WO2012143520A1

WO2012143520A1 - A method for decreasing the viscosity of flour

Info

Publication number: WO2012143520A1
Application number: PCT/EP2012/057289
Authority: WO
Inventors: Rowan MITCHELL; Jackie FREEMAN; Alison LOVEGROVE; Till PELLNY; Mark Wilkinson; Peter Shewry
Original assignee: Rothamsted Research Limited
Priority date: 2011-04-21
Filing date: 2012-04-20
Publication date: 2012-10-26
Also published as: GB2503598A; GB201106964D0; GB201317024D0; GB2503598B

Abstract

The present invention relates to a method for decreasing the viscosity of flour by decreasing soluble arabinoxylan content in a wheat grain, the method comprising reducing expression of Ta GT43_2, Ta GT47_2 or Ta GT61_1 in a wheat plant and wheat grains produced by said method.

Description

A METHOD FOR DECREASING THE VISCOSITY OF FLOUR

The present invention relates to a method for decreasing the viscosity of flour. There is a large market for wheat grain for fermentation (whisky, bioethanol) and for animal feed. However, current cultivars of wheat have viscous flour (because they are bred primarily for human food). Viscous flour is undesirable in fermentation as it results in additional cleaning costs and down time for machinery and undesirable for poultry feed as it results in sticky faeces, increasing cleaning costs.

It has now been surprisingly found that by reducing expression of TaGT43_2, TaGT47_2 or TaGT61_l genes in wheat, the flour produced from the wheat has a lower viscosity. The invention is particularly useful because wheat is hexaploid (bread wheat) or tetraploid (pasta wheat) and so a mutation in one homoeologue of one of these genes would only have a small effect on viscosity. The likelihood of one plant having functional mutations in two or more homoeologues is extremely low and therefore screening for low viscosity in a mutagenised population would not be effective.

According to a first aspect of the invention, there is provided a method of decreasing soluble arabinoxylan content in a wheat grain, the method comprising reducing expression of TaGT43_2, TaGT47_2 or TaGT61_l genes in a wheat plant. The TaGT43_2, TaGT47_2 or TaGT61_l genes all exist in two homoelogous forms in tetraploid wheat and in three homoelogous forms in hexaploid wheat. Reference to TaGT43_2, TaGT47_2 or TaGT61_l genes means both homoelogous forms of each gene in tetraploid wheat and all three homoelogous forms of each gene in hexaploid wheat.

Wheat grain produced according to a method of the invention has decreased soluble arabinoxylan content in comparison to wheat grain from control plants. Genetically modified plants according to a method of the invention may be prepared by any convenient procedure, examples of which are described below. The plants can be modified to express a nucleic acid sequence that targets TaGT43_2, TaGT47_2 or TaGT61_l, thereby decreasing expression of TaGT43_2, TaGT47_2 or TaGT61_l . A nucleic acid sequence that targets TaGT43_2, TaGT47_2 or TaGT61_l will be substantially complementary to the target gene.

The plants can also be treated in any other way to decrease the normal functioning of TaGT43_2, TaGT47_2 or TaGT61_l genes in the production of soluble arabinoxylan, for example, by mutagenesis, conveniently by chemical mutagenesis. Normal function can be decreased by reduced expression.

Reduced expression of a nucleic acid sequence includes expression below basal or endogenous levels, which can be defined with respect to levels of expression in an unaltered or control plant of the same species.

Normal function can also be decreased by a mutation in the coding sequence of the gene such that the protein sequence encoded by the gene differs from the normal sequence.

The nucleic acid sequence may be as shown in any one of Figures 1 to 3 or its complementary strand or a homologous sequence thereto. In the context of the present invention, the degree of identity between nucleic acid sequences may be at least 50%, suitably 60% or higher, e.g. 65%, 70%, 75%, 80%, 85%), 90%) or 95%). A homologous sequence according to the present invention may therefore have a sequence identity as described above. Sequence homology may be determined using any conveniently available protocol, for example using Clustal X™ from the University of Strasbourg and the tables of identities produced using Genedoc™ (Karl B. Nicholas). Also included within the scope of the present invention are nucleic acid sequences which hybridise to a sequence in accordance with the first aspect of the invention under stringent conditions, or a nucleic acid sequence which is homologous to or would hybridise under stringent conditions to such a sequence but for the degeneracy of the genetic code, or an oligonucleotide sequence specific for any such sequence.

