Direct Evidence that the Number and Location of Cysteine Residues affect Glutenin Polymer Structure

In this study, the possible roles of three well-characterised model prolamins in the structure of the glutenin macropolymer were examined. Model prolamins were labelled with fluorescein isothiocyanate (FITC), and incorporated into the glutenin macropolymer of a base flour using a partial reduction-oxidation scheme. The effect of incorporation of the model prolamins on dough behaviour was determined by assessing differences in polymer size distribution, mixing properties, and distribution of the model prolamins in a dough after incorporation. Using this approach, the prolamins capable of forming inter-chain disulphide bonds were shown to be incorporated into the glutenin macroÍpolymer, while prolamins that were not capable of forming inter-chain disulphide bonds were retained as monomers. The distribution of fluorescently-labelled prolamins after their incorporation into the glutenin macropolymer of the dough was examined by confocal light scanning microscopy, in order to determine the possible roles of ω-gliadins and glutenin-like subunits with varied cysteine residue compositions in the structural organisation. The role of the model prolamins was a function of the disulphide-bonding capabilities of the polypeptides. Model ω-gliadins were retained as monomers and functioned as space fillers; model glutenin-like subunits containing a single cysteine residue incorporated into the glutenin macropolymer but functioned as chain terminators; and model glutenin-like subunits containing two cysteine residues incorporated into the glutenin macropolymer and acted as chain extenders.     

Expression and Functional Analysis ofMr58 000 Peptides Derived from the Repetitive Domain of High Molecular Weight Glutenin Subunit 1Dx5

A highly repetitiveM_r58 000 peptide based on residues 102 to 643 of subunit 1Dx5 and forms containing one to four cysteine residues were expressed inE. coliand purified to homogeneity. Incorporation into dough using a 2 g Mixograph showed that most peptides resulted in reduced strength, which was possibly due to dilution or chain termination of glutenin polymers. However, a form containing four cysteines (two each close to the N-terminus and C-terminus) resulted in increased strength, indicating that the repetitive domains of the HMW subunits are sufficient to contribute to dough strength when incorporated into glutenin polymers.     

分子生物学技术在普通小麦谷蛋白研究中的应用

小麦谷蛋白是面筋的主要成分之一，对小麦食品的加工品质起着重要作用。本文从基因序列、分子结构、多态性、遗传转化、QTL研究和MAS等方面综述了国内外有关麦谷蛋白亚基的研究进展。这些研究结果表明，麦谷蛋白的等位基因变异十分丰富，多态性高．序列之间存在很高的同源性。麦谷蛋白的基因结构分为三部分：无重复结构的N-末端和C-末端以及中部重复区域，等位基因的变异主要由基因中部重复区域的序列大小、重复次数及该区域内DNA序列的插入或缺失所造成；Cys-残基的数目和位置影响麦谷蛋白聚合体内亚基间的相互作用，是影响亚基生化特性的重要因素；应用转基因技术已将HMW-GS基因(1Ax1、1Dx5和1Dy10)导入普通小麦中，有助于进行品质改良和麦谷蛋白结构与功能的深入研究。此外，对面筋强度性状的QTL分析和分子标记辅助育种也进行了阐述。     

Redox Reactions in Wheat Dough as Affected by Ascorbic Acid

Ascorbic acid (AA) is used as bread improver, as its addition to dough causes an increase in loaf volume and an improvement in crumb structure. To explain these effects we review the stereospecificity of the improver action and the properties of ascorbate oxidase and glutathione dehydrogenase and the occurrence of low molecular thiols in flour and their concentration changes during dough mixing in the presence and absence of AA. On the basis of the results the improver action of AA is explained by a reaction sequence leading to a rapid removal of endogenous GSH, which otherwise would cause dough weakening by sulphhydryl/disulphide interchange reactions with gluten proteins. To test this hypothesis the binding sites of endogenous GSH in gluten proteins have been determined by the addition of³⁵S-labelled GSH as a tracer to flour before dough mixing. The distribution of radioactivity in the gliadin and glutenin fractions of gluten obtained from dough indicates that the major portion of GSH is bound to glutenins. The isolation and sequence analysis of radioactive cystine peptides from an enzymatic digest of glutenins demonstrates that GSH is almost exclusively linked to those cysteine residues of LMW subunits that have been proposed to form intermolecular disulphide bonds.     

