In most systems C20-GAs containing a 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.
The growth-active GAs GA4 and GA1 are formed by 3ȕ-hydroxylation of GA9 and GA20, respectively, catalyzed by GA 3-oxidases (GA3ox). The major GA3ox of Arabidopsis (AtGA3ox1) is highly regiospecific, producing a single product, while enzymes from certain other species also oxidize neighboring C atoms to a small extent (26). For example, oxidation of both C-2 and C-3 produces a 2, 3-double bond, as in the conversion of GA20 to GA5. Further oxidation of GA5, initially on C-1 and then on C-3, by the same enzyme results in the formation of GA3 (2, 66). While most GA 3oxidases are specific for C19-GAs, some plants, and particularly seeds, produce 3ȕ-hydroxylated C20-GAs. For example, an enzyme from pumpkin endosperm 3ȕ-hydroxylates C20-GAs more readily than C19-GAs (41).
A third class of dioxygenase, GA 2-oxidase (GA2ox), is responsible for the irreversible deactivation of GAs by 2ȕ-hydroxylation, so ensuring GA turnover, which is necessary for effective regulation of GA concentration. In some tissues, such as the cotyledons and, particularly, the testae of