Correlations of the Amount of Gluten Protein Types to the Technological Properties of Wheat Flours Determined on a Micro-scale

Flour samples of 14 wheat cultivars previously characterised by rheological measurements and by baking tests on a micro-scale (Kieffer et al.: Journal of Cereal Science27 (1998) 53–60) were analysed for the relative amounts of gluten protein types using a combined extraction/HPLC procedure. Regression analysis was used to find relations between wheat properties and protein quantities. The results indicated that the maximum resistance of dough and gluten and the gluten index were strongly dependent on the quantity of glutenin subunits (GS) in flour; additionally they were influenced by the ratio of gliadin to glutenin subunits. Within the family of glutenin proteins, the correlation coefficients for high-molecular-weight (HMW) and low-molecular-weight (LMW) GS were in a similar range, but twice the amount of LMW GS was necessary to get the same resistance as with HMW GS. Among HMW GS, the contribution of x-type GS was more important than those of y-type GS. The extensibility of dough and gluten was mainly dependent on the ratio of gliadin to total glutenin subunits, to HMW GS and LMW GS. Dough development time showed the highest correlation with total HMW GS and x-type HMW GS. Bread volume was influenced by the total amount of gluten protein more than by the amount of protein in different groups or of different types, probably because of the rather low range of flour protein content (8·7–12·0 %) within the set studied. Significant differences between gliadins and glutenins with respect to their effects on bread volume could not be detected. The correlation between bread volumes and the quantity of gluten proteins was higher, when dough was mixed to optimum.     

Distribution and function of LMW glutenins,HMW glutenins,and gliadins in wheat doughs analyzed with ‘in situ’ detection and quantitative imaging techniques

The different gluten subunits, gliadins, LMW glutenins, and HMW glutenins have been reported to play different key roles in different type of wheat products. This paper studied the interaction between gliadin, LMW and HMW glutenins in soft, hard and durum semolina flour doughs during different stages of mixing. In order to see how do the gluten subunits (gliadin, LMW glutenin and HMW glutenin) redistribute during mixing, dough samples were taken at maximum strength and 10 min after maximum strength. The doughs have been mixed with the same level of added water (55%), therefore they all have different strengths values due to their changes in proteins content. Oscillatory rheological measurements were performed on the doughs. It has been found that HMW glutenins are relatively immobile because of their less molecular mobility and do no redistribute themselves especially at high strength for doughs such as hard wheat flour. LMW glutenins and gliadins on the other hand redistribute themselves at even at high dough strengths forming a more stable network. In weaker doughs such as soft wheat, the breakdown of the three proteins subunits is responsible for the decay in dough strength. We have also visualized how the greater amount of LMW glutenins in semolina is in constant interaction with HMW glutenins and gliadins allowing the dough to maintain a stable strength for an extended mixing time. Finally, we have found the ‘in situ’ detection and quantitative analysis techniques to be more sensitive to the changes occurring in the gluten network of the dough than the oscillatory rheological analysis.     

Disulphide Bonds in Wheat Gluten Proteins

Disulphide bonds play a key role in determining the structure and properties of wheat gluten proteins. Comparison of the sequences of monomeric gliadins and polymeric glutenin subunits allows the identification of conserved and variant cysteine residues. Direct disulphide bond determination demonstrates that the conserved cysteine residues present in S-rich prolamins (α-type gliadins, γ-type gliadins and LMW subunits) form intra-chain disulphide bonds while additional cysteines residues present only in the LMW subunits form inter-chain bonds with cysteines in HMW subunits and other LMW subunits. Conserved and variant cysteine residues are also present in the HMW subunits but their patterns of disulphide bond formation are less well understood. Further information on the abilities of individual cysteine residues to form intra- and inter-chain disulphide bonds has also been obtained by heterologous expression of wild type and mutant proteins inE. coliand, in the case of the HMW subunits, by examination of the patterns of dimers recovered on partial reduction of glutenin or resulting from the expression of subunits in transgenic tobacco plants. Wheat gluten proteins are folded and assembled within the lumen of the endoplasmic reticulum of the developing endosperm cells, where disulphide bond formation and exchange may be catalysed by the enzyme protein disulphide isomerase. Similarly, disulphide bond reduction, for example to facilitate mobilisation during germination, may be catalysed by thioredoxinh. Understanding the mechanism and specificity of disulphide bond formation in gluten is crucial for the manipulation of its functional properties by genetic engineering or chemical modification.     

