The alkaloid contents of sixty Nicotiana species

The alkaloid content (nicotine, nornicotine, anabasine, and anatabine) in leaves and roots of 60 Nicotiana species were analyzed by GC. All species contained alkaloids, the amounts varying with the species. There was no clearcut correlation between alkaloid amounts and subgeneric or sectional classification. The alkaloid content in the floral parts and immature and mature fruits of Nicotiana tabacum were also analyzed.     

The Only African Wild Tobacco,Nicotiana africana: Alkaloid Content and the Effect of Herbivory

Herbivory in some Nicotiana species is known to induce alkaloid production. This study examined herbivore-induced defenses in the nornicotine-rich African tobacco N. africana, the only Nicotiana species indigenous to Africa. We tested the predictions that: 1) N. africana will have high constitutive levels of leaf, flower and nectar alkaloids; 2) leaf herbivory by the African bollworm Helicoverpa armigera will induce increased alkaloid levels in leaves, flowers and nectar; and 3) increased alkaloid concentrations in herbivore-damaged plants will negatively affect larval growth. We grew N. africana in large pots in a greenhouse and exposed flowering plants to densities of one, three and six fourth-instar larvae of H. armigera, for four days. Leaves, flowers and nectar were analyzed for nicotine, nornicotine and anabasine. The principal leaf alkaloid was nornicotine (mean: 28 µg/g dry mass) followed by anabasine (4.9 µg/g) and nicotine (0.6 µg/g). Nornicotine was found in low quantities in the flowers, but no nicotine or anabasine were recorded. The nectar contained none of the alkaloids measured. Larval growth was reduced when leaves of flowering plants were exposed to six larvae. As predicted by the optimal defense theory, herbivory had a localized effect and caused an increase in nornicotine concentrations in both undamaged top leaves of herbivore damaged plants and herbivore damaged leaves exposed to one and three larvae. The nicotine concentration increased in damaged compared to undamaged middle leaves. The nornicotine concentration was lower in damaged leaves of plants exposed to six compared to three larvae, suggesting that N. africana rather invests in new growth as opposed to protecting older leaves under severe attack. The results indicate that the nornicotine-rich N. africana will be unattractive to herbivores and more so when damaged, but that potential pollinators will be unaffected because the nectar remains alkaloid-free even after herbivory.     

Modified alkaloid pattern in developing tobacco callus

Developing Nicotiana tabacum L. cv. Wisconsin-38 callus grown on modified Murashige-Skoog (MS) medium with Kao organic acids (pyruvic, citric, malic and fumaric acids) contains abnormally high levels of nornicotine and total alkaloids when compared with the leaves of the donor plant. Nornicotine/nicotine ratios observed during callus development suggest that nicotine is converted into nornicotine in the callus, with subsequent movement of alkaloids into roots formed on the callus and into the agar medium. Addition of Kao organic acids to the medium increases alkaloid levels, but cannot account for the abnormal increase in nicotine demethylation. This study thus reports two new findings: (a) that the total alkaloid content of tobacco callus can be greatly enhanced to 3.75% on a dry weight basis by exogenous organic acids, and (b) that endogenous nornicotine can accumulate in tobacco tissue cultures.     

Fast track to the trichome: induction of N-acyl nornicotines precedes nicotine induction in Nicotiana repanda

 Nicotiana repanda Wildenow ex Lehmann acylates nornicotine in its trichomes to produce N-acyl-nornicotine (NacNN) alkaloids which are dramatically more toxic than nicotine is to the nicotine-adapted herbivore, Manduca sexta. These NacNNs, like nicotine, were induced by methyl jasmonate (MeJA) and wounding, but the 2-fold increase in NacNN pools was much faster (within 6 h) than the MeJA-induced increase in nornicotine pools (24 h to 4 d), its parent substrate. When ¹⁵NO₃ ⁻ pulse-chase experiments with intact and induced plants were used to follow the incorporation of ¹⁵N into alkaloids in different plant parts over the plant's lifetime, it was found that the root nicotine pool was most rapidly labeled, followed by the shoot nornicotine and NacNN pools. After 3 d, 3.12% of ¹⁵N acquired was in nicotine (0.93%), nornicotine (0.32%) and NacNNs (1.73%) while only 0.14% was in anabasine. Once NacNNs are externalized to the leaf surface, they are not readily re-distributed within the plant and are lost with senescing leaves. The wound- and MeJA-induced N-acylation of nornicotine is independent of induced changes in nornicotine pools and the rapidity of the response suggests its importance in defense against herbivores. Received: 3 July 1999 / Accepted: 17 September 1999     

