Gibberellin-like activity in suspensors of Tropaeolum majus L. and Cytisus laburnum L.

Gibberellins in the embryo-suspensor system have been considered so far only in Phaseolus coccineus. We present in this report the localization of gibberellin-like substances in the suspensors of Tropaeolum majus L. and Cytisus laburnum L. The total gibberellin activity (expressed as gibberellic-acid equivalent in the -amylase bioassay) in 2000 suspensors (106 mg fresh weight; FW) of C. laburnum and in 600 suspensors (236 mg FW) of T. majus were 50.9 g g^-1 FW and 8.9 g g^-1 FW respectively.Abbreviation GA gibberellin     

Studies on the biosynthesis of tropane alkaloids by Datura stramonium L. transformed root cultures

Transformed root cultures of Datura stramonium, competent in tropane-alkaloid biosynthesis, have been treated with exogenous plant growth regulators. It was found that combinations of -naphthalene-acetic acid, kinetin (N⁶-furfurylaminopurine) and 2,4-dichlorophenoxyacetic acid induced de-differentiation, causing both the rooty phenotype and the hyoscyamine-biosynthetic capacity to be lost. Alkaloid biosynthesis disappeared rapidly and prior to the loss of morphological integrity. It was observed that the enzymes ornithine decarboxylase (EC 4.1.1.17), arginine decarboxylase (EC 4.1.1.19) and N-methylputrescine oxidase did not show the increase in level normally associated with subculturing the roots. The level of putrescine N-methyltransferase (EC 2.1.1.53) activity, the first enzyme fully committed to hyoscyamine biosynthesis, rapidly declined, about 80% being lost from the roots within 12h. This activity, although showing some temporary restoration, declined further after a few days, and was totally absent from fully dispersed cultures. N-Methylputrescine oxidase persisted at a low level. Following sub-culture of established de-differentiated lines to plant-growth-regulator-free medium, limited root regeneration occurred. The roots formed showed renewed competence in alkaloid biosynthesis and putrescine N-methyltransferase and N-methylputrescine oxidase activities were restored to their normal levels. The relationship between the morphological state and alkaloid-biosynthetic capacity of the cultures is discussed in relation to the overall control of alkaloid biosynthesis.Abbreviations ADC arginine decarboxylase - FW fresh weight - MPO N-methylputrescine oxidase - NAA -naphthalineacetic acid - ODC ornithine decarboxylase - pgr plant growth regulator - PMT putrescine N-methyltransferase We are most grateful to Abigael Peerless and Bridget Chapman for assistance with various part of this work.     

The purification and immunocharacterisation of N-methylputrescine oxidase from transformed root cultures of Nicotiana tabacum L. cv SC58

The enzyme N-methylputrescine oxidase which catalyses the conversion of N-methylputrescine to N-methylpyrrolinium salt has been purified to homogeneity from transformed roots of Nicotiana tabacum L. cv SC58. The enzyme has an apparent sub-unit molecular weight of 53 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with gel-filtration studies, indicating that the native form is a dimer. The K _m of the enzyme for N-methylputrescine has been estimated to be 0.1 mM. Polyclonal antibodies raised to the purified protein recognise one product in an immunoblot of a crude extract of transformed root tissue and will immunoprecipitate N-methylputrescine oxidase activity from such an extract. The antibodies also show a high degree of specificity in immunoblots of crude extracts of transformed root cultures from a range of other solanaceous and non-solanaceous species but do not cross-react with a partially purified preparation of pea-seedling diamine oxidase.Abbreviations MPO N-methylputrescine oxidase - PVDF polyvinylidene difluoride - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We would like to thank members of the Plant Cell Biotechnology Group, Institute of Food Research, Norwich Laboratory, for their helpful discussions during the preparation of this paper.     

Turnover and transport of quinolizidine alkaloids. Diurnal fluctuations of lupanine in the phloem sap,leaves and fruits of Lupinus albus L.