Effect of Allelic Variation of Glutenin Subunits and Gliadins on Baking Quality in the Progeny of Two Biotypes of Bread Wheat cv. Ulla

During the determination of the HMW glutenin subunit composition of Finnish varieties, the variety Ulla was observed to contain two biotypes which differed from each other at two loci:Glu-A1andGlu-A3/Gli-A1. One of them, called Ulla 1, contained subunit 2* (Glu-A1b) andGlu-A3o/Gli-A1o, and Ulla 2 contained the null allele (Glu-A1c) andGlu-A3a/Gli-A1c. In order to determine the effect of this allelic variation on quality, the two biotypes were crossed and random lines were produced from the progeny by single seed descent. In total, 95 F6 lines were analysed from four bulked Ulla progeny lines. Significant interaction between the allelic variants of HMW glutenins and LMW gluten proteins affected the SDS-sedimentation volume at the mean flour protein level of 13·1% (dmb); the effect of LMW gluten variants was larger in the lines deficient of a HMW glutenin subunit than in lines having a HMW glutenin subunit (2*). At the higher flour protein levels (mean=15·1%, dmb) the effect on SDS-sedimentation volume was additive; progeny carrying alleles b (subunit 2*) and o/o atGlu-A1andGlu-A3/Gli-A1had significantly greater sedimentation volumes than the progeny carrying alleles c (no subunit) and a/c, respectively. The SDS-sedimentation volumes indicated differences in the quantities of the polymeric glutenins, gel proteins which have been shown to reflect dough strength. In the four bulked Ulla progeny lines, the variation in HMW glutenin subunits affected the dough strength values of the Extensigraph. However, the variation in LMW glutenin subunits did not affect Extensigraph dough strength values, as was predicted by SDS-sedimentation volumes. In the Ulla progeny, adding a HMW glutenin subunit affected Extensigraph dough strength more than adding a LMW glutenin subunit, although both increased the SDS-sedimentation volumes. Moreover, the variation in LMW gluten proteins affected the dough mixing stability in the Farinograph and test baking results of the Ulla progeny.     

Combined nitrogen and sulphur fertilisation and its effect on wheat quality and protein composition measured by SE-FPLC and proteomics

Soil sulphur deficiency, which is increasingly prevalent in Western Europe, lowers wheat yields, and also affects the gluten quality of the flour. Differences in S availability may change the proportion of S-poor to S-rich gliadins and glutenin subunits. This may cause unpredictable and unwanted variations in wheat quality. The combined effects of nitrogen (N) and sulphur (S) fertilisers and split application of S and N on wheat gluten quality and composition were investigated. The results revealed effects of S fertilisation on dough quality. At high N fertilisation levels significant responses to S fertilisation were found which emphasised the need for precision application of S in intensive wheat production systems. Protein fractionation by SE-FPLC showed that quality differences were associated with changing proportions of high M_r polymeric proteins. Changes in protein composition of salt soluble proteins were also confirmed by proteomics. Glyceraldehyde-3-phosphate dehydrogenase and one of the serpin protein spots increased at high N, combined with the lower S level. The enzymes also increased in samples with increased S fertilisation combined with low N, but was not changed at higher N levels. Furthermore, at high N the serpin protein spot, and also a 27 K protein and one unidentified protein spot decreased with increasing S.     

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     

Biochemical Characterisation of a Novel Polymeric Protein Subunit from Bread Wheat (Triticum aestivum L.)

A new wheat endosperm protein subunit that was found in accessions belonging to different collections was identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Insoluble in 0·5 M NaCl, 70% ethanol, dimethyl sulphoxide (DMSO) and 50% propan-1-ol, it appeared in the pellet corresponding to the polymeric proteins along with high (HMW) and low molecular weight (LMW) glutenin subunits (GS). In the reduced form, it had an electrophoretic mobility between those two types of glutenin subunits. The apparent M_r of this novel protein was estimated by SDS-PAGE to be 71 000. N-terminal sequence and amino acid analyses indicated a composition similar to the ω-gliadins encoded by genes located on chromosome 1B. This protein can be ascribed to the D-subunits of LMW-GS with at least one cysteine residue that allows it to form part of the polymeric structure of glutenin, as shown by reaction with a fluorogenic reagent specific for sulphydryl groups. Fractions collected after size exclusion high-performance liquid chromatography (SE-HPLC) fractionation and further characterised by SDS-PAGE, confirm that the protein participates in the glutenin polymeric structure. An increase in its concentration was observed in fractions collected within the polymeric peak as elution time increased, implying that a larger amount of this protein is present in small size polymers. The role of this protein in the complex relationship between endosperm proteins and quality parameters is discussed in relation to its likely role as a chain terminator.     

