Stable Isotope Labeling, in Vivo, of d- and l-Tryptophan Pools in Lemna gibba and the Low Incorporation of Label into Indole-3-Acetic Acid

We present evidence that the role of tryptophan and other potential intermediates in the pathways that could lead to indole derivatives needs to be reexamined. Two lines of Lemna gibba were tested for uptake of [¹⁵N-indole]-labeled tryptophan isomers and incorporation of that label into free indole-3-acetic acid (IAA). Both lines required levels of l-[¹⁵N]tryptophan 2 to 3 orders of magnitude over endogenous levels in order to obtain measurable incorporation of label into IAA. Labeled l-tryptophan was extractable from plant tissue after feeding and showed no measurable isomerization into d-tryptophan. d-[¹⁵N]tryptophan supplied to Lemna at rates of approximately 400 times excess of endogenous d-tryptophan levels (to yield an isotopic enrichment equal to that which allowed detection of the incorporation of l-tryptophan into IAA), did not result in measurable incorporation of label into free IAA. These results demonstrate that l-tryptophan is a more direct precursor to IAA than the d isomer and suggest (a) that the availability of tryptophan in vivo is not a limiting factor in the biosynthesis of IAA, thus implying that other regulatory mechanisms are in operation and (b) that l-tryptophan also may not be a primary precursor to IAA in plants.     

Tryptophan Biosynthesis in Cell Cultures of Nicotiana tabacum

Some of the general features of the pathway for l-tryptophan biosynthesis in cell cultures of Nicotiana tabccum var. Wisc. 38 have been investigated. The results of both isotope competition and direct-labeling experiments show that shikimic acid, anthranilic acid, indoleglycerol phosphate, and indole can serve as precursors to l-tryptophan in these cells, indicating that, in terms of its biochemical intermediates, the pathway is similar to that described for the bacteria and fungi.     

Plant regeneration through somatic embryogenesis from mature leaf explants of Eryngium foetidum,a condiment

Eryngium foetidum L. is an important plant cultivated as a leafy vegetable and for its essential oil, which are of high economic value in international trade market. Plants were regenerated through somatic embryogenesis from mature leaf explants of field grown plants. Leaf explants produced dark brown, compact callus on Linsmaier and Skoog (LS) medium with the combination of 1.0 mg l^-1 2,4-dichlorophenoxy acetic acid (2,4-D) and 1.0 mg l^-1 benzylaminopurine (BAP). Somatic embryos were induced from embryo-forming callus cultures on Murashige and Skoog (MS) medium supplemented with 0.1 mg l^-1 2,4-D, 2.0 mg l^-1 BAP and 1.0 mg l^-1 gibberellic acid (GA₃). Subsequently, conversion of these somatic embryos into plantlets occurred on MS medium supplemented with 1.0 mg l^-1 GA₃ and/or 0.1 mg l^-1 BAP. The regenerated shoots were rooted and elongated on MS medium supplemented with 0.1 mg l^-1 IAA and 1.0 mg l^-1 GA₃. These plantlets were hardened and transferred to the soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Site-directed Mutagenesis Switching a Dimethylallyl Tryptophan Synthase to a Specific Tyrosine C3-Prenylating Enzyme

The tryptophan prenyltransferases FgaPT2 and 7-DMATS (7-dimethylallyl tryptophan synthase) from Aspergillus fumigatus catalyze C⁴- and C⁷-prenylation of the indole ring, respectively. 7-DMATS was found to accept l-tyrosine as substrate as well and converted it to an O-prenylated derivative. An acceptance of l-tyrosine by FgaPT2 was also observed in this study. Interestingly, isolation and structure elucidation revealed the identification of a C³-prenylated l-tyrosine as enzyme product. Molecular modeling and site-directed mutagenesis led to creation of a mutant FgaPT2_K174F, which showed much higher specificity toward l-tyrosine than l-tryptophan. Its catalytic efficiency toward l-tyrosine was found to be 4.9-fold in comparison with that of non-mutated FgaPT2, whereas the activity toward l-tryptophan was less than 0.4% of that of the wild-type. To the best of our knowledge, this is the first report on an enzymatic C-prenylation of l-tyrosine as free amino acid and altering the substrate preference of a prenyltransferase by mutagenesis.     

Biological Properties of d-Amino Acid Conjugates of 2,4-D

Some d-amino acid (glutamic acid, valine, or leucine) conjugates of 2,4-dichlorophenoxyacetic acid (2,4-D) at 10⁻⁵ molar, stimulated elongation of Avena sativa L. var Mariner coleoptile sections and growth of soybean (Glycine max. L. var Amsoy) tissue as much as did the l-amino acid conjugates at 10⁻⁶ molar. The d-methionine conjugate did not stimulate growth of soybean root callus tissue but did stimulate Avena elongation. The d-aspartic acid conjugate did not stimulate elongation of Avena coleoptiles but did stimulate growth of root callus tissue.     

