The impact of the indica rice SSIIa allele on the apparent high amylose starch from rice grain with downregulated japonica SBEIIb.

KEY MESSAGE: Catalytically active indica SSIIa allele in high amylose rice with down-regulated japonica SBEIIb can increase starch content and modify the starch structure and properties without changing its amylose content. Rice (Oryza sativa) genotypes with inactive starch synthase IIa (SSIIa) with recessive variants of starch branching enzyme IIb (SBEIIb) exhibit a range of alterations in grain phenotype, starch granule morphology, starch granule bound proteins, starch structure, and functional properties. However, the interactions between the two enzymes have not been thoroughly investigated yet. We analysed recombinant rice lines having down-regulated SBEIIb expression (SBEIIbDR) with either indica or japonica type SSIIa (SSIIaind or SSIIajap). In SBEIIbDR rice starch granules, the increased abundance of two protein bands (SSI and SSIIa) was found with eight additional protein bands not generally associated with starch granules. The amount of SSIIa was higher in SSIIaindSBEIIbDR than SSIIajapSBEIIbDR, which indicated that indica type SSIIa, possibly in the monomer form, was extensively involved in starch biosynthesis in the SBEIIbDR endosperm. Furthermore, SSIIaindSBEIIbDR grains had higher total starch content and higher starch swelling power than SSIIajapSBEIIbDR lines, but the amylopectin gelatinization temperatures and enthalpy and the apparent amylose content remained similar. In summary, this work suggests that SSIIaind can partly compensate for the alteration of starch synthesis resulting from the SBEIIb down-regulation in japonica background without reducing its amylose content. The study provides insight into the starch structural and textural improvements of high amylose starch.

Functional Genomic Validation of the Roles of Soluble Starch Synthase IIa in Japonica Rice Endosperm.

The enzyme starch synthase IIa (SSIIa) in cereals has catalytic and regulatory roles during the synthesis of amylopectin that influences the functional properties of the grain. Rice endosperm SSIIa is more active in indica accessions compared to japonica lines due to functional SNP variations in the coding region of the structural gene. In this study, downregulating the expression of japonica-type SSIIa in Nipponbare endosperm resulted in either shrunken or opaque grains with an elevated proportion of A-type starch granules. Shrunken seeds had severely reduced starch content and could not be maintained in succeeding generations. In comparison, the opaque grain morphology was the result of weaker down-regulation of SSIIa which led to an elevated proportion of short-chain amylopectin (DP 6-12) and a concomitant reduction in the proportion of medium-chain amylopectin (DP 13-36). The peak gelatinization temperature of starch and the estimated glycemic score of cooked grain as measured by the starch hydrolysis index were significantly reduced. These results highlight the important role of medium-chain amylopectin in influencing the functional properties of rice grains, including its digestibility. The structural, regulatory and nutritional implications of down-regulated japonica-type SSIIa in rice endosperm are discussed.

High wholegrain barley ß-glucan lowers food intake but does not alter small intestinal macronutrient digestibility in ileorectostomised rats.

Using barley cultivars differing widely in ß-glucan content, we aimed to determine their effects on small intestinal macronutrient digestion in 24 ileorectostomised rats. The rats were fed 1 of 4 experimental diets, each containing a different barley variety, for 11 d. The diets had a content of 0, 2.1, 2.6 and 4.3 g of ß-glucan/100 g. Feed intake and faecal excretion of fat, protein, starch, and non-starch polysaccharides were determined in the final 5 d of the study and apparent macronutrient digestibility calculated. Higher dietary levels of ß-glucan (2.6% and 4.3%) lowered feed intake (by 15 and 19%, respectively) but final body weight was only lowered by the 4.3% ß-glucan diet relative to rats fed the 0% ß-glucan diet (all ps < 0.05). Protein, lipid and starch digestibility was unrelated to the dietary ß-glucan content. Higher dietary levels of barley ß-glucan lower feed intake of ileorectostomised rats, which is independent of intestinal fermentation and unrelated to macronutrient digestibility.

Membrane pore architecture of the CslF6 protein controls (1-3,1-4)-ß-glucan structure.