Effects of gamma-irradiation of wheat on gluten proteins

The effects of ⁶⁰Co gamma-irradiation treatments (2·5, 5·0, 10·0 and 20·0 kGy) on the gluten proteins of two bread wheats and one durum wheat cultivar were investigated. Dough rheological properties of the flour processed from the irradiated wheat were also determined using a computerised micromixograph. Irradiation caused a significant deteriorating effect on all mixogram parameters. There was no observable effect of irradiation on gliadin proteins analysed by polyacrylamide gel electrophoresis. The 50% 1-propanol-insoluble (50 PI) glutenin fraction was highly affected by irradiation. By sodium dodecyl sulfate polyacrylamide gel electrophoresis, reduced 50 PI glutenin showed a noticeable reduction in band intensities of both high (HMW) and low molecular weight (LMW) glutenin subunits (GS) with increasing irradiation dosage greater than 5 kGy. The irradiation effect on 50 PI glutenin was further studied and quantified by reversed-phase high-performance liquid chromatography of glutenin subunits; there was a progressive decrease in the quantity of subunits with increasing irradiation dose level. Compared to non-irradiated wheat, the relative decline in total insoluble glutenin at the 20 kGy dosage level ranged from 34–49% depending on cultivar. Increasing levels of irradiation also progressively reduced the ratio of HMW:LMW-GS up to 13–15% at 20 kGy indicating that irradiation had a greater effect on the largest polymers of glutenin. The observed weakening of dough mixing properties and concomitant decline in the quantity of 50 PI glutenin with increasing levels of gamma-irradiation are consistent with a degradation of glutenin to a lower average molecular size by depolymerisation and/or disaggregation.     

Relationships between dough strength,polymeric protein quantity and composition for diverse durum wheat genotypes

Five different Glu-B1 HMW-GS patterns were identified among a collection of diverse durum wheat genotypes grown in 2001 in two locations in western Canada. The durum wheat lines exhibited a wide range of dough and gluten strength characteristics as measured by alveograph and 2 g mixograph parameters, gluten index (GI), and protein composition as measured by unextractable polymeric protein (UPP) content and the ratio of high-molecular weight (HMW) glutenin subunits (GS) to low-molecular weight (LMW) GS. HMW-GS subunits patterns represented within the genotypes were 6+8, 7+8, 7+16, 14+15 and 20. Two of the genotypes expressed Glu-A1 HMW-GS 2* in combination with other HMW-GS. Approximately 95% of the durum genotypes were γ-gliadin 45 types. Analysis of variance indicated that genotype was a greater source of variation in all measurements than was growing location, with the exception of protein content which showed less variation contributed by genotype and more contributed by location than for other quality parameters. UPP was strongly associated with all strength measurements. All of the γ-gliadin 42 types were low in UPP and weak. Among the γ-gliadin 45 types, those possessing HMW-GS 20 were typically in the lower half of the UPP and strength range. There was no clear evidence of an association between any of the other HMW-GS patterns and gluten strength. The majority exhibited HMW to LMW-GS ratios that were within the relatively narrow range of 0.15–0.25, yet there were wide variations in dough strength among genotypes within that range. Increasing proportions of HMW-GS resulting in ratios of greater than 0.30 were generally associated with weak dough and gluten and low UPP content.     

