Catabolism of glucose in pea stem section during root formation and its inhibition by kinetin and ethionine

The catabolism of specifically¹⁴C-labelled glucose during the root formation and its inhibition by kinetin and ethionine in the etiolated pea stem sections were studied. The formation of root meristematic foci in the pericycle region of sections was accompanied by the decrease of the C₆/C₁ ratio. Such a result and activation of pentose phosphate cycle, which was also checked by another method, suggested increased participation of pentose phosphate cycle in glucose oxidation. The above mentioned changes were also found after the prevention of root formation by ethionine and, therefore, do not seem to be specific for the meristematic foci formation. The growth of newly formed roots was closely connected with the rise of C₆/C₁ values. The increase of CO₂ release from the first carbon atom of glucose molecule was recorded after the inhibition of root formation by ethionine. The rise of C₆/C₁ values and decrease of pentose phosphate cycle activity was observed after the treatment of pea stem sections by kinetin in the first 64 hours after sectioning. In this case root formation was prevented and the growth of lateral buds was stimulated. The secondary xylem formation which took place later was accompanied by the activation of the pentose phosphate cycle. These phenomena are discussed in relation to cell division and biosynthesis of lignin-precursors.     

Root Formation in Pea Stem Sections and its Inhibition by Kinetin,Ethionine and Chloramphenicol

Root formation in the etiolated pea stem sections and inhibition of this process is described in the present paper. Sodium fluoride, iodoacetic acid, norvaline, phenylserine, 5-bromuracil and 2-thiouracil did not inhibit the root formation completely. Complete inhibition, however, was observed after treatment of pea stem sections by kinetin, ethionine and chloramphenicol (5 X 10^-5 M, 1 x 10^-2 M, and 1 x 10^-2 16 hours after sectioning). The concentration of kinetin which produced complete inhibition of root formation simultaneously stimulated the growth of the lateral buds. Root formation under the conditions described below can be divided into two stages. The first stage 64 hours from the beginning of the experiment, the second stage 64 hours later. Further, the first stage includes the formation of the meristematic cells in the pericycle areas. In the second stage are included the growth of roots and differentiation of root-tissues. Roots were formed, first of all, in the short vertical region of the sections near to the basal buds. Secondary xylem formation was also observed during the cultivation of the sections. This process was stimulated by kinetin.     

Fractionation of proteins of pea stem sections during root formation and its inhibition by kinetin and ethionine

Changes in the protein constituents in pea stem sections during root formation and its inhibition by kinetin and ethionine were studied. Only quantitative differences in the protein fractions separated on DEAE-cellulose column were noted. The formation of foci of meristematic cells in pericycle was accompanied by an increase in the amount of fraction “l”. This fraction disappeared rapidly in sections where root formation either did not occur (internodial sections without nodes) or was inhibited by ethionine (stem sections with basal and apical nodes). Incubation of stem sections in a kinetin solution for 16 hours after cutting of stems partially preserved fraction “l”. The increase in the amount of fraction “l” was one of the first metabolic changes in root-forming pea stem sections after cutting of stems.     

Carbohydrate Effects on Amino Acid Transport by Trypanosoma equiperdum*

SYNOPSIS. Uptake of ¹⁴C-labeled alanine, glutamate, lysine, methionine, proline, and phenylalanine by Trypanosoma equiperdum during 2-minute incubations occurred by diffusion and membrane-mediated processes. Amino acid metabolism was not detected by paper chromatography of trypanosome extracts. Most of 18 carbohydrates tested for ability to alter amino acid transport neither changed nor significantly inhibited transport. Glucose, however, stimulated glutamate, lysine and proline transport; fructose stimulated lysine uptake and 2-deoxy-D-glucose increased phenylalanine and methionine absorption. No evidence was found that the carbohydrates acted by binding to amino acid transport “sites.” Glucose inhibition of alanine, phenylalanine, and methionine uptake was linked to glycolysis. The rapid formation of alanine from glucose stimulated alanine release and, when glycolysis was blocked, glucose no longer inhibited alanine transport. Methionine and phenylalanine release was also stimulated by glucose. Glucose changed the ability of lysine, glutamate, and proline to inhibit each others’uptake, indicating that certain amino acids are preferentially absorbed by respiring cells. Analysis of free pool amino acid levels suggested that some amino acid transport systems in T. equiperdum are linked in such a way to glycolysis as to control the cell concentrations of these amino acids.     

