Dissecting the genome-wide genetic variants of milling and appearance quality traits in rice.

Higher head rice yield (HRY), which represents the proportion of intact grains that survive milling, and lower grain chalkiness (opacity) are key quality traits. We investigated the genetic basis of HRY and chalkiness in 320 diverse resequenced accessions of indica rice with integrated single- and multi-locus genome-wide association studies using 2.26 million single-nucleotide polymorphisms. We identified novel haplotypes that underly higher HRY on chromosomes 3, 6, 8, and 11, and that lower grain chalkiness in a fine-mapped region on chromosome 5. Whole-genome sequencing of 92 IRRI breeding lines was performed to identify the genetic variants of HRY and chalkiness. Rare and novel haplotypes were found for lowering chalkiness, but missing alleles hindered progress towards enhancing HRY in breeding material. The novel haplotypes that we identified have potential use in breeding programs aimed at improving these important traits in the rice crop.

Integrating a genome-wide association study with a large-scale transcriptome analysis to predict genetic regions influencing the glycaemic index and texture in rice.

Reliably generating rice varieties with low glycaemic index (GI) is an important nutritional intervention given the high rates of Type II diabetes incidences in Asia where rice is staple diet. We integrated a genome-wide association study (GWAS) with a transcriptome-wide association study (TWAS) to determine the genetic basis of the GI in rice. GWAS utilized 305 re-sequenced diverse indica panel comprising ~2.4 million single nucleotide polymorphisms (SNPs) enriched in genic regions. A novel association signal was detected at a synonymous SNP in exon 2 of LOC_Os05g03600 for intermediate-to-high GI phenotypic variation. Another major hotspot region was predicted for contributing intermediate-to-high GI variation, involves 26 genes on chromosome 6 (GI6.1). These set of genes included GBSSI, two hydrolase genes, genes involved in signalling and chromatin modification. The TWAS and methylome sequencing data revealed cis-acting functionally relevant genetic variants with differential methylation patterns in the hot spot GI6.1 region, narrowing the target to 13 genes. Conversely, the promoter region of GBSSI and its alternative splicing allele (G allele of Wxa ) explained the intermediate-to-high GI variation. A SNP (C˃T) at exon-10 was also highlighted in the preceding analyses to influence final viscosity (FV), which is independent of amylose content/GI. The low GI line with GC haplotype confirmed soft texture, while other two low GI lines with GT haplotype were characterized as hard and cohesive. The low GI lines were further confirmed through clinical in vivo studies. Gene regulatory network analysis highlighted the role of the non-starch polysaccharide pathway in lowering GI.

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.

Engineering high α-amylase levels in wheat grain lowers Falling Number but improves baking properties.

Late maturity α-amylase (LMA) and preharvest sprouting (PHS) are genetic defects in wheat. They are both characterized by the expression of specific isoforms of α-amylase in particular genotypes in the grain prior to harvest. The enhanced expression of α-amylase in both LMA and PHS results in a reduction in Falling Number (FN), a test of gel viscosity, and subsequent downgrading of the grain, along with a reduced price for growers. The FN test is unable to distinguish between LMA and PHS; thus, both defects are treated similarly when grain is traded. However, in PHS-affected grains, proteases and other degradative process are activated, and this has been shown to have a negative impact on end product quality. No studies have been conducted to determine whether LMA is detrimental to end product quality. This work demonstrated that wheat in which an isoform α-amylase (TaAmy3) was overexpressed in the endosperm of developing grain to levels of up to 100-fold higher than the wild-type resulted in low FN similar to those seen in LMA- or PHS-affected grains. This increase had no detrimental effect on starch structure, flour composition and enhanced baking quality, in small-scale 10-g baking tests. In these small-scale tests, overexpression of TaAmy3 led to increased loaf volume and Maillard-related browning to levels higher than those in control flours when baking improver was added. These findings raise questions as to the validity of the assumption that (i) LMA is detrimental to end product quality and (ii) a low FN is always indicative of a reduction in quality. This work suggests the need for a better understanding of the impact of elevated expression of specific α-amylase on end product quality.

Suppression of glucan, water dikinase in the endosperm alters wheat grain properties, germination and coleoptile growth.

