Redox interconversions of geraniol and nerol in higher plants

Use of ¹⁴C, ³H-labelled precursors showed that for feedings carried out in winter, isothujone (trans-thujan-3-one) was formed in Tanacetum vulgare from nerol (3,7-dimethyl-octa-cis-2,6-dien-1-ol) without loss of hydrogen from C-1 of the precursor. In contrast, formation from geraniol (the corresponding trans-isomer) involved stereospecific loss ofthe pro-(1S) hydrogen. This suggests that geraniol and nerol were interconverted by a redox system. Similar studies at other seasons with T. vulgare and on the biosynthesis of α- and β-pinenes (pin-3-ene; pin-2-(10)-ene) in Pinus pinaster; 1,8-cineole (1,8-oxidomethane) in Mentha piperita and Eucalyptus globulus; and carvone (menth-6,8(9)-dien-2-one) in M. spicata did not lead to such unambiguous conclusions. The results may be rationalized if (i) the redox system was reversible and/or (ii) tracer at C-1 of the phosphate esters of the precursors was scrambled by action of a phosphatase that induced CO bond fission.     

Redox interconversion of geraniol and nerol in Rosa damascena

Use of ¹⁴C, ³H-labelled precursors revealed that flowerheads of Rosa damascena converted geraniol (3,7-dimethylocta-trans-2,6-dien-1-ol) into nerol (the corresponding cis-isomer) with loss of the pro-(1S) hydrogen, whereas the reverse isomerization involved loss of the pro-(1R) atom. The inference that the interconversion proceeded by redox reactions with the formation of the corresponding aldehydes was supported by the preparation of cell-free extracts from R. damascena and R. dilecta that sustained such processes. These reactions were NADP⁺-NADPH dependent and had pH optima at 7.0 and 9.0, respectively.     

Biosynthetic origin of the gem-methyls of geraniol

Degradation of geraniol (3,7-dimethylocta-trans-2,6-dien-1-ol) biosynthesized in Rosa dilecta has proved that C-10 is exclusively derived from C-2 of mevalonate. This verifies the generally accepted but hitherto unproven view of the origin of the gem-methyls in this compound and of the corresponding groups in other terpenoids.     

Biosynthesis of geraniol and related monoterpenes in Pelargonium graveolens

Tracer studies on Pelargonium graveolens have shown that the extractable activity of geranyl pyrophosphate synthetase passed through a sharp maximu     

Biosynthesis of geraniol and nerol in cell-free extracts of Tanacetum vulgare

Cell-free extracts from leaves of Tanacetum vulgare synthesised geraniol and nerol (3,7-dimethylocta-trans-2-ene-1-ol and its cis isomer) in up to 11·9 and 2·4% total yields from IPP-[4-¹⁴C] and MVA-[2-¹⁴C] respectively. Optimum preparations were obtained from plant material just before the onset of flowering. The ratio of the monoterpenols varied 28-fold for different preparations under conditions where these products or their phosphate esters were not interconverted. Similar extracts incorporated α-terpineol-[¹⁴C] and terpinen-4-ol-[¹⁴C] (p-menth-1-en-8- and -4-ol respectively) in 0·05 to 2·2% yields into a compound tentatively identified as isothujone (trans-thujan-3-one), and preparations from flowerheads converted IPP-[4-¹⁴C] in 2·7% yield into geranyl and neryl β-d-glucosides. Inhibitors of IPP-isomerase had little effect on the incorporation of IPP into the monoterpenols in cell-free systems from which endogenous compounds of low molecular-weight had been removed. The inference that a pool of protein-bonded DMAPP or its biogenetic equivalent was present was supported by the demonstration that geraniol and nerol biosynthesised in the absence of the inhibitors were predominantly (65 to 100%) labelled in the moiety derived from IPP.     

Biosynthesis of artemisia ketone in higher plants

MVA-[2-¹⁴C], IPP-[4-¹⁴C] and DMAPP-[4-¹⁴C] were incorporated (optimum 0.04%–0.8 %) into artemisia ketone by Artemisia annua in a position-specific manner so that the C-5 moiety not containing the carbonyl group was preferentially (87–95 %) labelled. IPP and DMAPP, but not MVA, were similarly utilised in Santolina chamaecyparissus. Feeding of geraniol-[2-¹⁴C] to A.annua resulted in artemisia ketone being labelled in a position indicating extensive degradation of the precursor. ¹⁴C-labelled cis and trans-chrysanthemyl alcohols and chrysanthemates or DMVC were negligibly (< 5 × 10^?4 %) incorporated into artemisia ketone in both species over a range of feeding conditions. (+)-trans-Chrysanthemyl alcohol-[Me¹⁴C] was an effective (ca 2 % incorporation) precursor of the terpenoid part of pyrethrins I and II in flowers of Chrysanthemum cinerariaefolium but ¹⁴C-labelled artemisyl alcohol (3, 3, 6-trimethylheptan-1, 5-dien-4-ol) or (±)-cis-chrysanthemyl alcohol were not detectably incorporated. Although some of the negligible incorporations are probably attributable to compartmentation effects preventing access of precursors to biosynthetic sites, the experiments indicate some limitation of the previously proposed pathways of biogenesis of artemisia ketone and related irregular monoterpenes.     

