Transgenic Research 3, 3-12 (1994)

R E V I E W

Opportunities for manipulating the seed protein composition of wheat and barley in order to improve quality

P E T E R R . S H E W R Y I* , A R T H U R S. T A T H A M 1, N I G E L G . H A L F O R D 1, J A C K I E H . A . B A R K E R I, U L R I C H H A N N A P P E L ~'4, P . G A L L O I S 3, M. T H O M A S 2 and M A R T I N K R E I S e

:Department of A gricultural Sciences, University of Bristol, A FR C Institute of Arable Crops Research, Long A shton Research Station, Long A shton, Bristol, BS18 9AF, UK (Fax +44 02 75 3942 99) 2 Universit~ de Paris-Sud, Biologie du Developpement des Plantes, Batiment 430, F-91400 Orsay Cedex, France 3 Universit~ de Perpignan, Laboratoire de Physiologie et Biologie Mol~culaire V~g~tales, 52 A venue de Villeneuve, 66860 Perpignan Cedex, France 41nstitute of Plant Genetics and Crop Plant Research, Corrensstr. 3, D(0)-4325 Gatersleben, Germany

Received 20 May 1993; accepted 27 May 1993

Wheat and barley are the major temperate cereals, being used for food, feed and industrial raw material. However, in all cases the quality may be limited by the amount, composition and properties of the grain storage proteins. We describe how a combination of biochemical and molecular studies has led to an understanding of the molecular basis for breadmaking quality in wheat and feed quality in barley, and also provided genes encoding key proteins that determine quality. The control of expression of these genes has been studied in transgenic tobacco plants and by transient expression in cereal protoplasts, providing the basis for the production of transgenic cereals with improved quality characteristics.

Keywords: wheat; barley; gluten proteins; chymotrypsin inhibitors; nutritional quality; breadmaking quality

Introduction: cereal grain development and composition

The mature cereal grain arises from two initial nuclear fusions which give rise to the embryo and endosperm tissues. The embryo develops from the diploid zygotic nucleus, formed by fusion of the egg nucleus and one male nucleus, and differentiates to form the embryonic axis (consisting of the plumule and radicle enclosed by the coleoptile and coleorhiza respectively) and a single cotyledon or scutellum. The second male nucleus fuses with two polar nuclei to give the triploid primary endo- sperm nucleus. This divides, initially synchronously, to form a coenocyte containing over 2000 nuclei. Cell wall

*To whom correspondence should be addressed. This manuscript is based on a paper presented at a Phytochemical Society of Europe/Biochemical Society Meeting on Genetic Manipulation of Plant Growth and Metabolism, held at Norwich, UK, on 29-31 March, 1993.

0962-8819 �9 1994 Chapman & Hall

formation then occurs, followed by cell divisions which eventually become restricted to the outer layers of cells. These outer cells finally cease to divide and form the aleurone layer, consisting of several layers of cells in the mature barley grain, but only a single layer in wheat.

The mature grain therefore consists of four major tissues: the embryonic axis, scutellum, aleurone and starchy endosperm. Each of these contains different amounts and types of storage compounds, resulting from differential gene expression during grain development. In order to manipulate the amount and composition of storage compounds it is necessary to understand the control of gene expression in these tissues, as well as the properties and compositions of the different storage components and their biosynthetic pathways.

In wheat and barley the main storage tissue is the starchy endosperm, which gives rise to the white flour fraction when wheat is milled. This contains two major

groups of storage compound: starch' and alcohol-soluble prolamin storage proteins. However, smaller amounts of other storage compounds are also present, including albumin and globulin storage proteins and triacylglycerols. Despite their separate origins, the aleurone layer and scutellum have similar contents of storage compounds. Both tissues lack prolamins or appreciable amounts of starch, but are rich in triacylglycerols present as oil droplets. Both tissues also contain protein deposits in the form of protein bodies. In the aleurone, these consist of a protein matrix with two types of inclusion: globoid (phytin) and crystalline (densely packed protein and carbohydrate (Jacobsen et al., 1971)). The protein bodies in the scutellar parenchyma have a similar appearance to those in the aleurone, but contain only globoid inclusions (Gram, 1982). The proteins present in the aleurone and scutellar protein bodies have not been characterized, but it has been shown that the major protein type present in isolated aleurone layers and whole embryos is a globulin related to the vicilin-type 7S storage globulins of legumes and cottonseed (Yupsanis et al., 1990).

