The present invention relates generally to polysaccharide synthases. More particularly, the present invention relates to (1,3;1,4)-β-D-glucan synthases. The present invention provides, among other things, methods for influencing the level of (1,3;1,4)-β-D-glucan produced by a cell and nucleic acid and amino acid sequences which encode (1,3;1,4)-β-D-glucan synthases.

Barley has Only One CSLH Gene

Candidate CSLH genes in barley were initially identified by querying online EST databases, such as the discontinued Stanford cell wall website, NCBI, HarvEST, GrainGenes, Barley Gene Index and BarleyBase, with rice CSLH sequences. All CSLH-related ESTs from barley could be aligned into a single contiguous sequence of ˜1,500 bp that included the entire 3′ untranslated region (UTR) and a region encoding the COOH-terminal 488 (of an expected ˜750) amino acid residues of the protein (Table 2). This gene was designated HvCslH1. Screening of a barley BAC library with HvCslH1-derived probes identified several genomic clones all containing HvCslH1, from which the missing 5′ end was obtained (data not shown). A 2,430 bp HvCslH1 cDNA fragment was PCR-amplified, contains a single 2,256 bp ORF, and encodes a protein with a predicted MW of 82.6 kDa and a pI of 7.0 (FIG. 6A). Analysis of the conceptual translation of this sequence with ARAMEMNON found between five and nine transmembrane domains (TMDs), with the consensus among the different programs being two NH2-terminal and four COOH-terminal TMDs (FIG. 6B) and both termini of the mature protein predicted to be cytoplasmic. This topology also places the large, central domain containing the D,D,D,QFKRW motif within the cytoplasm (FIG. 6C). At the nucleotide level, HvCslH1 shares 68-74% identity (62-69% amino acid identity) (see Example 6) with the three rice CSLH genes (Hazen et al.,Plant Physiol128: 336-340, 2002). A phylogenetic reconstruction shows HvCslH1 to be the likely barley ortholog of OsCSLH1 (FIG. 7). Genetic mapping of HvCslH1 using a Sloop×Halcyon doubled haploid population (Read et al.,Aust J Agr Res54: 1145-1153, 2003) showed that HvCslH1 is on the short arm of chromosome 2H, approximately 1.5 cM from a cluster of four HvCSLF genes (HvCSLF3, 4, 8, 10) that Burton et al. (Plant Physiol146: 1821-1833, 2008) reported was within a major QTL controlling β-glucan content in ungerminated barley grain (Han et al.,Theor Appl Genet91: 921-927, 1995; FIG. 8).

Supporting Information

A BAC library screening was employed to obtain a complete set of full-length HvCslH family members. BAC filters containing 6.5 equivalents of the barley genome (cv. Morex) were screened and three clearly positive clones identified (data not shown). When a blot of BAC DNA from these clones digested with Hind III was probed, the same three clones, 3-5-10, 3-7-3 and 3-7-8, were verified as being positive. The digestion pattern of BACs 3-5-10 and 3-7-8 appeared identical and many bands were common to BAC 3-7-3, indicating that all 3 BACs cover identical or very similar regions of the barley genome. When a genomic DNA blot was hybridised with the same probe, single bands were observed in lanes digested with Hind III, Eco RI or Eco RV, corroborating the BAC digestion results. As all HvCslH ESTs are also derived from a single gene (Table 2), these data strongly suggest that there is only one CSLH gene in the barley genome.

An adaptor primer PCR method (Siebert et al.,Nucl Acids Res23: 1087-1088, 1995) was used to identify the 5′ end of HvCslH1. DNA was isolated from BACs 3-5-10 and 3-7-3, digested with a range of restriction enzymes producing blunt-ended DNA fragments to which adaptors were ligated. Nested PCR was then performed with adaptor- and HvCslH1-specific primers (Table 3) in order to amplify fragments containing the 5′ end of the gene. Amplification of BAC 3-7-3 DNA digested with Nru I using primers AP2 and H1R6 successfully amplified a 1.3 kbp fragment that contained all but ˜20 amino acids of the N-terminal sequence. Direct sequencing of BAC 3-7-3 DNA with the H1R10 primer, an antisense primer designed to anneal near the 5′ end of the 1.3 kb fragment, enabled the remainder of the open reading frame plus 748 bp of upstream sequence to be identified. As predicted from earlier results, the sequence obtained from BAC 3-5-10 was identical to BAC 3-7-3, confirming that there is only one CSLH gene within the barley genome.

TABLE 2List of ESTs derived from HvCslH1.ESTs are listed in order of alignment 5′ to 3′.Accession no.(5′ to 3′)CultivarSource tissueCA013594Barkeearly endosperm, 0-16 hours afterimbibitionBJ470984Haruna Nijo adulttop three leaves at heading stageBJ471865Haruna Nijo adulttop three leaves at heading stageBJ473288Haruna Nijo adulttop three leaves at heading stageBJ452043Akashinrikivegetative stage leavesBJ471909Haruna Nijo adulttop three leaves at heading stageAV932844Haruna Nijo adulttop three leaves at heading stageBJ469514Haruna Nijo adulttop three leaves at heading stageAV933503Haruna Nijo adulttop three leaves at heading stageAV933012Haruna Nijo adulttop three leaves at heading stageAV932649Haruna Nijo adulttop three leaves at heading stageAV932549Haruna Nijo adulttop three leaves at heading stageBJ475824Haruna Nijo adulttop three leaves at heading stageBJ476822Haruna Nijo adulttop three leaves at heading stageAV934650Haruna Nijo adulttop three leaves at heading stageBJ477472Haruna Nijo adulttop three leaves at heading stageAV935479Haruna Nijo adulttop three leaves at heading stageAV935951Haruna Nijo adulttop three leaves at heading stageAV832539Akashinrikivegetative stage leavesAV936586Haruna Nijo adulttop three leaves at heading stageCB881459Barkemale inflorescences (approx. 2 mm insize), green anther stageAV934667Haruna Nijo adulttop three leaves at heading stageBJ475744Haruna Nijo adulttop three leaves at heading stageBJ459600Akashinrikivegetative stage leavesAV832391Akashinrikivegetative stage leaves

Expression of HvCslH1 inArabidopsisResults in Deposition of (1,3;1,4)-β-D-Glucan

For heterologous expression inArabidopsis, the HvCslH1 ORF was cloned into the Gateway-enabled binary vector pGWB15 (Nakagawa et al.,J Biosci Bioeng104: 34-41, 2007; FIG. 15), which placed HvCslH1 under the control of the CaMV 35S promoter and added a 3×HA epitope tag to the encoded protein's NH2-terminal end (FIG. 1A). Initial selection of transformedArabidopsisseeds identified a number of putative transgenic seedlings which PCR confirmed contained HvCslH1. RNA blot analysis of these T1plants showed that approximately 90% accumulated HvCslH1 transcripts in rosette leaves (FIG. 1B). Western blotting using an anti-HA tag antibody was used to detect HvCslH1 protein in these lines (FIG. 1C). A mixed microsomal membrane fraction (50,000-100,000×g pellet) was prepared from pooled three-week old kanamycin-resistant T2 seedlings. Western blotting with the anti-HA antibody showed that only four of the 28 lines containing HvCslH1 transcripts accumulated a polypeptide of the expected size (˜90 kDa) (FIG. 1C). Occasionally proteins of higher and lower molecular mass were also detected (e.g. lane 11). The 90 kDa-protein was not observed in total protein extracts (data not shown) or in mixed-membrane fractions prepared from untransformedArabidopsisplants (FIG. 1C, Col-0 lane). It is not known why HA-tagged HvCslH1 accumulated in only some of the plant lines that expressed HvCslH1 mRNA or why no correlation was apparent between HvCslH1 protein levels and either HvCslH1 transcript levels (compareFIGS. 1Band C) or with the number of HvCslH1 transgenes present in a plant (data not shown), although this has been previously observed (Burton et al.,Science311: 1940-1742, 2006) Lines 8, 11, 16 and 24, which expressed the HA-tagged HvCslH1, and line 6, which did not express detectable levels of the protein (control), were selected for subsequent experimental work.