Discrimination of hybrid classes using cross-species amplification of microsatellite loci: methodological challenges and solutions in Daphnia

Microsatellite markers are important tools in population, conservation and forensic studies and are frequently used for species delineation, the detection of hybridization and introgression. Therefore, marker sets that amplify variable DNA regions in two species are required; however, cross-species amplification is often difficult, as genotyping errors such as null alleles may occur. To estimate the level of potential misidentifications based on genotyping errors, we compared the occurrence of parental alleles in laboratory and natural Daphnia hybrids (Daphnia longispina group). We tested a set of 12 microsatellite loci with regard to their suitability for unambiguous species and hybrid class identification using F(1) hybrids bred in the laboratory. Further, a large set of 44 natural populations of D. cucullata, D. galeata and D. longispina (1715 individuals) as well as their interspecific hybrids were genotyped to validate the discriminatory power of different marker combinations. Species delineation using microsatellite multilocus genotypes produced reliable results for all three studied species using assignment tests. Daphnia galeata × cucullata hybrid detection was limited due to three loci exhibiting D. cucullata-specific null alleles, which most likely are caused by differences in primer-binding sites of parental species. Overall, discriminatory power in hybrid detection was improved when a subset of markers was identified that amplifies equally well in both species.     

Sequestration of pyridine alkaloids anabasine and nicotine from Nicotiana (Solanaceae) by Orobanche ramosa (Orobanchaceae)

Plants of the root holoparasite Orobanche ramosa L. and four of its potential host species, Nicotiana glauca Graham, Nicotiana rustica L., Nicotiana sylvestris Speg. & S.Comes, and Nicotiana tabacum L., grown in the greenhouse in Kiel/Germany, were analyzed for their contents of pyridine alkaloids anabasine (1) and nicotine (2). All investigated samples contained both alkaloids in different amounts. The distribution of the alkaloids in the various plant organs of Nicotiana differed significantly between the species. The alkaloid contents of the Orobanche samples relative to the alkaloid contents of the roots of the respective host plants varied between 3.47 ± 1.08 and 28.8 ± 37.5%. Orobanche plants drain water and crucial nutrients from their hosts; also, some examples for the sequestration of specialized natural products have been reported. O. ramosa is not able to synthesize pyridine alkaloids anabasine (1) and nicotine (2) itself; therefore, the present study proves the sequestration of pyridine alkaloids by O. ramosa from the four investigated Nicotiana host species.     

Perturbation of alkaloid production by cadaverine in hairy root cultures of Nicotiana rustica

Cadaverine (1–10 mM) stimulated the production of anabasine by hairy root cultures of Nicotiana rustica transformed with Agrobacterium rhizogenes. In control cultures, nicotine accounted for at least 70–80% of the total alkaloid produced, whereas in cultures supplemented with 5 mM cadaverine about two-thirds of the alkaloid was anabasine and nicotine production was markedly diminished. Putrescine and agmatine caused some stimulation of alkaloid production, but the ratio of nicotine to anabasine was essentially unaffected. Lysine caused no substantial increase in anabasine formation.     