The cereal cell wall polysaccharide (1-3,1-4)-ß-glucan is a linear polymer of glucose containing both ß1-3 and ß1-4 bonds. The structure of (1-3,1-4)-ß-glucan varies between different cereals and during plant growth and development, but little is known about how this is controlled. The cellulose synthase-like CslF6 protein is an integral membrane protein and a major component of the (1-3,1-4)-ß-glucan synthase. I show that a single amino acid within the predicted transmembrane pore domain of CslF6 controls (1-3,1-4)-ß-glucan structure. A new mechanism for the control of the polysaccharide structure is proposed where membrane pore architecture and the translocation of the growing polysaccharide across the membrane control how the acceptor glucan is coordinated at the active site and thus the proportion of ß1-3 and ß1-4 bonds within the polysaccharide.

The different effects of starch synthase IIa mutations or variation on endosperm amylose content of barley, wheat and rice are determined by the distribution of starch synthase I and starch branching enzyme IIb between the starch granule and amyloplast stroma.

KEY MESSAGE: The distribution of starch synthase I and starch branching enzyme IIb between the starch granule and amyloplast stroma plays an important role in determining endosperm amylose content of cereal grains. Starch synthase IIa (SSIIa) catalyses the polymerisation of intermediate length glucan chains of amylopectin in the endosperm of cereals. Mutations of SSIIa genes in barley and wheat and inactive SSIIa variant in rice induce similar effects on the starch structure and the amylose content, but the severity of the phenotypes is different. This study compared the levels of transcripts and partitioning of proteins of starch synthase I (SSI) and starch branching enzyme IIb (SBEIIb) inside and outside the starch granules in the developing endosperms of these ssIIa mutants and inactive SSIIa variant. Pleiotropic effects on starch granule-bound proteins suggested that the different effects of SSIIa mutations on endosperm amylose content of barley, wheat and rice are determined by the distribution of SSI and SBEIIb between the starch granule and amyloplast stroma in cereals. Regulation of starch synthesis in ssIIa mutants and inactive SSIIa variant may be at post-translational level or the altered amylopectin structure deprives the affinity of SSI and SBEIIb to amylopectin.

Allelic effects on starch structure and properties of six starch biosynthetic genes in a rice recombinant inbred line population.

BACKGROUND: The genetic diversity of six starch biosynthetic genes (Wx, SSI, SSIIa, SBEI, SBEIIa and SBEIIb) in indica and japonica rices opens an opportunity to produce a new variety with more favourable grain starch quality. However, there is limited information about the effects of these six gene allele combinations on starch structure and properties. A recombinant inbred line population from a cross between indica and japonica varieties offers opportunities to combine specific alleles of the six genes. RESULTS: The allelic (indica vs japonica) effects of six starch biosynthetic genes on starch structure, functional properties, and abundance of granule bound proteins in rice grains were investigated in a common genetic background using a recombinant inbred line population. The indica Wx (Wxi) allele played a major role while indica SSI (SSIi), japonica SSIIa (SSIIaj) and indica SBEI (SBEIi) alleles had minor roles on the increase of amylose content. SSIIaj and japonica SBEIIb (SBEIIbj) alleles had a major and a minor role on high ratio of ∑DP ≤ 10 to ∑DP ≤ 24 fractions (RCL10/24), respectively. Both major alleles (Wxi and SSIIaj) reduced peak viscosity (PV), onset, peak and end gelatinization temperatures (GTs) of amylopectin, and increased amylose-lipid complex dissociation enthalpy compared with their counterpart-alleles, respectively. SBEIIai and SBEIIbj decreased PV, whereas SSIi and SBEIIbj decreased FV. SBEIi reduced setback viscosity and gelatinization enthalpy. RCL10/24 of chain length distribution in amylopectin is negatively correlated with PV and BD of paste property and GTs of thermal properties. We also report RILs with superior starch properties combining Wxi, SSIj, SSIIaj, SBEIi and SBEIIbj alleles. Additionally, a clear relation is drawn to starch biosynthetic gene alleles, starch structure, properties, and abundance of granule bound starch biosynthetic enzymes inside starch granules. CONCLUSIONS: Rice Wxi and SSIIaj alleles play major roles, while SSIi, SBEIi, SBEIIai and SBEIIbj alleles have minor roles in the determination of starch properties between indica and japonica rice through starch structural modification. The combination of these alleles is a key factor for starch quality improvement in rice breeding programs. RCL10/24 value is critical for starch structure and property determination.

