Participation of Mevalonate in the Biosynthetic Pathway of Ipomeamarone

1. The incorporation of mevalonate-2-¹⁴C into ipomeamarone in sweet potato root tissue infected by Ceratocystis fimbriata was demonstrated, but the rate was low when compared with acetate-2-¹⁴C. No dilution effect of mevalonate was noted during the incorporation of acetate-2-¹⁴C into ipomeamarone. This is very likely to result from the passive transfer of mevalonate into the cells.

2. No dilution effect of acetate during the incorporation of mevalonate-2-¹⁴C into ipomeamarone was noted. This indicates that mevalonate is not incorporated into ipomeamarone after its conversion to acetate.

3. Evidence for incorporation of acetate-2-¹⁴C into mevalonate was shown by the fact that the specific radioactivity of mevalonic acid benzhydrylamide was not lowered throughout repetitive crystallizations. These data also support the participation of mevalonate in ipomeamarone synthesis as an intermediate.

     

The Incorporation of Leucine-U-14C into Ipomeamarone

1. It was demonstrated by silica gel thin layer chromatography that leucine-U-¹⁴C was incorporated into furanoterpenes, e. g. ipomeamarone, in sweet potato root tissue infected by Ceratocystis fimbriata.

2. Further proof for ipomeamarone synthesis from leucine-U-¹⁴C was obtained by the constancy of the specific radioactivity of ipomeamarone semicarbazone through repetitive crystallization.

3. The synthetic pathway of ipomeamarone from leucine was found to be connected with the synthetic pathway from acetate at least at some steps.

4. Leucine-U-¹⁴C was incorporated into both saponifiable and non-saponifiable materials in the same way as acetate-2-¹⁴C.     

The Role of β-Hydroxypropionate in Ethylene Biosynthesis

To search precursors of ethylene in banana fruits, ethylene formation from acetate-2-¹⁴C and fumarate-2,3-¹⁴C by banana slices was studied. Ethylene-¹⁴C formation from acetate-2-^l4C was reduced by the addition of malonate or β-hydroxypropionate, and it was enhanced in a sealed chamber in comparison with the case in an aeration chamber. No label of fumarate-2,3-¹⁴C was incorporated into ethylene.

From these facts it was suggested that acetate-2-¹⁴C was incorporated into ethylene via malonate and β-hydroxypropionate. Participation of fumarate in ethylene biosynthesis of banana fruits was ruled out. β-Hydroxypropionate was postulated as an effective precursor of ethylene formation from acetate-2-^l4C.     

Effect of Ethrel and Ceratocystis fimbriata on the Synthesis of Fatty Acids and 6-Methoxy Mellein in Carrot Root

The rate of incorporation of ¹⁴C from acetate-1-¹⁴C into fatty acids by carrot root discs, 18 hours after inoculation with Ceratocystis fimbriata, was 9-fold greater than that in freshly cut discs. The rate in discs treated with water or Ethrel was 3-fold greater. The rate of incorporation of ¹⁴C from glucose-U-¹³C into fatty acids was 3-fold greater 18 hours after any of the above treatments. The rate of ¹⁴C incorporation from malonate-2-¹⁴C into fatty acids 24 hours after inoculation with C. fimbriata or treatment with water was 25 and 60%, respectively, of that in freshly cut discs. Linoleic acid was the principal fatty acid in carrot root, but incorporation of ¹⁴C from acetate-1-¹⁴C into the acid was low until 18 hours after inoculation with C. fimbriata or treatment with Ethrel. Turnover rates of the fatty acids appeared low and were similar for all treatments.     

Biosynthesis of the 3-benzylchroman-4-one eucomin in Eucomis bicolor

Feeding experiments have demonstrated the specific incorporation of radioactivity from dl-phenylalanine-[1-¹⁴C], l-phenylalanine-[U-¹⁴C], sodium acetate-[2-¹⁴C] and l-methionine-[methyl-¹⁴C] into the 3-benzylchroman-4-one eucomin in Eucomis bicolor. The labelling patterns indicate that eucomin is biosynthesized by the addition of a carbon atom derived from methionine onto a C₁₅ chalcone-type skeleton. Radioactivity from 2′,4′,4-trihydroxy-6′-methoxychalcone-[methyl-¹⁴C] and 2′,4′-dihydroxy-4,6′-dimethoxychalcone-[6′-methyl-¹⁴C] was incorporated into eucomin, the latter compound being the better precursor, demonstrating the feasibility that 2′-methoxychalcones are biosynthetic precursors of the “homoisoflavonoids”. Possible biosynthetic relationships in this class of compounds are discussed.     

