SMXL neofunctionalization and small RNAs underlie the recent evolution of a plant sex-determining system in...

Sexual systems in animals and plants are remarkably diverse. In plants, dioecy often evolved more recently than in well-studied animal systems, making plants especially important for understanding how separate sexes evolved independently from hermaphrodite ancestors. Although various dioecious plant systems have been studied, sex-determining genes have rarely been definitively identified. Here, to investigate flower sex determination in Sapindaceas, we sequenced the genome of Eurycorymbus cavaleriei, the only currently known dioecious species in this family and identified an XY sex-determining system. Integrating population and comparative genetics approaches, we demonstrated that SUN, an SMXL-family paralogue closely related to the Arabidopsis SMXL6/7 repressors of strigolactone signaling, has acquired a neofunctionalized role in flower sex determination in Sapindaceae. In this XY system, we identified a Y-linked non-coding allele, SUN^MAO, as the sex-determining factor; it promotes maleness by producing small RNAs that silence the intact X-linked SUN allele. In females, SUN stabilizes the HD-ZIP transcription factor KUN; in turn, KUN activates SUN expression, forming a positive autoregulatory loop that represses anther development and promotes female flower formation. A similar SUN-KUN regulatory relationship is also observed in litchi, a monoecious Sapindaceae species. Notably, KUN is orthologous to the persimmon sex-determining regulator MeGI, a distantly related dioecious plant, highlighting the involvement of conserved genetic elements in the evolution of plant sexual systems. Together, our findings provide a mechanistic framework for understanding flower sex determination and the transition from monoecy to dioecy in Sapindaceae and opens new avenues for precise biotechnological control of flower sex across plants.