Contribution of Nicotine and Nornicotine toward the Production of N'-Nitrosonornicotine in Air-Cured Tobacco (Nicotiana tabacum).

N'-Nitrosonornicotine (6) is a potent and organ-specific carcinogen found in tobacco and tobacco smoke in substantial amounts. Nicotine (1) and nornicotine (2) are proposed to be the precursors of 6 in tobacco. Since 1 can be rapidly demethylated to 2 in tobacco, to distinguish between the direct formation of 6 from these potential precursors is difficult. A gas chromatography/thermal energy analyzer method using two columns in series was developed to separate the enantiomers of 6, N'-nitrosoanabasine (7), and N'-nitrosoanatabine (8). Tobacco lines with different combinations of three nicotine demethylases inhibited were grown in the field. Air-cured leaves were analyzed for the enantiomeric composition of four main alkaloids and their corresponding tobacco-specific nitrosamines. The percentage of (R)-6 of total 6 varied from 7% to 69% in mutant lines. The measured 6 had the same enantiomeric composition as 2, rather than 1, even when the level of 2 was reduced to 0.6% of 1 in a triple mutant line. The pattern of the enantiomeric composition of 1, 2, and 6 demonstrated that the direct formation of 6 from 1, if it occurs, is negligible in air-cured tobacco. Since (S)-6 is more highly carcinogenic than its R form, the reduction of (S)-2 should be a priority for the reduction of 6.

Mitigation of Ergot Vasoconstriction by Clover Isoflavones in Goats (Capra hircus).

Ergot alkaloids produced by a fungal endophyte (Epichloë coenophiala; formerly Neotyphodium coenophialum) that infects tall fescue (Lolium arundinaceum) can induce persistent constriction of the vasculature in ruminants, hindering their capability to thermo-regulate core body temperature. There is evidence that isoflavones produced by legumes can relax the vasculature, which suggests that they could relieve ergot alkaloid-induced vasoconstriction and mitigate the vulnerability to severe heat stress in ruminants that graze tall fescue. To test if isoflavones can relieve alkaloid-induced vasoconstriction, two pen experiments were conducted with rumen-fistulated goats (Capra hircus) to determine with ultrasonograpy if isoflavones can (1) promote vascular compliance by countering alkaloid-induced vasoconstriction and (2) relieve already imposed alkaloid-induced vasoconstriction. Goats were fed ad libitum chopped orchardgrass (Dactylis glomerata)-timothy (Phleum pratense) hay prior to conducting the experiments. Measures of carotid and interosseous luminal areas were obtained pre- (baseline) and post-ruminal infusions in both experiments with goats being fed the hay, and for blood flow rate in the carotid artery in Experiment 2. Responses to infusion treatments were evaluated as proportionate differences from baseline measures. Peak systolic velocity, pulsatility index, and heart rate were measured on the last day on treatment (DOT) in Experiment 1, and on all imaging sessions during Experiment 2. For Experiment 1, rumens were infused with ground toxic fescue seed and isoflavones in Phase A and with only the toxic seed in Phase B. The infusion treatments were switched between phases in Experiment 2, which employed a fescue seed extract having an ergot alkaloid composition equivalent to that of the ground seed used in Experiment 1. During Experiment 1, luminal areas of carotid and interosseous arteries in Phase A did not deviate (P > 0.1) from baselines over 1, 2, 3, and 4 DOT, but the areas of both declined linearly from baselines over 1, 2, 3, and 4 DOT in Phase B. By 6, 7, and 8 DOT in Experiment 2, luminal areas of the arteries and flow rate declined from baselines with infusions with the only seed extract in Phase A, but luminal areas and flow rate increased over 4, 5, and 6 DOT with the additional infusion of isoflavones. Peak systolic velocity and heart rate were not affected by treatment in either experiment, but were highest when infused with only ergot alkaloids in both experiments. Treatment with isoflavones was demonstrated to relax the carotid and interosseous arteries and reduce resistance to blood flow. Results indicate that isoflavones can relax persistent vasoconstriction in goats caused by consumption of ergot alkaloids, and mitigate the adverse effect that ergot alkaloids have on dry matter intake.

Warming reduces tall fescue abundance but stimulates toxic alkaloid concentrations in transition zone pastures of the U.S.