The types of storage compounds present in the aleurone and embryo and their distributions within these tissues are of interest in relation to tissue-specific gene expression, but have little impact on the composition of the whole grain. This article will, therefore, focus on the starchy endosperm and, in particular, on opportunities to manipulate the proportions of storage proteins in this tissue using genetic transformation in order to improve aspects of grain quality.

Cereal seed proteins

The classification of cereal seed proteins is based largely on work carried out by T.B. Osborne around the turn of the century (see Osborne, 1924). He divided proteins into four groups on the basis of their extraction and solubility in a series of solvents. These are the albumins (water), globulins (dilute saline), prolamins (aqueous alcohols) and glutelins (dilute acids or alkalis). Whereas albumins, globulins and glutelins are present in most plant tissues, the prolamins are restricted to the grains of cereals and other grasses. They form the major storage protein fraction in all cereals except oats and rice (where they are relatively minor components), and usually account for about half of the total grain nitrogen. Although tradition- ally extracted in aqueous alcohols, studies carried out over the past twenty years have shown that some prolamins are present in alcohol-insoluble polymers stabilized by inter- chain disulphide bonds, and are only extracted with aqueous alcohol under reducing conditions (i.e. as single subunits) (see Shewry and Miflin, 1985).

The prolamin fractions of different cereals are given trivial names. In barley all are called hordeins, while in wheat the monomeric and polymeric components are

S h e w r y et al.

called gliadins and glutenins, respectively. The gliadins and glutenins together form the gluten proteins, which play a key role in determining the functionality of wheat flours (see below).

The prolamins are characterized by high contents of glutamine and proline, which account for over 50% of the total residues in whole prolamin fractions of wheat and barley (Byers et al., 1983; Kreis et al., 1984). In addition they are low in charged residues, including the essential amino acid lysine (about 1 mol %: see below).

The prolamin fractions are not single proteins, but complex polymorphic mixtures of over 50 proteins in hexaploid bread wheat and about 20 to 30 in barley. A number of individual proteins have been purified and characterized, and an increasing number of cDNA and/or genomic clones isolated. In addition, the structural genes for most prolamins have been mapped using classical or molecular genetic approaches. On the basis of these studies, the individual prolamins in the two species have been classified into three groups, and in some cases into families and sub-families within these groups (see Shewry and Miflin, 1985). The classification and properties of wheat and barley prolamins are summarized in Table 1.

Although prolamins form the major storage protein fraction, the starchy endosperms of wheat and barley also contain other storage proteins. These include the barley chymotrypsin inhibitor CI-2, which is located together with prolamins in the starchy endosperm protein bodies (Rasmussen et al., 1990), and accounts for about 0.4% of the total grain proteins (calculated from Hejgaard and Boisen, 1980).

Nutritional quality

A high proportion of the wheat and barley grown in Western Europe and North America is used for livestock feed, while both species are also used for human nutrition (either directly or after processing, for example, into bread). However, in both cereals the nutritional quality for humans and other monogastric animals (for example, pigs and poultry) is limited by the low contents of two essential amino acids: lysine and threonine. Of these, lysine is the more important, with levels in wheat and barley of about 1.98 and 3.26 g % respectively, compared to a WHO recommended level of 5.5 g % (Ewart, 1967; FAO, 1973). Because of this, it is necessary to mix cereals with more lysine-rich proteins in order to provide a mixed diet, resulting in Western Europe in the import of massive quantities of soyabeans and soyameal for livestock feed.

