Metabolism of Arginine and Ornithine in Suspension-Cultured Cells and Aseptic Roots of Intact Plants of Atropa belladonna

Suspension-cultured cells and aseptically cultured roots ofintact plants of Atropa belladonna L. removed tropane alkaloidprecursors arginine (Arg) and ornithine (Orn) at nearly an equalrate from the feeding medium. A great part of Arg- and Orn-derived¹⁴C-label was found in ethanol-insoluble compounds, mostly inproteins already after 2 h feeding. Ethanol-soluble label inthe roots was found mainly in amino acids (e.g. glutamine, Gln)after 2 h feeding, and after 20 h also in some intermediatesof the urea cycle (e.g. argininosuccinate). In suspension cultures, subculturing of the initiation callusdecreased both the uptake of the basic amino acids tested andtheir binding on to the apoplastic space. After 20 h feedingwith Arg more label was found in organic acids in stationaryphase suspension cultures with repressed alkaloid synthesisthan in roots producing alkaloids. The growth phase and passagenumber also affected into which amino acids the label was incorporated.When the initiation callus was young (the 3rd passage), theintermediates of the urea cycle were actively labelled, butwhen the initiation callus was older (the 8th passage) and thesuspension formed roots, especially Gln was labelled. Only tracesof -N-methylornithine were detected in feeding experiments withOrn and Arg. Considerable arginase activity with a high pH optimumwas observed in cell suspensions and roots of A. belladonna. Key words: Atropa, arginine, ornithine, roots, suspension culture     

Coculture of genetically transformed roots and shoots for synthesis, translocation, and biotransformation of secondary metabolites

Genetically transformed shooty teratomas of Atropa belladonna and a Duboisia leichhardtii x D. myoporoides hybrid were studied for biotransformation of tropane alkaloids in shake flasks and bioreactors. Although de novo synthesis of hyoscyamine and scopolamine was limited, shoots of both species were able to translocate and accumulate significant quantities of exogenous alkaloid. The maximum yield of scopolamine from hyoscyamine fed to the Duboisia hybrid shoots was 35% w/w; the yield of the scopolamine precursor, 6beta-hydroxyhyoscyamine, was 37% w/w. Biotransformation activity was poor in A. belladonna shooty teratomas provided with exogenous hyoscyamine; however, scopolamine levels comparable with those in leaves of the whole plant accumulated in shoots fed with hairy root extract. Coculture of A. belladonna shooty teratomas and hairy roots in the same hormone-free medium was investigated as a means of providing a continuous source of hyoscyamine for conversion to scopolamine. Of the biotransformation systems tested with A. belladonna, coculture produced the highest levels of scopolamine and the highest scopolamine: hyoscyamine ratios. Cocultured shoots accumulated up to 0.84 mg g(-1) dry weight scopolamine, or 3-11 times the average concentrations found in leaves of the whole plant. The scopolamine: hyoscyamine ratio in coculture ranged from 0.07 to 1.9, a significant improvement over levels of 0-0.03 normally found in A. belladonna hairy roots. Addition of Pluronic F-68 or copper sulfate to the medium and variation in initial medium pH did not improve hyoscyamine release from hairy roots. Scopolamine levels were increased using 1 muM copper sulfate or initial medium pH between 6.0 and 8.0; however, results from elicitation of hairy roots could not match the beneficial effect on scopolamine synthesis of root-shoot coculture. Addition of 0.001-1.0% (w/v) Pluronic F-68 to the roots reduced hyoscyamine release but postponed necrosis in the root tissue for up to 60 d. (c) 1996 John Wiley & Sons, Inc.     