Stringent conditions of hybridisation may be characterised by low salt concentrations or high temperature conditions. For example, highly stringent conditions can be defined as being hybridisation to DNA bound to a solid support in 0.5M NaHP0₄, 7% sodium dodecyl sulfate (SDS), ImM EDTA at 65°C, and washing in O. lxSSC/ 0.1%SDS at 68°C (Ausubel et al eds. "Current Protocols in Molecular Biology" 1, page 2.10.3, published by Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, (1989)). In some circumstances less stringent conditions may be required. As used in the present application, moderately stringent conditions can be defined as comprising washing in 0.2xSSC/0.1%SDS at 42°C (Ausubel et al (1989) supra). Hybridisation can also be made more stringent by the addition of increasing amounts of formamide to destabilise the hybrid nucleic acid duplex. Thus particular hybridisation conditions can readily be manipulated, and will generally be selected according to the desired results. In general, convenient hybridisation temperatures in the presence of 50% formamide are 42°C for a probe which is 95 to 100% homologous to the target DNA, 37°C for 90 to 95% homology, and 32°C for 70 to 90%) homology.

Mutagenesis of the plant may be conveniently achieved by any technique of chemical or radionucleide-induced mutagenesis Plants carrying mutations in the TaGT61_l, TaGT43_2 and TaGT47_2 genes may be identified from a population of mutagenised plants for example by TILLING (McCallum CM, et al, Plant Physiol. 123: 439-442; 2000). Other nucleic acid sequences in accordance with this aspect of the present invention may also comprise a nucleic acid sequence as previously defined in which the coding sequence is operatively linked to a promoter. The promoter may be constitutive and/or specific for expression in a particular plant cell or tissue, preferably in seeds.

Preferably, the nucleic acid sequence comprises a promoter which drives expression of a nucleic acid sequence described above. Such promoter sequences include promoters which occur naturally 5' to the coding sequence of the sequences shown in Figures 1 to 3. Promoters may also be selected to constitutively express the nucleic acid coding for the preferred gene sequences defined herein. Promoters that are induced by internal or external factors, such as chemicals, plant hormones, light or stress could also be used. Examples are the pathogenesis related genes inducible by salicylic acid, copper-controllable gene expression (Mett et al Proc. Nat'l. Acad. Sci. USA 90 4567-4571 (1993)) and tetracycline-regulated gene expression (Gatz et al Plant Journal 2 397-404 (1992)).

Suitable promoters for driving transgene expression in wheat grain include:

(i) High Molecular Weight Glutenin-l-Dl promoter from Wheat

(Figure 3; Lamacchia et al, 2001);

(ii) End-1 promoter from barley (Clarke BC, et al., Aust. J. Agric.

Res. 52: 1181-1193; 2001).

(iii) MAC1 promoter from maize (Sheridan et al., Genetics

142: 1009-1020, 1996);

(iv) Cat3 promoter from maize (GenBank No. L05934, Abler et al.,

Plant Mol. Biol. 22: 10131-1038, 1993);

(v) Atimycl from Arabidopsis (Urao et al., Plant J. Mol. Biol.

32:571-57, 1996; Conceicao et al, Plant 5:493-505, 1994); (vi) napA from Brassica napus (GenBank No. J02798);

(vii) Napin gene family from Brassica napus (Sjodahl et al., Planta

197:264-271,1995);

(viii) 2S storage protein promoter from Brassica napus (Dasgupta et al, Gene 133:301-302, 1993);

(ix) 2S seed storage protein gene family promoter from

Arabidopsis;

(x) Oleosin 20kD from Brassica napus (GenBank No. M63985); (xi) Oleosin A promoter (GenBank No. U09118) or Oleosin B promoter (GenBank No. U09119) from soybean;

(xii) Oleosin promoter from Arabidopsis (GenBank No. Z17657);

(xiii) Oleosin 18kD promoter from maize (GenBank No. J05212,

Lee, Plant Mol. Biol. 26: 1981-1987, 1994);

(xiv) Low molecular weight sulphur rich protein promoter from soybean (Choi et al, Mol. Gen. Genet. 246:266-268, 1995);

(xv) Promoters derived from zein-encoding genes (including the 15 kD, 16 kD, 19 kD, 22 kD, 27 kD, and gamma-zein genes, Pedersen et al, Cell 29: 1015-1026, 1982).