Heterologous Expression and Dough Mixing Studies of Wild-Type and Mutant C Hordeins

Wild type and mutant (cysteine-containing) forms of C hordein were expressed inEscherichia coli. Incorporation of a mutant form with N- and C-terminal cysteine residues into dough using a 2 g Mixograph showed similar positive effects on dough strength to the incorporation of HMW subunit 1Bx7. Co-incorporation showed that the effects of the two proteins were additive. In contrast, the incorporation of wild type C hordein or mutants with single cysteines at the N- or C-terminus resulted in decreased dough strength, with the two mutant forms inhibiting the positive effect of 1Bx7. Analysis of total protein extracts from the doughs indicate that the differences resulted from alterations in the proportions of gluten monomers, small gluten polymers and large gluten polymers.     

Effects of vacuum mixing and mixing time on the processing quality of noodle dough with high oat flour content

The effects of different mixing parameters (vacuum mixing and mixing time) on oat (70% oat flour) and wheat noodle dough were investigated on the basis of textural properties and gluten formation. The results showed that at a vacuum degree of −0.06 MPa and mixing time of 10 min, oat and wheat dough sheets exhibited the highest resistance to extension and glutenin macropolymer (GMP) content, and had the most compact and uniform gluten network. Compared with wheat noodle dough, oat dough had lower resistance to extension, lower tightly bound water content, and higher GMP content. Microstructural examination showed that oat noodle dough had a more aggregated distribution of gluten protein compared with wheat noodle dough under the optimum mixing parameters. Furthermore, the poor binding ability of vital wheat gluten with water molecules caused the indexes of oat noodle dough to be more strongly affected by the changes in mixing parameters than wheat noodle dough.     

Incorporation of high-molecular-weight glutenin subunits into doughs using 2 gram mixograph and extensigraphs

To study the contributions of high-molecular-weight glutenin subunits (HMW-GS) to the gluten macropolymer and dough properties, wheat HMW-GS (x- and y-types) are synthesized in a bacterial expression system. These subunits are then purified and used to supplement dough mixing and extensigraph experiments through dough partial reduction and reoxidation to allow these exogenously added HMW-GS to incorporate into gluten polymers. Detailed results are given for seven mixing and two extension parameters. HMW-GS synthesized in bacteria behaved similarly under these conditions to the same HMW-GS extracted from wheat flour. These experiments initially focused on the HMW-GS of the D-genome of hexaploid wheat encoded at the Glu-D1 locus; e.g. the Dx2, Dx5, Dy10, and Dy12 subunits. Experiments used five different flours and results are shown to be consistent when normalized to results from Dx5. The incorporation of Dx-type subunits into the gluten disulfide bonded network has greater effects on dough parameters than incorporation of Dy-type subunits. When Glu-D1 x- and y-type subunits are incorporated together, there are synergistic effects greater than those with either subunit type alone. This synergistic effect was greatest with approximately equal amounts of Dx- and Dy-type subunits - implying a 1:1 stoichiometric relationship.     

Developmental and environmental effects on the assembly of glutenin polymers and the impact on grain quality of wheat

Wheat kernel development can be divided into three phases i.e. cell division, cell enlargement and dehydration. Accumulation of gluten proteins continues till the end of the cell enlargement phase. During the dehydration phase, post-translational polymerization of the glutenin subunits occurs to form very large glutenin polymers. Assembly of the glutenin polymers has been monitored by increase in the unextractable polymeric protein. Lines possessing HMW-GS related to dough strength (e.g. 5 + 10) started accumulating large polymers several days earlier than lines with HMW-GS related to dough weakness (e.g. 2 + 12) and maintained their higher amounts till maturity. This may be explained by faster polymerization resulting from a higher concentration of cysteine residues in the x-type HMW-GS.     

Correlation of glutenin macropolymer with viscoelastic properties during dough mixing

Although significant correlations exist for glutenin macropolymer (GMP) quantity and rheological properties/bread making quality of dough, little information about these links is available. The relationship between GMP contents measured by UV absorption method/RP-HPLC and dough viscoelastic properties determined by TA-XT2i texturometer from three wheat varieties (Xiaoyan6, Yumai56 and Zhengnong8805) during mixing was investigated. GMP contents of doughs decrease significantly (P<0.05) during mixing. During the initial mixing stage, amounts of the HMW-GS and LMW-GS and GMP decrease significantly (P<0.05). Their contents begin to increase beyond peak dough development time (DDT). This indicates that during further mixing after peak DDT some glutenin subunits are incorporated into GMP by repolymerization. The HMW/LMW-GS ratio has a significant effect on load-deformation properties (area, resistance and extensibility) of dough. The varieties behaved differently in relation to the contribution of their HMW-LMW-GS ratio to the rheological properties.     