Amino acids and growth substances in barley root excretions (Hordeum distichon L.) and their biological effect

The biological effect of barley root excretions was studied during a 4 to 10 days cultivation period. The effect of root excretions changes according to the cultivation period of barley. It was ascertained by means of a bioassay that growth was either conclusively stimulated or the root excretions did not affect growth of the roots and of the upper part ofNasturtium. No significant inhibitory effect was observed. The effect of the single amino acids and of their mixtures found in the root excretions of barley was quite different. The following amino acids were determined by paper chromatography in root excretions: alanine, asparagine, phenylalanine, glycine, leucine, lysine, serine, tyrosine, glutamic acid and valine plus methionine. During cultivation their total quantity increased from 5·48. 10^?5 up to 1·13. 10^?3 mg per plant. Most of the 20 amino acids observed, displayed in theNasturtium test at a concentration from 0·1 to 1,000 mg/l an inhibitory effect onNasturtium growth. The effect of amino acid mixtures, corresponding qualitatively and quantitatively to the free amino acids in barley root excretions was dependent on their concentration. Growth regulators of the auxin type were found in culture solutions by chromatographic separation and with bioassay. As it may be seen from the results obtained, there are besides amino acids and indole derivatives other non-identified compounds involved in the effect of barley root excretions.     

Effect of Gibberellic Acid and Cycocel on Tryptophan Metabolism and Auxin Destruction in the Sunflower Seedling

A comparative study of tryptophan conversion in different regions of the sunflower seedling indicates that the regions most active in converting tryptophan on a pathway to auxin are the root apical segments and young leaves; next highest in activity is the cotyledonary tissue. The stem apex proper with leaf primordia is less active than the above regions in converting the auxin precursor. Hypocotyl tissue was observed to be least active. Pre-treatment of the apical bud region of the stem with gibberellic acid (GA) gives rise to tryptophan conversion rates which are 2.1 times those in untreated seedlings. The enhanced tryptophan conversion in the apical bud is followed by an increased elongation rate of the 1st internode which is 2.2 times that in the 1st internode of untreated seedlings. Treatment of the seedlings with Cycocel [(2-chloroethyl)trimethylamnionium chloride] does not reduce tryptophan conversion in the apical bud region of the seedling although elongation of the stem is greatly retarded. Indoleacetic acid (IAA) destruction in cell free preparations as well as in whole sections of the elongating region of the seedling stem was studied. IAA-1-¹⁴C destruction rates with the release of ¹⁴CO₂ in whole sections of 1st internode tissue were approximately 3 times those in cell free preparations of the same region. No significant changes in IAA destruction rates in seedlings pre-treated with GA or Cycocel were observed.     

The effects of electrical stimulation and ionic alterations on the metabolism of amino acids and proteins in excised superior cervical ganglia of the rat

Abstract— The effects of supramaximal electrical stimulation on the metabolism of amino acids and proteins in incubated superior cervical ganglia of the rat were studied by the use of a gas-liquid chromatographic (GLC) assay procedure. Stimulation at 5 Hz for 2 h caused an apparent increase in tissue levels of free amino acids, with alanine, serine, glycine, valine, threonine, isoleucine and aspartate (+ asparagine) most noticeably affected. The amino acid composition (partial) of the TCA-insoluble proteins of resting and stimulated ganglia was approximately the same after 60 min of incubation, but there was less TCA-insoluble protein in the stimulated ganglia. The addition of amino acids (at plasma concentrations) to the standard media had no apparent affect on the amino acid composition of this protein fraction. Stimulation for 0 , 5 h initially increased the efflux of alanine, valine, proline and ornithine into the incubation media but prolonged stimulation (for 4–0 h) decreased the efflux of alanine, serine, glycine and isoleucine and increased the efflux of lysine into the incubation media. The leakage of amino acids from the ganglia appeared to be a sodium-dependent process. The incorporation of ¹⁴C from [U-¹⁴C]glucose into glutamate (+ glutamine) and aspartate (+ asparagine) was greater in stimulated than in resting ganglia. However, the conversion of glutamate carbons from [U-¹⁴C]l -glutamate into aspartate was not affected by stimulation. Incorporation of ¹⁴C from [U-¹⁴C]glucose into glycine and serine was apparently not affected by stimulation during the 60 min of incubation. However, serine was the only amino acid which exhibited a higher specific radioactivity in stimulated ganglia than in resting ganglia incubated for 4 h in standard media. Lithium ions had the apparent specific effect of increasing the labelling with ¹⁴C from [U-¹⁴C]glucose into ornithine, and increasing the efflux and overall metabolism of serine in the ganglia. Incorporation of ¹⁴C from [U-¹⁴C]glucose into proteins was lower in the stimulated than in the resting ganglia if compensation was made for the higher radioactivity available in the total free amino acid pool of the stimulated ganglia. The rate of ¹⁴C incorporation from [U-¹⁴C]glutamate into the TCA-insoluble proteins of resting ganglia was greater when no other amino acids at concentrations approximating plasma levels were added to the bathing media; this rate was lower in stimulated than in resting ganglia.     