Immuno-EM was used to determine whether the walls of the transgenicArabidopsisplants accumulated detectable levels of β-glucan. Thin sections of mature leaf pieces from self-pollinated progeny of lines 8, 11, 16, 24 and 6 (T2 generation) were probed with a monoclonal antibody specific for β-glucan (Meikle et al.,Plant J5: 1-9, 1994), followed by detection using a secondary antibody conjugated to 18 nm gold particles. Gold particles were clearly evident in walls of the HA-tagged HvCslH1 positive lines 8, 11 and 16 (FIG. 2A, C, B, respectively) but not in the walls of either line 24, which also expressed HvCslH1 (data not shown), or line 6 (FIG. 2E) which had no detectable HvCslH1 protein. Each positive transgenic line showed a different pattern of tissue labeling. In line 8, patchy labeling was observed in the walls of epidermal cells and occasionally in xylem walls (FIG. 2A) whereas in line 11, epidermal and vascular tissue walls were only lightly labelled, but heavier (albeit more patchy) labeling was observed in mesophyll walls (FIG. 2C). Broadly distributed, light labeling was present in all walls of the mature leaf of line 16 (FIG. 2B). Irregular and inconsistent patterns of ectopic polysaccharide production by transgenicArabidopsislines expressing genes driven by the “constitutively”-expressed 35S promoter have been observed previously (Burton et al., 2006, supra). No labeling was seen in leaf sections of untransformedArabidopsis(FIG. 2D). Reduced levels of labeling were seen in leaf sections of transgenic plants that had been pre-incubated with aBacillus subtilisendo-hydrolase which specifically hydrolyses this β-glucan (Burton et al., 2006, supra; data not shown).

To provide biochemical confirmation of the presence of β-glucan in transgenicArabidopsiswalls and to examine the fine structure of the nascent β-glucan, leaf and/or stem material was pooled from the self-pollinated T3 and T4 progeny of lines derived from plants 8, 11 and 16. These lines were homozygous for the HvCslH1 transgene. Because β-glucan was found to accumulate with plant age, samples were taken when plants were in senescence. Walls were prepared and digested with a (1,3:1,4)-β-glucan-specific endo-hydrolase and the released oligosaccharides profiled by HPAEC and MALDI-TOF MS. (1,3;1,4)-β-D-Glucan endo-hydrolase specifically hydrolyses (1,4)-β-glucosidic linkages when these linkages are on the reducing-end side of a (1,3)-β-D-glucosyl residue. The action of this enzyme yields a series of oligosaccharides with different degrees of polymerization (DP). The diagnostic oligosaccharides in this series are the trisaccharide G4G3GRand the tetrasaccharide G4G4G3GR(where G is β-D-glucopyranose, 3 and 4 indicate (1,3) and (1,4) linkages, respectively, and GRrefers to the reducing terminal residue). Variable quantities of G4G3GRand G4G4G3GRwere released when walls prepared from leaf or leaf and stem from lines 8 and 11 and two independent lines derived from plant 16 (lines 16-1 and 16-2) were treated with (1,3;1,4)-β-D-glucan endo-hydrolase (FIGS. 3,9A). These oligosaccharides were not detected in the no-enzyme treatment control. The ratio of G4G3GRto G4G4G3GR(DP3:DP4) was estimated to be 3.5 in line 16-1, which is similar to the DP3:DP4 ratio of 3.6 obtained for β-glucan from the barley leaf sample. A peak that co-eluted with laminaribiose, a (1,3)-β-linked disaccharide of glucose (G3GR), was also observed in lines 8, 11 and 16-2 at varying levels across samples (FIG. 9A, data not shown). This product was absent from the barley and no-enzyme treatment control samples (FIG. 9A), verifying its appearance is not due to a contaminating enzyme in the (1,3;1,4)-β-D-glucan endo-hydrolase preparation or to endogenous disaccharide or enzyme activity withinArabidopsis. The identities of oligosaccharides in this profile were further confirmed by MALDI-TOF MS analysis, which showed the presence of Hex2, Hex3and Hex4in ratios similar to those observed in the HPAEC profile (FIG. 9B). The amounts of β-glucan in lines 16-1 and 16-2, as estimated from the areas of the G4G3GRpeaks, were 0.005% and 0.003% (w/w) of total wall, respectively.

HvCslH1 is Located in ER- and Golgi-Associated Vesicles but not the Plasma Membrane of TransgenicArabidopsisPlants Expressing HvCslH1

Sections of high pressure-frozen leaves from line 11 were incubated with the gold-labelled anti-HA antibody to determine the sub-cellular location of HvCslH1. Labelling was seen in the endoplasmic reticulum and in Golgi-derived vesicles but not in the plasma membrane (FIG. 4A, B). Similar results were observed in labelled sections of roots and seedlings (data not shown).

HvCslH1 is Transcribed in Barley at Low Levels in Developing Grain, Floral Tissues and Cells of the Leaf Undergoing Secondary Cell Wall Thickening

The levels of HvCslH1 transcripts in various barley tissues were determined using quantitative RT-PCR (QPCR). The gene-specific primers are presented in Table 4.

FIG. 5(A-C) shows that across a set of barley vegetative and floral tissue cDNAs, HvCslH1 transcripts were accumulated to levels that were routinely less than 1,000s copies/μl cDNA. This value is lower than some of the other barley CESAs and CSLs we have studied where values are typically in the range of 10,000s and 100,000s copies/μl cDNA. Levels of HvCslH1 transcripts were relatively low in tissues comprising rapidly elongating cells, including coleoptile and leaf base, which are those that are actively synthesising β-glucan.

The highest levels of HvCslH1 transcripts were in leaf tip, where cells are no longer actively growing and less β-glucan accumulates (FIG. 5C;2,4). HvCslH1 transcription in leaf was characterised further using RNAs isolated from six zones within the ˜13 cm-long leaves of 10 day-old seedlings, starting from the leaf tip. These zones comprises fully mature cells (zone A), to the leaf base comprising dividing cells (zone F). In situ PCR (see Example 5) was used to identify those cells in the leaf tip that contained the HvCslH1 mRNA. In this technique, cells in which gene transcripts are detected stain purple to dark brown (FIG. 5D, 18S RNA positive control). Cells where no transcription is detected stain light brown, as in the negative control (FIG. 5E). HvCslH1 was mostly transcribed in cells that are undergoing secondary wall thickening, such as interfascicular sclerenchymal fibre and xylem cells (FIG. 5F). Immuno-EM using sections taken from barley leaf and probed with the β-glucan antibody identified β-glucan in the walls of these cells.