Isolation and characterization of a novel variant of HMW glutenin subunit gene from the St genome of Pseudoroegneria stipifolia

The x- and y-type high molecular weight (HMW) glutenin subunits are conserved seed storage proteins in wheat and related species. Here we describe investigations on the HMW glutenin subunits from several Pseudoroegneria accessions. The electrophoretic mobilities of the HMW glutenin subunits from Pd. stipifolia, Pd. tauri and Pd. strigosa were much faster than those of orthologous wheat subunits, indicating that their protein size may be smaller than that of wheat subunits. The coding sequence of the Glu-1St1 subunit (encoded by the Pseudoroegneria stipifolia accession PI325181) was isolated, and found to represent the native open reading frame (ORF) by in vitro expression. The deduced amino acid sequence of Glu-1St1 matched with that determined from the native subunit by mass spectrometric analysis. The domain organization in Glu-1St1 showed high similarity with that of typical HMW glutenin subunits. However, Glu-1St1 exhibited several distinct characteristics. First, the length of its repetitive domain was substantially smaller than that of conventional subunits, which explains its much faster electrophoretic mobility in SDS-PAGE. Second, although the N-terminal domain of Glu-1St1 resembled that of y-type subunit, its C-terminal domain was more similar to that of x-type subunit. Third, the N- and C-terminal domains of Glu-1St1 shared conserved features with those of barley D-hordein, but the repeat motifs and the organization of its repetitive domain were more similar to those of HMW glutenin subunits than to D-hordein. We conclude that Glu-1St1 is a novel variant of HMW glutenin subunits. The analysis of Glu-1St1 may provide new insight into the evolution of HMW glutenin subunits in Triticeae species.     

Understanding the link between GMP and dough: from glutenin particles in flour towards developed dough

Clear correlations exist for glutenin macropolymer (GMP) quantity and rheological properties vs. wheat quality and dough rheological properties, but real insight in understanding these links is still missing. The observation that GMP consists of glutenin particles opens up new possibilities to reveal the underlying mechanism linking glutenin network properties with dough preparation. GMP was isolated from flour of three wheat varieties: Estica, Soissons and Baldus, strongly varying in their mixing requirements (expressed as time-to-peak, TTP). Decrease of GMP quantity and G′ vs. mixing energy was confirmed. More detail was obtained by studying the changes in GMP particles when mixing flour into dough. Mixing leads to a decrease in the average size of the particles. Interestingly, the TTP coincided with the work-input at which all particles just became soluble in SDS. At TTP, the average size of the GMP particles was the same for each variety. During mixing particles lost their globule shapes and appeared ruptured. Particle size analysis confirmed that particles were still present near TTP. Analysis of the change in particle size vs. energy input using physical principles revealed the following: (1) mixing energy is the predominant actuator in decreasing GMP particle size; (2) the initial GMP particle size in flour strongly determines the practical mixing requirements; and (3) the derived mixing energy vs. GMP particle size relationship was shown to be applicable for both Mixograph and Farinograph mixing. Our results demonstrate that, for the flour samples used, glutenin particle size determines TTP and GMP rheology, showing that glutenin particle properties could be a new key to understand the link between GMP and dough properties.     

Application of Polymer Science to Properties of Gluten

Much of the knowledge from polymer science studies can be usefully applied to increase understanding of the properties of gluten and how they are related to composition. Low solubility of gluten proteins compared to many other proteins that have been studied evidently arises from the low entropy of mixing of the largest-sized glutenins and a relatively high value for the Flory-Huggins interaction parameter mainly due to a low concentration of ionisable groups. Grain hardness is a property that appears to depend on the proteins that are concentrated at the starch/matrix interface and how they control adhesion. Dough mixing properties, dough strength and extensibility can be understood in terms of the extension of large glutenin molecules giving rise to rubber elasticity and to the presence of molecular entanglements which contribute strength and extensibility to dough systems.     

