The involvement of sucrose,glucose and other metabolites in the synthesis of triterpenes and dopa in the laticifers of Euphorbia lathyris

A quantitative triterpene analysis was made of latex stem tissue of Euphorbia lathyris. Young plants seedlings of E. lathyris were incubated with various labelled precursors. Incorporation into triterpenes was obtained from [2-¹⁴C]mevalonic acid, [1-¹⁴C]acetate, [3-¹⁴C]pyruvate, [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose, [U-¹⁴C]glyoxylate, [2,3-¹⁴C]succinic acid, [1-¹⁴C]glycerol [U-¹⁴C]serine. Both sugars tyrosine appeared to be effective precursors in DOPA synthesis inside the laticifers. Exogenously supplied mevalonic acid was only involved in triterpene synthesis outside the laticifers. GC-RC of triterpenes synthesized from [U-¹⁴C]glucose revealed the origin of these compounds in the latex. The labelled triterpenes obtained after incorporation of the other mentioned labelled precursors were only partly synthesized in the laticifers. For quantitative data on latex triterpene synthesis seedlings were incubated with [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose [1-¹⁴C]acetate in the presence of increasing amounts of unlabelled substrate. From the amount of ¹⁴C incorporated into the triterpenes the amount of substrate directly involved in triterpene synthesis was calculated, as was the absolute triterpene yield. Sucrose showed the highest triterpene yield, equivalent to the daily increase of the triterpene content of growing seedlings. The possible significance of the other precursors in triterpene synthesis in the laticifers is discussed.     

The biosynthesis,metabolism and translocation of β-amyrin in Sorghum bicolor

The biosynthesis of [¹⁴C] β-amyrin and three other labelled 3β-hydroxypentacyclic triterpenes from [2-^14C] acetate in leaves of Sorghum bicolor was demonstrated. Evidence for the metabolism of [¹⁴C] β-amyrin to the corresponding C-3 ketone (β-amyrone) and for the transport of [¹⁴C] β-amyrin in leaves was also obtained.     

The stereochemistry of biosynthesis of decaprenoxanthin in a cell-free system

Intact cells of Flavobacterium dehydrogenans grown on glucose or acetate did not incorporate mevalonic acid-[¹⁴C]. After treatment with lysozyme the protoplasts were lysed by sonication in a dilute medium containing mevalonic acid-[¹⁴C] and the cell-free system produced incorporated label into uncyclized C₄₀, monocyclic C₄₅ and bicyclic C₅₀ carotenoids of which decaprenoxanthin was the most abundant.With mevalonate-[2-¹⁴C,4R-4-³H₁] the ¹⁴C:³H ratios of the carotenoids showed that the hydrogen atoms at C-2 and C-6 of the ring and that at C-3 of the 1-hydroxy, 2-methyl but-2-ene-4-yl residues of decaprenoxanthin were derived from the 4-pro-R hydrogen atom of mevalonic acid.Mevalonate-[2-¹⁴C,2R-2-³H₁] and mevalonate-[2-¹⁴C,2S-2-³H₁] gave ratios which showed that the C-4 hydrogen atoms of decaprenoxanthin were derived from the 2-pro-S hydrogen atom of mevalonic acid.     

Lipid synthesis in the laticifers of Euphorbia lathyris L. seedlings

Various solutions of labeled precursors were absorbed by the cotyledons of etiolated Euphorbia lathyris L. seedlings. Incorporation of ¹⁴C into triterpenes from [2-¹⁴C]mevalonic acid, [1-¹⁴C]acetate, [3-¹⁴C]pyruvate, [U-¹⁴C]glyoxylate, [U-¹⁴C]glycerol, [U-¹⁴C]serine, [U-¹⁴C]xylose, [U-¹⁴C]glucose, and [U-¹⁴C]sucrose was obtained. The [¹⁴] triterpenes synthesized from [¹⁴C] sugars were mainly of latex origin. [¹⁴C]mevalonic acid was only involved in terpenoid synthesis outside the laticifers. Exogenously supplied glyoxylate, serine, and glycerol were hardly involved in lipid synthesis at all. The ¹⁴C-distribution over the various triterpenols was consistent with the mass distribution of these constituents in gas liquid chromatography when [¹⁴C]sugars, [¹⁴C]acetate, and [¹⁴C]pyruvate were used. These precursors were supplied to the seedlings in the presence of increasing amounts of unlabeled substrates. The amount of substrate directly involved in lipid synthesis as well as the absolute triterpenol yield was calculated from the obtained [¹⁴C]triterpenols. The highest yield was obtained in the sucrose incorporated seedlings, being 25% of the daily increase of latex triterpenes in growing seedlings.     

