**4. Impacts of environmental factors on MWD of prolamins**

The multiple agronomic studies which were done during the last 25 years indicate that environmental conditions affect the amount, composition and polymerization of the gluten proteins [109–119]. Furthermore, the impact of environmental components on the molecular weight distribution of the prolamins is significantly greater than that of genetic components (i.e. σ<sup>2</sup> E/σ<sup>2</sup> R > σ<sup>2</sup> G/σ<sup>2</sup> R) (**Table 2**) [120–122]. This is why, in a context of profound environmental changes [123], it is very important to better understand the mechanisms responsible for these effects in order to better anticipate them.

The availability of nutrients (nitrogen and/or sulphur availability) and the temperature (thermal regime) are the two main environmental factors responsible for these protein changes.


1 Quantity in mg/100 g DM.

2 M<sup>n</sup> = Molecular weight number-average and M<sup>w</sup> = Molecular weight-average (g.mol−1).

*\*\*\*F*-test significance at 0.1% level of probability; σ<sup>2</sup> G/σ<sup>2</sup> <sup>R</sup> = Genetic variance/Residual variance ratio and σ<sup>2</sup> E/ σ2 <sup>R</sup> = Environmental variance/Residual variance ratio.

NS: Not significant.

**Table 2.** Genetic (G) and environmental (E) influence on molecular weight distribution of storage proteins determined by analysis of variance (*F*-test) for 130 common French wheat genotypes cultivated in three different locations for 2005 and 2006 (from Aussenac et al. unpublished data).

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

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

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

The multiple agronomic studies which were done during the last 25 years indicate that environmental conditions affect the amount, composition and polymerization of the gluten proteins [109–119]. Furthermore, the impact of environmental components on the molecular weight distribution of the prolamins is significantly greater than that of genetic components

changes [123], it is very important to better understand the mechanisms responsible for these

The availability of nutrients (nitrogen and/or sulphur availability) and the temperature (thermal regime) are the two main environmental factors responsible for these protein changes.

**value**

M<sup>n</sup> = Molecular weight number-average and M<sup>w</sup> = Molecular weight-average (g.mol−1).

Total proteins<sup>1</sup> 8.700 15.100 11.187 11.411\*\*\* 271.577\*\*\* Total polymers<sup>1</sup> 2.785 5.755 4.016 11.104\*\*\* 187.462\*\*\* Polymer/monomer 0.321 0.700 0.561 14.845\*\*\* 72.611\*\*\*

<sup>2</sup> 0.730 × 10<sup>6</sup> 9.609 × 10<sup>6</sup> 0.972 × 10<sup>6</sup> 1.068NS 4.383\*\*\*

<sup>2</sup> 1.142 × 10<sup>6</sup> 22.970 × 10<sup>6</sup> 7.640 × 10<sup>6</sup> 3.370\*\*\* 38.974\*\*\*

G/σ<sup>2</sup>

**Table 2.** Genetic (G) and environmental (E) influence on molecular weight distribution of storage proteins determined by analysis of variance (*F*-test) for 130 common French wheat genotypes cultivated in three different locations for 2005

R) (**Table 2**) [120–122]. This is why, in a context of profound environmental

**Mean value σ<sup>2</sup>**

**G/σ<sup>2</sup>**

<sup>R</sup> = Genetic variance/Residual variance ratio and σ<sup>2</sup>

**<sup>R</sup> σ<sup>2</sup>**

**E/σ<sup>2</sup> R**

E/

**4. Impacts of environmental factors on MWD of prolamins**

desiccation totally inhibits the UPP deposition [104].

disulphide bridges.

146 Global Wheat Production

(i.e. σ<sup>2</sup>

Polymer M*<sup>n</sup>*

Polymer M<sup>w</sup>

Quantity in mg/100 g DM.

NS: Not significant.

1

2

σ2

E/σ<sup>2</sup> R > σ<sup>2</sup> G/σ<sup>2</sup>

effects in order to better anticipate them.

**Parameters Maximum value Minimum** 

*\*\*\*F*-test significance at 0.1% level of probability; σ<sup>2</sup>

<sup>R</sup> = Environmental variance/Residual variance ratio.

and 2006 (from Aussenac et al. unpublished data).

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 by higher nitrogen levels [127].

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 of H<sup>2</sup> O2 generated) is one of the most efficient. This essential cycle in chlorophyll tissues [139, 140] has also been studied in seeds [141, 142].

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 exchanges.

