Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat

(1,3;1,4)-beta-d-Glucan (beta-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative beta-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total beta-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable beta-glucan was also reduced by about 50% in the transgenic lines, and the M(r) distribution of the fraction was decreased from an average of 79 to 85 x 10(4) g/mol in the controls and 36 to 57 x 10(4) g/mol in the transgenics. Immunolocalization of beta-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a beta-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.

The assessment of enriched apoplastic extracts using proteomic approaches

In plant tissues the extracellular environment or apoplast, incorporating the cell wall, is a highly dynamic compartment with a role in many important plant processes including defence, development, signalling and assimilate partitioning. Soluble apoplast proteins from Arabidopsis thaliana, Triticum aestivum and Oryza sativa were separated by two-dimensional electrophoresis. The molecular weights and isoelectric points for the dominant proteins were established prior to excision, sequencing and identification by matrix-assisted laser-desorption ionisation time of flight mass spectrometry (MALDI - TOF MS). From the selected spots, 23 proteins from O. sativa and 25 proteins from A. thaliana were sequenced, of which nine identifications were made in O. sativa (39%) and 14 in A. thaliana (56%). This analysis revealed that: (i) patterns of proteins revealed by two-dimensional electrophoresis were different for each species indicating that speciation could occur at the level of the apoplast, (ii) of the proteins characterised many belonged to diverse families reflecting the multiple functions of the apoplast and (iii), a large number of the apoplast proteins could not be identified indicating that the majority of extracellular proteins are yet to be assigned. The principal proteins identified in the aqueous matrix of the apoplast were involved in defence, i.e. germin-like proteins or glucanases, and cell expansion, i.e. β-D-glucan glucohydrolases. This study has demonstrated that proteomic analysis can be used to resolve the apoplastic protein complement and to identify adaptive changes induced by environmental effectors.

Cell walls of developing wheat starchy endosperm: comparison of composition and RNA-Seq transcriptome

The transcriptome of the developing starchy endosperm of hexaploid wheat (Triticum aestivum) was determined using RNA-Seq isolated at five stages during grain fill. This resource represents an excellent way to identify candidate genes responsible for the starchy endosperm cell wall, which is dominated by arabinoxylan (AX), accounting for 70% of the cell wall polysaccharides, with 20% (1,3; 1,4)-beta-D-glucan, 7% glucomannan, and 4% cellulose. A complete inventory of transcripts of 124 glycosyltransferase (GT) and 72 glycosylhydrolase (GH) genes associated with cell walls is presented. The most highly expressed GT transcript (excluding those known to be involved in starch synthesis) was a GT47 family transcript similar to Arabidopsis (Arabidopsis thaliana) IRX10 involved in xylan extension, and the second most abundant was a GT61. Profiles for GT43 IRX9 and IRX14 putative orthologs were consistent with roles in AX synthesis. Low abundances were found for transcripts from genes in the acyl-coA transferase BAHD family, for which a role in AX feruloylation has been postulated. The relative expression of these was much greater in whole grain compared with starchy endosperm, correlating with the levels of bound ferulate. Transcripts associated with callose (GSL), cellulose (CESA), pectin (GAUT), and glucomannan (CSLA) synthesis were also abundant in starchy endosperm, while the corresponding cell wall polysaccharides were confirmed as low abundance (glucomannan and callose) or undetectable (pectin) in these samples. Abundant transcripts from GH families associated with the hydrolysis of these polysaccharides were also present, suggesting that they may be rapidly turned over. Abundant transcripts in the GT31 family may be responsible for the addition of Gal residues to arabinogalactan peptide.

Modest doses of beta-glucan do not reduce concentrations of potentially atherogenic lipoproteins

BACKGROUND: In 1997, the US Food and Drug Administration passed a unique ruling that allowed oat bran to be registered as the first cholesterol-reducing food at a dosage of 3 g beta-glucan/d. OBJECTIVE: The effects of a low dose of oat bran in the background diet only were investigated in volunteers with mild-to-moderate hyperlipidemia. DESIGN: The study was a double-blind, placebo-controlled, randomized, parallel study. Sixty-two healthy men (n = 31) and women (n = 31) were randomly allocated to consume either 20 g oat bran concentrate (OBC; containing 3 g beta-glucan) or 20 g wheat bran (control) daily for 8 wk. Fasting blood samples were collected at weeks -1, 0, 4, 8, and 12. A subgroup (n = 17) was studied postprandially after consumption of 2 meals (containing no OBC or wheat bran) at baseline and after supplementation. Fasting plasma samples were analyzed for total cholesterol, HDL cholesterol, triacylglycerol, glucose, and insulin. LDL cholesterol was measured by using the Friedewald formula. The postprandial samples were anlayzed for triacylglycerol, glucose, and insulin. RESULTS: No significant difference was observed in fasting plasma cholesterol, LDL cholesterol, glucose, or insulin between the OBC and wheat-bran groups. HDL-cholesterol concentrations fell significantly from weeks 0 to 8 in the OBC group (P = 0.05). There was a significant increase in fasting glucose concentrations after both OBC (P = 0.03) and wheat-bran (P = 0.02) consumption. No significant difference was found between the OBC and wheat-bran groups in any of the postprandial variables measured. CONCLUSIONS: A low dosage of beta-glucan (3 g/d) did not significantly reduce total cholesterol or LDL cholesterol in volunteers with plasma cholesterol concentrations representative of a middle-aged UK population.