Biosynthesis of mono- and sesquiterpenes in peppermint from mevalonate-2-C

The volatile oil from peppermint (Mentha piperita L.) is composed primarily of monoterpenes with less than 2% sesquiterpenes. However, radioactivity from mevalonate-2-¹⁴C is incorporated into caryophyllene and other sesquiterpene hydrocarbons much more extensively than into monoterpenes by peppermint cuttings. Both mono- and sesquiterpenes show maximum incorporation of label after 6 hr (0·03% vs. 0·33% of the physiological isomer) and lose 75% of the incorporated label after an additional 6 hr. Caryophyllene derived from mevalonate-2-¹⁴C after 6 hr of incorporation was chemically degraded. The isoprenoid origin of caryophyllene was confirmed, and preferential labelling of the isopentenyl pyrophosphate derived portions of the molecule was noted. On the basis of such evidence it appears that separate sites may exist for the biosynthesis of mono- and sesquiterpenes and that an endogenous dimethylallyl pyrophosphate pool may participate in the biosynthesis of sesquiterpenes in peppermint.     

Absence of rapid terpene turnover in several diverse species of terpene-accumulating plants

Terpenes are commonly believed to undergo rapid metabolic turnover in plants, but the evidence for this process comes largely from studies that used detached organs or applied radiolabeled precursors in unnatural ways. When ¹⁴CO₂ pulse labeling experiments were carried out with intact plants of four taxonomically distant, terpene-accumulating species, no significant turnover of monoterpenes, sesquiterpenes or diterpenes was detected in young foliage over a two week period after exposure to ¹⁴CO₂. These results are consistent with those of other investigations performed under physiologically realistic conditions, and caution against the uncritical incorporation of turnover into models or theories concerning plant chemical defense.     

Monoterpene formation by leucoplasts of Citrofortunella mitis and Citrus unshiu

Leucoplasts of immature calamondin and satsuma fruits were incubated with [1-¹⁴C] isopentenyl pyrophosphate under various conditions. Optimal incorporation of the tracer into geranyl pyrophosphate and monoterpene hydrocarbons occurred in the presence of exogenous dimethylallyl pyrophosphate and Mn²⁺ which was more effective than Mg²⁺. The dependence of dimethylallyl pyrophosphate showed that about 10 moles were required for 1 mole of isopentenyl pyrophosphate for the best recovery in monoterpene hydrocarbon biosynthesis. A time-course incorporation of isopentenyl pyrophosphate revealed that the C₁₀ hydrocarbon elaboration was dependent on the geranyl pyrophosphate production and at no time neryl pyrophosphate was synthesized by leucoplasts. The amount of labelled farnesyl pyrophosphate was rather low whatever the conditions used in the experiments and sesquiterpene hydrocarbon biosynthesis was never observed.Abbreviations DMAPP dimethylallyl pyrophosphate - FPP farnesyl pyrophosphate - GPP geranyl pyrophosphate - IPP isopentenyl pyrophosphate - LPP linalyl pyrophosphate - NPP neryl pyrophosphate     

Non-uniform labelling of geraniol biosynthesized from 14CO2 in Pelargonium graveolens

Geranium (Pelargonium graveolens) cuttings were exposed to a 2 hr pulse of ¹⁴CO₂ then allowed to metabolize the label in circulating air for an additional 22 hr. Geraniol isolated from cuttings 2, 4, 8, 12, 16, 20 and 24 hr after the start of the experiment revealed the label in this compound to suffer substantial turnover. Chemical degradation of the labelled geraniol to yield the C₃-isopropylidene fragment showed the distribution of label favored the isopentenyl pyrophosphate-derived half of the molecule. Between 2 and 12 hr of the time-course the distribution of label between the halves of the molecule showed the proportion of label associated with the isopentenyl pyrophosphate-derived half to increase to 78 %. From 12 to 24 hr this preferential labelling declined and approached an equal distribution between the halves. Hypotheses presented to rationalize these observations include the existence of a dimethylallyl pyrophosphate pool and multiple compartments of isoprenoid biosynthesis.     

