The Structure and Properties of Gluten an Elastic Protein From Wheat Grain

doi: 10.1098/rstb.2001.1024, 133-142357 2002 Phil. Trans. R. Soc. Lond. B

P. R. Shewry, N. G. Halford, P. S. Belton and A. S. Tatham wheat grainThe structure and properties of gluten: an elastic protein from

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Published online 25 February 2002

The structure and properties of gluten: an elasticprotein from wheat grain

Peter R. Shewry1*, Nigel G. Halford1, Peter S. Belton2 and Arthur S. Tatham1

1Institute of Arable Crops Research, Long Ashton Research Station, Department of Agricultural Science, University of Bristol,Long Ashton, Bristol BS41 9AF, UK

2School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, UK

The wheat gluten proteins correspond to the major storage proteins that are deposited in the starchyendosperm cells of the developing grain. These form a continuous proteinaceous matrix in the cells ofthe mature dry grain and are brought together to form a continuous viscoelastic network when flour ismixed with water to form dough. These viscoelastic properties underpin the utilization of wheat to givebread and other processed foods. One group of gluten proteins, the HMM subunits of glutenin, is parti-cularly important in conferring high levels of elasticity (i.e. dough strength). These proteins are presentin HMM polymers that are stabilized by disulphide bonds and are considered to form the ‘elastic back-bone’ of gluten. However, the glutamine-rich repetitive sequences that comprise the central parts of theHMM subunits also form extensive arrays of interchain hydrogen bonds that may contribute to the elasticproperties via a ‘loop and train’ mechanism. Genetic engineering can be used to manipulate the amountand composition of the HMM subunits, leading to either increased dough strength or to more drasticchanges in gluten structure and properties.

Keywords: wheat; gluten; protein elasticity; HMM subunits; transgenic plants

1. INTRODUCTION

Wheat is one of the three most important crops in theworld, together with maize and rice. Approximately 600million tonnes are harvested annually with cultivationextending over a vast geographical area, from Scandinaviato Argentina, including higher elevations in the tropics.Although the ability to give high yields under a range ofconditions has contributed to the success of wheat, themost important factor has been the unique properties ofwheat dough that allow it to be processed into a range offoodstuffs, notably bread, other baked products andpastas. These properties are usually described as viscoelas-ticity, with the balance between the extensibility and elas-ticity determining the end use quality. For example, highlyelastic (‘strong’) doughs are required for breadmaking butmore extensible doughs are required for making cakesand biscuits.

The grain proteins determine the viscoelastic propertiesof dough, in particular, the storage proteins that form anetwork in the dough called gluten (Schofield 1994).Consequently, the gluten proteins have been widely stud-ied over a period in excess of 250 yr, in order to determinetheir structures and properties and to provide a basis formanipulating and improving end use quality (see Shewryet al. 1995).

2. THE ORIGIN OF THE WHEAT GLUTENNETWORK

Gluten can be readily prepared by gently washing doughunder a stream of running water. This removes the bulk

* Author for correspondence ([email protected]).

Phil. Trans. R. Soc. Lond. B (2002) 357, 133–142 133 2002 The Royal SocietyDOI 10.1098/rstb.2001.1024

of the soluble and particulate matter to leave a protein-aceous mass that retains its cohesiveness on stretching(figure 1a). Gluten comprises some 75% protein on a dryweight basis, with most of the remainder being starch andlipids. Furthermore, the vast majority of the proteins areof a single type called prolamins.

Prolamins are a group of proteins that were initiallydefined based on their solubility in alcohol–water mixtures(Osborne 1924), typically 60–70% (v/v) ethanol. Thisdefinition has since been extended to include related pro-teins, which are not soluble in alcohol–water mixtures inthe native state, owing to their presence in polymers stabil-ized by interchain disulphide bonds. In wheat, thesegroups of monomeric and polymeric prolamins are knownas gliadins and glutenins, respectively, and together formgluten (Shewry et al. 1986).