The nucleic acid sequences of the present invention may also code for RNA which is antisense to the RNA normally found in a plant cell or may code for RNA which is capable of cleavage of RNA normally found in a plant cell. In such an approach, the whole cDNA or smaller fragments (>200bp) may be amplified by PCR and inserted into an appropriate expression vector in reverse orientation to the primer. Accordingly, the present invention also provides a nucleic acid sequence encoding a ribozyme capable of specific cleavage of RNA encoded by TaGT43_2, TaGT47_2 or TaGT61_l . Such ribozyme-encoding DNA would generally be useful in inhibiting expression of TaGT43_2, TaGT47_2 or TaGT61_l . Alternatively, the RNA may encode a short interfering RNA sequence capable of activating the RNAi cellular process for degrading TaGT43_2, TaGT47_2 or TaGT61_l .

RNAi can involve intron-spliced hairpin (ihpRNA) constructs (Smith, N.A., et al. (2000) Nature, 407:319-320), using 300-600 bp of the transcribed region of the target inserted in sense and antisense orientation flanking the intron of an ihpRNA vector such as pHELLSGATE (Wesley, S.V., et al. (2001) Plant J., 27:581-590.). Design of hammerhead ribozymes against target sequences, for example, GA2ox, may follow guidelines, for example Fritz, J.J., et al. (Methods (2002), 28:276-285). The ribozyme would be produced from synthetic oligonucleotides, annealed and inserted into an appropriate vector. It is preferable to use tissue-specific promoters for expression of antisense/RNAi/ribozymes in transgenic plants to avoid pleiotropic effects in other tissues. The promoters listed in the application are suitable. The constructs or RNAi fragments are introduced into the target species by routine methods in the art as described herein. Preparation of transgenic plants according to the present invention which have grain with reduced soluble arabinoxylan content may therefore be prepared by modification of a plant cell to express nucleic acid molecules that inhibit the expression of TaGT43_2, TaGT47_2 or TaGT61_l . Such nucleic acid sequences as herein defined can be introduced into plant cells by any suitable means.

Preferably, nucleic acid sequences of the present invention are introduced into plant cells by transformation using an appropriate vector. Alternatively, a binary vector can be used. Such plasmids may be then introduced into Agrobacterium tumefaciens by electroporation and can then be transferred into the host cell via a vacuum filtration procedure. Alternatively, transformation may be achieved using a disarmed Ti- plasmid vector and carried by Agrobacterium by procedures known in the art, for example as described in EP-A-0116718 and EP-A-0270822. Where Agrobacterium is ineffective, the foreign DNA could be introduced directly into plant cells using an electrical discharge apparatus alone. Any other method that provides for the stable incorporation of the nucleic acid sequence within the nuclear DNA or mitochondrial DNA of any plant cell would also be suitable.

Preferably, nucleic acid sequences as described herein for introduction into host cells also contain a second chimeric gene (or "marker" gene) that enables a transformed plant containing the foreign DNA to be easily distinguished from other plants that do not contain the foreign DNA. Examples of such a marker gene include antibiotic resistance (Herrera-Estrella et al EMBO J. 2 987-995 (1983)), herbicide resistance (EP-A-0242246) and glucuronidase (GUS) expression (EP-A-0344029). Expression of the marker gene is preferably controlled by a second promoter which allows expression in cells at all stages of development so that the presence of the marker gene can be determined at all stages of regeneration of the plant. A whole plant can be regenerated from a single transformed plant cell, and the invention therefore provides transgenic plants (or parts of them, such as propagating material, i.e. protoplasts, cells, calli, tissues, organs, seeds (grains), embryos, ovules, zygotes, tubers, roots, etc.) including nucleic acid sequences as described above.

In the context of the present invention, it should be noted that the term "Genetically modified" should not be taken to be limited in referring to an organism as defined above containing in their germ line one or more genes from another species, although many such organisms will contain such a gene or genes, i.e. a "transgenic" plant. Rather, the term "genetically modified" refers more broadly to any organism whose germ line has been the subject of technical intervention, for example by recombinant DNA technology or chemical mutagenesis. So, for example, an organism in whose germ line an endogenous gene has been deleted, duplicated, activated or modified is a genetically modified organism for the purposes of this invention as much as an organism to whose germ line an exogenous DNA sequence has been added.

Screening of plant cells, tissue and plants for the presence of specific DNA sequences may be performed by Southern analysis as described in Sambrook et al {Molecular Cloning: A Laboratory Manual, Second edition (1989)). This screening may also be performed using the Polymerase Chain Reaction (PCR) by techniques well known in the art.