HvCslH1 transcript levels were also investigated in more detail in a 24-day developing endosperm series (FIG. 5B). HvCslH1 expression was low throughout the starchy endosperm during development. Maximum transcript levels were reached at 4 DPA, approximately 1 day before β-glucan is first detected in endosperm walls. This transcription pattern is similar to that of several barley CSLF genes (HvCSLF3, 4, 7, 8 and 10) that are also expressed in developing grain, although HvCSLF9 and 6 show much higher transcript levels.

Discussion

The data presented here indicate that the product of HvCslH1, a member of the grass-specific CSLH gene family, mediates β-glucan biosynthesis inArabidopsis. Barley appears to have only a single CSLH gene based on EST database analyses, genomic DNA blot analysis and BAC library screening. EST analyses of other grasses such as bread wheat,Lolium multiflorum, Festuca arundinacaeandBrachypodium distachon(all subfamily Pooideae) have one identified CSLH gene, similar to barley, whereas maize, sorghum and sugar cane (all subfamily Panicoideae), like rice (subfamily Ehrhartoideae), appear to have multiple CSLH genes. When an epitope-tagged version of the HvCslH1 cDNA was heterologously expressed inArabidopsis, three of four plant lines in which protein was detected accumulated a polysaccharide in their walls that was recognized by a β-glucan-specific monoclonal antibody. When isolated walls of the transgenic lines were digested with a specific (1,3;1,4)-β-D-glucan endo-hydrolase, the characteristic trisaccharide (G4G3GR) and tetrasaccharide (G4G4G3GR) were detected at ratios similar to those found in β-glucans from barley endosperm, demonstrating that the walls from the transgenicArabidopsislines contained β-glucan. Furthermore, epitope-tagged HvCslH1 was found in the endoplasmic reticulum and in Golgi-derived vesicles in cells of transgenic plants. The morphological phenotype of the transgenicArabidopsislines that expressed HvCslH1 appeared identical to wild-type plants.

Although the overall proportion of (1,3)- and (1,4)-β-glucosyl linkages and the ratios of the G4G3GRand G4G4G3GRproducts from (1,3;1,4)-β-D-glucan endo-hydrolase digestion of walls derived from plant line 16-1 was similar to those observed in β-glucans isolated from barley tissues, one unusual feature that was observed was that the major oligosaccharide released by (1,3;1,4)-β-D-glucan endo-hydrolase from the walls of line 16-2 was laminaribiose (G3GR;FIG. 9A). The presence of G3GRin variable levels was also associated with increased levels of trisaccharide relative to the tetrasaccharide and, thus increased DP3:DP4 ratios. The presence of G3GRin wall digests of the majority of plant lines indicates a polysaccharide containing sections of alternating (1,3)-β- and (1,4)-β-linked glucosyl residues (-G3G4G3G4-). It is not known if these reside in a separate polysaccharide or constitute a portion of a β-glucan chain that also has the usual fine structural features. Alternating (1,3)-β-D-glucosyl and (1,4 β-D-glucosyl residues are not common in barley and other cereal β-glucans, but do represent a significant component of the β-glucan from the non-flowering plantEquisetumand are also detected in β-glucans from a number of fungi, including basidiomycetes and ascomycetes. It is possible that G3GRarises through misregulation of the β-glucan synthase in transgenicArabidopsis, possibly because its membrane micro-environment is different or because an unknown factor that in barley suppresses (1,3)-β glucosidic linkage formation (or alternatively promotes (1,4)-β glucosidic linkage formation) is present at suboptimal levels inArabidopsis. Minor variations in the level of this factor among the lines derived from plant 16 would account for the different structures that were obtained. Another possible explanation for the structural variability in the β-glucan may relate to subtle differences in post-assembly processing (see also Supporting Information below).

In barley, HvCslH1 was most highly transcribed in leaf tips, a tissue comprising fully mature cells. There is no evidence to indicate coordinate transcription of HvCslH1 and any of the barley CSLFs, suggesting that their encoded products are not components of a protein complex. HvCslH1 transcription, for example, was not high in elongating cells such as the coleoptile or developing endosperm, which in barley are the tissues where β-glucan is characteristically accumulated. Although usually found in primary cell walls of vegetative tissues where it is implicated in the control of cell expansion and possibly as a temporary store of glucose that can be mobilized as an energy source in the dark, β-glucan has also been found in the lignified cell walls of xylem tracheary elements and sclerenchyma fibres, where immuno-EM using the antibody to β-glucan shows labeling in both the middle lamella region (primary wall) and secondary wall of sclerenchyma cells. Because in situ PCR showing transcription of the HvCslH1 gene in the leaf was restricted to cells such as interfascicular sclerenchymal fibre and xylem cells, we suggest that a major role of this gene is in β-glucan synthesis during secondary wall development, although we cannot exclude a role in primary wall β-glucan synthesis elsewhere in the plant.

Regardless of how the fine structures of β-glucans are generated, it is clear that the CSLHs can mediate the synthesis of β-glucan inArabidopsis, a finding that has implications for our understanding of how this polysaccharide is synthesised. Any mechanism(s) being considered for the assembly of β-glucan must account for the synthesis of the predominant cellotriosyl and cellotetraosyl units, the random linking of these (1,4)-β-units together by single (1,3)-β-linkages and the means by which the molar ratio of tri- to tetra-saccharide units is regulated. At least two glycosyltransferase activities might act in concert: one that processively adds (1,4)-β-linked glucose residues to assemble the tri- and tetra-saccharides and the other that adds single (1,3)-β-linkages. Based on our current knowledge of polysaccharide synthases several mechanisms are hypothetically possible. The simplest explanation is that the one polypeptide is responsible for the synthesis of both glucosidic linkage types. Our transgenic experiments indicate that CSLH proteins are independently able to make a β-glucan and could therefore conceivably make both types of β-linkages. The CSLH family is classified by the Carbohydrate Active Enzymes (CAZy) database as members of glycosyltransferase family 2 (GT2) (http://www.cazy.org; Coutinho et al.,J Mol Biol328: 307-317, 2003), a family that includes enzymes capable of independently catalyzing the synthesis of either (1,3)-β- or (1,4)-β-linkages but also examples of bifunctional enzymes, i.e. enzymes that can synthesize two types of glycosidic linkages. For example, hyaluronan synthases (HAS) synthesize a repeating disaccharide of (1,4)-β-glucuronic acid-(1,3)-β-N-acetylglucosamine units and both transferase activities reside in the one polypeptide. In mouse HAS1, the region that includes the D,D,D,QXXRW motif is responsible for both these activities. The active site of the CSLHs, also containing the D,D,D,QXXRW motif, might be similarly bifunctional. Another possibility is that the CSLHs synthesise only one type of glucosidic linkage with another glucosyltransferase, common to monocots and dicots, responsible for synthesis of a second linkage.

