Characterization of lys3 mutants in three genetic backgrounds of Hordeum vulgare identifies the genes downstream of barley prolamin-box binding factor (BPBF)

Authors: Vinje, Marcus; Simmons, Carl

Submitted to: Molecular Genetics and Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/24/2023
Publication Date: 2/28/2024
Citation: Vinje, M.A., Simmons, C.H. 2024. Characterization of lys3 mutants in three genetic backgrounds of Hordeum vulgare identifies the genes downstream of barley prolamin-box binding factor (BPBF). Molecular Genetics and Genomics. 299, Article 17. https://doi.org/10.1007/s00438-024-02112-x.
DOI: https://doi.org/10.1007/s00438-024-02112-x

Interpretive Summary: The Lys3 locus in barley controls the expression of genes that influence numerous malting quality traits such as beta-glucan content, diastatic power, and protein levels. A mutant in this locus, termed lys3a, is known to downregulate the expression of a beta-amylase gene and many seed storage protein genes lowering diastatic power and protein levels. The underlying genetic causes responsible for the reduction of gene expression has not been completely elucidated. Previous research indicates that demethylation is required for endosperm gene expression and the lsy3a mutants are deficient in this enzyme that demethylates promoters in the developing endosperm allowing for gene transcription. The methylation status of the entire genome was determined to look for differences between three parents (Bomi, Bowman, Sloop) and five mutants (3 lys3a mutants, lys3b, and lys3c). There was no difference in the global methylation pattern between the parents and mutants. Looking specifically at the methylation patterns of CG regions in the promoter of selected highly expressed differentially expressed genes did not reveal any methylation trends between parents and the lys3a mutants that appeared to cause the observed downregulation in gene expression. Global gene expression was determined using next-generation sequencing and common differentially expressed genes were identified between all mutants and their parents. For several genes, the expression trends were the same between all three lys3a mutants. However, for other genes the expression trend was the same for Sloop and Bowman lys3a but opposite for Bomi lys3a. Furthermore, the causal mutation for the lys3a mutant is in the barley prolamin-binding factor gene and despite not carrying the causal lys3a mutation, the Bowman lys3a mutant still followed the same trend for numerous differentially expressed genes. These results revealed that the genetic background plays a large role in the way the lys3a mutant affects gene expression and that other mutations besides the SNP in the BPBF gene may also be playing a role.

Technical Abstract: The Lys3 locus in barley controls hordein (Hor1 and Hor2) and beta-amylase (Bmy1) gene expression in the developing endosperm and subsequently affects several malting quality traits due to these gene expression changes. There are three lys3 mutant alleles found in the Danish cultivar, Bomi - lys3a (Risø 1508), lys3b (Risø 18), and lys3c (Risø 19). Also, the lys3a locus has been introgressed into an Australian malting cultivar (Sloop) and a U.S. non-malting cultivar (Bowman). It is surmised that the lys3a locus inhibits the demethylation of the Hor2 promoter causing hypermethylation subsequently inhibiting gene expression. Recently, the causal mutation of the lys3a phenotype was determined to be caused by a SNP in the barley prolamin-binding factor gene (BPBF). Because of the similar gene expression patterns between Hor2 and Bmy1 in the lys3a mutants, we hypothesize that a similar problem with demethylation was occurring in the Bmy1 promoter. To test this hypothesis and to determine the downstream genes affected by the causal mutation in the BPBF gene, whole-genome bisulfite sequencing (WGBS) and mRNAseq were performed on developing endosperms from five lys3 mutants and their three parents. Overall, global methylation patterns were not different in the lys3 mutants and promoter methylation levels from Bmy1 did not explain differences in Bmy1 gene expression between the mutants and parents. There were 29 differentially expressed genes (DEGs) common between all the mutants and their parents and 118 DEGs common between all the lys3a mutants and their parents. Seed storage protein and Bmy1 gene expression in lys3a mutants were typically down-regulated but the other DEGs identified were predominantly up-regulated (e.g. beta-glucosidase, thaumatin-like protein) suggesting compensatory effects. The Bowman lys3a mutant line did not contain the causal SNP in the BPBF gene but still followed the same gene expression trends as the Sloop and Bomi lines carrying the lys3a locus. Furthermore, Sloop lys3a mutants followed the same gene expression trend as Bowman lys3a for several genes that had opposite patterns of expression in the Bomi lys3a. These analyses demonstrate that the genetic background plays an important role in lys3a mutants, and other SNPs might be responsible for the lys3a phenotype.