Plant Terpenoids Research Articles

Differential accumulation of dimethylallyl diphosphate in leaves and needles of isoprene- and methylbutenol-emitting and nonemitting species.

The biosynthesis and emission of volatile plant terpenoids, such as isoprene and methylbutenol (MBO), depend on the chloroplastic production of dimethylallyl diphosphate (DMAPP). To date, it has been difficult to study the relationship of cellular DMAPP levels to emission of these volatiles because of the lack of a sensitive assay for DMAPP in plant tissues. Using a recent DMAPP assay developed in our laboratories, we report that species with the highest potential for isoprene and MBO production also exhibit elevated light-dependent DMAPP production, ranging from 110% to 1,063%. Even species that do not produce significant amounts of volatile terpenoids, however, exhibit some potential for light-dependent production of DMAPP. We used a nonaqueous fractionation technique to determine the intracellular distribution of DMAPP in isoprene-emitting cottonwood (Populus deltoides) leaves; approximately 65% to 70% of the DMAPP recovered at midday occurred in the chloroplasts, indicating that most of the light-dependent production of DMAPP was chloroplastic in origin. The midday concentration of chloroplastic DMAPP in cottonwood leaves is estimated to be 0.13 to 3.0 mM, which is consistent with the relatively high K(m)s that have been reported for isoprene synthases (0.5-8 mM). The results provide support for the hypothesis that the light dependence of isoprene and MBO emissions is in part due to controls over DMAPP production.

Characterization and mass load estimates of organic compounds in agricultural irrigation runoff

Investigations of agricultural chemicals in surface runoff typically target nutrients or specific pesticides; however, numerous other organic compounds are regularly applied to agricultural fields in pesticide formulations, irrigation water, soil amendments and fertilizers. Many of these compounds have toxicological significance. We conducted a broad spectrum analysis of surface runoff from individual irrigated agricultural fields in coastal southern California to characterize organic compounds amenable to analysis by gas chromatography-mass spectrometry and to estimate the mass flux of selected chemicals. Aqueous phase extracts contained several pesticides, as well as personal care product ingredients and pharmaceutically active compounds apparently derived from treated wastewater used for irrigation. Several compounds potentially associated with pesticide adjuvants were also present in aqueous phase extracts. Dissolved NOM constituents in water phase extracts included n-fatty acids, aliphatic alcohols and plant terpenoids. Tentatively identified compounds sorbed to suspended particles included pesticides, a fecal sterol, aliphatic and alicyclic hydrocarbons, aliphatic alcohols, aldehydes, and C14 and C16 n-fatty acids and fatty acid esters. Bicyclic and polycyclic aromatic hydrocarbons were identified in both aqueous and suspended particle phases. Constituent concentrations, including total suspended solids (TSS), varied over the course of the sampled events by up to an order of magnitude, and typically were not correlated with flow. Variation in sorbed organic compound concentrations often did not parallel those for TSS concentration. Mass load estimates were strongly influenced by the choice of sampling interval.

Genomic organization of plant terpene synthases and molecular evolutionary implications.

Terpenoids are the largest, most diverse class of plant natural products and they play numerous functional roles in primary metabolism and in ecological interactions. The first committed step in the formation of the various terpenoid classes is the transformation of the prenyl diphosphate precursors, geranyl diphosphate, farnesyl diphosphate, and geranylgeranyl diphosphate, to the parent structures of each type catalyzed by the respective monoterpene (C(10)), sesquiterpene (C(15)), and diterpene synthases (C(20)). Over 30 cDNAs encoding plant terpenoid synthases involved in primary and secondary metabolism have been cloned and characterized. Here we describe the isolation and analysis of six genomic clones encoding terpene synthases of conifers, [(-)-pinene (C(10)), (-)-limonene (C(10)), (E)-alpha-bisabolene (C(15)), delta-selinene (C(15)), and abietadiene synthase (C(20)) from Abies grandis and taxadiene synthase (C(20)) from Taxus brevifolia], all of which are involved in natural products biosynthesis. Genome organization (intron number, size, placement and phase, and exon size) of these gymnosperm terpene synthases was compared to eight previously characterized angiosperm terpene synthase genes and to six putative terpene synthase genomic sequences from Arabidopsis thaliana. Three distinct classes of terpene synthase genes were discerned, from which assumed patterns of sequential intron loss and the loss of an unusual internal sequence element suggest that the ancestral terpenoid synthase gene resembled a contemporary conifer diterpene synthase gene in containing at least 12 introns and 13 exons of conserved size. A model presented for the evolutionary history of plant terpene synthases suggests that this superfamily of genes responsible for natural products biosynthesis derived from terpene synthase genes involved in primary metabolism by duplication and divergence in structural and functional specialization. This novel molecular evolutionary approach focused on genes of secondary metabolism may have broad implications for the origins of natural products and for plant phylogenetics in general.

