The biosynthesis of ent-kaurene in germinating seeds and the function of 2-oxoglutarate in gibberellin biosynthesis

The two topics of this paper deal with different parts of the gibberellin (GA) biosynthetic pathway. The diterpene hydrocarbon ent-kaurene is the first tetracyclic precursor of gibberellins. Its two-step formation from geranylgeranyl pyrophosphate through the action of kaurene synthetase A and B initiates the diversion of the GA pathway from the general terpenoid pathway, and is a likely point for GA biosynthesis regulation. The capacity of a tissue to biosynthesize ent-kaurene is usually measured by the conversion of labelled precursors, such as mevalonate or geranylgeranyl pyrophosphate, to ent-kaurene in cell-free systems. This method works well with immature seeds, which are often highly active for GA biosynthesis, but cell-free systems from germinating seeds and seedlings, which are less active, give erratic and often negative results. We report here a new method of measuring ent-kaurene formation in vivo. In this procedure, ent-kaurene oxidation is inhibited by the addition of a plant growth retardant, thus causing ent-kaurene to accumulate, presumably at the same rate as its normal biosynthesis. The accumulation is sufficient, even in seedlings, to be quantified by the use of isotope dilution and GC-SIM (Groselindemann et al., 1991).