The low levels of lysine in whole wheat and barley grain result from the low proportions of lysine in the prolamins. Thus the proportions of lysine in the individual prolamin groups vary from zero to about 1.5 mol %, while total hordeins and total gliadins + glutenins contain about 1 and 0.9 mol % respectively (Kreis et al., 1984; Bright and

Manipulat ing seed protein's composit ion o f wheat and barley

Table 1. Summary of the classification and properties of barley and wheat prolamins

Prolamin group and Prolamin % Total Subunit family Species name fraction structure

Amino acid Molecular composition weight (tool %)

High Barley D hordein 2-4 molecular weight Wheat HMW subunits 6-10

of glutenin

S-poor

S-rich y -type

Aggregated type

a-type

Barley C hordein t 10-20

Wheat c0-gliadins

Barley ~/-hordeins Wheat y-gliadins

Barley B hordein

Wheat LMWsubun~s ofglutenin

Barley Absent Wheat a-gliadins

70-80

Polymeric

Monomeric

Not clear Monomeric

Polymeric

Polymeric

Monomeric

30-35 Gin = 65 000 10-16 Pro

to 15-20 Gly 90000 0.5-1.5 Cys

0.7-1.4 Lys

40-50 Gin = 40000 20-30 Pro

to 8-9 Phe 70 000 0 Cys

0-0.5 Lys

= 30000 30-40 Gin to 15-20 Pro 45 000 2-3 Cys

< 1.0 Lys

Shewry, 1983). The nutritional quality of wheat grain has been largely ignored because of an emphasis on processing properties. However, because much of the barley in developed countries (particularly Northern Europe) is used for livestock feed, considerable efforts have been made to increase its content of lysine.

Most of the early work on barley improvement was based on the identification of mutant "high lysine" genes and their incorporation into breeding programmes. A number of such genes were identified in the 1970s, mostly by screening mutagenized populations (see Doll, 1983). However, one spontaneous mutant line (called Hiproly) was identified by screening about 2500 lines of the World Barley Collection (Munck et al., 1970). These mutant lines have been subjected to detailed biochemical analyses, which are discussed in several review articles (see for example Shewry et al., 1987). In general, the increased proportion of lysine is associated with a reduction in total prolamins, with concomitant increases in other more lysine-rich proteins. However, there are also associated effects on the amount of starch, resulting in decreased yield when compared with normal isolines. Consequently these traits have proved extremely difficult to incorporate into high yielding cultivars, although Munck (1992) has reported some success with Riso 1508.

An alternative strategy, to add additional lysine codons

to individual storage protein genes, is also unlikely to succeed since the prolamins of wheat and barley are encoded by complex multigene families, individual members of which account for about 2% of the total prolamin fraction or less (see below).

However, analysis of Hiproly has indicated an alternative strategy which may be more successful. The high lysine character in this line results from a single mutant gene (lys) which maps to barley chromosome 7 (Karlsson, 1976). This results in a 30% increase in total lysine, associated with a modest decrease in total prolamins (by about 10-20%) (Miflin and Shewry, 1979). The increased lysine content arises predominantly from increases in four specific lysine-rich proteins: B-amylase (5.0 g % Lys), protein Z (7.1 g % Lys) and chymotrypsin inhibitors CI-1 (9.5 g % Lys) and CI-2 (11.5 g % Lys) (Hejgaard and Boisen, 1980). Of these, CI-1 and CI-2 have received most attention because of their very high contents of lysine. Both are monomeric low M r ( = 8800 for CI-1, 9400 for CI-2) proteins with no cysteine residues. Their combined activities are increased from about 0.7 units g-1 in normal lines to 4.5-5.6 units g-i in Hiproly and derived lines (Hejgaard and Boisen, 1980), demonstrating that considerable increases can be tolerated without adversely affecting grain development. Their biological role is not known, but they could confer

6

protectiQn against invading micro-organisms and insect pests by inhibition of serine proteases.

Kreis and co-workers have isolated cDNAs for both inhibitors (Fig. 1) and studied the populations of mRNAs in developing endosperms and other tissues of normal and high lysine lines (Williamson et al., 1987, 1988). They have also isolated a gene for CI-2 (Peterson et al., 1991) in order to explore the control of its expression.