Influence of plant terpenoids on the permeability of mitochondria and lipid bilayers

Five sesquiterpene alcohol esters of the carotane series, from plants of the genus Ferula, were investigated with regard to their capacity to modify the ion permeability of both planar lipid bilayers and mitochondria. These compounds are subdivided into two structural groups that differ in their effects on membrane permeability. Complex esters of sesquiterpene alcohols with aliphatic acids, which constituted the first group (lapidin and lapiferin), do not possess ionophoric properties. The second group comprised complex esters of sesquiterpene alcohols with aromatic acids (ferutinin, tenuferidin and ferutidin), all of which increase cation permeability of lipid bilayers and mitochondria in a dose-dependent manner. A pronounced selectivity of the terpenoid-modified membranes for divalent cations versus monovalent cations was found. Evidence of a carrier mechanism for terpenoid-induced ion transport is demonstrated. A tentative complex composed of a divalent cation with two molecules of membrane-active terpenoid is proposed.

Specific biodegradation of polychlorinated biphenyls (PCBs) facilitated by plant terpenoids

The aim of this study was to examine how plant terpenoids, as natural growth substrates or inducers, would affect the biodegradation of PCB congeners. Various PCB degraders that could grow on biphenyl and several terpenoids were tested for their PCB degradation capabilities. Degradation activities of the PCB congeners, 4,4′-dichlorobiphenyl (4,4′-DCBp) and 2,2′-dichlorobiphenyl (2,2′-DCBp), were initially monitored through a resting cell assay technique that could detect their degradation products. The PCB degraders,Pseudomonas sp. P166 andRhodococcus sp. T104, were found to grow on both biphenyl and terpenoids ((S)-(−) limonene,p-cymene and α-terpinene) whereasArthrobacter sp. B1B could not grow on the terpenoids as a sole carbon source. The B1B strain grown on biphenyl exhibited good degradation activity for 4,4′-DCBp and 2,2′-DCBp, while the activity of strains P166 and T104 was about 25% that of the B1B strain, respectively. Concomitant GC analysis, however, demonstrated that strain T104, grown on (S)-(−) limonene,p-cymene and α-terpinene, could degrade 4,4′-DCBp up to 30%, equivalent to 50% of the biphenyl induction level. Moreover, strain T104 grown on (S)-(−) limonene, could also degrade 2,2′-DCBp up to 30%. This indicates that terpenoids, widely distributed in nature, could be utilized as both growth and/or inducer substrate(s) for PCB biodegradation in the environment.

ChemInform Abstract: Simple and Versatile Synthesis of Branched Polyols: (+)-2-C-Methylerythritol and (+)-2-C-Methylthreitol.

The paper reports a new approach for the enantioselective synthesis of 2-C-methyl tetrols. The procedure has been utilized for preparing methylthreitol and methylerythritol, a putative intermediate in the mevalonate-independent biosynthesis of terpenoids in bacteria, algae and higher plants. The methodology offers straightforward access to related compounds and isotopically labeled derivatives.

Simple and versatile synthesis of branched polyols: (+)-2- C-methylerythritol and (+)-2- C-methylthreitol

The paper reports a new approach for the enantioselective synthesis of 2- C-methyl tetrols. The procedure has been utilized for preparing methylthreitol and methylerythritol, a putative intermediate in the mevalonate-independent biosynthesis of terpenoids in bacteria, algae and higher plants. The methodology offers straightforward access to related compounds and isotopically labeled derivatives.

Crystal Structure Determination of Aristolochene Synthase from the Blue Cheese Mold, Penicillium roqueforti*

The 2.5-A resolution crystal structure of recombinant aristolochene synthase from the blue cheese mold, Penicillium roqueforti, is the first of a fungal terpenoid cyclase. The structure of the enzyme reveals active site features that participate in the cyclization of the universal sesquiterpene cyclase substrate, farnesyl diphosphate, to form the bicyclic hydrocarbon aristolochene. Metal-triggered carbocation formation initiates the cyclization cascade, which proceeds through multiple complex intermediates to yield one exclusive structural and stereochemical isomer of aristolochene. Structural homology of this fungal cyclase with plant and bacterial terpenoid cyclases, despite minimal amino acid sequence identity, suggests divergence from a common, primordial ancestor in the evolution of terpene biosynthesis.