Utilization of exogenously supplied 14C-saccharose into primary metabolites and alkaloid production in Catharanthus roseus

Partitioning of exogenously supplied U-¹⁴C-saccharose into primary metabolic pool as sugars, amino acids, and organic acids was analyzed and simultaneous utilization for production of alkaloid by leaf, stem, and root in twigs and rooted plants of Catharanthus roseus grown in hydroponic culture medium was determined. Twigs revealed comparable distribution of total ¹⁴C label in leaf and stem. Stems contained significantly higher ¹⁴C label in sugar fraction and in alkaloids [47 kBq kg⁻¹(DM)] than leaf. In rooted plants, label in ¹⁴C in metabolic fractions in root such as ethanol-soluble, ethanol-insoluble, and chloroform-soluble fractions and in components such as sugars, amino acids, and organic acids were significantly higher than in stems and leaves. This was related with significantly higher content of ¹⁴C in alkaloids in stems and leaves. ¹⁴C contents in sugars, amino acids, and organic acids increased from leaf to stem and roots. Roots are the major accumulators of metabolites accompanied by higher biosynthetic utilization for alkaloid accumulation.     

Nutritional and Hormonal Requirements for the Growth of Vigna sinensis Callus in vitro

Nutritional and hormonal requirements for in vitro growth of callus tissue of Vigna sinensis Endl. were studied. Callus was formed on hypocotyl and root sections, when they were cultured on Linsmaier and Skoog's basal medium solidified with 10 g/1 agar and supplemented with only 0.5 mg/12,4-D or 2 mg/1 IAA. Further addition of 0.2 mg/1 kinetin and 1 g/l yeast extract resulted in more active callus formation. For unlimited vigorous growth of subcultured callus which was originally isolated from root sections, yeast extract was indispensable besides 2,4-D and kinetin. Such growth-promoting activity was observed also in malt extract and Ebios (dried cell powder of brewery yeast). Of known compounds tested, nicotinic acid, nicotinamide., methyl nicotinate and NAD were promotive to the growth of the callus, although much less effective than yeast extract. Other pyridine derivatives, vitamins and amino acids tested were ineffective or slightly effective. Sucrose was the most suitable carbon source. Fructose, glucose and maltose also supported the growth. Kinetin stimulated cell proliferation of the callus and cell differentiation to tracheary element.     

The Uptake of Cytokinins by Protoplasts and Root Sections of Brassica campestris

The effect of cytokinins was studied on the incorporation of ¹⁴C-labelled precursors into the nucleic acid fraction of protoplasts isolated from callus or roots of Brassica campestris. Protoplasts from callus and roots took up ¹⁴C-uridine from the incubation medium and incorporated this precursor into the ribonucleic acid fraction during the experimental period of 16 h. Low concentrations of kinetin (10^?8-5 × 10^?6M) did not stimulate the incorporation, and kinetin inhibited this process at higher concentrations (5 × 10^?5M). This result led to an investigation on the uptake of cytokinins by protoplasts of roots. In contrast to a rapid uptake of radio-actively labelled adenine and uridine. protoplasts from roots took up only small amounts of labelled kinetin. zeatin, zeatin riboside and zeatin nucleotides from the incubation medium. Root sections took up far more adenine and kinetin than protoplasts from roots. The ratio between the amount of kinetin taken up and applied was much higher for the sections than for protoplasts, indicating that intact root cells took up kinetin far more rapidly than protoplasts. It is suggested that the plasmalemma and cell wall play an essential role in the uptake of cytokinins or that the differences in the uptake rates are related to differences between the rates of metabolism of cytokinins in root sections and in protoplasts.     