Biosynthesis of sitosterol in barley seedlings

A method is described for the chemical synthesis of stigmasta-5,24-dien-3β-ol-[26-¹⁴C] and (24S)-24-ethylcholesta-5,25-dien-3β-ol-[26-¹⁴C] (clerosterol). 28-Isofucosterol-[7-³H₂] fed to developing barley seedlings (Hordeum vulgare) was incorporated into sitosterol and stigmasterol confirming the utilisation of a 24-ethylidene sterol intermediate in 24α-ethyl sterol production in this plant. Also, the use of mevalonic acid-[2-¹⁴C(4R)-4-³H₁] verified the loss of the C-25 hydrogen of 28-isofucosterol during its conversion into sitosterol and stigmasterol in agreement with the previously postulated isomerisation of the 24-ethylidene sterol to a Δ²⁴⁽²⁵⁾-sterol prior to reduction. However, feeding stigmasta-5,24-dien-3β-ol [26-¹⁴C] to barley seedlings gave very low incorporation into sitosterol. Attempts to trap radioactivity from mevalonic-[2-¹⁴C(4R)-4-³H₁] in stigmasta-5,24-dien-3β-ol when this unlabelled sterol was administered to barley seedlings gave only a very small incorporation although both 28-isofucosterol and sitosterol were labelled.     

Effect of Light on Mevalonic Acid Incorporation into the Phytosterols of Nicotiana tabacum L. Seedlings

Mevalonic acid-2-¹⁴C was readily incorporated into the free, esterified, and glycosidic sterol fractions of tobacco (Nicotiana tabacum L. var. Burley 21) seedlings. The time course of mevalonic acid-2-¹⁴C incorporation was different for the various individual sterols. Campesterol and sitosterol (group I) became radioactive as the free sterol and subsequently as the steryl ester. The reverse order was observed for cholesterol and stigmasterol (group II). Light stimulated the incorporation of mevalonic acid-2-¹⁴C into the group I free sterols and during the first 6 to 9 hours into the steryl esters of group II. The increase in specific radioactivity of the group II steryl esters was followed by a decline. Based on time course studies it is suggested that the group II steryl esters turn over rapidly and that light influences the rate of turnover.     

Comparative study of in vivo stigmasterol biosynthesis in Nicotiana tabacum and Hordeum vulgare

Six-day-old tobacco (Nicotiana tabacum) and barley (Hordeum vulgare) seedlings rapidly incorporated and metabolized exogenously supplied [4-¹⁴C]sitosterol but neither plant was able to convert it into stigmasterol. However, a sterol metabolite was isolated from both species and the acetate derivative was slightly more polar, on AgNO₃—silica gel TLC, than stigmasteryl acetate. A similar metabolite was also obtained with [4-¹⁴C]cholesterol, indicating a general metabolic reaction of plants to exogenous sterols. Both species incorporated [2-¹⁴C]mevalonic acid into sitosterol and stigmasterol. We suggest that in vascular plants, whether monocotyledons or dicotyledons, the pathway of stigmasterol biosynthesis is not via sitosterol but through a common precursor which is derived from mevalonic acid.     

The Origin of Carbons of Dihydrofuran Ring and C-6 in the Biosynthesis of Rotenone

For the investigation of rotenone biosynthesis, acetate-2-¹⁴C, mevalonic acid-2-¹⁴C lactone and methionine-methyl-¹⁴C were administered to Derris elliptica plants, respectively, and the distribution of carbon-14 in the labeled rotenone was determined by degradation. When mevalonic acid-2-¹⁴C lactone was incorporated into rotenone, the radioactivity was found equally in the carbons at both C-7′ and C-8′, indicating that these carbons are derived from the carbon-2 of mevalonic lactone. In the case of methionine-methyl-¹⁴C about 80% of the total radioactivity was found to enter two methoxyl groups. This result demonstrates that methionine is an efficient precursor of the methoxyl group. Furthermore, it is also suggested that methionine may be a precursor of the carbon at C-6.     

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.     

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.     