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

(PPSSG) [159]. Even if glutathione is able to bind to the storage proteins during grain filling, the formation of PSSG is not however correlated with the accumulation of the storage proteins in the grain but coincide rather with the grain desiccation during which the major wheat storage proteins residing in protein bodies undergo redox change (i.e. become oxidized) and UPP

Storage Proteins Accumulation and Aggregation in Developing Wheat Grains

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

149

**Figure 12.** The relationship between the content of high aggregated polymeric proteins (PP with M<sup>w</sup> > 2.0×10<sup>6</sup>

cultivars (harvest 2005 and 2006 in three locations) (from Aussenac et al. Unpublished data).

common French wheat cultivars (harvest 2005 and 2006 in three locations). σ<sup>2</sup>

ratio and σ<sup>2</sup>

E/σ<sup>2</sup>

and the content of polymeric proteins conjugated to glutathione (PPSSG) for five different common French wheat

**Figure 13.** Variation of the content of polymeric proteins conjugated to glutathione (PPSSG) for a significant set of

<sup>R</sup> = environmental variance/residual variance ratio (from Aussenac et al. Unpublished data).

G/σ<sup>2</sup>

<sup>R</sup> = genetic variance/Residual variance

g.mol−1)

are accumulated [103, 159, 160].

form (i.e. high GSH/GSSH ratio) which has the effect both for maintaining the SH status of proteins (to maintain enzymatic activities [147]) and continue to trap H<sup>2</sup> O2 .

Under the influence of oxidative stress, the redox status of glutathione will be modified; GSSG dimer will accumulate due to either an increase in GSH oxidation and/or a decrease in GSSG reduction activity (i.e. decrease of GSH/GSSH ratio). Such changes in the SH/SS status have already been widely observed in response to oxidative stress, especially during seed desiccation [148]. Glutathione which is able to bind to protein thiols is considered a "protective" element of these protein compounds since it prevents the formation of intramolecular disulphide (S-S) bridges during the desiccation phenomena [149]. In this way, GSH contributes both to limit the protein denaturation phenomena and to modulate enzymatic activity [150]. In contrast to desiccation, the imbibition phenomenon preceding germination causes the reduction of the disulphide bonds (SS) of a large number of compounds such as, GSSG [151, 152], protein-SSG conjugates [153], α-amylases [154] or the storage proteins [155, 156]. A synthesis of the presumed role of glutathione can be postulated, referring in particular to the hypothesis formulated by Kranner and Grill [150] (**Figure 11**).

Glutathione may occur endogenously in wheat flour in the free reduced glutathione (GSH) and free oxidized glutathione disulphide (GSSG) forms as well as in the form of proteinglutathione mixed disulphides (PSSG) [146–159]. Moreover, approximately 85% of PSSG in mature wheat grains are represented by polymeric proteins (PP) conjugated to glutathione

**Figure 11.** SH/SS interchange during dehydration/rehydration phenomenon. (*Dh*) dehydration step, (*Rh*) rehydration step, (*GP*) glutathione peroxidase, (*GR*) glutathione reductase (from Kranner and Grill [150]).

(PPSSG) [159]. Even if glutathione is able to bind to the storage proteins during grain filling, the formation of PSSG is not however correlated with the accumulation of the storage proteins in the grain but coincide rather with the grain desiccation during which the major wheat storage proteins residing in protein bodies undergo redox change (i.e. become oxidized) and UPP are accumulated [103, 159, 160].

**Figure 12.** The relationship between the content of high aggregated polymeric proteins (PP with M<sup>w</sup> > 2.0×10<sup>6</sup> g.mol−1) and the content of polymeric proteins conjugated to glutathione (PPSSG) for five different common French wheat cultivars (harvest 2005 and 2006 in three locations) (from Aussenac et al. Unpublished data).

**Figure 13.** Variation of the content of polymeric proteins conjugated to glutathione (PPSSG) for a significant set of common French wheat cultivars (harvest 2005 and 2006 in three locations). σ<sup>2</sup> G/σ<sup>2</sup> <sup>R</sup> = genetic variance/Residual variance ratio and σ<sup>2</sup> E/σ<sup>2</sup> <sup>R</sup> = environmental variance/residual variance ratio (from Aussenac et al. Unpublished data).

**Figure 11.** SH/SS interchange during dehydration/rehydration phenomenon. (*Dh*) dehydration step, (*Rh*) rehydration

form (i.e. high GSH/GSSH ratio) which has the effect both for maintaining the SH status of

Under the influence of oxidative stress, the redox status of glutathione will be modified; GSSG dimer will accumulate due to either an increase in GSH oxidation and/or a decrease in GSSG reduction activity (i.e. decrease of GSH/GSSH ratio). Such changes in the SH/SS status have already been widely observed in response to oxidative stress, especially during seed desiccation [148]. Glutathione which is able to bind to protein thiols is considered a "protective" element of these protein compounds since it prevents the formation of intramolecular disulphide (S-S) bridges during the desiccation phenomena [149]. In this way, GSH contributes both to limit the protein denaturation phenomena and to modulate enzymatic activity [150]. In contrast to desiccation, the imbibition phenomenon preceding germination causes the reduction of the disulphide bonds (SS) of a large number of compounds such as, GSSG [151, 152], protein-SSG conjugates [153], α-amylases [154] or the storage proteins [155, 156]. A synthesis of the presumed role of glutathione can be postulated, referring in particular to