Effect of incorporation of an i-type low-molecular-weight glutenin subunit and a modified γ-gliadin in durum and in bread wheat doughs as measured by micro-mixographic analyses

The effects of incorporation of an i-type low-molecular-weight glutenin subunit (LMW-i) and of a modified γ-gliadin showing an additional cysteine residue, on 2 g Mixograph parameters of durum (biotypes 42 and 45 of the Italian cv. Lira) and bread wheat (Australian cv. Kukri) doughs were studied. In bread wheat flour incorporation of the modified γ-gliadin resulted in a significant decrease in dough strength (decreased mixing time and peak resistance), but at the same time it produced a slight increase in dough stability (decreased resistance to breakdown). The incorporation of the LMW-i type into bread wheat dough had minimal effects on dough mixing requirements. The incorporation of both LMW-i type and modified γ-gliadin in durum wheat doughs produced a significant decrease in the overall dough strength, especially in Lira 45 biotype doughs. Reversed phase high-performance liquid chromatography (RP-HPLC), size exclusion high-performance liquid chromatography (SE-HPLC) and two-dimensional gels analyses of control and reconstituted semolina doughs showed that the two polypeptides were in the polymeric fraction. The effect of the incorporation of the two polypeptides in durum and bread wheat doughs showed remarkable differences and the reasons for this is discussed in terms of both intrinsic differences between wheat flour and durum semolina and in methodological approaches.     

小麦贮藏蛋白特性及其遗传转化

小麦籽粒贮藏蛋白由醇溶蛋白和谷蛋白组成。醇溶蛋白在组成上以单体形式存在 ,具有高度的异质性和复杂性。它决定小麦面筋的粘性。谷蛋白是由多个亚基组成的高分子聚合体 ,决定面筋的弹性。它可分为低分子量谷蛋白亚基和高分子量谷蛋白亚基 (HMW- GS)。HMW- GS具有相似的分子结构 ,即由中央重复序列、无重复的 N端和 C端组成。HMW- GS对小麦烘烤品质起着决定性作用 ,但因 HMW- GS类型不同而对加工品质的贡献大小各异。许多 HMW- GS基因已被揭示。实践证明 ,利用基因枪法 ,将 HMW- GS基因导入普通小麦的细胞核内 ,能够达到改良小麦烘焙品质的目的。随着分子生物学技术的不断发展 ,可望从营养和加工角度来改良小麦品质的特性     

Impact of ethanol,succinic acid,and the combination thereof at levels produced during sponge fermentation on hard wheat,soft wheat,and durum wheat farinograph rheology

The sponge and dough mixing process is one of the most common in the world, yet the mechanistic understanding of this process has yet to be sufficiently explored. In this study, aqueous solutions of ethanol, succinic acid, and their combination were prepared at concentrations intended to replicate fermentation times of 3, 4 and 6 h. These solutions were added to a farinograph mixer to make dough using hard wheat, soft wheat, and durum wheat flour. The results indicate that these yeast metabolites (ethanol, succinic acid) impact the mixing resistance, peak mixing value, and dough mixing stability in each of the flour types, likely primarily affected by the ratios of gliadin to glutenin and LMW glutenin in each flour type. Results suggest a stabilizing non-covalent interaction imparted by gliadin at peak mixing time, a stabilizing effect of HMW glutenin during break down, and synergistic effects of ethanol and succinic acid that leads to a faster rate of breakdown in later stages of mixing. It also suggests an increase in mixing resistance when acidulants are added to durum wheat dough. Taken together, this study adds new insights on the sponge and dough mixing process in a way that has not previously been conducted.     

Evidence for the Presence of Only One Cysteine Residue in the D-type Low Molecular Weight Subunits of Wheat Glutenin

D-type low molecular weight subunits of bread wheat glutenin have ω-gliadin type N-terminal amino acid sequences, but are incorporated into the glutenin polymers because of the presence of cysteine residues. In order to determine the number and position of cysteine residues, ID-encoded D-type low molecular weight subunits of wheat glutenin were purified from the cv. Chinese Spring using a procedure that allowed high recovery. Comparison of the molecular weights of alkylated and unalkylated subunits by MALDI mass spectrometry indicated the presence of only one cysteine residue per molecule. This was supported also by the detection of dimers of D subunits in gluten. An internal sequence of 62 amino acids preceding the cysteine was obtained, but it was not possible to identify the cysteine residue, either because it was not within the range of N-terminal sequencing of peptides obtained, or because it was present in one of the two unidentified positions.     