A genetic strategy generating wheat with very high amylose content.

Resistant starch (RS), a type of dietary fibre, plays an important role in human health; however, the content of RS in most modern processed starchy foods is low. Cereal starch, when structurally manipulated through a modified starch biosynthetic pathway to greatly increase the amylose content, could be an important food source of RS. Transgenic studies have previously revealed the requirement of simultaneous down-regulation of two starch branching enzyme (SBE) II isoforms both located on the long arm of chromosome 2, namely SBEIIa and SBEIIb, to elevate the amylose content in wheat from ~25% to ~75%. The current study revealed close proximity of genes encoding SBEIIa and SBEIIb isoforms in wheat with a genetic distance of 0.5 cM on chromosome 2B. A series of deletion and single nucleotide polymorphism (SNP) loss of function alleles in SBEIIa, SBEIIb or both was isolated from two different wheat populations. A breeding strategy to combine deletions and SNPs generated wheat genotypes with altered expression levels of SBEIIa and SBEIIb, elevating the amylose content to an unprecedented ~85%, with a marked concomitant increase in RS content. Biochemical assays were used to confirm the complete absence in the grain of expression of SBEIIa from all three genomes in combination with the absence of SBEIIb from one of the genomes.

Wholegrain barley ß-glucan fermentation does not improve glucose tolerance in rats fed a high-fat diet.

Fermentation of oat and barley ß-glucans is believed to mediate in part their metabolic health benefits, but the exact mechanisms remain unclear. In this study, we sought to test the hypothesis that barley ß-glucan fermentation raises circulating incretin hormone levels and improves glucose control, independent of other grain components. Male Sprague-Dawley rats (n = 30) were fed a high-fat diet for 6 weeks and then randomly allocated to 1 of 3 dietary treatments for 2 weeks. The low- (LBG, 0% ß-glucan) and high- (HBG, 3% ß-glucan) ß-glucan diets contained 25% wholegrain barley and similar levels of insoluble dietary fiber, available carbohydrate, and energy. A low-fiber diet (basal) was included for comparison. Immediately prior to the dietary intervention, gastric emptying rate (using the (13)C-octanoic breath test) and postprandial glycemic response of each diet were determined. At the end of the study, circulating gut hormone levels were determined; and a glucose tolerance test was performed. The rats were then killed, and indices of cecal fermentation were assessed. Diet did not affect live weight; however, the HBG diet, compared to basal and LBG, reduced food intake, tended to slow gastric emptying, increased cecal digesta mass and individual and total short-chain fatty acid pools, and lowered digesta pH. In contrast, circulating levels of glucose, insulin, gastric-inhibitory peptide, and glucagon-like peptide-1, and glucose tolerance were unaffected by diet. In conclusion, wholegrain barley ß-glucan suppressed feed intake and increased cecal fermentation but did not improve postprandial glucose control or insulin sensitivity.

Rice starch granule amylolysis--differentiating effects of particle size, morphology, thermal properties and crystalline polymorph.

The underlying mechanism of amylolysis of rice starch granules was investigated using isolated starch granules from wild-type, as well as SBEIIb mutant and down-regulated lines. Fused granule agglomerates isolated from mutant and transgenic lines were hydrolysed at similar rates by amylases, and had similar crystalline patterns and thermal properties as individual granules. Surface pores, a feature previously only reported for A-polymorphic starch granules, were also observed in B- and C-polymorphic rice starch granules. Although the microscopic patterns of hydrolysis among granules with different crystalline polymorphs were qualitatively similar, the extent and the rate of amylolysis were different, suggesting that B-type crystalline polymorphs are intrinsically more resistant to enzymatic hydrolysis than A-type in rice starch granules. It is proposed that the slightly longer branch lengths of amylopectin which leads to the formation of more stable B-type double helical structures compared to their A-type counterparts is the major parameter, with other factors such as granule size, surface pores and interior channels having secondary roles, in determining the rate of enzymatic hydrolysis of rice starch granules.

Suppression of starch synthase I expression affects the granule morphology and granule size and fine structure of starch in wheat endosperm.