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.     

Monoterpene variation in two Achillea ageratum chemotypes

Artemisia ketone and artemisia acetate are the main monoterpene components in both the flowers and leaves of A. ageratum growing in central Italy, but are replaced by 1,8-cineole in plants growing in Sardinia (Italy).     

On the structure of a novel ether from Artemisia tridentata

1,6,6-Trimethyl-4-ethenyl-2-oxabicyclo-[3. 1.0]hexane was unambiguously synthesized in seven steps from 4,5-dihydro-2-methylfuran. The product possessed IR, ¹H NMR and MS which differed from those of a compound isolated from Artemisia tridentata. This compound has been assigned the above structure on the basis of spectral evidence: consequently the proposed structure is erroneous.     

Evidence for geraniol as an obligatory precursor of isothujone

Isothujone (trans-thujan-3-one) was formed from MVA-[¹⁴C, ³H] in Tanacetum vulgare with retention of the pro-(4R) hydrogen of precursor, but with loss of the pro-(4S) hydrogen and of one hydrogen from C-5. Cell-free extracts could not sustain the formation of isothujone from MVA but yielded geraniol and nerol (3,7-dimethylocta-trans-2,6-dien-1-ol and its cis isomer) with retention of the pro-(4R) and loss of the pro-(4S) hydrogen in each case: no hydrogen was lost from C-5 of MVA in formation of geraniol, but one such atom was lost in the formation of nerol. These results support the sequence: geraniol → nerol → isothujone: in which the first two compounds (or their biogenetic equivalents) are interconverted by a redox process involving their derived aldehydes. They are not consistent with a direct pathway to nerol from C₅ intermediates or with routes involving cyclisation of linalol (3,7-dimethylocta-1,6-dien-3-ol) formed directly from the C₅ compounds or from geraniol. The cell-free preparations could not interconvert geraniol and nerol, their phosphates or pyrophosphates. This may be due to the inability of a prenyltransferase-isomerase multi-enzyme system to accept exogenously-supplied intermediates under these (in vitro) conditions.     

1,2 Hydrogen-shifts in the biosynthesis of the thujane skeleton

Degradation of (+)-isothujone (trans-thujan-3-one) biosynthesized in Tanacetum vulgare from (3RS)-mevalonic acid (MVA)-[2-¹⁴C, 2-³H₂] showed that one hydrogen from C-2 of the precursor was specifically incorporated at C-4 of product whereas the other was lost. Feeding of α-terpineol-[9-¹⁴C, 4-³H₁, 10-³H₃] (p-menth-1-en-8-ol) yielded isothujone with the same isotope ratios as in precursor. These results indicate 1,2 hydrogen-shifts at two locations in the construction of the the thujane skeleton from α-terpineol or its biogenetic equivalent, and are consistent with a mechanism involving direct cyclization of the latter to a product that by-passes the formation of the biogenetic equivalent of terpinen-4-ol (p-menth-1-en-4-ol) as an intermediate. (3R)-MVA-[¹⁴C, ³H] was more effectively incorporated (up to 1.5 %) into (+)-isothujone in vivo during autumn or winter than in summer (up to 0.02%).     

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.     

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.     

3-Epideoxyradicinol and the biosynthesis of deoxyradicinin

A novel phytotoxic compound, in addition to the known phytotoxin deoxyradicinin, has been isolated from Alternaria helianthi and its structure elucidated as 3-epideoxyradicinol. Its epimer is also briefly described. The polyacetate biosynthetic origin of deoxyradicinin has been demonstrated.     

The role of strictosidine in monoterpenoid indole alkaloid biosynthesis

In contrast to previous reports that vincoside was the sole precursor for indole alkaloids in Vinca rosea, the 3α epimer strictosidine has been incorporated into tetrahydroalstonine, ajmalicine, catharanthine and vindoline; the anomalous 3β to 3α inversion is no longer required.     

Metabolism of phenylpropanoids in Hydrangea serrata var. thunbergii and the biosynthesis of phyllodulcin

DL-Phenylalanine-[3-¹⁴C] and cinnamic acid-[3-¹⁴C] were fed to this plant and the label from cinnamic acid was incorporated into gallic acid, phyllodulcin and quercetin. By feeding p- coumaric acid-[U-³H], caffeic acid-[U-³H] and hydrangea glucoside A-[U-³H], it was possible to show that hydroxylation at C-3′in phyllodulcin occurs after the ring closure of dihydroisocoumarin. The biosynthetic pathway of phyllodulcin in this plant is thus: phenylalanine → cinnamic acid → p- coumaric acid → hydrangenol → phyllodulcin.     