Wheat prolamins are the major storage proteins presentin the starchy endosperm cells of the grain, where they aresynthesized and deposited via the secretory system. Thus,the individual polypeptides are synthesized on ribosomeson the RER and pass via the usual translocation machin-ery into the lumen, with the loss of an N-terminal signalpeptide. Once within the lumen it is probable that proteinfolding and disulphide bond formation occur with nofurther post-translational modifications taking place (i.e.no glycosylation or proteolysis as may occur with othertypes of seed storage protein).

The subsequent fate of the proteins may also vary withthe protein type and with the age and stage of develop-ment of the tissue. Some of the proteins appear to betransported via the Golgi apparatus into the vacuole,where they form protein deposits (see Shewry 1999).However, others appear to accumulate directly within the



134 P. R. Shewry and others Wheat gluten proteins

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Figure 1. (a) A sheet of gluten stretched to demonstrate itscohesive properties. (b) Two-dimensional analysis (isoelectricfocusing followed by SDS–PAGE) of wheat gluten proteinsshows multiple components.

lumen of the ER to form a second population of proteinbodies. Galili (1997) has proposed that vesicles sub-sequently engulf the latter, with the contents being‘internalized’ into vacuoles but to date this has not beenconfirmed by other workers. What is known is that duringthe later stages of grain maturation the starchy endospermcells become disrupted and die and the protein bodies fuseto form a continuous matrix, which surrounds the starchgranules and engulfs other organelles and membranes.

Galili (1997) has also suggested that the gliadins arepreferentially transported to the vacuole via the Golgiapparatus, which is consistent with the lack of a classicalER retention sequence. We have also shown that a γ-gliadin is rapidly degraded in leaves and seeds of trans-genic tobacco (presumably in the vacuole) unless a C-ter-minal ER retention sequence (the tetrapeptide His–Asp–Glu–Leu or Lys–Asp–Glu–Leu) is added (Napier et al.1997). However, the glutenins also lack an obvious ERretention sequence but, nevertheless, Galili (1997) hasproposed that they are preferentially retained in the ER.In this case, their rapid assembly into high Mr polymers,which precipitate and accumulate directly within the ERlumen, could determine retention. It is also possible toenvisage how the relative rates of trafficking via the ERand Golgi routes could vary with the level of protein syn-thesis and age of the tissue.

Phil. Trans. R. Soc. Lond. B (2002)

As a result of the formation of a protein matrix, individ-ual cells of wheat flour contain networks of glutenproteins, which are brought together during dough mix-ing. The precise changes that occur in the dough duringmixing are still not completely understood, but an increasein dough stiffness occurs that is generally considered toresult from ‘optimization’ of protein–protein interactionswithin the gluten network. In molecular terms, this ‘opti-mization’ may include some exchange of disulphide bondsas mixing in air, oxygen and nitrogen result in differenteffects on the sulphydryl and disulphide contents of dough(Tsen & Bushuk 1963; Mecham & Knapp 1966).

Of course, the natural fate of the wheat grain is not toprovide flour for humankind but to germinate to producea new plant. The biological role of the gluten protein is,therefore, to provide a store of carbon, nitrogen and sul-phur to support seed germination and seedling growth.The gluten proteins have no other known biological roleand their viscoelastic properties appear to be a purely for-tuitous consequence of their sequences and interactions.

3. THE HMM GLUTENIN SUBUNITS

Wheat gluten is a highly complex mixture of proteinswith at least 50 individual components being separated bytwo-dimensional isoelectric focusing/SDS–PAGE ofreduced total fractions (figure 1b). Furthermore, there isgreat variation in the component proteins present in differ-ent genotypes. This high level of polymorphism initiallylimited attempts to isolate and characterize individualcomponents, but details of the structures and sequencesof all of the major gluten protein types are now known(see Shewry et al. 1999). However, much of the work overthe past 20 years has focused on one group of proteins,which are the subject of the remainder of this article.These are the HMM subunits of wheat glutenin (alsocalled the HMW subunits).