Biosynthesis of the prenyl side chains of plastiquinone and related compounds in maize and barley shoots

The incorporation of ¹⁴C by etiolated maize and barley shoots exposed to light of ¹⁴CO₂ and [2-¹⁴C]mevalonic acid into phylloquinone, plastoquinone, ubiquinone, α-tocopherolquinone and α-tocopherol was examined. In maize (the principal tissue studied) it was demonstrated that ¹⁴C from [2-¹⁴C]mevalonic acid is incorporated into phylloquinone, plastoquinone and ubiquinone. α-Tocopherol and α-tocopherolquinone, although undoubtedly labelled from this substrate, were not purified completely. As expected, ¹⁴C from ¹⁴CO₂ was incorporated into all components examined. Ozonolytic degradation studies showed that ¹⁴C from [2-¹⁴C]mevalonic acid was incorporated specifically into the prenyl side chains of plastoquinone and ubiquinone, and from this it was inferred that mevalonic acid can be regarded as the specific distal precursor to the prenyl portions of all terpenoid quinones occurring in plant tissues. From a comparison of the relative incorporation of ¹⁴C from ¹⁴CO₂ and [2-¹⁴C]mevalonic acid into the intra- and extra-chloroplastidic terpenoids evidence was obtained consistent with the tenet that the prenyl portions of the chloroplastidic quinones phylloquinone and plastoquinone, along with β-carotene, are biosynthesized within the confines of the chloroplast, the side chain of the extraplastidic ubiquinone and phytosterols being synthesized elsewhere within the cell. The results obtained for the incorporation of ¹⁴C from ¹⁴CO₂ and [2-¹⁴C]mevalonic acid into α-tocopherol and α-tocopherolquinone were not readily interpretable with regard to the site of synthesis of these compounds.     

UDPG: p-Coumarate glucosyltransferase activity in enzyme extracts from higher plants

Leaves of Coleus, Pilea, Cistus and Cestrum, and ripe tomatoes were all able to convert trans-cinnamic acid-[3-¹⁴C]into glucose esters of cinnamic acids. The pool sizes of these esters were measured by the radioisotopic dilution method, and they were found to be of the order of a few μg/g fresh plant material. 1-O-Caffeoyl-β-d-glucose in Cestrum leaves amounted to 70μg/g fresh plant material. Enzyme extracts from Cestrum leaves were able to convert trans-p-coumaric acid-[3-¹⁴C] to 1-O-p-coumaroyl-β-d- glucose, using UDPG as a source of glucose. This enzyme activity could be measured only by trapping techniques, due to the presence of considerable hydrolase activity in crude enzyme extracts.     

The stereospecificity of biosynthesis of squalene and β-amyrin in Pisum sativum

The distribution of deuterium in squalene and β-amyrin, biosynthesized from mevalonic acid-6,6,6-d₃ in Pisum sativum, has been examined b     

Removal of the acetate-moiety of 2,4-dichlorophenoxyacetic acid in Ribes sativum

Ten minutes after uptake of 2,4-dichlorophenoxyacetic acid-1-¹⁴C(2,4-D-1-¹⁴C) by excised Ribes sativum leaves, 37·8 % of the radioactivity in water-soluble metabolites was in glyoxylic acid. When 2,4-D- 2-¹⁴C was supplied under the same conditions, 23·0 % of the radioactivity of the water-soluble rnetabolites was in glyoxylic acid. Radioactive glycine and glyoxylic acid, isolated from Ribes sativum 6 hr after uptake of 2,4-D-1-¹⁴C, contained essentially all of the ¹⁴C in the carboxyl-carbon atoms. When 2,4-D-2-¹⁴C was the precursor, the glycine isolated contained 64·8 % of its radioactivity in C₂, while 60·0 % of the radioactivity in glyoxylic acid was in C₂. The side-chain label of 2,4-D-2-¹⁴C-4-³⁶Cl was more efficiently incorporated into ethanol-insoluble plant residue than the ring-label. The metabolism of glyoxylic acid-1-¹⁴C and 2,4-D-1-¹⁴C in excised Ribes sativum leaves were compared. The data suggest a cleavage of the acetate-moiety of 2,4-D resulting in a C₂ compound, perhaps glyoxylate.     

Kinetic study of possible intermediates in the biosynthesis of chlorogenic acid in Cestrum poeppigii

p-Coumaric and 3-O-p-coumarylquinic acid seem to be important precursors of chlorogenic acid in the leaves of Cestrum poeppigii. 3-O-Cinnamylquinic acid, which has a very small metabolic activity, is of little importance in this respect. The kinetics of incorporation of radioactivity from t-cinnamic acid-3-[¹⁴C] into p-coumaric, 3-O-p-coumarylquinic, chlorogenic and 3-O-cinnamylquinic acid showed that the biosynthetic rates for these products decrease in the order shown. For p-coumaric acid, which has a markedly high metabolic activity, a turnover rate of 28 μg/hr and per gram fresh plant leaf, was calculated. Some trapping experiments with caffeic acid, and the acids mentioned above and using either t-cinnamic acid-3-[¹⁴C] or p-coumaric acid-2-[¹⁴C] as precursor, are discussed. A HPLC method for the rapid determination of phenolic acids in plant extracts, is described.     