Tall fescue pastures cover extensive acreage in the eastern half of the United States and contribute to important ecosystem services, including the provisioning of forage for grazing livestock. Yet little is known concerning how these pastures will respond to climate change. Tall fescue's ability to persist and provide forage under a warmer and wetter environment, as is predicted for much of this region as a result of climate change, will likely depend on a symbiotic relationship the plant can form with the fungal endophyte, Epichloë coenophiala. While this symbiosis can confer environmental stress tolerance to the plant, the endophyte also produces alkaloids toxic to insects (e.g., lolines) and mammals (ergots; which can cause "fescue toxicosis" in grazing animals). The negative animal health and economic consequences of fescue toxicosis make understanding the response of the tall fescue symbiosis to climate change critical for the region. We experimentally increased temperature (+3°C) and growing season precipitation (+30% of the long-term mean) from 2009-2013 in a mixed species pasture, that included a tall fescue population that was 40% endophyte-infected. Warming reduced the relative abundance of tall fescue within the plant community, and additional precipitation did not ameliorate this effect. Warming did not alter the incidence of endophyte infection within the tall fescue population; however, warming significantly increased concentrations of ergot alkaloids (by 30-40%) in fall-harvested endophyte-infected individuals. Warming alone did not affect loline alkaloid concentrations, but when combined with additional precipitation, levels increased in fall-harvested material. Although future warming may reduce the dominance of tall fescue in eastern U.S. pastures and have limited effect on the incidence of endophyte infection, persisting endophyte-infected tall fescue will have higher concentrations of toxic alkaloids which may exacerbate fescue toxicosis.

Changes in TSNA contents during tobacco storage and the effect of temperature and nitrate level on TSNA formation.

Samples of burley, sun-cured, and flue-cured tobacco from the main producing areas of relevant tobacco types in China were collected to study the changes in tobacco-specific nitrosamine (TSNA) contents during storage and to investigate the effect of storage temperature and tobacco nitrate level on TSNA formation of cured tobacco. Contents of TSNAs in burley and sun-cured tobacco increased substantially during 1 year under natural storage environment, with total TSNA content increasing about 215% for both tobacco types. The most rapid increase occurred during the high temperature season. Temperature had a significant promoting effect on TSNA formation during storage. Storage temperature as high as 27 °C for 12 days was enough to induce the increase of TSNA formation, while the most significant effect was shown when the temperature was above 30 °C. The increased rate of accumulation became greater as the temperature increased. Total TSNA content in air-cured burley tobacco after the treatment of 60 °C for 24 days was 772% higher than that in the low temperature control. Different types of tobacco showed different results in terms of the response of TSNA formation to high temperature. TSNA formation in flue-cured tobacco did not increase after high-temperature treatment for 36 days, while burley and sun-cured tobacco saw a dramatic increase of TSNA content. This difference could be explained by the fact that burley tobacco and sun-cured tobacco usually had more than 10 times the nitrate content than flue-cured tobacco. As the nitrate nitrogen increased in cured burley tobacco, TSNA formation during leaf storage at high temperature significantly increased. Addition of nitrate onto flue-cured tobacco to the level equivalent to burley tobacco followed by high-temperature treatment increased the TSNA concentration comparable to burley tobacco. The interaction between high temperature and abundant nitrate content in cured tobacco could be responsible for TSNA formation during storage.

(R)-nicotine biosynthesis, metabolism and translocation in tobacco as determined by nicotine demethylase mutants.

Nicotine is a chiral compound and consequently exists as two enantiomers. Since (R)-nicotine consists of less than 0.5% of total nicotine pool in tobacco, few investigations relating to (R)-nicotine have been reported. However, previous studies of nicotine demethylases suggested there was substantial amount of (R)-nicotine at synthesis in the tobacco plant. In this study, the accumulation and translocation of (R)-nicotine in tobacco was analyzed. The accumulation of nicotine and its demethylation product the nornicotine enantiomers, were investigated in different tobacco plant parts and at different growth and post-harvest stages. Scion/rootstock grafts were used to separate the contributions of roots (source) from leaves (sink) to the final accumulation of nicotine and nornicotine in leaf tissue. The results indicate that 4% of nicotine is in the (R) form at synthesis in the root. After the majority of (R)-nicotine is selectively demethylated by CYP82E4, CYP82E5v2 and CYP82E10 in the root, nicotine and nornicotine are translocated to leaf, where more nicotine becomes demethylated. Depending on the CYP82E4 activity in senescing leaf, constant low (R)-nicotine remains in the tobacco leaf and variable nornicotine composition is produced. These results confirmed the enantioselectivity of three nicotine demethylases in planta, could be used to predict the changes of nicotine and nornicotine composition, and may facilitate demethylase discovery in the future.

