5-epi-aristolochene is a common precursor of the sesquiterpenoid phytoalexins capsidiol and debneyol

Whitehead, Ian M.; Threlfall, David R.; Ewing, David F.

doi:10.1016/0031-9422(89)80114-1

Cited by 88 publications

(61 citation statements)

References 14 publications

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“…The most conclusive evidence for this was the comigration of the cyclase enzyme activity and the mAB reactive polypeptides on native polyacrylamide gels. Whether these molecular forms represent distinct cyclase enzymes, enzymes which catalyze distinct steps of the cyclase reaction (35), or processed forms of a single polypeptide cannot be discerned from our current results. A determination and characterization of the cyclase gene number and expression will be necessary to distinguish between these possibilities.…”

Section: Comparison Of the Tobacco Sesquiterpene Cyclase To Other Termentioning

confidence: 84%

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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Section: Comparison Of the Tobacco Sesquiterpene Cyclase To Other Termentioning

confidence: 84%

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

1990

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“…One of the induced proteins has been identified as a sesquiterpene cyclase, an enzyme that catalyzes a reaction committing carbon to the production of extracellular, antimicrobial sesquiterpenoids (25,29). As previously discussed, an additional physiological consequence of this induction is the possible diversion of carbon from the general isoprenoid biosynthetic pathway, and hence, the depletion of isoprenoids necessary for actively growing cells, such as sterols (23,25).…”

Section: Discussionmentioning

confidence: 97%

Regulation of a Sesquiterpene Cyclase in Cellulase-Treated Tobacco Cell Suspension Cultures

Vögeli

Chappell²

1990

Plant Physiol.

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The regulation of an elicitor-inducible sesquiterpene cyclase in tobacco (Nicotiana tabacum) cell suspension cultures was investigated. Sesquiterpene cyclase activity was absent from control cell cultures but induced to a maximum within 15 hours of cellulase addition to the cell cultures. When fungal elicitors are added to tobacco cell suspension cultures, the cultures cease sterol accumulation and instead synthesize and secrete sesquiterpenoids (23,25). Previous work correlated the decline in sterol biosynthesis with a suppression of squalene synthetase enzyme activity, and the induction of sesquiterpenoid biosynthesis with an induction of a sesquiterpene cyclase enzyme activity (23,25). Because these two enzymes are positioned at a putative branch point in the isoprenoid biosynthetic pathway, the induction of one enzyme and the suppression of the other were interpreted as an important mechanism controlling carbon flow and hence, end product formation (25). This simplistic model assumed that FPP3 was equally available to both enzymes and that sesquiterpenoid and sterol biosynthesis were not localized to different subcellular compartments.

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“…Capsidiol, the principal sesquiterpene phytoalexin found in tobacco and other Solanaceous plants, is not detectable in healthy (control) plant tissues but rapidly accumulates in tobacco plants challenged with viral, bacterial, or funga1 pathogens (Bailey et al, 1975;Guedes et al, 1982;Ricci et al, 1989) and in cell cultures treated with a variety of elicitors (Brooks et al, 1986;Vogeli and Chappell, 1988;Whitehead et al, 1989;Milat et al, 1991 Transgenic lines not having any GUS expression, control or inducible, were excluded from these calculations (see Fig. 7).…”

Section: Discussionmentioning

confidence: 99%

“…pathogen-derived elicitors such as cellulase, funga1 cell wall hydrolysates, and cryptogein, an extracellular protein produced by Pkytophthora cryptogea (Brooks et al, 1986;Vogeli and Chappell, 1988;Ricci et al, 1989;Whitehead et al, 1989;Milat et al, 1991). Sesquiterpene phytoalexins are characteristic of the Solanaceae and are 15-C derivatives arising from the isoprenoid biosynthetic pathway (Chappell, 1995;Ku'c, 1995).…”

mentioning

confidence: 99%

Regulation of Sesquiterpene Cyclase Gene Expression (Characterization of an Elicitor- and Pathogen-Inducible Promoter)

et al. 1997

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The promoter for a tobacco (Nicofiana tabacum) sesquiterpene cyclase gene, a key regulatory step in sesquiterpene phytoalexin biosynthesis, has been analyzed. The EAS4 promoter was fused to the P-glucuronidase (CUS) reporter gene, and the temporal and spatial expression patterns of C U S activity were examined in stably transformed plants and in transient expression assays using electroporated protoplasts of tobacco. N o C U S activity was observed i n any tissues under normal growth conditions. A low level of C U S activity was detected in wounded leaf, root, and stem tissues, whereas a much higher level was observed when these tissues were challenged with elicitors or microbial pathogens. l h e CUS expression pattern directed by the EAS4 promoter was identical to the induction patterns observed for the endogenous sesquiterpene cyclase genes. Neither exogenous salicylic acid nor methyl iasmonate induced C U S expression; and H,O, induced GUS expression to only a limited extent. Although the EAS4 promoter contains cissequences resembling previously identified transcriptional control motifs, other cis-sequences important for quantitative and qualitative gene expression were identified by deletion and gain-offunction analyses. The EAS4 promoter differs from previously described pathogen-/elicitor-inducible promoters because it only supports inducible gene expression and directs unique spatial expression patterns.

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5-epi-aristolochene is a common precursor of the sesquiterpenoid phytoalexins capsidiol and debneyol

Cited by 88 publications

References 14 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

Regulation of a Sesquiterpene Cyclase in Cellulase-Treated Tobacco Cell Suspension Cultures

Regulation of Sesquiterpene Cyclase Gene Expression (Characterization of an Elicitor- and Pathogen-Inducible Promoter)

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