α-and β-Glycosidases in maize roots

Four glycosidases were analyzed in 10 mm apical segments prepared from growing roots (15 mm) of Zea mays L. The pH optima were found to be 5.8 for -glucosidase, 4.4 for -galactosidase, 6.4 for -glucosidase and 6.0 for -galactosidase. The -glucosidase showed 4-fold higher activity than the -galactosidase. The distribution of the -glucosidase activity was signifcantly different from that of the -galactosidase, -glucosidase and -galactosidase.Abbreviations -Glu -glucosidase - -Gal -galactosidase - -Glu -glucosidase - -Gal -galactosidase     

Detection and partial characterization of two antigenically distinct β-amylases in developing kernels of wheat

A -amylase (EC 3.2.1.2) was identified in the outer pericarp (P) of developing seeds of wheat (Triticum aestivum L.) and compared with the well known -amylase which is synthesized during seed development in the starchy endosperm (E). The enzyme P already exists in the tissues before anthesis and vanishes at the time when E starts to accumulate. The isoelectric-focusing patterns of P and E are very similar. The relative molecular weight (M_r) of P is slightly higher than that of E (66 and 64.5 kDa, respectively). Both P and E exhibit common epitopes in addition to epitopes specific for each of them. The two enzymes were identified in small amounts in the green tissues of the developing seeds (inner pericarp and testa). No antigenic difference was detected between P and the -amylases of roots and leaves.Abbreviations P pericarp -amylase - E endosperm -amylase - IS1 anti--amylase immune serum - IS2 anti- and anti- amylase immune serum - IS3 anti- amylase immune serum - IEF isoelectric focusing - IgG immunoglobulin G The authors thank Dr. P. Ziegler (Universität Bayreuth, FRG) for stimulating discussion and for useful suggestions during the writing of the text. The authors thank Miss C. Mayer for her skillful technical assistance.     

Glutamine synthetase isoenzymes of Phaseolus vulgaris L.: subunit composition in developing root nodules and plumules

In the legume Phaseolus vulgaris L., glutamine synthetase (GS) (EC.6.3.1.2.) occurs as three cytosolic polypeptides, , and , and a plastidic polypeptide, . This paper describes the subunit composition of active octameric GS isoenzymes from root nodules and plumules using ionexchange high-performance liquid chromatography followed by two-dimensional denaturing gel electrophoresis and Western immunodetection. Root nodules contained four separable GS activities, three of which were composed mainly of cytosolic , / and GS polypeptides, whereas the fourth activity, consisted of plastidic GS polypeptides. The increase in GS activity during nodulation was due largely to the appearance of -containing isoenzymes, and to a lesser extent on the isoenzyme, whereas the -isoenzyme activity remained approximately constant throughout. Plumule GS from imbibed seeds was found to be composed of separate and isoenzymes, but 2 d after germination, plumule GS consisted of a mixture of , / and isoenzymes. The results from both nodules and plumules indicate that different cytosolic GS polypeptides in P. vulgaris are able to assemble into both homo-octameric and heterooctameric isoenzymes. Moreover, the changes in the patterns of isoenzymes observed during nodule development and plumule growth are interpreted to be caused both by temporal changes in the denovo synthesis of the polypeptides and also by their spatial separation in different cell types.Abbreviations 1D, 2D one-, two-dimensional - GS glutamine synthetase - GS_s GS semibiosynthetic activity - GS_t GS transferase activity - IEX-HPLC ion-exchange high-performance liquid chromatography - kDa kilodaltons - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Variability in the 11S globulin fraction of seed storage proteins ofHelianthus (Asteraceae)

The seed storage globulins from sixHelianthus and four hybrids were studied using mono and bidimensional gel SDS electrophoresis (+ 2 mercaptoethanol). The polypeptide composition of each subunit was determined. Different pairs are specifically expressed according to the species studied. Three typical patterns were discriminated. All the studied species exhibit five subunits: two of them are expressed in all the species (₁₁ and ₂₂). The subunit corresponding to the ₁₁ pair is present inH. petiolaris and in the three populations ofH. annuus studied. The _2b2 pair is common toH. annuus andH. argophyllus. H. petiolaris presents two specific _2a2 and ₄₄ pairs andH. annuus a specific ₃₃ pair. InH. argophyllus _{11 33} or ₄₄ are never observed but are replaced by ₁₃ and ₃₁ pairs. Some globulins, poorly represented, are of forms but present chains of higher molecular weights (in the range 54–56 kDa). Expressing variations in the banding patterns between these species by the use of a similarity index reveals complete identity between the three populations ofH. annuus. Identity between the twoH. petiolaris studied is also observed.H. annuus andH. argophyllus appear to be closer to each other thanH. petiolaris concerning the seed storage globulins.     

