Unraveling Betalain Suppression in Pitaya: Insights from Co-activator HuMYB9 Binding at HuCYP76AD1-1, HuADH1, and HuDODA1 Super-Enhancers

Abstract Myeloblastosis (MYB) proteins, recognized as crucial transcriptional regulators, play an integral role in orchestrating the range of plant colors. Pitaya (Hylocereus spp.), a distinctive commercially cultivated fruit, stands apart due to its unique betalains, water-soluble nitrogen-enriched pigments that confer its vibrant hues. Although betalain biosynthesis has been extensively explored, the potential influence of co-activators that might disrupt pigment production remains relatively unexplored. In this study we found that HuMYB9, an R2R3-MYB repressor, exhibited a remarkable decreasing during pitaya maturation. Sequence alignment analyses showed conserved R2 and R3 domains within the C-terminal of HuMYB9. HuMYB9's regulatory activities were found to be nuclear localized and it interacted with specific elements within the promoters of HuDODA1, HuADH1 and HuCYP76AD1-1, thus influencing the transcriptional activities in vitro. HuMYB9 transiently down-regulated the expressions of key betalain biosynthetic genes with a corresponding effect on the levels of pitaya pulp betalains. Our results propose that HuMYB9 operates as a suppressor, specifically down-regulating the expression of HuCYP76AD1-1, HuDODA1 and HuADH, thereby modulating betalain biosynthesis in pitaya. Collectively, our findings provide invaluable insights on the regulation of betalain accumulation in pitaya.