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     

Incorporation of 14CO2, into cardenolide and sapogenin steroids of Digitalis purpurea

The relative rates of biosynthesis of cardenolide and sapogenin steroids of Digitalis purpurea were estimated by their uptake of ¹⁴CO₂. The incorporation of label into both groups, although initially slow, indicated that biosynthesis occurs even at the end of the growing season. The sapogenins were produced more rapidly than the cardenolides at this stage of plant development. Within the group of sapogenins, digitogenin, the trihydroxy compound, was produced at a greater rate than the dihydroxy steroid gitogenin. In the case of the cardenolides, the trihydroxy gitoxigenin was produced at a slower rate than the dihydroxy digitoxigenin.     

Incorporation of 14CO2, 14C-phenylalanine and 14C-cinnamate into soluble and insoluble cinnamic derivatives in Mentha arvensis

With the successful development of methods for the isolation and purification of ethanol-insoluble cinnamic acid esters in mint it became possible to initiate kinetic, isotopic studies on purified, ‘insoluble’ derivatives of caffeic acid, ferulic acid and p-coumaric acid. Pulse-feeding experiments were conducted with ¹⁴CO₂, phenylalanine-U-¹⁴C and cinnamic acid-3-¹⁴C. The ferulic acid derivative exhibited a significant turnover as compared to the other insoluble derivatives which were relatively stable. Time-course tracer studies were performed to compare the turnover of soluble caffeic acid derivatives with ‘insoluble’ forms of caffeic acid. Caffeic acid associated with a macromolecular fraction consistently showed a higher specific activity than either soluble caffeic acid or the caffeic acid associated with a second insoluble derivative.     

Biosynthesis of mono- and sesqui-terpenes in peppermint from glucose-C and CO2

Labeled glucose and CO₂ are more efficient precursors of monoterpenes in peppermint (Mentha piperita L.) cuttings than is mevalonate, which is the best precursor of sesquiterpenes in this plant. Metabolic turnover of the labeled monoterpenes was observed, in agreement with previous observations. Pulegone derived from ¹⁴CO₂ after 1, 3, 6, 9 and 12 hr of incubation was chemically degraded, and in every case at least 90% of the ¹⁴C-label was found in the seven-carbon fragment containing the isopentenyl pyrophosphate-derived portion of the molecule. The isopropylidene side chain, containing three carbons hypothetically derived from dimethylallyl pyrophosphate, was found to be essentially unlabeled. The results suggest that an endogenous dimethylallyl pyrophosphate pool participates in monoterpene biosynthesis, much as earlier work had suggested that a similar pool participates in sesquiterpene biosynthesis in this plant. These findings are of particular interest because it appears, based on the differential utilization of labeled precursors, that monoterpenes and sesquiterpenes are produced at separate sites in the plant.     

Biosynthesis of sterols and triterpenes in Pelargonium hortorum

Although all parts of the geranium plant (Pelargonium hortorum) are capable of synthesizing sterols and triterpenes and their esters in vitro from mevalonic acid-[2-¹⁴C], the aerial portions are more active than other tissues. All plant parts were shown to incorporate mevalonic acid-[2-¹⁴C] into isoprenoids for at least 3 days. The leaves and petioles had the greatest incorporation on a wet weight basis. Chopped preparations showed comparable incorporations of mevalonate whereby rootlets incorporated about one half as much as most parts; the flower petals incorporated five times the average amount. In leaves the principal sterol synthesized was sitosterol. Metabolic studies with isolated leaves indicated a fairly rapid conversion of free tetracyclic triterpenes to 4-desmethyl-sterols, while β-amyrin was synthesized at a different rate than α-amyrin. Esterified tetracyclic triterpenes exhibited only a slight amount of conversion to 4-desmethylsterols.     

Automatic collector of expired 14CO2 for liquid scintillation counting

A filtration technique was employed to trap ¹⁴CO₂ continuously for liquid scintillation counting. Devices for delivering scintillator and ethanolamine solutions were combined symmetrically with two fritted-glass aspirators for altenating operation. The collector was regulated by a fraction collector timer. Trial and animal tests indicated that the described method was efficient, reliable, and more convenient for frequent collection over long periods than alternative methods. The automatic collector was used for metabolic studies of [1-¹⁴C] arachidonic acid in rats kept in metabolic cages and the results were processed by multicompartmental analysis.     

14CO2 dark fixation in the halophytic species mesembryanthemum crystallinum

The time course of ¹⁴CO₂ dark fixation was studied in leaves of the facultatively halophytic plant species Mesembryanthemum crystallinum cultivated with and without 400 mM NaCl in the nutrient medium. It is generally known from the literature that plants grown under saline conditions incorporate ¹⁴C predominately into amino acids. By contrast in leaves of M. crystallinum grown on NaCl and exposed to ¹⁴CO₂ in the dark, relatively more radioactivity is incorporated in the organic acids (especially malate) than in amino acids. The data obtained are discussed in relation to the NaCl induced Crassulacean acid metabolism in M. crystallinum reported earlier.     

