Targeted mutation of barley (1,3;1,4)-β-glucan synthases reveals complex relationships between the storage and cell wall polysaccharide content.

Barley (Hordeum vulgareL) grain is comparatively rich in (1,3;1,4)-beta-glucan, a source of fermentable dietary fibre that protects against various human health conditions. However, low grain (1,3;1,4)-beta-glucan content is preferred for brewing and distilling. We took a reverse genetics approach, using CRISPR/Cas9 to generate mutations in members of theCellulose synthase-like(Csl) gene superfamily that encode known (HvCslF6andHvCslH1) and putative (HvCslF3andHvCslF9) (1,3;1,4)-beta-glucan synthases. Resultant mutations ranged from single amino acid (aa) substitutions to frameshift mutations causing premature stop codons, and led to specific differences in grain morphology, composition and (1,3;1,4)-beta-glucan content. (1,3;1,4)-beta-Glucan was absent in the grain ofcslf6knockout lines, whereascslf9knockout lines had similar (1,3;1,4)-beta-glucan content to wild-type (WT). However,cslf9mutants showed changes in the abundance of other cell-wall-related monosaccharides compared with WT. Thousand grain weight (TGW), grain length, width and surface area were altered incslf6knockouts, and to a lesser extent TGW incslf9knockouts. cslf3andcslh1mutants had no effect on grain (1,3;1,4)-beta-glucan content. Our data indicate that multiple members of theCslF/Hfamily fulfil important functions during grain development but, with the exception ofHvCslF6,do not impact the abundance of (1,3;1,4)-beta-glucan in mature grain.