Biosynthesis of squalene and other triterpenes in Mentha piperita from mevalonate-2-14C

Unlike mono- and sesqui-terpenes, squalene and other triterpenes in peppermint readily incorporate mevalonate-2-¹⁴C label (greater than 30% incorporation of R-mevalonate in 4 hr). The labelled squalene produced turns over rapidly. Squalene derived from mevalonate-2-¹⁴C in incorporation times of 1, 4 and 7 hr was degraded chemically and shown to be equivalently labelled, according to theory, in the isopentenyl pyrophosphate-derived and dimethylallyl pyrophosphate-derived portions of the molecule. This contrasts with earlier studies on the biosynthesis of mono- and sesqui-terpenes in peppermint from ¹⁴C-precursors, in which the isopentenyl pyrophosphate-derived portions of the terpene molecules were found to be preferentially labelled, suggesting the presence of endogenous dimethylallyl pyrophosphate pools. The kinetics of squalene biosynthesis, and the labelling pattern of squalene, suggest that sites of triterpene biosynthesis are readily accessible to exogenous mevalonate and that endogenous dimethylallyl pyrophosphate pools do not participate in triterpene biosynthesis to any appreciable extent. The triterpene biosynthetic sites in peppermint thus appear to differ significantly from the monoterpene and sesquiterpene biosynthetic sites.     

Role of acyclic compounds in monoterpene biosynthesis in Pinus pinaster

The biosynthesis of monoterpene hydrocarbons was studied in maritime pine needles by incorporation of ¹⁴CO₂. It was shown that the acyclic terpenes β-myrcene and trans-β-ocimene, act as transitory compounds before the biosynthesis of cyclic monoterpenes such as α- and β-pinene. The mechanisms of biosynthesis are genetically controlled.     

Metabolic pools associated with monoterpene biosynthesis in higher plants

Partial degradations of (+)-isothujone biosynthesised in Tanacetum vulgare after feeding IPP-[4-¹⁴C], DMAPP-[4-¹⁴C] or 3,3-dimethylacrylate-[Me-¹⁴C], and of geraniol and (+)-pulegone formed in Pelargonium graveolens and Mentha pulegium respectively after uptake of 3,3-dimethylacrylate-[Me-¹⁴C], indicated that none of these metabolites was a direct source of the part of the monoterpene skeleton derived hypothetically from DMAPP. Uptake of glucose-[U¹⁴C] into P. graveolens led, in contrast, to both IPP and DMAPP-derived moieties of geraniol being extensively labelled. Feeding of l-valine-[U-¹⁴C] and l-leucine-[U-¹⁴C] to all three plants resulted in negligible incorporation of tracer into monoterpenes. A soluble enzyme system prepared from foliage of T. vulgare that had been exposed to CO₂-[¹⁴C] for 20 days converted isotopically-normal IPP into GPP with the DMAPP-derived portion containing essentially all (>98%) of the radioactivity present. These observations and those previously obtained from feeding experiments with other [¹⁴C]-labelled precursors on the same plant species are consistent with the occurrence of two metabolic pools of intermediates for monoterpene biosynthesis, one of which is probably protein-bonded.     

Biosynthesis and metabolism of steryl glycosides in Sinapis alba seedlings

With ¹⁴CO₂, d-glucose-[U-¹⁴C] and dl-mevalonate-[4R-4-³H₁] used as precursors, a study was made of the labelling dynamics of the steryl glucosides (SG) and steryl acylglucosides (ASG) in Sinapis alba seedlings. The radioactivity of the sterol and sugar moieties, as well as of the fatty acid moieties in the case of ASG, was analysed separately. The course of incorporation of ¹⁴C from ¹⁴ CO₂ and glucose-[U-¹⁴C] into the sugar part of SG and ASG indicated that about $\text{2}\text{3}$ of the whole pool of the newly synthesized sterol glycosides of both types underwent rapid deglucosylation. Likewise, fatty acids in the ASG pool were rapidly exchanged. The present results point to a high metabolic activity of the sterol glycoside derivatives in plant cells.     