Patterns of biosynthesis and accumulation of hydrocarbons and contact sex pheromone in the female german cockroach,Blattella germanica

De novo synthesis of contact female sex pheromone and hydrocarbons in Blattella germanica was examined using short in vivo incubations. Accumulation of pheromone on the epicuticular surface and the internal pheromone titer were related to age-specific changes in hydrocarbon synthesis and accumulation in normal and allatectomized females. The incorporation of radiolabel from [1-¹⁴C]propionate into the cuticular methyl ketone pheromone fraction was positively related to corpora allata activity during two gonotrophic cycles. During peak pheromone production the total internal lipid fraction contained greater titers of pheromone than the cuticular surface, and it too exhibited a cycle internally, preceding the rise in external pheromone. This suggests that synthesis and accumulation of pheromone internally are followed by transport of pheromone to the epicuticular surface where it accumulates. Radiolabel was incorporated efficiently into both cuticular and internal hydrocarbons after the imaginal molt and until the peak of pheromone synthesis, but it declined to lower levels before ovulation and throughout pregnancy. The internal hydrocarbon titer decreased 58% after oviposition, suggesting deposition in the egg case. It remained relatively unchanged during pregnancy and increased again during the second gonotrophic cycle. In allatectomized females, hydrocarbon synthesis was reduced relative to control females until oviposition in the latter. However, subsequent rates of hydrocarbon synthesis in allatectomized females (without oothecae) exceeded the rates in sham-operated females (with oothecae). In the absence of ovarian uptake of hydrocarbons, the internal titer increased without the decline found in control females at oviposition. As internal hydrocarbons increased, so did cuticular hydrocarbons and both internal and cuticular methyl ketone pheromones. These patterns corresponded well with feeding patterns in sham-operated and allatectomized females, suggesting that pheromone production is normally regulated by stage-specific feeding-induced hydrocarbon synthesis (precursor accumulation internally) and juvenile hormoneinduced conversion of hydrocarbon to pheromone. They also suggest that both the cuticle and the ovaries might be target sites for hydrocarbon and possibly methyl ketone deposition. © 1994 Wiley-Liss, Inc.     

Induction of anthocyanin formation and of enzymes related to its biosynthesis by UV light in cell cultures of Haplopappus gracilis

Only UV light below 345 nm stimulates anthocyanin formation in dark grown cell suspension cultures of Haplopappus gracilis. A linear relationship between UV dose and flavonoid accumulation, as found previously with parsley cell cultures, was not observed with the H. gracilis cells. Only continuous irradiation with high doses of UV was effective. Drastic increases in the activities of the enzymes phenylalanine ammonia-lyase, chalcone isomerase and flavanone synthase were observed under continuous UV light. The increase in enzyme activities paralleled anthocyanin formation.     

Biosynthesis of marine natural products: isolation and characterization of a myrcene synthase from cultured tissues of the marine red alga Ochtodes secundiramea

The acyclic monoterpene myrcene is the likely progenitor of the unusual cytotoxic halogenated monoterpenes that are found in marine algae and that function as feeding deterrents to herbivores. Myrcene synthase was isolated from suspension cultures of the marine red alga Ochtodes secundiramea, representing the first enzyme of this type from a marine organism. The algal myrcene synthase produces exclusively myrcene from the natural substrate geranyl diphosphate (GDP), utilizes Mg(+2) as the required divalent metal ion cofactor, has a molecular mass of about 69 kDa, and exhibits a pH optimum near 7.2. These features are similar to those of monoterpene synthases from terrestrial organisms. When incubated with neryl diphosphate (the cis-isomer of GDP), the O. secundiramea myrcene synthase produces the cyclic monoterpene limonene, whereas incubation with (+/-)linalyl diphosphate (the tertiary allylic isomer of geranyl diphosphate) yields both acyclic and cyclic monoterpenes. These results suggest that the enzyme is incapable of isomerizing geranyl diphosphate to linalyl diphosphate, a feature common to all monoterpene cyclases from terrestrial sources. The limited catalytic capability of the myrcene synthase may reflect the ancient evolutionary origin of the producing organism. The ability to assay this enzyme in cultured algae, grown under strictly defined conditions, provides an unparalleled opportunity to delineate factors eliciting the biosynthesis of this class of secondary metabolites, to investigate the metabolic pathway leading to the halogenated monoterpenes, and to determine their role in the chemical ecology of marine algae.