The CI-2 gene contains a single 90 bp intron at 18 bp upstream of the coding region, with CAT and TATA boxes. In addition, two putative 5' regulatory sequences are present. A sequence homologous with the ' - 300 element' ('prolamin box') of cereal prolamin genes (Forde et al., 1985) is present at - 538 bp upstream of the transcription start site, and a sequence homologous with the '7-11-7 element' at - 4 0 4 bp. The latter is also present in the prolamin (zein) genes of maize (Boronat et al., 1986), but not in those of wheat or barley.

Expression of chimaeric C I - 2 / g u s genes in transgenic tobacco plants demonstrated that either 4300 or 1100 bp of 5' upstream sequence was sufficient to confer tissue-specific expression, but such expression was not observed when only 263 bp of upstream sequence was present. However, transient expression was observed when the construct with 1100 bp of 5' sequence was delivered into protoplasts derived from barley endosperm cells (Lee et al., 1991) or tobacco mesophyll cells (Fig. 2).

CI- IAIB ~S ~!~R

CI - 2A S .... 2B

CI - IA A S S IB AS G

CI - 2A D R H 2B D C Q

CI - IA ~ i I 1B I CI - 2A V

2B V

CI - IA M~H IB M

CI - 2A I 2B I

CI - IBIA i~ QRR CI - 2A Q

2B

G S V L ~ Y ~ P T E G S I G GSV P y PT E G S I G

K GVNTGAG *DCLC

a Rs GMNaNK N . TE GK~V~E~ N T~ EX~V~E~

RID K PIDIA QII E I A QID K PIEIA QII I L T QID K PIE JA QI I I L T

LNF DPN LDFNPN MEYRID MEYRID

RL DKLDN RL DRLDN

* sequence incomplete

Fig. 1. The amino acid sequences deduced from cDNA clones for barley chymotrypsin inhibitors CI-1 and CI-2. In each case two sub-families of clones were identified, and were designated A and B. Note the high content of lysine residues (K) and absence of cysteine. Based on data in Williamson et al. (1987, 1988). Standard single letter abbreviations for amino acids are used.

S h e w r y et al.

A. Barley Endosperm Protoplasts

E _ m t'- Q. O O

O "

4.0

N \ ' q

N \ N I

r x \ ~ l

x \ ' , l

r / / / e .4 ,,,',

c, x,9- ,,,,'

f----]

B. Tobacco Mesophyll Protoplasts

2 . 0

= ~" 1.5 ~ / >' ~ / /

0 . 5

/ II111tl

Fig. 2. Transient expression of gus constructs with the CaMV 35S promoter (CaMV-GUS), with 1100 bp of 5" upstream sequ- ence from the CI-2 gene (pCI-2-SP-Gus), with 688 bp of 5' upstream sequence from the wheat gene encoding HMW glutenin subunit 1Dx5 (pGlu-lDx5-Gus 1) or with no promoter (p-Gus) in protoplasts derived from barley starchy endosperm cells (part A) or tobacco mesophyll cells (part B) (taken from Lee et al., 1991; Kreis and Shewry, 1992).

These results contrasted with those obtained using a similar construct with the 5' upstream region from a wheat high molecular weight (HMW) glutenin subunit gene, which was only expressed in the endosperm-derived protoplasts (Fig. 2). The expression of the CI-2 constructs in the tobacco mesophyll protoplasts may relate to tissue damage during protoplast preparation as similar levels of expression were observed when protoplasts were prepared from transgenic tobacco plants with the same chimaeric

Manipulating seed protein's composition of wheat and barley

gene construct (Fig. 3). Also, related protease inhibitors are synthesised in tomato fruit, where they are induced by wounding (Graham et al., 1986). There is no evidence for induction of CI-2 expression when barley is wounded, so it must be assumed that the wound-inducibility observed in tobacco does not occur in the barley plant.

