The calcium-dependent protein kinase CmaCPK4 regulates sex determination in pumpkin (<i>Cucurbita maxima</i> D.)

Pumpkin (Cucurbita maxima D.) is typically monoecious with individual male and female flowers, and its yield is associated with the degree of femaleness, i.e. the ratio of female to male flowers produced by the plant. Subgynoecy represents a sex form with a high degree of femaleness, but the regulatory mechanisms in pumpkin remain poorly understood. In this study, using the F2 population crossed from the subgynoecious line 2013-12 and the monoecious line 9-6, we initially identified a recessive locus to control the subgynoecious trait and named it sg1. After bulked segregant analysis with whole-genome resequencing and molecular marker linkage analysis, the sg1 locus was mapped to pumpkin Chromosome 2. Genetic sequence analysis found a pumpkin calcium-dependent protein kinase (CPK) gene, CmaCPK4, in the mapping interval as the candidate gene. A retrotransposon insertion identified within the promoter elevated CmaCPK4 expression in 2013-12. Morphological characterization of near-isogenic lines containing the sg1 allele showed increases in the ratio of female flowers and high ethylene contents in terminal buds compared with the receptor parent. Heterologous overexpression of CmaCPK4 significantly increased the ratio of female flowers in cucumber (Cucumis sativus). Furthermore, CmaCPK4 directly interacts with and phosphorylates 1-aminocyclopropane-1-carboxylate synthase 5 (CmaACS5) and 1-aminocyclopropane-1-carboxylate synthase 7 (CmaACS7), resulting in increased ethylene content in 2013-12, which affected pumpkin sex determination. These findings provide insights into the role of the CmaCPK4-CmaACS5/CmaACS7 module in ethylene-induced sex determination in pumpkin.