Published online 25 February 2002

The structure and properties of gluten: an elastic

protein from wheat grain

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

1
Institute of Arable Crops Research, Long Ashton Research Station, Department of Agricultural Science, University of Bristol,
Long Ashton, Bristol BS41 9AF, UK
2
School 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 starchy
endosperm cells of the developing grain. These form a continuous proteinaceous matrix in the cells of
the mature dry grain and are brought together to form a continuous viscoelastic network when flour is
mixed with water to form dough. These viscoelastic properties underpin the utilization of wheat to give
bread 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 present
in 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 the
HMM subunits also form extensive arrays of interchain hydrogen bonds that may contribute to the elastic
properties via a loop and train mechanism. Genetic engineering can be used to manipulate the amount
and composition of the HMM subunits, leading to either increased dough strength or to more drastic
changes in gluten structure and properties.
Keywords: wheat; gluten; protein elasticity; HMM subunits; transgenic plants

1. INTRODUCTION of the soluble and particulate matter to leave a protein-

Wheat is one of the three most important crops in the aceous mass that retains its cohesiveness on stretching
world, together with maize and rice. Approximately 600 (figure 1a). Gluten comprises some 75% protein on a dry
million tonnes are harvested annually with cultivation weight basis, with most of the remainder being starch and
extending over a vast geographical area, from Scandinavia lipids. Furthermore, the vast majority of the proteins are
to Argentina, including higher elevations in the tropics. of a single type called prolamins.
Although the ability to give high yields under a range of Prolamins are a group of proteins that were initially
conditions has contributed to the success of wheat, the defined based on their solubility in alcoholwater mixtures
most important factor has been the unique properties of (Osborne 1924), typically 6070% (v/v) ethanol. This
wheat dough that allow it to be processed into a range of definition has since been extended to include related pro-
foodstuffs, notably bread, other baked products and teins, which are not soluble in alcoholwater mixtures in
pastas. These properties are usually described as viscoelas- the native state, owing to their presence in polymers stabil-
ticity, with the balance between the extensibility and elas- ized by interchain disulphide bonds. In wheat, these
ticity determining the end use quality. For example, highly groups of monomeric and polymeric prolamins are known
elastic (strong) doughs are required for breadmaking but as gliadins and glutenins, respectively, and together form
more extensible doughs are required for making cakes gluten (Shewry et al. 1986).
and biscuits. Wheat prolamins are the major storage proteins present
The grain proteins determine the viscoelastic properties in the starchy endosperm cells of the grain, where they are
of dough, in particular, the storage proteins that form a synthesized and deposited via the secretory system. Thus,
network in the dough called gluten (Schofield 1994). the individual polypeptides are synthesized on ribosomes
Consequently, the gluten proteins have been widely stud- on the RER and pass via the usual translocation machin-
ied over a period in excess of 250 yr, in order to determine ery into the lumen, with the loss of an N-terminal signal
their structures and properties and to provide a basis for peptide. Once within the lumen it is probable that protein
manipulating and improving end use quality (see Shewry folding and disulphide bond formation occur with no
et al. 1995). further post-translational modifications taking place (i.e.
no glycosylation or proteolysis as may occur with other
2. THE ORIGIN OF THE WHEAT GLUTEN types of seed storage protein).
NETWORK The subsequent fate of the proteins may also vary with
the protein type and with the age and stage of develop-
Gluten can be readily prepared by gently washing dough
ment of the tissue. Some of the proteins appear to be
under a stream of running water. This removes the bulk
transported via the Golgi apparatus into the vacuole,
where they form protein deposits (see Shewry 1999).
*
Author for correspondence ([email protected]). However, others appear to accumulate directly within the

Phil. Trans. R. Soc. Lond. B (2002) 357, 133142 133 2002 The Royal Society
DOI 10.1098/rstb.2001.1024
134 P. R. Shewry and others Wheat gluten proteins

(a) As a result of the formation of a protein matrix, individ-

ual cells of wheat flour contain networks of gluten
proteins, which are brought together during dough mix-
ing. The precise changes that occur in the dough during
mixing are still not completely understood, but an increase
in dough stiffness occurs that is generally considered to
result from optimization of proteinprotein interactions
within the gluten network. In molecular terms, this opti-
mization may include some exchange of disulphide bonds
as mixing in air, oxygen and nitrogen result in different
effects 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 to
provide flour for humankind but to germinate to produce
a new plant. The biological role of the gluten protein is,
therefore, to provide a store of carbon, nitrogen and sul-
(b) phur to support seed germination and seedling growth.
IEF
The gluten proteins have no other known biological role
and their viscoelastic properties appear to be a purely for-
tuitous consequence of their sequences and interactions.
SDSPAGE