These studies of CI-2 gene expression provide interesting information on the control of gene expression in the developing barley grain, and also open the way to eventually improving the nutritional quality of barley and wheat by genetic engineering. To do this it may be necessary to express the CI-2 coding region under the control of a stronger endosperm-specific promoter, for example from an HMW glutenin subunit gene (see below). With such a promoter, only one additional CI-2 gene copy could have an impact on the lysine content of a transgenic cereal grain.

Malting quality The malting quality of barley is a complex character, which is affected by a number of interacting factors. These

Protoplasts from p-Gus

-- ~ 2 , 0 "Protoplasts from SP-35 transgenic O ~ transgenic plants plants

1.o-

" "

" @ 0 M H

o," o," o,- .oQ ,oQ ,oQ .| ^a _,,| / ' / /

Fig. 3. Expression of gus activity in protoplasts derived from mesophyll cells of transgenic tobacco plants with the pCI-2-SP- Gus construct (Plants SP-35) or the p-Gus construct (see legend to Fig. 2). The SP-35 protoplasts are assayed without electropor- ation and after electroporation with or without the p-Gus construct (unpublished data of M. Kreis).

include endosperm texture, ~-glucan content and the levels of hydrolytic enzymes (notably a-amylase and 13- glucanase) produced during germination. Most of the hydrolases that digest storage reserves during malting and germination are synthesised de novo. However, ~-amylase is synthesised during grain development, and acts as a storage protein in that its amount is increased in response to nitrogen availability (Giese and Hejgaard, 1984). It is located principally in the cytosolic fraction (Nishimura et al., 1987) rather than protein bodies, and appears to become associated with the periphery of starch granules during seed desiccation. Kreis et al. (1987) isolated a cDNA clone for barley 13-amylase, and Thomas et al. (unpublished results) a genomic clone. Analysis of chimaeric ~-amylase /gus constructs in transgenic tobacco showed that about 400 bp of 5' sequence was sufficient to confer seed-specific expression, while further transient expression studies in tobacco mesophyll protoplasts identified two regions affecting gene expression. Only 120 bp of sequence upstream of the ATG initiation codon were required for gene expression, but the level was increased by about 50% when additional sequences between 1100 and 6000 bp were present (Fig. 4). The availability of this cDNA and gene provides an oppor- tunity to explore the consequences of up- or down- regulation of ~-amylase synthesis for malting quality by transformation of barley plants with sense and antisense constructs.

10

~ 8

c~

c 6 .9 E O. x: 4

0

<

30 IJg DNA per million protoplasts

pGUS pG10 pG9 pG8 pG7 pG5 pG4 pG3 pG1 pCGUS 0 -70 -120 -340 -450 -850 -1100-1250 -6kb 355

Fig. 4. gus activity in tobacco mesophyll protoplasts electropo- rated with constructs in which the GUS reporter gene is placed downstream of increasing lengths of the [3-amylase promoter. pGUS and pCGUS are negative and positive controls, respect- ively (taken from IACR Annual Report, 1989).

Shewry et al.

Breadmaking quality Although wheat is used to make noodles, pasta, cakes, biscuits, pastries and a range of other food products, its utilization for breadmaking has been of overwhelming importance in determining the direction of research, particularly in Western Europe and North America. The present discussion will therefore focus on this aspect of wheat quality.

Although the precise molecular interactions in wheat doughs are still largely unknown, it is clear that the quality of wheat for breadmaking is determined largely by the amount and properties of the gluten proteins (i.e. the gliadins and glutenins). These are present as a network in dough, but can be isolated in a form that is substantially pure by washing doughs to remove water-s01uble com- ponents and starch. The resulting gluten consists of about 70% (dry weight) protein, and exhibits an unusual combination of two physical properties: extensibility and elasticity. A precise combination of these properties is essential to enable the entrapment in the dough of carbon dioxide produced by fermentation, resulting in a light porous crumb structure. Elasticity is known to be associ- ated with the glutenins, which consist of HMW subunits and low molecular weight (LMW) subunits assembled into high M r (in excess of 1 x 106) polymers stabilized by inter-chain disulphide bonds. In contrast, extensibility (viscous flow) is associated with the gliadins (subdivided into a-type, y-type, and 00-gliadins), which interact with each other and with the glutenin polymers by non- covalent hydrogen bonds and hydrophobic interactions.