Materials and Methods

Binary Vector Construction and Plant Transformation

The HvCslH1 ORF was amplified from barley cv. Schooner mature leaf tip cDNA with Herculase® (Stratagene) using primers HvH1TOPOf and HvH1TOPOr (Table 3) and the PCR product cloned into pENTR/D-TOPO (Invitrogen). Using the manufacturer's protocol (Invitrogen), an LR reaction was used to clone the cDNA into the destination vector pGWB15 containing an NH2-terminal 3×HA tag (Nakagawa et al.,J Biosci Bioeng104: 34-41, 2007) and the predicted sequence confirmed by DNA sequencing. The HvCslH1::pGBW15 construct was transferred fromEscherichia coliintoAgrobacterium tumefaciensstrain AGL1 via triparental mating using the helper plasmid pRK2013.Arabidopsis thalianaCol-0 plants were transformed using the floral dip method (Clough and Bent,Plant J16: 735-743, 1998).

RNA Blot Analysis

Samples of ˜10 μg total RNA extracted from mature rosette leaves of T1 plants using TRIzol® (Invitrogen) were prepared and separated on a 1% w/v agarose-formaldehyde gel (Farrell,RNA methodologies: A laboratory guide for isolation and characterization, Academic Press, Inc., San Diego,1993). RNA was transferred to Hybond™ N+membranes, pre-hybridised and hybridised according to the method outlined in the Gene Images CDP-Star detection module (Amersham-Biosciences). A HvCslH1 fragment amplified with primers H1F2 and HvH1TOPOr (Table 3) was labeled using the Gene Images Random Prime labeling module (Amersham) following the manufacturer's protocol and used as the probe.

Quantitative PCR Analysis

RNA extractions, cDNA syntheses and QPCR were carried out as described in Burton et al. (Science311, 1940-1942, 2006;Plant Physiol134, 224-236, 2004) with the modifications listed in Burton et al. (Plant Physiol146, 1821-1833, 2008). The primer sequences of the barley control genes are listed in Table 4.

In Situ PCR

In situ PCRs were conducted according to the method of Koltai & Bird (Plant Physiol123: 1203-1212, 2000) with the following modifications. After tissue sectioning, genomic DNA was removed by treatment for 6 h at 37° C. in 1× DNase buffer and 4 U RNase-free DNase (Promega). cDNA synthesis was carried out using Thermoscript™ RT (Invitrogen) except that the RNase H step was omitted and a gene-specific primer (1 μg, Table 3) used for reverse transcription. PCRs were carried out in a final volume of 50 μL containing 1×PCR buffer, 200 μm dNTPs (Promega), 0.2 nmol digoxigenin-11-dUTP (Roche), 2 mM MgCl2, 200 ng of each primer and 2 U Taq DNA polymerase (Invitrogen). Cycling parameters were as follows: initial denaturation at 96° C. for 2 min, then 40 cycles of 94° C. for 30 sec, 59° C. for 30 sec, 72° C. for 1 min. Sections were then washed, incubated with 1.5 U alkaline phosphatase-conjugated anti-digoxigenin Fab fragments (Roche) and developed for 10-20 min as outlined by Koltai & Bird (2000, supra). For negative control sections, reverse transcriptase was omitted and all the Hv 18S rRNA primers included to check whether there was any amplification from genomic DNA.

Preparation of Mixed Microsomal Membranes

T1 seed of HvCslH1 transgenic plants was collected and ˜100 seeds sown onto 1×MS agar media containing 50 mg/L kanamycin (Sigma). After 3 weeks, kanamycin-resistant seedlings were pooled, frozen in liquid N2and ground at 4° C. in a mortar and pestle containing homogenising buffer (50 mM NaPO4buffer, pH 7.5, 0.5 M sucrose, 20 mM KCl, 10 mM DTT, 0.2 mM PMSF, 83 μL plant protease inhibitor cocktail (Sigma, P9599)). Homogenate was filtered through a 50 μM mesh and the S/N centrifuged at 6,000×g for 10 min at 4° C. The S/N was decanted and centrifuged at 50,000×g for 30 min at 4° C. in 4.5 ml ultracentrifuge tubes (Beckmann). The 50,000×g S/N was decanted and the pellet resuspended in 10 mM Tris-MES buffer, pH 7.5 using a glass-teflon homogenizer. The resuspended pellet was diluted to 4.5 mL with Tri-MES buffer and centrifuged at 100,000×g for 1 h at 4° C. The pellet was resuspended in 0.25 M sucrose, 10 mM Tris-MES buffer, pH 7.5, as outlined above. Protein concentration was measured using Bradford assay reagent (BioRad) using bovine serum albumin as the standard.

Western Blotting

Samples of membrane protein (30 μg) were incubated at 60° C. for 20-60 min in 200 mM dithiothreitol and sample buffer (37.5 mM Tris-HCl, pH 7.0, 10% glycerol, 3% sodium dodecylsulphate (SDS), 0.025% bromophenol blue) to give an SDS:protein ratio of 1.5 mg SDS to 30 μg protein before loading onto an 8% SDS-PAGE gel. After electrophoresis, gels were blotted onto nitrocellulose (OSMONIC™ Nitropure 22 μm) in Towbin buffer (25 mM Tris base, 192 mM glycine, 20% methanol) containing 0.05% SDS at 100 V for 90 min at 4° C. Membranes were then blocked overnight in Tris-buffered saline (TBS; 20 mM Tris base, 150 mM NaCl) containing 3% w/v milk powder before incubation for 1 h at RT in rat anti-HA polyclonal antibody (Roche) diluted 1:1000 in TBS containing 1% BSA. Membranes were washed 3× in TBS containing 0.05% SDS (TBST), then incubated in anti-rat IgG HRP-conjugated antibody (Dako) diluted 1:1000 in TBS containing 3% w/v nonfat milk powder. Membranes were washed 3× in TBST before signal was detected with the SuperSignal® West Pico chemiluminescent substrate (Pierce).

Arabidopsistissues were fixed and labeled with anti-(1,3;1,4)-β-D-glucan specific antibody (Meikle et al.,Plant J5: 1-9, 1994) according to Burton et al. (Science311, 1940-1942, 2006). For labeling with anti-HA antibody, plant tissue was placed between two copper planchets and rapidly frozen in a Leica EM high pressure freezer (set at 2.7×105kPa and at an approximate rate of −10,000° C. s−1). The planchets were transferred into 100% ethanol in a Leica automated freeze-substitution unit set at −50° C. for 72 h. Samples were brought to room temperature (RT) overnight, removed and infiltrated with LR White resin and embedded in gelatin capsules as detailed in Burton et al. (2006, supra). Thin sections of embedded leaf tissue were collected on formvar-coated gold grids and incubated in a 1:200 dilution of the rat anti-HA polyclonal antibody in phosphate buffered saline (PBS; 137 mM NaCl, 10 mM NaPO4, 2.7 mM KCl, pH 7.4) containing 1% w/v BSA for 1 h at RT and then overnight at 4° C. Grids were washed several times in PBS, then incubated in a 1:20 dilution of anti-rat secondary antibody conjugated to 18 nm gold (Jackson ImmunoResearch) in PBS containing 1% w/v BSA for 1 h at RT. The grids were then washed, post stained and viewed under the TEM as described by Burton et al. (2006, supra)

Preparation of Cell Wall Material

Alcohol insoluble residue (AIR) was prepared by grinding plant material in liquid N2using a mortar and pestle. Five volumes of 80% ethanol was added to the homogenate prior to mixing by rotation for 1 h at 4° C. After centrifugation at 3,400×g for 5 min, the supernatant was removed and the residue was refluxed twice at RT in 80% ethanol for 1 h, followed by refluxing in 50% ethanol twice for 1 h. The ethanol-soluble fraction was removed and the AIR was washed once in 100% ethanol prior to drying at 40° C. under vacuum.