Alteration of the alkaloid profile in genetically modified tobacco reveals a role of methylenetetrahydrofolate reductase in nicotine N-demethylation.

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of the tetrahydrofolate (THF)-mediated one-carbon (C1) metabolic network. This enzyme catalyzes the reduction of 5,10-methylene-THF to 5-methyl-THF. The latter donates its methyl group to homocysteine, forming methionine, which is then used for the synthesis of S-adenosyl-methionine, a universal methyl donor for numerous methylation reactions, to produce primary and secondary metabolites. Here, we demonstrate that manipulating tobacco (Nicotiana tabacum) MTHFR gene (NtMTHFR1) expression dramatically alters the alkaloid profile in transgenic tobacco plants by negatively regulating the expression of a secondary metabolic pathway nicotine N-demethylase gene, CYP82E4. Quantitative real-time polymerase chain reaction and alkaloid analyses revealed that reducing NtMTHFR expression by RNA interference dramatically induced CYP82E4 expression, resulting in higher nicotine-to-nornicotine conversion rates. Conversely, overexpressing NtMTHFR1 suppressed CYP82E4 expression, leading to lower nicotine-to-nornicotine conversion rates. However, the reduced expression of NtMTHFR did not affect the methionine and S-adenosyl-methionine levels in the knockdown lines. Our finding reveals a new regulatory role of NtMTHFR1 in nicotine N-demethylation and suggests that the negative regulation of CYP82E4 expression may serve to recruit methyl groups from nicotine into the C1 pool under C1-deficient conditions.

Variable nornicotine enantiomeric composition caused by nicotine demethylase CYP82E4 in tobacco leaf.

Nornicotine is the demethylation product of nicotine and the precursor of tobacco-specific nitrosamine N'-nitrosonornicotine (NNN) in tobacco (Nicotiana tabacum L.). There is an inconsistent enantiomer fraction (EF) of nornicotine reported in the literature. The objective of this study was to explore possible mechanisms to account for the variable EF(nnic) in tobacco. A survey of tobacco with different demethylating capabilities confirmed that there was variable EF(nnic). Experiments of induction and inhibition of the major nicotine demethylase CYP82E4 activity in tobacco demonstrated that CYP82E4 selectively demethylated (S)-nicotine and resulted in different EF(nnic) in tobacco leaves. Results from plants with silenced demethylases by RNAi suggested that other demethylases selectively used (R)-nicotine and resulted in high EF(nnic). In summary, enantioselective demethylation likely plays an important role in contributing to a large and variable EF(nnic) observed in tobacco.

Enantioselective demethylation of nicotine as a mechanism for variable nornicotine composition in tobacco leaf.

Nicotine and its N-demethylation product nornicotine are two important alkaloids in Nicotiana tabacum L. (tobacco). Both nicotine and nornicotine have two stereoisomers that differ from each other at 2'-C position on the pyrrolidine ring. (S)-Nicotine is the predominant form in the tobacco leaf, whereas the (R)-enantiomer only accounts for ∼0.2% of the total nicotine pool. Despite considerable past efforts, a comprehensive understanding of the factors responsible for generating an elevated and variable enantiomer fraction of nornicotine (EF(nnic) of 0.04 to 0.75) from the consistently low EF observed for nicotine has been lacking. The objective of this study was to determine potential roles of enantioselective demethylation in the formation of the nornicotine EF. Recombinant CYP82E4, CYP82E5v2, and CYP82E10, three known tobacco nicotine demethylases, were expressed in yeast and assayed for their enantioselectivities in vitro. Recombinant CYP82E4, CYP82E5v2, and CYP82E10 demethylated (R)-nicotine 3-, 10-, and 10-fold faster than (S)-nicotine, respectively. The combined enantioselective properties of the three nicotine demethylases can reasonably account for the nornicotine composition observed in tobacco leaves, which was confirmed in planta. Collectively, our studies suggest that an enantioselective mechanism facilitates the maintenance of a reduced (R)-nicotine pool and, depending on the relative abundances of the three nicotine demethylase enzymes, can confer a high (R)-enantiomer percentage within the nornicotine fraction of the leaf.

Impact of alleles at the Yellow Burley (Yb) loci and nitrogen fertilization rate on nitrogen utilization efficiency and tobacco-specific nitrosamine (TSNA) formation in air-cured tobacco.