Poor breadmaking quality is often associated with low gluten elasticity, especially in European wheats. Two types of study indicate that the HMW subunits play a key role in determining gluten elasticity. Firstly, the studies of Payne and his followers (see Payne, 1987) have clearly demon- strated that differences in the breadmaking quality of wheats are often correlated with allelic variation in the number (three, four or five) and SDS-PAGE mobilities of the HMW subunits. On the basis of these studies it has been possible to assign quality scores to all the commonly occurring subunits, and to predict the breadmaking performance of many cultivars on the basis of HMW subunit composition. Secondly, the HMW subunits are only present in high M r polymers, the amounts of which are also correlated with breadmaking quality (Field et aL, 1983). Attention has therefore been focused on this group of proteins in order to determine their role in gluten structure and functionality.

The HMW subunits are encoded by the Glu-1 loci on the long arms of the group 1 chromosomes of bread wheat (chromosomes 1A, 1B, 1D) (Payne, 1987). Each locus consists of two genes, encoding a low M r y-type and high M r x-type subunit, giving a theoretical maximum of six subunits per cultivar (Harberd et aL, 1986). However,

specific gene silencing results in the expression of only three, four or five genes. As a result, all cultivars contain 1Bx, 1Dx and 1Dy subunits, while 1By and l a x subunits are also present in some cultivars, lAy subunits are never expressed in cultivars of bread wheat. Variation in quality is correlated particularly with the absence or presence of lAx subunits, and with allelic variation in 1Dx + 1Dy subunits. In the latter case, the widely occurring subunit pair 1Dx5 + 1Dyl0 is associated with good quality and the allelic pair 1Dx2 -t- 1Dyl2 with poor quality. Because recombination between the iDx a n d 1Dy genes occurs only rarely, it is not possible to conclude whether the quality derives from subunit 1Dx5, subunit 1Dyl0 or the specific combination of these two subunits.

Studies in several laboratories have led to the isolation of nine genes encoding HMW subunits, including those for lAx subunits 1 and 2", the allelic pairs 1Dx2 + 1Dyl2 and 1Dx5 4- 1Dyl0, and a silent l a y gene (see Shewry et aL, 1989, 1992). The deduced amino acid sequences have provided a basis for physico-chemical analyses of protein structure, including detailed studies of a single subunit (1Bx20) from tetraploid pasta wheat.

These studies show that all the HMW subunits have a characteristic three domain structure, with a central repetitive domain flanked by non-repetitive domains. The repeats are based on hexapeptide and nonapeptide repeat motifs, with a tripeptide motif present in the x-type subunits only (Fig. 5).

The N-terminal and C-terminal domains show limited variation in length (81-104 residues and always 42 residues respectively) and sequence between individual subunits. In contrast the repetitive domain varies from 481 (1Dyl0) to 696 (1Dx5) residues, and is largely responsible for the differences between the molecular weights of the whole proteins (67495 to 88137). The distribution of cysteine residues is of particular interest as

x-type (1Dx5)

89 785 827 NH2 - ~ REPEATS

PGQGQQ+GYYPTSPQQ+GQQ ~ - COOH / 1 \ \ I

SH SHSHSH SH

y- type (1 Dy 10) 104 585 627

NH2 " ~ REPEATS ~-COOH PGQGQQ+GYYPTSLQQ

/ / A \ ~ I SHSHSHSHSH SH SH

(~not present in 1Dx2)

Fig. 5. Summary of the sequences of x- and y-type HMW subunits of glutenin, based on data reported in Anderson et aL (taken from Shewry et aL, 1992).

Manipulat ing seed protein's composi t ion o f wheat and barley

these are potential cross-linking sites for the formation of glutenin polymers. They are predominantly located in the non-repetitive domains, with three (x-type subunits) or five (y-type subunits) in the N-terminal domain and one in the C-terminal domain. However, additional cysteines are also present in the repetitive domains of some subunits: towards the C-terminal ends in y-type subunits (e.g. 1Dyl0) and close to the N-terminal end in 1Dx5 only (Fig. 5).

