Co-ordinated inhibition of squalene synthetase and induction of enzymes of sesquiterpenoid phytoalexin biosynthesis in cultures of Nicotiana tabacum

Threlfall, David R.; Whitehead, Ian M.

doi:10.1016/0031-9422(88)87028-6

Cited by 135 publications

(70 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The induction of a sesquiterpene cyclase, 5-epi-aristoloch-cultures has also been described (30,31). This cyclase enzyme activity was not detectable in control cell cultures but was induced to a maximum within 12 h of elicitor addition.…”

mentioning

confidence: 87%

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

1990

View full text Add to dashboard Cite

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, '

show abstract

mentioning

confidence: 87%

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

1990

View full text Add to dashboard Cite

show abstract

“…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).…”

Section: Squalene Synthasesmentioning

confidence: 99%

Biosynthesis of secondary metabolites in sugarcane

et al. 2001

View full text Add to dashboard Cite

A set of genes related to secondary metabolism was extracted from the sugarcane expressed sequence tag (SUCEST) database and was used to investigate both the gene expression pattern of key enzymes regulating the main biosynthetic secondary metabolism pathways and the major classes of metabolites involved in the response of sugarcane to environmental and developmental cues. The SUCEST database was constructed with tissues in different physiological conditions which had been collected under varied situation of environmental stress. This database allows researchers to identify and characterize the expressed genes of a wide range of putative enzymes able to catalyze steps in the phenylpropanoid, isoprenoid and other pathways of the special metabolic mechanisms involved in the response of sugarcane to environmental changes. Our results show that sugarcane cDNAs encoded putative ultra-violet induced sesquiterpene cyclases (SC); chalcone synthase (CHS), the first enzyme in the pathway branch for flavonoid biosynthesis; isoflavone synthase (IFS), involved in plant defense and root nodulation; isoflavone reductase (IFR), a key enzyme in phenylpropanoid phytoalexin biosynthesis; and caffeic acid-O-methyltransferase, a key enzyme in the biosynthesis of lignin cell wall precursors. High levels of CHS transcripts from plantlets infected with Herbaspirillum rubri or Gluconacetobacter diazotroficans suggests that agents of biotic stress can elicit flavonoid biosynthesis in sugarcane. From this data we have predicted the profile of isoprenoid and phenylpropanoid metabolism in sugarcane and pointed the branches of secondary metabolism activated during tissue-specific stages of development and the adaptive response of sugarcane to agents of biotic and abiotic stress, although our assignment of enzyme function should be confirmed by careful biochemical and genetic supporting evidence.

show abstract

“…Tobacco cell suspension cultures respond to treatment with elicitors such as fungal cell wall hydrolysates or cellulase by the de novo synthesis and secretion of antibiotic sesquiterpenoids, primarily the phytoalexin capsidiol (9,10 (9). The induction of sesquiterpene cyclase enzyme activity and, hence, sesquiterpenoid biosynthesis has also been correlated with the suppression of squalene synthetase enzyme activity and consequent decline in sterol biosynthesis (9,10).…”

Section: Introductionmentioning

confidence: 99%

“…Central to capsidiol biosynthesis is the induction of a sesquiterpene cyclase [5-epi-aristolochene synthase (EAS)] that catalyzes the cyclization of trans,trans-famesyl diphosphate (FPP) to the bicyclic intermediate 5-epi-aristolochene (9). The induction of sesquiterpene cyclase enzyme activity and, hence, sesquiterpenoid biosynthesis has also been correlated with the suppression of squalene synthetase enzyme activity and consequent decline in sterol biosynthesis (9,10). The induction of one enzyme and the suppression of the other are interpreted as an important regulatory mechanism controlling end-product formation, since the two enzymes are positioned at putative branch points in isoprenoid metabolism (9).…”

mentioning

confidence: 99%

Gene family for an elicitor-induced sesquiterpene cyclase in tobacco.

Facchini

Chappell

1992

Proc. Natl. Acad. Sci. U.S.A.

237

174

View full text Add to dashboard Cite

The initial step in the conversion of the isoprenoid intermediate farnesyl diphosphate to the sesquiterpenoid phytoalexin capsidiol in elicitor-treated tobacco tissues is catalyzed by an inducible sesquiterpene cyclase [5-epiaristolochene synthase (EAS)]. Two independent cDNA clones (cEAS1 and cEAS2) encoding EAS were isolated from an elicitor-induced tobacco cDNA library by differential hybridization and subsequently were characterized by hybrid selection-in vitro translation. Insertion of cEAS1, a partial cDNA clone encoding 175 C-terminal amino acids, into an Escherchia coli expression vector resulted in accumulation of a fusion protein immunodetectable with EAS-specific polyclonal antibodies. The cDNA clones were used to isolate two full-length EAS genes that mapped 5 kilobases (kb) apart on one 15-kb genomic clone. The nucleotide sequences of the structural gene components were identical from 388 base pairs (bp) upstream of the transcription initiation site to 40 bp downstream of the translation termination codon, suggesting a relatively recent duplication event. The genes consist of 1479-bp open reading frames, each containing five introns and specifying 56,828-Da proteins. The N-terminal amino acid sequence deduced from the genomic clones was identical to the first 16 amino acids of the EAS protein identifuible by Edman degradation. RNA blot hybridization with cEAS1 demonstrated a mRNA induction time course consistent with the induction of the EAS mRNA translational activity with maximum levels 4-6 h after elicitation. EAS mRNA was not detected in control cells. DNA blot-hybridization analysis of genomic DNA revealed a copy number of '12-15 for EAS-like genes in the tetraploid tobacco genome. The conservation of a putative allelic prenyl diphosphate binding motif is also discussed.Sesquiterpenoids are a structurally diverse class of isoprenoids found in plants, some fungi, and bacteria, but not in vertebrates, which have important implications in plantplant (1), plant-insect (2, 3), and plant-pathogen (4, 5) interactions. Despite an extensive appreciation for the structure-function relationships of plant sesquiterpenoids, the sesquiterpenoid biosynthetic pathway and its regulation are not well understood (4, 6). Although many enzymes of general isoprenoid biosynthesis have been measured in plants, few enzymes suspected of being rate-limiting have been identified (4, 6). Moreover, little is known about the mechanisms regulating the activity and absolute levels of plant isoprenoid biosynthetic enzymes with the exception of a diterpene cyclase (7). This is in contrast to the extensive molecular analysis of regulatory mechanisms controlling isoprenoid metabolism in mammalian systems (8).Tobacco cell suspension cultures respond to treatment with elicitors such as fungal cell wall hydrolysates or cellulase by the de novo synthesis and secretion of antibiotic sesquiterpenoids, primarily the phytoalexin capsidiol (9, 10). Central to capsidiol biosynthesis is the induction of a sesquiterpene cyclase [5-epi...

show abstract

Co-ordinated inhibition of squalene synthetase and induction of enzymes of sesquiterpenoid phytoalexin biosynthesis in cultures of Nicotiana tabacum

Cited by 135 publications

References 26 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

Biosynthesis of secondary metabolites in sugarcane

Gene family for an elicitor-induced sesquiterpene cyclase in tobacco.

Contact Info

Product

Resources

About