Biosynthesis of C6-C3 acids in Datura innoxia

Datura innoxia plants were fed via the roots with cinnamic acid-[2¹⁴C], (±)-phenyllactic (2-hydroxy-3-phenylpropanoic) acid-[2¹⁴C] and phenylalanine-[2-¹⁴C]. In each case apohyoscine, hyoscine, hyoscyamine and littorine were isolated from the aerial parts, and hyoscine, hyoscyamine and littorine from the roots. Cinnamic acid was not incorporated into the acid moieties of the alkaloids. Phenyllactic acid served as a better precursor than phenylalanine for tropic acid (hyoscine and hyoscyamine) and atropic acid (apohyoscine). Phenylalanine served as an effective precursor for the phenyllactic acid moiety of Littorine.     

Biosynthesis of p-hydroxybenzoic acid in poplar lignin

Biosynthetic pathways to p-hydroxybenzoic acid in polar lignin were examined by tracer experiments. High incorporation of radioactivity to the acid was observed when shikimic acid-[1-¹⁴C], phenylalanine-[3-¹⁴C], trans-cinnamic acid-[3-¹⁴C], p-coumaric acid-[3-¹⁴C] and p-hydroxybenzoic acid-[COOH-¹⁴C] were administered, while incorporation was low from shikimic acid-[COOH-¹⁴C], phenylalanine-[1-¹⁴C], phenylalanine-[2-¹⁴C], tyrosine-[3-¹⁴C], benzoic acid-[COOH-¹⁴C], sodium acetate-[1-¹⁴C] and d-glucose-[U-¹⁴C]. Thus p-hydroxybenzoic acid in poplar lignin is formed mainly via the pathway: shikimic acid → phenylalanine → trans-cinnamic acid → p-coumaric acid → p-hydroxybenzoic acid.     

Triterpenoid biosynthesis in tissue cultures of Glycyrrhiza glabra var. glandulifera

The incorporation of [1-¹⁴C]acetate and [2¹⁴ C]mevalonate into free and esterified triterpen-3-ols was examined in original plant organs and tissue cultures of Glycyrrhiza glabra var. glandulifera. Both substrates labeled -amyrin, an oleanane-type triterpene, and cycloartenol and 24-methylenecycloartanol, both of which are intermediates of phytosterol biosynthesis. The label in esterified triterpenes was distributed mainly in phytosterol intermediates, but not in -amyrin. The ratio of amyrin formation among the three triterpenes from [2-¹⁴C]mevalonate was relatively high in stolon segments and in root cultures, but negligible in callus cultures. Administration of a specific inhibitor of squalene-2, 3-epoxide:cycloartenol (lanosterol) cyclase caused a marked increase of -amyrin synthesis in root suspension cultures, and of 24-methylenecycloartanol synthesis in cell suspension cultures, from [2-¹⁴C]mevalonate.Abbreviations GL glycyrrhizin - GLA glycyrrhetic acid - MVA mevalonic acid - CCI squalene-2, 3-epoxide:cycloartenol cyclase inhibitor This paper is Part 72 in the series Studies on Plant Tissue Cultures from the laboratory at Kitasato University. For Part 71, see Furuya T, Koge K, Orihara Y (submitted for publication).     

Time Course of Steroid Biosynthesis and Metabolism in Haplopappus heterophyllus

A single dose of mevalonic acid-2-¹⁴C was administered simultaneously to 6 Haplopappus heterophyllus plants. They were harvested at intervals ranging from 3 days to 6 months. Four groups of biosynthetically related sterols were found to be radioactive in each plant, and the changes in radioactivity with time were studied. The most striking finding was a radioactive phenolic material present only in the 6-month plant, which had flowered.     

Biosynthesis of the tigloyl esters of Datura: Cis-trans isomerism

Datura innoxia plants were wick fed with angelic acid-[1-¹⁴C] and l-isoleucine-[U-¹⁴C] to act as a positive control. After 7 days the root alkaloids 3α-tigloyloxytropane, 3α,6β-ditigloyloxytropane, and 3α,6β-ditigloyloxytropan-7β-ol were isolated and it was determined that angelic acid is not a precursor for the tigloyl moiety of these alkaloids. Tiglic acid-[1-¹⁴C] which was fed via the roots to hydroponic cultures of Datura innoxia, was incorporated to a considerable degree after 8 days.