Influence of gibberellic acid on <Superscript>14</Superscript>CO<Subscript>2</Subscript> metabolism,growth, and production of alkaloids in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Changes in growth parameters, carbon assimilation efficiency, and utilization of ¹⁴CO₂ assimilate into alkaloids in plant parts were investigated at whole plant level by treatment of Catharanthus roseus with gibberellic acid (GA). Application of GA (1 000 g m⁻³) resulted in changes in leaf morphology, increase in stem elongation, leaf and internode length, plant height, and decrease in biomass content. Phenotypic changes were accompanied by decrease in contents of chlorophylls and in photosynthetic capacity. GA application resulted in higher % of total alkaloids accumulated in leaf, stem, and root. GA treatment produced negative phenotypic response in total biomass production but positive response in content of total alkaloids in leaf, stem, and roots. ¹⁴C assimilate partitioning revealed that ¹⁴C distribution in leaf, stem, and root of treated plants was higher than in untreated and variations were observed in contents of metabolites as sugars, amino acids, and organic acids. Capacity to utilize current fixed ¹⁴C derived assimilates for alkaloid production was high in leaves but low in roots of treated plants despite higher content of ¹⁴C metabolites such as sugars, amino acids, and organic acids. In spite of higher availability of metabolites, their utilization into alkaloid production is low in GA-treated roots.     

Turnover of14C-labelled oat residues and small molecular organic compounds in two soils under different levels of mineral nutrition

Mineralization and redistribution of carbon from¹⁴C-labelled oat shoots and [¹⁴C(U)] labelled glucose, leucine, acetate and phenylacetate were studied in light loamy sand and medium clay loam under different levels of mineral nutrition. Losses of mineralized¹⁴C as CO₂ were greater in the sandy soil than in the clay soil. NPK and NPK+Ca fertilization increased the rates of decay of the introduced plant organic matter. Among the small molecular organic compounds glucose was degraded fastest and phenylacetate slowest. Incorporation of radioactive carbon into humus fractions varied and depended on the nature of the compound introduced and on the soil type. Carbon of glucose, phenylacetate and acetate was mainly incorporated into fulvic acids, whereas¹⁴C of leucine was almost evenly distributed between humic and fulvic acids and¹⁴C of oat residues in fulvic acids and humin fractions. There was significantly higher incorporation of¹⁴C into humic acids and lower incorporation into humins in the sandy soil compared to the clay soil. NPK+Ca decreased the conversion of¹⁴C from phenylacetate and acetate to bitumens and increased its content in humic acids, particularly in the clay soil. The incorporation of¹⁴C from phenylacetate to humins benefitted from mineral fertilization during the first 30 days of the experiment in both soils.     

The influence of some inhibitors on the formation of caffeic acid in cultures ofPerilla cell suspensions

A cell suspension culture, prepared fromPerilla frutescens var.crispa callus induced by Murashige and Skoog (1962) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 ml/l) and kinetin (0.1 mg/l), contained caffeic acid derivatives as the phenolic components. Fresh and dry weights of the cells increased exponentially for about 11 days after transfer to a fresh medium. The contents of caffeic acid and protein also reached a maximum on the 11th day, but α-amino nitrogen phenylalanine and tyrosine continued to increase in amount until the 20th to 23rd day. Caffeic acid formation in the cells was increased by lowering the concentration of 2,4-D. The administration ofl-2-aminooxy-3-phenylpropionic acid (l-AOPP), 2-aminooxyacetic acid (AOA) andN-(phosphonomethyl)glycine (glyphosate) to the cells inhibited caffeic acid formation to a large extent. An 80% inhibition of caffeic acid formation was caused by 10⁻⁴Ml-AOPP whereas phenylalanine and tyrosine contents of the cells became 7.5 and 2.3 times higher at thisl-AOPP concentration than those in the control. An 85% inhibition of caffeic acid formation was achieved at 10⁻³M glyphosate concentration, while 10⁻³M AOA inhibited caffeic acid formation by 95% and also growth rate by 80%. The influence of inhibitors on caffeic acid formation is discussed in relation to the level of α-amino nitrogen, particularly aromatic amino acids, in the cell suspension cultures.     

