Biosynthesis of avenacins and phytosterols in roots of Avena sativa cv. Image

In keeping with the proposal that avenacin biosynthesis is restricted to the tips of primary roots of oat seedlings, the incorporation of radioactivity from R-[2-(14)C]mevalonic acid (MVA) into avenacins and beta-amyrin by serial sections of primary roots was found to be more-or-less restricted to root tip sections. Squalene synthase (SQS) (EC 2.5.1.21) and 2,3-oxidosqualene:beta-amyrin cyclase (OS beta AC) (EC 5.4.99) were also most active in these sections. The incorporation of radiolabel from R-[2-(14)C]MVA into cycloartenol and 24-methylene cycloartanol by, and the 2,3-oxidosqualene:cycloartenol cyclase (OSCC) (EC 5.4.99) activity in, the various serial sections were consistent with phytosterol biosynthesis occurring in all the sections of the root with some tailing-off in the rate of synthesis in the more distal sections.     

Sterol and triterpene synthesis in the developing and germinating pea seed

Developing and germinating pea seeds were compared with respect to their capacity to incorporate mevalonate into sterols and triterpenes. The capacity for sterol synthesis is greatest in the least mature fruits and decreases during their development. Label is shown, by gas–liquid chromatography and counting the radioactivity of trapped fractions, to be associated with campesterol, β-sitosterol and isofucosterol. During early stages of germination sterol synthesis is insignificant. The triterpene fraction becomes heavily labelled during both development and germination. The label is associated almost exclusively with β-amyrin during germination but with cycloartenol and 24-methylenecycloartanol during development. It is only in the terminal stages of maturation that β-amyrin becomes significantly labelled. At the same time an unidentified radioactive polar compound appears. The possible significance of the appearance of this polar compound and the regulation of the synthesis of these higher terpenoids is discussed.     

N-[(1,5,9)-trimethyl-decyl]-4 alpha,10-dimethyl-8-aza-trans-decal-3 beta-ol a novel potent inhibitor of 2,3-oxidosqualene cycloartenol and lanosterol cyclases

N-[(1,5,9)-trimethyl-decyl)]-4 alpha,10-dimethyl-8-aza-trans-decal-3 beta-ol 9 was designed to mimic the C9 or C8 high energy carbocationic intermediates postulated during the enzymic cyclization of 2,3-oxidosqualene to different triterpenes. The structurally new molecule 9 inhibits strongly cycloartenol and lanosterol cyclases in maize seedlings and rat liver microsomes respectively, whereas it does not inhibit beta-amyrin cyclase in the plant system. For the first time 2,3-oxidosqualene cycloartenol cyclase and beta-amyrin cyclase have been differentiated in the same plant material by use of a specific inhibitor.     

Investigation of avenacin-deficient mutants of Avena strigosa

The biosynthesis of cyclic triterpenoids in ten saponin-deficient (sad) mutant varieties of the diploid oat Avena strigosa is reported. Two mutants were found to be deficient in 2,3-oxidosqualene:beta-amyrin cyclase (OSbetaAC) (EC 5.4.99) and thus unable to produce the beta-amyrin necessary for the production of avenacins. The other mutants studied had post beta-amyrin lesions. 2,3-Oxidosqualene:cycloartenol cyclase (OSCC) (EC 5.4.99.8) needed for sterol formation was present in all ten mutants.     

Biosynthesis of phytosterols. Kinetic mechanism for the enzymatic C-methylation of sterols

Cloned soybean sterol methyltransferase was purified from Escherichia coli to gel electrophoretic homogeneity. From initial velocity experiments, catalytic constants for substrates best suited for the first and second C1 transfer activities, cycloartenol and 24(28)-methylenelophenol, were 0.01 and 0.001 s-1, respectively. Two-substrate kinetic analysis using cycloartenol and S-adenosyl-l-methionine (AdoMet) generated an intersecting line pattern characteristic of a ternary complex kinetic mechanism. The high energy intermediate analog 25-azacycloartanol was a noncompetitive inhibitor versus cycloartenol and an uncompetitive inhibitor versus AdoMet. The dead end inhibitor analog cyclolaudenol was competitive versus cycloartenol and uncompetitive versus AdoMet. 24(28)-Methylenecycloartanol and AdoHcy generated competitive and noncompetitive kinetic patterns, respectively, with respect to AdoMet. Therefore, 24(28)-methylenecycloartanol combines with the same enzyme form as does cycloartenol and must be released from the enzyme before AdoHcy. 25-Azacycloartanol inhibited the first and second C1 transfer activities with about equal efficacy (Ki = 45 nm), suggesting that the successive C-methylation of the Delta 24 bond occurs at the same active center. Comparison of the initial velocity data using AdoMet versus [2H3-methyl]AdoMet as substrates tested against saturating amounts of cycloartenol indicated an isotope effect on VCH3/VCD3 close to unity. [25-2H]24(28)-Methylenecycloartanol, [28E-2H]24 (28)-methylenelanosterol, and [28Z-2H]24(28)-methylene lanosterol were prepared and paired with AdoMet or [methyl-3H3]AdoMet to examine the kinetic isotope effects attending the C-28 deprotonation in the enzymatic synthesis of 24-ethyl(idene) sterols. The stereochemical features as well as the observation of isotopically sensitive branching during the second C-methylation suggests that the two methylation steps can proceed by a change in chemical mechanism resulting from differences in sterol structure, concerted versus carbocation; the kinetic mechanism remains the same during the consecutive methylation of the Delta 24 bond.     