3. THE HMM GLUTENIN SUBUNITS

Wheat gluten is a highly complex mixture of proteins
with at least 50 individual components being separated by
two-dimensional isoelectric focusing/SDSPAGE of
reduced total fractions (figure 1b). Furthermore, there is
great variation in the component proteins present in differ-
ent genotypes. This high level of polymorphism initially
limited attempts to isolate and characterize individual
components, but details of the structures and sequences
Figure 1. (a) A sheet of gluten stretched to demonstrate its of all of the major gluten protein types are now known
cohesive properties. (b) Two-dimensional analysis (isoelectric
(see Shewry et al. 1999). However, much of the work over
focusing followed by SDSPAGE) of wheat gluten proteins
shows multiple components. the 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 (also
called the HMW subunits).
lumen of the ER to form a second population of protein Bread wheat is a hexaploid species with three genomes
bodies. Galili (1997) has proposed that vesicles sub- (called A, B and D) derived from related wild grass spe-
sequently engulf the latter, with the contents being cies. Single loci encoding HMM subunits are present on
internalized into vacuoles but to date this has not been the long arms of the group 1 chromosomes (1A, 1B, 1D),
confirmed by other workers. What is known is that during each locus comprising two genes encoding subunits that
the later stages of grain maturation the starchy endosperm differ in their properties and are called x-type and y-type
cells become disrupted and die and the protein bodies fuse subunits (Payne 1987). Although bread wheats could
to form a continuous matrix, which surrounds the starch theoretically contain six HMM subunits (1Ax, 1Ay, 1Bx,
granules and engulfs other organelles and membranes. 1By, 1Dx, 1Dy), the silencing of specific genes results in
Galili (1997) has also suggested that the gliadins are the presence of only three (1Bx, 1Dx, 1Dy) to five (1Ax,
preferentially transported to the vacuole via the Golgi 1Bx, 1By, 1Dx, 1Dy) subunits (Payne et al. 1987).
apparatus, which is consistent with the lack of a classical The HMM subunits have been reported to account, on
ER retention sequence. We have also shown that a - average, for about 12% of the total grain protein, corre-
gliadin is rapidly degraded in leaves and seeds of trans- sponding to 11.7% of the flour dry weight (Seilmeier et
genic tobacco (presumably in the vacuole) unless a C-ter- al. 1991; Halford et al. 1992; Nicolas 1997). However,
minal ER retention sequence (the tetrapeptide HisAsp variation in the amount of HMM subunits (associated
GluLeu or LysAspGluLeu) is added (Napier et al. with the differences in gene silencing discussed above) and
1997). However, the glutenins also lack an obvious ER in the properties of expressed subunits have been reported
retention sequence but, nevertheless, Galili (1997) has to account for between 45 and 70% of the variation in
proposed that they are preferentially retained in the ER. breadmaking performance within European wheats
In this case, their rapid assembly into high Mr polymers, (Branlard & Dardevet 1985; Payne et al. 1987, 1988).
which precipitate and accumulate directly within the ER These correlative studies are supported by the develop-
lumen, could determine retention. It is also possible to ment and analysis of near-isogenic lines that differ only in
envisage how the relative rates of trafficking via the ER their HMM subunit composition. Analyses of such lines
and Golgi routes could vary with the level of protein syn- have confirmed that the subunits are largely responsible
thesis and age of the tissue. for determining dough viscoelasticity and that specific

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

 Wheat gluten proteins P. R. Shewry and others 135

SH SH SH SH SH SH SH SH
(1Dx5 only) (1By-and
deleted in x-type 1Dy-type only)

N-terminal domain repetitive domain C-terminal

domain
81-104 residues 481-696 residues 42 residues
3 cysteine (x-type) comprises hexapeptides nonapeptides 1 cysteine
5 cysteine (y-type) and tripeptides (x-type only)
0/1 cysteine