Analysis of the Potential Use of Androgenic Datura innoxia for the Development of Cell Cultures Producing High Amounts of Tropane Alkaloids

Normal and androgenic diploid Datura innoxia plants were selfedand the progeny was analysed for its leaf alkaloid content.Since the androgenic lines had originally produced very differentamounts of the tropane alkaloids, scopolamine and hyoscyamine,we were interested in determining whether this trait is transmittedby self-fertilization. The alkaloid content of the progeny wasfound to correlate well with that of the parental plants. Also,calli were initiated from leaf discs derived from plants withdifferent capacities for alkaloid biosynthesis. These were furthersubcultured for 2 years. Again, the same correlations in hyoscyamineand scopolamine content were observed. This indicates that itis possible to initiate callus with a high alkaloid contentstarting from actively alkaloid-producing androgenic Daturainnoxia plants. Key words: Datura innoxia, tropane alkaloids, androgenic plants, callus culture     

Effects of calcium,alginate, and calcium-alginate immobilization on growth and tropane alkaloid levels of a stable suspension cell line of Datura innoxia Mill

Summary A stabilized two-year old suspension of a Datura innoxia cell line, producing small amounts of tropane alkaloids (scopolamine and hyoscyamine) was used in this study. Calcium alginate immobilization has been shown to be able to increase secondary metabolite (i. e. alkaloid) production. The effects of calcium and ungellified alginate were both beneficial for tropane alkaloid synthesis; a 10mM calcium chloride supply gave the best results, with a 10-fold yield increase.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - S standard cell culture medium - HPLC high performance liquid chromatography - FW fresh weight - FDA fluorescein diacetate - FW fresh weight     

Alkaloids in plants and root cultures of Atropa belladonna overexpressing putrescine N-methyltransferase

Putrescine N-methyltransferase (PMT) is the first alkaloid-specific enzyme for nicotine and tropane alkaloid formation. The pmt gene from Nicotiana tabacum was fused to the CaMV 35S promoter and integrated into the Atropa belladonna genome. Transgenic plants and derived root cultures were analysed for gene expression and for levels of alkaloids and their precursors. Scopolamine, hyoscyamine, tropine, pseudotropine, tropinone, and calystegines were found unaltered or somewhat decreased in pmt-overexpressing lines compared to controls. When root cultures were treated with 5% sucrose, calystegine levels were elevated in control roots, but were not affected in pmt-overexpressing roots. 1 microM auxin reduced calystegine levels in control roots, while in pmt-overexpressing roots all alkaloids remained unaltered. Expression level of pmt alone is apparently not limiting for tropane alkaloid formation in A. belladonna.     

Effects of Oxygen on Nicotine and Tropane Alkaloid Production in Cultured Roots Duboisia myoporoides

The effects of oxygen on nicotine and tropane alkaloid production in root cultures of Duboisia myoporoides were investigated. Duboisia roots cultured in air produced both nicotine and tropane alkaloids equally. However, when roots were cultured in pure oxygen, the metabolic flux to tropane alkaloids increased, and that to nicotine alkaloids decreased. Intermediate product analysis by GC-MS showed an increase in tropine, but decreases in acetyl derivatives of tropane alkaloids and tropine esters with low-class fatty acids. Furthermore, hyoscyamine 6β-hydroxylase (H6H, EC 1.14.11.11, the key enzyme in the pathway from hyosyamine to scopolamine) also increased. These results suggest that pure oxygen contributes to scopolamine production not only by activating the biosynthetic steps for scopolamine, but also by inactivating the biosynthetic steps for nicotine and other tropine derivatives.     

Root, Callus, and Cell Suspension Cultures, from Atropa belladonna, L. and Atropa belladonna, Cultivar lutea Doll

Root, callus, and cell suspension cultures have been establishedfrom seedlings of Atropa belladonna, L. and Atropa belladonna,cultivar lutea Döll. The growth of these cultures is described.Callus cultures transferred to auxin (-naphthaleneacetic acid)-freemedium initiated roots and shoots. Excised root cultures havebeen established from such roots and plants from such shoots.Extracts of the cultures have been submitted to the Vitali—Morinreaction and following chromatography, to the Dragendorff reaction.Cultured excised roots and plants raised from shoots initiatedon cultured callus were shown to contain atropine (hyoscyamine)and reactive substances corresponding in Rf to hyoscine andcuscohygrine. These alkaloids were absent from cultured callusand cultured cell suspensions and from leaves when initiatedwithout roots on callus. The cultured calluses and cell suspensionscontained choline (0.022–0.027 g per 100 g dry weightof root callus). The growth of cell suspension cultures wasnot inhibited by incorporating atropine sulphate, L-hyoscyamine,L-hyoscine hydrobromide, or DL-scopoline nitrate in the culturemedium at 250 mg/I. These alkaloids were absorbed by the cells,a high proportion of the added alkaloid could be recovered fromthe cultures even after 4 weeks' growth and no evidence wasobtained of the presence of degradation products of the alkaloids.The suppression of alkaloid formation in actively growing callusand cell suspension cultures is discussed.     

