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

Barley (Hordeum vulgare L) grain is comparatively rich in (1,3;1,4)-ß-glucan, a source of fermentable dietary fibre that protects against various human health conditions. However, low grain (1,3;1,4)-ß-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)-ß-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)-ß-glucan content. (1,3;1,4)-ß-Glucan was absent in the grain of cslf6 knockout lines, whereas cslf9 knockout lines had similar (1,3;1,4)-ß-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)-ß-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)-ß-glucan in mature grain.

Identification of candidate MYB transcription factors that influence CslF6 expression in barley grain.

(1,3;1,4)-ß-Glucan is a non-cellulosic polysaccharide required for correct barley grain fill and plant development, with industrial relevance in the brewing and the functional food sector. Barley grains contain higher levels of (1,3;1,4)-ß-glucan compared to other small grain cereals and this influences their end use, having undesirable effects on brewing and distilling and beneficial effects linked to human health. HvCslF6 is the main gene contributing to (1,3;1,4)-ß-glucan biosynthesis in the grain. Here, the transcriptional regulation of HvCslF6 was investigated using an in-silico analysis of transcription factor binding sites (TFBS) in its putative promoter, and functional characterization in a barley protoplast transient expression system. Based on TFBS predictions, TF classes AP2/ERF, MYB, and basic helix-loop-helix (bHLH) were over-represented within a 1,000 bp proximal HvCslF6 promoter region. Dual luciferase assays based on multiple HvCslF6 deletion constructs revealed the promoter fragment driving HvCslF6 expression. Highest HvCslF6 promoter activity was narrowed down to a 51 bp region located -331 bp to -382 bp upstream of the start codon. We combined this with TFBS predictions to identify two MYB TFs: HvMYB61 and HvMYB46/83 as putative activators of HvCslF6 expression. Gene network analyses assigned HvMYB61 to the same co-expression module as HvCslF6 and other primary cellulose synthases (HvCesA1, HvCesA2, and HvCesA6), whereas HvMYB46/83 was assigned to a different module. Based on RNA-seq expression during grain development, HvMYB61 was cloned and tested in the protoplast system. The transient over-expression of HvMYB61 in barley protoplasts suggested a positive regulatory effect on HvCslF6 expression.

Barley grain (1,3;1,4)-ß-glucan content: effects of transcript and sequence variation in genes encoding the corresponding synthase and endohydrolase enzymes.

The composition of plant cell walls is important in determining cereal end uses. Unlike other widely consumed cereal grains barley is comparatively rich in (1,3;1,4)-ß-glucan, a source of dietary fibre. Previous work showed Cellulose synthase-like genes synthesise (1,3;1,4)-ß-glucan in several tissues. HvCslF6 encodes a grain (1,3;1,4)-ß-glucan synthase, whereas the function of HvCslF9 is unknown. Here, the relationship between mRNA levels of HvCslF6, HvCslF9, HvGlbI (1,3;1,4)-ß-glucan endohydrolase, and (1,3;1,4)-ß-glucan content was studied in developing grains of four barley cultivars. HvCslF6 was differentially expressed during mid (8-15 DPA) and late (38 DPA) grain development stages while HvCslF9 transcript was only clearly detected at 8-10 DPA. A peak of HvGlbI expression was detected at 15 DPA. Differences in transcript abundance across the three genes could partially explain variation in grain (1,3;1,4)-ß-glucan content in these genotypes. Remarkably narrow sequence variation was found within the HvCslF6 promoter and coding sequence and does not explain variation in (1,3;1,4)-ß-glucan content. Our data emphasise the genotype-dependent accumulation of (1,3;1,4)-ß-glucan during barley grain development and a role for the balance between hydrolysis and synthesis in determining (1,3;1,4)-ß-glucan content, and suggests that other regulatory sequences or proteins are likely to be involved in this trait in developing grain.