Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings

Abstract

ABSTRACICasbene is a macrocyclic diterpene hydrocarbon that is produced in young castor bean (Ricinus communis L.) seedlings after they are exposed to Rhizopus stolonifer or other fungi. The activities of enzymes that participate in casbene biosynthesis were measured in cell-free extracts of 67-hour castor bean seedlings (a) that had been exposed to R. stolonifer spores 18 hours prior to the preparation of extracts, and (b) that were maintained under aseptic conditions throughout. Activity for the conversion of mevalonate to isopentenyl pyrophosphate does not change significantly after infection. On the other hand, the activities of farnesyl pyrophosphate synthetase (geranyl transferase), geranylgeranyl pyrophosphate synthetase (farnesyl transferase), and casbene synthetase are all substantially greater in infected tissues in comparison with control seedlings maintained under sterile conditions. The subcellular localization of these enzymes of casbene biosynthesis was investigated in preparations of microsomes, mitochondria, glyoxysomes, and proplastids that were resolved by centrifugation in linear and step sucrose density gradients of homogenates of castor bean endosperm tissue from both infected and sterile castor bean seedlings. Isopentenyl pyrophosphate isomerase and geranyl transferase activities are associated with proplastids from both infected and sterile seedlings. Significant levels of farnesyl transferase and casbene synthetase are found only in association with the proplastids of infected tissues and not in the proplastids of sterile tissues. From these results, it appears that at least the last two steps of casbene biosynthesis, geranylgeranyl pyrophosphate synthetase and casbene synthetase, are induced during the process of infection, and that the enzymes responsible for the conversion of isopentenyl pyrophosphate to casbene are localized in proplastids.

Developmental Regulation of Monoterpene Biosynthesis in the Glandular Trichomes of Peppermint

“…The close correspondence between in vitro enzyme activity and the rate of monoterpene biosynthesis measured in vivo suggests that monoterpene formation in peppermint oil gland cells is tightly controlled by the levels of biosynthetic enzymes. Similar correlations have been noted for the formation of a variety of isoprenoids during flower (Pichersky et al, 1994), leaf (Croteau et al, 1981), and seedling (Green and Baisted, 1972) development, and after induction by pathogens (Dudley et al, 1986;Hanley et al, 1992), but this is the first time that a related array of isoprenoid biosynthetic enzymes has been investigated in a single specialized cell type.…”

Terpenoid metabolism.

“…Funga1 infection of castor bean seedlings results in no measurable change in the rate of conversion of mevalonate to IPP. However, the activities of FPP synthase, GGPP synthase, and the diterpene cyclase casbene synthase all increase markedly (Dudley et al, 1986). The increase in casbene synthase activity is preceded by an increase in the level of mRNA encoding the enzyme (Moesta and West, 1985).…”

“…GGPP synthase activity has been demonstrated in proplastids of castor bean (Dudley et al, 1986) and in the stroma of daffodil chromoplasts (Laferrikre and Beyer, 1991). The enzyme has been partially purified from fruit chromoplasts of tomato (Spurgeon et al, 1984) and purified to homogeneity from C. annuum chromoplasts (Dogbo and Camara, 1987) and the stroma of mustard (Sinapis aba) etioplasts (Laferrikre and Beyer, 1991).…”

“…The cyclase activity responsible for converting GGPP to casbene is associated with proplastids of castor bean (Dudley et al, 1986). The enf-kaurene synthase activity, which catalyzes an early step of gibberellin (GA) biosynthesis, is associated with chloroplasts (Simcox et al, 1975;Moore and Coolbaugh, 1976;Railton et al, 1984).…”

“…The enzyme has been partially purified from fruit chromoplasts of tomato (Spurgeon et al, 1984) and purified to homogeneity from C. annuum chromoplasts (Dogbo and Camara, 1987) and the stroma of mustard (Sinapis aba) etioplasts (Laferrikre and Beyer, 1991). GGPP synthase has been directly localized to the plastid stroma by immunocytochemical methods (Cheniclet et al, 1992;Kuntz et al, 1992).The cyclase activity responsible for converting GGPP to casbene is associated with proplastids of castor bean (Dudley et al, 1986). The enf-kaurene synthase activity, which catalyzes an early step of gibberellin (GA) biosynthesis, is associated with chloroplasts (Simcox et al, 1975;Moore and Coolbaugh, 1976;Railton et al, 1984).…”