Bread wheat is a hexaploid species with three genomes(called A, B and D) derived from related wild grass spe-cies. Single loci encoding HMM subunits are present onthe long arms of the group 1 chromosomes (1A, 1B, 1D),each locus comprising two genes encoding subunits thatdiffer in their properties and are called x-type and y-typesubunits (Payne 1987). Although bread wheats couldtheoretically contain six HMM subunits (1Ax, 1Ay, 1Bx,1By, 1Dx, 1Dy), the silencing of specific genes results inthe presence of only three (1Bx, 1Dx, 1Dy) to five (1Ax,1Bx, 1By, 1Dx, 1Dy) subunits (Payne et al. 1987).

The HMM subunits have been reported to account, onaverage, for about 12% of the total grain protein, corre-sponding to 1–1.7% of the flour dry weight (Seilmeier etal. 1991; Halford et al. 1992; Nicolas 1997). However,variation in the amount of HMM subunits (associatedwith the differences in gene silencing discussed above) andin the properties of expressed subunits have been reportedto account for between 45 and 70% of the variation inbreadmaking performance within European wheats(Branlard & Dardevet 1985; Payne et al. 1987, 1988).These correlative studies are supported by the develop-ment and analysis of near-isogenic lines that differ only intheir HMM subunit composition. Analyses of such lineshave confirmed that the subunits are largely responsiblefor determining dough viscoelasticity and that specific



Wheat gluten proteins P. R. Shewry and others 135

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Figure 2. Schematic summary of the sequences of x-type and y-type HMM subunits.

allelic subunit pairs are associated with either high or lowdough strength (Popineau et al. 1994).

A number of genes encoding HMM subunits have beenisolated from bread wheat (see Shewry et al. 1992;Reddy & Appels 1993) and from related wheat species andwild relatives (Mackie et al. 1996; Wan et al. 2001). Theseshow that the HMM subunits have conserved amino-acidsequences, comprising three distinct parts or domains(figure 2). The central domains of the proteins consist ofrepeated peptides, based on two or three short peptidemotifs. They vary in length from about 420 to 700 resi-dues and account for between 74 and 84% of the wholeprotein. These domains are flanked by short non-repeti-tive domains, which vary in length from 81 to 104 residuesat the N-terminus but always comprise 42 residues at theC-terminus.

4. SEQUENCES OF THE REPETITIVE DOMAINS

The x-type and y-type subunits have essentially similarrepeat structures, comprising mainly nonapeptide andhexapeptide motifs. Also, whereas tandem blocks of hexa-peptides may be present, the nonapeptides are alwaysinterspersed with hexapeptides. Consequently, it is con-venient to consider them as forming a 15 amino-acidmotif. The x-type subunits also differ from the y-type inhaving additional tripeptide motifs, which also only occurin tandem with hexapeptides, forming a second nonapep-tide motif. Figure 3 shows the sequences of the repetitivedomains of typical x-type and y-type subunits (1Dx5 and1Dy10, respectively) arranged to show their repeatedblock structure. The repeat motifs are rich in glutamine,proline and glycine, which together account for over 70%of the total amino-acid residues. No major differences areapparent between the homeoallelic proteins of breadwheat (A, B, D) or related genomes present in other wheatspecies or wild relatives (A, C and D), so combined datafor five x-type and seven y-type subunits are presented intable 1.

Comparison of the patterns of amino-acid substitutionsshows that some positions of the motifs appear to be morehighly conserved than others. In particular, glutaminetends to be more highly conserved at specific positionsthan other consensus amino acids: at positions 3, 5 and 6


of the hexapeptide, 8 and 9 of the nonapeptide and 2 and3 of the tripeptide. This may relate to the role of glutamineresidues in stabilizing the structures and interactions ofthe subunits. Similarly, serine is conserved at position 6of the nonapeptides. In contrast, positions 1 and 4 of thehexapeptide are poorly conserved, as is position 7 of thenonapeptide.