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

allelic subunit pairs are associated with either high or low of the hexapeptide, 8 and 9 of the nonapeptide and 2 and
dough strength (Popineau et al. 1994). 3 of the tripeptide. This may relate to the role of glutamine
A number of genes encoding HMM subunits have been residues in stabilizing the structures and interactions of
isolated from bread wheat (see Shewry et al. 1992; the subunits. Similarly, serine is conserved at position 6
Reddy & Appels 1993) and from related wheat species and of the nonapeptides. In contrast, positions 1 and 4 of the
wild relatives (Mackie et al. 1996; Wan et al. 2001). These hexapeptide are poorly conserved, as is position 7 of the
show that the HMM subunits have conserved amino-acid nonapeptide.
sequences, comprising three distinct parts or domains Some differences are also observed between the x-type
(figure 2). The central domains of the proteins consist of and y-type subunits. Thus, replacement of Pro with Ser
repeated peptides, based on two or three short peptide at position 1 of the hexapeptides is more common in x-
motifs. They vary in length from about 420 to 700 resi- type subunits, as is replacement of Gln with Pro at pos-
dues and account for between 74 and 84% of the whole ition 6. However, the latter only occurs in hexapeptides
protein. These domains are flanked by short non-repeti- within a 15 residue (6 + 9) motif, rather than in the tan-
tive domains, which vary in length from 81 to 104 residues demly-arranged hexapeptides. Similarly, replacement of
at the N-terminus but always comprise 42 residues at the Tyr with His at position 2 and Thr with Ala at position 5
C-terminus. of the nonapeptides are more common in y-type subunits
and these two substitutions usually occur together, giving
the two consensus motifs GYYPTSLQQ and GHY-
4. SEQUENCES OF THE REPETITIVE DOMAINS
PASLQQ compared with GYYPTSPQQ for x-type sub-
The x-type and y-type subunits have essentially similar units.
repeat structures, comprising mainly nonapeptide and There is no evidence that amino-acid substitution leads
hexapeptide motifs. Also, whereas tandem blocks of hexa- to replacement with similar amino-acid residues (i.e. con-
peptides may be present, the nonapeptides are always servative substitutions). Instead, analysis of codons indi-
interspersed with hexapeptides. Consequently, it is con- cates that most replacements are due to single nucleotide
venient to consider them as forming a 15 amino-acid changes, with substitutions resulting from double nucleot-
motif. The x-type subunits also differ from the y-type in ide changes occurring more rarely. For example, proline
having additional tripeptide motifs, which also only occur (CCA) occurs at position 1 in 55% of x-type hexapeptides,
in tandem with hexapeptides, forming a second nonapep- with single nucleotide changes leading to the occurrence
tide motif. Figure 3 shows the sequences of the repetitive of leucine (CTA, 12%) and serine (TCA, 30%) and two
domains of typical x-type and y-type subunits (1Dx5 and nucleotide changes to isoleucine (ATA, 3%).
1Dy10, respectively) arranged to show their repeated The failure to detect any appreciable differences
block structure. The repeat motifs are rich in glutamine, between the consensus motifs and degree of conservation
proline and glycine, which together account for over 70% of the repetitive sequences present in the HMM subunits
of the total amino-acid residues. No major differences are of cultivated and wild species (Wan et al. 2001) indicates
apparent between the homeoallelic proteins of bread that selection by plant breeders for dough strength, which
wheat (A, B, D) or related genomes present in other wheat has been carried out systematically for the last century and
species or wild relatives (A, C and D), so combined data perhaps unconsciously over the 10 000 year life of bread
for five x-type and seven y-type subunits are presented in wheat, has had little or no impact on the sequences (and
table 1. hence structure) of the subunits. However, it is possible
Comparison of the patterns of amino-acid substitutions that the differences in degree of conservation within the
shows that some positions of the motifs appear to be more motifs and the precise amino-acid residues that are present
highly conserved than others. In particular, glutamine as substitutions at different positions within the motifs
tends to be more highly conserved at specific positions may relate to their role in determining the structure
than other consensus amino acids: at positions 3, 5 and 6 adopted by the domain.

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

136 P. R. Shewry and others Wheat gluten proteins

Cysteine residues occur only rarely in the repetitive

sequences, with single cysteine residues present towards
the C-terminal end of the repetitive domains (at position
73) of 1By and 1Dy subunits only. In addition, subunit
1Dx5 differs from all other subunits whose sequences are
known in that a single additional cysteine is present at
position 8 relative to the N-terminal end of the domain.

