HvTT8 Regulates Proanthocyanidin Accumulation in the Barley Testa Layer by Transcriptionally Activating the Flavonoid Biosynthesis Pathway

ABSTRACT Barley accumulates proanthocyanidins (PA), a type of flavonoid compound, in the testa layer. The accumulated PA have a detrimental effect on the quality of beer and pearled barley. Therefore, controlling PA accumulation in the testa layer is an important breeding objective in barley. PA accumulation is regulated by a ternary complex consisting of MYB, basic helix–loop–helix (bHLH), and WD40 transcription factors (MBW complex) in various plant species. The MBW complex regulates the transcription of structural genes encoding enzymes in the flavonoid biosynthesis pathway. Although HvMYB10 and HvWD40‐1 have been identified as regulators of PA accumulation in the barley testa layer, the bHLH transcription factor regulating it remains unclear. In this study, we isolated a novel PA‐defective mutant, TK113, using sodium azide mutagenesis. Expression analysis revealed that the expression levels of multiple structural genes decreased in the seed coat of TK113. Single nucleotide polymorphism (SNP)‐index analysis with a candidate gene approach identified a mutation causing amino acid substitution in HvTT8 , which encodes the bHLH transcription factor, of TK113 associated with the PA‐defective phenotype. The mutation was located in a highly conserved domain among TT8 orthologs, indicating that it is the most likely causal mutation for the PA‐defective phenotype of TK113.