In vitro induction of pollen embryos and plantlets in Aesculus carnea Hayne through anther culture

Uninuclear microspores in red horse chestnut anther cultures formed pollen embryos and plantlents in MS agar medium supplemented with varying 2,4-D concentrations (1.0, 1.5 or 2.0 mg l^-1) and 1.0 mg l^-1 Kin. The highest number of embryogenic anthers (38%) was obtained in MS medium containing 1.0 mg l^-1 of each 2,4-D and Kin. The ability of pollen embryos to germinate was closely correlated with normal embryo morphology and was influenced by hormone content in the medium (MS+;1.0 mg l^-1 IAA+1.0 mg l^-1 GA₃+0.1 mg l^-1 Kin+400 mg l^-1 glutamine). Pollen embryos and plantlets had the haploid chromosome number (x=n=40). Cytological examinations demonstrated pollen dimorphism of this Aesculus species.Abbreviations AC activated charcoal - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin 6-furfurylaminopurine - GA₃ gibberellic acid - MS Murashige and Skoog     

Properties of an Aminotransferase of Pea (Pisum sativum L.)

A transaminase (aminotransferase, EC 2.6.1) fraction was partially purified from shoot tips of pea (Pisum sativum L. cv. Alaska) seedlings. With α-ketoglutarate as co-substrate, the enzyme transaminated the following aromatic amino acids: d,l-tryptophan, d,l-tyrosine, and d,l-phenylalanine, as well as the following aliphatic amino acids: d,l-alanine, d,l-methionine, and d,l-leucine. Of other α-keto acids tested, pyruvate and oxalacetate were more active than α-ketoglutarate with d,l-tryptophan. Stoichiometric yields of indolepyruvate and glutamate were obtained with d,l-tryptophan and α-ketoglutarate as co-substrates. The specific activity was three times higher with d-tryptophan than with l-tryptophan.     

Plant regeneration through somatic embryogenesis on Holostemma ada-kodien,a rare medicinal plant

Plant regeneration through indirect somatic embryogenesis has been established on Holostemma ada-kodien Schult. Type of auxin significantly influenced somatic embryogenesis. Friable callus, developed from leaf, internode and root explants on Murashige and Skoog (MS) medium supplemented with 2,4-D (1.0 mg l^–1), was most effective for the induction of somatic embryos. Subculture of the friable callus developed on 2,4-D (1.0 mg l^–1) onto solid or liquid 1/2 MS medium with 0.1 or 0.5 mg l 2,4-D turned the callus embryogenic. Suspension cultures were superior to static cultures (solid medium) for the induction of somatic embryos. Transfer of embryogenic callus to liquid 1/2 or 1/4 MS medium with lower levels of 2,4-D (0.05–0.1 mg l^–1) induced the highest number of somatic embryos. An average of 40 embryos were obtained from 10 mg callus. Fifty per cent embryos exhibited maturation and conversion upon transfer to 1/10 MS basal solid medium. Plantlets were established in field conditions and 90 per cent survived.     

3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase from Carrot Root (Daucus carota) Is a Hysteretic Enzyme

Roots of carrots (Daucus carota) contain three activities of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase, the enzyme that catalyzes the first step of the shikimate pathway. The three activities, enzymes I, II, and III, are separated by chromatography on phosphocellulose. Enzyme III, purified to electrophoretic homogeneity, has a native molecular weight of 103,000 and consists of two identical subunits of 53,000 daltons each. Double reciprocal plots of reaction velocity versus substrate concentration yield K_m values of 0.03 and 0.07 millimolar for P-enolpyruvate and erythrose-4-P, respectively. Both products, DAHP and orthophosphate, inhibit the enzyme. Enzyme III is a hysteretic enzyme that is activated by physiological concentrations of l-tryptophan and Mn²⁺, both of which also partially eliminate the hysteretic lag. Feedback activation of carrot DAHP synthase by tryptophan is interpreted to be an early regulatory signal for polyphenol biosynthesis. The three carrot DAHP synthase isoenzymes share antigenic determinants.     