Starch phosphate ester content is known to alter the physicochemical properties of starch, including its susceptibility to degradation. Previous work producing wheat (Triticum aestivum) with down-regulated glucan, water dikinase, the primary gene responsible for addition of phosphate groups to starch, in a grain-specific manner found unexpected phenotypic alteration in grain and growth. Here, we report on further characterization of these lines focussing on mature grain and early growth. We find that coleoptile length has been increased in these transgenic lines independently of grain size increases. No changes in starch degradation rates during germination could be identified, or any major alteration in soluble sugar levels that may explain the coleoptile growth modification. We identify some alteration in hormones in the tissues in question. Mature grain size is examined, as is Hardness Index and starch conformation. We find no evidence that the increased growth of coleoptiles in these lines is connected to starch conformation or degradation or soluble sugar content and suggest these findings provide a novel means of increasing coleoptile growth and early seedling establishment in cereal crop species.

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.

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.

Protein-protein interactions among enzymes of starch biosynthesis in high-amylose barley genotypes reveal differential roles of heteromeric enzyme complexes in the synthesis of A and B granules.

The present study investigated the role of protein phosphorylation, and protein complex formation between key enzymes of amylopectin synthesis, in barley genotypes exhibiting "high amylose" phenotypes. Starch branching enzyme (SBE) down-regulated lines (ΔSBEIIa and ΔSBEIIb), starch synthase (SS)IIa (ssiia(-), sex6) and SSIII (ssiii(-), amo1) mutants were compared to a reference genotype, OAC Baxter. Down-regulation of either SBEIIa or IIb caused pleiotropic effects on SSI and starch phosphorylase (SP) and resulted in formation of novel protein complexes in which the missing SBEII isoform was substituted by SBEI and SP. In the ΔSBEIIb down-regulated line, soluble SP activity was undetectable. Nonetheless, SP was incorporated into a heteromeric protein complex with SBEI and SBEIIa and was readily detected in starch granules. In amo1, unlike other mutants, the data suggest that both SBEIIa and SBEIIb are in a protein complex with SSI and SSIIa. In the sex6 mutant no protein complexes involving SBEIIa or SBEIIb were detected in amyloplasts. Studies with Pro-Q Diamond revealed that GBSS, SSI, SSIIa, SBEIIb and SP are phosphorylated in their granule bound state. Alteration in the granule proteome in ΔSBEIIa and ΔSBEIIb lines, suggests that different protein complexes are involved in the synthesis of A and B granules.

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.

Resistant starch alters colonic contractility and expression of related genes in rats fed a Western diet.

BACKGROUND AND AIM: Dietary fiber shortens gut transit time, but data on the effects of fiber components (including resistant starch, RS) on intestinal contractility are limited. We have examined RS effects in male Sprague-Dawley rats fed either a high-amylose maize starch (HAMS) or a wholemeal made from high-amylose wheat (HAW) on ileal and colonic contractility ex vivo and expression of genes associated with smooth muscle contractility. METHODS: Rats were fed diets containing 19 % fat, 20 % protein, and either low-amylose maize starch (LAMS), HAMS, wholemeal low-amylose wheat (LAW) or HAW for 11 week. Isolated ileal and proximal colonic sections were induced to contract electrically, or by receptor-independent (KCl) or receptor-dependent agents. Colonic gene expression was assessed using an Affymetrix microarray. RESULTS: Ileal contractility was unaffected by treatment. Maximal proximal colonic contractility induced electrically or by angiotensin II or carbachol was lower for rats fed HAMS and LAW relative to those fed LAMS (P < 0.05). The colonic expression of genes, including cholinergic receptors (Chrm2, Chrm3), serotonin receptors (Htr5a, Htr7), a protease-activated receptor (F2r), a prokineticin receptor (Prokr1), prokineticin (Prok1), and nitric oxide synthase 2 (Nos2), was altered by dietary HAMS relative to LAMS (P < 0.05). HAW did not significantly affect these genes or colonic contractility relative to effects of LAMS. CONCLUSIONS: RS and other fiber components could influence colorectal health through modulation of stool transit time via effects on muscular contractility.

Molecular, mesoscopic and microscopic structure evolution during amylase digestion of extruded maize and high amylose maize starches.