高分子量麦谷蛋白亚基评分系统的改进及应用

对 191个冬、春小麦品种的品质分析结果表明 :拉伸曲线的最大抗延阻力能比较客观地反映面筋的强度 ,但该项试验所需的样品量较大 (至少需要 30 0g面粉 )。高分子量麦谷蛋白亚基 (HMW GS)组成受遗传基因的控制 ,麦谷蛋白电泳分析所需的样品量少 (只需半粒小麦即可 ) ,适宜育种的早世代分析。选用各高分子量麦谷蛋白亚基的最大抗延阻力均值的差异建立的HMW GS组成评分系统 ,可以对小麦品种的面筋强度进行早期预测     

小麦高、低分子量麦谷蛋白亚基对品质性状的影响

为给小麦品质改良提供理论依据,以6个国家的532个品种(系)为材料,分析了小麦高、低分子量麦谷蛋白亚基与品质性状的关系。结果表明,各位点上不同的等位变异对品质性状的效应存在显著差异,亚基1、2*、17+18、5+10、GluA3f、GluB3b和GluB3g对面团形成时间和稳定时间具有显著的正向效应;亚基N、1、14+15、17+18、7+8、2+12、3+12、4+12、GluB3d、GluB3f、GluB3h和GluB3g对蛋白质和湿面筋含量具有显著正向效应;从亚基对品质性状的综合影响来看,1、17+18、GluA3f、GluB3g和GluB3f可作为优势亚基。具有1、17+18、2+12和1、17+18、5+10高分子量麦谷蛋白亚基组合的品种(系)综合品质性状显著优于含有N、14+15、2+12亚基组合的品种(系);具有低分子量麦谷蛋白亚基组合GluA3c、GluB3g,GluA3d、GluB3g和GluA3f、GluB3b品种(系)的综合品质性状显著优于含有GluA3b、GluB3j亚基组合的品种(系)。     

The impact of heating and cooling on the physico-chemical properties of wheat gluten–water suspensions

The rapid visco analysis (RVA) system was used to measure rheological behaviour in 20% (w/v) gluten-in-water suspensions upon applying temperature profiles. The temperature profiles included a linear temperature increase, a holding step, a cooling step with a linear temperature decrease to 50 °C, and a final holding step at 50 °C. Temperature and duration of the holding phase both affected RVA viscosity and protein extractability. Size-exclusion and reversed-phase HPLC showed that increasing the temperature (up to 95 °C) mainly decreased glutenin extractability. Holding at 95 °C resulted in polymerisation of both gliadin and glutenin. Above 80 °C, the RVA viscosity steadily increased with longer holding times while the gliadin and glutenin extractabilities decreased. Their reduced extractability in 60% ethanol showed that γ-gliadins were more affected after heating than α-gliadins and ω-gliadins. Enrichment of wheat gluten in either gliadin or glutenin showed that both gliadin and glutenin are necessary for the initial viscosity in the RVA profile. The formation of polymers through disulphide bonding caused a viscosity rise in the RVA profile. The amounts of free sulphydryl groups markedly decreased between 70 and 80 °C and when holding the temperature at 95 °C.     

Disulphide structure of high-molecular-weight (HMW-) gliadins as affected by terminators

This study aimed at elucidating SS-bonds of HMW-gliadins (HGL) from wheat with the focus on terminators of glutenin polymerisation. HGL from wheat flour extracts non-treated or treated with the S-alkylation reagent N-ethylmaleinimide (NEMI) were compared. HGL from wheat flour Akteur were isolated, hydrolysed with thermolysin and the resulting peptides pre-separated by gel permeation chromatography and analysed by liquid chromatography/mass-spectrometry using alternating electron transfer dissociation/collision-induced dissociation. Altogether, 22 and 28 SS-peptides from samples without and with NEMI treatment, respectively, were identified. Twenty-six peptides included standard SS-bonds of α- and γ-gliadins, high-molecular-weight and low-molecular-weight glutenin subunits. Eleven SS-bonds were identified for the first time. Fifteen peptides unique to HGL contained cysteine residues from gliadins with an odd number of cysteines (ω5-, α- and γ-gliadins). Thus, gliadins with an odd number of cysteines, glutathione and cysteine had acted as terminators of glutenin polymerisation. Decisive differences between samples without and with NEMI treatment were not obvious showing that the termination of polymerisation was already completed in the flour. The two HGL samples, however, were different in the majority of ten peptides that included disulphide-linked low-molecular-weight (LMW) thiols such as glutathione and cysteine with the former being enriched in the non-treated HGL-sample.     

