Regulation of (1,3;1,4)-β-glucan synthesis in barley (Hordeum vulgare L.)

(1,3;1,4)-β-Glucan is the most abundant non-cellulosic polysaccharide that accumulates in the cell walls of barley grain. This polysaccharide is of importance because barley grains contain higher levels of (1,3;1,4)-β-glucan compared to other small grain cereals. Grain (1,3;1,4)-β-glucan concentration influences the use of barley, having undesirable effects on brewing and distilling and beneficial effects linked to human health. These health benefits are because (1,3;1,4)-β-glucan is a soluble dietary fibre, reducing the risk of developing cardiovascular disease, colorectal cancer and type II diabetes. Previous research identified members of the Cellulose synthase-like HvCslF/H gene families as (1,3;1,4)-β-glucan synthases differentially expressed across barley tissues (Burton et al., 2006; Doblin et al., 2009). Amongst them, HvCslF6 is thought to be the main driver of (1,3;1,4)-β-glucan biosynthesis in the grain (Burton et al., 2011) with other putative (1,3;1,4)-β-glucan synthases, remodelling and hydrolytic enzymes potentially affecting variation in (1,3;1,4)-β-glucan content. Despite limited sequence variation in the HvCslF6 CDS and promoter, this gene and HvGlbI, a putative (1,3;1,4)-β-glucan endohydrolase, were found to be differentially expressed across elite barleys exhibiting different levels of grain (1,3;1,4)-β-glucan. The transcriptional regulation of HvCslF6 was investigated by its putative promoter region in a barley protoplast transient expression system. Dual luciferase assays based on multiple HvCslF6 deletion constructs revealed the essential promoter fragment driving HvCslF6 expression. This finding was combined with an in silico analysis of putative TF binding sites which allowed the identification of three candidate MYB TFs. The transient overexpression of HvMyb61 in barley protoplasts suggested positive regulatory effect on HvCslF6 expression. In addition, the functional characterization of CRISPR/Cas9 knockout mutants of putative (1,3;1,4)-β-glucan synthases revealed that HvCslF9 is not essential for (1,3;1,4)-β-glucan synthesis in the grain, whereas HvCslF6 disruption led to decreased (1,3;1,4)-β-glucan content and altered grain size and shape. The collection of CRISPR/Cas9-induced mutants generated for HvCslF/H demonstrated the effectiveness of genome editing technology in barley and constitute a useful genetic resource to study barley cell walls.