Extrusion processing of cereal starch granules with high (>50%) amylose content is a promising approach to create nutritionally desirable resistant starch, i.e. starch that escapes digestion in the small intestine. Whilst high amylose content seems to be required, the structural features responsible for the slow digestion of extrudates are not fully understood. We report the effects of partial enzyme digestion of extruded maize starches on amylopectin branch length profiles, double and single helix contents, crystallinity and lamellar periodicity. Comparing results for three extruded maize starches (27, 57, and 84% apparent amylose) that differ in amylase-sensitivity allows conclusions to be drawn concerning the rate-determining features operating under the digestion conditions used. Enzyme resistance is shown to originate from a combination of molecular and mesoscopic factors, including both recrystallization and an increase in very short branches during the digestion process. This is in contrast to the behaviour of the same starches in the granular form (Shrestha et al., 2012) where molecular and mesoscopic factors are secondary to microscopic structures in determining enzyme susceptibility. Based on the structure of residual material after long-time digestion (>8h), a model for resistant starch from processed high amylose maize starches is proposed based on a fringed micelle structure with lateral aggregation and enzyme susceptibility both limited by attached clusters of branch points.

Engineering α-amylase levels in wheat grain suggests a highly sophisticated level of carbohydrate regulation during development.

Wheat starch degradation requires the synergistic action of different amylolytic enzymes. Our spatio-temporal study of wheat α-amylases throughout grain development shows that AMY3 is the most abundant isoform compared with the other known α-amylases. Endosperm-specific over-expression of AMY3 resulted in an increase of total α-amylase activity in harvested grains. Unexpectedly, increased activity did not have a significant impact on starch content or composition but led to an increase of soluble carbohydrate (mainly sucrose) in dry grain. In AMY3 overexpression lines (A3OE), germination was slightly delayed and triacylglycerol (TAG) content was increased in the endosperm of mature grain. Despite increased AMY3 transcript and protein content throughout grain development, alterations of α-amylase activity and starch granule degradation were not detected until grain maturation, suggesting a post-translational inhibition of α-amylase activity in the endosperm during the starch filling period. These findings show unexpected effects of a high level of α-amylase on grain development and composition, notably in carbon partitioning and TAG accumulation, and suggest the presence of a hitherto unknown regulatory pathway during grain filling.

New perspectives on the role of α- and ß-amylases in transient starch synthesis.

Transient starch in leaves is synthesized by various biosynthetic enzymes in the chloroplasts during the light period. This paper presents the first mathematical model for the (bio)synthesis of the chain-length distribution (CLD) of transient starch to aid the understanding of this synthesis. The model expresses the rate of change of the CLD in terms of the actions of the enzymes involved. Using this to simulate the experimental CLD with different enzyme combinations is a new means to test for enzymes that are significant to the rate of change of the CLD during synthesis. Comparison between the simulated CLD from different enzyme combinations and the experimental CLD in the leaves of the model plant Arabidopsis thaliana indicate α-amylase, in addition to the core starch biosynthetic enzymes, is also involved in the modification of glucans for the synthesis of insoluble starch granules. The simulations suggest involvement of ß-amylase, in the absence of α-amylase in mutants, slows the rate of attaining a crystalline-competent CLD for crystallization of glucans to form insoluble starch. This suggests a minor role of ß-amylase in shaping normal starch synthesis. The model simulation predicts that debranching of glucans is an efficient mechanism for the attainment of crystalline-competent CLD; however, attaining this is still possible, albeit slower, through combinations of α- and ß-amylase in the absence of isoamylase-type debranching enzyme. In Arabidopsis defective in one of the isoamylase-type debranching enzymes, the impact of α-amylase in starch synthesis is reduced, while ß-amylase becomes significantly involved, slowing the rate of synthesis in this mutant. Modeling of transient starch CLD brings to light previously unrecognized but significant effects of α- and ß-amylase on the rate of transient starch synthesis.

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.

Use of a large multiparent wheat mapping population in genomic dissection of coleoptile and seedling growth.