Changes of 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid concentrations in plant tissue culture media in the presence of activated charcoal

Somatic embryos and embryogenic callus were initiated from immature zygotic embryos of ginseng (Panax ginseng C.A. Meyer). These somatic embryos were multiplied by adventitious (secondary and tertiary) embryogenesis and their growth and development were dependent on growth hormones in the medium. Auxins, 2,4-d, NAA, and IAA at 1.0 mg l^-1 were effective in inducing secondary and tertiary somatic embryos, which proliferated directly from the apical or cotyledonary portions of the primary somatic embryos. Single somatic embryos or clusters or embryos developed from the explanted primary embryos. Cytokinin (Kn, BA) inhibited adventitious embryogenesis. Secondary somatic embryos developed to maturation and later regenerated into plantlets in two stage process; firstly elongation of the shoot axes on MS +1.0 mg l^-1 Kn, secondly formation of root on 1.0 mg l^-1 Kn+1.0 mg^-1 GA₃ medium.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - IAA in-doleacetic acid - Kn kinetin - BA benzylaminopurine - PSE primary somatic embryo - SSE secondary somatic embryo - TSE tertiary somatic embryo     

Somatic embryogenesis in cell cultures of Thapsia garganica

Cell cultures from different species of the genus Thapsia (Apiaceae) have been investigated. In one 4-yearold line of T. garganica L. spontaneous somatic embryogenesis up to the globular stage occurred in a suspension culture containing 1 mg l^–12,4-dichlorophenoxyacetic acid (2,4-D). Also callus cultures of this line, previously maintained on a medium containing 1 mg l^–1 2,4-D, when transferred to various media deprived of 2,4-D, produced somatic embryos that developed into plantlets. Cell culture, embryos and regenerated organs were analysed for their content of thapsigargins. The undifferentiated cell culture did not synthezise thapsigargins, but was found to produce a yet unidentified compound not present in planta. White embryos in the pre-cotyledonary stage did not synthezise thapsigargins either, but when the embryos developed to the cotyledonary stage and became green, the synthesis started. Regenerated roots and shoots also contained thapsigargins.Abbreviations BAP Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - EtOAc ethyl acetate - FDA fluorescein diacetate - IAA Indole-3-acetic acid - IBA indole-3-butyric acid - 2-iP 2-isopentenyladenine - NAA 1-Napthaleneacetic acid     

In vitro regeneration from excised leaves of Flaveria trinervia (Sprengel) C. Mohr

Flaveria trinervia (Compositae) leaves are used for the treatment of jaundice and fever. From the leaf callus cultures regeneration of plantlets has been achieved. The results showed that BAP greatly stimulated the bud formation in concentrations ranging from 2–5 mg l^–1 than at very low concentrations (0.2–1.0 mg l^–1). Roots developed on the regenerated shoots, over a range of treatments, but were most prolific in the medium containing 1 mg l^–1 IAA. Histological observations revealed that cultured spongy cells of the mesophyll were greatly enlarged and underwent repeated cell divisions leading to the formation of hard nodular callus from which shoot buds differentiated. The shoots obtained were readily rooted and transplanted into glass houses. Cytological studies of the callus showed abnormalities such as bridges, endomitosis and multinucleolate conditions. Root tip squashes of the regenerated plants showed no variations and were diploid in chromosome number.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA napthalene acetic acid - IAA indole acetic acid - BAP 6-benzyl aminopurine - Kn kinetin     

Human Indoleamine 2,3-Dioxygenase Is a Catalyst of Physiological Heme Peroxidase Reactions: IMPLICATIONS FOR THE INHIBITION OF DIOXYGENASE ACTIVITY BY HYDROGEN PEROXIDE*

The heme enzyme indoleamine 2,3-dioxygenase (IDO) is a key regulator of immune responses through catalyzing l-tryptophan (l-Trp) oxidation. Here, we show that hydrogen peroxide (H₂O₂) activates the peroxidase function of IDO to induce protein oxidation and inhibit dioxygenase activity. Exposure of IDO-expressing cells or recombinant human IDO (rIDO) to H₂O₂ inhibited dioxygenase activity in a manner abrogated by l-Trp. Dioxygenase inhibition correlated with IDO-catalyzed H₂O₂ consumption, compound I-mediated formation of protein-centered radicals, altered protein secondary structure, and opening of the distal heme pocket to promote nonproductive substrate binding; these changes were inhibited by l-Trp, the heme ligand cyanide, or free radical scavengers. Protection by l-Trp coincided with its oxidation into oxindolylalanine and kynurenine and the formation of a compound II-type ferryl-oxo heme. Physiological peroxidase substrates, ascorbate or tyrosine, enhanced rIDO-mediated H₂O₂ consumption and attenuated H₂O₂-induced protein oxidation and dioxygenase inhibition. In the presence of H₂O₂, rIDO catalytically consumed nitric oxide (NO) and utilized nitrite to promote 3-nitrotyrosine formation on IDO. The promotion of H₂O₂ consumption by peroxidase substrates, NO consumption, and IDO nitration was inhibited by l-Trp. This study identifies IDO as a heme peroxidase that, in the absence of substrates, self-inactivates dioxygenase activity via compound I-initiated protein oxidation. l-Trp protects against dioxygenase inactivation by reacting with compound I and retarding compound II reduction to suppress peroxidase turnover. Peroxidase-mediated dioxygenase inactivation, NO consumption, or protein nitration may modulate the biological actions of IDO expressed in inflammatory tissues where the levels of H₂O₂ and NO are elevated and l-Trp is low.     