Phytoecdysteroid Turnover in Spinach: Long-term Stability Supports a Plant Defense Hypothesis

Using short (8-day) and long-term (28-day) experiments, we examined the stability of 20-hydroxyecdysone (20E) and the dominant phytosterols synthesized from a pulse of [2-14C]mevalonic acid ([14C]MVA) in hydroponically grown spinach (Spinacia oleracea). In the short-term experiment, plant dry mass and shoot 20E pools steadily increased. Root uptake of [14C]MVA resulted in the stable incorporation of 14C radiolabel into whole plant 20E pools, with no significant changes over time. Levels of free and saponifiable phytosterols increased in the shoots while 14C-labeled shoot phytosterols remained constant. Unexpectedly, both 14C-labeled and unlabeled pools of root phytosterols decreased over time. In the long-term experiment, plant dry mass and shoot 20E levels increased over time, while total 14C-labeled 20E pools remained constant. Both root and shoot phytosterol pools increased over time while the 14C incorporation in these pools remained constant. Together these experiments indicate that 20E in spinach is metabolically stabile and thus shares this characteristic with plant terpenoids of known defensive function. While little is known about phytosterol turnover in plants, our results suggest that phytosterols can indeed exist in a very dynamic state but may also be stable over time.

Cytidine 5'-triphosphate-dependent biosynthesis of isoprenoids: YgbP protein of Escherichia coli catalyzes the formation of 4-diphosphocytidyl-2-C-methylerythritol.

2-C-methylerythritol 4-phosphate has been established recently as an intermediate of the deoxyxylulose phosphate pathway used for biosynthesis of terpenoids in plants and in many microorganisms. We show that an enzyme isolated from cell extract of Escherichia coli converts 2-C-methylerythritol 4-phosphate into 4-diphosphocytidyl-2-C-methylerythritol by reaction with CTP. The enzyme is specified by the hitherto unannotated ORF ygbP of E. coli. The cognate protein was obtained in pure form from a recombinant hyperexpression strain of E. coli harboring a plasmid with the ygbP gene under the control of a T5 promoter and lac operator. By using the recombinant enzyme, 4-diphosphocytidyl-[2-(14)C]2-C-methylerythritol was prepared from [2-(14)C]2-C-methylerythritol 4-phosphate. The radiolabeled 4-diphosphocytidyl-2-C-methylerythritol was shown to be efficiently incorporated into carotenoids by isolated chromoplasts of Capsicum annuum. The E. coli ygbP gene appears to be part of a small operon also comprising the unannotated ygbB gene. Genes with similarity to ygbP and ygbB are present in the genomes of many microorganisms, and their occurrence appears to be correlated with that of the deoxyxylulose pathway of terpenoid biosynthesis. Moreover, several microorganisms have genes specifying putative fusion proteins with ygbP and ygbB domains, suggesting that both the YgbP protein and the YgbB protein are involved in the deoxyxylulose pathway. A gene from Arabidopsis thaliana with similarity to ygbP carries a putative plastid import sequence, which is well in line with the assumed localization of the deoxyxylulose pathway in the plastid compartment of plants.

Gibberellin biosynthesis in Gibberella

The gibberellins are a group of terpenoid plant hormones with applications in agriculture and brewing. Present in low amounts in plants, they are abundantly produced by some strains of the ascomycete Gibberella fujikuroi. Gibberellin production in this fungus is repressed by nitrogen and, to a less extent, by glucose. A simple screening procedure has allowed a systematic search of Gibberella mutants with alterations in the gibberellin pathway. The pheno- types of these and other mutants indicate indépendant regulations for the biosyntheses of gibberellins and other terpenoids, such as carotenoids or sterols. The recent cloning of the first fungal gibberellin gene, the one coding for kaurene synthase in Phaeosphaeria, will facilitate the molecular characterization of the Gibberella genes involved in gibberellin biosynthesis.