Studies in Arabidopsis and rice suggest that manipulation of starch synthase I (SSI) expression in wheat may lead to the production of wheat grains with novel starch structure and properties. This work describes the suppression of SSI expression in wheat grains using RNAi technology, which leads to a low level of enzymatic activity for SSI in the developing endosperm, and a low abundance of SSI protein inside the starch granules of mature grains. The amylopectin fraction of starch from the SSI suppressed lines showed an increased frequency of very short chains (degree of polymerization, dp 6 and 7), a lower proportion of short chains (dp 8-12), and more intermediate chains (dp 13-20) than in the grain from their negative segregant lines. In the most severely affected line, amylose content was significantly increased, the morphology of starch granules was changed, and the proportion of B starch granules was significantly reduced. The change of the fine structure of the starch in the SSI-RNAi suppression lines alters the gelatinization temperature, swelling power, and viscosity of the starch. This work demonstrates that the roles of SSI in the determination of starch structure and properties are similar among different cereals and Arabidopsis.

Biomolecular analyses of starch and starch granule proteins in the high-amylose rice mutant Goami 2.

Elevated proportions of amylose in cereals are commonly associated with either the loss of starch branching or starch synthase activity. Goami 2 is a high-amylose mutant of the temperate japonica rice variety Ilpumbyeo. Genotyping revealed that Goami 2 and Ilpumbyeo carry the same alleles for starch synthase IIa and granule-bound starch synthase I genes. Analyses of granule-bound proteins revealed that SSI and SSIIa accumulate inside the mature starch granules of Goami 2, which is similar to the amylose extender mutant IR36ae. However, unlike the amylose extender mutants, SBEIIb was still detectable inside the starch granules of Goami 2. Detection of SBEIIb after protein fractionation revealed that most of the SBEIIb in Goami 2 accumulates inside the starch granules, whereas most of it accumulates at the granule surface in Ilpumbyeo. Exhaustive mass spectrometric characterisations of granule-bound proteins failed to detect any peptide sequence mutation or major post-translational modifications in Goami 2. Moreover, the signal peptide was found to be cleaved normally from the precursor protein, and there is no apparent N-linked glycosylation. Finally, no difference was found in the SBEIIb structural gene sequence of Goami 2 compared with Ilpumbyeo. In contrast, a G-to-A mutation was detected in the SBEIIb gene of IR36ae located at the splice site between exon and intron 11, which could potentially introduce a premature stop codon and produce a truncated form of SBEIIb. It is suggested that the mutation responsible for producing high amylose in Goami 2 is not due to a defect in SBEIIb gene as was observed in IR36ae, even though it produces a phenotype analogous to the amylose extender mutation. Understanding the molecular genetic basis of this mutation will be important in identifying novel targets for increasing amylose and resistant starch contents in rice and other cereals.

A SHORT INTERNODES (SHI) family transcription factor gene regulates awn elongation and pistil morphology in barley.

The awn, an apical extension from the lemma of the spikelet, plays important roles in seed dispersal, burial, and photosynthesis. Barley typically has long awns, but short-awn variants exist. The short awn 2 (lks2) gene, which produces awns about 50% shorter than normal, is a natural variant that is restricted to Eastern Asia. Positional cloning revealed that Lks2 encodes a SHI-family transcription factor. Allelism tests showed that lks2 is allelic to unbranched style 4 (ubs4) and breviaristatum-d (ari-d), for which the phenotypes are very short awn and sparse stigma hairs. The gene identity was validated by 25 mutant alleles with lesions in the Lks2 gene. Of these, 17 affected either or both conserved regions: the zinc-binding RING-finger motif and the IGGH domain. Lks2 is highly expressed in awns and pistils. Histological observations of longitudinal awn sections showed that the lks2 short-awn phenotype resulted from reduced cell number. Natural variants of lks2 were classified into three types, but all shared a single-nucleotide polymorphism (SNP) that causes a proline-to-leucine change at position 245 in the IGGH domain. All three lks2 natural variants were regarded as weak alleles because their awn and pistil phenotypes are mild compared with those of the 25 mutant alleles. Natural variants of lks2 found in the east of China and the Himalayas had considerably different sequences in the regions flanking the critical SNP, suggesting independent origins. The available results suggest that the lks2 allele might have a selective advantage in the adaptation of barley to high-precipitation areas of Eastern Asia.

