Synthesis of l-(+)-Tartaric Acid from l-Ascorbic Acid via 5-Keto-d-Gluconic Acid in Grapes

5-Keto-l-idionic acid (5-keto-d-gluconic acid, d-xylo-5-hexulosonic acid) was found as a metabolic product of l-ascorbic acid in slices of immature grapes, Vitis labrusca L. cv `Delaware'. Specifically labeled compounds, recognized as metabolic products of l-ascorbic acid in grapes, were fed to young grape tissues to investigate the metabolic pathway from l-ascorbic acid to l-(+)-tartaric acid.     

Loss of Hydrogen from Carbon 5 of d-Glucose during Conversion of d-[5-H,6-C]Glucose to l-Ascorbic Acid in Pelargonium crispum (L.) L'Hér

Conversion of d-[5-³H,6-¹⁴C]glucose to l-ascorbic acid in detached apices of Pelargonium crispum (L.) L'Hér cv Prince Rupert (lemon geranium) was accompanied by complete loss of tritium in the product. Chemical degradation of d-glucose which was recovered from the labeled apices yielded d-glyceric acid (corresponding to carbons 4, 5, and 6 of glucose) with a ³H:¹⁴C ratio of 4 to be compared with 9, the ratio in d-[5-³H,6-¹⁴C]glucose initially. Conversion of d-[6-³H,6-¹⁴C]glucose in the same tissue was accompanied by retention of tritium in l-ascorbic acid with a ³H:¹⁴C ratio comparable to that of compounds from the hexose pool. Results indicate that during l-ascorbic acid biosynthesis from glucose in Pelargonium crispum hydrogen at carbon 5 undergoes exchange with the medium, suggesting an epimerization at this carbon atom.     

l-Ascorbic Acid Metabolism in Vitaceae: Conversion to (+)-Tartaric Acid and Hexoses

The metabolic fate of l-ascorbic acid-1-¹⁴C and -6-¹⁴C has been investigated in two species in two genera of Vitaceae. Results suggest that ascorbic acid metabolism in the Vitaceae involves splitting the 6-carbon chain into 4- and 2-carbon fragments. The former, corresponding to C1 through C4 of ascorbic acid, is further oxidized to tartaric acid while the latter, corresponding to C5 and C6, is recycled into hexose phosphate metabolism. Comparison of these findings with previous observations on the conversion of ascorbic acid to (+)-tartaric acid in Pelargonium crispum clearly reveals two distinct processes of tartaric acid biosynthesis in those plants identified as tartaric acid accumulators.     

The Biosynthesis of (+)-Tartaric Acid in Pelargonium crispum

Metabolic conversion of l-galactono-1, 4-lactone and l-ascorbic acid to (+)-tartaric acid and oxalic acid has been studied in Pelargonium crispum, cv. Prince Rupert. Experiments with specifically labeled substrates suggest a path of conversion involving cleavage of l-ascorbic acid, or a metabolic product of l-ascorbic acid, between C₂ and C₃, such that oxalic acid arises from the two carbon fragment and (+)-tartaric acid from the four carbon fragment.     

d-Glucosone and l-Sorbosone, Putative Intermediates of l-Ascorbic Acid Biosynthesis in Detached Bean and Spinach Leaves

d-[6-¹⁴C]Glucosone that had been prepared enzymically from d-[6-¹⁴C]glucose was used to compare relative efficiencies of these two sugars for l-ascorbic acid (AA) biosynthesis in detached bean (Phaseolus vulgaris L., cv California small white) apices and 4-week-old spinach (Spinacia oleracea L., cv Giant Noble) leaves. At tracer concentration, ¹⁴C from glucosone was utilized by spinach leaves for AA biosynthesis much more effectively than glucose. Carbon-14 from [6-¹⁴C]glucose underwent considerable redistribution during AA formation, whereas ¹⁴C from [6-¹⁴C]glucosone remained almost totally in carbon 6 of AA. In other experiments with spinach leaves, l-[U-¹⁴C]sorbosone was found to be equivalent to [6-¹⁴C]glucose as a source of ¹⁴C for AA. In the presence of 0.1% d-glucosone, conversion of [6-¹⁴C] glucose into labeled AA was greatly repressed. In a comparable experiment with l-sorbosone replacing d-glucosone, the effect was much less. The experiments described here give substance to the proposal that d-glucosone and l-sorbosone are putative intermediates in the conversion of d-glucose to AA in higher plants.     