The conformations of HMW subunits have been studied using a combination of small angle x-ray scattering and hydrodynamic measurements to determine size and shape, and spectroscopic analyses (circular dichroism and Fourier transform infra-red) of whole proteins and synthetic peptides based on consensus repeat motifs to determine secondary structure contents. In addition, the protein secondary structure has also been predicted from the amino acid sequences. These studies show that the repetitive sequences form regular [3-turns, which are organized to form a loose spiral structure. The result is an extended rod-shaped molecule of about 20/k diameter, and scanning tunneling microscopy STM of a purified HMW subunit has shown aligned rods of 19/k diameter with spiral striations with a periodicity of 14.9/k (Fig. 6) (Miles et al., 1991). These molecules are imaged in the hydrated state after deposition from a solution in trifluoroethanol, and the conformation observed may therefore correspond to that present in protein bodies and in gluten. The non-repetitive domains are not imaged by STM, and are probably globular.

The relationship between HMW subunit structure and gluten elasticity is still uncertain, but two features may be relevant. The first is the spiral conformation adopted by the repetitive domains. A similar [3-spiral structure is formed by a synthetic polypentapeptide based on a repeat motif in elastin (an elastomeric protein present in mammalian connective tissues), and has been shown to be intrinsically elastic (Urry et al., 1983). It has similarly been suggested that the wheat HMW glutenin subunits are intrinsically elastic, and that this contributes to the elastic mechanism of gluten (Tatham et al., 1984; 1985). Furthermore, differences in this property between subunits 1Dyl0 and 1Dy12 have been proposed to account for the quality differences associated with the allelic pairs 1Dx2 + 1Dyl2 and 1Dx5 + 1Dyl0 (Flavell et al., 1989).

The second feature is the number and distribution of cross-links (disulphide bonds), which would be expected to affect the elastic modulus, as in rubbers (Flory, 1953). It is interesting in this respect that subunit 1Dx5 contains an additional cysteine residue compared to subunit 1Dx2, which could result in the formation of more highly cross-linked (and hence more elastic) polymers.

Variation in the degree of cross-linking may account for the qualitative effects of subunits 1Dx5 + 1Dyl0 and

9

Fig. 6. STM of a purified HMW glutenin subunit (1Bx20) from durum wheat reveals a regular spiral structure, with a diameter of about 19.5A and a periodicity of about 14.9A (the STM image is taken from Miles et al. (t991) and the figure from Shewry et al. (1992)).

1Dx2 + 1Dyl2 on quality, but it is likely that variation in breadmaking quality also arises from quantitative effects of the amounts of HMW subunit. In particular, Halford et a/. (1992) compared the combined proportions of HMW subunit protein in cultivars which differed in the absence or presence of subunit 1Axl or lAx2*. They showed that HMW subunits accounted for about 8% of the total extracted protein in cultivars with four subunits and about 10.2% in cultivars with five subunits. In addition, smaller differences were observed between cultivars with subunits 1Axl and lAx2*, and between cultivars with 1By8 and 1By9.

These results indicate two possible approaches to manipulating gluten elasticity by transformation of wheat. The first is to increase the total amount of HMW subunits (and hence the amount of high M r polymers) by inserting additional copies of HMW subunit genes. The second is to insert additional HMW subunit genes with extra cysteine residues, in order to produce more highly cross-linked

10

polymers. As each expressed HMW subunit gene accounts for about 2% of the total grain protein (Halford et al., 1992), it should be possible to have a significant impact by inserting a single gene copy.