Protein and Nucleic Acid Metabolism in Germinating Peas

Protease, peptidase, and ribonuclease activities were demonstratedin germinating pea cotyledons and axis tissues. These activitiesindicate that the enzymatic machinery for the hydrolysis ofprotein and nucleic acid reserves are present in the germinatingcotyledon. The fate of hydrolytic products was determined byinjecting leucine-¹⁴C or adenine-8-¹⁴C into the cotyledons.At most, 20 per cent of the leucine-¹⁴C and 10 per cent of theadenine-8-¹⁴C administered were transported from the cotyledonto axis tissues. Both compounds were extensively metabolizedand the labelling patterns suggest that different metabolicpathways are in operation in the two organs. The amounts ofadenine incorporated into nucleic acids and of leucine incorporatedinto protein in the cotyledons suggest that synthesis and turnoverwere occurring at a rapid rate. The adenine transported fromthe cotyledon was not readily available for nucleic acid synthesisin the axis whereas transported leucine was readily incorporatedinto axis protein.     

S-Adenosyl-l-methionine–cycloartenol methyltransferase activity in cell-free systems from Trebouxia sp. and Scenedesmus obliquus

1. Homogenates prepared from Trebouxia sp. 213/3 and Scenedesmus obliquus exhibited S-adenosyl-l-methionine-cycloartenol methyltransferase activity. 2. The products of the reaction, with cycloartenol as the substrate, were 24-methylenecycloartanol and cyclolaudenol. 3. Optimal enzyme activity was found in homogenates prepared at pH7.6 and the transmethylase was distributed between the supernatant and microsomal fractions of the Trebouxia homogenate. 4. The relevance of these results is discussed in relation to C(28) and C(29) sterol production in the algae.     

The Metabolism of Ethanol in Germinating Pea Seedlings

Considerable losses of ethanol occurred during the germinationof green pea seeds which could not be ascribed to losses dueto the volatility of the alcohol. Changes in the contents ofacetaldehyde, acetone, organic acids, and in the gas exchangessuggested that the alcohol was oxidatively metabolized. Feedingethanol to slices of pea cotyledon tissue also indicated ethanolconversion to acetaldehyde and the interconversion of acetaldehydeand acetone. Feeding ethanol 2:¹⁴C to the slices confirmed thatthe ethanol was metabolized, giving similar changes in the contentof carbonyl compounds and organic acids to those observed inthe intact germinating pea seedlings. Thus the endogenous ethanolwhich accumulated when pea seed imbibed water prior to germinationmay be metabolized subsequently by the germinating seedling.     

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).     

Studies in phytosterol biosynthesis. Mechanism of biosynthesis of cycloartenol

1. The mechanism of cycloartenol biosynthesis in leaves of Solanum tuberosum was investigated with the use of [2-¹⁴C,(4R)-4-³H₁]mevalonic acid. 2. The ³H/¹⁴C atomic ratio in cycloartenol was 6:6, the same as that in squalene; this eliminates lanosterol as a possible biosynthetic precursor of cycloartenol, and indicates that a hydrogen migration from C-9 to C-8 occurs. 3. Chemical isomerization of the cycloartenol to lanosterol (³H/¹⁴C ratio 5:6) and parkeol (³H/¹⁴C ratio 6:6) confirms the hydrogen migration from C-9 to C-8. 4. Possible mechanisms for the biosynthesis of cycloartenol and parkeol are discussed. 5. The ³H/¹⁴C ratio for 24-methylenecycloartanol was 6:6, demonstrating that the hydrogen atom at C-24 is retained during alkylation of the cycloartenol side chain.     