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.     

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.     

Inositol Metabolism in Plants. V. Conversion of Myo-inositol to Uronic Acid and Pentose Units of Acidic Polysaccharides in Root-tips of Zea mays

The metabolism of myo-inositol-2-¹⁴C, d-glucuronate-1-¹⁴C, d-glucuronate-6-¹⁴C, and l-methionine-methyl-¹⁴C to cell wall polysaccharides was investigated in excised root-tips of 3 day old Zea mays seedlings. From myo-inositol, about one-half of incorporated label was recovered in ethanol insoluble residues. Of this label, about 90% was solubilized by treatment, first with a preparation of pectinase-EDTA, then with dilute hydrochloric acid. The only labeled constituents in these hydrolyzates were d-galacturonic acid, d-glucuronic acid, 4-O-methyl-d-glucuronic acid, d-xylose, and l-arabinose, or larger oligosaccharide fragments containing these units. Medium external to excised root-tips grown under sterile conditions in myo-inositol-2-¹⁴C contained labeled polysaccharide.     

Metabolic Studies on Intermediates in the myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen: III. Polysaccharidic Origin of Labeled Glucose

myo-Inositol-linked glucogenesis in germinated lily (Lilium longiflorum Thunb., cv. Ace) pollen was investigated by studying the effects of added l-arabinose or d-xylose on metabolism of myo-[2-³H]inositol and by determining the distribution of radioisotope in pentosyl and hexosyl residues of polysaccharides from pollen labeled with myo-[2-¹⁴C]inositol, myo-[2-³H]inositol, l-[5-¹⁴C]arabinose, and d-[5R,5S-³H]xylose.     

l-Canavanine Transport and Utilization in Developing Jack Bean, Canavalia ensiformis (L.) DC. [Leguminosae

l-Canavanine, the guanidinooxy structural analog of l-arginine, is an important nonprotein amino acid of many leguminous plants with nitrogen storage a major proported role. l-[Guanidinooxy-¹⁴C]canavanine, [¹⁴C] urea, and [¹⁵N]urea were injected separately into the fleshy, green cotyledons of 9-day old jack bean plants, Canavalia ensiformis (L.) DC. [Leguminosae]. There was significant transport of canavanine from the cotyledons to the aboveground portions of the plant, but not to the roots. Within 1.5 hours of isotope administration, the remaining labeled canavanine was divided equally between the cotyledons and the aboveground portions of the plant. During the 48-hour postinjection period, the contribution of l-[guanidinooxy-¹⁴C]canavanine to the total ¹⁴carbon of the cotyledons decreased rapidly while it increased in the aboveground portions of the plant.     

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.     

The production and efflux of 4-aminobutyrate in isolated mesophyll cells

The pathway of 4-aminobutyric acid (GABA) production and efflux was investigated in suspensions of mesophyll cells isolated from asparagus (Asparagus sprengeri Regel) cladophylls. Analysis of free amino acids demonstrated that, on a molar basis, GABA represented 11.4, 19, and 6.5% of the xylem sap, intact cladophyll tissue, and isolated mesophyll cells, respectively. l-Glu, a GABA precursor, was abundant in intact cladophylls and isolated cells but not in xylem sap. When cells were incubated with l-[U-¹⁴C]Glu, intracellular GABA contained less than 10% of the radioactivity found in intracellular Glu. However, GABA in the medium contained 78% of the radioactivity found in extracellular l-Glu metabolites. Incubation with l-[1-¹⁴C]Glu resulted in the appearance of unlabeled GABA, demonstrating its production through decarboxylation at carbon 1. GABA released to the medium from cells incubated with l-[U-¹⁴C]Glu had a specific activity of 18 nanocuries per nanomole, whereas GABA remaining in the cell had a specific activity of 2.25 × 10⁻¹ nanocuries per nanomole. In the presence of exogenous l-Glu, amino acid analysis and cell volume measurements indicated intracellular Ala and GABA concentrations of 4.2 and 1.4 millimolar, respectively. In the medium, however, the corresponding concentrations were 2 and 57 micromolar. The data indicate that l-Glu entering the cell is decarboxylated to GABA, and that specific and passive efflux is from this pool of recently synthesized GABA and not from a previously synthesized unlabeled pool of GABA.     

