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

(1,3;1,4)-β-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)-β-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. is the main gene contributing to (1,3;1,4)-β-glucan biosynthesis in the grain. Here, the transcriptional regulation of was investigated using an 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 promoter region. Dual luciferase assays based on multiple deletion constructs revealed the promoter fragment driving expression. Highest 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: and as putative activators of expression. Gene network analyses assigned to the same co-expression module as and other primary cellulose synthases (, , and ), whereas was assigned to a different module. Based on RNA-seq expression during grain development, was cloned and tested in the protoplast system. The transient over-expression of in barley protoplasts suggested a positive regulatory effect on expression.