Uptake of amino acids by plants from the soil: A comparative study with castor bean seedlings grown under natural and axenic soil conditions

Castor bean seedlings grown in different media (soil, quartz sand, or liquid culture) under natural or axenic conditions take up¹⁴C labelled proline when offered to the rooting medium at concentrations similar to those occuring in the soil. Most of the absorbed proline was transferred through the root into the xylem without metabolic conversion, though some conversion to glutamine and alamine occurred.It is concluded that roots successfully compete with microorganisms for free amino acids in the soil for the following reasons: (a) The initial rate of appearance of radioactivity in the xylem sap was the same in plants grown in natural or in axenic soil, and (b) the specific activity of proline in the xylem sap was approximately the same in plants grown in natural conditions and in axenic soil (even somewhat higher under natural condition).The role of soil microorganisms became evident however in long-term experiments (e.g. 5h), because the soil solution was much more rapidly depleted of labelled amino acids in natural soil than in axenic soil. Therefore after 20 hours roots grown in sterilized soil or quartz sand always contained more¹⁴C label than those grown in natural soil.It is suggested that viable roots use free amino acids from the soil and that the main flux of carbon to the rhizosphere might be in the form of organic acids.     

Permeability Characteristics and Amino Acid Incorporation during Senescence (Ripening) of Banana Tissue

Changes in free space of banana tissue during ripening were measured using radioisotopes. Free space increased significantly about 44 hours before the onset of, and rose exponentially during the respiratory climacteric. The increase in free space indicates a progressive increase in the proportion of cells which becomes completely permeable to solutes in the ambient solution by simple diffusion. At the respiratory peak the tissue was essentially 100% free space to mannitol, sucrose, fructose and chloride.

The capacity for active uptake of solutes declined about one day before the onset of the respiratory rise and fell to a very low level by the respiratory peak.

There was no change in the level of protein or amino acids during ripening. Assays of tissue sections before and after washing indicated an increased rate of leakage of amino acids during ripening.

Studies of incorporation of 3 concentrations of ¹⁴C-labeled leucine and phenylalanine indicated marked changes in the size and specific activity of the amino acid pool at the site of protein synthesis just prior to and during the climacteric rise, due to a diffusive mixing of the labeled substrate with the previously sequestered endogenous, unlabeled pool of substrate. The use of a high concentration of exogenous substrate (above saturation for uptake) resulted in an apparent constant specific activity of the metabolic pool through the ripening period. Data from these studies indicated a decline in amino acid incorporation during the climacteric.

It was concluded that the initiation of permeability changes marks the onset of senescence in banana. The causative relations between alterations in permeability, the respiratory rise and other chemical changes attending fruit ripening are discussed.

     

BIOSYNTHESIS OF FREE AMINO ACIDS IN THE BRAIN OF JIMPY MICE

—The metabolism of free amino acids: γ-aminobutyric acid (GABA), glutamine, glycine and glutathione has been studied. The labelling of these free amino acids in normal and in myelin-deficient brains of Jimpy mice was followed after intraperitoneal injection of ¹⁴C-labelled glucose precursor. The quantitative distribution of these amino acids in the two kinds of mouse brain has been compared. A higher level of GABA and a faster labelling of the amino acids in Jimpy than in normal mouse brain was observed.     

Incorporation of carbon from glucose into cerebral amino acids, proteins and lipids, and alterations during recovery from hypoglycaemia

Abstract— [U-¹⁴C]Glucose was given to dogs by intravenous infusion to maintain a fixed level of specific radioactivity of the plasma glucose. The time course of incorporation of ¹⁴C into free amino acids, proteins and lipids of the cerebrum was observed for periods up to 2 h. Labelling of amino acids closely related to the tricarboxylic acid cycle increased progressively throughout the infusion, approaching specific radioactivities per μg-atom of carbon equal to that of the plasma glucose. No significant dilution by unlabelled carbon entering the metabolic pathways was apparent. In dogs subjected to profound insulin hypoglycaemia, [U-¹⁴C]glucose mixed with unlabelled glucose was given to bring about recovery. The incorporation of ¹⁴C into glutamate, glutamine and aspartate during a 40-min period was greater than during a comparable period in control animals, whereas the incorporation into serine was reduced. When considered in relation to alterations in amino acid levels, the data suggest that during recovery from hypoglycaemia the rates of synthesis of amino acids related to the tricarboxylic acid cycle are increased. During the period of recovery the rates of incorporation of ¹⁴C from glucose into proteins exceeded the pre-insulin rates to a degree surpassing the increased incorporation into free amino acids. The labelling of the ganglioside, cerebroside-sulphatide and cephalin fractions was also increased, The rates of incorporation into the lecithin-sphin-gomyelin and cholesterol fractions during recovery were the same as in the pre-insulin period. Hypoglycaemia decreased the cerebral content of the phospholipid fractions; the lecithin-sphingomyelin fraction returned toward normal during recovery, whereas the cephalins did not increase significantly. The electrographic patterns and the occurrence of convulsive activity are discussed, both in insulin hypoglycaemia and during recovery.     