Production of tropane alkaloids in cultured cells of Hyoscyamus niger

Tests for calluses rich in tropane alkaloids were made with newly induced calluses of Atropa belladonna, Datura stramonium and Hyoscyamus niger. Only calluses of H. niger gave an alkaloid-positive test.A Hyoscyamus cell line had the highest total alkaloid content of all the calluses screened by the cell-squash alkaloid assay. Both hyoscyamine and scopolamine were identified in the cultured cells of this line by TLC, GLC and GC-MS.Abbreviations NAA 1-Naphthaleneacetic acid - BA Benzyladenine - BSA N,O-Bis(trimethylsilyl) acetamide     

Simultaneous determination of scopolamine, hyoscyamine and littorine in plants and different hairy root clones of Hyoscyamus muticus by micellar electrokinetic chromatography

Hyoscyamus muticus hairy root clones were established following infection with Agrobacterium rhizogenes strains A4, LBA-9402 and 15834 and with A. tumefaciens strain C58C1pRTGus104. The accumulation of tropane alkaloids hyoscyamine, littorine and scopolamine was evaluated by micellar electrokinetic capillary electrophoresis. Littorine was reported for the first time in these clones as well as in the roots of the intact plant and confirmed by collision induced dissociation-mass spectrometry. Tropane alkaloid content in hairy roots was compared with leaves and roots of normal plants at two vegetative stages. Significant differences appeared between the alkaloid contents of the different clones. In particular, all the hairy root clones and the roots of the intact plant produced 1.5-3 and 4.5-9 times more littorine than scopolamine, respectively. The only exception was clone KB7, carrying the h6h gene, which overproduced scopolamine. The aerial parts of H. muticus plants did not contain any littorine, thus indicating different transportation or translocation mechanisms of the various tropane alkaloids.     

Effect of pmt gene overexpression on tropane alkaloid production in transformed root cultures of Datura metel and Hyoscyamus muticus

In order to increase the production of the pharmaceuticals hyoscyamine and scopolamine in hairy root cultures, a binary vector system was developed to introduce the T-DNA of the Ri plasmid together with the tobacco pmt gene under the control of CaMV 35S promoter, into the genome of Datura metel and Hyoscyamus muticus. This gene codes for putrescine:SAM N-methyltransferase (PMT; EC. 2.1.1.53), which catalyses the first committed step in the tropane alkaloid pathway. Hairy root cultures overexpressing the pmt gene aged faster and accumulated higher amounts of tropane alkaloids than control hairy roots. Both hyoscyamine and scopolamine production were improved in hairy root cultures of D. metel, whereas in H. muticus only hyoscyamine contents were increased by pmt gene overexpression. These roots have a high capacity to synthesize hyoscyamine, but their ability to convert it into scopolamine is very limited. The results indicate that the same biosynthetic pathway in two related plant species can be differently regulated, and overexpression of a given gene does not necessarily lead to a similar accumulation pattern of secondary metabolites.     

Tropinone reductases, enzymes at the branch point of tropane alkaloid metabolism

Two stereospecific oxidoreductases constitute a branch point in tropane alkaloid metabolism. Products of tropane metabolism are the alkaloids hyoscyamine, scopolamine, cocaine, and polyhydroxylated nortropane alkaloids, the calystegines. Both tropinone reductases reduce the precursor tropinone to yield either tropine or pseudotropine. In Solanaceae, tropine is incorporated into hyoscyamine and scopolamine; pseudotropine is the first specific metabolite on the way to the calystegines. Isolation, cloning and heterologous expression of both tropinone reductases enabled kinetic characterisation, protein crystallisation, and structure elucidation. Stereospecificity of reduction is achieved by binding tropinone in the respective enzyme active centre in opposite orientation. Immunolocalisation of both enzyme proteins in cultured roots revealed a tissue-specific protein accumulation. Metabolite flux through both arms of the tropane alkaloid pathway appears to be regulated by the activity of both enzymes and by their access to the precursor tropinone. Both tropinone reductases are NADPH-dependent short-chain dehydrogenases with amino acid sequence similarity of more than 50% suggesting their descent from a common ancestor. Putative tropinone reductase sequences annotated in plant genomes other that Solanaceae await functional characterisation.     

