Natural variations in the MYB transcription factor MYB31 determine the evolution of extremely pungent peppers

Plants produce countless specialized metabolites crucial for their development and fitness, and many are useful bioactive compounds. Capsaicinoids are intriguing genus-specialized metabolites that confer a pungent flavor to Capsicum fruits, and they are widely applied in different areas. Among the five domesticated Capsicum species, Capsicum chinense has a high content of capsaicinoids, which results in an extremely hot flavor. However, the species-specific upregulation of capsaicinoid-biosynthetic genes (CBGs) and the evolution of extremely pungent peppers are not well understood. We conducted genetic and functional analyses demonstrating that the quantitative trait locus Capsaicinoid1 (Cap1), which is identical to Pun3 contributes to the level of pungency. The Cap1/Pun3 locus encodes the Solanaceae-specific MYB transcription factor MYB31. Capsicum species have evolved placenta-specific expression of MYB31, which directly activates expression of CBGs and results in genus-specialized metabolite production. The capsaicinoid content depends on MYB31 expression. Natural variations in the MYB31 promoter increase MYB31 expression in C. chinense via the binding of the placenta-specific expression of transcriptional activator WRKY9 and augmentation of CBG expression, which promotes capsaicinoid biosynthesis. Our findings provide insights into the evolution of extremely pungent C. chinense, which is due to natural variations in the master regulator, and offers targets for engineering or selecting flavor in Capsicum.