Heterogeneity of the hemoglobin of the Ohrid trout (Salmo L. typicus)

We have analyzed the hemoglobins of five individual trout from the Ohrid Lake (Salmo L. typicus) by electrophoretic methods, by reversed-phase high-performance liquid chromatography, and by limited structural analyses. The two major classes of hemoglobin are type I (35% of total) and type IV (65%). Type IV is the major oxygen-transporting hemoglobin; it consists of three types of chain (in about equal quantities) and three types of chain (one major and two minor types). Several structural differences have been observed between these three (IV) chains and between the three (IV) chains, suggesting a complex genetic system governing the synthesis of these proteins. Moreover, a few amino acid substitutions occur at positions involved in contacts between chains, which suggests that differences in oxygen affinity may exist between these various type IV hemoglobins. Type I hemoglobin is less complex because it contains one type of chain and two chains; the latter two differ in numerous positions, suggesting duplications of the (I)-globin gene. The and chains of type I hemoglobin differ considerably from the and chains of type IV hemoglobin, indicating the existence of (I)- and (I)-globin genes separate from the (IV)- and (IV)- globin genes.This study was supported in part by the Yugoslav-American Joint Funds, pp 812 (to G.D.E.), and by United States Public Health Service Research Grant HLB-05168 (to T.H.J.H.).     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

Steroid-induced regulation of 3α,20β- and 3β,17β-hydroxysteroid dehydrogenase activity in wild type and mutants of Streptomyces hydrogenans

The effect of estradiol, hydrocortisone and progesterone on 3,20-and 3,17-hydroxysteroid dehydrogenase (HSD) in mutants of Streptomyces hydrogenans was compared to the steroid response of the wild type. Mutants were defective in arginine biosynthesis and/or aerial mycelial formation and lacked both enzymes or only 17-HSD. Some 17-HSD mutants had lost the ability to be induced by estradiol, by progesterone or by both. Some 20-HSD mutants had lost the ability to be induced by hydrocortisone, by progesterone or by both. Non-inducibility of 17-and 20-HSD by progesterone was not co-ordinate. An additional study of the growth phase-dependent enzyme activity of the wild type after induction with estradiol, hydrocortisone and progesterone was performed.Non-standard abbreviations 17-HSD 3,17-Hydroxysteroid dehydrogenase (EC 1.1.1.51) - 20-HSD 3,20-hydroxysteroid dehydrogenase (EC 1.1.1.53) - AO acridine orange - EBr ethidium bromide - EMS ethyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine     

Carbohydrates of influenza virus. Structure of the oligosaccharides linked to asparagines 406 and 478 in the hemagglutinin of fowl plague virus,strain dutch

Fowl plague virus, strain Dutch, was metabolically labeled withd-[2-³H]mannose, or withd-[6-³H]glucosamine, and the small subunit (HA₂; 0.8 mg in total) of the viral hemagglutinin was isolated by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis. After proteolytic digestion, the radioactive oligosaccharides were sequentially liberated from the glycopeptides by treatment with different endo--N-acetylglucosaminidases and with peptide:N-glycosidase or, finally, by hydrazinolysis. In this manner, four groups of glycans could be obtained by consecutive gel filtrations and were subfractionated by HPLC. The structures of the individual oligosaccharides were analyzed by micromethylation, by acetolysis or by digestion with exoglycosidases. The major species amongst the high mannose glycans at Ans-406 of the viral glycopolypeptide were found to be Man1-2Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNac1-4GlcNAc and Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNAc1-4GlcNAc, while the complex glycans at Asn-478 are predominantly GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc (lacking, in part, one of the outerN-acetylglucosamine residues) and GlcNAc1-2Man1-3(Gal1-4GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc.Abbreviation BSA bovine serum albumin - endo D (F,H) endo--N-acetyl-d-glucosaminidase D (F,H) - HA hemagglutinin (HA₁, large subunit of HA - HA₂ small subunit - FPV fowl plague virus - PNGase F peptide:N-glycosidase F - SDS sodium dodecylsulfate     