The Conversion of d-Tryptophan to l-Tryptophan in Cell Cultures of Tobacco

d-Tryptophan was converted to l-tryptophan in tissue cultures of tobacco, in whole cells treated with dimethylsulfoxide, and in cell-free extracts treated by Sephadex G-25 filtration. Evidence was obtained that tryptophanase, tryptophan pyrrolase, and transaminase activities were not involved. The data were best explained by the presence of a tryptophan racemase as the enzyme catalyzing the reaction. The possible role of d-tryptophan in the biosynthesis of indoleacetic acid is discussed.     

Emission of Hydrogen Sulfide by Leaf Tissue in Response to l-Cysteine

Leaf discs and detached leaves exposed to l-cysteine emitted a volatile sulfur compound which was proven by gas chromatography to be H₂S. This phenomenon was demonstrated in all nine species tested (Cucumis sativus, Cucurbita pepo, Nicotiana tabacum, Coleus blumei, Beta vulgaris, Phaseolus vulgaris, Medicago sativa, Hordeum vulgare, and Gossypium hirsutum). The emission of volatile sulfur by cucumber leaves occurred in the dark at a similar rate to that in the light. The emission of leaf discs reached the maximal rate, more than 40 picomoles per minute per square centimeter, 2 to 4 hours after starting exposure to l-cysteine; then it decreased. In the case of detached leaves, the maximum occurred 5 to 10 h after starting exposure. The average emission rate of H₂S during the first 4 hours from leaf discs of cucurbits in response to 10 millimolar l-cysteine, was usually more than 40 picomoles per minute per square centimeter, i.e. 0.24 micromoles per hour per square decimeter. Leaf discs exposed to 1 millimolar l-cysteine emitted only 2% as much as did the discs exposed to 10 millimolar l-cysteine. The emission from leaf discs and from detached leaves lasted for at least 5 and 15 hours, respectively. However, several hours after the maximal emission, injury of the leaves, manifested as chlorosis, was evident. H₂S emission was a specific consequence of exposure to l-cysteine; neither d-cysteine nor l-cystine elicited H₂S emission. Aminooxyacetic acid, an inhibitor of pyridoxal phosphate dependent enzymes, inhibited the emission. In a cell free system from cucumber leaves, H₂S formation and its release occurred in response to l-cysteine. Feeding experiments with [³⁵S]l-cysteine showed that most of the sulfur in H₂S was derived from sulfur in the l-cysteine supplied and that the H₂S emitted for 9 hours accounted for 7 to 10% of l-cysteine taken up. ³⁵S-labeled SO₃²⁻ and SO₄²⁻ were found in the tissue extract in addition to internal soluble S²⁻. These findings suggest the existence of a sulfur cycle which converts l-cysteine to SO₄²⁻ through cysteine desulfhydration.     

Relationship of indole-3-acetic acid and tryptophan concentrations in normal and 5-methyltryptophan-resistant cell lines of wild carrots

A 5-methyltryptophan(5-MT)-resistant cell line of wild carrot (Daucus carota L.), W001, that exhibited auxin-independent callus growth, was found to accumulate indole-3-acetic acid (IAA) and tryptophan (trp). Anthranilate-synthetase activity in W001 cell extract was less sensitive to feedback inhibition by trp than in the original 5-MT-sensitive cell lines. It is hypothesized that the resistant enzyme allowed more trp synthesis and accumulation which, in turn, affected the IAA concentration in the cell. Since carrot cultures cannot regenerate in the presence of exogenous auxin, the elevated IAA concentration in W001 may be responsible for its drastically reduced capacity to regenerate. The relationship between trp and IAA levels was further investigated by examining the effect of 2,4-dichlorophenoxy acetic acid (2,4-D) on the endogenous concentration of trp and IAA. In general, the IAA level was reduced but the trp concentration was elevated when 2,4-D was present in the culture medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 5-MT 5-methyltryptophan - 5-MT^r 5-MT-resistant - 5-MT^s 5-MT-sensitive - trp tryptophan     