Transformation of plant cells includes separating transformed cells from those that have not been transformed. One convenient method for such separation or selection is to incorporate into the material to be inserted into the transformed cell a gene for a selection marker. As a result only those cells which have been successfully transformed will contain the marker gene. The translation product of the marker gene will then confer a phenotypic trait that will make selection possible. Usually, the phenotypic trait is the ability to survive in the presence of some chemical agent, such as an antibiotic, e.g. kanamycin, G418, paromomycin, etc, which is placed in a selection media. Some examples of genes that confer antibiotic resistance, include for example, those coding for neomycin phosphotransferase kanamycin resistance (Velten et al EMBO J. 3 2723-2730 (1984)), hygromycin resistance (van den Elzen et al Plant Mol. Biol. 5 299-392 (1985)), the kanamycin resistance (NPT II) gene derived from Tn5 (Bevan et al Nature 304 184-187 (1983); McBride et al Plant Mol. Biol. 14 (1990)) and chloramphenicol acetyltransferase. The PAT gene described in Thompson et al {EMBO J. 6 2519-2523 (1987)) may be used to confer herbicide resistance.

An example of a gene useful primarily as a screenable marker in tissue culture for identification of plant cells containing genetically engineered vectors is a gene that encodes an enzyme producing a chromogenic product. One example is the gene coding for production of β-glucuronidase (GUS). This enzyme is widely used and its preparation and use is described in Jefferson {Plant Mol. Biol. Reporter 5 387-405 (1987)).

Once the transformed plant cells have been cultured on the selection media, surviving cells are selected for further study and manipulation. Selection methods and materials are well known to those of skill in the art, allowing one to choose surviving cells with a high degree of predictability that the chosen cells will have been successfully transformed with exogenous DNA.

After transformation of the plant cell or plant using, for example, the Agrobacterium Ti-plasmid, those plant cells or plants transformed by the Ti-plasmid so that the enzyme is expressed, can be selected by an appropriate phenotypic marker. These phenotypic markers include, but are not limited to, antibiotic resistance. Other phenotypic markers are known in the art and may be used in this invention.

Positive clones are regenerated following procedures well-known in the art. Subsequently transformed plants are evaluated for the presence of the desired properties and/or the extent to which the desired properties are expressed. A first evaluation may include, for example, the level of bacterial/fungal resistance of the transformed plants, stable heritability of the desired properties, field trials and the like. The methods of present invention extend to methods for the preparation of transgenic plants and the sexual and/or asexual progeny thereof, which have been transformed with a recombinant DNA sequence as defined herein. The regeneration of the plant can proceed by any known convenient method from suitable propagating material either prepared as described above or derived from such material.

The expression "asexual or sexual progeny of transgenic plants" includes by definition according to the invention all mutants and variants obtainable by means of known process, such as for example cell fusion or mutant selection and which still exhibit the characteristic properties of the initial transformed plant, together with all crossing and fusion products of the transformed plant material.

The methods of the invention also concern the proliferation material of transgenic plants. The proliferation material of transgenic plants is defined relative to the invention as any plant material that may be propagated sexually in vivo or in vitro. Particularly preferred within the scope of the present invention are protoplasts, cells, calli, tissues, organs, seeds, embryos, egg cells, zygotes, together with any other propagating material obtained from transgenic plants.

Reduction of soluble arabinoxylan content as described herein encompasses the expression of a nucleic acid sequence which inhibits the expression of TaGT43_2, TaGT47_2 or TaGT61_l . Inhibition of expression includes expression below basal or endogenous levels. Preferably, reduction of soluble arabinoxylan content is caused by the expression of nucleic acid sequences as herein defined in the grain.

Reduction or abolition of gene expression as described above can be achieved using antisense or sense suppression, RNAi or the identification of mutants with reduced expression. Reduction or abolition of enzyme activity can be achieved through the identification of mutagen-induced or existing lines with altered properties, for example by TILLING (McCallum CM, et al, Plant Physiol. 123: 439-442; 2000) or any other method of screening for mutants. Suppressing expression of TaGT43_2, TaGT47_2 or TaGT61_l leads to reduced soluble arabinoxylan content of grains. Soluble arabinoxylan content decreases by at least 5%, suitably in the range of from 5% to 90%, preferably from 20% to 80%>, most preferably from 20% to 60%, can be achieved, compared to grain from control plants grown under normal conditions which have not been subject to genetic modification. A decrease of at least 5% is statistically significant and represents a measurable and real improvement to flour viscosity. The invention also include a method of decreasing flour viscosity comprising producing wheat grain according to the method described above and processing the grain to produce flour.