Beta-1,3-glucanase from unfertilized eggs of the sea urchin Strongylocentrotus intermedius. Comparison with beta-1,3-glucanases of marine and terrestrial mollusks

-1,3-Glucanase (Lu) was isolated from unfertilized eggs of the sea urchin Strongylocentrotus intermedius. A comparative study of some properties of -1,3-glucanase Lu and -1,3-glucanases with different action types—endo--1,3-glucanase from crystalline style of the marine mollusk Spisula sachalinensis (LIV) and exo--1,3-glucanase from the terrestrial snail Eulota maakii (LII)—was performed. It was found that -1,3-glucanase Lu hydrolyzes laminaran with a high yield of glucose in the reaction products. The enzyme hydrolyzes substrates with retention of the glycosidic bond configuration, is able to cleave modified substrates, and exhibits transglycosylating activity. All properties of -1,3-glucanase from S. intermedius were more similar to those of the endo--1,3-glucanase from the marine mollusk (LIV) than exo--1,3-glucanase LII from the terrestrial snail. The differences in the effect of LIV and Lu on laminaran are probably related to the functions of -1,3-glucanase Lu from sea urchin eggs (which, in contrast to LIV, is not a digestive enzyme).     

A quantitative method for separating reaction components of CMP-sialic acid: Lactosylceramide sialyltransferase using Sep Pak C18 cartridges

A rapid procedure is described for the separation of CMP-sialic acid:lactosylceramide sialyltransferase reaction components using Sep Pak C₁₈ cartridges. The quantitative separation of the more polar nucleotide sugar, CMP-sialic acid, and its free acid from the less polar G_M3-ganglioside is simple and rapid relative to previously described methods. Recovery of G_M3 is optimized by the addition of phosphatidylcholine to the reaction mixture prior to the chromatographic step. Using rat liver Golgi membranes as a source of CMP-sialic acid: lactosylceramide sialyltransferase activity (G_M3 synthase; ST-1), the transfer of [¹⁴C] sialic acid from CMP-[¹⁴C] sialic acid to lactosylceramide can be quantified by this assay. The procedure is reliable and may be applicable to the isolation of ganglioside products in otherin vitro glycosyltransferase assays.Abbreviations G_M3 G_M3-ganglioside - II³NeuAc-LacCer NeuAc2-3Gal1-4Glc1-1Cer - G_D1a G_D1a-ganglioside, IV³NeuAc, II³NeuAc-GgOse₄Cer, NeuAc2-3Gal1-3GalNac1-4(NeuAc2-3)Gal1-4Glc1-1Cer - G_D3 G_D3-ganglioside, II³(NeuAc)₂LacCer, NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer - GgOse₄Cer asialo-G_M1 Gal1-3GalNAc1-4Gal1-4Glc1-1Cer - FucG_MI fucosyl-G_MI-ganglioside, Fuc1-2Gal1-3GalNAc1-4Gal1-4 Glc1-1Cer - ST-1 G_M3 synthase, CMP-sialic acid:lactosylceramide sialyltransferase - LacCer lactosylceramide, Gal1-4Glc1-1Cer - CMP-NeuAc cytidine 5-monophospho-N-acetylneuraminic acid - PC phosphatidylcholine - PMSF phenylmethylsulfonyl fluoride     

The Structural Basis for the Susceptibility of Gangliosides to Enzymatic Degradation

The conformational properties of GM2, GalNac-4(Neu5Ac-3) Gal-4Glc-1Cer have been compared to those of 6-GM2, in which the linkage between the GalNAc and Gal was altered from GalNac-4Gal- to GalNac-6Gal-, and to those of GD1a, Neu5Ac-3Gal-3GalNAc-4(Neu5Ac-3)Gal-4Glc-1Cer, and GalNAc-GD1a.Our results revealed that unlike the compact and rigid oligosaccharide head group found in GM2, where the Neu5Ac and the GalNAc residues interact, the sugar chain of 6-GM2 is in an open spatial arrangement, with the Neu5Ac no longer interacting with GalNAc, freely accessible to external interactions.The structure of GD1a can be regarded as that of GM2 with an extension of the terminal Neu5Ac-3Gal-disaccharide. The inner portion of GD1a is that of GM2 comprising the very rigid GalNAc-[Neu5Ac-]Gal trisaccharide. The terminal Neu5Ac-Gal linkage is flexible and fluctuates between two limiting conformations. In GalNAc-GD1a the outer sialic acid gains conformational rigidity due to the presence of the outer GalNAc in position 4 of galactose. This ganglioside has two core GalNAc-[Neu5Ac-]Gal trisaccharide linked in tandem.     

Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata

Of 49 microbial strains screened for their capabilities to transform ginsenoside Rb₁, Rhizopus stolonifer and Curvularia lunata produced four key metabolites: 3-O-[-d-glucopyranosyl-(1,2)--d-glucopyranosyl]- 20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ene (1), 3-O-[-d-glucopyranosyl-(1,2)--d- glucopyranosyl]-20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ol (2), 3-O-[-d-gluco- pyranosyl-(1,2)--d-glucopyranosyl]-3, 12, 20(S)-trihydroxydammar-24-ene (3), and 3-O--d-glucopyranosyl-3, 12, 20(S)-trihydroxydammar-24-ene (4), identified by TOF-MS, ¹H- and ¹³C-NMR spectral data. Metabolites 1, 3 and 4 were from the incubation with R. stolonifer, and 1 and 2 from the incubation with C. lunata. Compound 2 was identified as a new compound.     

Characterization of two beta-tubulin genes from Geotrichum candidum.

Summary The -tubulin genes G1 and G2 from the phytopathogenic hemiascomycete Geotrichum candidum were found to be highly diverged in amino acid sequence from those of other filamentous fungi. G1 and G2 were also divergent from each other, with the coding regions sharing only 66% nucleotide sequence homology and 64% amino acid identity. However, the proteins shared 82% similarity and only 25 of the 161 non-identical amino acid substitutions were non-conservative. The organization of G1 is similar to other fungal -tubulin genes, but G2 has several unusual features; it has 2 amino acid additions in the N-terminal 40 residues and must employ an uncommon 5 splice junction sequence in preference to an overlapping perfect consensus. The amino acid change found to confer benomyl resistance in Neurospora crassa was also present in G2. G1 has four introns which are located similarly to those of -tubulin genes in other fungi. G2, however, has a single intron in a unique location. Translational fusions employing the 5 non-coding regions of the two Geotrichum -tubulin genes were made with the hygromycin phosphotransferase gene and shown to function in Schizosaccharomyces pombe and Trichoderma hamatum. However, G. candidum could not be transformed with these or other tested plasmids commonly used for fungal transformation.     

Erratum: Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis

The genes were cloned for the two apoprotein subunits, and ,of phycocyanin from the cyanobacterium Spirulina maxima = Arthrospiramaxima) strain F3. The - and -subunit gene-coding regionscontain 489 bp and 519 bp, respectively. The -subunit gene is upstreamfrom the -subunit gene, with a 111-bp segment separating them.Similarities between the -subunits of S. maxima and nine othercyanobacteria were between 58% and 99%, as were those between the -subunits. The maximum similarity between the - and -subunits from S. maxima was 27%.     

Polymorphism of human liver alcohol dehydrogenase: Identification of ADH2 2-1 and ADH2 2-2 phenotypes in the Japanese by isoelectric focusing

Liver homogenate-supernatants from most Japanese exhibit an atypical pH optimum for ethanol oxidation at pH 8.8 instead of 10.5, the typical pH-activity optimum. It has been proposed that atypical livers contain alcohol dehydrogenase isozymes with ₂ subunits while typical livers contain isozymes with ₁ subunits, both produced by the ADH ₂ gene. Because it is difficult to differentiate the atypical ADH₂ 2-2 phenotype from the ADH₂ 2-1 phenotype by starch gel electrophoresis, an agarose isoelectric focusing procedure was developed that clearly separated the atypical Japanese livers into two groups, A1 and A2. The isozymes in A1 and A2 livers were purified. Type A1 livers contained a single isozyme with an atypical pH-rate profile; it was designated ₂₂. Three isozymes were isolated from A2 livers, two of which corresponded to ₁₁ and ₂₂. A third, absent from the typical and the atypical A1 livers, had an intermediate mobility; it was designated ₂₁. Type A1 livers are, therefore, the homozygous ADH₂ 2-2 phenotype, and type A2 livers, the heterozygous ADH₂ 2-1 phenotype. The ADH₂ 2-2 phenotype was found in 53% of 194 Japanese livers, and the ADH₂ 2-1 phenotype, in 31%. Accordingly, the frequency of ADH ₂ ² was 0.68.This study was supported by U.S. Public Health Service Grant AA 02342.     

