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.

Fig. 1.

Frequency distribution of (1,3;1,4)-β-D-glucan content in 104 F₂ plants from the cross between ‘Bowman’ and OUM125. F₂ plants were classified into three classes (mutant homozygous, heterozygous, and wild-type homozygous) according to the genotypes of the HvCslF6 gene.

Fig. 2.

(1,3;1,4)-β-D-Glucan content in the grains of wild-type ‘Nishinohoshi’ (Ni) and mutant bgl-Ni and their reciprocal F₁ hybrids. Bars indicate standard deviations.

Fig. 3.

Plant phenotype after exposure to winter chilling in the field. Left is ‘Nishinohoshi’ (Ni), and right is bgl-Ni.

Fig. 4.

Structure of the HvCslF6 gene, which is predicted to encode 947 amino acid (aa) residues. Mutation points of three mutants (bgl.a, bgl.b, and bgl.c) are indicated. The positions of introns are indicated by the triangles and the lengths of the introns (in base pairs) are indicated within each triangle. The blue boxes show the positions of eight times of trans-membrane helices. The red bars indicate the positions of the D, D, D, QxxRW motifs.

Fig. 5.

Changes of (1,3;1,4)-β-D-glucan synthase activity and the sugar composition of cell walls during development of endosperms of SH84 and bgl-SH84. (A, C) Enzyme activity was determined with microsomes prepared from endosperms of SH84 (A) or bgl-SH84 (C) at different developing stages for 7–35 d after flowering (DAF). The [¹⁴C]Glc transfer products were digested with lichenase, separated by paper chromatography, and analysed with a fluoroimage analyser. Note that the spots detected between tri- (G₄G₃G) and tetrasaccharides (G₄G₄G₃G) are a contaminant contained in the commercial UDP-[¹⁴C]Glc specimen. (B, D) Enzyme activities are expressed based on the fresh weight of endosperms. The sugar composition of the cell walls was analysed as described in the Materials and methods. Endosperms at 7 and 35 DAF were tightly attached to pericarps. Hence, the values shown reflect the involvement of pericarps and are connected to other data by dotted lines. Data for activity and sugar composition are averages of duplicate or triplicate assays.

Fig. 6.

FACE analysis of oligosaccharides released from lichenase-digested cell walls. Wild-type ‘Akashinriki’ (A), mutant OUM125 (B), and KM27 (C) HvCSlF6 genes were transiently expressed in Nicotiana benthamiana leaves and oligosaccharides released from cell wall preparations after lichenase digestion were analysed by 8-amino-1,3,6-pyrenetrisulphonic acid (APTS) fluorescence labelling and separation by capillary electrophoresis. Lichenase digests of barley leaf cell walls from the wild type (‘Akashinriki’, green line) and mutant (OUM125, pink line) are shown for comparison in (D). The degree of polymerization (DP) of the oligosaccharides is indicated. The large peak at the beginning of the trace (4.5 min) and smaller peak at approximately 7.75 min are unlabelled APTS and a non-specific labelled product as they appear in minus lichenase controls (data not shown).

Fig. 7.

Phylogenetic analysis of the natural variation of the HvCslF6 gene in 29 barley accessions. The tree is generated through genomic sequences. The materials are grouped into three clades (I, II, and III). Numbers indicate bootstrap values. Accessions with (1,3;1,4)-β-D-glucan content information from the literature are denoted either by L (low content) or H (high content) after the cultivar name.