According to a further aspect of the invention, there is provided the use of a nucleic acid sequence targeting TaGT43_2, TaGT47_2 or TaGT61_l in the preparation of wheat grain with decreased soluble arabinoxylan content.

According to a further aspect of the invention, there is provided a wheat grain produced according to the method of the invention.

In a yet further aspect, a genetically modified wheat plant comprising grain with reduced soluble arabinoxylan content is provided. The grain with reduced soluble arabinoxylan content is also included. A further aspect relates to a method of selecting a wheat plant comprising grain with reduced soluble arabinoxylan content, comprising screening wheat plants for a mutated sequence of TaGT43_2, TaGT47_2 or TaGT61_l .

A mutated sequence means a sequence altered from the wild-type sequence. A mutated or altered sequence can lead to reduced expression of the gene. As is clear from the discussion above, reference to TaGT43_2, TaGT47_2 or TaGT61_l is to both homoelogous forms in tetraploid wheat and is to all three homoelogous forms in hexaploid wheat. So the method of selecting according to the invention means that wheat plants are screened for a mutated sequence of TaGT43_2, TaGT47_2 or TaGT61_l in all homoelogous forms.

By way of illustration and summary, the following scheme sets out a typical process by which genetically modified plant material, including whole plants, may be prepared according to a method of the present invention for decreasing soluble arabinoxylan content. The process can be regarded as involving five steps:

(1) Design a single antisense RNA, RNAi or ribozyme construct that targets TaGT43_2, TaGT47_2 or TaGT61_l;

(la) For antisense RNA, amplify the whole cDNA or smaller

fragments (>200bp) by PCR and insert in an appropriate expression vector in reverse orientation to the promoter; or

(lb) Design an intron-spliced hairpin (ihpRNA) construct; or

(lc) Design a hammerhead ribozyme and insert into an appropriate vector;

(2) transforming the construct of step (1) into plant material by means of known processes and expressing it therein;

(3) screening of the plant material treated according to step (3) for reduced expression of TaGT43_2, TaGT47_2 or TaGT61_l : and

(4) optionally regenerating the plant material transformed according to step (2) to a whole plant.

Another example of an approach for identifying a suitable DNA source is to identify loss-of -function or reduced- function variants of the target genes using TILLING or other sequence variant detection methods. TILLING can identify sequence variants in the target gene(s) in natural or induced populations of crop species (McCallum, CM., et al. (2000) Plant Physiol, 123:439-442; Comai, L., et al. (2004) Plant J., 37:778- 786; Slade, A. J., et al. (2005) Nature Biotechnology, 23:75-81). A simplified protocol could be:

(1) Design sequence specific primers to amplify conserved exon-rich

regions from genomic DNA using homoeologue-specific primers for wheat

(2) Carry out TILLING method, involving PCR, heteroduplex annealing, cell cleavage and product detection, to identify sequence variants and confirm this by DNA sequencing; and

(3) Back-cross to remove unwanted mutations and assess the effects.

Alternatively, as described above, the genetically modified plant may be produced through the action of chemically induced mutagenesis of a subject plant or plant tissue, followed by any other method screening to identify plants with mutations in the TaGT43_2, TaGT47_2 or TaGT61_l genes. For example, high throughput sequencing of DNA isolated from a population of plants may be searched for sequences corresponding to those in Figures 1-3. Sequences which show evidence of mutations which are likely to alter function of encoded enzyme may be selected and the plants which carry these mutations identified. Plants which carry a functional mutation in one homoeologue of the gene will be crossed with another plant which carries a mutation in another homoeologue of the same gene. Progeny carrying mutations in all three homoeologues may thus be identified and these may be expected to have the greatest effect on flour viscosity.