Enhanced production of scopolamine by bacterial elicitors in adventitious hairy root cultures of Scopolia parviflora

In an attempt to increase productivity, the effect of elicitation on tropane alkaloids (TA) biosynthesis was studied in adventitious hairy root cultures of Scopolia parviflora. Two Gram-positive strains and one Gram-negative strain of bacteria were used as biotic elicitors. The raw bacterial elicitors affected the tropane alkaloid profile by increasing the scopolamine concentration, while the autoclaved bacterial elicitors produced similar effects on the control. The conversion ratio of hyoscyamine to scopolamine was increased following elicitation using raw bacterial elicitors. The bacterial elicitor inhibited the expression of H6H (hyoscyamine 6β-hydoxylase) whereas the expression of PMT (putrescine N-methyltransferase) was raised by elicitation. These results have important implications for the large-scale production of tropane alkaloids.     

UV-B辐射对颠茄氮代谢及次生代谢产物含量的影响

有害的中波紫外线（ultraviolet B,UV-B;280～320 nm）辐射影响植物的生长与发育。但也有研究证明,UV-B辐射可诱导生物碱合成。然而,UV-B辐射能否提高颠茄(Atropa belladonna L.)中托品烷类生物碱（tropane alkaloids,TAs）的含量尚未见报道。本研究以颠茄实生苗为材料,研究UV-B不同照射度强、时间（d数）对颠茄的氮代谢、生物碱含量及TAs代谢途径中的几个关键酶基因表达量的影响。结果表明,随着辐射天数的增加（5～30 d）,低强度（LU,5 μW/cm2）UV-B处理与对照（无辐射）比较,硝态氮、莨菪碱、东莨菪碱含量无显著差异。然而,中等强度（MU,10 μW/cm2）和高强度（HU,15 μW/cm2）UV-B辐射,明显增加硝态氮含量,谷氨酰胺合成酶（glutamine synthetase,GS）、谷氨酸脱氢酶（glutamine dehydrogenase,GDH）活性明显高于对照。重要的是,中、高强度UV-B辐射显著降低了颠茄的叶片与茎中莨菪碱和东莨菪碱含量。荧光定量PCR揭示,莨菪碱合成的关键酶腐胺N 甲基转移酶（putrescine N methyltransferase,PMT）编码基因、莨菪碱-6-β-羟化酶（hyoscyamine-6-β-hydroxylase,H6H）基因表达呈高度组织特异性,主要是在根部表达。与对照比较,低强度照射25 d引起pmt在根部的表达量显著上调,而中、高强度照射导致其下调;h6h在根部的相对表达量随着处理强度的增加逐渐降低;托品酮还原酶Ⅰ（tropinone reductaseⅠ, TRⅠ）编码基因在叶片中的表达量较高,随照射强度的增加而升高。上述结果表明,低强度UV-B辐射促进氮代谢,有利于莨菪碱合成;而长期中、高强度UV-B辐射,尽管促进了谷氨酸代谢,但却使pmt和h6h表达降低,不利于莨菪碱和东莨菪碱的积累。总之,本研究结果显示,不同UV-B辐射强度和时间,对颠茄合成TAs的影响不同,可为大田试验生产莨菪碱提供有益的参考。     

Production of tropane alkaloids by small-scale bubble column bioreactor cultures of Scopolia parviflora adventitious roots

The mass production of tropane alkaloids from adventitious root cultures of Scopolia parviflora, in small-scale bubble column bioreactor (BCB) was attempted. Adventitious roots of S. parviflora produced relatively enhanced levels of scopolamine and hyoscyamine in bioreactor compared to flask type cultures, and rapidly produced root clumps, with continuously increasing biomass throughout the culture period. The production of scopolamine and hyoscyamine in the top and bottom regions of root clumps were higher than in the core region. The adventitious root cultures of S. parviflora in the BCB required a relatively high level of aeration. The optimized conditions for the bioreactor culture growth and alkaloid production were found to be 3g of inoculum, on a fresh weight basis, a 15-day culture period and 0.4vvm of airflow. The elicitation by Staphylococus aureus increased the specific compound of scopolamine, while the production of hyoscyamine was slightly inhibited in BCB cultures.     