Inhibition of the Non-Mevalonate 1-Deoxy-ᴅ-xylulose-5-phosphate Pathway of Plant Isoprenoid Biosynthesis by Fosmidomycin

Abstract Various bacterial and plastidic plant terpenoids are synthesized via the non-mevalonate1-deoxy-ᴅ-xylulose-5-phosphate (DOXP) pathway. The antibiotic and herbicidal compound fosmidomycin is known to inhibit growth of several bacteria and plants, but so far its mode of action was unknown. Here we present data which demonstrate that the DOXP pathway of isoprenoid biosynthesis is efficiently blocked by fosmidomycin. The results point to the DOXP reductoisom erase as the probable target enzyme of fosmidomycin.

Molecular engineering of monoterpene production.

The terpenoids (often referred to as isoprenoids) constitute the largest group of natural products, with more than 22, 000 individual structures now known (1). They are all derived from the sequential addition of branched, five-carbon isopentenoid (isoprenoid) units, and range in size from isoprene (C5H8) to natural rubber (molecular weight greater than 106). In plants, the terpenoids and meroterpenoids (in which the terpenoid comprises a part of a larger molecule) play a broad range of metabolic and physiological roles, for example in photosynthesis (chlorophyll side chains, carotenoids, phylloquinones, plastoquinones), respiration (ubiquinone), cell membrane architecture (sterols), regulation of growth and development (gibberellins, abscisic acid, brassinosteroids), defense mechanisms (as phytoalexins, insect feeding and oviposition deterrents, competitive phytotoxins), and reproduction (as attractants of pollinators and seed dispersing animals). Additionally, isoprenylation of proteins by addition of a C15 or C20 olefinic chain provides a means of membrane anchoring, for example of cell cycle-regulating proteins. In this chapter, we focus on one family of industrially and agronomically significant plant terpenoids, the monoterpenes, and on the potential for the molecular genetic manipulation of the biosynthesis of this group of C10 compounds formed by the head-to-tail condensation of two isoprenoid units.

Higher plant terpenoids: A phytocentric overview of their ecological roles

Characteristics of higher plant terpenoids that result in mediation of numerous kinds of ecological interactions are discussed as a framework for this Symposium on Chemical Ecology of Terpenoids. However, the role of terpenoid mixtures, either constitutive or induced, their intraspecific qualitative and quantitative compositional variation, and their dosage-dependent effects are emphasized in subsequent discussions. It is suggested that little previous attention to these characteristics may have contributed to terpenoids having been misrepresented in some chemical defense theories. Selected phytocentric examples of terpenoid interactions are presented: (1) defense against generalist and specialist insect and mammalian herbivores, (2) defense against insect-vectored fungi and potentially pathogenic endophytic fungi, (3) attraction of entomophages and pollinators, (4) allelopathic effects that inhibit seed germination and soil bacteria, and (5) interaction with reactive troposphere gases. The results are integrated by discussing how these terpenoids may be contributing factors in determining some properties of terrestrial plant communities and ecosystems. A terrestrial phytocentric approach is necessitated due to the magnitude and scope of terpenoid interactions. This presentation has a more broadly based ecological perspective than the several excellent recent reviews of the ecological chemistry of terpenoids.

Potential sweetening agents of plant origin. Part 27. Stimulation of the gerbil's gustatory receptors by some potently sweet terpenoids

The gerbil was investigated as a model for sweet taste among several highly sweet plant terpenoids and the sweet dihydroisocoumarin phyllodulcin. Although the gerbil's chorda tympani nerve did not respond in electrophysiological experiments to rebaudiosides B and C, steviolbioside, and phyllodulcin, concentration-response curves were obtained for the stimulatory sweeteners hernandulcin, mogroside V, periandrin III, rebaudioside A, and stevioside. These compounds were more effective stimuli in the gerbil than sucrose, in the following order of potency: rebaudioside A = stevioside = periandrin III > hernandulcin > mogroside V > sucrose. In conditioned-taste aversion studies, gerbils trained to avoid these five stimulatory compounds generalized an avoidance to sucrose but not to hydrochloric acid, and except for the perception of a concomitant salty taste, our data show that these substances taste like sucrose to gerbils, as in humans. Support is thus provided for the potential involvement of this methodology to guide the purification of natural sweeteners from plant extracts.

