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.     

Biosynthesis of bakuchiol from cinnamic and p-coumaric acids

Specific incorporation of l-[U-¹⁴C]phenylalanine, [U-¹⁴C]cinnamic acid and p[2-¹⁴C]coumaric acid into bakuchiol has been observed in Psoralea corylifolia. Our findings show that the aromatic moiety along with two carbon atoms of the side chain are biosynthetically derived via phenylpropane pathway and not by the alternate pathway proposed earlier.     

Incorporation of various putative precursors into cuticular 3,4-dihydroxyphenylacetic acid of adult Tenebrio molitor

Post-emergence levels of 3,4-dihydroxyphenylacetic acid (DOPAC) and ketocatechol were determined in cuticle from adult Tenebrio molitor. Possible pathways for biosynthesis of DOPAC were studied by comparing the incorporation of injected [U-¹⁴C]tyrosine, [7-¹⁴C]dopamine, [7-¹⁴C]DOPA, [7-¹⁴C]tyramine, [U-¹⁴C]p-hydroxyphenylpyruvic acid (p-HPPA) and [ring-³H]p-hydroxyphenylacetic acid (p-HPAA) into cuticular DOPAC during its period of maximal increase 1–3 days after adult emergence. Increased incorporation of [U-¹⁴C]tyrosine between days 0 and 3 suggests rapid de novo biosynthesis of DOPAC from this primary precursor. Of the putative intermediates tested, only p-HPPA had a pattern of incorporation similar to that seen with tyrosine. Since p-HPAA was poorly incorporated into both cuticle and DOPAC, a tentative pathway tyrosine → p-HPPA → 3,4-dihydroxyphenylpyruvic acid → DOPAC is proposed.     

Biosynthesis of the tigloyl esters in Datura: The role of 2-methylbutyric acid

Datura innoxia plants were wick fed with (±)-2-methylbutyric acid-[1-¹⁴C] and harvested after 7 days. The root alkaloids 3α,6β-ditigloyloxytropane and 3α,6β-ditigloyloxytropan-7β-ol were isolated and degraded. In each case the radioactivity was located in the ester carbonyl group indicating that this acid is an intermediate in the biosynthesis of tiglic acid from l-isoleucine. On the other hand, (±)-2-hydroxy-2-methylbutyric acid-[1-¹⁴C], which was fed to hydroponic cultures of Datura innoxia alongside isoleucine[U-¹⁴C] positive control plants, is not an intermediate.     

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.     

Participation of C6C1 unit in the biosynthesis of ephedrine in Ephedra

Feeding of benzoic acid-[7-¹⁴C], benzaldehyde-[7-¹⁴C] and cinnamic acid-[3-¹⁴C] to Ephedra distachya resulted in efficient incorporations of ¹⁴C into the α-carbon atom of the side chain of l-ephedrine. Thus ephedrine was shown to be biosynthesized by the condensation of a C₆C₁ portion which is derived from phenylalanine via cinnamate and an unidentified C₂-N fragment.     

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 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.     

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.     

Failure of 3-hydroxy-3-phenylpropanoic acid and cinnamic acid to serve as precursors of tropic acid in Datura innoxia

Datura innoxia plants were fed the R- and S-isomers of [3-¹⁴C]-3-hydroxy-3-phenylpropanoic acid, and [3-¹⁴C]cinnamic acid along with dl-[4-³H]phenylalanine. The hyoscyamine and scopolamine isolated from the plants 7 days later were labeled with tritium, but devoid of ¹⁴C, indicating that 3-hydroxy-3-phenylpropanoic acid and cinnamic acid are not intermediates between phenylalanine and tropic acid. The [³H] tropic acid obtained by hydrolysis of the hyoscyamine was degraded and shown to have essentially all its tritium located at the para position of its phenyl group, a result consistent with previous work.     

Degradation of phenylalanine and tyrosine by Sporobolomyces roseus

Ammonia-lyase activity for l-phenylalanine, m-hydroxyphenylalanine and l-tyrosine was demonstrated in cell-free extracts of Sporobolomyces roseus. Cultures of this organism converted dl-[ring-¹⁴C]phenylalanine and l-[U-¹⁴C]tyrosine into the corresponding cinnamic acid. Tracer studies showed that these compounds were further metabolized to [¹⁴C]protocatechuic acid. Benzoic acid and p-hydroxybenzoic acid were intermediates in this pathway. Washed cells of the organism readily utilized cinnamic acid, p-coumaric acid, caffeic acid, benzoic acid and p-hydroxybenzoic acid. Protocatechuic acid was the terminal aromatic compound formed during the metabolism of these compounds. The cells of S. roseus were able to convert m-coumaric acid into m-hydroxybenzoic acid, but the latter compound, which accumulated in the medium, was not further metabolized. 4-Hydroxycoumarin was identified as the product of o-coumaric acid metabolism by this organism.     

Ascorbic acid metabolism in geranium and grape

l-Ascorbic acid-[UL-¹⁴C] has been used to follow the appearance of ¹⁴C-labeled oxalic acid and tartaric acid as metabolic products of oxidative cleavage of ascorbic acid in geranium apices (Pelargonium crispum). The enantiomeric specificity of ascorbic acid metabolism was established in geranium by comparing the incorporation of d- and l-ascorbic acid-[6-¹⁴C] in the presence of l-ascorbic acid-[4-³H]. l-Ascorbic acid-[4-³H] has been used to demonstrate the retention of ³H during biosynthesis of l-(+)-tartaric acid in the geranium and its exchange with water during biosynthesis of l-( +)-tartaric acid in the grape.     

