A bHLH-mediated misexpression of SPL/NZZ underpins the anther developmental arrest of female flowers in sex differentiation in monoecious litchi

Sex differentiation is a crucial developmental process accompanied by tightly regulated anther development or its selective abortion. In litchi (Litchi chinensis), anthers develop normally in male flowers but are defective in female flowers, with impaired pollen development. However, the underlying regulatory mechanism remains unclear. We report here that transcription factor SPOROCYTELESS/NOZZLE (SPL/NZZ) is critical for anther and pollen development in litchi. Notably, LcSPL/NZZ shows persistently high expression throughout anther development in female flowers. Ectopic overexpression of LcSPL/NZZ suppresses filament elongation, reduces anther size, impairs pollen development, and ultimately causes male sterility. We further show that the misexpression of LcSPL/NZZ in female flower is likely mediated by a bHLH transcription factor (bHLH91). Mechanistically, LcbHLH91 binds the LcSPL/NZZ promoter to modulate its expression and physically interacts with LcSPL/NZZ in the nucleus, thereby enhancing its expression and protein activity. Phylogenetic and promoter analyses further indicate that the bHLH91-SPL/NZZ regulatory module is conserved within the Sapindaceae. Results obtained in this study imply a novel regulatory circuit controlling sex differentiation and anther development in litchi and related species.