Using <i>aco</i> multiple mutants to elucidate the differential regulation of ethylene in sex determination and fruit elongation in cucumber

The determination of sex morphs and the optimization of fruit shape are key challenges in cucumber cultivation and breeding, with ethylene recognized as a crucial regulatory factor in both processes. Ethylene is synthesized through the coordinated actions of 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) and ACC oxidase (ACO). While previous studies have highlighted the significant role of CsACSs in cucumber sex determination, investigations on CsACOs remain limited. In this study, we generated mutants of all members of the ACO gene family involved in ethylene synthesis during cucumber flower development and systematically analyzed their functions. Our results demonstrate that CsACO2 plays a dual role: it is involved in the early induction of carpel initiation during flower development and subsequently regulates ovary elongation. Mutations in CsACO2 affect ovary elongation, as evidenced by the shorter ovaries of female flowers in the cswip1/csaco2 double mutants. In developing female buds, after carpel initiation, we identified CsACO3 as the core regulatory gene responsible for stamen arrest, in cooperation with either CsACO2 or CsACO4. Notably, the cswip1/csaco2/csaco3 triple mutants produce hermaphrodite flowers with shorter ovaries. In contrast, the cswip1/csaco3/csaco4 triple mutants exhibit hermaphrodite flowers with normal ovaries and fruit shapes, and more importantly, their fruit-setting ability is comparable to that of the subgynoecious control. Our results enrich the ethylene regulatory network of cucumber female/hermaphrodite flower development and fruit shape, and provide an optional breeding pathway for high-yield varieties.