Anthocyanin3 is an R3-MYB repressor that inhibits the anthocyanin pathway in maize

Anthocyanins are colorful molecules receiving recent attention due to their numerous health benefits and applications as natural colorants and nutraceuticals. Purple corn in particular is being investigated as a more economical source of natural colorants. Anthocyanin3 (A3) is a known recessive intensifier of anthocyanin pigmentation in maize. In this study, anthocyanin content was elevated 100-fold in mutant a3 plants. Two genetic approaches were used to pinpoint the gene involved with the a3 intense purple plant phenotype. First, a large-scale transposon-tagging population was created with a Dissociation (Ds) insertion in the nearby Anthocyanin1 gene. A de novo a3-m1::Ds mutant was generated and the transposon insertion was found to be located in Mybr97, which has homology to R3-MYB repressor CAPRICE in Arabidopsis. Second, a bulk segregant RNA-sequencing population found expression differences between pools of green A3 plants and purple mutant a3 plants. All characterized anthocyanin biosynthetic genes were upregulated in mutants along with several genes of the lignin pathway. Mybr97 was highly downregulated in mutant plants, confirming its role as a negative regulator of the anthocyanin pathway. Photosynthesis-related gene expression was reduced in mutant plants through an unknown mechanism. Novel transcription factors and biosynthetic genes were also upregulated and need further investigation. The mechanism of A3 inhibition is most likely through the interaction with basic-helix-loop-helix transcription factors like Booster1. Overall, A3 has a profound effect on the maize plant and reducing its expression has many favorable implications for crop protection, human health, and natural colorant production.