Incorporation of arginine, ornithine and phenylalanine into tropane alkaloids in suspension-cultured cells and aseptic roots of intact plants oi Atropa belladonna

Label from U-¹⁴C-arginine (Arg), -ornithine (Orn) and -phenylalanine(Phe) was incorporated into hyoscyam-ine and scopolamine byboth dissected roots of intact plants and homogeneous or aggregatingsuspension cultures of Atropa belladonna L. The early biosyntheticcompounds (hygrine, tropinone, tropanol) were found only inthe roots, and alkaloid synthesis proceeded as far as to scopolaminethere. In the synthesis of the tropane skeleton, Orn was usedmore efficiently than Arg. Phe was quickly metabolized bothin roots and suspension cultures, and the label was incorporatedinto both ethanol-insoluble compounds, particularly proteinsand different ethanol-soluble compounds, especially phenolics. When the callus, used to initiate suspension cultures, was repeatedlysubcultured, the degree of organization of the suspension-culturedcells (second suspension passage) grown in the presence of 2.5µM 2-naphthaleneacetic acid (NAA), changed first fromhomogeneous to aggregating and later to heavily rooty. Simultaneouslywith an increasing degree of organization, alkaloid synthesisdecreased. At first, the rate of incorporation of Arg and Phe,and later Orn into alkaloids decreased. In heavily rooty suspensionsneither hyoscyamine nor scopolamine were found, but traces oftropanol were detected. Hence, the synthesis of tropane alkaloidsseems to be regulated at the later biosynthetic steps. Key words: Arginine, Atropa, hyoscyamine, omithine, phenylalanine, roots, scopolamine, suspension culture, tropane alkaloids     

Engineering tropane alkaloid production and glyphosate resistance by overexpressing AbCaM1 and G2-EPSPS in Atropa belladonna

Atropa belladonna is an important industrial crop for producing anticholinergic tropane alkaloids (TAs). Using glyphosate as selection pressure, transgenic homozygous plants of A. belladonna are generated, in which a novel calmodulin gene (AbCaM1) and a reported EPSPS gene (G2-EPSPS) are co-overexpressed. AbCaM1 is highly expressed in secondary roots of A. belladonna and has calcium-binding activity. Three transgenic homozygous lines were generated and their glyphosate tolerance and TAs’ production were evaluated in the field. Transgenic homozygous lines produced TAs at much higher levels than wild-type plants. In the leaves of T2GC02, T2GC05, and T2GC06, the hyoscyamine content was 8.95-, 10.61-, and 9.96 mg/g DW, the scopolamine content was 1.34-, 1.50- and 0.86 mg/g DW, respectively. Wild-type plants of A. belladonna produced hyoscyamine and scopolamine respectively at the levels of 2.45 mg/g DW and 0.30 mg/g DW in leaves. Gene expression analysis indicated that AbCaM1 significantly up-regulated seven key TA biosynthesis genes. Transgenic homozygous lines could tolerate a commercial recommended dose of glyphosate in the field. In summary, new varieties of A. belladonna not only produce pharmaceutical TAs at high levels but tolerate glyphosate, facilitating industrial production of TAs and weed management at a much lower cost.     

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.     

Influence of hairy root ecotypes on production of tropane alkaloids in Brugmansia candida

Hyoscyamine and scopolamine are tropane alkaloids widely applied in medicine. Differences in alkaloid production and growth kinetics have been observed in Argentinian and Colombian ecotypes of Brugmansia candida hairy roots. The aim of this work was to analyze the production of key intermediates in tropane alkaloid synthesis in both ecotypes to determine differences in the biosynthetic pathway. Additionally, rolC gene expression was analyzed to determine its correlation with hairy root growth. The results showed a higher accumulation of polyamines in Colombian hairy roots, suggesting that there may be a rate-limiting enzyme in the last steps of hyoscyamine biosynthesis. Additionally, rolC gene expression was correlated with an improvement in hairy root growth, which supports the function of rol genes as growth modulators and suggests that metabolic engineering approaches involving rolC manipulation may be useful for the development of more efficient B. candida hairy root cultures for biotechnological applications.     

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.     

Norlittorine and norhyoscyamine identified as products of littorine and hyoscyamine metabolism by 13C-labeling in Datura innoxia hairy roots

The presence of two compounds, norlittorine and norhyoscyamine, has been reported in leaves and roots of Datura innoxia; however their metabolic origin in the tropane alkaloid pathway has remained unknown. Precise knowledge of this pathway is a necessary pre-requisite to optimize the production of hyoscyamine and scopolamine in D. innoxia hairy root cultures. The exact structure of norlittorine and norhyoscyamine was confirmed by LC–MS/MS and NMR analyses. Isotopic labeling experiments, using [1-¹³C]-phenylalanine, [1′-¹³C]-littorine and [1′-¹³C]-hyoscyamine, combined with elicitor treatments, using methyl jasmonate, coronalon and 1-aminocyclopropane-1-carboxylic acid, were used to investigate the metabolic origin of the N-demethylated tropane alkaloids. The results suggest that norlittorine and norhyoscyamine are induced under stress conditions by conversion of littorine and hyoscyamine. We propose the N-demethylation of tropane alkaloids as a mechanism to detoxify cells in overproducing conditions.     

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     

Biochemical and structural characterization of recombinant hyoscyamine 6β-hydroxylase from Datura metel L.