l-Glutamate-Dependent Medium Alkalinization by Asparagus Mesophyll Cells : Cotransport or Metabolism?

Mechanically isolated Asparagus sprengeri Regel mesophyll cells cause alkalinization of the suspension medium on the addition of l-glutamate or its analog l-methionine-d,l-sulfoximine. Using a radiolabeled pH probe, it was found that both compounds caused internal acidification whereas l-aspartate did not. Fusicoccin stimulated H⁺ efflux from the cells by 111% and the uptake of l-[U-¹⁴C]glutamate by 55%. Manometric experiments demonstrated that, unlike l-methionine-d,l-sulfoximine, l-glutamate stimulated CO₂ evolution from nonilluminated cells. Simultaneous measurements of medium alkalinization and ¹⁴CO₂ evolution upon the addition of labeled l-glutamate showed that alkalinization was immediate and reached a maximum value after 45 minutes whereas ¹⁴CO₂ evolution exhibited a lag before its appearance and continued in a linear manner for at least 100 minutes. Rates of alkalinization and uptake of l-[U-¹⁴C]glutamate were higher in the light while rates of ¹⁴CO₂ evolution were higher in the dark. The major labeled product of glutamate decarboxylation, γ-aminobutyric acid, was found in the cells and the suspension medium. Its addition to the cell suspension did not result in medium alkalinization and evidence indicates that it is lost from the cell to the medium. The data suggest that the origin of medium alkalinization is co-transport not metabolism, and that the loss of labeled CO₂ and γ-aminobutyric acid from the cell result in an overestimation of the stoichiometry of the H⁺/l-glutamate uptake process.     

Proline fed to intact soybean plants influences acetylene reducing activity and content and metabolism of proline in bacteroids

Supplying l-proline to the root system of intact soybean (Glycine max [L.] Merr.) plants stimulated acetylene reducing activity to the same extent as did supplying succinate. Feeding l-proline also caused an increase in bacteroid proline dehydrogenase activity that was highly correlated with the increase in acetylene-reducing activity. Twenty-four hours after irrigating with l-proline, endogenous proline content had increased in host cell cytoplasm and bacteroids, about three- and eightfold, respectively. In bacteroids, proline concentration was calculated to be at least 3.5 millimolar. In experiments in which [U-¹⁴C]l-proline was supplied to uprooted, intact plants incubated in aerated solution, ¹⁴C-labeled products of proline metabolism, as well as [¹⁴C]proline itself, accumulated in both host cells and bacteroids. When plants were incubated in aerated solutions containing [5-³H]l-proline, ³H-labeled proline was found in host cells and bacteroids. [³H] Pyrroline-5-carboxylate was found in bacteroids, but not host cells, after a 2-hour incubation in [5-³H]l-proline. When [U-¹⁴C]l-proline was supplied for 24 hours, a significant amount of [¹⁴C] pyrroline-5-carboxylate was found in the host cells, in contrast with the results from the shorter incubation in [5-³H]proline, although the amount in the host cells was only about half the quantity found in the bacteroids. Taken as a whole, these results indicate that proline crosses both plant and bacterial membranes under the in vivo experimental conditions utilized and are consistent with a significant role for proline as an energy source in support of bacteroid functioning. In spite of the increase in acetylene-reducing activity when proline was supplied to the root system of intact plants, proline application did not rescue stemgirdled plants from loss of acetylene-reducing activity, although succinate application did. This suggests a nonphloem route for succinate, but not proline, from roots to nodules.     

