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

Abstract

(1,3;1,4)-β-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)-β-D-glucanless (betaglucanless; bgl) mutants of barley which completely lack (1,3;1,4)-β-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)-β-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)-β-D-glucans, whereas the mutant HvCslF6 proteins completely lack the ability to synthesize (1,3;1,4)-β-D-glucans. The fine structure of the (1,3;1,4)-β-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)-β-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)-β-D-glucans in accordance with the purposes of use.

Heterosis and genetic parameters for grain quality in oat segregating populations

“…According to previous reports, the β-glucan content in oat grains appears to be controlled by genes with predominant additive effects (Cervantes- Martinez et al, 2001). In barley, a diploid species, it was suggested that only the gene HvCslF6 has high influence on the synthesis of β-glucans in the grains (Taketa et al, 2012).…”

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

“…In addition to employing the N. benthamiana leaf system to view the subcellular distribution of CSLF6/H1, we also sought to use it as a functional assay system (Taketa et al, 2012;Vega-Sánchez et al, 2012). Given the abnormal accumulation pattern of VENUS-CSLF6 in large aggregates at the periphery of infiltrated epidermal cells (Supplemental Figure 8), we questioned whether the fluorescently tagged CSLF6 protein was catalytically active.…”

“…Protein gel blot analysis confirmed that the intact ;125-kD VENUS-CSLF6 fusion protein accumulated to detectable levels in these leaves ( Figure 10A). Upon digestion of AIR samples with lichenase, a MLG-specific endohydrolase, the diagnostic oligosaccharides of grass MLGs, the tri-(G4G3G R ) and the tetra-(G4G4G3G R ) saccharides, were detected ( Figure 10B), indicating that the VENUStagged CSLF6, like its untagged counterpart (Taketa et al, 2012;Vega-Sánchez et al, 2012), is active in N. benthamiana leaves.…”

“…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.…”

“…It is the most highly expressed CSLF gene in most tissues of barley (Hordeum vulgare), wheat (Triticum aestivum), Brachypodium distachyon, and rice (Oryza sativa), including developing seedling leaf, coleoptiles, and endosperm (Burton et al, 2008;Kimpara et al, 2008;Doblin et al, 2009;Nemeth et al, 2010;Pellny et al, 2012;Vega-Sánchez et al, 2012;Suliman et al, 2013;Trafford et al, 2013;Schreiber et al, 2014). When CSLF6 expression is reduced either by knockdown or knockout via mutation or T-DNA insertion (Tonooka et al, 2009;Nemeth et al, 2010;Taketa et al, 2012;Vega-Sánchez et al, 2012;Hu et al, 2014), a significant reduction in MLG is observed in both vegetative and floral tissues, indicating its gene product is responsible for the synthesis of the majority of MLG in grasses.…”