The Gluten Proteins

By D. Lafiandra, S. Masci, R. D’Ovidio

The Royal Society of Chemistry

Copyright © 2004 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-0-85404-633-1
CHAPTER 1

THE USE OF BIOTECHNOLOGY TO STUDY WHEAT ENDOSPERM DEVELOPMENT AND IMPROVE GRAIN QUALITY

P.R. Shewry, H.D. Jones, M.J. Holdworth , J.R. Lenton and K.J. Edwards

1. INTRODUCTION

Pollination of bread wheat results in a double fertilization event within the embryo sac. One pollen nucleus fuses with the egg cell which subsequently gives rise to the zygote which has the normal hexaploid constitution of 42 chromosomes. At the same time the second pollen nucleus fuses with two polar nuclei in the embryo sac to give rise to the endosperm which consequently has three copies of each chromosome. Therefore, although the endosperm of bread wheat is often referred to as triploid, it actually has 63 chromosomes, nine of each homoeologous group.

The primary endosperm nucleus divides mitotically with the products of the first two divisions establishing the right and left halves and the distal and proximal poles of the endosperm, respectively. Further nuclear divisions then occur, which are initially synchronous, to give a syncytium which, in wheat may have 1000 to 2000 nuclei. This stage is usually reached by about 72 hours, after which cell wall formation occurs. The newly formed cells then divide and differentiate with division becoming restricted to the outer layer of cells which form the aleurone.

Storage products, starch and protein, are first observed in the starchy endosperm cells at about 14 days after anthesis with maximum accumulation occurring over the following 14 days. Subsequently, the grain dries down, the starchy endosperm cells become disorganised and die and the embryo and aleurone enter a state of dormancy. Consequently, the development of wheat under UK conditions can be divided into three phases of approximately equal duration. Phase 1 (0 – 14 days) is when the patterns of cell division and differentiation essentially establish the basic structure and organisation of the tissue. This will include genetically determined differences in size, shape and architecture (e.g. crease structure). Phase 2 (15 – 28 days) is grain filling which determines the final yield and quality of the grain. Phase 3 (29 – 42 days) is desiccation and dormancy development. However, it must be noted that the duration of these phases will be greatly affected by environmental conditions, being shortened under high temperatures

It is clear that we need to understand how events taking place during grain development are regulated if we wish to manipulate the yield and quality of the grain

2. TRANSCRIPTOMICS

The identification and quantization of the whole range of transcripts expressed in specific cells, tissues and stages of development has become a standard tool for molecular biologists as it allows transcripts which are associated with specific characteristics (events, mutations, environmental impacts etc.) to be identified. The standard system is to use arrays of DNA sequences corresponding to specific genes for hybridization against cDNA fractions from the tissue of interest.

In order to generate a resource for transcriptional analysis of wheat development we constructed 35 cDNA libraries, using mRNA fractions from various stages of grain development as well as from vegetative tissues grown under normal and stress conditions. Over 26,000 of these cloned “expressed sequence tags” (ESTs) have been subjected to single pass sequencing (ie, one strand only) and their sequences made publicly available in the IGF (Investigating Gene Function) database (http://www.cerealsdb.uk.net).

10,000 of these EST have also been arrayed on glass slides to give a unigene set which is publicly available for high throughput gene expression studies. We are currently using this array for several projects, including determining the effects of crop nutrition and environmental factors (temperature, water availability) on grain development and quality and comparison of the “substantial equivalence” of GM and non-GM wheat.

3. TRANSFORMATION AND GENE IDENTIFICATION

High throughput transformation is an essential prequisite for determining the functions of transcripts identified by transcriptome analysis and confirming the identities of genes identified by tagging or mutagenesis.

We have focused on developing a routine biolistics (particle bombardment) system which can be applied to a wide range of wheat genotypes , as discussed in a separate chapter in this volume. In addition, we are focusing on two lines of wheat as tools for functional genomics studies.

Firstly, the cultivar Cadenza, which was grown recently in the UK as a winter wheat, although vernalisation is not strictly required. It is hard with moderate breadmaking quality and is classed as NABIN Group 2 with a NIAB score of 6. We have found that Cadenza gives higher rates of regeneration and transformation than other commercial cultivars which have been grown in the UK in recent years, averaging about 10 % but ranging up to 20 %. We therefore routinely use Cadenza as a “model” commercial bread wheat for transformation.

Secondly, the diploid cultivated species Triticum monococcum (einkorn)which is related to the ancestral donor of the A genome of polyploid wheats but is free threshing and has plump seeds and good agronomic performance. As a diploid it is more appropriate for mutagenesis, gene tagging and transformation to determine gene function than is hexaploid bread wheat. We have therefore screened a number of accessions of T monococcum from the collections held by the John Innes Centre (Norwich, UK) and the Vavilov Institute (St. Petersburg, Russia) and selected one line which exhibits good regeneration capacity for transformation. The first transformed plants in this line have recently been generated by particle bombardment.