Sugar-nucleotide precursors of arabinopyranosyl, arabinofuranosyl, and xylopyranosyl residues in spinach polysaccharides

Cultured spinach (Spinacia oleracea L. cv Monstrous Viroflay) cells incorporated exogenous l-[³H]arabinose sequentially into β-l-arabinopyranose-1-phosphate, uridine diphospho-β-l-arabinopyranose, uridine diphospho-α-d-xylopyranose and (in some experiments) α-d-xylopyranose-1-phosphate. The amount of ³H in each of these compounds reached a plateau after a few minutes, and could be rapidly chased with nonradioactive l-arabinose, demonstrating rapid turnover. After a few minutes' lag, incorporation of ³H into the arabinofuranosyl, arabinopyranosyl, and xylopyranosyl residues of polysaccharides was linear with respect to time. The kinetics of labeling were compatible with UDP-β-l-arabinopyranose and UDP-α-d-xylopyranose being the immediate precursors of arabians (both the pyranose and the furanose residues) and xylans, respectively. No other radioactive nucleotides were formed; in particular, UDP-arabinofuranose was absent. There was no evidence for conversion of arabinopyranose to arabinofuranose within the polysaccharides, suggesting that this conversion occurs during polymer synthesis. The glycolipids detected showed too slow a turnover to be intermediates of pentosan synthesis.     

Facile Preparation of Deuterium-Labeled Standards of Indole-3-Acetic Acid (IAA) and Its Metabolites to Quantitatively Analyze the Disposition of Exogenous IAA in Arabidopsis thaliana

[2′,2′-²H₂]-indole-3-acetic acid ([2′,2′-²H₂]IAA) was prepared in an easy and efficient manner involving base-catalyzed hydrogen/deuterium exchange. 1-O-([2′,2′-²H₂]-indole-3-acetyl)-β-D-glucopyranose, [2′,2′-²H₂]-2-oxoindole-3-acetic acid, and 1-O-([2′,2′-²H₂]-2-oxoindole-3-acetyl)-β-D-glucopyranose were also successfully synthesized from deuterated IAA, and effectively utilized as internal standards in the quantitative analysis of IAA and its metabolites in Arabidopsis thaliana by using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). The use of this technique shows that these metabolites were accumulated in the roots of Arabidopsis seedlings. Dynamic changes in the metabolites of IAA were observed in response to exogenous IAA, revealing that each metabolic action was regulated differently to contribute to the IAA homeostasis in Arabidopsis.     

Quantitative studies of embryogenesis in normal and 5-methyltryptophan-resistant cell lines of wild carrot

The frequency of embryo formation was determined in normal and 5-methyltryptophan-resistant (5-MT^r) cell lines of wild carrot (Daucus carota L.) grown in the presence or absence of 2-isopentenyladenine (2-ip) and 2,4-dichlorophenoxyacetic acid (2,4-D). 2-ip stimulated the intitation of embryo formation and also accelerated embryo development. 2.4-D inhibited embryo differentiation at several stages: at 0.1 mg/l, it stopped regeneration at the earliest stage, resulting in callus growth instead of embryo formation; at 0.04 mg/l 2,4-D, some globular embryos were produced, but they did not develop into more advanced embryos. Variant cell lines with higher levels of auxin (indole-3-acetic acid, IAA) were used to study the effect of an elevated endogenous concentration of auxin on embryogenesis. IAA at these concentrations suppressed regeneration in the same manner as the exogenous auxin, 2,4-D, did. This result confirms the hypothesis that high levels of IAA are responsible for the suppression of regeneration in the 5-MT^r cell lines.     

Fucosylation of membrane proteins in soybean cultured cells : effects of tunicamycin and swainsonine

Cultures of soybean cells incorporate [5,6-³H]-l-fucose into various cellular components including lipids and proteins. The membrane glyco-proteins were digested with pronase to produce glycopeptides, and the glycopeptides were isolated on columns of Biogel P-4. The major fucoselabeled glycopeptide sized as a Hexose_15-17-N-acetylglucosamine₂ (GlcNAc₂) on columns of Biogel P-4. Fucose incorporation was also examined in the presence of the processing inhibitor swainsonine, and the glycosylation inhibitor tunicamycin. In the presence of swainsonine, the incorporation of fucose was not reduced but the glycopeptides were smaller in size and migrated like Hexose_12-13-GlcNAc₂ structures. On the other hand, tunicamycin inhibited the incorporation of fucose into the glycopeptides by 70 to 80%, indicating that the l-fucose was present in N-linked oligosaccharides.