Production of tropane alkaloids in diploid and tetraploid plants and in vitro hairy root cultures of Egyptian henbane (Hyoscyamus muticus L.)

In this study, the effects of ploidy level and culture medium were studied on the production of tropane alkaloids. We have successfully produced stable tetraploid hairy root lines of Hyoscyamus muticus and their ploidy stability was confirmed 30?months after transformation. Tetraploidy affected the growth rate and alkaloid accumulation in plants and transformed root cultures of Egyptian henbane. Although tetraploid plants could produce 200% higher scopolamine than their diploid counterparts, this result was not observed for corresponding induced hairy root cultures. Culture conditions did not only play an important role for biomass production, but also significantly affected tropane alkaloid accumulation in hairy root cultures. In spite of its lower biomass production, tetraploid clone could produce more scopolamine than the diploid counterpart under similar growth conditions. The highest yields of scopolamine (13.87?mg?l^?1) and hyoscyamine (107.7?mg 1^?1) were obtained when diploid clones were grown on medium consisting of either Murashige and Skoog with 60?g/l sucrose or Gamborg??s B5 with 40?g/l sucrose, respectively. Although the hyoscyamine is the main alkaloid in the H. muticus plants, manipulation of ploidy level and culture conditions successfully changed the scopolamine/hyoscyamine ratio towards scopolamine. The fact that hyoscyamine is converted to scopolamine is very important due to the higher market value of scopolamine.     

Organogenesis, Differentiation and Histolocalization of Alkaloids in Cultured Tissues and Organs of Duboisia myoporoides R. Br.

Duboisia myoporoides R. Br. shoots were regenerated from non-organogenicand organogenic calli induced with nine different cytokinin/auxincombinations. Alkaloid colour reagents localized tropane alkaloidsin the vascular regions which had large cells in the secondaryxylem of the basal stem sections of the non-rooted shoots. Tropanealkaloids were localized in shoots regenerated from calli inducedwith two different cytokinin/auxin combinations. No alkaloidswere localized in shoots regenerated from calli induced withother cytokinin/auxin combinations. However, only nicotine wasdetected by GC-MS in the non-rooted shoots regenerated fromcalli induced with three different cytokinin/auxin combinations.Tropane alkaloids were also localized in xylem cells of rootsregenerated from calli induced with two different cytokininand auxin combinations independently. The presence or absenceof nicotine, hyoscyamine and scopolamine in different culturedplant materials was confirmed by GC-MS, indicating that althoughthe root is the main site for alkaloid biosynthesis, with suitablecell differentiation, alkaloid biosynthesis may take place incultured shoots without root initiation. Copyright 2000 Annalsof Botany Company Duboisia myoporoides, Corkwood tree, Solanaceae, tropane alkaloid, alkaloid localization, shoot culture, root culture, iodoplatinate     

Aluminum elicits tropane alkaloid production and antioxidant system activity in micropropagated Datura innoxia plantlets

One of the methods which enhances production of plant secondary metabolites including alkaloids is the use of abiotic elicitors in vitro. The aim of the study was the employment of aluminum (Al) as an abiotic elicitor in such concentrations, which were not stressful for the plant, to avoid intense growth limiting, but could elicit tropane alkaloid biosynthesis. The in vitro propagated plantlets of Datura innoxia were exposed to different concentrations of AlCl₃ (0, 25, 75 and 225 μM) to determine its effects on hyoscyamine and scopolamine contents in roots and shoots, which were analyzed by HPLC. Antioxidant enzyme activities were determined by spectrophotometer. Results showed that aluminum reduced shoots’ and roots’ fresh weights and the reduction in roots were more than the shoots. In addition, Al had significant positive effects on hyoscyamine and scopolamine contents especially in roots of the plantlets and AlCl₃ caused ROS production in shoots. These findings suggest that aluminum can likely elicit the tropane alkaloid biosynthesis in D. innoxia plantlets in vitro.