AIR (100 mg, prepared as described above) was incubated in 5 mL 20 mM NaPO4buffer, pH 6.5 for 2 h at 50° C. with continuous mixing in an incubator with shaking at 200 rpm. After 2 h, the suspension was centrifuged (3,400×g, 5 min) and the supernatant (S/N) removed. Another 5 mL of buffer was added and the incubation and centrifugation repeated. The S/N from this second incubation was used as the no enzyme negative control. The pelleted AIR was resuspended in 5 mL NaPO4buffer to which 100 μl (1,3;1,4)-β-D-glucan endo-hydrolase (McCleary et al.,J Inst Brew91: 285-295, 1985) was added. The mixture was incubated for 2 h at 50° C. with continuous mixing after which the S/N was collected as the (1,3;1,4)-β-D-glucan endo-hydrolase-released oligosaccharides. The negative control and (1,3;1,4)-β-D-glucan endo-hydrolase-treated S/Ns were desalted on a graphitised carbon cartridge as described by Packer et al. (Glycoconj J15: 737-747, 1998) and dried.

The dried (1,3;1,4)-β-D-glucan endo-hydrolase-released oligosaccharides were dissolved in 100 μL Milli H2O and 20 μL injected onto a CarboPac PA1 column (Dionex) equilibrated with 50 mM NaOAc in 0.2 M NaOH using a Dionex BioLC ICS 300 system (Dionex) equipped with a pulsed amperometric detector (PAD) and autosampler. Oligosaccharides were eluted at 1 mL/min with a linear gradient of NaOAc from 50 mM in 0.2M NaOH to 350 mM in 0.2 M NaOH over 15 min. Laminaribiose (Seigaku), maltose and cellobiose (both from Sigma) were run as standards.

MALDI-TOF MS Analysis

Aliquots (30 μL) of the remaining (1,3;1,4)-β-D-glucan endo-hydrolase-released oligosaccharides were lyophilised, dissolved in DMSO and methylated using the NaOH method (Ciucanu and Kerek,Carb Research131: 209-217, 1984). Methylated oligosaccharides were partitioned into dichloromethane (DCM) and the DCM phase washed 3× with MilliQ water. The DCM phase was dried under a N2stream before re-dissolving in 10 μL 50% acetonitrile. A 1 μL aliquot was mixed with 1 μL 2,5-dihydroxy benzoic acid matrix (10 mg/mL dissolved in 50% acetonitrile) and 1 μL of the mix was spotted onto a MALDI plate for analysis in a MALDI TOF mass spectrometer (Voyager DSTR, Applied Biosystems).

EST Analyses, Contig Assembly and Bioinformatics

CSLH ESTs were obtained by querying public databases including the now discontinued Stanford Cell Wall website, NCBI (http://www.ncbi.nlm.nih.gov/), HarvEST (http://harvest.ucr.edu/), GrainGenes (http://wheat.pw.usda.gov/GG2/index.shtml), Barley Gene Index (http://compbio.dfci.harvard.edu/tgi/plant.html) and BarleyBase (www.barleybase.org) using the BLAST search tool (Altschul et al.,Nucl Acids Res25: 3389-3402, 1997). Sequences were assembled into contigs using either Sequencer™ 3.0 (GeneCodes) or ContigExpress, a module of Vector NTI® Advance 9.1.0 (Invitrogen). DNA or protein sequences were aligned using ClustalX (Thompson et al.,Nucl Acids Res24: 4876-4882, 1997). Phylogenetic analysis was carried out using the in-built neighbour joining algorithm and tree robustness assessed using 1000 bootstrapped replicates. Sequence similarities were calculated using MatGat 2.02 (http://bitincka.com/ledion/matgat/) (Campanella et al.,BMC Bioinformatics4: 29, 2003). Transmembrane domains were predicted using the suite of programs described in ARAMEMNON (http://aramemnon.botanik.uni-koeln.de) (Schwacke et al.,Plant Physiol131: 16-26, 2003). Motifs predicting post-translational modifications were identified using the programs listed in ExPasy under the Tools menu (http://www.expasy.org/tools/#pattern). Protein parameters were calculated using ProtParam at ExPasy (http://www.expasy.org/cgi-bin/protparam).

Barley BAC Screening

BAC filters containing 6.5 equivalents of the barley genome from the non-Yd2 cv. Morex (Clemson University Genomics Institute, CUGI) were blocked for 6 h at 65° C. in prehybidisation solution (0.53 M NaPO4buffer pH 7.2, 7.5% w/v SDS, 1 mM EDTA, 11 μg/ml salmon sperm DNA). The radiolabeled cDNA and gDNA fragment amplified with primers H1F1 and H1R1 or H1R5 (Table 3) was added and incubated for 24 h at 65° C. Filters were washed 3× with 2×SSC, 0.1% SDS at RT. Final washes were done with 1×SSC, 0.1% SDS. Filters were exposed to X-ray film for 2 d. Positive BAC clones were identified and ordered as directed on the CUGI website (http://www.genome.clemson.edu). Clones were streaked onto LB agar containing 25 μg/ml chloramphenical and grown overnight at 37° C. Colonies for each clone were picked, placed on gridded nylon membranes resting on LB agar containing 25 μg/ml chloramphenicol and incubated overnight at 37° C. DNA was fixed to the membrane and denatured by placing on filter paper soaked in 0.4 M NaOH for 20 min, then neutralized by placing on filter paper soaked in neutralizing solution (1.5 M NaCl, 0.5 M Tris-HCl pH 7.2, 1 mM EDTA). Membranes were then washed 3× in 2×SSC, 0.1% SDS and hybridized using standard conditions (Sambrook et al.,Molecular cloning: a laboratory manual, Cold Spring Harbour Laboratory Press, New York, 1989).

BAC DNA Isolation

Positive clones were cultured overnight in LB broth containing 25 μg/ml chloramphenicol at 37° C. Cells were pelleted by centrifugation (12,000×g, 3 min) and the pellet resuspended in 90 μL TES buffer (25 mM Tris-HCl pH 8.0, 10 mM EDTA, 15% w/v sucrose). An aliquot (180 μL) of lysis solution (0.2 M NaOH, 1% SDS) was added and mixed gently, followed by 135 μL 3 M NaOAc pH 4.6. The chromosomal DNA was pelleted by centrifugation (12,000×g, 15 min). The S/N was collected and 2 μL RNase A (10 mg/mL) added and incubated for 1 h at 37° C. A 400 μL aliquot of Tris-saturated phenol-chloroform (1:1 ratio) was added and the samples centrifuged again (12,000×g, 5 min). The S/N was collected and BAC DNA precipitated using 2-3 volumes chilled 95% ethanol for 10 min at RT. The BAC DNA was pelleted by centrifugation (15,000×g, 15 min), washed in 70% ethanol, resuspended in 20-50 μL TE and stored at 4° C.

