same total polymer amount (**Figure 9A**), the wheat genotype Soissons, which is characterized by a HMW-GS/LMW-GS ratio twice that of the wheat genotype Thesée, has a SDS-insoluble polymer/total polymer ratio twice as large that of Thésée (**Figure 9B**).