The incorporation of [5,6-13C2]Nicotinic acid into the tobacco alkaloids examined by the use of 13C nuclear magnetic resonance

[5,6-¹⁴C,¹³C₂]Nicotinic acid was prepared from [¹⁴C,¹³C]methyl iodide via nitromethane, 2-nitroacetaldehyde oxime, 3-nitroquinoline, 3-aminoquinoline, and quinoline in 20% overall yield. Administration of this material to Nicotiana tabacum and N. glauca afforded labeled anabasine, anatabine, nicotine, and nornicotine. Qualitative and quantitative incorporation (0.07–4.5% specific incorporation) was determined by radioactive assay and by examination of the ¹³C NMR spectra of these alkaloids. Satellites due to spin-spin coupling of the incorporated contiguous ¹³C atoms were observed at the resonances due to C-5 and C-6 in anabasine, nicotine, and nornicotine. In anatabine, satellites were found at C-5, C-6, C-5′, and C-6′.     

Separation and measurement of plant alkaloid enantiomers by RP-HPLC analysis of their Fmoc-Alanine analogs

Introduction . Ammodendrine ( 1 ), anabasine ( 2 ) and coniine ( 3 ) can cause congenital malformations in livestock. They appear naturally in both enantiomeric forms, and can cause variable physiological responses. A method to measure the enantiomeric ratio of these natural toxins is needed. Objective . To develop a simple and economical method in order to determine the enantiomeric ratios of piperidine and pyrrolidine alkaloids in small samples of plant material. Methodology . Mixtures of isolated or purified plant alkaloids were converted to their Fmoc‐l ‐Ala‐alkaloid analogues forming diastereomeric mixtures, which were then analysed by high pressure liquid chromatography (HPLC) with mass spectrometry (MS) and ultraviolet (UV) detection to determine enantiomeric ratios. Results . The diastereomeric analogs for ammodendrine, anabasine and nornicotine could be separated and the enantiomeric ratios determined. The Fmoc‐l ‐Ala‐coniine analogue was not resolved under the HPLC conditions studied. The enantiomeric ratios of the selected plant alkaloids were measured and found to differ between both location within a species and location between species. Conclusion . A low‐cost HPLC method to analyse the enantiomeric ratio of plant alkaloids containing primary or secondary amine nitrogens via conversion to their respective diastereomeric analogues has been developed. Published in 2008 by John Wiley & Sons.     

Enantiomeric analysis of anatabine, nornicotine and anabasine in commercial tobacco by multi-dimensional gas chromatography and mass spectrometry

A fully automated multi-dimensional gas chromatography (MDGC) system with a megabore precolumn and cyclodextrin-based analytical column was developed to analyze the enantiomeric compositions of anatabine, nornicotine and anabasine in commercial tobacco. The enantiomer abundances of anatabine and nornicotine varied among different tobacco. S-(-)-anatabine, as a proportion of total anatabine, was 86.6% for flue-cured, 86.0% for burley and 77.5% for oriental tobacco. S-(-)-nornicotine, as a proportion of total nornicotine, was 90.8% in oriental tobacco and higher than in burley (69.4%) and flue-cured (58.7%) tobacco. S-(-)-anabasine, as a proportion of total anabasine, was relatively constant for flue-cured (60.1%), burley (65.1%) and oriental (61.7%) tobacco. A simple solvent extraction with dichloromethane followed by derivatisation with trifluoroacetic anhydride gave relative standard deviations of less than 1.5% for the determination of the S-(-)-isomers of all three alkaloids. The study also indicated that, a higher proportion of S-(-)-nornicotine is related to the more active nicotine demethylation in the leaf.     

