We identified six mutations among which three were silent and three led to G33C, S238F and S249F missense mutations. Among the CsACS2 induced missense mutations, G33C correspond to one of the natural mutations previously identified. The G33C mutation occurs in a highly conserved amino acid position and was predicted, using the SIFT program, to affect the function of the protein . Consistent with this prediction, X-ray crystallography analysis located the residue G33 in the ACS hydrophobic pocket, where the aminoethoxyvinylglycine, a structural analog of the S-adenosylmethionine substrate, is positioned. Thus the G33 amino acid is likely to interact directly with the ACS substrate, SAM and thus G33C mutation should strongly affect the function of the protein. In contrast,LY294002 the S238F mutation involved a conserved amino acid that is predicted to have lower impact on the protein function. Consistent with this prediction, X-ray crystallography analysis located the S238 in the b6 strand of the protein away from the active site of the enzyme. The third mutation, S249F, implicated a non-conserved amino acid position and was predicted to not affect the function of the protein, using the SIFT program and X-ray crystallography analysis. To test whether the induced mutations in CsACS2 affect the cucumber sexual phenotype,Masitinib we first backcrossed the three mutant lines to the wild type and we followed the segregation of the mutations with the sexual transition in more than 500 F2 plants for each cross. Consistent with the crystallography analysis, the S238F and S249F mutations predicted to not affect the function of the protein had no impact on the sexual type of the plant. In contrast, plants homozygous for G33C mutation showed a sexual transition from monoecious to andromonoecious and as predicted the female flowers were transformed to hermaphrodite. Based on the TILLinG experiment and our previous genetic dissection of the M locus, we concluded that the M locus encodes for CsACS2. In the G33C homozygote mutant, the hermaph-rodite flowers result from the release of stamen development in carpel-bearing buds.