Several studies of HMW subunit gene expression in transgenic tobacco plants have been reported. Robert et a/. (1989) transferred a wheat genomic clone consisting of the coding region for subunit 1Dx12, 2.6 kb of 5' sequence and 1.5 kb of 3' sequence. HMW subunit protein accumulated only in the seeds and at levels up to 0.1% of the total endosperm protein. The endosperm-specificity of the HMW subunit promoters was confirmed by the same workers and by Halford et al. (1989), using chimaeric constructs with CAT and gus reporter genes respectively. These studies also demonstrated that endosperm-specificity was conferred by only 433 bp (Robert et al., 1989) or 280 bp (Halford et a/., 1989) of 5' upstream sequence. In both cases reporter gene activity was first detected at 8-10 days after fertilization (Fig. 7), and persisted until maturity. Halford et al. (1989) also demonstrated that a similar length of sequence from a silent lAy subunit gene isolated from the

S h e w r y et al.

same cultivar of wheat failed to give expression. Comparison of the nucleotide sequence of this region from the silent gene with those from six expressed genes showed only ten unique single base pair substitutions in the former, and it was concluded that one or more of these was probably responsible for the gene silencing. More recently, Thomas and Flavell (1990) tested the ability of different sequences from the HMW subunit promoter region to enhance the transcription from an adjacent truncated CaMV 35S RNA promoter, again using gus as a reporter gene. This indicated that an enhancer element for endosperm-specific expression was present in a 40 bp region about 170 bp upstream of the transcription start site. It is of interest that none of the unique substitutions in the silent l a y gene of Halford et al. (1989) fall within this region.

Comparisons with the CaMV 35S RNA promoter (Halford et al., 1989) and with promoters from other cereal seed protein genes (Halford, Shewry, Gallois, Thomas and Kreis unpublished results) have shown that HMW subunit gene promoters give unusually high levels of endosperm-specific expression, which is consistent with

50

r-

E ~. 40 o

O

c~ 30 ~o

O E r

v 20

, m

o 10

CO

(.9

10 20 30

" ~ Glu- 1Dx5-Gus 2 no. 1

~I, Glu- 1Dx5-Gus 1 no.3 \

~ o Glu- 1Dx5-Gus 3 no.3

Days after pollination

Fig. 7. Expression of the chimaeric Glu-1 Dx5 gus constructs in developing seeds of transgenic tobacco plants. The Gus 1, Gus 2 and Gus 3 constructs contain 649, 378 and 277 bp respectively of sequence upstream of the transcription start site of the glu- lDX5 gene. (taken from Halford et al. (1989)).

Manipu la t ing seed prote in ' s compos i t ion o f whea t a n d barley 11

their products each accounting for about 2% of the total grain protein (Halford et al., 1992). They should, therefore, prove to be of considerable value in directing endosperm-specificity of transgenes in cereals.

Conclusions

Several aspects of barley and wheat quality (notably nutritional and breadmaking quality) are currently amen- able to manipulation by transformation, in that the molec- ular basis is understood in some detail, and suitable genes are available for transfer. In other cases (for example, malting quality), the molecular basis for the quality trait is insufficiently understood, but transformation could provide valuable insights by allowing the up or down regu- lation of specific gene products.

Studies in transgenic tobacco have also identified suitable promoters for conferring endosperm-specific expression. In particular, the HMW subunit gene promo- ters are particularly strong, both in wheat (where each gene accounts for about 2% of the total grain protein) and in transgenic tobacco. These would therefore be the first choice for future attempts to manipulate endosperm protein composition in transgenic cereals.

The major limitation in such applications is, of course, the transformation of wheat and barley. However, recent work in several laboratories has resulted in stably-trans- formed fertile transgenic plants of wheat (Vasil et al., 1992; Weeks et al., 1993) and Tritordeum (a hybrid between pasta wheat and a wild species of barley) (Barcelo et al., 1993), using particle gun bombardment of embryogenic cultures. In addition, the cell electroporation method recently developed for maize (D'Halluin et al., 1992) may also prove applicable to barley and wheat. It is, therefore, only a matter of time before this technology is brought to bear on the manipulation of grain quality.

Acknowledgements

U. Hannappel was supported as part of the EC BRIDGE Training Network 'Seeds of Tomorrow Through .Genetic Engineering'.

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