Quinolizidine alkaloids formed in the leaves of Lupinus albus L. are translocated via the phloem to the other plant organs, especially the maturing fruits. Compared with amino-acid transport in the phloem, the alkaloids contribute about 8% to the overall nitrogen being exported from the leaf. Since it is likely that the alkaloids are subsequently degraded in the target tissues a minor role of quinolizidine alkaloids might be nitrogen transport. A marked diurnal fluctuation of alkaloids was observed in the leaves, the phloem sap, the roots and the fruits with an increase during the day and an amplitude of several hundred percent thus providing evidence for a rapid turnover of endogenous alkaloids.Abbreviations QA quinolizidine alkaloids - GLC gas-liquid chromatography     

Protein bodies from the cotyledons of Cytisus scoparius L. (Link). Ultrastructure,isolation, and subunit composition of albumin,legumin and vicilin

The structure of protein bodies differs in the upper and lower parts of the cotyledons of mature seeds of Cytisus scoparius L. The palisade-mesophyll cells contain essentially homogeneous protein bodies, without globoids, but the protein bodies of the spongy-mesophyll cells are heterogeneous, with numerous globoids. Albumins, legumins and vicilins were selectively extracted from isolated protein bodies and their subunits separated by SDS-PAGE, under non-reducing and reducing conditions.Abbreviations SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

The distribution of strictosidine-synthase activity and alkaloids in Cinchona plants

The relation between the total alkaloid content and the activity of strictosidine synthase (EC 4.3.3.2), a key enzyme in alkaloid biosynthesis, was studied in distinct parts of six-month-old plants of Cinchona ledgeriana Moens. Strictosidine-synthase activity was present in the tops of the stems, including the young developing leaflets, and in the roots. The highest alkaloid contents of the plant were also found in these parts; however, the types of alkaloids differed, cinchophyllines being present in the aerial parts and quinoline alkaloids in the roots. In the stem and in old leaves, both strictosidine-synthase activity and alkaloid content were low. These results indicate that in young Cinchona plants the alkaloids are mainly synthesized in the axial extremities of the plant and that they are stored at the site of their synthesis.Abbreviations HPLC high-performance liquid chromatography - SSS strictosidine synthase We wish to thank Wim Snoeijer for excellent technical assistance, and Dr. H.J.v.d. Meulen, Multiplant Holding B.V. (Maarssen), for kindly providing us with Cinchona ledgeriana Moens seeds. Financial support by Multiplant Holding B.V. (Maarssen) is gratefully acknowledged.To whom correspondence should be addressed.     

Metabolic links between the biosynthesis of pyrrolizidine alkaloids and polyamines in root cultures of Senecio vulgaris

Isotope feeding and inhibitor experiments were performed in order to elucidate the pathway common to polyamine and alkaloid biosynthesis in root cultures of Senecio vulgaris L. -Difluoromethylarginine, a specific inhibitor of arginine decarboxylase, prevented completely the incorporation of radioactivity from [¹⁴C]arginine and [¹⁴C]ornithine into spermidine and the pyrrolizidine alkaloid senecionine N-oxide. In contrast, -difluoromethylornithine, a specific ornithine-decarboxylase inhibitor, had no effect on the flow of radioactivity from labelled ornithine and arginine into polyamines and alkaloids. Thus, putrescine, the common precursor of polyamines and pyrrolizidine alkaloids, is exclusively derived via the arginine-agmatine route. Ornithine is rapidly transformed into arginine. Recycling of the guanido moiety of agmatine back to ornithine can be excluded. Putrescine and spermidine were found to be reversibly interconvertable and to excist in a highly dynamic state. In contrast, senecionine N-oxide did not show any turnover but accumulated as a stable metabolic product. In-vivo evidence is presented that the carbon flow from arginine into the polyamine/alkaloid pathway may be controlled by spermidine. The possible importance of the metabolic coupling of pyrrolizidine-alkaloid biosynthesis to polyamine metabolism is discussed.Abbreviations DFMA D,l--difluoromethylarginine - DFMO D,l--difluoromethylornithine - FW fresh weight     

Purification and properties of putrescine N-methyltransferase from transformed roots of Datura stramonium L.