Characterisation of an s-type low molecular weight glutenin subunit of wheat and its proline and glutamine-rich repetitive domain

The low molecular weight glutenin subunits (LMW-GS) are major components of the glutenin polymers which determine the elastomeric properties of wheat (Triticum aestivum L.) gluten and dough. They comprise a complex mixture of components and have proved to be difficult to purify for detailed characterisation. The mature LMW subunit proteins comprise two structural domains, with one domain consisting of repeated sequences based on short peptide motifs. DNA sequences encoding this domain and a whole subunit were expressed in Escherichia coli and the recombinant proteins purified. Detailed comparisons by spectroscopy (CD, FT-IR) and dynamic light scattering indicated that the repetitive and non-repetitive domains of the proteins formed different structures with the former having an extended conformation with an equilibrium between poly-L-proline II-like structure and type II' β-turns, and the latter a more compact globular structure rich in α-helix. Although the structures of these two domains appear to form independently, dynamic light scattering of the whole subunit dissolved in trifluoroethanol (TFE) suggested that they interact, leading to a more compact conformation. These observations may have relevance to the role of the LMW-GS in gluten structure and functionality.     

Effects of specific domains of high-molecular-weight glutenin subunits on dough properties by an in vitro assay

An in vitro system for incorporating bacterially produced high-molecular-weight glutenin subunits (HMW-GS) into doughs was used to study the effects of specific domains of the HMW-GS. Synergistic effects of incorporating into doughs both the Dx5 and Dy10 subunits are localized to the N-terminal domains. All single and pair-wise combinations of original subunits and hybrid subunits with their N-terminal domains exchanged between Dx5 and Dy10 finds three classes of respondents: the greatest response is when the N-termini of both Dx5 and Dy10 are present, followed by presence of the Dx5 N-terminus alone, and the least response by the presence of the Dy10 N-terminus alone. In addition, studies of Dx5 variants possessing repetitive domains of different length and composition find evidence that the length of the HMW-GS repetitive domain is important for dough properties and that the exact composition of the repeat domain has a detectible, though lesser contribution. Finally, in this experimental system, the Glu-D1 x- and y-subunits function in the mixing experiments as if they were a fused dimer, although the exact molecular basis of the effect is not known.     

The effect of mixing on glutenin particle properties: aggregation factors that affect gluten function in dough

Previously we reported that the SDS insoluble gel-layer: the Glutenin Macro Polymer (GMP) can be considered as a gel consisting of protein particles. These glutenin particles have a size of about 10⁻¹–10² μm and consist of HMW-GS and LMW-GS only. In GMP isolates from flour, the particles are spherical. In isolates from dough, glutenin particles have lost this shape. This seems relevant, since mixing disrupts the particles and the mixing energy required for dough development correlated with the glutenin particle size in flour. The question studied in this paper is how changes at a glutenin particle level affected the subsequent process of gluten network formation during dough rest and if this could be used to explain resulting dough rheological properties. To this end, we studied how various mixing regimes affected the dough properties during and after resting (elasticity). We cannot fully explain the differences in the final dough properties observed using parameters such as the quantity of GMP in flour, the quantity of re-assembled GMP in dough and the size of re-assembled glutenin particles. However, other parameters were found to be important: (1) the Huggins constant K′ reflecting the tendency of glutenin particles to interact at level II of the Hyperaggregation model; (2) the composition of glutenin particles affecting the potential to form smaller or larger particles and (3) for over-mixed dough, covalent re-polymerisation at the so-called level I of hyperaggregation. Using these parameters we can better explain dough viscoelasticity after resting.     

Factors Governing Levels and Composition of the Sodium Dodecyl Sulphate-Unextractable Glutenin Polymers During Straight Dough Breadmaking

The effect of several additives (1·215 μmol KIO₃, 0·892 μmol cysteine, endo-xylanase and 0·5% (w/w) rye-water-extractable arabinoxylans) on changes in the level and glutenin subunit composition of the sodium dodecyl sulphate (SDS)-unextractable protein during breadmaking was investigated. Protein extractability drastically increased during dough mixing and was enhanced both by cysteine and KIO₃. The mixing-induced increase in protein extractability was partly reversed during fermentation. Fermenting doughs containing endo-xylanase had a higher level of SDS-unextractable protein than control doughs, while with KIO₃the amount of SDS-unextractable protein remained very low. During baking most protein became SDS-unextractable. Bread baked from doughs with added KIO₃contained a significantly higher level of SDS-extractable protein. Changes in subunit composition of the SDS-unextractable glutenin polymers, determined with RP-HPLC, coincided with changes in protein extractability during dough processing. Mixing decreased the ratio of high to lowM_rglutenin subunits. Simultaneously, the relative proportions of the different highM_rglutenin subunits in the unextractable glutenin polymers changed. During fermentation changes in subunit composition of the SDS-unextractable glutenin were opposite to those during mixing.