We are currently using T. monococcum for two projects aimed at discovering new genes; mutagenesis using chemical and physical mutagens and gene tagging using a system based on the Ac/Ds transposable element system of maize.

4. APPLICATION OF TRANSFORMATION TO IMPROVING GRAIN PROCESSING QUALITY

Much of our work over the past 10 years has focused on understanding the molecular basis for wheat gluten visco-elasticity. This has included the transformation of bread and durum wheats to express additional genes for the quality-associated HMW subunits 1Ax 1 and 1Dx5, including analysis of their effects on dough mixing characteristics and breadmaking quality. These studies have shown that the two subunits have dramatically different effects on dough properties Whereas subunit 1Ax1 gave the expected increase in dough strength and gluten elasticity, the expression of subunit 1Dx5 often resulted in low water absorption and the failure of the flour to form a normal dough, giving a dense loaf of low volume. Fractionation of the gluten proteins demonstrated that this was associated with a high proportion of highly cross-linked insoluble glutenin polymers. This could have resulted from an additional cysteine residue present within the repetitive domain of subunit 1Dx5 (when compared with subunit 1Ax1 and all other characterised x-type subunits).

Current work with HMW subunit transgenes is focusing on determining their impact on quality parameters of “general purpose” cultivars grown in the UK and western Europe. This is being achieved by direct transformation of cultivars (notably cvs. Cadenza, Canon and Imp) and by introgression of transgenes unto cultivars by crossing with the transgenic lines reported by Barro et al.

CHAPTER 2

AGRONOMIC, BIOCHEMICAL AND QUALITY CHARACTERISTICS OF WHEATS CONTAINING HMW-GLUTENIN TRANSGENES

A.E. Blechl, P. Bregitzer, K. O’Brien, J. Lin, S.B. Nguyen and O.D. Anderson

1 INTRODUCTION

Bread dough strength is primarily dependent on its composition of high-molecular-weight glutenin subunits (HMW-GS), a class of storage proteins that typically comprises 5-10% of flour proteins. We have made a set of transgenic wheats that differ both quantitatively and qualitatively in their HMW-GS compositions. All the transgenics were derived from the cultivar Bobwhite, a hard white spring wheat that contains HMW-GS Ax2*, Bx7, Dx5, By9 and Dy10, and also the 1BL/1RS rye translocation. The introduced transgenes included native wheat genes for Dx5 and/or Dy10, or modified genes that encode a Dx5 variant with an extra-long repeat region or a Dy10:Dx5 hybrid subunit. The majority of 28 lines exhibit increases in HMW-GS levels due to additive expression of the transgenes and endogenous genes. However, five lines show transgene-mediated suppression of endogenous HMW-glutenin genes. In this paper, we present results of a single field trial for 32 different transgenic wheats and mixing characteristics for transgenic wheats that over-express native subunits Dx5 and/or Dy10.

2 METHODS AND RESULTS

2.1 Field Evaluations

Thirty-two different transgenic wheats and their non-transformed parent Bobwhite were planted in Aberdeen, Idaho, in the spring of 2001. The planting was a randomized complete block design with four replicates of each entry. All the transgenic wheats contained the transformation marker gene, bialaphos resistance encoded by the BAR gene under control of the maize Ubiquitinl promoter. Twenty-eight also contained transgenes that encoded HMW-glutenin subunits. Figure 1 shows the average yields of these 33 entries plotted as a frequency diagram. None of the four lines that contained only the marker gene had significantly different yields compared to the non- transformed parent. Most of the lines were phenotypically similar to Bobwhite, although several showed variability for height, vigor, and/or time of maturity (Figure 2). Eleven transgenics with added HMW-glutenin genes had reduced yields (significant at the 5% level in a Dunnett’s t test). Four of the latter lines were shorter than the control (significant at the 1% level in a Dunnett’s t test) and one of those was also delayed in maturing (Figure 2). Another low-yielding line had a lower test weight than non-transformed Bobwhite.

2.2 Mixing and Baking Tests

Flours from these field-grown wheats were milled in a Quadramat Senior mill and subjected to quality analyses. Figure 3 shows traces from 10-gram mixographs for the non-transformed control and five of the transgenic lines with increased levels of the natural Dx5 and/or Dy10 subunits. All such lines had improved mixing tolerance compared to the control, as indicated by decreased slope and/or increased bandwidth after peak resistance was achieved. Lines that contained more than a doubling of Dy10 (A) or Dx5 (B) or both (D) had mixing curves characterized by more rapid development and lower peak resistances, compared to those of the parental cultivar (C), and long stabilities (beyond the seven minutes over which the experiments were conducted). Lines with lesser amounts of transgene-encoded Dx5 and Dy10 increases (E and F) had curves characterized by longer dough development times than the control. Mixograms of flours with unbalanced amounts of Dy10 and Dx5 (A and B) had narrow bandwidths while those with subunit ratios near 1 (D, E and F) had broad bandwidths. This may indicate that more energy can be applied to doughs with more balanced x and y compositions before they break or disengage from the mixing pins. This property may reflect differences in the structure and/or size of the gluten macropolymer when x- and y-type subunits levels are balanced.

