AUTHOR=Garcia-Gimenez Guillermo , Schreiber Miriam , Dimitroff George , Little Alan , Singh Rohan , Fincher Geoffrey B. , Burton Rachel A. , Waugh Robbie , Tucker Matthew R. , Houston Kelly 

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

JOURNAL=Frontiers in Plant Science

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.883139

DOI=10.3389/fpls.2022.883139

ISSN=1664-462X

ABSTRACT=<p>(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. <italic>HvCslF6</italic> is the main gene contributing to (1,3;1,4)-β-glucan biosynthesis in the grain. Here, the transcriptional regulation of <italic>HvCslF6</italic> was investigated using an <italic>in-silico</italic> 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 <italic>HvCslF6</italic> promoter region. Dual luciferase assays based on multiple <italic>HvCslF6</italic> deletion constructs revealed the promoter fragment driving <italic>HvCslF6</italic> expression. Highest <italic>HvCslF6</italic> 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: <italic>HvMYB61</italic> and <italic>HvMYB46/83</italic> as putative activators of <italic>HvCslF6</italic> expression. Gene network analyses assigned <italic>HvMYB61</italic> to the same co-expression module as <italic>HvCslF6</italic> and other primary cellulose synthases (<italic>HvCesA1</italic>, <italic>HvCesA2</italic>, and <italic>HvCesA6</italic>), whereas <italic>HvMYB46/83</italic> was assigned to a different module. Based on RNA-seq expression during grain development, <italic>HvMYB61</italic> was cloned and tested in the protoplast system. The transient over-expression of <italic>HvMYB61</italic> in barley protoplasts suggested a positive regulatory effect on <italic>HvCslF6</italic> expression.</p>