Herbivory induces systemic production of plant volatiles that attract predators of the herbivore: Extraction of endogenous elicitor

It was previously shown that in response to infestation by spider mites (Tetranychus urticae), lima bean plants produce a volatile herbivoreinduced synomone that attracts phytoseiid mites (Phytoseiulus persimilis) that are predators of the spider mites. The production of predator-attracting infochemicals was established to occur systemically throughout the spider mitein-fested plant. Here we describe the extraction of a water-soluble endogenous elicitor from spider mite-infested lima bean leaves. This elicitor was shown to be transported out of infested leaves and was collected in water in which the petiole of the infested leaf was placed. When the petioles of uninfested lima bean leaves were placed in water in which infested leaves had been present for the previous seven days, these uninfested lima bean leaves became highly attractive to predatory mites in an olfactometer when an appropriate control of uninfested lima bean leaves was offered as alternative. The strength of this effect was dependent on the number of spider mites infesting the elicitor-producing leaves. Higher numbers of spider mites resulted in an elicitor solution with a stronger effect. In addition, spider mite density was important. The elicitor obtained from one leaf with 50 spider mites had a stronger effect on the attractiveness of uninfested leaves than the elicitor obtained from three leaves with 17 spider mites each. This suggests that the stress intensity imposed on a plant is an important determinant of the elicitor quantity. While the elicitor has a strong effect on the attractiveness of uninfested leaves, spider mite-infested leaves are still much more attractive to predatory mites than elicitor-exposed leaves. The data are discussed in the context of systemic effects in plant defense and the biosynthesis of herbivore-induced terpenoids in plants.

Esters of Ferula karakalensis. Structure and stereochemistry of karaferin and karaferinin

Two new guaiane esters — karaferin and karaferinin — have been isolated fromFerula karakalensis, and their structures and stereochemistry have been established. It has been shown that theFerula sesquiterpene alcohols angrendiol, ugamdiol, and karaferol are biogenetically interconnected and are products of a single chain of the biogenesis of terpenoids in plants.

Ecological Chemistry and Biochemistry of Plant Terpenoids.

Ecological Chemistry and Biochemistry of Plant Terpenoids.

Identification of a cDNA for the plastid-located geranylgeranyl pyrophosphate synthase from Capsicum annuum: correlative increase in enzyme activity and transcript level during fruit ripening.

Geranylgeranyl pyrophosphate synthase is a key enzyme in plant terpenoid biosynthesis. Using specific antibodies, a cDNA encoding geranylgeranyl pyrophosphate synthase has been isolated from bell pepper (Capsicum annuum) ripening fruit. The cloned cDNA codes for a high molecular weight precursor of 369 amino acids which contains a transit peptide of approximately 60 amino acids. In-situ immunolocalization experiments have demonstrated that geranylgeranyl pyrophosphate synthase is located exclusively in the plastids. Expression of the cloned cDNA in E. coli has unambiguously demonstrated that the encoded polypeptide catalyzes the synthesis of geranylgeranyl pyrophosphate by the addition of isopentenyl pyrophosphate to an allylic pyrophosphate. Peptide sequence comparisons revealed significant similarity between the sequences of the C. annuum geranylgeranyl pyrophosphate synthase and those deduced from carotenoid biosynthesis (crtE) genes from photosynthetic and non-photosynthetic bacteria. In addition, four highly conserved regions, which are found in various prenyltransferases, were identified. Furthermore, evidence is provided suggesting that conserved and exposed carboxylates are directly involved in the catalytic mechanism. Finally, the expression of the geranylgeranyl pyrophosphate synthase gene is demonstrated to be strongly induced during the chloroplast to chromoplast transition which occurs in ripening fruits, and is correlated with an increase in enzyme activity.

Compound co-occurrence and biosynthetic inference

Abstract Zavarin suggested that co-occurrence of terpenoids in plants provides an indication of likely biosynthetic relationships when enzymatic and tracer results are unavailable to determine the matter. Zavarin's proposal is here evaluated with data from another class of secondary metabolites, the glucosinolates, where biosynthetic pathways are well known. Three datasets from taxa of Cakile and Streptanthus (Cruciferae) were analyzed statistically with SPSS programs. Pair-wise comparisons of compounds tested their assignment to one of three possible categories: related by one-carbon chain extension; related by cleavage of terminal methylthio-group to produce a double bond; or no close relationship. While one dataset showed a tendency for chain-extended compounds to correlate significantly, the data exhibited no strong indication overall that co-occurring compounds were any more likely to be biosynthetically linked than not. Furthermore, contrasting positive and negative correlations for biosynthetically related pairs indicated that co-occurrence did not track mechanisms of biosynthesis. Hence, compound co-occurrence by itself provides no clear guide to biosynthetic relationships. By analogy, similar results can be expected for other classes of diverse secondary metabolites such as flavonoids and terpenoids.