**3.3. Unextractable polymeric protein (UPP) accumulation**

The formation and accumulation of polymeric protein fractions characterized by high levels of aggregation (indifferently qualified in the literature of SDS-insoluble polymeric proteins, unextratable polymeric proteins (UPP) and glutenin macro polymers (GMP)) have been the focus

**Figure 9.** Accumulation of total polymers, SDS-soluble and SDS-insoluble polymers as a function of the days after anthesis. (A) Total polymers (mg.kernel−1) for (●) Soissons and (O) Thésée. (B) Open symbols are for Thésée and closed symbols are for Soissons. ( ) SDS-soluble polymers and (O) SDS-insoluble polymers. Stages: (P1) cell division; (P2) cell enlargement and (P3) grain desiccation and maturation (from Carceller and Aussenac [67]).

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.

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. 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

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

The formation and accumulation of polymeric protein fractions characterized by high levels of aggregation (indifferently qualified in the literature of SDS-insoluble polymeric proteins, unextratable polymeric proteins (UPP) and glutenin macro polymers (GMP)) have been the focus

**Figure 9.** Accumulation of total polymers, SDS-soluble and SDS-insoluble polymers as a function of the days after anthesis. (A) Total polymers (mg.kernel−1) for (●) Soissons and (O) Thésée. (B) Open symbols are for Thésée and closed symbols are for Soissons. ( ) SDS-soluble polymers and (O) SDS-insoluble polymers. Stages: (P1) cell division; (P2) cell

enlargement and (P3) grain desiccation and maturation (from Carceller and Aussenac [67]).

to lose its water balance (i.e. end of the "water plateau") [67, 92, 96] (**Figure 9B**).

**3.3. Unextractable polymeric protein (UPP) accumulation**

(**Figure 9B**).

144 Global Wheat Production

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

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 polymers [103].

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) (from Rhazi et al. [103]).

subunits and particularly LMW-GS have a large amount of free SH groups and become oxidized during grain desiccation which coincided with the accumulation of UPP. Moreover, monobromobimane (mBBr) derivatized of free glutenin SH groups before the artificial grain desiccation totally inhibits the UPP deposition [104].

High nitrogen availability translates into high protein contents in the grain and flour but also by changes in protein composition. With increasing protein content, gliadins tend to increase at a greater rate than other proteins. This can lead to MWD alterations which results from decreases in the polymeric-to-monomeric protein ratio and/or increases in the HMW-GS to

Storage Proteins Accumulation and Aggregation in Developing Wheat Grains

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

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When sulphur fertilization is limited, the molecular distribution of glutenins is strongly affected insofar as this limitation results in a significant modification of the HMW-GS/ LMW-GS ratio [109, 126]. The increase in the HMW-GS/LMW-GS ratio which is linked to the fact that the high molecular weight glutenin subunits are much less affected by a sulphur limitation because they are poorer in corresponding amino acids, therefore results in an increase in the average molecular weight of the polymers. Finally, sulphur deficiency is accentuated

Temperature (i.e. daily mean temperature, temperature regime and temperature application stage) can induce very large changes in the association state of polymers during grain filling [110, 128–130]. Thus, in the great majority of the work carried out in recent years, various researchers have shown that the increase in temperature and/or the sudden change in the thermal regime during grain filling could lead to a significant decrease in the association status of prolamins resulting in a decrease of MWD (or solubility) of glutenins [131–133]. In the majority of the work to which we have just referred to above, the effects observed are most often attributed to modifications in the synthesis activities of the different storage proteins (i.e. gliadins vs. glutenins and/or HMW-GS vs. LMW-GS) resulting from modulation of the expression of storage protein genes [85]. Today, it seems that other phenomena could also be reasonably involved. These phenomena could be based, in particular, on important variations in the cellular redox status in response to environmental stimuli (i.e. environmental stress).

It has long been established that desiccation of plant tissues causes the appearance of free radicals. Although this phenomenon is a very general mechanism, a large number of observations have been made from seeds of various species [134–137]. In the majority of these studies, the presence of free radicals has been correlated with viability losses [138]. Among these implemented detoxification mechanisms, the ascorbate/glutathione cycle (i.e. trapping

At a cellular level, thiols are the first compounds affected by oxidative stress in general because of the high sensitivity to the oxidation of sulfhydryl (SH) groups. The predominant non-protein thiol in most plant species is glutathione (GSH). This tripeptide ensures the maintenance of the redox status at a cellular level but also the storage and transport of the reduced sulphur necessary for the synthesis of proteins [143–145]. The first compound resulting from the oxidation of glutathione is its dimer (GSSG) which is produced in vivo largely thanks to SH/SS exchanges with proteins (noted P) [146]. The reactions below illustrate these

The GSSG dimer is normally reduced in GSH by glutathione reductase (GR) activity. Thus, under normal conditions, glutathione is very much present at a cellular level in its reduced

generated) is one of the most efficient. This essential cycle in chlorophyll tissues [139,

LMW-GS ratio [113, 124, 125].

by higher nitrogen levels [127].

140] has also been studied in seeds [141, 142].

of H<sup>2</sup> O2

exchanges.

In our hypothesis which is very close to the model proposed by Hamer and van Vliet [105] for the gluten structure termed "hyper aggregation" model, the grain desiccation promotes the aggregation of polymers already present (i.e. SDS-soluble polymers or level I aggregates in the "hyper aggregation" model) by facilitating specifically the formation of interchain hydrogen bonding between the repeat regions of glutenin subunits [106–108], which can bring glutenin free accessible SH groups into close proximity to form additional intermolecular disulphide bridges.