Cyanide as an asparagine precursor in corn roots

K¹⁴CN is efficiently converted to asparagine in corn roots with asparagine accounting for 26% of the total radioactivity after 2 hr. Additions of glucose, cysteine or serine do not affect the reaction. Cysteine-¹⁴C(U) is normally a poor precursor of asparagine, but in the presence of 10⁻⁶ M KCN becomes a significant source. Cyanide does not promote the incorporation of serine-¹⁴C(U) or acetate-2-¹⁴C into asparagine. The antibiotic cycloheximide is a potent inhibitor of asparagine formation in the root tips when acetate-2-¹⁴C or aspartate-¹⁴C(U) serve as precursors. However, when K¹⁴CN is the precursor it is without effect. The results, therefore, show that cyanide is a potential asparagine precursor in maize root tips and suggest that normally the availability of cyanide and the synthesis of cysteine from serine are major rate limiting reactions in this pathway.     

Biosynthesis of the sesquiterpenoid,capsidiol, in sweet pepper fruits inoculated with fungal spores

Capsicum frutescens fruits inoculated with spore suspensions of Monilinia fructicola incorporated 1–4% of sodium acetate-[2-¹⁴C] or RS-mevalonolactone-[2-¹⁴C] into the phytoalexin, capsidiol. Labelled capsidiol was characterized by GC-RC, TLC-RC, gel chromatography (in conjunction with liquid scintillation counting) and GC-MS. The mode of incorporation of sodium acetate-[1,2-¹³C₂] into capsidiol, as indicated by the pattern of ¹³C-¹³C coupling from ¹³C NMR data, supports the hypothesis that the angular methyl group of the capsidiol skeleton arises by migration from the C-10 position of a eudesmane-type intermediate.     

The mechanism supplying acetyl-CoA for terpene biosynthesis in sweet potato with black rot: Incorporation of acetate-2-14C, pyruvate-3-14C and citrate-2,4-14C into ipomeamarone

Silica gel thin layer chromatography showed that acetate-2-¹⁴C,pyruvate-3-¹⁴C and citrate-2,4-¹⁴C were incorporated into ipomeamaronein sweet potato root tissues infected by Ceratocystis fimbriata.Rates of incorporation of ¹⁴C, from these 3 substances, intothe CHCl³-CH₃OH-soluble lipid fraction and ipomeamarone wereof the followingder: acetate > pyruvate > citrate ¹This paper constitutes Part 82 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury (Received December 11, 1969; )     

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.     

Participation of farnesol in the biosynthesis of ipomeamarone

Farnesol-2-¹⁴C was readily incorporated into ipomeamarone, oneof the furanoterpenoids produced in sweet potato infected withthe black rot fungus, Ceratocystis fumbriata. This was demonstratedby isolating labeled ipomeamarone and analyzing its radioauto-gramby silica gel thin layer chromatography of the extracts solublein chloroformmethanol (1: 1 , v/v), after farnesol-2-¹⁴C feedingto the tissue. Further proof for farnesol-2-¹⁴C incorporationinto ipomeamarone comes from the fact that the specific radioactivityof ipomeamarone semicarbazone was constant throughout the crystallizations.Fractionation of the label of farnesol-2-¹⁴C showed that radioactivitywas little distributed in the methanol-water fraction and wasmainly incorporated into ipomeamarone. Accordingly, it is notlikely that farnesol is incorporated into ipomeamarone afterits degradation to a small molecule(s) such as acetate. An additionalexperiment indicatedthat the incorporation of farnesol-2-¹⁴Cinto ipomeamarone markedly decreased under strict anaerobicconditions. This shows that some oxidative reactions are involvedin ipomeamarone biosynthesis from farnesol. ¹ This paper constitutes Part 91 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. (Received February 3, 1971; )     

Lipogenesis in Fatty Liver by Amino Acid Imbalance

Lipogenesis in the fatty liver of rat which was induced by feeding an amino acid-irnbalanced diet containing 8% casein supplemented with 0.3% dl-methionine has been investigated by measuring the incorporation of acetate-1-¹⁴C into various lipid fractions during in vitro incubation of liver slices.