A method for the estimation of amino acid radioactivity in biological samples

A method for quantitative estimation of total radioactivity present in the free amino acid fraction of tissue samples has been described. Samples deproteinized with cold acetone were extracted, in acidic medium, with ethyl (peroxide free); after centrifugation, the aqueous phase was used for amino acid derivatization at 40°C for 15 h with 1-flouro-2,4-dinitrobenzene in bicarbonate-buffered medium. Aliquots of the derivatized samples were acidified and extracted twice again with ethyl ether. The combined organic phases were placed in glass scintilation vials, dried, and used for the determination of its radiactivity, corresponding to the radioactivity present in the free amino acid fraction of the sample. Deproteinized samples of rat blood plasma, as well as hen egg white and yolk were tested after addition of known quantities of ¹⁴C-labelled amino acids or glucose, for validation of the method. No glucose radioactivity was found in any of the extracted samples. All radioactivity added to the samples in the form of ¹⁴C-labelled alanine, glutamic acid, leucine and phenylalanine was quantitatively recovered in the derivatized fraction; only a fraction of arginine radioactivity was recovered.     

Metabolic discrimination of sucrose starvation from<Emphasis Type="Italic">Arabidopsis</Emphasis> cell suspension by<Superscript>1</Superscript>H NMR based metabolomics

Whole cell extracts ofArabidopsis cell cultures maintained on various sucrose concentrations (0,3, and 6%) were analyzed by¹H NMR spectroscopy to determine the comprehensive metabolic change in these cultures during sucrose starvation. The amount of sucrose, glucose, and fructose in the cells decreased to almost nothing after 12 h of culture in medium without sucrose. In contrast, the total free amino acid content of the cells increased as the culture proceeded. Among the free amino acids, phenylalanine and malic acid increased the most, followed by asparagine and alanine, whereas glutamic acid did not change significantly. These results are in agreement with previous studies using HPLC.¹H NMR spectroscopy enabled measurement of changes in the sugar and free amino acid content of whole cell extracts without fractionation and complicated sample preparation. These results indicate that comprehensive metabolic changes in the cells can be determined by a simple, rapid method using whole cell extracts and¹H NMR spectroscopy.     

Biosynthesis of gallic and ellagic acids with14C-labeled compounds inAcer andRhus leaves

The biosynthetic pathway for gallic and ellagic acids in young, mature and autumn leaves ofAcer buergerianum andRhus succedanea was examined by tracer experiments, and also by isotope competition, withd-shikimic acid-¹⁴C,l-phenylalanine-U-¹⁴C,l-phenyllactic acid-U-¹⁴C, gallic acid-G-¹⁴C and their unlabeled compounds. In young leaves of both plants, the incorporation rate of labeled shikimic acid into gallic acid was significantly higher than that of labeled phenylalanine, whereas in the mature and autumn leaves the latter was a good precursor rather than the former for the gallic acid biosynthesis. Therefore, two pathways for gallic acid formation, through β-oxidation of phenylpropanoid and through dehydrogenation of shikimic acid, could be operating inAcer andRhus leaves, and the preferential pathway is altered by leaf age. In both plants, the incorporation rate of labeled phenyllactic acid during a 24 hr metabolic period was almost the same as that of labeled phenylalanine. The incorporation ofd-skikimic acid-G-¹⁴C,l-phenylalanine-U-¹⁴C andl-phenyllactic acid-U-¹⁴C into ellagic acid was very similar to the case of the radioactive gallic acid formation. Furthermore, regardless of the presence of unlabeled shikimic acid and/or phenylalanine, incorporation of the radioactivity of labeled gallic acid into ellagic acid occurred at a very high rate, suggesting the reciprocal radical reaction of gallic acid for the ellagic acid formation. The incorporation of labeled compounds into ellagitanins was also examined and their biosynthesis discussed further.