Plant Terpenoids： Biosynthesis and Ecological Functions

Among plant secondary metabolites terpenolds are a structurally most diverse group; they function as phytoalexins In plant direct defense, or as signals In Indirect defense responses which involves herbivores and their natural enemies. In recent years, more and more attention has been paid to the Investigation of the ecological role of plant terpenolds. The biosynthesis pathways of monoterpenes, sesquiterpenes, and diterpenes Include the synthesis of C5 precursor isopentenyl diphosphate （IPP） and Its allylic isomer dlmethylallyl dlphosphate （DMAPP）, the synthesis of the immediate diphosphate precursors, and the formation of the diverse terpenoids. Terpene synthases （TPSs） play a key role In volatile terpene synthesis. By expression of the TPS genes, significant achievements have been made on metabolic engineering to Increase terpenoid production. This review mainly summarizes the recent research progress In elucidating the ecological role of terpenoids and characterization of the enzymes Involved in the terpenold biosynthesis. Spatial and temporal regulations of terpenoids metabolism are also discussed.     

In vitro biosynthesis of isopentenylacetophenones in Eupatorium rugosum

A cell free homogenate of Eupatorium rugosum leaves was prepared and utilized to study the biosynthesis of dehydrotremetone (1). Homogenates of young leaves were most efficient in the formation of 1. The furan ring and its side chain were derived from an isoprenoid unit, which appeared to be isopentenyl pyrophosphate and not dimethylallyl pyrophosphate. Potential aromatic precursors such as 4-hydroxyacetophenone and 4-hydroxy-3[isopenten-(2)-yl]-acetophenone were poorly utilized. However, tremetone, especially in the presence of the coenzyme NADP, was very efficiently converted to 1. An apparent intermediate in the pathway leading to 1 was isolated and appeared similar to 4-hydroxy-3[isopenten-(2)-yl]-acetophenone. It is proposed that the aromatic moiety of 1 is derived from acetate via a polyketide intermediate, which undergoes isoprenylation by isopentenyl pyrophosphate, followed by aromatization and furan ring closure.     

Zur Kompartimentierung der Synthese von Mono- und Sesqui-Terpenen des ätherischen Öls beiPoncirus trifoliata

Zusammenfassung In der Frucht vonPoncirus trifoliata liegen in der Außenschale Drüsenzellkomplexe, die ein monoterpenreiches ätherisches Öl mit geringem Anteil an Sesquiterpenen und O-haltigen Substanzen produzieren. Ähnlich aussehende Exkretzellkomplexe aus den Saftschläuchen enthalten hauptsächlich Sesquiterpenkohlenwasserstoffe (STKW) und O-haltige Komponenten und sehr wenig Monoterpenkohlenwasserstoffe (MTKW). Im Schalenöl konnten nach gaschromatographischer Trennung mit Hilfe der Massenspektrometrie 19 Komponenten identifiziert werden, im Saftschlauchöl 25.Elektronenmikroskopische Aufnahmen der jüngsten Drüsenzellen beider Drüsenkomplexe lassen erkennen, daß beide Terpenklassen wahrscheinlich hauptsächlich bzw. ausschließlich plastidär entstehen.Exogen angebotenes¹⁴CO₂ wird zunächst überwiegend in die MTKW eingebaut, erst später nimmt die Markierung der STKW und O-haltigen Komponenten stark zu. Über den Ferntransportweg angebotenes¹⁴C-Leucin führt anfangs zu einer starken Markierung der STKW und O-haltigen Komponenten, erst später verschiebt sich der Einbau etwas mehr in Richtung MTKW. Als Hauptursache für den differenten Einbau wird das Vorhandensein zweier Typen von Drüsenzellkomplexen mit unterschiedlichen Syntheseleistungen angesehen.Die aus dem¹⁴CO₂ in der Außenrinde gebildeten Assimilate werden zuerst in das MTKW-reiche Öl der Schalenexkretbehälter eingebaut. Die überwiegend STKW erzeugenden Saftschlauchbehälter werden erst später beliefert. Beim Leucinangebot über die Fruchtstiele scheint es gerade umgekehrt zu verlaufen. Die aufeinanderfolgenden Maxima der Ölproduktion in den beiden Drüsenzellkomplex-Typen und die Änderung des Komponentenspektrums ihres ätherischen Öls im Verlauf der Vegetationsperiode tragen ebenfalls zu einem je nach Jahreszeit unterschiedlichen Einbau in die MTKW und STKW bei.