The interaction of endogenous gibberellins in correlation with cotyledons and axillary buds in the pea (Pisum sativum L.)

After the decapitation and amputation of one cotyledon in germinating pea seedlings, the axillary bud of the amputated cotyledon always grows and the growth of the axillary bud of the remaining cotyledon is inhibited. Before morphological differences appear between the axillary bud of the amputated and preserved cotyledon, a higher endogenous gibberellin content can be demonstrated chromatographically in the axillary bud of the amputated cotyledon. This indicates that the increased growth of the axillary bud of the amputated cotyledon is in connection with an earlier increase in the activation of endogenous gibberellins.     

不同含水量的豌豆种子萌发时物质动员及代谢研究

不同含水量的豌豆种子在饱和水蒸气中保持7d过程中,含水量低于萌动临界含水量时,子叶中贮藏蛋白质和淀粉的动员不能启动;含水量达到或超过萌动临界含水量,贮藏物质的动员被启动,豌启种子萌动后,子叶中蛋白质和淀动员程度与种子含水量呈正相关,前3d物质动员的程度比后4d强烈得多,因此,含水量是豌豆种子萌发时物质动员的启动因子和调节因子,同时,豌豆种子的含水量直接影响胚轴的生长状况。     

The stereospecific biosynthesis of plant sterols and α- and β-amyrin

1. A preparation of pea seedlings has been obtained that will incorporate [2-(14)C]mevalonate into squalene, alpha- and beta-amyrin and the phytosterols. 2. The (14)C/(3)H ratio in alpha- and beta-amyrin biosynthesized in the presence of [2-(14)C,4R-(3)H]-mevalonate is the same as in the starting material and in squalene; this gives experimental support to the mechanism for the cyclization of squalene proposed by Ruzicka for the formation of these pentacyclic triterpenoids. 3. The (14)C/(3)H ratio for beta-sitosterol was 5:3, the same as that in cholesterol in liver. 4. As the absence of (3)H from C-3 in beta-sitosterol was demonstrated (3)H must be present in the side chain and thus the H at C-24 is not lost during alkylation of the side chain; it probably migrates to C-25.     

Regulation of diamine oxidase activity in germinating pea seeds

Diamine oxidase present in the cotyledons of germinating pea seeds is induced by putrescine, spermidine and ornithine. Auxins inhibit enzyme synthesis in cotyledons only in the presence of embryo. Cycloheximide inhibits the synthesis of the cotyledon enzyme but has no effect on the embryo enzyme. 5-Fluorouracil inhibits the synthesis of both cotyledon and embryo enzymes.     

Comparative responses of the yeast mutant strain GL7 to lanosterol,cycloartenol, and cyclolaudenol

Under anaerobic growth conditions the isomeric 4,4′,14-trimethylcholestane derivatives lanosterol and, more efficiently, cycloartenol satisfy the sterol requirement of the yeast sterol auxotroph $\text{Saccharomyces cerevisiae}$ strain GL7. Aerobic mutant growth is supported only by cycloartenol and not by lanosterol, suggesting different structural requirements for aerobic and anaerobic cells. It is proposed that the non-planar conformation imposed by the 9,19-cyclopropane ring of cycloartenol moderates the adverse membrane effects of the nuclear methyl groups at C-4 and C-14. Under both aerobic and anaerobic conditions cyclolaudenol, a C-24-methyl derivative of cycloartenol, is a significantly more effective sterol source for strain GL7 than cycloartenol. This result is in keeping with the predominance of C-24-methyl sterols (ergosterol) in wild-type yeast.     

Cyclization of squalene-2,3-epoxide to 10α-cucurbita-5,24-dien-3β-ol by microsomes from Cucurbita maxima seedlings

Labelled 10α-cucurbita-5,24-dien-3β-ol was obtained from [3-³H]squalene-2,3-epoxide incubated with microsomes of Cucurbita maxima seedlings. By contrast the lanostane triterpenoids [2-³H]cycloartenol, [2-³H]parkeol and their corresponding derivatives [2,12-³H]11-ketocycloartenol and [2-³H]24,25-dihydro-90α,11a-epoxyparkeol, incubated under the same conditions, gave no rearranged products with a cucurbitane skeleton. These results suggest that biosynthesis of cucurbitane triterpenoids from squalene-2,3-epoxide occurs through direct cyclization without the intermediacy of lanostane-type triterpenoids such as cycloartenol or parkeol. Labelled cycloartenol, α-amyrin, β-amyrin and 24-methylenecycloartanol were also obtained from [3-³H]squalene-2,3-epoxide in the same enzymatic system yielding labelled 10α-cucurbitadienol.     