Preferred features for the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis. The invention will now be further described by way of reference to the following Examples and Figures which are provided for the purposes of illustration only and are not to be construed as being limiting on the invention. Reference is made to a number of Figures in which:

FIGURE 1 illustrates the nucleotide sequence of three different forms (putative homoelogues) of the TaGT47_2 gene. The sequences are cDNA sequences derived from mRNA isolated from endosperm tissue of hexaploid wheat, variety Cadenza. The sequences include the complete coding sequence for the encoded enzyme;

FIGURE 2 illustrates the nucleotide sequence of two different forms (putative homoelogues) of the TaGT43_2 gene. The sequences are cDNA sequences derived from mRNA isolated from endosperm tissue of hexaploid wheat, variety Cadenza. The sequences include the complete coding sequence for the encoded enzyme;

FIGURE 3 illustrates the nucleotide sequence of three different forms (putative homoelogues) of the TaGT61_l gene. The sequences are cDNA sequences derived from mRNA isolated from endosperm tissue of hexaploid wheat, variety Cadenza. The sequences include the complete coding sequence for the encoded enzyme;

FIGURE 4 illustrates High Performance Anion-Exchange Chromatography (HPAEC) peak area corresponding to total digestible arabinoxylan content from cell wall fractions of flour from null (N) and transgenic (T) wheat grain. Results are from flour from transgenic wheat lines carrying TaGT43_2 RNAi transgene (red bars) and TaGT47_2 RNAi transgene (yellow bars) with null segregant controls (grey bars). Bars are mean of four biological reps; error bars are 1 SE;

FIGURE 5 illustrates HPAEC peak area corresponding to amount of arabinoxylan monosubstituted by arabinose at 3 position from digested cell wall fractions of flour from null (N) and transgenic (T) wheat grain from six lines transformed with the TaGT61_l RNAi construct. Bars are mean of 4 biological reps with error bars of 1 SE; FIGURE 6 illustrates total arabinoxylan content of flour samples measured by monosaccharide analyses by gas chromatography. The samples were divided to estimate total (TOT) and water-unextractable (WU) arabinoxylan content; water- extractable was then calculated as TOT - WU. Flour samples were derived from the transgenic (T2, T3) and null segregant controls (N2, N3) of lines transformed with TaGT61_l RNAi construct. Bars are mean of four biological reps; error bars are 1 SE and

FIGURE 7 illustrates relative viscosity for water extractions of flour samples from control and transgenic wheat lines. Control lines are the null segregant controls which do not contain the transgene (grey columns) compared with the transgenic line (red columns) which contains the RNAi transgene indicated. Bars are mean of three (TaGT61_l RNAi) or two (TaGT43_2 RNAi and TaGT47_2 RNAi) biological reps of one line for each transgene; error bars are 1 SE.

Example 1

We conducted a transcriptome analysis of pure endosperm samples (the tissue which gives rise to flour) taken from developing grain of hexaploid wheat, Triticum aestivum L. (var. Cadenza) and identified the particular GT43, GT47 and GT61 wheat genes which are most highly expressed. (Note that here the term 'gene' is used to encompass all three homoeologous forms on the three genomes of hexaploid wheat, unless otherwise stated). These three wheat genes are designated TaGT43_2, TaGT47_2 and TaGT61_l .