Putrescine-N-methyltransferase (PMT; EC 2.1.1.53), the first enzyme in the biosynthetic pathway leading from putrescine to tropane and pyrrolidine alkaloids, has been purified about 700-fold from root cultures of Datura stramonium established following genetic transformation with Agrabacterium rhizogenes. The native enzyme had a molecular weight estimated by gel-permeation chromatography on Superose-6 of 40 kDa; sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the peak fractions from Superose-6 chromatography revealed a band of 36 kDa molecular weight. Kinetic studies of the purified enzyme gave K _m values for putrescine and S-adenosyl-l-methionine of 0.31 mM and 0.10 mM, respectively, and K _i values for S-adenosyl-l-homocysteine and N-methylputrescine of 0.01 mM and 0.15 mM, respectively. The enzyme was active with some derivatives and analogous of putrescine, including 1,4-diamino-2-hydroxybutane and 1,4-diamino-trans-but-2-ene. Little activity was observed with 1,4-diamino-cis-but-2-ene and none with 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), indicating a requirement for substrate activity of two amino groups in a trans conformation, separated by four carbon atoms. A large number of monoamines were inhibitors of the enzyme. Though not a substrate, cadaverine was a competitive inhibitor of the enzyme, with a K _i of 0.04 mM; the significance of this in relation to the biosynthesis of cadaverine-derived alkaloids is discussed.Abbreviations PEG polyethylene glycol - PMT putrescine-N-methyltransferase - SAH S-adenosyl-l-homocysteine - SAM S-adenosyl-l-methionine - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We are grateful to C.R. Waspe, M.G. Hilton and P.D.G. Wilson for assistance with the provision of roots from fermenters. We thank W. Martin and S.D. Barr, Chemistry Department, University of Glasgow, and T.A. Smith, Long Ashton Research Station, Bristol, for the supply of compounds not commercially available, as indicated in the text. For helpful discussion and comment, we are grateful to A.J. Parr, W.R. McLauchlan and P. Bachmann. H.D.B, thanks the Science and Engineering Research Council for a research studentship and the Agricultural and Food Research Council Institute of Food Research for additional support.     

Production of plumbagin from cell cultures of Plumbago rosea L.

Callus and suspension cultures derived from leaf explants of Plumbago rosea were established and plumbagin, a naphthoquinone, was isolated from them and confirmed by ¹H NMR and electron-ionization mass spectroscopy. Maximum content of plumbagin was obtained in the stationary phase of growth (4.3 mg g^–1 dry cell wt). Media pH, phytohormones and carbon sources were optimized for biomass and plumbagin accumulation. Cell aggregates, measuring 500 m in diam, produced 8.2 g dry cell wt l^–1, but larger aggregates (above 500 m) favored plumbagin accumulation with an yield of 4.5 mg g^–1 dry cell wt.     

Factors influencing artemisinin production from shoot cultures of Artemisia annua L.

Artemisinin, an anti-malarial drug isolated from the annual wormwood Artemisia annua L., has a marked activity against chloroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum, and is useful in treatment of cerebral malaria. Shoot cultures of Artemisia annua L. were established on Murashige and Skoog basal medium which contained (per litre) 30 g sucrose, 0.5 mg 6-benzyladenine and 0.05 mg naphthaleneacetic acid. Using an optimized combination of sucrose (30 g/l), nitrate (45 mM), inorganic phosphate (200 mg/l), gibberellic acid (7 mg/l) and the ratio of NH₄ ⁺-N to NO₃ ^–-N of 1:3, artemisinin production reached 26.7 mg/l after 30 days. This procedure provides a potential alternative for production of artemisinin from in vitro tissue cultures.     

Alkaloid biosynthesis in somatic hybrids of Duboisia leichhardtii F. Muell. and Nicotiana tabacum L.

Somatic hybrids of Duboisia leichhardtii and Nicotiana tabacum were obtained by electrofusion followed by individual cloning. The hybrid nature of the cloned cells and regenerated shoots was confirmed by cytological investigation and ribosomal-DNA analysis, respectively. The hybrid plantlets predominantly produced nicotine, while Duboisia plantlets produced both tropane and nicotine alkaloids. Activities involved in tropane-alkaloid biosynthesis were examined in a series of precursor-feeding experiments. The presence in the hybrid plants of activities responsible for the reduction of tropinone, the hydroxylation and epoxidation of hyoscyamine, and the conversion of nicotine to nornicotine demonstrated the presence of the Duboisia genes for these enzyme activities.We thank Mr. T. Shikanai, Kyoto University, for the preparation of rice rRNA. We also appreciate Dr. J.H. Fitchen, Kyoto University, for critical discussion and English correction.     