Technological quality of field grown transgenic lines of commercial wheat cultivars expressing the 1Ax1 HMW glutenin subunit gene

Ten transgenic lines were studied which expressed a transgene encoding HMW subunit 1Ax1 in three elite spring wheat cultivars: Imp, Canon and Cadenza. These lines contained one to five copies of the transgene and the 1Ax1 subunit was expressed as 1–20% of the total glutenin protein. These lines were grown in field trials in a continental, arid climate (Martonvásár, Hungary) over two years (2004, 2005). The expression of the transgenes and their effects on the grain properties were stably inherited over the two years. Significant differences in yield were observed between three of the transgenic lines and the original genotypes, but no differences were found in their adaptiveness. Clear differences were found in the technological and rheological properties of four lines, with all the parameters characterising dough strength and extensibility (GI, W, G, Re, Ext, A) changing significantly. These differences were associated with increases in the ratio of HMW/LMW subunits and decreases in the ratios of 1Dx/1Dy and 1Bx/1By subunits. Two transgenic lines of cv Imp had high over-expression of the 1Ax1 subunit which in one line resulted in an overstrong type of dough, similar to that described previously for lines over-expressing HMW subunit 1Dx5. Transformation of cvs. Canon and Cadenza resulted in two lines with increased dough stability due to the significantly improved gluten quality. It is concluded that significant changes in the structure of the glutenin polymers caused by the altered ratio of x-type to y-type HMW subunits led to the changes in flour functional properties.     

Comparative Studies of HighMrSubunits of Rye and Wheat. I. Isolation and Biochemical Characterisation and Effects on Gluten Extensibility

The structural features of highM_rglutenin subunits of wheat were compared with those of analogous proteins from rye. Subunits of two rye cultivars (Danko and Halo) and of the wheat cultivar Rektor were isolated from defatted flours by extraction with 50% (v/v) aqueous propan-1-ol under reducing conditions at 60°C followed by precipitation using a 60% concentration of propan-1-ol. The yields of dialysed and freeze-dried subunits were 0·33% and 0·32% (w/w of flour), respectively (rye cultivars), and 0·91% (Rektor). SDS–PAGE revealed that the rye cultivars contained at least five subunits with mobilities corresponding to the x-type subunits of wheat. Separation by RP–HPLC indicated that the rye cultivars did not differ in the qualitative composition of subunits, but in their quantitative proportions. The surface hydrophobicities of the rye subunits were significantly lower than those of wheat subunits. The amino acid compositions of single rye subunits were characterised by high contents of Glx, Gly and Pro, and they were closely related to those of wheat subunits, except that the Glx content was generally lower and the Cys content higher. Notable differences between rye and wheat subunits were found in their contributions to gluten strength. Whereas wheat subunits, reoxidised with potassium bromate and mixed with a standard wheat flour, caused a significant increase in gluten strength, reoxidised rye subunits had the opposite effect.     

Silencing of γ-gliadins by RNA interference (RNAi) in bread wheat

RNA silencing is a sequence-specific RNA degradation system that is conserved in a wide range of organisms. The elucidation of the mechanism of RNA silencing has stimulated its use as a reverse genetics tool, because RNA silencing strongly down-regulates the expression of the target gene in a sequence-specific manner. The major protein fraction of wheat grain is gluten which is largely responsible for the functional properties of dough. Gliadins contribute mainly to the extensibility and viscosity of gluten and dough, with the polymeric glutenins being responsible for elasticity. The aim of this work was therefore to silence the expression of specific γ-gliadins by RNA interference, to demonstrate the feasibility of systematically silencing specific groups of gluten proteins. The sequence of a γ-gliadin gene was used to construct the pghp8.1 plasmid. The hpRNA silencing fragment was designed on the basis of 169 base pairs (bp) in sense and antisense orientation with the sequence of the Ubi1 intron as spacer region between the repeats. Two lines of bread wheat were transformed by particle bombardment. Gliadins were extracted from 30 mg of flour, separated by acid-PAGE and determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Seven transgenic lines were obtained and all of them showed reduced levels of γ-gliadins. All seven transgenic plants were fully fertile and their grain morphology and seed weight were comparable to the control lines. MALDI-TOF MS showed that six peaks, present in the untransformed line, were missing in transgenic lines of the BW208 genotype whereas three peaks were missing in the BW2003 genotypes. The proportion of γ-gliadins was reduced, by about 55–80% in the BW208 lines and by about 33–43% in the BW2003 lines. The ELISA assay based on the R5 antibody showed reductions in total gliadins (μg/mg flour) in three of the BW208 lines and in one BW2003 line, but an increase in one BW208 line (C613).     