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Compartmentation of mono- and sesqui-terpene biosynthesis of the essential oil inPoncirus trifoliata

Summary The fruit ofPoncirus trifoliata shows glandular cell complexes in the exocarp, which produce a volatile oil rich in monoterpenes but poor in sesquiterpenes and oxigenated compounds. The juice vesicles of the endocarp possess similar cell complexes mainly containing sesquiterpenes and oxigenated compounds, whereas monoterpenes only occur in small amounts. By the use of combined gas chromatography-mass spectrometry 19 components of the rind oil and 15 compounds of the endocarp oil could be identified.As demonstrated by electron microscopy the terpenes most probably are synthesized predominantly, if not exclusively in plastids. As shown by gasradiochromatography radioactive precursors (¹⁴CO₂ and¹⁴C-leucine) are incorporated into mono- and sesqui-terpenes to a different extent.This is due to two gland types producing essential oils of different composition with regard to their mono- and sesqui-terpene percentage. In fruit development the exocarp glands differentiate earlier than the endocarp glands do. The activity of exogenously applied¹⁴CO₂ first reaches the peripheral glands and later on appears in the interior glands. Depending upon the growth season, labelled leucine transported by the conducting tissues from lower plant parts leads to a high specific activity of the sesqui-terpenes and oxigenated compounds. It could be argued that in this instance the glands of the pulp are better provided with precursors than the exocarp glands. The successive maxima of essential oil production in both glandular complexes, and the changes in the concentration of individual oil constituents during the ontogeny of the fruit also contribute to different incorporation ratios of radioactive precursors into mono- and sesqui-terpenes.

     

Biosynthesis of halogenated monoterpenes in Plocamium cartilagineum

The biosynthesis of halogenated monoterpenes in this alga has been studied by feeding experiments using labelled bicarbonate, acetate and mevalonate. Seasonal effects have been observed in these studies and the incorporation of H¹⁴CO₃^? into primary metabolites is consistent with the expected reductive pentose pathway. The incorporations of [³H, ¹⁴C]mevalonates and limited chemical degradations are consistent with the expected cyclization to these rearranged monoterpenes. Attempts to obtain cell free systems to carry out all or part of the biosynthetic sequence or to halogenate various substrates have been unsuccessful, although a classical peroxidase has been isolated, characterized and partially purified.     

Dimethylallyl diphosphate and geranyl diphosphate pools of plant species characterized by different isoprenoid emissions

Dimethylallyl diphosphate (DMADP) and geranyl diphosphate (GDP) are the last precursors of isoprene and monoterpenes emitted by leaves, respectively. DMADP and GDP pools were measured in leaves of plants emitting isoprene (Populus alba), monoterpenes (Quercus ilex and Mentha piperita), or nonemitting isoprenoids (Prunus persica). Detectable pools were found in all plant species, but P. persica showed the lowest pool size, which indicates a limitation of the whole pathway leading to isoprenoid biosynthesis in nonemitting species. The pools of DMADP and GDP of nonemitting, isoprene-emitting, and monoterpene-emitting species were partially labeled (generally 40%-60% of total carbon-incorporated (13)C) within the same time by which volatile isoprenoids are fully labeled (15 min). This indicates the coexistence of two pools for both precursors, the rapidly labeled pool presumably occurring in chloroplasts and thereby synthesized by the methylerythritol phosphate pathway and the nonlabeled pool presumably located in the cytosol and synthesized by the mevalonic pathway. In M. piperita storing monoterpenes in specialized leaf structures, the GDP pool remained totally unlabeled, indicating either that monoterpenes are totally formed by the mevalonic pathway or that labeling occurs slowly in comparison to the large pool of stored monoterpenes in this plant. The pools of DMADP and GDP increased during the season (from May to July) but decreased when the leaf was darkened or exposed to very high temperature. In the dark, the pool of DMADP of the isoprene-emitting species decreased faster than the pool of GDP. However, after 6 h of darkness, both pools were depleted to about 10% of the pool size in illuminated leaves. This indicates that both the chloroplastic and the cytosolic pools of precursors are depleted in the dark. When comparing measurements over the season and at different temperatures, an inverse correlation was observed between isoprene emission by P. alba and the DMADP pool size and between monoterpene emission by Q. ilex and the GDP pool size. This suggests that the pool size does not limit the emission of isoprenoids. Rather, it indicates that the flux of volatile isoprenoids effectively controls the size of their pools of precursors.     