Tobacco-specific nitrosamine (TSNA) formation in tobacco is influenced by alkaloid levels and the availability of nitrosating agents. Tobacco types differ in their potential for TSNA accumulation due to genetic, agronomic, and curing factors. Highest TSNA concentrations are typically measured in burley tobaccos. One of the main genetic differences between burley and all other tobacco types is that this tobacco type is homozygous for recessive mutant alleles at the Yellow Burley 1 (Yb(1)) and Yellow Burley 2 (Yb(2)) loci. In addition, burley tobacco is typically fertilized at higher nitrogen (N) rates than most other tobacco types. This study utilized nearly isogenic lines (NILs) differing for the presence of dominant or recessive alleles at the Yb(1) and Yb(2) loci to investigate the potential influence of genes at these loci on TSNA accumulation. Three pairs of NILs were evaluated at three different nitrogen fertilization rates for alkaloid levels, nitrogen physiology measures, and TSNA accumulation after air-curing. As previously observed by others, positive correlations were observed between N application rates and TSNA accumulation. Recessive alleles at Yb(1) and Yb(2) were associated with increased alkaloid levels, reduced nitrogen use efficiency, reduced nitrogen utilization efficiency, and increased leaf nitrate nitrogen (NO(3)-N). Acting together, these factors contributed to significantly greater TSNA levels in genotypes possessing the recessive alleles at these two loci relative to those carrying the dominant alleles. The chlorophyll-deficient phenotype conferred by the recessive yb(1) and yb(2) alleles probably contributes in a substantial way to increase available NO(3)-N during curing and, consequently, increased potential for TSNA formation.

Effects of multiple climate change factors on the tall fescue-fungal endophyte symbiosis: infection frequency and tissue chemistry.

• Climate change (altered CO(2) , warming, and precipitation) may affect plant-microbial interactions, such as the Lolium arundinaceum-Neotyphodium coenophialum symbiosis, to alter future ecosystem structure and function. • To assess this possibility, tall fescue tillers were collected from an existing climate manipulation experiment in a constructed old-field community in Tennessee (USA). Endophyte infection frequency (EIF) was determined, and infected (E+) and uninfected (E-) tillers were analysed for tissue chemistry. • The EIF of tall fescue was higher under elevated CO(2) (91% infected) than with ambient CO(2) (81%) but was not affected by warming or precipitation treatments. Within E+ tillers, elevated CO(2) decreased alkaloid concentrations of both ergovaline and loline, by c. 30%; whereas warming increased loline concentrations 28% but had no effect on ergovaline. Independent of endophyte infection, elevated CO(2) reduced concentrations of nitrogen, cellulose, hemicellulose, and lignin. • These results suggest that elevated CO(2) , more than changes in temperature or precipitation, may promote this grass-fungal symbiosis, leading to higher EIF in tall fescue in old-field communities. However, as all three climate factors are likely to change in the future, predicting the symbiotic response and resulting ecological consequences may be difficult and dependent on the specific atmospheric and climatic conditions encountered.

RNA interference (RNAi)-induced suppression of nicotine demethylase activity reduces levels of a key carcinogen in cured tobacco leaves.

Technologies for reducing the levels of tobacco product constituents that may contribute to unwanted health effects are desired. Target compounds include tobacco-specific nitrosamines (TSNAs), a class of compounds generated through the nitrosation of pyridine alkaloids during the curing and processing of tobacco. Studies have reported the TSNA N'-nitrosonornicotine (NNN) to be carcinogenic in laboratory animals. NNN is formed via the nitrosation of nornicotine, a secondary alkaloid produced through enzymatic N-demethylation of nicotine. Strategies to lower nornicotine levels in tobacco (Nicotiana tabacum L.) could lead to a corresponding decrease in NNN accumulation in cured leaves. The major nicotine demethylase gene of tobacco has recently been isolated. In this study, a large-scale field trial was conducted to evaluate transgenic lines of burley tobacco carrying an RNA interference (RNAi) construct designed to inhibit the expression of this gene. Selected transgenic lines exhibited a six-fold decrease in nornicotine content relative to untransformed controls. Analysis of cured leaves revealed a commensurate decrease in NNN and total TSNAs. The inhibition of nicotine demethylase activity is an effective means of decreasing significantly the level of a key defined animal carcinogen present in tobacco products.

Importance of host plant species, Neotyphodium endophyte isolate, and alkaloids on feeding by Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae.