Analysis of the arabinoxylan arabinofuranohydrolase gene family in barley does not support their involvement in the remodelling of endosperm cell walls during development.

Arabinoxylan arabinofuranohydrolases (AXAHs) are family GH51 enzymes that have been implicated in the removal of arabinofuranosyl residues from the (1,4)-ß-xylan backbone of heteroxylans. Five genes encoding barley AXAHs range in size from 4.6 kb to 7.1 kb and each contains 16 introns. The barley HvAXAH genes map to chromosomes 2H, 4H, and 5H. A small cluster of three HvAXAH genes is located on chromosome 4H and there is evidence for gene duplication and the presence of pseudogenes in barley. The cDNAs corresponding to barley and wheat AXAH genes were cloned, and transcript levels of the genes were profiled across a range of tissues at different developmental stages. Two HvAXAH cDNAs that were successfully expressed in Nicotiana benthamiana leaves exhibited similar activities against 4-nitrophenyl α-L-arabinofuranoside, but HvAXAH2 activity was significantly higher against wheat flour arabinoxylan, compared with HvAXAH1. HvAXAH2 also displayed activity against (1,5)-α-L-arabinopentaose and debranched arabinan. Western blotting with an anti-HvAXAH antibody was used to define further the locations of the AXAH enzymes in developing barley grain, where high levels were detected in the outer layers of the grain but little or no protein was detected in the endosperm. The chromosomal locations of the genes do not correspond to any previously identified genomic regions shown to influence heteroxylan structure. The data are therefore consistent with a role for AXAH in depolymerizing arabinoxylans in maternal tissues during grain development, but do not provide compelling evidence for a role in remodelling arabinoxylans during endosperm or coleoptile development in barley as previously proposed.

Loss of Cellulose synthase-like F6 function affects mixed-linkage glucan deposition, cell wall mechanical properties, and defense responses in vegetative tissues of rice.

Mixed-linkage glucan (MLG) is a cell wall polysaccharide containing a backbone of unbranched (1,3)- and (1,4)-linked ß-glucosyl residues. Based on its occurrence in plants and chemical characteristics, MLG has primarily been associated with the regulation of cell wall expansion due to its high and transient accumulation in young, expanding tissues. The Cellulose synthase-like F (CslF) subfamily of glycosyltransferases has previously been implicated in mediating the biosynthesis of this polymer. We confirmed that the rice (Oryza sativa) CslF6 gene mediates the biosynthesis of MLG by overexpressing it in Nicotiana benthamiana. Rice cslf6 knockout mutants show a slight decrease in height and stem diameter but otherwise grew normally during vegetative development. However, cslf6 mutants display a drastic decrease in MLG content (97% reduction in coleoptiles and virtually undetectable in other tissues). Immunodetection with an anti-MLG monoclonal antibody revealed that the coleoptiles and leaves retain trace amounts of MLG only in specific cell types such as sclerenchyma fibers. These results correlate with the absence of endogenous MLG synthase activity in mutant seedlings and 4-week-old sheaths. Mutant cell walls are weaker in mature stems but not seedlings, and more brittle in both stems and seedlings, compared to wild type. Mutants also display lesion mimic phenotypes in leaves, which correlates with enhanced defense-related gene expression and enhanced disease resistance. Taken together, our results underline a weaker role of MLG in cell expansion than previously thought, and highlight a structural role for MLG in nonexpanding, mature stem tissues in rice.

Functional characterization of barley betaglucanless mutants demonstrates a unique role for CslF6 in (1,3;1,4)-ß-D-glucan biosynthesis.