Glutathione may occur endogenously in wheat flour in the free reduced glutathione (GSH) and free oxidized glutathione disulphide (GSSG) forms as well as in the form of proteinglutathione mixed disulphides (PSSG) [146–159]. Moreover, approximately 85% of PSSG in mature wheat grains are represented by polymeric proteins (PP) conjugated to glutathione

O2 .

proteins (to maintain enzymatic activities [147]) and continue to trap H<sup>2</sup>

148 Global Wheat Production

the hypothesis formulated by Kranner and Grill [150] (**Figure 11**).

step, (*GP*) glutathione peroxidase, (*GR*) glutathione reductase (from Kranner and Grill [150]).

Low molecular weight endogenous thiols such as glutathione, which mainly act as "protein protectors" [149] through the formation of PSSG during tissue desiccation, are responsible in wheat grains during its desiccation to a significant reduction of the MWD of the polymeric proteins by the formation of PPSSG (**Figure 12**). This action is all the more important because it is very targeted because GSH was bound almost exclusively to those cysteine residues that have been proposed to form intermolecular disulphide bonds (in particular, cysteines Cb\* and Cx, which are responsible for the aggregative nature of LMW-GS) as Köhler et al. [161] has been able to demonstrate it by using 35Slabelled GSH.

**Author details**

Beauvais, France

**References**

Aussenac Thierry\* and Rhazi Larbi

DOI: 10.1006/jcrs.1994.1018

1907; Pub. N° 84: 119 pp

S0733-5210(86)80012-1

jcrs.1994.1020

Address all correspondence to: thierry.aussenac@unilasalle.fr

wheats. Cereal Chemistry. 1948;**25**:291-312

Transformations and Agro-Resources (UP 2018.C103), Institut Polytechnique UniLaSalle,

Storage Proteins Accumulation and Aggregation in Developing Wheat Grains

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

151

[1] Finney KF, Barmore MA. Loaf volume and protein content of hard winter and spring

[2] Cornec M, Popineau Y, Lefebvre J. Characterization of gluten subfractions by SE-HPLC and dynamic rheological analysis in shear. Journal of Cereal Science. 1994;**19**:131-139.

[3] Hoseney RC, Rogers DE. The formation and properties of wheat flour doughs. Critical Reviews in Food Science and Nutrition. 1990;**29**:73-93. DOI: 10.1080/10408399009527517

[4] Gupta RB, Batey IL, MacRitchie F. Relationships between protein composition and func-

[5] Osborne TB. The proteins of the wheat kernel. Carnegie Institution of Washington D.C.

[6] Shewry PR, Tatham AS, Forde J, Kreis M, Miflin BJ. The classification of wheat gluten proteins; a reassessment. Journal of Cereal Science. 1986;**4**:97-106. DOI: 10.1016/

[7] Singh NK, Shepherd KW. Linkage mapping of genes controlling endosperm storage proteins in wheat. I – Genes on the short arms of group 1 chromosomes. Theoretical and

[8] Wieser H, Seilmeier W, Belitz HD. Quantitative determination of gliadin subgroups from different wheat cultivars. Journal of Cereal Science. 1994;**19**:149-155. DOI: 10.1006/

[9] Brown JWS, Flavell RB. Fractionation of wheat gliadin and glutenin subunits by two dimensional electrophoresis and the role of group 6 and group 2 chromosomes in gliadin synthesis. Theoretical and Applied Genetics. 1981;**59**:349-359. DOI: 10.1007/BF00276448

[10] Autran JC, Bourdet A. L'identification des variétés de blé : établissement d'un tableau général de détermination fondé sur le diagramme électrophorétique des gliadines du

[11] Gupta RB, Shepherd KW. Two step one dimensional SDS-PAGE analysis of LMG subunits of glutenin. I - Variation and genetic control of the subunits in hexaploid wheats.

Theoretical and Applied Genetics. 1990;**80**:65-74. DOI: 10.1007/BF00224017

tional properties of wheat flour. Cereal Chemistry. 1992;**69**:125-131

Applied Genetics. 1988;**75**:628-641. DOI: 10.1007/BF00289132

grain. Annales de l'Amélioration des Plantes. 1975;**25**:277-301

Consequently, it is now clear that glutathione conjugation with polymeric proteins during the grain development resulting in drastic changes of the cellular redox status (largely due to environmental factors - **Figure 13**) plays a crucial role in controlling the MWD of the polymeric proteins which has been shown to be important in determining baking performance.