Genome Walking

The adaptor ligation method of Siebert et al. (Nucl Acids Res23: 1087-1088, 1995) was used to amplify fragments of genomic DNA upstream of known CSLH EST sequence. Restriction enzymes used to digest barley genomic DNA were Eco RV, Nru I, Pvu II, Sca I or Ssp I. Primary PCR reactions were conducted in 25 μL volumes containing 2 μL ligated DNA (1:10 dilution), 1×PCR buffer, 2 mM MgCl2, 100 ng each of adaptor primer AP1 and antisense primer H1R7 (Table 3), 0.4 mM dNTPs and 1 unit Taq polymerase (Invitrogen). Cycle parameters were as follows: 96° C. for 2 min then 40 cycles of 94° C. for 30 sec, 59° C. for 30 sec, 72° C. for 1 min, and a final step at 72° C. for 7 min. A secondary PCR reaction was conducted with 1 μL of the primary PCR using 100 ng each of adaptor primer AP2 and the nested primer H1R6. Reaction composition and cycle parameters were the same as above except that an annealing temperature of 61° C. was used.

BAC Sequencing

For sequencing, between 0.5 and 1 μg of isolated BAC DNA was combined with 5 pmol primer and 1× Big Dye Terminator v 3.1 (BDT) mix (Applied Biosystems, USA) in a final volume of 20 μL. Cycle parameters were as follows: 96° C. for 15 min, then 65 cycles of 96° C. for 10 sec, 55° C. for 10 sec and 60° C. for 4 min. DNA was precipitated with 0.1 vol 3M NaOAc pH 5.2 and 2.5 vol 95% ethanol on ice for 10 min, then pelleted by spinning at 12,000×g for 30 min. The pellet was rinsed with 70% ethanol, dried and sent to AGRF (Brisbane, Australia) for sequencing.

Mapping of HvCslH1

Genetic mapping was done using a Sloop×Halcyon doubled haploid (DH) mapping population of 60 lines (Read et al.,Aust J Agric Res54: 1145-1153, 2003). Using standard methods of DNA blot hybridization (Sambrook et al., 1989, supra) a HvCslH1 probe PCR-amplified using primers H1F1and H1R5 (Table 3) was hybridized to membranes containing parental line genomic DNA digested with one of six restriction enzymes (Bam HI, Dra I, Eco RI, Eco RV, Hind III, Xba I). The dihybrid population was then digested with enzymes that gave a clear polymorphism (Dra I). Polymorphisms were scored and HvCslH1 map location determined using the ‘find best location’ function of MapManager QT version 0.30 (Manly et al.,Mamm Genome12: 930-932, 2001). Map locations were correlated with QTL data using resources available at http://www.barleyworld.org/.

Arabidopsisseeds were surface-sterilized in a sterilization solution (sodium hypochlorite (2% available chlorine), drop of Tween-20) for 15 min then rinsed 4× with sterile MilliQ water.

Surface-sterilized seed was spread onto 85×25 mm Petri dishes containing 50 mL of sterile 1×MS medium (4.33 g/L Murashige and Skoog basal salts (Phytotechnology Laboratories), 2% w/v sucrose, 1% w/v bactoagar). For selection of transformants, 50 mg/L kanamycin (Sigma) was added to the medium. Plates were placed in a cold room for 3-5 days at 4° C. to synchronize germination. Cold-stratified plates were then transferred into controlled environment growth cabinets (Thermoline L+M model TPG 1260 TO-5×400, Smithfield, NSW, Australia) with day and night temperatures of 23° C. and 17° C., respectively. The average light intensity at rosette leaf level was ˜70 μE m−2sec−1supplied by 3-foot fluorescent tubes (Sylvania Standard F30W/133-T8 Cool White) during a 16 h light cycle. After 3 weeks on MS plates, individual plantlets were transferred into hydrated 42 mm diameter Jiffy pellets. Nine rows of six pellets were arranged in trays with three trays being housed on each 2×3.5-foot wire rack shelf. Relative humidity was measured to be between 60 and 70%. Plants were watered with tap water supplemented with Peter's Professional™ General Purpose plant fertilizer (Scotts Australia) by sub-irrigation every 2-3 days.

Genomic DNA Extraction and PCR Analysis ofArabidopsisTransgenics

DNA was extracted from a singleArabidopsisleaf according to the method described in Edwards et al. (Nucl Acids Res19: 1349, 1991). A 1 μL aliquot of genomic DNA was used as template in PCR screens of transgenic plants using primers H1F2and HvCslH1TOPOr (Table 3) with the following cycling regime: 94° C. for 2 min followed by 35 cycles of 94° C. for 20 sec, 57° C. for 30 sec, 72° C. for 30 sec.

Alignment of CslH DNA and Amino Acid Sequences from Rice and Barley

An alignment of the DNA and amino acid sequences for the CslH sequences in both rice and barley was performed to calculate the percent identity and similarity between the sequences, the results of which are shown inFIG. 10. The DNA and protein sequences were aligned and compared using the default parameters in MatGAT version 2.02 downloaded from http://bitincka.com/ledion/matgat/.

Multiple sequence alignments and phylogenetic tree generation was performed using the ClustalX program as described by Thompson et al. (Nucl Acids Res25: 4876-4882, 1997). The protein alignment and resultant phylogenetic tree are shown inFIGS. 11 and 12, respectively.

Cross of HvCslH1 and OsCSLF2 TransgenicArabidopsisLines

Two transgenicArabidopsislines, 15-8 and 15-11, in which the tagged HvCslH1 protein was detected using an anti-HA antibody, were chosen to genetically cross with two other transgenicArabidopsislines containing OsCslF2, H37 and H17-4, as described by Burton et al. (Science311: 1940-1942, 2006). It was thought that by expressing the HvCslH1 and OsCSLF2 proteins in the same cell types, higher levels of (1,3;1,4)-β-D-glucan above those observed in single gene (CSLH or CSLF only) transgenicArabidopsisplants, could potentially be deposited into cell walls. In addition, this would aid in detecting (1,3;1,4)-β-D-glucan in immuno-electron microscopy studies as well as in chemical cell wall analyses.

All four of the parental lines were confirmed to contain (1,3;1,4)-β-D-glucan in their cell walls by immuno-electron microscopy (FIG. 13). Individuals from each of the four populations were used as male and female parents. Flowers of the female parent (e.g. individual H37-5) were emasculated prior to anther dehiscence and pollinated using dehisced anthers from the male parent (e.g. individual 15-8-3). Each crossed flower was labelled and the resulting seed pods collected upon dehydration.

The progeny of each cross were sown in soil and their genotypes determined by PCR using leaf genomic DNA as template and HvCslH1-specific primers and, in a separate reaction, OsCslF2-specific primers. Mature leaves were fixed, embedded, sectioned and labeled with (1,3;1,4)-β-D-glucan monoclonal antibody. A number of the progeny were found to have greater levels of labelling than the parental lines, as observed inFIG. 14. For example, the labelling in the epidermal cells of the individual shown in Panel D is much heavier than compared to its 15-8-3×H37-7 parents (FIG. 13). A sib with the same genotype (FIG. 14, panel C) showed consistent yet lower levels of epidermal cell wall labeling.

Cloning of CslH cDNA and Genomic Sequences from Barley Cultivar Himalaya and Wheat

A full length cDNA sequence of the CslH1 gene was isolated from barley cultivar Himalaya using a combination of barley EST sequences, PCR from cDNA using primers based on the rice CslH1 gene sequence (LOC_Os10g20090) and 5′RACE.

The 2333 bp consensus sequence designated HvCslH1(Him) (SEQ ID NO: 69) is shown inFIG. 16. There is a single long open reading frame of 751 amino acids (SEQ ID NO: 70).