Some differences are also observed between the x-typeand y-type subunits. Thus, replacement of Pro with Serat position 1 of the hexapeptides is more common in x-type subunits, as is replacement of Gln with Pro at pos-ition 6. However, the latter only occurs in hexapeptideswithin a 15 residue (6 + 9) motif, rather than in the tan-demly-arranged hexapeptides. Similarly, replacement ofTyr with His at position 2 and Thr with Ala at position 5of the nonapeptides are more common in y-type subunitsand these two substitutions usually occur together, givingthe two consensus motifs GYYPTSLQQ and GHY-PASLQQ compared with GYYPTSPQQ for x-type sub-units.

There is no evidence that amino-acid substitution leadsto replacement with similar amino-acid residues (i.e. con-servative substitutions). Instead, analysis of codons indi-cates that most replacements are due to single nucleotidechanges, with substitutions resulting from double nucleot-ide changes occurring more rarely. For example, proline(CCA) occurs at position 1 in 55% of x-type hexapeptides,with single nucleotide changes leading to the occurrenceof leucine (CTA, 12%) and serine (TCA, 30%) and twonucleotide changes to isoleucine (ATA, 3%).

The failure to detect any appreciable differencesbetween the consensus motifs and degree of conservationof the repetitive sequences present in the HMM subunitsof cultivated and wild species (Wan et al. 2001) indicatesthat selection by plant breeders for dough strength, whichhas been carried out systematically for the last century andperhaps unconsciously over the 10 000 year life of breadwheat, has had little or no impact on the sequences (andhence structure) of the subunits. However, it is possiblethat the differences in degree of conservation within themotifs and the precise amino-acid residues that are presentas substitutions at different positions within the motifsmay relate to their role in determining the structureadopted by the domain.




Figure 3. Amino-acid sequences of the repetitive domains oftypical x-type (1Dx5,(a)) and y-type (1Dy10,(b)) HMMsubunits arranged to show their repeat unit structures.


Cysteine residues occur only rarely in the repetitivesequences, with single cysteine residues present towardsthe C-terminal end of the repetitive domains (at position�73) of 1By and 1Dy subunits only. In addition, subunit1Dx5 differs from all other subunits whose sequences areknown in that a single additional cysteine is present atposition 8 relative to the N-terminal end of the domain.

5. STRUCTURE OF THE HMM SUBUNITREPETITIVE DOMAIN

Although several workers have attempted to determinethe structure adopted by the HMM subunit repeats by X-ray crystallography of whole subunits or repetitive pep-tides, the crystals produced have failed to give clear dif-fraction patterns. Similarly, analysis of synthetic peptidesbased on the repetitive sequence motifs has not yet led tothe determination of three-dimensional structures. Conse-quently, our current view of HMM subunit structurecomes from a range of indirect studies.

Early hydrodynamic studies of subunit 1Bx20 purifiedfrom pasta wheat indicated that it had an extended rod-shaped conformation in solution, the dimensions rangingfrom ca. 500 × 17.5 to 620 × 15 A depending on the sol-vent (Field et al. 1987). Detailed spectroscopic studies ofwhole subunits, of recombinant repetitive peptides and oflinear and circular synthetic peptides (Tatham et al. 1985;Field et al. 1987; van Dijk et al. 1997a,b; Gilbert et al.2000) have also been reported. The results are consistentwith the repetitive sequences forming β-reverse turnswhich may be in equilibrium with poly-L-proline II struc-ture, the latter predominating at low temperature (Gilbertet al. 2000). It has also been proposed that the β-turns areorganized to give a regular spiral structure (termed a β-spiral) similar to that demonstrated for a synthetic polyp-entapeptide based on a repeat motif of elastin (Urry1988). Molecular modelling can be used to generate suchspiral structures (figure 4) whose dimensions (diameter,pitch and length) are consistent with those determined byviscometric analysis and revealed by STM of purified pro-teins in the hydrated solid state (Miles et al. 1991). How-ever, Kasarda et al. (1994) have proposed that analternative type of spiral structure is formed, based on γ-turns rather than β-turns.

