Gender-specific expression of GIBBERELLIC ACID INSENSITIVE is critical for unisexual organ initiation in dioecious Spinacia oleracea.

While unisexual flowers have evolved repeatedly throughout angiosperm families, the actual identification of sex-determining genes has been elusive, and their regulation within populations remains largely undefined. Here, we tested the mechanism of the feminization pathway in cultivated spinach (Spinacia oleracea), and investigated how this pathway may regulate alternative sexual development. We tested the effect of gibberellic acid (GA) on sex determination through exogenous applications of GA and inhibitors of GA synthesis and proteasome activity. GA concentrations in multiple tissues were estimated by enzyme-linked immunosorbent assay analysis. Gene function was investigated and pathway analysis was performed through virus-induced gene silencing. Relative gene expression levels were estimated by quantitative reverse transcription-polymerase chain reaction. Inhibition of GA production and proteasome activity feminized male flowers. However, there was no difference in GA content in tissues between males and females. We characterized a single DELLA family transcription factor gene (GIBBERELLIC ACID INSENSITIVE (SpGAI)) and observed inflorescence expression in females two-fold higher than in males. Reduction of SpGAI expression in females to male levels phenocopied exogenous GA application with respect to flower development. These results implicate SpGAI as the feminizing factor in spinach, and suggest that the feminizing pathway is epistatic to the masculinizing pathway. We present a unified model for alternative sexual development and discuss the implications for established theory.