A general procedure for the preparation of labeled l-ascorbic acid from labeled d-glucose

A simple, three-step conversion of 1,2-O-isopropylidene-α-d-glucofuranose into l-ascorbic acid, originally described by Bakke and Theander, was used to prepare l-[4-¹⁴C]ascorbic acid from milligram amounts of d-[3-¹⁴C]glucopyranose in 28% radioisotopic yield. In addition, l-[6-¹⁴C]- and l-[U-¹⁴C]-ascorbic acid were prepared from d-[1-¹⁴C]- and d-[U-¹⁴C]-glucopyranose, respectively. The procedure is useful for the synthesis of l-ascorbic acid bearing isotopic hydrogen, carbon, or oxygen atoms at specific positions, subject only to the availability of starting material.     

Conversion of labeled substrates to sugars, cell wall polysaccharides, and tartaric Acid in grape berries

[U-¹⁴C]Sucrose, myo-[U-¹⁴C]inositol, [6-¹⁴C]- and [U-¹⁴C]glucuronate, UDP-[U-¹⁴C]glucuronate, [U-¹⁴C]gluconate, and l-[1-¹⁴C]ascorbic acid were fed into grape berries, Vitis labrusca L. cv. Delaware, at intervals throughout the ripening process and incorporation of ¹⁴C into several metabolites was studied.     

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.     

Non-reversibility of the isoleucine-acetate pathway in Datura

Five-month-old Datura meteloides plants were fed via the roots with 3-hydroxy-2-methylbutanoic acid-[1-¹⁴C] and isoleucine-[U-¹⁴C] as a positive control. After 5 days the plants were collected and in each case the root alkaloids 3α,6β-ditigloyloxytropane, 3α,6β-ditigloyloxytropan-7β-ol, meteloidine, hyoscine and hyoscyamine were isolated. Whereas isoleucine served as a precursor for the tiglic acid moieties 3-hydroxy-2-methylbutanoic acid did not.     

Time-course tracer studies on the metabolism of cinnamic acid in Cestrum poeppigii

Time-course tracer studies were performed on the metabolism of trans-cinnamic acid-[3-¹⁴C] and trans-p-coumaric acid-[2-¹⁴C] in the     

The pentose phosphate pathway as a source of NADPH for lignin synthesis

The aim of this work was to investigate whether the pentose phosphate pathway provides reducing power for lignin synthesis. Explants of the stem of Coleus blumei and the storage tissue of Helianthus tuberosus were cultured for 4 days on media which caused extensive lignification. [3-³H]-glucose and either [3-¹⁴C]- or [U-¹⁴C]-glucose were supplied to such 4-day-cultured explants, and also to the roots of 5-day-old seedlings of Pisum sativum. Significant amounts of ³H and ¹⁴C were recovered in syringaldehyde, vanillin, p-hydroxybenzaldehyde, and ligothio-glycollic acid from the explants of Coleus and Helianthus; and in vanillin, p-hydroxybenzaldehyde, and milled-wood lignin from pea roots. The ³H/¹⁴C ratios in these derivatives and preparations of lignin are held to indicate that much of the reducing power for lignin synthesis comes from the pentose phosphate pathway.     

The biosynthesis of ubiquinone in isolated rat heart cells

Beating heart cells were isolated from the adult rat and the biosynthesis of ubiquinone was studied. These cells were able to incorporate p-hydroxy[U-¹⁴C]benzoate into ubiquinone and some unidentified compounds, presumably intermediates in the biosynthesis of ubiquinone. The unidentified compounds were labile to alkali and were also labeled by [5-³H]-mevalonate and [methyl-³H]methionine, but not by p-hydroxy[carboxy-¹⁴C]benzoate. They appear to be chromatographically different from 5-demethoxy ubiquinone and 5-desmethyl ubiquinone. Addition of unlabeled mevalonate stimulated the incorporation of p-hydroxy [U-¹⁴C]benzoate into ubiquinone and the other compounds. The addition of dimethylsulfoxide to the isolated cells or the isolation medium caused inhibition of ubiquinone biosynthesis. Adriamycin was not inhibitory to the biosynthesis of ubiquinone in the cells. The advantages of these cells are the rapidity and ease in studying the biosynthesis of ubiquinone from various precursors and its regulation.     

Decreased labeling of amino acids by inhibition of the utilization of [3H,14C]glucose via the hexosemonophosphate shunt in rat brain in vivo

Treatment of rats with 6-aminonicotinamide showed a small but significant decrease in the labeling of amino acids in the brain after injection of [³H]acetate. The results of these experiments also gave evidence of the presence of [³H]glucose and [³H]lactate, and an increase in [³H]glucose content in the brain of 6-aminonicotinamide treated rats. To apportion the contribution of [³H]glucose formed by gluconeogenesis from [³H]acetate to the labeling of amino acids a method was formulated based on the measurement of radioactivity of amino acids, lactate and free sugars in brain after injection of [6-³H]glucose or [1-³H]glucose relative to that after co-injection of [U-¹⁴C]glucose or [2-¹⁴C]glucose. In contrast to the expected formation of [1, 6-³H]glucose by gluconeogenesis from [³H]acetate,³H-labeled glucose isolated from brain, blood and liver showed the presence of [6-³H]glucose only. The values corrected for the presence of [6-³H]glucose showed that treatment with 6-aminonicotinamide had no effect on the labeling of amino acids by oxidation of [³H]acetate. These findings indicated that a significant decrease in the labeling of amino acids from [U-¹⁴C]glucose reported previously and again confirmed using [1-³H], [6-³H], [2-¹⁴C] or [U-¹⁴C]glucose in the present investigation was not due to the inhibition of the activities of enzymes of the citric acid cycle. These results support the postulated role of the hexosemonophosphate shunt for the utilization of glucose in providing neurotransmitter amino acids glutamate and -aminobutyrate.Dedicated to Professor K. A. C. Elliott on his 80th birthday.