Nicotine Synthesis Research Articles

Optimal Defense Theory Predicts the Ontogeny of an Induced Nicotine Defense

Optimal Defense (OD) theory predicts that the within-plant allocation of secondary metabolites that function as defenses will be positively correlated with the fitness value of particular plant parts. Here, we experimentally examine this prediction by exploiting our understanding of the mechanisms of wound-induced nicotine production in Nicotiana sylvestris (Solanaceae) to manipulate the patterns of nicotine allocation and to determine their fitness consequences. In two perturbation experiments conducted over three stages of ontogeny (rosette, elongation, and flowering), we wounded or removed leaves of different ages (young, mature, or old) and determined the effects on nicotine allocation (whole-plant and within-plant) and fitness (lifetime viable seed production). OD theory predicts that, as leaves age and their fitness value decreases, the allocation of defense to particular leaves and the fitness consequences of their removal should be positively correlated. We found that (1) leaf removal results in a significant decrease in seed mass at the elongation stage, but not at the rosette or flowering stages; (2) the relative value of leaves decreases from young and mature to old leaves; (3) leaf damage significantly increases the whole-plant nicotine contents of rosette-stage plants, but not of elongation- or flowering-stage plants, and after damage, younger leaves are more heavily defended than older leaves at the elongation and flowering stages; and (4) regardless of ontogenetic stage, plants distribute nicotine among leaves in accordance with their relative fitness value, thus supporting OD theory predictions. Leaf value increases after N fertilization at the flowering stage, but is not changed if adjacent leaves are removed at earlier growth stages. Moreover, plants are capable of sending their root-synthesized nicotine to specific leaves after damage; at the elongation stage, the new and young leaves receive greater proportional allocations of nicotine than other leaves. The cessation of significant whole-plant nicotine inductions at later stages in ontogeny is not due to the root's decreased ability to respond to the plant's wound signal, jasmonic acid (JA) with increased nicotine biosynthesis, but rather to the decline in a leaf's sensitivity to wounding and in its ability to export JA from the leaves to roots. Ontogeny has profound effects on the use of this induced defense. Plants mount systemic nicotine inductions at the rosette stage that rely on large increases in de novo nicotine synthesis, switch to the selective targeting of nicotine to the new and young leaves at the elongation stage without large increases in de novo nicotine synthesis, and allocate to reproductive structures, but not leaves, at the flowering stage. These changes are consistent with the predictions of OD theory.

Allocation of nitrogen to an inducible defense and seed production in Nicotiana attenuata.