The effect of all-trans-retinoic acid on the synthesis of epidermal cell-surface-associated carbohydrates

1. all-trans-Retinoic acid at concentrations greater than 10⁻⁷m stimulated the incorporation of d-[³H]glucosamine into 8m-urea/5% (w/v) sodium dodecyl sulphate extracts of 1m-CaCl₂-separated epidermis from pig ear skin slices cultured for 18h. The incorporation of ³⁵SO₄²⁻, l-[¹⁴C]fucose and U-¹⁴C-labelled l-amino acids was not significantly affected. 2. Electrophoresis of the solubilized epidermis showed increased incorporation of d-[³H]glucosamine into a high-molecular-weight glycosaminoglycan-containing peak when skin slices were cultured in the presence of 10⁻⁵m-all-trans-retinoic acid. The labelling of other epidermal components with d-[³H]glucosamine, ³⁵SO₄²⁻, l-[¹⁴C]fucose and U-¹⁴C-labelled l-amino acids was not significantly affected by 10⁻⁵m-all-trans-retinoic acid. 3. Trypsinization dispersed the epidermal cells and released 75–85% of the total d-[³H]glucosamine-labelled material in the glycosaminoglycan peak. Thus most of this material was extracellular in both control and 10⁻⁵m-all-trans-retinoic acid-treated epidermis. 4. Increased labelling of extracellular epidermal glycosaminoglycans was also observed when human skin slices were treated with all-trans-retinoic acid, indicating a similar mechanism in both tissues. Increased labelling was also found when the epidermis was cultured in the absence of the dermis, suggesting a direct effect of all-trans-retinoic acid on the epidermis. 5. Increased incorporation of d-[³H]-glucosamine into extracellular epidermal glycosaminoglycans in 10⁻⁵m-all-trans-retinoic acid-treated skin slices was apparent after 4–8h in culture and continued up to 48h. all-trans-Retinoic acid (10⁻⁵m) did not affect the rate of degradation of this material in cultures `chased' with 5mm-unlabelled glucosamine after 4 or 18h. 6. Cellulose acetate electrophoresis at pH7.2 revealed that hyaluronic acid was the major labelled glycosaminoglycan (80–90%) in both control and 10⁻⁵m-all-trans-retinoic acid-treated epidermis. 7. The labelling of epidermal plasma membranes isolated from d-[³H]glucosamine-labelled skin slices by sucrose density gradient centrifugation was similar in control and 10⁻⁵m-all-trans-retinoic acid-treated tissue. 8. The results indicate that increased synthesis of mainly extracellular glycosaminoglycans (largely hyaluronic acid) may be the first response of the epidermis to excess all-trans-retinoic acid.     

Carbohydrates Stimulate Ethylene Production in Tobacco Leaf Discs : II. Sites of Stimulation in the Ethylene Biosynthesis Pathway

Galactose, sucrose, and glucose (50 millimolar) applied to tobacco leaf discs (Nicotiana tabacum L. cv `Xanthi') during a prolonged incubation (5-6 d) markedly stimulated ethylene production which, in turn, could be inhibited by aminoethoxyvinylglycine (2-amino-4-(2′-aminoethoxy)-trans-3-butenoic acid) (AVG) or Co²⁺ ions. These three tested sugars also stimulated the conversion of l-[3,4-¹⁴C]methionine to [¹⁴C]1-amino-cyclopropane-1-carboxylic acid (ACC) and to [¹⁴C]ethylene, thus indicating that the carbohydrates-stimulated ethylene production proceeds from methionine via the ACC pathway. Sucrose concentrations above 25 mm considerably enhanced ACC-dependent ethylene production, and this enhancement was related to the increased respiratory carbon dioxide. However, sucrose by itself could directly promote the step of ACC conversion to ethylene, since low sucrose concentrations (1-25 mm) enhanced ACC-dependent ethylene production also in the presence of 15% CO₂.     