Identification of alleles towards the selection for improved seedling vigour is a key objective of many wheat breeding programmes. A multiparent advanced generation intercross (MAGIC) population developed from four commercial spring wheat cultivars (cvv. Baxter, Chara, Westonia and Yitpi) and containing ca. 1000 F(2) -derived, F(6:7) RILs was assessed at two contrasting soil temperatures (12 and 20 °C) for shoot length and coleoptile characteristics length and thickness. Narrow-sense heritabilities were high for coleoptile and shoot length (h(2) = 0.68-0.70), indicating a strong genetic basis for the differences among progeny. Genotypic variation was large, and distributions of genotype means were approximately Gaussian with evidence for transgressive segregation for all traits. A number of significant QTL were identified for all early growth traits, and these were commonly repeatable across the different soil temperatures. The largest negative effects on coleoptile lengths were associated with Rht-B1b (-8.2%) and Rht-D1b (-10.9%) dwarfing genes varying in the population. Reduction in coleoptile length with either gene was particularly large at the warmer soil temperature. Other large QTL for coleoptile length were identified on chromosomes 1A, 2B, 4A, 5A and 6B, but these were relatively smaller than allelic effects at the Rht-B1 and Rht-D1 loci. A large coleoptile length effect allele (a = 5.3 mm at 12 °C) was identified on chromosome 1AS despite the relatively shorter coleoptile length of the donor Yitpi. Strong, positive genetic correlations for coleoptile and shoot lengths (r(g) = 0.85-0.90) support the co-location of QTL for these traits and suggest a common physiological basis for both. The multiparent population has enabled the identification of promising shoot and coleoptile QTL despite the potential for the confounding of large effect dwarfing gene alleles present in the commercial parents. The incidence of these alleles in commercial wheat breeding programmes should facilitate their ready implementation in selection of varieties with improved establishment and early growth.

A parameterized model of amylopectin synthesis provides key insights into the synthesis of granular starch.

A core set of genes involved in starch synthesis has been defined by genetic studies, but the complexity of starch biosynthesis has frustrated attempts to elucidate the precise functional roles of the enzymes encoded. The chain-length distribution (CLD) of amylopectin in cereal endosperm is modeled here on the basis that the CLD is produced by concerted actions of three enzyme types: starch synthases, branching and debranching enzymes, including their respective isoforms. The model, together with fitting to experiment, provides four key insights. (1) To generate crystalline starch, defined restrictions on particular ratios of enzymatic activities apply. (2) An independent confirmation of the conclusion, previously reached solely from genetic studies, of the absolute requirement for debranching enzyme in crystalline amylopectin synthesis. (3) The model provides a mechanistic basis for understanding how successive arrays of crystalline lamellae are formed, based on the identification of two independent types of long amylopectin chains, one type remaining in the amorphous lamella, while the other propagates into, and is integral to the formation of, an adjacent crystalline lamella. (4) The model provides a means by which a small number of key parameters defining the core enzymatic activities can be derived from the amylopectin CLD, providing the basis for focusing studies on the enzymatic requirements for generating starches of a particular structure. The modeling approach provides both a new tool to accelerate efforts to understand granular starch biosynthesis and a basis for focusing efforts to manipulate starch structure and functionality using a series of testable predictions based on a robust mechanistic framework.

Genome-wide comparative diversity uncovers multiple targets of selection for improvement in hexaploid wheat landraces and cultivars.

Domesticated crops experience strong human-mediated selection aimed at developing high-yielding varieties adapted to local conditions. To detect regions of the wheat genome subject to selection during improvement, we developed a high-throughput array to interrogate 9,000 gene-associated single-nucleotide polymorphisms (SNP) in a worldwide sample of 2,994 accessions of hexaploid wheat including landraces and modern cultivars. Using a SNP-based diversity map we characterized the impact of crop improvement on genomic and geographic patterns of genetic diversity. We found evidence of a small population bottleneck and extensive use of ancestral variation often traceable to founders of cultivars from diverse geographic regions. Analyzing genetic differentiation among populations and the extent of haplotype sharing, we identified allelic variants subjected to selection during improvement. Selective sweeps were found around genes involved in the regulation of flowering time and phenology. An introgression of a wild relative-derived gene conferring resistance to a fungal pathogen was detected by haplotype-based analysis. Comparing selective sweeps identified in different populations, we show that selection likely acts on distinct targets or multiple functionally equivalent alleles in different portions of the geographic range of wheat. The majority of the selected alleles were present at low frequency in local populations, suggesting either weak selection pressure or temporal variation in the targets of directional selection during breeding probably associated with changing agricultural practices or environmental conditions. The developed SNP chip and map of genetic variation provide a resource for advancing wheat breeding and supporting future population genomic and genome-wide association studies in wheat.

