Characterization of barley (Horduem vulgare) lys3 mutants identifies genes under the regulation of the prolamin-box binding transcription factor and elucidates its role in endosperm promoter methylation during grain development

Abstract

Abstract The barley Lys3 locus controls hordein (Hor1 and Hor2) and b-amylase (Bmy1) gene expression in the developing endosperm and affects several malting quality traits due to these and other gene expression changes. The Lys3 locus was determined to regulate Bmy1, Hor1, and Hor2 genes using a chemically induced mutant from the Danish cultivar Bomi. The causal mutation in lys3.a mutants is a SNP in the barley prolamin-box binding factor gene (BPBF). It is hypothesized that the lys3.a locus inhibits demethylation at the Hor2 promoter causing hypermethylation that subsequently inhibits gene expression. Because of the similar gene expression patterns between Hor2 and Bmy1 in the lys3.a mutants, we hypothesize that hypermethylation was occurring in the Bmy1 promoter. To test this hypothesis and to determine the downstream genes affected by the lys3.a mutation, whole-genome bisulfite sequencing (WGBS) and mRNA-seq were performed on developing endosperms from two lys3 mutants (lys3.a from Risø 1508; lys3.b from Risø 18) and their parent Bomi along with the lys3.a locus introgressed into Sloop, an Australian malting cultivar. Overall, global and genic DNA methylation levels were not significantly different between the lys3 mutants and parents. Promoter DNA methylation levels did not explain differences in gene expression between mutants and their parents. RNAseq analysis identified 306 differentially expressed genes (DEGs) shared between all mutants and their parents and 185 DEGs shared between both lys3.a mutants and their parents. The majority of DEGs were downregulated (e.g., B- and C-hordeins and Bmy1), but some DEGs were upregulated (e.g., b-glucosidase, D-hordein) suggesting compensatory effects and potentially explaining the low β-glucan phenotype observed in lys3.a germplasm.