Resource-based tradeoffs in the allocation of a limiting resource are commonly invoked to explain negative correlations between growth and defense in plants, but critical examinations of these tradeoffs are lacking. To rigorously quantify tradeoffs in a common currency, we grew Nicotiana attenuata plants in individual hydroponic chambers, induced nicotine production by treating roots with methyl jasmonate (MJ) and standardized leaf puncturing, and used 15N to determine whether nitrogen-based tradeoffs among nicotine production, growth, and seed production could be detected. Plants were treated with a range of MJ quantities (5, 45 or 250 μg plant-1) to effect a physiologically realistic range of changes in endogenous jasmonic acid levels and increases in nicotine production and accumulation; MJ treatments were applied to the roots to target JA-induced nicotine production, since nicotine biosynthesis is restricted to the roots. Leaf puncturing and 5 μg MJ treatments increased de novo nicotine synthesis and whole-plant (WP) nicotine pools by 93 and 66%, while 250 μg MJ treatments increased these values 3.1 and 2.5-fold. At these high rates of nicotine production, plants incorporated 5.7% of current nitrogen uptake and 6.0% of their WP nitrogen pools into nicotine. The 15N-labeled nicotine pools were stable or increased for the duration of vegetative growth, indicating that the N-nicotine was not metabolized and re-used for growth. Plants with elevated nicotine production grew more slowly and the differences in plant biomass gain between MJ-treated plants and controls were linearly related to the differences in nicotine accumulation. Despite the reductions in rosette-stage growth associated with nicotine production, estimates of lifetime fitness (cumulative lifetime seed production, mass/seed, seed viability) were not affected by any treatment. Only two treatments (leaf puncturing and 250 μg MJ) increased the allocations of 15N acquired at the time of induction to seed production. On average, plants used only 14.9% of their WP nitrogen pool for seed production, indicating that either the nitrogen requirements for seed production or the reproductive effort of these hydroponically-grown plants are low. To determine if seed production is strongly influenced by the amount of vegetative biomass attained before reproduction, the experiment was repeated with plants that had 44% of their leaf area (or 29% of their WP biomass) removed before MJ treatments with a removal technique that minimized the nicotine response. MJ treatments of these plants dramatically increased nicotine production and accumulation, but these plants also suffered no measurable fitness consequences from either the leaf removal or MJ treatments. We conclude that when N. attenuata plants are grown in these individual hydroponic chambers, their allocation to reproduction is sufficiently buffered to obscure the large increases in nitrogen allocations to an inducible defense. To determine whether soil-grown plants are similarly buffered, we grew two genotypes of plants in the high-nutrient soil from a 1-year-old burn in a piñyon-juniper forest (the plants' natural habitat) and in low-nutrient soil from an adjacent unburned area, and induced nicotine production in half of the plants with a 500 μg root MJ treatment. Plants grown in burned soils had an estimated lifetime fitness that was on average 2.8-fold greater than that of plants grown in unburned soils. MJ treatment reduced fitness estimates by 43% and 71% in the burned and unburned soils, respectively. We conclude that while hydroponic culture allows one to rigorously quantitate nitrogen allocation to growth, reproduction and defense, the allocation patterns of plants grown in hydroponic culture differ from those of plants grown in soil. Under hydroponic conditions, plants have low reproductive allocations and reproductive-defense tradeoffs are not detected. Reproductive-defense tradeoffs are readily discernible in soil-grown plants, but under these growing conditions, the nitrogen-basis for the tradeoff is difficult to quantify.

Effect of Regenerated Roots and Shoots on Nicotine Production in Tobacco Tissue Culture

The accumulation of nicotine in regenerated shoot and root tissues of Nicotiana tabacum L. cv MD 872 grown from callus culture has been examined by high pressure liquid chromatography (HPLC) procedures. The presence of roots on callus tissue dramatically increased the accumulation of nicotine in both callus without shoots and in callus which had regenerated shoots. When shoots were regenerated, the concentration of nicotine that accumulated in callus decreased whether roots were present or not. When varying concentrations of the nicotine precursors ornithine and putrescine were fed to callus derived tissue with and without regenerated roots, the rooted tissue was able to take up the precursors and accumulate nicotine in the shoots in amounts greater than controls. The half-life turnover rate for ’H-nicotine absorbed by the roots and transported into shoots was approximately 48 hours. These experiments indicate that although the root is the major site of synthesis of nicotine in tobacco, the accumulation of nicotine in various parts of the plant is regulated by the interaction of both root and shoot tissue.

Synthesis and crystal structure of 2-nitroxyethyl nicotinate and its complex with PdCl2

The reaction of nicotinoyl chloride with ethylene glycol mononitrate yielded the previously unknown 2-nitroxyethyl nicotinate. The resulting ester was used as a ligand in the reaction with PdCl2 for preparing a new complex,trans-bis(2-nitroxyethyl nicotinate-N)dichloropalladium(ii). The structures of the ligand and the complex were established by X-ray structural analysis.