Oligonucleotide primers SJ91 and SJ85 were designed from the 5′ and 3′ ends of the consensus sequence and used to amplify a 3203 bp DNA fragment from genomic DNA designated HvCslH1gHim (SEQ ID NO: 71) inFIG. 17.

Alignment of the barley cDNA sequence and genomic sequences indicated that the CslH gene has eight small (approximately 100 bp) introns each flanked by the consensus GT . . . AG splice donor/acceptor sites (FIG. 17).

A wheat homolog of CslH1 was identified in the TIGR database as TC255929. Three classes of sequences made up this tentative consensus as exemplified by ESTs CJ614392, CJ609729 and CJ721204. PCR primers were designed from the barley sequence surrounding the ATG initiation codon (SJ163) and from the consensus sequence of all three EST types at the 3′ end (SJ164) and used to amplify a full length genomic fragment from wheat cultivar Chinese Spring. Two sequence types were identified and designated TaCslH1-1 (SEQ ID NO: 78) and TaCslH1-2 (SEQ ID NO: 79). The third homeolog designated TaCslH1-3 (SEQ ID NO: 80) was isolated using primers SJ204 and SJ164 as described in more detail in materials and methods.

Comparison with the barley sequences indicated that the intron-exon junctions were conserved in all three genes (FIG. 17). The three wheat genes are 94.8-96.1% identical.

The predicted coding region sequences of the three wheat CslH1 genes (SEQ ID NO: 72, SEQ ID NO: 73 and SEQ ID NO: 74) each encode a polypeptide of 752 amino acids (SEQ ID NO: 75, SEQ ID NO: 76 and SEQ ID NO: 77).

The DNA coding sequences and amino acids sequences of the barley and wheat CSLH1 genes were aligned using the muscle alignment program and the percentage identity and similarity was calculated using a PAM250 matrix. A table showing the percentage identity and similarity is show inFIG. 27.

As shown inFIG. 27, the wheat proteins are about 94-95.0% identical to each other and about 92.6-93.1% identical to the barley proteins.

CslH Gene Expression in Barley and Wheat

Expression of the CslH1 gene was examined by semi quantitative (RT-PCR and gel electrophoresis) and quantitative (real time PCR) methods.

The coleoptile is a good tissue to examine expression of genes related to beta glucan biosynthesis since the levels of beta glucan increase as the coleoptile grows and then decline after growth has stopped. The CslH1 gene shows maximum expression only after growth has ceased and is high in the oldest tissues (6-8 days old, as shown in FIG.19A/B).

Other tissues were also examined. In developing leaf, the CslH1 gene shows differential and maximum expression in the oldest tissue at the tip of the leaf (FIG. 20). It appears from these results that the CslH1 gene is preferentially (although not exclusively) expressed in cells that have stopped dividing and elongating and are thus differentiating. Cells in the maturing endosperm would be in a similar phase of development, ie. cell division would have stopped, cell enlargement would be slowing with the cells differentiating into specialised starch storage parenchyma.

In barley endosperm tissue, CslH1 gene expression peaked around 4 days post anthesis and then increased during later stages to reach a maximum at 28 days (FIG. 21).

There was a large difference in CslH1 gene expression in wheat where expression peaked at 4 days post anthesis after which expression was very low. These results were confirmed by real time PCR which showed that at 28 days post anthesis, the CslH gene was expressed about 10 fold higher levels in barley than in wheat (FIG. 22).

Overexpression of the Barley CslH Gene in Wheat Grain

Transgenic wheat plants were generated by biolistics transformation with the full length genomic HvCslH1 (cv. Himalaya) gene under control of the glutenin promoter such that expression should only occur in endosperm tissues (FIG. 23). Lines were screened for the presence or absence of the transgene by PCR of young leaf material. Twelve PCR positive lines and three PCR negative lines (H1-2, -7 and -11) were grown to maturity in the glasshouse. RNA was isolated from developing grain at approximately 15 days post anthesis and cDNA was made using Superscript III. Expression of the barley transgene was then analysed by real time PCR. Table 5 shows the relative expression levels compared to the endogenous wheat CslH gene as the primers used amplify both the wheat and barley genes.

Most of the lines expressed the barley CslH gene at several hundred fold higher levels than the controls with line 9, 10, 12 and 14 showing the highest expression (greater than one thousand fold higher).

At maturity, single grains from were analysed for beta glucan content and a summary of the results are shown in Table 6:

The PCR negative lines all had the lowest beta glucan contents averaging 0.69% of grain weight, whereas grain from the PCR positive lines had an increased average beta glucan content of 0.97%. The last column of Table 6 shows the maximum beta glucan content of any single grain from a given line—the highest PCR negative line was 1.0% (and most grains were much lower than this) but several of the PCR positive lines had grains with significantly increased beta glucan levels with line 9 and line 10 (the highest expressers) having grains with up to 1.9% beta glucan. These levels of beta glucan have never been seen before in wheat.

The heads from these T0 plants contain T1 seed which are segregating for the transgene. If the DNA is inserted at a single locus a ratio of three transgenic to one wild type seed should be observed.FIG. 25shows the beta glucan levels of individual T1 seeds from the H1 transgenic line 10 from which it can be seen that approximately three quarters (47/61) have higher beta glucan levels than the average of the PCR negative lines (0.7%). From the ratio of the highest beta glucan level (1.9%) to the average PCR negative level (0.7%) the increase in beta glucan content is 2.7 times that normally seen in wild type wheat grains. A further significant observation is that a high proportion of the grains have at least 1.4% beta glucan.

It is expected that further increases in beta glucan will be seen in these grains when the lines are made homozygous and gene dosage increases.

Materials and Methods for Examples 9 to 11

Plant Material

Primer Sequences

The primer sequences referred to in Examples 9 to 11 and this example are shown below in Table 7:

Plant DNA was isolated from fully expanded leaf tissue using a CTAB based method (Murray and Thompson,Nucleic Acids Res.8: 4321-4325, 1980). Total RNA was isolated from leaf and coleoptile tissues using an RNAeasy kit from Qiagen. RNA was isolated from developing endosperm using a phenol SDS method and LiCl precipitation (Clarke et al.,Functional and Integrative Genomics8, 211-221, 2007). RNA was treated with DNAse using a “DNA-free” kit from Ambion and then cDNA was synthesised using SuperscriptIII reverse transcriptase according to the manufacturer's instructions (Clontech).

Cloning of CslH Genes

The methods for cloning CslH genes were similar to those described in the cloning and characterisation of CslF genes (Burton et al.,Plant Physiol146: 1821-1833, 2008). A 1.8 kb tentative consensus sequence (TC140327) of a barley homolog of the rice Cellulose synthase like H1 gene (LOC_Os10g20090) was identified in the TIGR database. PCR primer pairs (SJ27-SJ73 and SJ72-SJ75) were designed based on the rice CslH1 sequence and used to amplify sequences from cDNA. The 5′ end of the gene was then amplified by 5′RACE using a SMART cDNA library and nested CslH1 primers SJ28 and SJ79 according to the manufacturer's instructions (Clontech).

A full length genomic clone was isolated by amplification with primers SJ91 and SJ85 and Phusion Taq polymerase (Finnzymes) according to the manufacturers recommend cycling conditions (denature 30 sec at 98° C. followed by 35 cycles of 98° C. for 5 sec, 63° C. for 7 sec and 72° C. for 3 min) and cloned into the pCRBluntII TOPO cloning vector (Invitrogen).

