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

Abstract

Cell walls in commercially important cereals and grasses are characterized by the presence of (1,3;1,4)-β-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)-β-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)-β-d-glucan content in grain of transgenic barley. Analyses of (1,3;1,4)-β-d-glucan fine structure indicate that individual CslF enzymes might direct the synthesis of (1,3;1,4)-β-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)-β-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)-β-d-glucans in grain, but not in leaves. Thus, the effects of CslF genes on (1,3;1,4)-β-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)-β-d-glucan levels in grain and vegetative tissues will have potential applications in human health, where (1,3;1,4)-β-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.

Determining the Subcellular Location of Synthesis and Assembly of the Cell Wall Polysaccharide (1,3; 1,4)-β-d-Glucan in Grasses

“…We propose that these large PM-associated CSLF6 protein aggregates are a consequence of incomplete fusion of secretory vesicles with the PM, with the host cell potentially not having the capacity to correctly deliver CSLF6 protein to the PM and/or regulate its levels (and/or other regulatory components) at this location in the normal manner. Aberrant phenotypes associated with CSLF6 overexpression have also been identified in barley (Burton et al, 2011) implying this is a common occurrence in CSLF6 overexpression studies. However, while CSLF6 cellular distribution is partially disrupted, it is catalytically active as shown by the large ectopic deposits of MLG observed within these regions ( Figure 10C).…”

“…We used multiple experimental approaches to determine the location of the CSLF6 and CSLH1, the catalytic subunits of the MLG synthase Doblin et al, 2009;Burton et al, 2011;Taketa et al, 2012;Vega-Sánchez et al, 2012). Intriguingly, the two classes of CSL proteins showed distinct subcellular locations.…”

The Dynamics of Transcript Abundance during Cellularization of Developing Barley Endosperm

“…Total RNA was prepared from tissue homogenates using a commercially prepared phenol-guanidine reagent (Trizol; Life Technologies, Carlsbad, CA) according to the manufacturer's guidelines and pretreated with the DNA-free kit (Ambion, Austin, TX) prior to its use as the template for cDNA synthesis using Superscript III reverse transcriptase (Life Technologies), as previously described in Burton et al, 2011. …”

Pattern of Deposition of Cell Wall Polysaccharides and Transcript Abundance of Related Cell Wall Synthesis Genes during Differentiation in Barley Endosperm

“…The quantitation of the monosaccharides Xyl, arabinose, and Man was performed after acid hydrolysis by separating the monosaccharides by reversed phase HPLC using a Hypersil C18 5 mm, 2.1 3 250 mm column as described in Burton et al (2011). Twenty-five milligrams of ground, freeze-dried endosperm was hydrolyzed with sulfuric acid (1 M, 1 mL) for 3 h at 100°C, cooled, and centrifuged at 20,000g for 5 min.…”

“…Twenty-five milligrams of ground, freeze-dried endosperm was hydrolyzed with sulfuric acid (1 M, 1 mL) for 3 h at 100°C, cooled, and centrifuged at 20,000g for 5 min. The samples were diluted 20-fold before derivatization with 1-phenyl-3-methyl-5-pyrazolone (Burton et al, 2011). Talose was included with each sample as an internal standard.…”