We suppressed expression of TaGT43_2 and TaGT47_2 genes by transformation with an RNAi construct designed specifically to the target gene. Flour from the resulting transgenic plants was then analysed for the content and structure of arabinoxylan and compared with non-transgenic controls. The procedures used to suppress expression of TaGT43_3 and TaGT47_2 genes followed protocols for making the RNAi constructs, plant transformation, identification of genotypes, isolation of flour and analysis of flour for cell wall polysaccharide composition by enzyme digestion and HPAEC as set out in Nemeth et al. (2010), which describes similar experiments in our lab, but on a different gene (the TaCSLF6 gene). Data in Figure 1 show that a decrease in arabinoxylan content was achieved in the transgenic wheat lines with TaGT43_2 RNAi transgene and in some of those with the TaGT47_2 transgene. Figure 4 shows a 70-80% decrease in digestible arabinoxylan content in some lines (TaGT43_2 RNAi lines 1, 3, 5 and 6; TaT47_2 RNAi lines 1 and 4) and all these effects are highly statistically significant (P < 0.001). (The TaGT47_2 RNAi lines 2 and 3, which show no effect, probably do not express the transgene). We also suppressed expression of TaGT61_l . The procedures used to suppress expression of the TaGT61_l gene followed protocols for making the RNAi construct, plant transformation, identification of genotypes, isolation of flour and analysis of flour for cell wall polysaccharide composition by enzyme digestion and HPAEC as set out in Nemeth et al. (2010),. This changed the molecular structure of arabinoxylan, such that the amount of arabinosyl substitution on the xylan backbone was decreased. Using transgenic wheat plants with RNAi designed specifically against TaGT61_l, we have shown that mono-substitution of 3 -linked arabinose of xylan is decreased by 53%, 76%, 75%, 57%, 82% and 61% in the six lines, respectively (P<0.001 for line 1, P<0.0001 for all other lines) in the transgenic plants compared with null segregant controls which lack the RNAi transgene (Figure 5). There was no significant difference between transgenics and controls on grain weight, nor any visible phenotype in the transgenics. The change in arabinoxylan structure makes the arabinoxylan less water-extractable, which decreases flour viscosity. Figure 6 shows that the amount of water-extractable arabinoxylan is decreased in the two TaGT61_l RNAi transgenic lines that have been tested. It is the amount of water- extractable arabinoxylan that is important in determining viscosity; it can be seen that whereas water-unextractable arabinoxylan content is unaffected by the TaGT61_l RNAi transgene, the water-extractable arabinoxylan is specifically decreased by 68% and 52% (P<0.05) in the two lines (Figure 6). Viscosity has been determined for water extractions of flour samples from some of the transgenic and control lines following the method of Saulnier et al. (1995). The results of this are shown in Figure 7; the viscosity is expressed relative to pure water ("relative viscosity") therefore the minimum possible value in the absence of any flour is 1.0. Therefore the additional viscosity contributed by the flour can be calculated as relative viscosity - 1. The additional viscosity contributed by flour from all the transgenic lines tested was markedly decreased relative to their null segregant controls; the change was -80%, -77% and -49% for flour samples from lines carrying the TaGT43_2 R Ai, TaGT47_2 RNAi and TaGT61_l R Ai transgenes, respectively.

References:

Barcelo P, & Lazzeri P. (1995). In Methods in Molecular Biology: Plant Gene Transfer and Expression Protocols, p. 113-123. Eds H. Jones. Humana Press: Totowa NJ.

Christensen A H, & Quail P H. (1996). Transgenic Research. 5: 213-218. Lamacchia et al, (2001) J. Exp. Bot. 52: 243-250.

Nemeth, C, Freeman, J., Jones, H.D., Sparks, C, Pellny, T.K., Wilkinson, M.D., Dunwell, J., Andersson, A.A.M., Aman, P., Guillon, F., Saulnier, L., Mitchell, R.A.C. and Shewry, P.R. (2010). Plant Physiol, 152, 1209-1218.

Pastori et al., (2001) Journal of Experimental Botany : 52: 857-863. Phillips et al, (1995) Plant Physiol. 108: 1049-1057. Rasco-Gaunt et al., (2001) Journal of Experimental Botany. 52: 865-874.

Sparks C A, & Jones H D. (2004). Transformation of wheat by biolistics, In Transgenic Crops of the World - Essential Protocols. Ed I.S. Curtis. Kluwer: Dordrecht: Netherlands.

Saulnier L, Peneau N, Thibault JF (1995). Journal of Cereal Science. 22: 259 264.

Claims

1. A method of decreasing soluble arabinoxylan content in a wheat grain, the method comprising reducing expression of TaGT43_2, TaGT47_2 or TaGT61_l in a wheat plant.

2. The method of claim 1, comprising reducing expression of TaGT43_2, TaGT47_2 or TaGT61_l by expression of a small interfering nucleic acid sequence.

3. A method as claimed in claim 1 or claim 2, in which the decrease in soluble arabinoxylan content is by at least 5%

4. A method as claimed in claim 3, in which the decrease in soluble arabinoxylan content is in the range of from 5% to 90%

5. A method of decreasing flour viscosity comprising producing wheat grain according to the method of any one of claims 1-4 and processing the grain to produce flour.

6. The use of a nucleic acid sequence targeting TaGT43_2, TaGT47_2 or TaGT61_l in the preparation of wheat grain with decreased soluble arabinoxylan content.

7. A wheat grain produced according to the method of any one of claims 1-4.

8. A genetically modified wheat plant comprising grain with reduced soluble arabinoxylan content.

9. A genetically modified wheat grain with reduced soluble arabinoxylan content.

10. A method of selecting a wheat plant comprising grain with reduced soluble arabinoxylan content, comprising screening wheat plants for a mutated sequence of TaGT43_2, TaGT47_2 or TaGT61_l .