In the incorporation of acetate-1-¹⁴C into the total lipid per one g of the slices, no significant difference for the imbalance group was observed. However, the total radioactivity of liver lipid per 100 g of the body and the incorporation into triglyceride in the lipids were significantly higher in the imbalance group than in the control group. Conversion of acetate-1-¹⁴C to CO₂ was not impaired in the imbalance group.

It is evident from these results that the induction of this type of fatty liver is due mainly to the synthesis of triglyceride.     

In vivo Biosynthesis of isopentenylacetophenones in Eupatorium rugosum

The biosynthesis of dehydrotremetone in Eupatorium rugosum has been investigated by feeding radioactive precursors to intact plants. The carbon atoms of acetate-[1-¹⁴C] and acetate-[2-¹⁴C] were identified in dehydrotremetone by degradation of the molecule. From the pattern of labeling it was concluded that the acetophenone moiety was derived from acetate via the polyacetate pathway. From the incorporation of mevalonate it appeared that the furan ring and its side chain were formed from an isoprenoid compound. Potential aromatic intermediates were chemically synthesized and also fed to plants but only tremetone was found to be efficiently incorporated into dehydrotremetone. Neither 4-hydroxyacetophenone nor 4-hydroxy-3[isopenten-(2)-yl]-acetophenone were efficiently incorporated into dehydrotremetone.     

Polyprenyl quinones and α-tocopherol in Calendula officinalis

In various cellular subfractions of Calendula officinalis leaves a study was made of the distribution of polyprenyl quinones and α-tocopherol and the dynamics of their labelling with ¹⁴CO₂ and acetate-[1-¹⁴C] and incorporation of mevalonate-[2-¹⁴C] after 3 hr. It was confirmed that plastoquinone occurs only in the chloroplasts, ubiquinone only in the mitochondria and α-tocopherol in both these subfractions. Phylloquinone was found in the chloroplast and mitochondrial fractions as well as in the post-mitochondrial supernatant. Studies of the dynamics of radioactive precursor incorporation indicated that α-tocopherol is metabolized more rapidly than the polyprenyl quinones studied; the incorporation of mevalonate-[2-¹⁴C] suggests that the side chain of plastoquinone can be synthesized in the cytoplasm and transported to the chloroplasts.     

Lysine Biosynthesis in Barley (Hordeum vulgare L.)

Lysine biosynthesis in seedlings of barley (Hordeum vulgare L. var. Emir) was studied by direct injection of the following precursors into the endosperm of the seedlings: acetate-1-¹⁴C; acetate-2-¹⁴C; pyruvate-1-¹⁴C; pyruvate-2-¹⁴C; pyruvate-3-¹⁴C; alanine-1-¹⁴C; aspartic acid-1-¹⁴C; aspartic acid-2-¹⁴C; aspartic acid-3-¹⁴C; aspartic acid-4-¹⁴C; α-aminoadipic acid-1-¹⁴C; and α, ε-diaminopimelic acid-1-(7)-¹⁴C. The distribution of activity in the individual carbon atoms of lysine in the different biosynthetic experiments was determined by chemical degradation. The incorporation percentages and labeling patterns obtained are in agreement with the occurrence of the diaminopimelic acid pathway. The results do not fit the incorporation percentages and labeling patterns expected if the α-aminoadipic acid pathway was operating. However, the results show that barley seedlings are able to convert a small part of the α-aminoadipic acid administered directly to lysine.     

The Biosynthesis of Some Isothiocyanates and Oxazolidinethiones in Rape (Brassica campestris L.)

The incorporation of the radioactivity from acetate-1-¹⁴C, acetate-2-¹⁴C, dl-methionine-1-¹⁴C, dl-methionine-2-¹⁴C, dl-methionine-3,4-¹⁴C, dl-homomethionine-2-¹⁴C, dl-allyl-glycine-2-¹⁴C, and dl-2-amino-5-hydroxyvalerate-2-¹⁴C into the aglycones of progoitrin, gluconapin, and glucobrassicanapin of maturing rape plants (Brassica campestris L.) was investigated. Radioactivity from dl-methionine-2-¹⁴C, dl-methionine-3,4-¹⁴C, dl-homomethionine-2-¹⁴C, and acetate-2-¹⁴C were incorporated into the 3 major thioglucosides. The other organic compounds were poorly incorporated except for dl-allylglycine-2-¹⁴C into glucobrassicanapin. The results obtained suggest that the rape plant can synthesize amino acids by the condensation of acetate (as acetyl CoA) to α-keto acids to yield a homologue of the original amino acid. These newly formed amino acids are then employed to synthesize the 3 major thioglucosides.     