Labelling of lupine nodule metabolites with 14CO2 assimilated from the leaves

On feeding ¹⁴CO₂ to the shoots of lupine (25 mCi per plant) 30 min was the minimal time needed to determine the incorporation of label into bacteroid compounds. The predominant incorporation, exhibited in all root, nodule and bacteroid samples after 30 min exposure, was into sucrose (45–90% of the corresponding fraction radioactivity) of the neutral fraction; into malate (30–40%) of the acid fraction; into aspartic acid and asparagine (60–80% in sum) of the basic fraction. The composition of carbon compounds containing the greatest amount of ¹⁴C in the cytosol of nodules and in bacteroids was similar. Their radioactivity after 30 min exposure was for bacteroids (nCi per g of bacteroid fr. wt): sucrose 5.73, glucose 1.00, malate 0.15, succinate 0.11; for the nodule cytosol (nCi per g of nodule fr. wt): sucrose 200.00, glucose 8.40, malate 9.34, succinate 8.50. Thus it was demonstrated that in lupine, sucrose is the main photoassimilate entering not only into nodules but also into bacteroids. The biosynthesis of aspartic acid and asparagine occurs during nitrogen fixation in bacteroids.     

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.     

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.     

Biosynthesis of radiolabelled alkaloids from 14C-tyrosine in Erythrina crista-galli

Alkaloid biosynthesis has been demonstrated in the young expanding tissues of Erythrina crista-galli shoots, the stem part of which elaborated, from [2-^14C]-tyrosine, alkaloid of specific radioactivity 2μuCi mmol^?1, 10-fold greater than previously reported.     

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.     

[14C]epicatechin and [14C]procyanidins from seed shells of Aesculus hippocastanum

Direct injection of sodium-[1-¹⁴C]acetate into growing fruits of horse chestnut provides a convenient route to [¹⁴C]labelled epicatechin and procyanidins.     

An improved method for the liquid scintillation counting of 14CO2

A method has been described which permits the determination of the specific activity of ¹⁴CO₂ with an accuracy limited only by the accuracy of the liquid scintillation counter. Its principal idea is the selective conversion of CO₂ to nonvolatile organic compounds, such as benzoic acid. While this method is applicable quite generally it has been applied with particular success in connection with the Schmidt degradation of samples as small as 0.2 mmole where contamination of the evolved CO₂ is serious due to the necessarily large excess of reactants.     

Amo 1618 effects on incorporation of 14C-MVA and 14C-acetate into sterols in Nicotiana and Digitalis seedlings and cell-free preparations from Nicotiana

Incorporation of radioactivity from acetate-[¹⁴C] and MVA-[¹⁴C] into sterols and sterol precursors in tobacco was inhibited by Amo 1618; differing patterns of accumulation were obtained with the two precursors, suggesting more than one point of inhibition. This was borne out with cell-free preparations with which it was demonstrated that both HMG-CoA reductase and squalene-2,3-epoxide cyclase were inhibited, the latter more strongly than the former. GLC analysis of gross sterol and hydrocarbon fractions confirmed previous indications that incorporation of radioactivity into individual sterols was inhibited by Amo 1618. Finally, incorporation of MVA-[¹⁴C] into sterols and sterol precursors of Digitalis was significantly altered by the retardant, thus expanding the generality of the relationship between sterol (particularly 4-desmethylsterol) biosynthesis inhibition and retardant effect.     

Biosynthesis of monoterpenes in plants from 14C-labelled acetate and CO2

Degradation of (+)-isothujone biosynthesized by Tanacetum vulgare or Thuja plicata from acetate-[1-¹⁴C], -[2-¹⁴C] and -[2-³H₃] or from CO₂-[14C] at physiological concentration revealed a pattern of asymmetric labelling whereby tracer predominantly (72–98% resided in that part of the skeleton derived from IPP. This is similar to the patterns previously obtained for uptake of MVA-[2-¹⁴C] but differed from those reported in other species with acetate-[¹⁴C] as precursor. Within the IPP-derived moiety the 3 parts derived from acetate units were not equivalently labelled. Partial degradations of geraniol and (+)-pulegone formed in Pelargonium graveolens and Mentha pulegium after uptake of ¹⁴C-labelled acetate or CO₂ showed that the C-2 units of the skeletons of these monoterpenes were also labelled to widely differing extents and these patterns persisted over a range of feeding and seasonal conditions. These results suggest that metabolic pools of acetyl-CoA and/or acetoacetyl-CoA exist in these plants. The general occurrence of such pools and the consequent nonequivalent labelling patterns in secondary metabolism could invalidate biosynthetic conclusions drawn from partial degradations of labelled natural products.     

Preparation of [14C]-gibberellic acid

A combination of a chemical and a microbiological method is described for the preparation of [¹⁴C]-gibberellic acid.     

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.     

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.