Characterization of starch phosphorylases in barley grains.

BACKGROUND: Starch is synthesized in both leaves and storage tissues of plants. The role of starch syntheses and branching enzymes is well understood; however, the role of starch phosphorylase is not clear. RESULTS: A gene encoding Pho1 from barley was characterized and starch phosphorylases from both developing and germinating grain were characterized and purified. Two activities were detected: one with a molecular mass of 110 kDa and the other of 95 kDa. It was demonstrated through the use of antisera that the 110 kDa activity was located in the amyloplast and could correspond to the polypeptide encoded by the Pho1 gene cloned. The 95 kDa activity was localized to the cytoplasm, most strongly expressed in germinating grain, and was classified as a Pho2-type sequence. Using RNAi technology to reduce the content of Pho1 in the grain to less than 30% of wild type did not lead to any visible phenotype, and no dramatic alterations in the structure of the starch were observed. CONCLUSION: Two starch phosphorylase activities were identified and characterized in barley grains, and shown to be present during starch synthesis. However, their role in starch synthesis still remains to be elucidated.

Fructan extracts from wheat stem and barley grain stimulate large bowel fermentation in rats.

The benefits of inulin-type fructans for bowel health are well established, but less so for other fructan sources. In vitro data suggest that fructans extracted from cereals are readily fermented and produce favorable short-chain fatty acid profiles; however, whether this occurs in vivo is unknown. We hypothesized that in rats, fructans extracted from wheat stem and barley grain would have similar effects on fermentation as oligofructose (OF). Fifty-six male Sprague-Dawley rats were randomly assigned to 1 of 7 dietary treatments that contained either 2% or 5% fructan, provided by a barley grain fructan extract (BGFE), a wheat stem fructan extract, or OF or no added fructan (control). The duration of the feeding study was 14 days. Rats fed diets containing 5% fructan had higher cecal digesta weights; larger acetate, propionate, and total short-chain fatty acid pools; and lower pHs in comparison with the control group. In addition, only the 5% OF and 5% BGFE groups increased cecal butyrate pools, and 5% BGFE was the only group in which colonic digesta pH was lower than that of the control. Diets containing 2% fructan did not affect any of these fermentation end points. Whereas bifidobacteria numbers in cecal digesta of 2% and 5% OF were higher than that in the control group, they were not different from those in rats fed diets containing BGFE and wheat stem fructan extract. Barley grain and wheat stem fructans produced similar large bowel fermentation patterns to OF when fed to rats at 5% of the diet.

Glucan affinity of starch synthase IIa determines binding of starch synthase I and starch-branching enzyme IIb to starch granules.

The sugary-2 mutation in maize (Zea mays L.) is a result of the loss of catalytic activity of the endosperm-specific SS (starch synthase) IIa isoform causing major alterations to amylopectin architecture. The present study reports a biochemical and molecular analysis of an allelic variant of the sugary-2 mutation expressing a catalytically inactive form of SSIIa and sheds new light on its central role in protein-protein interactions and determination of the starch granule proteome. The mutant SSIIa revealed two amino acid substitutions, one being a highly conserved residue (Gly522→Arg) responsible for the loss of catalytic activity and the inability of the mutant SSIIa to bind to starch. Analysis of protein-protein interactions in sugary-2 amyloplasts revealed the same trimeric assembly of soluble SSI, SSIIa and SBE (starch-branching enzyme) IIb found in wild-type amyloplasts, but with greatly reduced activities of SSI and SBEIIb. Chemical cross-linking studies demonstrated that SSIIa is at the core of the complex, interacting with SSI and SBEIIb, which do not interact directly with each other. The sugary-2 mutant starch granules were devoid of amylopectin-synthesizing enzymes, despite the fact that the respective affinities of SSI and SBEIIb from sugary-2 for amylopectin were the same as observed in wild-type. The data support a model whereby granule-bound proteins involved in amylopectin synthesis are partitioned into the starch granule as a result of their association within protein complexes, and that SSIIa plays a crucial role in trafficking SSI and SBEIIb into the granule matrix.