6. SEQUENCES AND STRUCTURES OF THENON-REPETITIVE DOMAINS

The N-terminal domains vary in length, being 81–89residues in the x-type subunits and 104 residues in they-type. This difference results from a deletion in the x-type subunits compared with the y-type, which involvesthe loss of two cysteine residues. Consequently, the N-termini of the x-type subunits usually contain three cyst-eine residues and those of the y-type subunits five. Struc-ture prediction and molecular modelling studies indicatethat this domain is ‘globular’ with one or more α-helices(Tatham et al. 1984, 1985; Van Dijk et al. 1998; Kohleret al. 1997).

The C-terminal domains of all of the subunits comprise42 residues with single cysteine residues at position �13with respect to the C-terminus. Structure prediction indi-cates that this domain may be α-helical (Tatham et al.




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1984) and NMR spectroscopy of a synthetic peptidedissolved in 40% (v/v) aqueous trifluoroethanol (a ‘struc-ture-inducing’ solvent) allowed a low-resolution structurecontaining two α-helices to be determined (Bekkers etal. 1996).

7. HMM SUBUNIT STRUCTURE AND GLUTENELASTICITY

The HMM subunits are only present in glutenin poly-mers, particularly in high Mr polymers, the amounts ofwhich are positively correlated with dough strength (Fieldet al. 1983). This provides support for the genetic evidence(see § 3) that the HMM subunits are the major determi-nants of dough and gluten elasticity.

Two features of HMM subunit structure may be rel-evant to their role in glutenin elastomers: the number anddistribution of disulphide bonds and the properties andinteractions of the repetitive domains.

Direct sequence analysis of disulphide-linked peptidesreleased by enzymic digestion of glutenin or gluten frac-tions has revealed a number of inter- and intrachaindisulphide bonds involving HM W subunits (Kohler etal. 1991, 1993, 1994; Tao et al. 1992; Keck et al. 1995).These are summarized diagrammatically in figure 5 andinclude one interchain disulphide bond within the N-terminal domain of an x-type subunit, two parallel disul-phide bonds between the N-termini of y-type subunits,an interchain bond between a y-type subunit and aLMM glutenin subunit and a bond linking y-type andx-type subunits in a ‘head-to-tail’ fashion. The latter is


consistent with the results obtained by partial reductionof glutenin, which leads to the release of dimers compris-ing x-type + y-type subunits (Lawrence & Payne 1983;Tao et al. 1992). Such dimers have therefore been pro-posed to form the ‘building blocks’ of glutenin(Graveland et al. 1985). However, our knowledge of thedetailed disulphide structure of glutenin is not suf-ficiently complete to allow us to relate disulphide distri-bution to biomechanical properties.

Although it is now widely accepted that disulphide-linked glutenin chains provide an ‘elastic backbone’ to glu-ten, evidence from spectroscopic studies (using NMR andFTIR spectroscopy) of HMM subunits and of model pep-tides based on the repeat motifs suggests that non-covalenthydrogen bonding between glutenin subunits and poly-mers may also be important (Belton et al. 1994, 1995,1998; Wellner et al. 1996; Gilbert et al. 2000). These stud-ies have shown that the dry proteins are disordered withlittle regular structure, but that their mobility increasesand β-sheet structures form on hydration. Further changesoccur if hydration continues, with a further increase inprotein mobility and the formation of turn-like structuresat the expense of β-sheet.

These observations led to the development of a ‘loopand train’ model (Belton 1999), which is summarized infigure 6. This proposes that the low hydration state hasmany protein–protein interactions, via hydrogen bondingof glutamine residues in the β-spiral structures. As thehydration level increases the system is platicized, allowingthe orientation of the β-turns in adjacent β-spirals to formstructures that resemble an ‘interchain’ β-sheet. Furtherhydration leads to the breaking of some of the interchainhydrogen bonds in favour of hydrogen bonds between glu-tamine and water, which then leads to the formation ofloop regions. However, it does not result in the completereplacement of interchain hydrogen bonds, and hence sol-ution of the protein, as the number of glutamine residuesis high and the statistical likelihood of all the interchainbonds breaking simultaneously is therefore low. The resultis an equilibrium between hydrated ‘loop’ regions andhydrogen-bonded ‘chain’ regions, with the ratio betweenthese being dependent on the hydration state.