Effect of hydrostatic pressure and temperature on the chemical and functional properties of wheat gluten: Studies on gluten, gliadin and glutenin

The effect of hydrostatic pressure (0.1–800 MPa) in combination with various temperatures (30–80 °C) on the chemical and physical properties of wheat gluten, gliadin and glutenin was studied. Chemical changes of proteins were determined by extraction, reversed-phase high-performance liquid chromatography (HPLC), sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD) spectroscopy, thiol measurement and studies on disulphide bonds. Rheological changes were measured by extension tests and dynamic stress rheometry. Treatment of gluten with low pressure (200 MPa) and temperature (30 °C) increased the proportion of the ethanol-soluble fraction (ESF) and decreased gluten strength. The enhancement of both pressure and temperature provoked a strong reduction of the ESF and the thiol content of gluten. Within gliadin types, cysteine containing α- and γ-gliadins, but not cysteine-free ω-gliadins were sensitive to pressure and were transferred to the ethanol-insoluble fraction. Disulphide peptides isolated from treated gluten confirmed that cleavage and rearrangement of disulphide bonds were involved in pressure-induced reactions. Increased pressure and temperature induced a significant strengthening of gluten, and under extreme conditions (e.g. 800 MPa, 60 °C), gluten cohesivity was lost. Isolated gliadin and glutenin reacted differently: solubility, HPLC and SDS-PAGE patterns of gliadin having a very low thiol content were not influenced by pressure and heat treatment; only conformational changes were detected by CD spectroscopy. In contrast, the properties of isolated glutenin having a relatively high thiol content were strongly affected by high pressure and temperature, similar to the effects on total gluten.     

Effects of post-anthesis fertilizer on the protein composition of the gluten polymer in a US bread wheat

Both genetic and environmental factors influence the types and amounts of wheat proteins that link together to form polymers essential for flour quality. To understand how plant growth conditions might influence gluten polymer formation, protein fractions containing small and large polymers were separated from flour from the US wheat Butte 86 grown in the absence or presence of post-anthesis fertilizer. Proteins in the polymer fractions were analyzed by quantitative two-dimensional gel electrophoresis (2-DE). The ratio of high molecular weight glutenin subunits (HMW-GS) to low molecular weight glutenin subunits (LMW-GS) increased in both fractions in response to fertilizer, due in part to small increases in the proportions of individual HMW-GS. There were also changes within the LMW-GS. In particular, omega and alpha chain terminators increased in proportion in both polymer fractions, but changes were more pronounced in the large polymer fractions. Serpins also increased in both polymer fractions. Additionally, the study revealed differences in the proportions of traditional LMW-GS in small and large polymer fractions. LMW-s type proteins were more abundant in the large polymers while LMW-i type proteins were more prevalent in the small polymers, suggesting that these proteins may play different roles in the gluten polymer.     

Quality-related Epitopes of High Mr Subunits of Wheat Glutenin

Three hundred and eighty four immobilised overlapping nonapeptides, corresponding to the full amino acid sequences of three high M_r subunits of glutenin from bread wheat (Triticum aestivum) grain, were used to determine the linear epitopes recognised by four monoclonal antibodies. These antibodies were selected on the basis of significant and positive correlations between their binding to wheat flour extracts in a two-site ('sandwich') enzyme immunoassay and rheological measures of dough strength, an important aspect of bread wheat quality. The antibodies did not bind to a single, specific sequence but bound a series of related peptides in each high M_r glutenin subunit examined. The sequences recognised were not identical for the four antibodies, but in each case were in the central repeating domain of the high M_r glutenin subunits, and usually comprised regions that overlapped the degenerate repeat nonamer and hexamer sequences. High M_r glutenin subunits that have been associated with greater dough strength, such as the D-genome allelic products 1Dx5 and 1Dy10, displayed an increased number of the epitope sequences. The location of the epitopes in sequences of overlapping β-turns in the repetitive region supports the hypothesis that dough elasticity arises partly from β-turn-forming secondary structure in the repeat regions of the M_r glutenin subunits. Additional β-turn within high M_r subunits may extend their structure to allow increased interaction between the glutenin subunits and with the other proteins of the gluten complex, thus improving dough strength.