The solution structure of rat Aβ-(1–28) and its interaction with zinc ion: insights into the scarcity of amyloid deposition in aged rat brain

The amyloid -peptide (A) is a major component of insoluble amyloid deposits in Alzheimers disease, and the ability of the -peptide to exist in different conformations is dependent on residues 1–28 [-(1–28)]. However, different from humans, no A amyloid deposition has been found in aged rats brains. Studying the three-dimensional solution structure of rat A-(1–28) and the binding circumstance of Zn²⁺ is beneficial to a clear understanding of the potential role of Zn²⁺ in Alzheimer-associated neuropathogenesis and to suggest why there is no amyloid deposition in aged rats brains. Here we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of rat A-(1–28) and the binding constant of Zn²⁺ to rat A-(1–28). Our results suggest that (1) the three-dimensional solution structure of rat A-(1–28) is more stable than that of human A-(1–28) in DMSO-d₆ and that a helical region from Glu16 to Val24 exists in the rat A-(1–28); (2) the affinity of Zn²⁺ for rat A-(1–28) is lower than that for human A-(1–28) and the NMR data suggest that Arg13, His6, and His14 residues provide the primary binding sites for Zn²⁺; and (3) the proper binding of Zn²⁺ to rat A-(1–28) can induce the peptide to change to a more stable conformation.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0556-xAbbreviations A amyloid -peptide - AD Alzheimers disease - hA-(1–28) human A-(1–28) - rA-(1-28) rat A-(1–28) - REM restrained energy minimization     

Modification of domains of α and β subunits of F1-ATPase from the thermophylic bacterium PS3, in their isolated and associated forms,by 3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (BzATP)

Photoaffinity labeling by 3-O-(4-benzoyl)benzoyl adenosine 5-triphosphate (BzATP) of the adenine nucleotide binding site(s) on isolated and complexed and subunits of F₁-ATPase from the thermophilic bacterium PS3 (TF₁) is described. BzATP binds to both isolated and subunits, to complexed subunit but not to complexed subunit. Amino acid sequence determination of radiolabeled peptides obtained by proteolytic digestion of [-³²P]BzATP-labeled subunit indicates that residues on both the amino-terminal (residues A41-E67) and carboxy-terminal (residues Q422–Q476) were modified by BzATP. One of the residues in the carboxy-terminal modified by BzATP is most probably Q422. Although the binding stoichiometry of 1 mol of BzATP incorporated by either isolated or complexed subunit was maintained, the spatial conformation of the polypeptide determines which amino acid residue(s) is more accessible to the reactive radical. CNBr derived fragments G10-M64, E75-M233, and D390-M469 were labeled with the isolated subunit. With complexed subunit the label was found only in CNBr fragments: E75-M233 and G339-M389. The locations where the covalently bound BzATP was found, in the soluble and assembled subunits, indicate that different conformational states exist. In the isolated form of the and subunits the amino- and carboxy-termini can fold and reach the central domain of the polypeptide, the domain containing the adenine nucleotide binding site. When combines with to form the ₃₃ core complex the new conformation of the subunits is such that covalent labeling by BzATP of and of the amino terminal of subunit is excluded.     

Theβ-subunit of human chorionic gonadotropin containsN-glycosidic trisialo tri- and tri′-antennary carbohydrate chains

TheN-linked carbohydrate chains of the-subunit of highly purified urinary human chorionic gonadotropin have been re-investigated. The oligosaccharides were released enzymatically by peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F, and fractionated by a combination of FPLC and HPLC. As a result of the application of improved fractionation methods, apart from the earlier reported carbohydrate chains, also small amounts of trisialo tri- and tri-antennary oligosaccharides were found. The primary structures of the latter carbohydrate chains have been determined by 500-MHz¹H-NMR spectroscopy to beAbbreviations hCG human chorionic gonadotropin - hCG- -subunit - hCG- -subunit - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F (E.C. 3.5.1.52) - endo-F endo--N-acetylglucosaminidase-F (E.C. 3.2.1.96) - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - CBB coomassie brilliant blue R 250 - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - Man mannose - Gal galactose - Fuc fucose     