Wheat CslH genomic clones were isolated by PCR with Phusion polymerase from the cultivar Chinese Spring using primers SJ163 and SJ164 and an annealing temperature of 70° C. A genome walking kit was used according to the manufacturers instructions (Clontech) to obtain sequences extending upstream of the coding region of all three wheat CslH homeologs from the variety Bob White (data not shown). A primer (SJ204) was designed that was specific to the third homeolog and used with SJ164 to isolate the third full length genomic clone. It was confirmed that the predicted exon/intron boundaries could be spliced correctly by sequencing cDNA fragments (data not shown).

Expression Analysis of CslH Gene in Wheat and Barley by RT-PCR Total RNA was isolated from sections of the first leaf of a 7 day old plant, from dark grown coleoptiles of different ages, and from developing grain collected at 4 day intervals post anthesis (DPA), DNAse treated and reverse transcribed with Superscript III according to the manufacturer's instructions (Invitrogen). PCR reactions were performed using HotStarTaq (Qiagen). The cDNA was diluted and used in PCR reactions at a level equivalent to 1 ng of original RNA per microlitre. For semi-quantitative RT-PCR, CslH1 primers SJ72 and SJ74, for the CslF genes, primer pairs were as follows; (CslF6; SJ107-SJ82), (CslF4; SJ94-SJ95), (CslF9; SJ97-SJ93), (CslF3; SJ44-SJ38), (CslF8; SJ96-SJ37). An annealing temperature of 59° C. was used. Test amplifications were performed to ensure that the amplification was not saturated (approx 32-35 cycles except tubulin 24 cycles) and the products were analysed by ethidium bromide staining after agarose gel electrophoresis. Real time PCR was performed on triplicate samples on a Rotorgene 6000 machine (Corbett Life Sciences, AU) using HotStarTaq (Qiagen), SybrGreen and primers SJ183 and SJ164 and an annealing temperature of 60° C. Relative expression levels were calculated using the machine software with wheat 0 dpa samples as the comparator (set to one). The Ct value of this sample was 25.5 cycles. For analysis of transgenic grain at 15 dpa, the relative expression values were normalised against tubulin and compared to the lowest expression line (H1-13).

Expression Analysis of CslH Gene in Barley by Q-PCR

HvCslH1 transcript was measured in developing coleoptile 0.5 to 7 days post germination. HvCslH1 transcript was shown to accumulate only after the completion of the elongation phase and the emergence of the leaf. Highest levels of expression were seen at 7 days when the coleoptile is senescing (twisting and shrinking) (Gibeaut et al.,Planta221:729-738, 2005).

Production of Transgenic Wheat Plants Overexpressing the Barley CslH Gene in Endosperm

The full length barley cv. Himalaya genomic CslH sequence (SEQ ID NO: 71) was amplified using primers SJ91 and SJ85, was inserted as an EcoRI fragment between a 1.9 kb fragment of the high molecular weight glutenin Bx17 promoter and the nopaline synthase terminator (FIG. 23). The Bx17 promoter confers high level expression in developing endosperm (Reddy and Appels,Theor Appl Genet85: 616-624, 1993).

Bob White 26 wheat plants were transformed using the biolistics method (Pellegrineschi et al.,Genome45: 421-430, 2002) with 50 mg/L G418 as the selection agent. The HvCslH expression vector (pZLBx17HvgH1 and a second plasmid with the CaMV 35S promoter driving expression of the NPTII selectable marker (pCMSTLSneo,FIG. 24) were mixed in equimolar amounts and co bombarded into immature embryos.

Transgenic plants were screened for the presence of the transgene using young leaf tissue and the RedExtractnAmp™ kit from Sigma with primers SJ244 and SJ79.

At anthesis (emergence of the anthers and shedding of pollen) heads were tagged to enable grain to be sampled at approximately 15 dpa. Three grains from a head were pooled, RNA extracted and reverse transcribed and levels of transgene expression were analysed by real time PCR using primers SJ183 and SJ85. Expression levels were normalised against alpha tubulin (primers TUB and TUB2F) and finally expressed as a ratio compared to the lowest expresser.

Flour from mature single grains was analysed for beta glucan content using a scaled down version of the lichenase enzymatic method (AACC Method 32-33, Megazyme assay kit, McCleary and Glennie-Holmes,J. Inst Brewing91: 285-295, 1985). Beta glucan contents are expressed as a percentage (w/w) of the milled whole grain flour.

Overexpression of the Barley CslH Gene in Barley Cv. Golden Promise

The full-length coding region of the barley CslH cDNA (SEQ ID NO: 1) was transferred into two Gateway-enabled barley transformation vectors. The vector pRB474 contains the oat globulin promoter (Vickers et al.,Plant Mol Biol62: 195-214, 2006) which provides endosperm specific expression and the vector pMDC32 (Curtis and Grossniklaus,Plant Physiol.133: 462-9, 2003) contains a double 35S promoter which drives constitutive expression in all plant tissues.

Barley Transformation

The vectors were transferred intoAgrobacterium tumefaciensand immature scutella of the barley cultivar Golden Promise were transformed using established protocols to produce two populations of transgenic plants. Insertion of the transgene was confirmed by Southern blotting. Plants 236-1 to 236-18 contain the barley CslH gene driven by the oat globulin promoter. Plants 237-1 and -2 contain the barley CslH gene driven by the 35S promoter. Plants 208-2, -3, -5 and -7 are control plants and are transgenic for the empty vector pRB474 carrying the oat globulin promoter only.

Transcript Analysis

Leaf and developing grain samples, from 7 and 14 days after pollination (DAP) were collected from the 236 plants. Total RNA was extracted using TRIzol reagent (Invitrogen) following a standard protocol and cDNA was synthesized according to Burton et al., (Plant Physiol 146: 1821-1833, 2008). Quantitative real-time PCR (QPCR) was carried out according to Burton et al. (2008, supra). The transcript levels of the CslH gene were compared in the endosperm of the transgenic grain to wild type endosperm levels which are generally very low.

As shown inFIG. 26, the empty vector control lines (208) have typical wild type levels of CslH transcript. The transgenic lines (236) show significantly increased HvCslH1 mRNA levels at 7 days (7 D) and further increases at 14 days (14 D) after pollination.

The T1 seed from the transgenic plants were collected. A sample of the bulked T1 grain from each individual plant was ground to flour and the amount of beta-glucan present was assayed using Megazyme method (described supra). The data from each plant are presented as the mean value of two replicates and the amount of beta-glucan as a percentage of grain weight is shown in Table 8, below:

TABLE 8(1,3;1,4)-β-D-glucan content of bulked transgenic barley flour

The empty vector control lines (208) have a (1,3;1,4)-β-D-glucan content around 4% which is typical for wild type Golden Promise grain. Even though the T1 grain is bulked (and therefore contains null-segregant grains) a significant number of the transgenic lines (shaded) show an overall (1,3;1,4)-β-D-glucan content greater than the control, with the highest value at 5.9%.

Also, it must be noted that, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context already dictates otherwise. Thus, for example, reference to “a transgene” includes a single transgene as well as two or more transgenes; “a plant cell” includes a single cell as well as two or more cells; and so forth.