A single amino acid substitution in the R2R3 conserved domain of the BrPAP1a transcription factor impairs anthocyanin production in turnip (Brassica rapa subsp. rapa)

Abstract

The purple pigmentation in the epidermis of swollen roots of ‘Tsuda’ turnip (Brassica rapa subsp. rapa) is induced by light, providing a good system to investigate the genetic mechanism of light-dependent anthocyanin biosynthesis in B. rapa. Here, we identified the R2R3 MYB transcription factor gene PRODUCTION OF ANTHOCYANIN PIGMENT1 (BrPAP1a) as the critical gene in the anthocyanin-defective mutant w68. A nucleotide mutation in the turn region of the R3 domain was screened, which caused an amino acid substitution from glycine to serine (G94S). Functional analysis showed that the interaction of BrPAP1a with two bHLH factors ENHANCER OF GLABRA 3 (BrEGL3) and TRANSPARENT TESTA 8 (BrTT8) were impaired by the mutation. Expression of BrTT8 was activated by BrPAP1a and enhanced by MYB-bHLH-WDR (MBW) complexes, but blocked by the mutation. Furthermore, BrPAP1a directly bound the MYB-recognizing element (MRE) in the BrTT8 promoter, while the G94S substitution caused a loss of DNA-binding activity. Our findings indicate that G94 is required for protein interaction with BrTT8 and BrEGL3 and DNA-binding of BrPAP1a to activate BrTT8 expression, which leads to anthocyanin biosynthesis. Collectively, our data indicate the importance of the highly conserved amino acids within R2R3 MYB proteins in regulating anthocyanin biosynthesis and could aid programs to increase anthocyanins in turnip roots.