Structural Requirements of Jasmonates and Mimics for Nicotine Induction inNicotiana sylvestris

Jasmonic acid (JA) is strongly implicated in the long-distance signal transduction cascade increasing nicotine synthesis in the roots of plants after leaf wounding. In order to explore the structural requirements of the inducing signal, we examined jasmonates, mimics, and a biosynthetic precursor for nicotine-inducing activity (NIA). We examine the importance of the keto group on the five-membered ring and the double bond in then-pentenyl chain by comparing the NIA of methyl jasmonate (MJ) with that of cucurbic acid, 1,3-dithiolane-MJ, 1,3-dioxolane-MJ, methyl dihydrojasmonate (DHMJ), 1,3-dioxolane-DHMJ, 1-oxo-indan-4-carboxylic acid ILE-methyl ester, and 1-hydroxyl-indan-4-carboxylic acid ILE-methyl ester. We found that: 1,3-dioxolane MJ, cucurbic acid, and 1,3-dioxolane DHMJ were less active than MJ and that the isoleucine (ILE) conjugates of 1-oxo- and l-hydroxyindanon-4-carboxylic acid had the same NIA as MJ. The activities of these indanon amino acid conjugates may be due to the structural similarity of their keto or hydroxyl groups on the five-membered ring to MJ or to the keto-enolized MJ. These results support the hypothesis that the enolization of the keto group during or prior to its interaction with the putative JA receptor is required for activity. We explore the importance of the esterification of the carboxyl functional group by comparing the NIAs of cucurbic acid and cucurbic acid methyl ester, l-oxo-indan-4-carboxylic acid, 1-oxo-indan-4-carboxylic acid methyl ester, and l-oxo-indan-4-carboxylic acid ILE-methyl ester. In all cases, the esters were more active than the free acids. We compared the NIA of MJ of different epimeric composition (8% and 20% 3R,7S-MJ); 12-oxophytodienoic acid (12-oxo-PDA) methyl ester, an important precursor of JA; and coronatine (a well-known phytotoxin and putative structural mimic of 12-oxo-PDA).We found that: (1) the epimeric composition of MJ did not affect its NIA; (2) 12-oxo-PDA methyl ester had lower NIA than MJ; and (3) coronatine significantly inhibited plant growth but did not increase nicotine biosynthesis. In summary, JA, rather than its biosynthetic precursor, 12-oxo-PDA, is likely the endogenous signal inNicotiana sylvestris, and the keto functional group on the five-membered ring and the double bond in then-pentenyl side chain are crucial components of JA for NIA.

Transport of [2- 14 C]jasmonic acid from leaves to roots mimics wound-induced changes in endogenous jasmonic acid pools in Nicotiana sylvestris

Jasmonic acid (JA) is part of a long-distance signal-transduction pathway that effects increases in de-novo nicotine synthesis in the roots of Nicotiana sylvestris Speg et Comes (Solanaceae) after leaf wounding. Elevated nicotine synthesis increases whole-plant nicotine pools and makes plants more resistant to herbivores. Leaf wounding rapidly increases JA pools in damaged leaves, and after a 90-min delay, root JA pools also increase. The systemic response in the roots could result from either: (i) the direct transport of JA from wounded leaves, or (ii) JA synthesis or its release from conjugates in roots in response to a second, systemic signal. We synthesized [2-14C]JA, and applied it to a single leaf in a quantity (189 μg) known to elicit both a whole-plant nicotine and root JA response equivalent to that found in plants subjected to leaf wounding. We quantified radioactive material in JA, and in metabolites both more and less polar than JA, from treated and untreated leaves and roots of plants in eight harvests after JA application. [2-14C]Jasmonic acid was transported from treated leaves to roots at rates and in quantities equivalent to the wound-induced changes in endogenous JA pools. The [2-14C]JA that had been transported to the roots declined at the same rate as endogenous JA pools in the roots of plants after leaf wounding. Most of the labeled material applied to leaves was metabolized or otherwise immobilized at the application site, and the levels of [2-14C]JA in untreated leaves did not increase over time. We measured the free JA pools before and after four different hydrolytic extractions of root and shoot tissues to estimate the size of the potential JA conjugate pools, and found them to be 10% or less of the free JA pool. We conclude that the direct transport of wound-induced JA from leaves to roots can account for the systemic increase in root JA pools after leaf wounding, and that metabolism into less polar structures determines the duration of this systemic increase. However, the conclusive falsification of this hypothesis will require the suppression of all other signalling pathways which could have shoot-to-root transport kinetics similar to that of endogenous JA.