Steroid Polyols from the Far Eastern Starfish Henricia sanguinolenta and H. leviuscula leviuscula

Two new asterosaponins, (20R)-3-O--D-(2-O-methylxylopyranosyl)-24-propylcholest-4-ene-3,6,8,15,16,29-hexaol (sanguinoside A) and (20R,24S)-3-O--D-(2,3,4-tri-O-methylxylopyranosyl)-5-cholestane-3,4,6,8,15,24-hexaol (sanguinoside B), were isolated from two species of Pacific Far Eastern Starfish Henricia sanguinolenta and H. leviuscula leviuscula, collected in the Sea of Okhotsk. Both glycosides contain aglycones with pentahydroxysteroid nuclei of similar structures, which are substituted at the 3-hydroxy group with differently methylated -D-xylosyl residues. Sanguinoside A has an unusual structure of its aglycone side chain, whereas sanguinoside B has a unique permethylated carbohydrate chain. In addition, laevisculoside G, a known glycoside, was identified in the H. leviuscula starfish. The structures of the isolated glycosides were established by interpreting their spectral data and by comparing their spectral characteristics with those of known compounds.     

Effects of supplied cinnamic acids and biosynthetic intermediates on the anthocyanins accumulated by wild carrot suspension cultures

Anthocyanins isolated and characterized from the wild carrot suspension cultures used here were 3-O--D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D<-galactopyranosylcyanidin (1), 3-O-[-D- xylopyranosyl-(12)--D-galactopyranosyl]cyanidin (2), 3-O-(6-O-sinapoyl)--D-glucopyranosyl-(16)-[-D- xylopyranosyl-(12)-]-D-galactopyranos ylcyanidin (3), 3-O-(6-O-feruoyl)--D-glucopyranosyl-(16)-[- D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (4), 3-O-(6-O-coumaroyl)--D-glucopyranosyl-(16)- [-D-xylopyranosyl-(12)-]-D-galactopyrano sylcyanidin (5), 3-O-[6-O-(3,4,5-trimethoxycinnamoyl)]-- D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (6), 3-O-[6-O-(3,4-dime- thoxycinnamoyl)]--D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (7), 3-O-[(6-O-sinapoyl)--D-glucopyranosyl-(16)--D-galactopyranosyl]cyanidin (8), and 3-O-(-D-galactopyranosyl)cyanidin (9). Except when cinnamic acids were provided in the culture medium, the major anthocyanin present in the two clones examined was 2. When the naturally occurring and some non-naturally occurring cinnamic acids were provided individually in the medium, 1 and 2 were minor components and the anthocyanin acylated with the supplied cinnamic acid, namely 3, 4, 5, 6, or 7 was the major anthocyanin present in the tissue. When caffeic acid was provided the major anthocyanin in the tissue was 4, thereby suggesting that the caffeic acid was methylated before its use in anthocyanin biosynthesis. Other cinnamic acids supplied had limited effects on the anthocyanins accumulated and appeared not to result in the accumulation of new anthocyanins by the tissue. Thus the tissue can use some but not all analogues of sinapic acid to acylate anthocyanins. Additional anthocyanins were detected in extracts of the wild carrot tissue cultures using mass spectrometry (both MS/MS and HPLC/MS). The additional compounds detected have also been found in cultures of black carrot, an Afghan cultivar of Daucus carota ssp. sativa and the flowers of wild carrot giving no evidence for qualitative differences in the anthocyanins synthesized by subspecies, cell cultures from subspecies, or clones from cell cultures. There are major differences in the amounts of individual anthocyanins found in cultures from different subspecies and in different clones from cell cultures. Here anthocyanins without acyl groups were usually found in the tissues and their accumulation is discussed. On the basis of the structures of the isolated anthocyanins, a likely pathway from cyanidin to the accumulated anthocyanins is proposed and discussed.Abbreviations Sin sinapoyl - Fer feruoyl - 4-Coum. 4-coumaroyl - 3,4-MeO₂Cin 3,4-dimethoxyeinnamoyl - 3,4,5-MeO₃Cin 3,4,5-trimethoxycinnamoyl - Cya cyanidin