Induction of Sesquiterpene Cyclase and Suppression of Squalene Synthetase Activities in Plant Cell Cultures Treated with Fungal Elicitor

Abstract

Addition of elicitor, cell wall fragments of the fungus Phytophthora parasitica, to tobacco cell suspension cultures (Nicotiana tabacum) resulted in the rapid synthesis and secretion of large amounts of antibiotic sesquiterpenoids. Pulse-labeling experiments with I'4Ciacetate and PHI mevalonate demonstrated that the induction of sesquiterpenoid biosynthesis, maximal by 6 to 9 hours after elicitor addition to the cell cultures, was paralleled by a rapid and large decline in the incorporation rate of radioactivity into sterols. Consequently, sterol accumulation was also inhibited upon addition of elicitor to the cell cultures. Sesquiterpene cyclase activity was absent from control cell cultures but induced to a maximum within 10 hours of elicitor addition to the cell cultures. The cyclse activity remained elevated for an additional 30 hours before declining. In contrast, squalene synthetase activity was suppressed to less than 15% of that found in control cells within 7 hours of elicitor addition. confined to plants, fungi, and select bacteria (5). Although sesquiterpenoids have been found in these organisms at many stages of their life cycles, a dramatic accumulation of antibiotic sesquiterpenoids has been observed in plants challenged with microbial and viral pathogens (20). The induction of sesquiterpenoid accumulation in pathogen-challenged plants thus provides an opportunity for evaluating the mechanisms regulating the availability ofFPP for the competing branch pathways in the important biological context of plant-pathogen interactions.Extensive studies with mammalian cells have demonstrated that sterol metabolism is regulated primarily by controlling the enzyme activity level of HMGR (6). However, control of isoprenoid metabolism at such an early step in the pathway would be expected to also reduce the synthesis rate of other isoprenoids by making the FPP levels limiting. A secondary level of control was hence suspected, imposed by a modulation of the branch point enzymes, especially squalene synthetase. Faust et al. (12) have shown that when mammalian cell cultures are provided exogenous sterols, FPP utilization for sterol synthesis is dramatically reduced with little or no effect on the in vivo synthesis rate of ubiquinone. In subsequent work, they (13) were able to show that squalene synthetase activity, the first committed step in the sterol biosynthetic branch pathway, was indeed modulated in fibroblast cells responding to an exogenous supply of sterols. Their study also provided evidence that the prenyltransferase activity responsible for ubiquinone synthesis has a lower affinity constant (Km) for FPP than squalene synthetase. Therefore, the limited amount of FPP produced by cells incubated in the presence ofsterols would be utilized preferentially for ubiquinone formation. James and Kandutsch (15)

Biosynthesis of the Monoterpenes Limonene and Carvone in the Fruit of Caraway1

“…In contrast, the rapid switches in terpenoid metabolism that occur upon pathogen infection have been extensively investigated. For example, when cell cultures of tobacco or potato are treated with fungal elicitors, there is an induction of sesquiterpenoid phytoalexin biosynthesis and a repression of sterol formation (Brindle et al, 1988;Vö geli and Chappell, 1988;Chappell, 1995). Enzymatic analyses have revealed that fungal elicitors activate farnesyl diphosphate-utilizing enzymes in phytoalexin biosynthesis while reducing the activity of squalene synthase, a branchpoint enzyme in sterol formation that also competes for farnesyl diphosphate.…”

Biosynthesis of secondary metabolites in sugarcane

“…In cell cultures of Nicotiana tabacum treated with a fungal elicitor, biotic elicitation can also cause coordinated inhibition of squalene synthase and induction of sesquiterpene cyclase resulting in a rapid decrease in phytosterol biosynthesis coincident with the induction and accumulation of novel terpenoids involved in the plant defense response (Threlfall and Whitehead, 1988;Voegeli and Chappell, 1988).…”

Kinetic and Molecular Analysis of 5-Epiaristolochene 1,3-Dihydroxylase, a Cytochrome P450 Enzyme Catalyzing Successive Hydroxylations of Sesquiterpenes

“…Capsidiol biosynthesis is regulated by the expression of two key enzymes (see Scheme 1). 5-Epiaristolochene synthase catalyzes the cyclization of farnesyl diphosphate to the bicyclic intermediate 5-epiaristolochene (EA) 1,2 and is responsible for the diversion of farnesyl diphosphate from the mevalonate pathway toward antimicrobial compound biosynthesis (12,13). Recently, two highly homologous cDNAs for EA dihydroxylase (EAH) were isolated from Nicotiana tabacum and functionally expressed in yeast, and the encoded proteins were characterized as cytochrome P450 enzymes catalyzing the stereo-and regiospecific hydroxylation of EA at C-1 and C-3 (see Scheme 1) (14).…”