Molecular cloning and expression of Chimonanthus praecox farnesyl pyrophosphate synthase gene and its possible involvement in the biosynthesis of floral volatile sesquiterpenoids

Farnesyl pyrophosphate (FPP) synthase catalyzes the biosynthesis of FPP, which is the precursors of sesquiterpenoids such as floral scent volatiles, from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). cDNA encoding wintersweet (Chimonanthus praecox L.) FPP synthase was isolated by the RT-PCR and RACE methods. The deduced amino acid sequence showed a high identity to plant FPP synthases. Expression of the gene in Escherichia coli yielded FPPS activity that catalyzed the synthesis of FPP as a main product. Tissue-specific and developmental analyses of the mRNA levels of CpFPPS and volatile sesquiterpenoids levels in C. praecox flowers revealed that the FPPS may play a regulatory role in floral volatile sesquiterpenoids of wintersweet.     

Biosynthesis of monoterpenes by Ceratocystis moniliformis

Ceratocystis moniliformis produced and excreted monoterpenes when grown on potato-dextrose broth. Geraniol, nerol, citronellol, linalol, α-terpineol, geranial and neral were identified by GC-MS. Their production commenced with the depletion of nitrogen in the growth medium and their combined concentration peaked at about 50 μg/ml on the 5th day of growth. The pathway for the biosynthesis of the identified monoterpenes was studied by supplying the radioactive precursors mevalonic acid-[2-¹⁴C], l-leucine-[4,5-³H(N)], and acetate- [2-¹⁴C] to C. moniliformis. For each precursor, the extent of incorporation into the above monoterpenes and the distribution of radioactivity in geraniol was determined. It was concluded that monoterpenes were formed via the mevalonate pathway, previously established for higher terpenes in other organisms. This represents the first information available on the biosynthetic pathway for free monoterpenes in a microbial system.     

Biosynthesis of artemisinin – revisited

Artemisinin is a well-known antimalarial drug isolated from the Artemisia annua plant. The biosynthesis of this well-known molecule has been reinvestigated by using [1-¹³C]acetate, [2-¹³C]acetate, and [1,6-¹³C₂]glucose. The ¹³C peak enrichment in artemisinin was observed in six and nine carbon atoms from [1-¹³C]acetate and [2-¹³C]acetate, respectively. The ¹³C NMR spectra of ¹³C-enriched artemisinin suggested that the mevalonic acid (MVA) pathway is the predominant route to biosynthesis of this sesquiterpene. On the other hand, the peak enrichment of five carbons of ¹³C-artemisinin including carbon atoms originating from methyls of dimethylallyl group of geranyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP) was observed from [1,6-¹³C₂]glucose. This suggested that GPP which is supposed to be biosynthesized in plastids travels from plastids to cytosol through the plastidial wall and combines with isopentenyl pyrophosphate (IPP) to form the (E,E)-FPP which finally cyclizes and oxidizes to artemisinin. In this way the DXP pathway also contributes to the biosynthesis of this sesquiterpene.     

Gibberellin estimation and biosynthesis in germinating Hordeum distichon

Gibberellic acid (GA₃) and 13-deoxy-gibberellic acid (GA₇) were identified in extracts of germinating barley as their ¹⁴C-methyl esters. The maximal level of GA₃ was estimated by an isotopic dilution procedure to be 1·5 ng per grain. Germinating barley incorporated 2-¹⁴C-mevalonic acid into several terpenes, whose specific radioactivities were measured, but incorporation into GA₃ could not be detected. Cell-free embryo extracts from germinating barley converted 2-¹⁴C-mevalonic acid into isopentenol, dimethylallyl alcohol, farnesol and squalene, while ¹⁴C-isopentenyl pyrophosphate was incorporated into geraniol, farnesol, geranylgeraniol and squalene. There was no detectable incorporation into the gibberellin intermediate ent-kaurene.     

