Targeted mutation of barley (1,3;1,4)‐β‐glucan synthases reveals complex relationships between the storage and cell wall polysaccharide content
Garcia‐Gimenez, Guillermo | Barakate, Abdellah | Smith, Pauline | Stephens, Jennifer | Khor, Shi F. | Doblin, Monika S. | Hao, Pengfei | Bacic, A. (Antony) | Fincher, Geoffrey B. | Burton, Rachel A. | Waugh, Robbie | Tucker, Matthew R. | Houston, Kelly
Barley (Hordeum vulgare L) grain is comparatively rich in (1,3;1,4)‐β‐glucan, a source of fermentable dietary fibre that protects against various human health conditions. However, low grain (1,3;1,4)‐β‐glucan content is preferred for brewing and distilling. We took a reverse genetics approach, using CRISPR/Cas9 to generate mutations in members of the Cellulose synthase‐like (Csl) gene superfamily that encode known (HvCslF6 and HvCslH1) and putative (HvCslF3 and HvCslF9) (1,3;1,4)‐β‐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)‐β‐glucan content. (1,3;1,4)‐β‐Glucan was absent in the grain of cslf6 knockout lines, whereas cslf9 knockout lines had similar (1,3;1,4)‐β‐glucan content to wild‐type (WT). However, cslf9 mutants 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 in cslf6 knockouts, and to a lesser extent TGW in cslf9 knockouts. cslf3 and cslh1 mutants had no effect on grain (1,3;1,4)‐β‐glucan content. Our data indicate that multiple members of the CslF/H family fulfil important functions during grain development but, with the exception of HvCslF6, do not impact the abundance of (1,3;1,4)‐β‐glucan in mature grain.
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