Inhibition of flavonoid biosynthesis by gibberellic acid in cell suspension cultures of Daucus carota L.

In carrot cells (Daucus carota L.), cultured in the presence of gibberellic acid, anthocyanin synthesis is blocked at the level of chalcone synthase. By feeding suitable precursors for anthocyanins (naringenin, eriodictyol, dihydroquercetin) biosynthesis of cyanidin glycosides can be restored. After addition of these substrates to the culture medium in the presence of gibberellic acid, the activity of chalcone synthase remained as low as in the control without precursors. The highest increase in anthocyanin content was achieved using dihydroquercetin as the added precursor. The time course of this supplementation showed a rapid response; within 4 h a substantial increase in anthocyanin could be observed. In contranst, the flavonol quercetin is not a precursor for cyanidin. The fact that naringenin was also accepted for cyanidin synthesis leads to the conclusion that hydroxylation in 3-position of ring B in Daucus carota takes place at the flavonoid stage.Abbreviations CHI Chalcone isomerase - CHS chalcone synthase - DMSO dimethylsulfoxide - GA₃ gibberellic acid - PAL phenylalanine ammonia-lyase     

Enzymatic synthesis of quinolizidine alkaloid esters: a tigloyl-CoA: 13-hydroxylupanine O-tigloyltransferase from Lupinus albus L.

A tigloyl-CoA: 13-hydroxylupanine O-tigloyl-transferase could be demonstrated in crude enzyme preparations from Lupinus albus seedlings. The enzyme activity increases concomitantly with for formation of 13-tigloyloxylupanine in developing lupin seedlings. The transferase catalyzes specifically the transfer of an acyl group to 13-hydroxylupanine. The apparent Km-values are 140 M for tigloyl-CoA and 18 M for 13-hydroxylupanine. Other hydroxylated compounds, e.g., lupinine, 4-hydroxylupanine, and cholesterol are not acylated. The transferase shows optimal activity at pH 7–8 and at 30°C. It is activated by dithioerythritol and inhibited by thiol reagents. Tigloyl-CoA can be replaced as acyl donor by benzoyl-CoA and to a lesser extent by valeroyl-CoA, 3-methylbutyryl-CoA, butyryl CoA and propionyl-CoA but not acetyl-CoA. Preliminary evidence indicates that the transfer of the tigloyl and benzoyl moieties is catalyzed by different enzymes. The transferase activity could only be demonstrated in plants which produce quinolizidine alkaloids.Abbreviations DIECA diethyldithiocarbamate - DTE dithioerythritol - GLC gas-liquid chromatography     

Regeneration and characterization of plants obtained from anther cultures in Medicago sativa L

Summary The androgenic ability of four Medicago sativa L. genotype (Boynitza 5, Byala, 494, and 3815) was tested. Callus and organogenesis were induced in all lines studied. The percentage of anthers producing calluses and organogenesis showed wide variation (calluses—from 11% up to 77%; organogenesis—4.8% to 15.2%). It has been established that genotype, nutrient medium composition, and stage of pollen development considerably affected both callus production and organogenesis. Androgenesis in M. sativa could be achieved via callus and direct embryogenesis. About 500 morphologically different regenerants were obtained. Wide variability in chromosome number of regenerated plants was observed by cytological studies. Haploid, dihaploid, as well as mixoploid plants were obtained.     

Embryogenesis of photoautotrophic cell cultures of Daucus carota L.