None of the transgenic wheats exhibited significant improvements in loaf volume compared to their parent. Of the lines depicted in Figure 3, baking results were obtained for flours C (control), D, E and F (extra Dx5 and Dy10). The control had a loaf volume of 1025 cc, as did line F, while the volume of a loaf made from the flour in E was slightly reduced (1000 cc). The loaf made from flour D was less than 500 cc. The decreases in loaf volumes for D and E could not be accounted for by their flour protein contents, which were greater than that of the control (12.2 and 11.4, respectively, vs 11%).

3 CONCLUSION

We have used genetic transformation to change mixing and baking characteristics of wheat flour. Our field data show that such changes can be achieved without necessarily changing the agronomic characteristics of the wheat plant. Increases in HMW-GS content increase dough mixing tolerances. This is desirable because a wide range of mixing times will produce doughs with optimum development. However, overly strong doughs may require more work input to achieve even hydration and mixing. They also typically produce smaller loaves. As more transgenic lines are characterized by biochemical and quality analyses, we will learn more about the relationship between dough functionality and HMW-glutenin subunit composition.

CHAPTER 3

CHARACTERIZATION OF GLUTENIN POLYMERS IN A TRANS GENIC BREAD WHEAT LINE OVER-EXPRESSING A LMW-GS

S. Masci, R. D’Ovidio’, F. Scossa, C. Patacchini, D. Lafiandra, O.D. Anderson, A.E. Blechl

1 INTRODUCTION

Technological properties of wheat flour depend mainly on the proteins that make up the polymeric network called gluten. High and low molecular weight glutenin subunits (HMW-GS and LMW-GS, respectively) are the most abundant components of gluten and both contribute to the formation of the glutenin polymers, whose size is directly correlated with flour rheological properties. HMW-GS play the most prominent role in bread wheat, whereas the LMW-GS are relatively more important for durum wheat properties. However, LMW-GS also have a role in bread-making quality.

Because LMW-GS are the most common polypeptides in the glutenin polymer and because their relative amount and/or allelic forms are known to influence dough visco-elasticity properties, we are investigating the effects of increasing the LMW-GS fraction. Here we present the results of analyses of flours from a transgenic bread wheat line that over-expresses a LMW-GS gene controlled by its own promoter. We compare these results to those obtained from transgenic wheats that over-express HMW-GS.

2 MATERIALS AND METHODS

Immature embryos of the bread wheat cultivar Bobwhite were used for transformation experiments. UBI:BAR and pLMWF23A plasmid DNAs, the latter containing a LMW-GS gene cloned from the Glu-D3 locus of the bread wheat cultivar Cheyenne, were used for wheat transformation. The alcohol-soluble and insoluble seed protein fractions were analysed by one and two dimensional SDS-PAGE under reducing and non-reducing conditions, by RP-HPLC, and by SE-HPLC. Southern blotting of genomic DNA were probed with a cloned LMW-GS gene. Protein content was determined by the Kjeldahl method (N X 5.7) and the SDS sedimentation test was performed according to Dick and Quick.

3 RESULTS

One out of the eleven bialaphos-resistant transformed plants showed detectable over-expression of the transgenic LMW-GS (Figure 1). Plants were grown from T1 seeds that showed over-expression. The integration of the transgene in these wheat plants was demonstrated by Southern blotting which showed the presence of the expected 3.7 kbp fragment derived from the plasmid used in the transformation experiment (data not shown). Plants over-expressing the transgenic LMW-GS were propagated by selfing for three further generations.

Seed proteins were fractionated by ethanol solubility. Comparing the soluble and insoluble fractions showed that the transgenic LMW-GS is incorporated into the glutenin polymers, and it is not present as a monomer (data not shown). Moreover, two-dimensional SDS-PAGE analysis showed that the transgenic LMW-GS is the main component of the oligomers present in the alcohol-soluble fraction (data not shown).

Densitometric analysis performed on the SDS-PAGE patterns of total endosperm protein extracted from T seeds indicated a twelve-fold increase of the transgenic product compared to native LMW-GS with similar molecular weights, whereas RP-HPLC of the reduced insoluble fraction indicated a sixteen-fold increase (Figure 2).

(Continues…)Excerpted from The Gluten Proteins by D. Lafiandra, S. Masci, R. D’Ovidio. Copyright © 2004 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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