Horizontal Transfer of Genetic Material among Saccharomyces Yeasts

The genus Saccharomyces consists of several species divided into the sensu stricto and the sensu lato groups. The genomes of these species differ in the number and organization of nuclear chromosomes and in the size and organization of mitochondrial DNA (mtDNA). In the present experiments we examined whether these yeasts can exchange DNA and thereby create novel combinations of genetic material. Several putative haploid, heterothallic yeast strains were isolated from different Saccharomyces species. All of these strains secreted an a- or alpha-like pheromone recognized by S. cerevisiae tester strains. When interspecific crosses were performed by mass mating between these strains, hybrid zygotes were often detected. In general, the less related the two parental species were, the fewer hybrids they gave. For some crosses, viable hybrids could be obtained by selection on minimal medium and their nuclear chromosomes and mtDNA were examined. Often the frequency of viable hybrids was very low. Sometimes putative hybrids could not be propagated at all. In the case of sensu stricto yeasts, stable viable hybrids were obtained. These contained both parental sets of chromosomes but mtDNA from only one parent. In the case of sensu lato hybrids, during genetic stabilization one set of the parental chromosomes was partially or completely lost and the stable mtDNA originated from the same parent as the majority of the nuclear chromosomes. Apparently, the interspecific hybrid genome was genetically more or less stable when the genetic material originated from phylogenetically relatively closely related parents; both sets of nuclear genetic material could be transmitted and preserved in the progeny. In the case of more distantly related parents, only one parental set, and perhaps some fragments of the other one, could be found in genetically stabilized hybrid lines. The results obtained indicate that Saccharomyces yeasts have a potential to exchange genetic material. If Saccharomyces isolates could mate freely in nature, horizontal transfer of genetic material could have occurred during the evolution of modern yeast species.     

Three nicotine demethylase genes mediate nornicotine biosynthesis in Nicotiana tabacum L.: functional characterization of the CYP82E10 gene

In most tobacco (Nicotiana tabacum L.) plants, nornicotine is a relatively minor alkaloid, comprising about 2-5% of the total pyridine alkaloid pool in the mature leaf. Changes in gene expression at an unstable locus, however, can give rise to plants that produce high levels of nornicotine, specifically during leaf senescence and curing. Minimizing the nornicotine content in tobacco is highly desirable, because this compound serves as the direct precursor in the synthesis of N'-nitrosonornicotine, a potent carcinogen in laboratory animals. Nornicotine is likely produced almost entirely via the N-demethylation of nicotine, in a process called nicotine conversion that is catalyzed by the enzyme nicotine N-demethylase (NND). Previous studies have identified CYP82E4 as the specific NND gene responsible for the unstable conversion phenomenon, and CYP82E5v2 as a putative minor NND gene. Here, by discovery and characterization of CYP82E10, a tobacco NND gene, is reported. PCR amplification studies showed that CYP82E10 originated from the N. sylvestris ancestral parent of modern tobacco. Using a chemical mutagenesis strategy, knockout mutations were induced and identified in all three tobacco NND genes. By generating a series of mutant NND genotypes, the relative contribution of each NND gene toward the nornicotine content of the plant was assessed. Plants possessing knockout mutations in all three genes displayed nornicotine phenotypes that were much lower (～0.5% of total alkaloid content) than that found in conventional tobacco cultivars. The introduction of these mutations into commercial breeding lines promises to be a viable strategy for reducing the levels of one of the best characterized animal carcinogens found in tobacco products.     

Studies on the Chemical Regulation of Alkaloid Biosynthesis in Tobacco Plants

Effect of auxin treatment upon the alkaloid content of Nicotiana was examined using hydroponically grown plants. Nicotine content of Nicotiana tabacum was reduced by the addition of 3-indoleacetic acid or 2, 4-dichlorophenoxyacetic acid to nutrient solution, an the incorporation of nitrate-¹⁵N into nicotine was decreased by the addition of 2, 4-dichloro-phenoxyacetic acid. Contents of nicotine, nornicotine, anabasine and anatabine in Nicotiana glutinosa were decreased by 2, 4-dichlorophenoxyacetic acid added to the nutrient solution. Foliar applications of eleven kinds of synthetic auxins were effective in reducing nicotine content of Nicotiana tabacum and the effectiveness of the compounds seemed to be concerned with their auxin activities. Foliar application of auxin tended to increase the dry weight of stems whereas decreased that of roots, and, moreover, higher content of soluble sugars and lower content of starch in leaves were observed by the treatment. A possibility of a practical application of auxin to reduce the nicotine content of tobacco plants were drawn from this work.