Comparison ofEnt-kaurene synthetase A and B activities in cell-free extracts from young tomato fruits of wild-type andgib-1, gib-2, andgib-3 tomato plants

The nonallelicgib-1 andgib-3 tomato (Lycopersion esculentum Mill.) mutants are gibberellin deficient and exhibit a dwarfed growth habit. Previous work has shown that this dwarfed growth pattern can be reversed by the application of a number of gibberellins and their precursors, includingent-kaurene (ent-kaur-16-ene). This indicates that they are blocked in gibberellin biosynthesis at a step prior toent-kaurene metabolism. The normal accumulation of carotenoids observed in these mutants suggests a functionally normal isoprenoid pathway.Ent-kaurene is synthesized from geranylgeranyl pyrophosphate in a two-step process with copalyl pyrophosphate as an intermediate.In vitro assays using young fruit extracts from wild-type andgib-2 plants resulted in the conversion of geranylgeranyl pyrophosphate to copalyl pyrophosphate, and the conversion of copalyl pyrophosphate toentkaurene. Similar assays usinggib-1 plants indicated a reduced ability for synthesis of copalyl pyrophosphate from geranylgeranyl pyrophosphate, and thus a reducedent-kaurene synthetase A activity. Furthermore,gib-3 extracts demonstrated a reduced ability to synthesizeent-kaurene from copalyl pyrophosphate, and thus a reducedent-kaurene synthetase B activity. These results establish the enzymatic conversion of geranylgeranyl pyrophosphate to copalyl pyrophosphate, and copalyl pyrophosphate toent-kaurene, as the sites of the mutations ingib-1 andgib-3 tomatoes, respectively. We also note that tomato fruit extracts contain components which are inhibitory toent-kaurene synthesis.