Deciphering a Novel Function of the WOX Family: SpWOX1-Mediated Male Suppression in Dioecious Spinach

Spinach, a dioecious species, serves as an important model for studying sex determination and differentiation in plants. However, the molecular mechanisms underlying these processes remain largely unresolved. The WUSCHEL-related homeobox (WOX) gene family plays critical roles in meristem maintenance, cell fate determination, and plant development, yet remains uncharacterized in spinach. In this study, a genome-wide comparative analysis identified 10-11 WOX genes across the genus Spinacia. Transcriptome analysis across floral developmental stages, combined with weighted gene co-expression network analysis, revealed that SpWOX1 exhibits female-biased expression and is associated with transcription factors involved in floral development. Functional analysis showed that silencing of SpWOX1 using virus-induced gene silencing resulted in the conversion of female flowers into hermaphroditic flowers in spinach, whereas ectopic expression in Arabidopsis thaliana led to reduced pollen viability and impaired stamen development. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation assays demonstrate that SpWOX1 interacts with the masculine factor SpMS1 and three key regulators of floral development, including AFO, AS2 and ARF3. Collectively, these findings suggest that SpWOX1 functions as a suppressor of male organ development and may play a central role in the spinach sex-determination pathway. This study provides new insights into the molecular basis of sex differentiation in spinach.