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.     

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.     

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.     

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.     

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.     

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.     

[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.     

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.     

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 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.     

An 18O2 study of the biosynthesis and metabolism of rishitin

An ¹⁸O₂ study has shown that the hydroxyl oxygen atoms of lubimin, rishitin and two metabolites of rishitin, 13-hydroxyrishitin and 11,12-dihydro-13 (or 12)-hydroxyrishitin, are derived from molecular oxygen. It could not be shown that the aldehyde oxygen of lubimin was derived from molecular oxygen, probably due to its exchange with the oxygen atom of water. These findings have established that mono-oxygenases are involved in all of the hydroxylation reactions and that, contrary to a previous proposal, 11,12-dihydro-13 (or 12)-hydroxyrishitin is not formed from rishitin by hydration.     

Interconversion of gibberellin A5 to gibberellin 3 in seedlings of dwarf Pisum sativum

[³H]-Gibberellin A₅ ([³H]-GA₅) applied to seedlings of dark-grown dwarf pea (Pisum sativum L. cv. Meteor), was converted to two acidic compounds, GA₃ and a chromatographically similar unknown. Identification of GA₃ was made by gas-liquid radiochromatography using three stationary phases.     

Light-dependent monoterpene synthesis in Pinus radiata cultures

Callus cultures of Pinus radiata that synthesized monoterpenes de novo and which were stable for at least 1 year have been established. The products differed from those of parent plants in that α-pinene (87–100%) rather than β-pinene was the main component. The best lines accumulated monoterpenes (ca 2 × 10^?3% wt/wet wt)in yields 40–20% of that in the parent stem and needles. The composition of the extractable oil depended on the light regime. After culture in total darkness toluene and acetone accumulated. These compounds also occurred (at low levels) in dark-grown seedlings and in seeds of P. radiata and a route for their formation from α-pinene is suggested. Cell-free extracts of the culture lines converted [¹⁴C] IPP into geraniol, nerol and α- and β-pinenes in up to 46% total yield. These are the most active crude extracts for monoterpene biosynthesis that have been reported from either tissue cultures or higher plants.     

Formation of neoxanthin,diadinoxanthin and peridinin from [14C]zeaxanthin by a cell-free system from Amphidinium carterae

A cell-free system prepared from an axenic culture of the alga Amphidinium carterae converted [¹⁴C]zeaxanthin into neoxanthin and then into peridinin (62%) and diadinoxanthin (38%). Peridinin, therefore, is made by the excision of three carbon atoms from a C₄₀ carotenoid and the acetylene group of diadinoxanthin is formed from the allene of neoxanthin, rather than the reverse.