5. STRUCTURE OF THE HMM SUBUNIT

REPETITIVE DOMAIN
Although several workers have attempted to determine
the 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 peptides
based on the repetitive sequence motifs has not yet led to
the determination of three-dimensional structures. Conse-
quently, our current view of HMM subunit structure
comes from a range of indirect studies.
Early hydrodynamic studies of subunit 1Bx20 purified
from pasta wheat indicated that it had an extended rod-
shaped conformation in solution, the dimensions ranging
from ca. 500 17.5 to 620 15 A depending on the sol-
vent (Field et al. 1987). Detailed spectroscopic studies of
whole subunits, of recombinant repetitive peptides and of
linear 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 consistent
with the repetitive sequences forming -reverse turns
which may be in equilibrium with poly-L-proline II struc-
ture, the latter predominating at low temperature (Gilbert
et al. 2000). It has also been proposed that the -turns are
organized to give a regular spiral structure (termed a -
spiral) similar to that demonstrated for a synthetic polyp-
entapeptide based on a repeat motif of elastin (Urry
1988). Molecular modelling can be used to generate such
spiral structures (figure 4) whose dimensions (diameter,
pitch and length) are consistent with those determined by
viscometric 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 an
alternative type of spiral structure is formed, based on -
turns rather than -turns.

6. SEQUENCES AND STRUCTURES OF THE

NON-REPETITIVE DOMAINS
The N-terminal domains vary in length, being 8189
residues in the x-type subunits and 104 residues in the
y-type. This difference results from a deletion in the x-
type subunits compared with the y-type, which involves
the 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 indicate
that this domain is globular with one or more -helices
(Tatham et al. 1984, 1985; Van Dijk et al. 1998; Kohler
et al. 1997).
Figure 3. Amino-acid sequences of the repetitive domains of The C-terminal domains of all of the subunits comprise
typical x-type (1Dx5,(a)) and y-type (1Dy10,(b)) HMM 42 residues with single cysteine residues at position 13
subunits arranged to show their repeat unit structures. with respect to the C-terminus. Structure prediction indi-
cates that this domain may be -helical (Tatham et al.

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

 Table 1. Frequency of occurrence of different amino acid residues in each position of: (a) The 347 hexapeptide, 103 nonapeptide and 81 tripeptide repeat motifs of x-type HMW subunits
1Ax1, 1Bx7 and 1Dx5 (T. aestivum); 1Ax (T. timopheevi ); 1Dx (A. cylindrica). (b) The 339 hexapeptide and 123 nonapeptide repeat motifs of y-type HMW subunits 1Ay (not expressed),

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

1By 9 and 1Dy10 (T. aestivum); 1Ay (T. timopheevi ); 1Cy and 1Dy (A. cylindrica); 1Dy (T. tauschii ). (Percentages may not add up to 100 because of rounding. Residues present at less
than 1% are either included as other if together they add up to 1%, or are not shown.)

hexapeptides (%) tripeptides (%) nonapeptides (%)

(a) 1 2 3 4 5 6 1 2 3 1 2 3 4 5 6 7 8 9

Pro 62 Gly 84 Gln 99 Gly 75 Gln 94 Gln 80 Gly 89 Gln 99 Gln 99 Gly 84 Tyr 98 Tyr 97 Pro 90 Thr 96 Ser 100 Pro 70 Gln 88 Gln 94
Ser 26 Ala 7 Other 1 Trp 9 Leu 3 Pro 15 Asp 5 Arg 1 Arg 1 Arg 6 His 2 Asp 2 Leu 8 Ile 4 Ser 13 Leu 8 Leu 4
Leu 10 Glu 4 Leu 7 Other 3 Ser 2 Ala 2 Glu 3 Phe 1 Ser 2 Leu 11 Trp 3 Glu 2
Ile 1 Arg 3 Glu 4 Arg 1 Arg 2 Trp 3 Ala 2 Arg 1
Other 2 Thr 2 Arg 2 Leu 1 His 1 Val 2 Glu 2
Ala 1 Other 2 Ala 1 2
Other 1 Lys 1 Arg 1