Hyoscyamine 6β-hydroxylase (H6H; EC 1.14.11.11), an important enzyme in the biosynthesis of tropane alkaloids, catalyzes the hydroxylation of hyoscyamine to give 6β-hydroxyhyoscyamine and its epoxidation in the biosynthetic pathway leading to scopolamine. Datura metel produces scopolamine as the predominant tropane alkaloid. The cDNA encoding H6H from D. metel (DmH6H) was cloned, heterologously expressed and biochemically characterized. The purified recombinant His-tagged H6H from D. metel (DmrH6H) was capable of converting hyoscyamine to scopolamine. The functionally expressed DmrH6H was confirmed by HPLC and ESI-MS verification of the products, 6β-hydroxyhyoscyamine and its derivative, scopolamine; the DmrH6H epoxidase activity was low compared to the hydroxylase activity. The K_m values for both the substrates, hyoscyamine and 2-oxoglutarate, were 50 μM each. The CD (circular dichroism) spectrum of the DmrH6H indicated a preponderance of α-helicity in the secondary structure. From the fluorescence studies, Stern–Volmer constants for hyoscyamine and 2-oxoglutarate were found to be 0.14 M^?1 and 0.56 M^?1, respectively. These data suggested that the binding of the substrates, hyoscyamine and 2-oxoglutarate, to the enzyme induced significant conformational changes.     

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.     

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.     

Alkaloid production in cultured roots of three species of Duboisia

Cultured roots were obtained from calluses of Duboisia leichhardtii, D. myoporoides and D. hopwoodii. Cultured roots of all these species produced both tropane and pyridine-type alkaloids. The selected cultured roots of D. leichhardtii showed high contents of tropane alkaloids (hyoscyamine 0.53%, scopolamine 1.16%, on a dry weight basis).     

δ-N-Methylornithine: a natural constituent of Atropa belladonna

δ-N-Methylornithine, a tropane alkaloid precursor, is shown for the first time to be a natural plant constituent; it was isolated in radioactive form after feeding [5-¹⁴C]- and [5-³H]ornithine to Atropa belladonna. This finding supports the deduced role of δ-N-methylornithine in tropane alkaloid biosynthesis.     

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.     

Enhanced production of hyoscyamine and scopolamine from genetically transformed root culture of <Emphasis Type="Italic">Hyoscyamus reticulatus</Emphasis> L. elicited by iron oxide nanoparticles

The medicinal plant Hyoscyamus reticulatus L. is a rich source of hyoscyamine and scopolamine, the tropane alkaloids. The use of hairy root cultures has focused significant attention on production of important metabolites such as stable tropane alkaloid production. Elicitation is an effective approach to induce secondary metabolite biosynthetic pathways. Hairy roots were derived from cotyledon explants inoculated with Agrobacterium rhizogenes and elicited by iron oxide nanoparticles (FeNPs) at different concentrations (0, 450, 900, 1800, and 3600 mg L^?1) for different exposure times (24, 48, and 72 h). The highest hairy root fresh and dry weights were found in the medium supplemented with 900 mg L^?1 FeNPs. Antioxidant enzyme activity was significantly increased in induced hairy roots compared to non-transgenic roots. The highest hyoscyamine and scopolamine production (about fivefold increase over the control) was achieved with 900 and 450 mg L^?1 FeNPs at 24 and 48 h of exposure time, respectively. This is the first report of the effect of FeNP elicitor on hairy root cultures of a medicinal plant. We suggest that FeNPs could be an effective elicitor in hairy root cultures in order to increase tropane alkaloid production.     

Metabolism of hygrine in Atropa,Hyoscyamus and Physalis

Three-month-old plants of Physalis alkekengi, Atropa belladonna and Hyoscyamus niger were fed via the roots with either d( + ) or l( - )-hygrine-[2′- ¹⁴C]. The feeding experiments were terminated after 7 days when the following alkaloids were isolated from the paired groups of plants: tigloidine, 3α-tigloyloxytropane and cuscohygrine from Physalis; hyoscyamine from Hyoscyamus and from Atropa. In contrast to Datura these genera appear to use both hygrine enantiomers in the biosynthesis of the tropane ring.     

Traits of transgenic Atropa belladonna doubly transformed with different Agrobacterium rhizogenes strains

Hairy root cultures of Atropa belladonna L. were established by infection either with Agrobacterium rhizogenes ATCC 15834 or MAFF 03-01724, and transgenic plants were obtained from both hairy root cultures. Doubly transformed roots were induced by re-infection of the leaf segments of transgenic Atropa belladonna plants (A. rhizogenes 15834) with MAFF 03-01724. Shoots and viviparous leaves were regenerated from the doubly transformed roots. The genetic transformation was determined by the opine assay (agropine, mannopine and/or mikimopine) and polymerase chain reaction. Physiological changes and tropane alkaloid biosynthesis in the hairy roots (singly and doubly transformed) were investigated. The alkaloid content in the doubly transformed root strain was intermediate as compared to the root strains which were singly transformed. On the other hand endogenous IAA levels in doubly transformed roots were significantly decreased compared to both singly transformed roots.Abbreviations BA benzyladenine - IAA indoleacetic acid - NAA naphthaleneacetic acid - PCR polymerase chain reaction - t-ZR trans-zeatin