Conversion of l-Sorbosone to l-Ascorbic Acid by a NADP-Dependent Dehydrogenase in Bean and Spinach Leaf

An NADP-dependent dehydrogenase catalyzing the conversion of l-sorbosone to l-ascorbic acid has been isolated from Phaseolus vulgaris L. and Spinacia oleracea L. and partially purified. It is stable at −20°C for up to 8 months. Molecular masses, as determined by gel filtration, were 21 and 29 kilodaltons for bean and spinach enzymes, respectively. K_m for sorbosone were 12 ± 2 and 18 ± 2 millimolar and for NADP⁺, 0.14 ± 0.05 and 1.2 ± 0.5 millimolar, for bean and spinach, respectively. Lycorine, a purported inhibitor of l-ascorbic acid biosynthesis, had no effect on the reaction.     

Arginine Metabolism in Developing Soybean Cotyledons: III. Utilization

Tracerkinetic experiments were performed using l-[guanidino-¹⁴C]arginine, l-[U-¹⁴C]arginine, l-[ureido-¹⁴C]citrulline, and l-[1-¹⁴C]ornithine to investigate arginine utilization in developing cotyledons of Glycine max (L.) Merrill. Excised cotyledons were injected with carrier-free ¹⁴C compounds and incubated in sealed vials containing a CO₂ trap. The free and protein amino acids were analyzed using high performance liquid chromatography and arginine-specific enzyme-linked assays. After 4 hours, 75% and 90% of the ¹⁴C metabolized from [guanidino-¹⁴C]arginine and [U-¹⁴C]arginine, respectively, was in protein arginine. The net protein arginine accumulation rate, calculated from the depletion of nitrogenous solutes in the cotyledon during incubation, was 17 nanomoles per cotyledon per hour. The data indicated that arginine was also catabolized by the arginase-urease reactions at a rate of 5.5 nanomoles per cotyledon per hour. Between 2 and 4 hours ¹⁴CO₂ was also evolved from carbons other than C-6 of arginine at a rate of 11.0 nanomoles per cotyledon per hour. It is suggested that this extra ¹⁴CO₂ was evolved during the catabolism of ornithine-derived glutamate; ¹⁴C-ornithine was a product of the arginase reaction. A model for the estimated fluxes associated with arginine utilization in developing soybean cotyledons is presented.     

Evidence for a specific glutamate/h cotransport in isolated mesophyll cells

Mechanically isolated Asparagus sprengeri Regel mesophyll cells were suspended in 1 millimolar CaSO₄. Immediate alkalinization of the medium occured on the addition of 1 millimolar concentrations of l-glutamate (Glu) and its analog l-methionine-d,l-sulfoximine (l-MSO). d-Glu and the l isomers of the protein amino acids did not elicit alkalinization. l-Glu dependent alkalinization was transient and acidification resumed after approximately 30 to 45 minutes. At pH 6.0, 5 millimolar l-Glu stimulated initial rates of alkalinization that varied between 1.3 to 4.1 nmol H⁺/10⁶ cells·minute. l-Glu dependent alkalinization was saturable, increased with decreasing pH, was inhibited by carbonyl cyanide-p-trichloromethoxyphenyl hydrazone (CCCP), and was not stimulated by light. Uptake of l-[U-¹⁴C]glutamate increased as the pH decreased from 6.5 to 5.5, and was inhibited by l-MSO. l-Glu had no influence on K⁺ efflux. Although evidence for multiple amino acid/proton cotransport systems has been found in other tissues, the present report indicates that a highly specific l-Glu/proton uptake process is present in Asparagus mesophyll cells.     

Identification and Quantitative Analysis of Indole-3-Acetyl-l-Aspartate from Seeds of Glycine max L

Indole-3-acetyl-l-aspartate (IAAsp) was isolated from seeds of Glycine max L. cv. Hark and its identity established by its chromatographic performance and its mass spectral fragmentation. Following acid hydrolysis, the aspartate moiety was shown to be the l-enantiomer by reverse phase high performance liquid chromatographic retention time of the bisethyl ester derivatized with 2,3,4,6-tetra-O-acetyl-β-d-glycopyranosyl isothiocyanate. Isotope dilution analysis using [¹⁴C]IAAsp as internal standard showed that soybean seed contained 10 μmol/kg IAAsp and this accounted for one-half of the total indoleacetic acid of the seed.