The effects of levulinic Acid and 4,6-dioxoheptanoic Acid on the metabolism of etiolated and greening barley leaves

Application of levulinic acid (LA), a competitive inhibitor of δ-aminolevulinic acid (ALA) dehydratase, to greening plant tissues causes ALA to accumulate at the expense of chlorophyll. 4,6-Dioxoheptanoic acid (DA), which has been reported to be an effective inhibitor of this enzyme in animal systems, has a similar but more powerful effect on ALA and chlorophyll metabolism in greening leaves of Hordeum vulgare L. var. Larker. Both LA and DA also inhibit the uptake of [¹⁴C]amino acids into etiolated and greening barley leaves and reduce their incorporation into protein. Treatment of etiolated and greening leaves with these compounds results in the inhibition of ¹⁴CO₂ evolution from labeled precursors, including amino and organic acids. Inhibition of ¹⁴CO₂ evolution by these compounds is more effective in greening leaves than in etiolated leaves when [4-¹⁴C]ALA or [1-¹⁴C]glutamate are employed as precursors. Both LA and DA also inhibit the uptake and increase the incorporation of ³²Pi into organophosphorus by etiolated barley leaves. These results indicate that LA and DA have more far-reaching effects upon plant metabolism than was previously believed.     

The Biosynthetic Origin of Irregular Monoterpenes in Lavandula: ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF A NOVEL cis-PRENYL DIPHOSPHATE SYNTHASE GENE,LAVANDULYL DIPHOSPHATE SYNTHASE*

Lavender essential oils are constituted predominantly of regular monoterpenes, for example linalool, 1,8-cineole, and camphor. However, they also contain irregular monoterpenes including lavandulol and lavandulyl acetate. Although the majority of genes responsible for the production of regular monoterpenes in lavenders are now known, enzymes (including lavandulyl diphosphate synthase (LPPS)) catalyzing the biosynthesis of irregular monoterpenes in these plants have not been described. Here, we report the isolation and functional characterization of a novel cis-prenyl diphosphate synthase cDNA, termed Lavandula x intermedia lavandulyl diphosphate synthase (LiLPPS), through a homology-based cloning strategy. The LiLPPS ORF, encoding for a 305-amino acid long protein, was expressed in Escherichia coli, and the recombinant protein was purified by nickel-nitrilotriacetic acid affinity chromatography. The approximately 34.5-kDa bacterially produced protein specifically catalyzed the head-to-middle condensation of two dimethylallyl diphosphate units to LPP in vitro with apparent K_m and k_cat values of 208 ± 12 μm and 0.1 s⁻¹, respectively. LiLPPS is a homodimeric enzyme with a sigmoidal saturation curve and Hill coefficient of 2.7, suggesting a positive co-operative interaction among its catalytic sites. LiLPPS could be used to modulate the production of lavandulol and its derivatives in plants through metabolic engineering.     

Conversion of geranylgeranyl pyrophosphate to ent-kaurene in enzyme extracts of sonicated chloroplasts

Farnesyl pyrophosphate-[¹⁴C] and geranylgeranyl pyrophosphate-[¹⁴C] were biosynthesized from mevalonic acid-[2-¹⁴C] by cell-free enzyme extracts of pea (Pisum sativum) cotyledons containing MgCl₂, MnCl₂, ATP and AMO-1618. Maximum yields of farnesyl pyrophosphate were obtained after 30 min incubation while geranylgeranyl pyrophosphate was the primary product after 180 min. Biosynthesized geranylgeranyl pyrophosphate-[¹⁴C] served as an efficient substrate for ent-kaurene biosynthesis in reaction mixtures containing cotyledon enzymes when AMO-1618 was omitted. Enzyme extracts from green pea shoot tips and chloroplasts also converted geranylgeranyl pyrophosphate to ent-kaurene in very low yields. Ent-kaurene production from mevalonic acid-[2-¹⁴C] in extracts of pea shoot tips was also enhanced by addition of chloroplast enzymes. This evidence indicates that kaurene synthetase is present in pea chloroplasts and adds to the possibility that some gibberellin biosynthesis may be compartmentalized in those organelles.