The incorporation of ornithine-[2,3-13C2] into nicotine and nornicotine established by NMR

dl-Ornithine-[2,3-¹³C₂] was synthesized from acetate-[1-¹³C] and ethyl acetamidocyanoacetate-[2-¹³C]. This labelled material was mixed with dl-ornithine-[5-¹⁴C] and fed to Nicotiana glutinosa plants by the wick method. After 10 days the plants were harvested affording radioactive nicotine and nornicotine (0.14% and 0.051% specific incorporations, respectively). Even at these low specific incorporations an examination of their ¹³C NMR spectra established the incorporation of ornithine symmetrically into the pyrrolidine rings of these alkaloids. Satellites were observable at the signals due to C-2′, 3′, 4′ and 5′ positions, arising by the presence of contiguous carbons at C-2′, 3′ and C-4′, 5′.     

Nature of DNA and RNA Degradation in Escherichia coli Induced by Colicin E2

Lipogenesis in the fatty liver of rat, which was induced by feeding an amino acid unbalanced diet containing 8% casein supplemented with 0.3% dl-methionine, has been studied by measuring the incorporation of glycerol-1-¹⁴C, palmitate-1-¹⁴C, citrate-1,5-¹⁴C, pyruvate-1-¹⁴C and pyruvate-2-¹⁴C into various lipid fractions and ¹⁴CO₂ during in vitro incubation of liver slices.

The total radioactivity of liver lipid per 100 g of the body and the incorporation of each substrate into triglyceride in the lipid were significantly higher in the imbalance group than the control group. Conversion of each substrate to ¹⁴CO₂ was not imparied in the imbalance group.

It is evident from these results that the induction of this type of fatty liver is due mainly to the triglyceride synthesis by both the fatty acid synthesis and the transesterification of fatty acid.

These results are considered to support the previous assumption in which acetate-1-¹⁴C was used as a precursor.     

Sodium-1,2-C Acetate Incorporation in Roots of Frost-hardy and Less Hardy Alfalfa Varieties under Hardening Conditions

When the temperature of incorporation of sodium acetate-1, 2-¹⁴C into lipids of alfalfa (Medicago media Pers. var. Rambler and Medicago sativa L. var. Caliverde) roots was lowered from 22 C to 1 C, elongation and desaturation of fatty acids and the labeling of phosphatidylcholine were strongly stimulated.     

In vivo incorporation of radioactive metabolites by Golgi apparatus and other cell fractions of onion stem

Incorporation in vivo of various ¹⁴C-labeled substrates into dictyosomes of onion (Allium cepa) stem was determined, and comparisons were made with other cell fractions on a nitrogen basis. Tissue explants were incubated for varying times in the presence of the radioactive metabolites supplied in the external medium. Fractions were then obtained from homogenates stabilized with glutaraldehyde. Purified fractions containing dictyosomes (individual stacks of cisternae) of the Golgi apparatus were obtained by centrifugation in a sucrose gradient also yielding a smooth membrane fraction free of dictyosomes. Dictyosomes were preferentially labeled with choline-1,2-¹⁴C and acetate-2-¹⁴C, suggesting that plant Golgi apparatus participate in the synthesis or modification of membrane lipids. Dictyosomes were also labeled with glucose-U-¹⁴C and leucine-U-¹⁴C, but on a molar basis incorporation was less than with choline or acetate.     

FORMATION OF LIGNIN IN TISSUE CULTURE OF PINUS STROBUS

The formation of shikimic acid and lignin from glucose in thecambium tissue was investigated. Glucose-1-¹⁴C, shikimic acid-G-¹⁴C, sodium acetate-1-¹⁴C and sodium acetate-2-¹⁴C were administeredto the tissue culture of strob pine. Glucose was well incorporatedinto shikimic acid, but acetic acid was less effective. Shikimicacid was very efficient as a precursor of aromatic nucleus andglucose was also converted efficiently to lignin. The extentof incorporation of acetic acid, however, was considerably low.A possibility was discussed that in the cultured tissue ligninand its precursor were synthesized from glucose via the shikimicacid pathway. (Received May 14, 1960; )