In this paper photoautotrophic carrot (Daucus carota L.) suspension cultures are described which are able to produce somatic embryos. The development of somatic embryos, however, requires a sucrose supplement. Although an elevation of the CO₂ concentration up to 2.3% results in the same level of dry weight production as with sucrose in the medium, somatic embryos could not be observed.Results on the influence of sucrose on some aspects of the photosynthetic apparatus of cultured cells are discussed.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DW dry weight - ELISA enzyme-linked-immunosorbent-assay - FW fresh weight - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - PEPCase phosphoenol-pyruvate carboxylase - Rubisco Ribulose- 1,5-bisphosphate carboxylase/oxygenase - se somatic embryogenesis     

Identification of an UDP-glucose: Flavonol 3-O-glucosyl-transferase from cell suspension cultures of soybean (Glycine max L.)

A glucosyltransferase, which catalyses the glucosylation of flavonols, using uridine diphosphate-D-glucose as glucose donor, has been isolated and purified about 5–10 fold from cell suspension cultures of soybean (Glycine max L., var. Mandarin). The pH optimum for this reaction was ca. 8.5 in glycine-NaOH buffer, and no additional cofactors were required. The enzyme glucosylated the following flavonols predominantly at the 3-position: quercetin (Km 126 M), kaempferol (Km 172 M), isorhamnetin (Km 200 M) and fisetin (Km 270 M). With quercetin as substrate, the apparent Km value for uridine diphosphate-D-glucose was 0.3 M. Glucosylation of flavonols and flavones by this preparation occurred weakly also at the 7-position. No activity was found with dihydroquercetin, naringenin, 4,2,4-trihydroxychalcone, daidzein or texasin. The enzyme was specific for flavonoid compounds, since no activity was observed towards cinnamic acids or simple phenols. However, the preparation was contaminated by a vanillic acid glucosyltransferase, from which it could be partially separated by ionexchange chromatography. The specific activity of the flavonol 3-O-glucosyltransferase increased with age of the culture, reaching a maximum late in the growth cycle of the culture.Abbreviations SAM S-adenosyl-L-methionine - CMT, SAM caffeate 3-O-methyltransferase - FMT, SAM flavonoid O-methyltransferase - UDP-glucose uridine diphosphate-D-glucose - PAL phenylalanine ammonia-lyase     

Molecular analysis of plants regenerated from embryogenic cultures of wheat (Triticum aestivum L.)

Total DNAs of plants regenerated from immature embryo-derived 2-month-old embryogenic calli of wheat (cultivars Florida 302, Chris, Pavon, RH770019) were probed with six maize mitochondrial genes (atpA, atp6, apt9, coxI, coxII, rrn18-rrn5), three hypervariable wheat mitochondrial clones (K, K3, X2), five random pearl millet mitochondrial clones (4A9, 4D1, 4D12, 4E1, 4E11) and the often-used wheat Nor locus probe (pTA71), in order to assess the molecular changes induced in vitro. In addition, protoplast-derived plants, and 24-month-old embryogenic and non-embryogenic calli and cell suspension cultures of Florida 302 were also analyzed. No variation was revealed by the wheat or millet mitochondrial clones. Qualitative variation was detected in the nonembryogenic suspension culture by three maize mitochondrial genes (coxI, rrn18-rrn5, atp6). A callus-specific 3.8-kb Hind III fragment was detected in all four cultivars after hybridization with the coxI gene. The organization of the Nor locus of the plants regenerated from Florida 302 and Chris was stable when compared to their respective control plants and calli. The Nor locus in regenerants of Pavon and RH, on the other hand, was found to be variable. However, Nor locus variability was not observed in 14 individual seed-derived control plants from either Pavon or RH sources. In Pavon, a 3.6-kb Taq I or a 5.6-kb Bam HI+ Eco RI fragment was lost after regeneration. In one of the RH regenerants, which lost a fragment, an additional fragment was observed.     

Elicitation studies in cell suspension cultures of Cannabis sativa L.

Cannabis sativa L. plants produce a diverse array of secondary metabolites. Cannabis cell cultures were treated with biotic and abiotic elicitors to evaluate their effect on secondary metabolism. Metabolic profiles analysed by ¹H NMR spectroscopy and principal component analysis (PCA) showed variations in some of the metabolite pools. However, no cannabinoids were found in either control or elicited cannabis cell cultures. Tetrahydrocannabinolic acid (THCA) synthase gene expression was monitored during a time course. Results suggest that other components in the signaling pathway can be controlling the cannabinoid pathway.