(b) 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9

Pro 65 Gly 92 Gln 96 Gly 76 Gln 94 Gln 94 Gly 96 Tyr 54 Tyr 85 Pro 91 Thr 60 Ser 97 leu 54 Gln 97 Gln 90
Ser 12 Glu 6 Lys 4 Glu 7 His 2 Glu 2 Trp 2 His 41 Cys 4 Leu 5 Ala 37 Tyr 2 Pro 21 His 3 His 7
Leu 10 Lys 2 Trp 7 Lys 2 His 2 Arg 1 Gln 5 Asp 2 Arg 2 Ser 2 Phe 1 Gln 19 Glu 2
Ile 7 Other 1 Arg 4 Other 1 2 Tyr 1 His 2 Ser 1 Ile 1 Val 4 Stop 1
Thr 4 Ala 2 Ile 2 Thr 1 Gly 2
Gln 1 Val 2 Phe 2 Ser 2
Other 1 Lys 1 Arg 1 Ala 1
Other 2 Asn 1
Glu 1
Wheat gluten proteins P. R. Shewry and others
137
138 P. R. Shewry and others Wheat gluten proteins

consistent with the results obtained by partial reduction

of 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 the
detailed 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 and
FTIR spectroscopy) of HMM subunits and of model pep-
tides based on the repeat motifs suggests that non-covalent
hydrogen 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 with
little regular structure, but that their mobility increases
and -sheet structures form on hydration. Further changes
occur if hydration continues, with a further increase in
protein mobility and the formation of turn-like structures
at the expense of -sheet.
These observations led to the development of a loop
and train model (Belton 1999), which is summarized in
Figure 4. Molecular model developed for a -spiral structure
figure 6. This proposes that the low hydration state has
based on the amino-acid sequence of a repetitive domain of
many proteinprotein interactions, via hydrogen bonding
a HMM subunit. The backbone structure only is shown
(D. J. Osguthorpe, O. Parchment, P. R. Shewry & A. S. of glutamine residues in the -spiral structures. As the
Tatham, unpublished results). hydration level increases the system is platicized, allowing
the orientation of the -turns in adjacent -spirals to form
structures that resemble an interchain -sheet. Further
hydration leads to the breaking of some of the interchain
1984) and NMR spectroscopy of a synthetic peptide hydrogen bonds in favour of hydrogen bonds between glu-
dissolved in 40% (v/v) aqueous trifluoroethanol (a struc- tamine and water, which then leads to the formation of
ture-inducing solvent) allowed a low-resolution structure loop regions. However, it does not result in the complete
containing two -helices to be determined (Bekkers et replacement of interchain hydrogen bonds, and hence sol-
al. 1996). ution of the protein, as the number of glutamine residues
is high and the statistical likelihood of all the interchain
bonds breaking simultaneously is therefore low. The result
7. HMM SUBUNIT STRUCTURE AND GLUTEN
is an equilibrium between hydrated loop regions and
ELASTICITY
hydrogen-bonded chain regions, with the ratio between
The HMM subunits are only present in glutenin poly- these being dependent on the hydration state.
mers, particularly in high Mr polymers, the amounts of The equilibrium between loops and trains may also
which are positively correlated with dough strength (Field contribute to the elasticity of glutenin, as an extension of
et al. 1983). This provides support for the genetic evidence the dough will result in stretching of the loops and
(see 3) that the HMM subunits are the major determi- unzipping of the trains. The resulting formation of
nants of dough and gluten elasticity. extended chains may be a mechanism by which elastic
Two features of HMM subunit structure may be rel- energy is stored in the dough, thus providing an expla-
evant to their role in glutenin elastomers: the number and nation for the increased resistance to extension that occurs
distribution of disulphide bonds and the properties and during dough mixing. The formation of interchain hydro-
interactions of the repetitive domains. gen bonds between glutamine residues may also account
Direct sequence analysis of disulphide-linked peptides for the observations that the esterification of glutamine
released by enzymic digestion of glutenin or gluten frac- residues results in decreased resistance to extension, while
tions has revealed a number of inter- and intrachain mixing in the presence of deuterium oxide (D2O) rather
disulphide bonds involving HM W subunits (Kohler et than water results in increased resistance (Beckwith et al.
al. 1991, 1993, 1994; Tao et al. 1992; Keck et al. 1995). 1963; Mita & Matsumoto 1981; Bushuk 1998).
These are summarized diagrammatically in figure 5 and
include one interchain disulphide bond within the N-
8. MANIPULATION OF HMM SUBUNIT
terminal domain of an x-type subunit, two parallel disul-
COMPOSITION IN TRANSGENIC WHEAT
phide bonds between the N-termini of y-type subunits,
an interchain bond between a y-type subunit and a The major aim of determining the structures of the
LMM glutenin subunit and a bond linking y-type and HMM subunits and their role in gluten and dough elas-
x-type subunits in a head-to-tail fashion. The latter is ticity is to facilitate the improvement of the end use

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

 Wheat gluten proteins P. R. Shewry and others 139

LMW

y-type

LMW x-type
x-type
LMW
y-type
y-type
y-type

1Dx5
LMW x-type
HMW?

