Induction of Sesquiterpenoid Biosynthesis in Tobacco Cell Suspension Cultures by Fungal Elicitor

Chappell, Joseph; Nable, Ross O.

doi:10.1104/pp.85.2.469

Cited by 133 publications

(87 citation statements)

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“…Nonetheless, sesquiterpenoids have been implicated as both positive and negative mediators of plant-plant (15), plant-insect (23), and plant-microbe (15,27) interactions. Of particular interest for the current work, the accumulation of species-specific, antimicrobial, bicyclic sesquiterpenoids has been demonstrated for pathogen-or elicitor-challenged plants (2,17,27,33) and cell cultures (4,5,9,34) within the Solanaceae.…”

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confidence: 99%

Purification and Characterization of an Inducible Sesquiterpene Cyclase from Elicitor-Treated Tobacco Cell Suspension Cultures

1990

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An elicitor-inducible sesquiterpene cyclase, which catalyzes the conversion of farnesyl diphosphate to 5-epi-aristolochene (IM Whitehead, DR Threlfall, DF Ewing [1989] Phytochemistry 28:775-779) and representing a committed step in the phytoalexin biosynthetic pathway in tobacco, was purified by a combination of hydrophobic interaction, anion exchange, hydroxylapatite, and chromatofocusing chromatography. From 2 kilograms of elicited tobacco (Nicotiana tabacum) cells, approximately 500 micrograms of cyclase protein was purified, representing greater than a 130-fold increase in the specific activity of the enzyme and a 4% recovery of the starting activity. The purified enzyme resolved as two major polypeptides of 60 and 62 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Biochemical characterization of the enzyme activity included an absolute requirement for magnesium, an isoelectric point of 4.5 to 4.9, and a Km for famesyl diphosphate of 2 to 5 micromolar. The purified cyclase protein was used to generate mouse polyclonal antibodies which efficiently inhibited cyclase activity in an in vitro assay. Electrophoresis of extracts from elicitor-treated cells or purified cyclase enzyme on native polyacrylamide gels separated the cyclase enzyme into four polypeptides as shown by immunoblot analysis using poly-and monoclonal antibodies. Proportionate cyclase enzyme activity comigrated with those polypeptides. No cyclase polypeptides were detectable in extracts of control cells by immunoblot analysis. However, immunoblot analysis of proteins from elicitor-treated cells using four independent monoclonal antibody lines and the polyclonal antibodies detected the same polypeptides, regardless of whether the proteins were separated by native or SDS-PAGE. The results suggest an induction of multiple cyclase polypeptides in elicitortreated cells resulting from either the expression of multiple genes or multiple post-translational processing events.Sesquiterpenoids comprise a large and diverse class of isoprenoids found in plants, fungi, and select bacteria, and insects (6, 22). In plants, sesquiterpenoids are often associated with essential oils (22), and except for a limited number of cases, '

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Purification and Characterization of an Inducible Sesquiterpene Cyclase from Elicitor-Treated Tobacco Cell Suspension Cultures

1990

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“…W5, a structural analog of W7 possessing a much lower affinity for calmodulin (18), did not interfere with elicitor-induced capsidiol accumulation at these concentrations. W7 and TFP also reduced the intracellular capsidiol content, which normally represents 5 to 10% of the total extra-and intracellular capsidiol found in elicitor-treated cultures (8), paralleling the effect observed on the capsidiol content in the media (data not shown). The antagonists by themselves (without elicitor) did not induce capsidiol accumulation under any experimental conditions.…”

Section: Elicitor-induced Capsidiol Accumulation Is Suppressed By Calmentioning

confidence: 53%

“…None of these studies, however, investigated whether calcium/calcium-binding proteins interacted directly with enzymes for phytoalexin biosynthesis or possibly affected the expression of the genes coding for these enzymes. Tobacco plants challenged with a variety of pathogens accumulate large amounts of sesquiterpene phytoalexins (8). Similar sesquiterpenoids are synthesized and secreted into the media of tobacco cell-suspension cultures upon addition of cellulase (9) or fungal cell wall fragments prepared from Phytophthora parasitica (8). Previous work correlated the induction of sesquiterpenoid accumulation with an induction of a sesquiterpene cyclase, 5-epi-aristolochene synthase (34,36).…”