(1,3;1,4)-ß-D-glucans (mixed-linkage glucans) are found in tissues of members of the Poaceae (grasses), and are particularly high in barley (Hordeum vulgare) grains. The present study describes the isolation of three independent (1,3;1,4)-ß-D-glucanless (betaglucanless; bgl) mutants of barley which completely lack (1,3;1,4)-ß-D-glucan in all the tissues tested. The bgl phenotype cosegregates with the cellulose synthase like HvCslF6 gene on chromosome arm 7HL. Each of the bgl mutants has a single nucleotide substitution in the coding region of the HvCslF6 gene resulting in a change of a highly conserved amino acid residue of the HvCslF6 protein. Microsomal membranes isolated from developing endosperm of the bgl mutants lack detectable (1,3;1,4)-ß-D-glucan synthase activity indicating that the HvCslF6 protein is inactive. This was confirmed by transient expression of the HvCslF6 cDNAs in Nicotiana benthamiana leaves. The wild-type HvCslF6 gene directed the synthesis of high levels of (1,3;1,4)-ß-D-glucans, whereas the mutant HvCslF6 proteins completely lack the ability to synthesize (1,3;1,4)-ß-D-glucans. The fine structure of the (1,3;1,4)-ß-D-glucan produced in the tobacco leaf was also very different from that found in cereals having an extremely low DP3/DP4 ratio. These results demonstrate that, among the seven CslF and one CslH genes present in the barley genome, HvCslF6 has a unique role and is the key determinant controlling the biosynthesis of (1,3;1,4)-ß-D-glucans. Natural allelic variation in the HvCslF6 gene was found predominantly within introns among 29 barley accessions studied. Genetic manipulation of the HvCslF6 gene could enable control of (1,3;1,4)-ß-D-glucans in accordance with the purposes of use.

The barley amo1 locus is tightly linked to the starch synthase IIIa gene and negatively regulates expression of granule-bound starch synthetic genes.

In this study of barley starch synthesis, the interaction between mutations at the sex6 locus and the amo1 locus has been characterized. Four barley genotypes, the wild type, sex6, amo1, and the amo1sex6 double mutant, were generated by backcrossing the sex6 mutation present in Himalaya292 into the amo1 'high amylose Glacier'. The wild type, amo1, and sex6 genotypes gave starch phenotypes consistent with previous studies. However, the amo1sex6 double mutant yielded an unexpected phenotype, a significant increase in starch content relative to the sex6 phenotype. Amylose content (as a percentage of starch) was not increased above the level observed for the sex6 mutation alone; however, on a per seed basis, grain from lines containing the amo1 mutation (amo1 mutants and amo1sex6 double mutants) synthesize significantly more amylose than the wild-type lines and sex6 mutants. The level of granule-bound starch synthase I (GBSSI) protein in starch granules is increased in lines containing the amo1 mutation (amo1 and amo1sex6). In the amo1 genotype, starch synthase I (SSI), SSIIa, starch branching enzyme IIa (SBEIIa), and SBEIIb also markedly increased in the starch granules. Genetic mapping studies indicate that the ssIIIa gene is tightly linked to the amo1 locus, and the SSIIIa protein from the amo1 mutant has a leucine to arginine residue substitution in a conserved domain. Zymogram analysis indicates that the amo1 phenotype is not a consequence of total loss of enzymatic activity although it remains possible that the amo1 phenotype is underpinned by a more subtle change. It is therefore proposed that amo1 may be a negative regulator of other genes of starch synthesis.

Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA- and hairpin RNA-mediated RNA silencing.

The inactivation of starch branching IIb (SBEIIb) in rice is traditionally associated with elevated apparent amylose content, increased peak gelatinization temperature, and a decreased proportion of short amylopectin branches. To elucidate further the structural and functional role of this enzyme, the phenotypic effects of down-regulating SBEIIb expression in rice endosperm were characterized by artificial microRNA (amiRNA) and hairpin RNA (hp-RNA) gene silencing. The results showed that RNA silencing of SBEIIb expression in rice grains did not affect the expression of other major isoforms of starch branching enzymes or starch synthases. Structural analyses of debranched starch showed that the doubling of apparent amylose content was not due to an increase in the relative proportion of amylose chains but instead was due to significantly elevated levels of long amylopectin and intermediate chains. Rices altered by the amiRNA technique produced a more extreme starch phenotype than those modified using the hp-RNA technique, with a greater increase in the proportion of long amylopectin and intermediate chains. The more pronounced starch structural modifications produced in the amiRNA lines led to more severe alterations in starch granule morphology and crystallinity as well as digestibility of freshly cooked grains. The potential role of attenuating SBEIIb expression in generating starch with elevated levels of resistant starch and lower glycaemic index is discussed.

Over-expression of specific HvCslF cellulose synthase-like genes in transgenic barley increases the levels of cell wall (1,3;1,4)-ß-d-glucans and alters their fine structure.