N-terminal domain repetitive domain LMW subunits

C-terminal domain disulphide bonds

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)

subunit genes. The model lines form part of a near iso-

genic series, which have been produced by crossing lines
differing in their expression of HMM subunit genes. Thus,
line L88-31 expresses only two HMM subunit genes
(encoding subunits 1Bx17 and 1By18), while L88-6
also expresses genes encoding subunits 1Ax, 1Dx5 and
1Dy10 (Lawrence et al. 1988). The two genes used for
transformation encode subunits 1Ax1 and 1Dx5, the latter
always occurring as part of an allelic pair with subunit
1Dy10. The three transgenic lines that have been studied
in detail express the 1Ax1 subunit in L88-31 at a level of
about 5.7% of the total protein (compared with 0% in the
control line) and the 1Dx5 subunit in L88-31 and L88-6
at 8.7% (compared with 0%) and 17% (compared with
4.2%) of the total protein, respectively (Barro et al. 1997;
Popineau et al. 2001).
The effects of the transgenes on dough strength were
determined using a Mixograph. This measures the energy
input during the mixing of dough and is routinely used
for quality testing in a number of countries. When dough
is mixed the resistance increases up to a certain level, after
which it decreases. The increase in resistance may result
Figure 6. Model for the effect of hydration on the loop to from limited exchange of disulphide bonds (see 2) and
train ratio of HMM subunits. (a) Low hydration, disordered, formation of the most stable patterns of hydrogen bonding
close interactions; (b) intermediate hydration, low loop to (i.e. to form extensive train regions). In contrast, the sub-
train ratio; (c) high hydration, high loop to train ratio. sequent decrease in resistance is thought to result from
disruption of these interactions by overmixing. Conse-
quently, beneficial effects of the transgenes on dough
properties of wheat. Substantial improvement in the pro- strength and stability should be observed as increases in
cessing performance of wheat has already been achieved the PR (i.e. the maximum resistance that is observed) and
by a combination of classical plant breeding and optimiz- the MT (i.e. the time taken to mix to PR) and a decrease
ation of the agronomic and processing conditions. How- in RBD (i.e. the rate of decrease in the resistance on over-
ever, it is unlikely that these approaches will be sufficient mixing beyond PR).
in the long term and genetic engineering therefore pro- The results obtained with expression of the two trans-
vides an important additional approach. We are, therefore, genes in the L88-31 background are summarized in figure
using genetic engineering of wheat in order to further 7 (Popineau et al. 2001). The control line has low dough
study the role of the HMM subunits in determining pro- strength, which is consistent with the expression of only
cessing properties and to define strategies for the pro- two endogenous HMM subunit genes, and the expression
duction of improved germplasm for incorporation into of the 1Ax1 transgene results in substantial increases in
plant breeding programmes. PR and MT. In contrast, expression of the 1Dx5 trans-
Most of our work, to date, has focused on transformation gene in the same line was clearly detrimental to the mixing
of two model lines of wheat with two different HMM properties. An even more extreme effect was observed

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

140 P. R. Shewry and others Wheat gluten proteins

80
(a) (b)
60

torque (%)
1
5 40
17+18
10 20
(i) (ii) (iii) (iv) (v)
0
100 200 300 400 500 600

80 80
(c) (d)
60 60
torque (%)

torque (%)
40 40

20 20

0 0
100 200 300 400 500 600 100 200 300 400 500 600

80 80
(e) (f)
60 60
torque (%)

torque (%)

40 40

20 20

0 0
100 200 300 400 500 600 100 200 300 400 500 600
time (s) time (s)