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“…More recently, Knight et al (21) demonstrated that elicitation of tobacco (Nicotiana tabacum) resulted in a rapid but transient increase in intracellular calcium levels. None of these studies, however, investigated whether calcium/calcium-binding proteins interacted directly with enzymes for phytoalexin biosynthesis or possibly affected the expression of the genes coding for these enzymes. Tobacco plants challenged with a variety of pathogens accumulate large amounts of sesquiterpene phytoalexins (8). Similar sesquiterpenoids are synthesized and secreted into the media of tobacco cell-suspension cultures upon addition of cellulase (9) or fungal cell wall fragments prepared from Phytophthora parasitica (8).…”

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“…Previous work correlated the induction of sesquiterpenoid accumulation with an induction of a sesquiterpene cyclase, 5-epi-aristolochene synthase (34,36). Regulation of the cyclase enzyme activity was recently demonstrated to be controlled primarily by the transcription rate of the respective gene (37) KY 14 were maintained in Murashige-Skoog medium as previously described (8). Cell cultures in their rapid phase of growth, corresponding to 3 d after subculturing, were used for all the experiments presented.…”

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Inhibition of Phytoalexin Biosynthesis in Elicitor-Treated Tobacco Cell-Suspension Cultures by Calcium/Calmodulin Antagonists

Vögeli¹,

Vögeli‐Lange²,

Chappell³

1992

Plant Physiol.

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A role for calcium/calcium-binding proteins in a mechanism of signaling elicitor-inducible phytoalexin biosynthesis was investigated. Two classes of calcium/calmodulin antagonists, phenothiazines and naphthalenesulfonamides, inhibited sesquiterpene phytoalexin accumulation in tobacco (Nicotiana tabacum) cellsuspension cultures when added 1 h before elicitor. The antagonists also inhibited the induction of sesquiterpene cyclase enzyme activity, a key regulatory enzyme for sesquiterpene biosynthesis. The antagonists suppressed the induction of sesquiterpene cyclase only if added before or simultaneously with elicitor. Additionally, the antagonists inhibited (a) accumulation of the cyclase protein as measured in immunoblots; (b) the in vivo synthesis rate of the cyclase protein, measured as the incorporation of [35S]methionine into immunoprecipitable cyclase protein; and (c) the cyclase mRNA translational activity, measured as the incorporation of [35S]-methionine into immunoprecipitable cyclase protein synthesized by in vitro translation of RNA isolated from antagonist-treated, elicitor-induced cells. In contrast, elicitor-inducible phenylalanine ammonia lyase enzyme activity, the level of the enzyme protein, the in vivo synthesis rate, and the mRNA translational activity were not affected by any of the antagonist treatments. Uptake and incorporation of [35S]methionine into total cellular proteins and total in vitro translation products were also not indiscriminately altered by the antagonist treatments. The current results suggest that calcium and/or calmodulin-like proteins may be elements of a signal transduction pathway mediating elicitor-induced accumulation of phytoalexins in tobacco.Plants can respond to pathogen challenge by the induction of a number of biochemical responses (3), including phytoalexin accumulation (11, 13), synthesis and secretion of hydrolytic enzymes (4), and the putative reinforcement of their cell walls with lignin and/or structural glycoproteins (32).

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A novel membrane reactor design for controlled studies of interacting populations (simulation of the interaction between microorganism and plant suspension cultures)

Pestchanker¹,

Ercoli

1997

Biotechnol. Bioeng.

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Induction of Sesquiterpenoid Biosynthesis in Tobacco Cell Suspension Cultures by Fungal Elicitor

Abstract: ABSTRACI

Cited by 133 publications

References 23 publications

Purification and Characterization of an Inducible Sesquiterpene Cyclase from Elicitor-Treated Tobacco Cell Suspension Cultures

Purification and Characterization of an Inducible Sesquiterpene Cyclase from Elicitor-Treated Tobacco Cell Suspension Cultures

Inhibition of Phytoalexin Biosynthesis in Elicitor-Treated Tobacco Cell-Suspension Cultures by Calcium/Calmodulin Antagonists

A novel membrane reactor design for controlled studies of interacting populations (simulation of the interaction between microorganism and plant suspension cultures)

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