The equilibrium between ‘loops’ and ‘trains’ may alsocontribute to the elasticity of glutenin, as an extension ofthe dough will result in stretching of the ‘loops’ and‘unzipping’ of the ‘trains’. The resulting formation ofextended chains may be a mechanism by which elasticenergy is stored in the dough, thus providing an expla-nation for the increased resistance to extension that occursduring dough mixing. The formation of interchain hydro-gen bonds between glutamine residues may also accountfor the observations that the esterification of glutamineresidues results in decreased resistance to extension, whilemixing in the presence of deuterium oxide (D2O) ratherthan water results in increased resistance (Beckwith et al.1963; Mita & Matsumoto 1981; Bushuk 1998).

8. MANIPULATION OF HMM SUBUNITCOMPOSITION IN TRANSGENIC WHEAT

The major aim of determining the structures of theHMM subunits and their role in gluten and dough elas-ticity is to facilitate the improvement of the end use




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Figure 5. Schematic model of the structure of HMM subunit polymers, based on mapped disulphide bonds (Kohler et al.1991, 1993, 1994; Tao et al. 1992; Keck et al. 1995)

Figure 6. Model for the effect of hydration on the loop totrain ratio of HMM subunits. (a) Low hydration, disordered,close interactions; (b) intermediate hydration, low loop totrain ratio; (c) high hydration, high loop to train ratio.

properties of wheat. Substantial improvement in the pro-cessing performance of wheat has already been achievedby a combination of classical plant breeding and optimiz-ation of the agronomic and processing conditions. How-ever, it is unlikely that these approaches will be sufficientin the long term and genetic engineering therefore pro-vides an important additional approach. We are, therefore,using genetic engineering of wheat in order to furtherstudy the role of the HMM subunits in determining pro-cessing properties and to define strategies for the pro-duction of improved germplasm for incorporation intoplant breeding programmes.

Most of our work, to date, has focused on transformationof two ‘model’ lines of wheat with two different HMM


subunit genes. The model lines form part of a near iso-genic series, which have been produced by crossing linesdiffering in their expression of HMM subunit genes. Thus,line L88-31 expresses only two HMM subunit genes(encoding subunits 1Bx17 and 1By18), while L88-6also expresses genes encoding subunits 1Ax, 1Dx5 and1Dy10 (Lawrence et al. 1988). The two genes used fortransformation encode subunits 1Ax1 and 1Dx5, the latteralways occurring as part of an ‘allelic pair’ with subunit1Dy10. The three transgenic lines that have been studiedin detail express the 1Ax1 subunit in L88-31 at a level ofabout 5.7% of the total protein (compared with 0% in thecontrol line) and the 1Dx5 subunit in L88-31 and L88-6at 8.7% (compared with 0%) and 17% (compared with4.2%) of the total protein, respectively (Barro et al. 1997;Popineau et al. 2001).

The effects of the transgenes on dough strength weredetermined using a Mixograph. This measures the energyinput during the mixing of dough and is routinely usedfor quality testing in a number of countries. When doughis mixed the resistance increases up to a certain level, afterwhich it decreases. The increase in resistance may resultfrom limited exchange of disulphide bonds (see § 2) andformation of the most stable patterns of hydrogen bonding(i.e. to form extensive ‘train’ regions). In contrast, the sub-sequent decrease in resistance is thought to result fromdisruption of these interactions by overmixing. Conse-quently, beneficial effects of the transgenes on doughstrength and stability should be observed as increases inthe PR (i.e. the maximum resistance that is observed) andthe MT (i.e. the time taken to mix to PR) and a decreasein RBD (i.e. the rate of decrease in the resistance on over-mixing beyond PR).