Figure 7. Analysis of the mixing properties of transgenic wheats expressing additional HMM subunits using the 2g Mixograph.
(a) SDSPAGE of the HMM subunits from (i ), control line L88-31 (comigrating subunits 1Bx17 + 1By18); (ii ), L88-31
expressing 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. (bf ) Mixographs of (b), L88-31; (c), L88-31 expressing
the 1Ax1 transgene; (d ), L88-31 expressing the 1Dx5 transgene; (e), L88-6; ( f ), L88-6 expressing the 1Dx5 transgene. The
resistance 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 the 6 transgenic line. These subunits can be assumed to be
L88-6 line which had much stronger mixing properties present in insoluble glutenin polymers. In contrast,
(figure 7f ). In fact, both lines expressing the 1Dx5 trans- expression of the 1Ax1 transgene was associated with a
gene failed to absorb water and form a normal dough in modest increase in the amount of subunits present in poly-
the mixing bowl. mers that were extracted by sonication in the absence of
Rheological studies were also carried out on gluten frac- reducing agent, but had no effect on the amount of sub-
tions from the transgenic lines, showing that the units present in insoluble polymers.
expression of subunit 1Dx5 resulted in large increases in These results suggest that the proteins encoded by the
elasticity (measured as the storage and loss moduli, G two transgenes had fundamentally different effects on the
and G, and the viscoelastic plateau, Gn) while only a structure of the glutenin polymers in the two lines, with
small increase was associated with expression of subunit the 1Dx5 protein leading to the formation of highly cross-
1Ax1. In fact, the effect of subunit 1Dx5 was similar to linked polymers that resulted in high gluten strength,
that previously observed when gluten was modified by unusual hydration behaviour and failure to form a
treatment with transglutaminase to introduce interchain homogeneous network during mixing. In contrast, the
lysylglutamyl cross-links (Popineau et al. 2001). expression of subunit 1Ax1 resulted in similar effects on
The expression of the subunit 1Dx5 transgene was also gluten composition and properties to those observed when
associated with an increase in the amounts of glutenin comparing near-isogenic lines differing in HMM subunit
subunits that were only extracted from flour by sonication composition.
with detergent (2% sodium dodecylsulphate) in the pres- As discussed in 4, the 1Dx5 subunit protein differs
ence of reducing agent (1% dithiothreitol) from 23% from other characterized subunits in the presence of an
of the total flour proteins in the control lines to over 18% additional cysteine residue within the repetitive domain
in the L88-31 transgenic line and almost 30% in the L88- and this may be responsible for the formation of highly

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

 Wheat gluten proteins P. R. Shewry and others 141

cross-linked polymers in the transgenic lines. However, it Gilbert, S. M., Wellner, N., Belton, P. S., Greenfield, J. A.,
must also be borne in mind that, in non-transgenic wheat, Siligardi, G., Shewry, P. R. & Tatham, A. S. 2000
subunit 1Dx5 is always found together with 1Dy10 and Expression and characterisation of a highly repetitive peptide
that dimers of these subunits are released by partial derived from a wheat seed storage protein. Biochim. Biophys.
Acta 1479, 135146.
reduction of glutenin. Consequently, a precise molar bal-
Graveland, A., Bosveld, P., Lichtendonk, W. J., Marseille,
ance of these two subunits may be required to give a nor- J. P., Moonen, J. H. E. & Scheepstra, A. 1985 A model
mal glutenin polymer structure. for the molecular structure of the glutenins from wheat flour.
These results demonstrate, therefore, that transform- J. Cereal Sci. 3, 116.
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may have fundamentally different effects on gluten struc- Robert, L., Thompson, R., Flavell, R. B., Tatham, A. S. &
ture and properties, which may relate to the expression Shewry, P. R. 1992 Analysis of HMW glutenin subunits
levels, structures and interactions of the individual pro- encoded by chromosome 1A of bread wheat (Triticum aesti-
teins. vum L.) indicates quantitative effects on grain quality. Theor.
Appl. Genet. 83, 373378.
Kasarda, D. D., King, G. & Kumosinski, T. F. 1994 Compari-
I. A. C. R. and I. F. R. receive grant-aided support from the
Biotechnology and Biological Sciences Research Council son of spiral structures in wheat high-molecular-weight glu-
(BBSRC) of the United Kingdom. tenin subunits and elastin by molecular modeling. In
Molecular modeling: from virtual tools to real problems. Amer-
ican Chemical Society Symp. series no. 576 (ed. T. F.
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Phil. Trans. R. Soc. Lond. B (2002)