Cell walls in commercially important cereals and grasses are characterized by the presence of (1,3;1,4)-ß-d-glucans. These polysaccharides are beneficial constituents of human diets, where they can reduce the risk of hypercholesterolemia, type II diabetes, obesity and colorectal cancer. The biosynthesis of cell wall (1,3;1,4)-ß-d-glucans in the Poaceae is mediated, in part at least, by the cellulose synthase-like CslF family of genes. Over-expression of the barley CslF6 gene under the control of an endosperm-specific oat globulin promoter results in increases of more than 80% in (1,3;1,4)-ß-d-glucan content in grain of transgenic barley. Analyses of (1,3;1,4)-ß-d-glucan fine structure indicate that individual CslF enzymes might direct the synthesis of (1,3;1,4)-ß-d-glucans with different structures. When expression of the CslF6 transgene is driven by the Pro35S promoter, the transgenic lines have up to sixfold higher levels of (1,3;1,4)-ß-d-glucan in leaves, but similar levels as controls in the grain. Some transgenic lines of Pro35S:CslF4 also show increased levels of (1,3;1,4)-ß-d-glucans in grain, but not in leaves. Thus, the effects of CslF genes on (1,3;1,4)-ß-d-glucan levels are dependent not only on the promoter used, but also on the specific member of the CslF gene family that is inserted into the transgenic barley lines. Altering (1,3;1,4)-ß-d-glucan levels in grain and vegetative tissues will have potential applications in human health, where (1,3;1,4)-ß-d-glucans contribute to dietary fibre, and in tailoring the composition of biomass cell walls for the production of bioethanol from cereal crop residues and grasses.

The genetics and transcriptional profiles of the cellulose synthase-like HvCslF gene family in barley.

Cellulose synthase-like CslF genes have been implicated in the biosynthesis of (1,3;1,4)-beta-d-glucans, which are major cell wall constituents in grasses and cereals. Seven CslF genes from barley (Hordeum vulgare) can be divided into two classes on the basis of intron-exon arrangements. Four of the HvCslF genes have been mapped to a single locus on barley chromosome 2H, in a region corresponding to a major quantitative trait locus for grain (1,3;1,4)-beta-d-glucan content. The other HvCslF genes map to chromosomes 1H, 5H, and 7H, and in two cases the genes are close to other quantitative trait loci for grain (1,3;1,4)-beta-d-glucan content. Spatial and temporal patterns of transcription of the seven genes have been defined through quantitative polymerase chain reaction. In developing barley coleoptiles HvCslF6 mRNA is most abundant. Transcript levels are maximal in 4- to 5-d coleoptiles, at a time when (1,3;1,4)-beta-d-glucan content of coleoptile cell walls also reaches maximal levels. In the starchy endosperm of developing grain, HvCslF6 and HvCslF9 transcripts predominate. Two peaks of transcription are apparent. One occurs just after endosperm cellularization, 4 to 8 d after pollination, while the second occurs much later in grain development, more than 20 d after pollination. Marked varietal differences in transcription of the HvCslF genes are observed during endosperm development. Given the commercial importance of cereal (1,3;1,4)-beta-d-glucans in human nutrition, in stock feed, and in malting and brewing, the observation that only two genes, HvCslF6 and HvCslF9, are transcribed at high levels in developing grain is of potential relevance for the future manipulation of grain (1,3;1,4)-beta-d-glucan levels.

Immunomodulation of enzyme function in plants by single-domain antibody fragments.

Immunomodulation involves the use of antibodies to alter the function of molecules and is an emerging tool for manipulating both plant and animal systems. To realize the full potential of this technology, two major obstacles must be overcome. First, most antibodies do not function well intracellularly because critical disulfide bonds cannot form in the reducing environment of the cytoplasm or because of difficulties in targeting to subcellular organelles. Second, few antibodies bind to the active sites of enzymes and thus they generally do not neutralize enzyme function. Here we show that the unique properties of single-domain antibodies from camelids (camels and llamas) can circumvent both these obstacles. We demonstrate that these antibodies can be correctly targeted to subcellular organelles and inhibit enzyme function in plants more efficiently than antisense approaches. The use of these single-domain antibody fragments may greatly facilitate the successful immunomodulation of metabolic pathways in many organisms.