The results obtained with expression of the two trans-genes in the L88-31 background are summarized in figure7 (Popineau et al. 2001). The control line has low doughstrength, which is consistent with the expression of onlytwo endogenous HMM subunit genes, and the expressionof the 1Ax1 transgene results in substantial increases inPR and MT. In contrast, expression of the 1Dx5 trans-gene in the same line was clearly detrimental to the mixingproperties. An even more extreme effect was observed




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Figure 7. Analysis of the mixing properties of transgenic wheats expressing additional HMM subunits using the 2g Mixograph.(a) SDS–PAGE of the HMM subunits from (i ), control line L88-31 (comigrating subunits 1Bx17 + 1By18); (ii), L88-31expressing the 1Ax1 transgene; (iii ), L88-31 expressing the 1Dx5 transgene; (iv), control line L88-6 (subunits 1Ax1, 1Dx5 +1Dy10, 1Bx17 + 1By18); (v), L88-6 expressing the 1Dx5 transgene. (b–f ) Mixographs of (b), L88-31; (c), L88-31 expressingthe 1Ax1 transgene; (d), L88-31 expressing the 1Dx5 transgene; (e), L88-6; ( f ), L88-6 expressing the 1Dx5 transgene. Theresistance is given as torque (%) and the MT in seconds (s). Taken from Popineau et al. (2001), with permission.

when the subunit 1Dx5 transgene was expressed in theL88-6 line which had much stronger mixing properties(figure 7f ). In fact, both lines expressing the 1Dx5 trans-gene failed to absorb water and form a normal dough inthe mixing bowl.

Rheological studies were also carried out on gluten frac-tions from the transgenic lines, showing that theexpression of subunit 1Dx5 resulted in large increases inelasticity (measured as the storage and loss moduli, G�and G�, and the viscoelastic plateau, Gn°) while only asmall increase was associated with expression of subunit1Ax1. In fact, the effect of subunit 1Dx5 was similar tothat previously observed when gluten was modified bytreatment with transglutaminase to introduce interchainlysyl–glutamyl cross-links (Popineau et al. 2001).

The expression of the subunit 1Dx5 transgene was alsoassociated with an increase in the amounts of gluteninsubunits that were only extracted from flour by sonicationwith detergent (2% sodium dodecylsulphate) in the pres-ence of reducing agent (1% dithiothreitol) – from 2–3%of the total flour proteins in the control lines to over 18%in the L88-31 transgenic line and almost 30% in the L88-


6 transgenic line. These subunits can be assumed to bepresent in insoluble glutenin polymers. In contrast,expression of the 1Ax1 transgene was associated with amodest increase in the amount of subunits present in poly-mers that were extracted by sonication in the absence ofreducing agent, but had no effect on the amount of sub-units present in insoluble polymers.

These results suggest that the proteins encoded by thetwo transgenes had fundamentally different effects on thestructure of the glutenin polymers in the two lines, withthe 1Dx5 protein leading to the formation of highly cross-linked polymers that resulted in high gluten strength,unusual hydration behaviour and failure to form ahomogeneous network during mixing. In contrast, theexpression of subunit 1Ax1 resulted in similar effects ongluten composition and properties to those observed whencomparing near-isogenic lines differing in HMM subunitcomposition.

As discussed in § 4, the 1Dx5 subunit protein differsfrom other characterized subunits in the presence of anadditional cysteine residue within the repetitive domainand this may be responsible for the formation of highly




cross-linked polymers in the transgenic lines. However, itmust also be borne in mind that, in non-transgenic wheat,subunit 1Dx5 is always found together with 1Dy10 andthat dimers of these subunits are released by partialreduction of glutenin. Consequently, a precise molar bal-ance of these two subunits may be required to give a ‘nor-mal’ glutenin polymer structure.

These results demonstrate, therefore, that transform-ation of bread wheat with different HMW subunit genesmay have fundamentally different effects on gluten struc-ture and properties, which may relate to the expressionlevels, structures and interactions of the individual pro-teins.

I. A. C. R. and I. F. R. receive grant-aided support from theBiotechnology and Biological Sciences Research Council(BBSRC) of the United Kingdom.

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