Three grass host species--tall fescue, Festuca arundinacea Schreber; meadow fescue, Festuca pratensis Hudson; and perennial ryegrass, Lolium perenne L.--each infected with a number of different Neotyphodium endophyte isolates, were investigated for their effects on fall armyworm, Spodoptera frugiperda (J.E. Smith). Alkaloid profiles varied among associations. Choice and no-choice tests comparing feeding and early development of S. frugiperda larvae on endophyte-infected and endophyte-free leaf blade material were performed. Endophyte-mediated resistance to S. frugiperda was greatest in meadow fescue and weakest in tall fescue. Some endophyte isolates, particularly in perennial ryegrass and meadow fescue, had a major effect on feeding and development of S. frugiperda, whereas others had no effect or were only weakly efficacious. In tall fescue, some associations deterred S. frugiperda from feeding in choice tests but had no effect on development, whereas larvae reared on other associations weighed significantly more than control larvae fed endophyte-free grass. It was concluded that the deleterious consequences of endophyte infection were easily masked by other factors in tall fescue. Relative leaf age had no effect on feeding preferences in the three host species. Chemical analysis of herbage from the plants used, and results from a no-choice study using spiked artificial diets, failed to individually implicate any of the major known alkaloids (peramine, lolitrem B, ergovaline, and lolines) in the observed effects on S. frugiperda. Hypotheses explaining these observations, and their impact on creating desirable grass-endophyte associations for use in pastures, are discussed.

Identification and synthesis of novel alkaloids from the root system of Nicotiana tabacum: affinity for neuronal nicotinic acetylcholine receptors.

A novel pyridine derivative, 3,5-bis-(1-methyl-pyrrolidin-2-yl)-pyridine, and a pair of diastereomers of 1,1'-dimethyl-[2,3']bipyrrolidinyl were isolated from the root of Nicotiana tabacum plants and identified as novel alkaloids by GC-MS analysis. The structures of these new alkaloids were confirmed by total synthesis. The affinities of these novel alkaloids, and other structurally related compounds for alpha4beta2*, alpha7* neuronal nicotinic acetylcholine receptors (nAChRs), and for nAChRs mediating nicotine-evoked dopamine release from rat striatum were also assessed. The results indicate that these compounds do not interact with alpha7* nAChRs, but inhibit [3H]nicotine binding to the alpha4beta2* nAChR subtype. The results also demonstrate that these compounds act as antagonists at nAChRs mediating nicotine-evoked dopamine release from rat striatum.

Biosynthetic precursors of fungal pyrrolizidines, the loline alkaloids.

Loline alkaloids are saturated pyrrolizidines with a substituted 1-amino group and an oxygen bridge between C2 and C7, and are insecticidal metabolites of plant-symbiotic fungi (endophytes). Cultures of the endophyte, Neotyphodium uncinatum, incorporated labeled L-proline and L-homoserine into the 1-aminopyrrolizidine, N-formylloline. The A-ring carbons C1-C3 and the N1 were derived from L-homoserine; the B-ring carbons C5-C8 and the ring nitrogen were derived from L-proline. Incorporation of both deuterium atoms from L-[4,4-(2H2)]homoserine and feeding tests with labeled L-methionine indicated that L-homoserine incorporation was not achieved via aspartyl semialdehyde or S-adenosylmethionine, but probably involved a highly novel N--C bond-forming gamma-substitution reaction.

Peramine alkaloid variation in Neotyphodium-infected Arizona fescue: effects of endophyte and host genotype and environment.

We determined concentrations of peramine, the only alkaloid produced by Neotyphodium-infected (E+) Arizona fescue plants (of the four major types typically assayed in infected grasses), in a long-term field experiment. Four plant genotypes with (E+) and without (experimentally removed, E-) their respective haplotypes (two haplotypes in two plant genotypes) of Neotyphodium were grown in the field under manipulated soil moisture and nutrients. Peramine production required the presence of the endophyte; plants without their endophytes did not contain peramine. Whereas the endophyte was necessary for peramine production, levels of peramine did not vary by Neotyphodium haplotype within plant genotypes. Furthermore, peramine levels did not differ among soil moisture and nutrient treatments, and growth and reproductive measures of the host grass explained little of variation in peramine levels. Instead, peramine levels differed significantly between plant genotypes harboring the same endophyte haplotype, suggesting that plant genotype, rather than endophyte haplotype or environment, largely determines levels of peramine in Arizona fescue. The results suggest that whereas the endophyte is required for peramine production, the plant genotypic background in which the endophyte is embedded, rather than endophyte haplotype or environmental factors, mostly influences peramine levels within this population of Arizona fescue.