This enzyme may serve to ensure formation when activity is low, as in vegetative tissues, when lactone formation may compete with further oxidation of the alcohol intermediate. In most systems C20-GAs containing a 20-carboxylic acid group are formed by GA20ox as minor biologically inactive by-products, which are not converted to C19-GAs. However, these tricarboxylic acid GAs are major products of the GA20ox present in endosperm and immature embryos of C. maxima that was the first GA20ox to be cloned (40). The function of this pumpkin enzyme is considered abnormal as this type of activity has not been encountered in other species or, indeed, in vegetative tissues of pumpkin. The chemical mechanism for the loss of C-20 has not been elucidated. There is evidence that it is lost as CO2 and that both O atoms in the lactone function originate from the 19-carboxyl group. Direct removal of C-20 as CO2 requires the formation of an intermediate between the aldehyde and final C19-GA product, but none has been identified and it may remain bound to the enzyme.