Effect of ageing on sterol metabolism in potato tuber slices

When fresh potato tuber slices were incubated with [1-¹⁴C]-sodium acetate, cycloartenol was heavily labelled but no radioactivity was recovered in 24-methylene cycloartanol and free sterols. If potato slices were aged for 0–24 hr before feeding with radioactive acetate, a rapid increase of the label in the sterol precursors and the free sterols was observed. The free sterol content was 5 × higher after ageing for 24 hr. Isofucosterol synthesis was especially stimulated. The synthesis of sterols during the ageing process seems to be related to the appearance of a cycloartenol C24-methylase and may be linked to a biogenesis of membranes.Nomenclature: (1) 4,4,14α-trimethyl 9β, 19β-cyclo-5α-cholest-24-en 3β-ol; (2) 4,4,14α-trimethyl 9β, 19β-cyclo-5α-ergost-24(28)-en 3β-ol; (3) 4α,14α-dimethyl 9β,19β-cyclo 5α-ergost 24(28)-en 3β-ol; (4) 4α, 14α-dimethyl 5α-ergosta 8.24(28)-dien 3β-ol; (5) 4α-methyl 5α-ergosta 7,24(28)-dien 3β-ol; (6) ergosta 5,24(28)-dien 3β-ol; (7) stigmasta 5,Z-24(28)-dien 3β-ol; (8) (24R)-24 methyl cholest 5-en 3β-ol; (9) (24R)-24 ethyl cholest 5-en 3β-ol; (10) (24S)-24 ethyl cholesta 5,E-22(23)-dien 3β-ol; (11) cholest 5-en 3β-ol.     

Triterpene alcohols in the seeds of solanaceae

Lanost-8-en-3β-ol, lanosterol and 24-methylenelanost-8-en-3β-ol in addition to cycloartanol, cycloartenol, 24-methylenecycloartanol, lupeol, β-amyrin, daturaolone and daturadiol were identified in the seeds of Solanaceae plants. The distribution of the first eight triterpene alcohols in the seeds of eleven plants belonging to seven genera of Solanaceae family was determined.     

Sitosterol biosynthesis in Hordeum vulgare

Excised barley embryos cultured on a nutrient medium containing methionine-[CD₃] incorporated deuterium into the newly biosynthesized sterols. Two deuterium atoms were present in 24-methylenecycloartanol, 24-methylenelophenol and campesterol and a maximum of four deuterium atoms were incorporated into 24-ethylidenelophenol, stigmasterol and sitosterol. Mevalonic acid-[2-¹⁴C(4R)4-³H₁] was utilized by the barley embryos to give 28-isofucosterol with a ³H-¹⁴C atomic ratio of 3:5 and stigmasterol and sitosterol with a ³H-¹⁴C atomic ratio of 2:5. 24-Methylenelophenol and 24-ethylidenelophenol were isolated from barley seed and 24-ethylidenelophenol-[2,4-³H₃] was incorporated into sitosterol by barley seedlings. These results show that in the production of sitosterol a 24-ethylidenesterol intermediate is produced and it is suggested that this is isomerized to give a Δ^24,(25) sterol prior to reduction to the saturated C₂₉ sterol side chain.     

Up-regulation of soyasaponin biosynthesis by methyl jasmonate in cultured cells of Glycyrrhiza glabra

Exogenously applied methyl jasmonate (MeJA) stimulated soyasaponin biosynthesis in cultured cells of Glycyrrhiza glabra (common licorice). mRNA level and enzyme activity of beta-amyrin synthase (bAS), an oxidosqualene cyclase (OSC) situated at the branching point for oleanane-type triterpene saponin biosynthesis, were up-regulated by MeJA, whereas those of cycloartenol synthase, an OSC involved in sterol biosynthesis, were relatively constant. Two mRNAs of squalene synthase (SQS), an enzyme common to both triterpene and sterol biosyntheses, were also up-regulated by MeJA. In addition, enzyme activity of UDP-glucuronic acid: soyasapogenol B glucuronosyltransferase, an enzyme situated at a later step of soyasaponin biosynthesis, was also up-regulated by MeJA. Accumulations of bAS and two SQS mRNAs were not transient but lasted for 7 d after exposure to MeJA, resulting in the high-level accumulation (more than 2% of dry weight cells) of soyasaponins in cultured licorice cells. In contrast, bAS and SQS mRNAs were coordinately down-regulated by yeast extract, and mRNA accumulation of polyketide reductase, an enzyme involved in 5-deoxyflavonoid biosynthesis in cultured licorice cells, was induced transiently by yeast extract and MeJA, respectively.