Quantification, correlations and manipulations of wound-induced changes in jasmonic acid and nicotine in Nicotiana sylvestris

Jasmonic acid (JA) is thought to be part of a signal-transduction pathway which dramatically increases de-novo nicotine synthesis in the roots and increases whole-plant (WP) nicotine pools in response to the wounding of the leaves in Nicotiana sylvestrisSpegazzini and Comes (Solanaceae). We report the synthesis of a doubly labeled JA ([1, 2-13C]JA) and use it as an internal standard to quantify by gas chromatography-mass spectrometry the changes in root and shoot JA pools in plants subjected to differing amounts of standardized leaf wounding. Wounding increased JA pools 10-fold locally in damaged leaves within 90 min and systemically in the roots (3.5-fold) 180 min after wounding. If JA functions as an intermediary between stimulus and response, quantitative relationships among the stimulus, JA, and the response should exist. To examine these relationships, we varied the number of punctures in four leaves and quantified both the resulting JA in damaged leaves after 90 min and the resulting WP nicotine concentration after 5 d. We found statistically significant, positive relationships among number of leaf punctures, endogenous JA, and WP nicotine accumulation. We used two inhibitors of wound-induced nicotine production, methyl salicylate and indole-3-acetic acid, to manipulate the relationships between wound-induced changes in JA and WP nicotine accumulation. Since wounding and the response to wounding occur in widely separated tissues, we applied inhibitors to different plant parts to examine their effects on the local and systemic components of this response. In all experiments, inhibition of the wound-induced increase in leaf JA 90 min after wounding was associated with the inhibition of the nicotine response 5 d after wounding. We conclude that wound-induced increases in leaf JA are an important component of this long-distance signal-transduction pathway.

Methyl jasmonate‐induced nicotine production in Nicotiana attenuata: inducing defenses in the field without wounding

AbstractThe functional significance of herbivore‐induced plant traits known to directly or indirectly influence herbivore performance remains largely untested under field conditions due to the difficulty of uncoupling the response to herbivory from the act of herbivory. The signals that activate many of the induced responses in plants are endogenously produced in response to wounding, unlike many of the predator‐induced responses found in aquatic invertebrates (which are activated by exogenous cues derived from predators). Jasmonates, endogenously‐produced damage signals, activate diverse wound‐induced responses in plants including induced nicotine production in Nicotiana sylvestris. The results presented here are from two experiments which illustrate the use of jasmonates to uncouple induced nicotine production in Nicotiana attenuata (Torrey ex. Watson) from wounding. The exogenous addition of methyl jasmonate (MJ) in small quantities (11 μg for a 1.4 g dry mass plant) to roots of hydroponically‐grown plants induces de novo nicotine synthesis and increases whole‐plant nicotine concentrations just as wounding does. The MJ‐induced changes were proportional to the quantity of MJ given. Moreover, the effects of MJ were additive to the effects of damage. Applications of MJ to shoots were less effective. Root treatments also worked with plants growing in a field plot. The application of MJ represents a promising tool for examining the functional significance of induced nicotine responses in plants growing in their native environments.

Effects of octadecanoid metabolites and inhibitors on induced nicotine accumulation inNicotiana sylvestris

We examined the effects of inhibitors of the octadecanoid pathway (n-propyl gallate, acetosalicylic acid, salicylhydroxamic acid, methyl salicylate, and antipyrine) on wound- and jasmonate-induced nicotine accumulation and compared the nicotine-inducing ability of exogeneous additions of linolenic acid (18:3) and its methyl ester, linoleic acid (18:2), abscisic acid, traumatic acid, and methyl dihydrojasmonate to the nicotine-inducing ability of exogenous additions of methyl jasmonate (MJ). The first four of these inhibitors significantly reduced wound-induced nicotine accumulation when applied in a lanolin paste to wounded tissues immediately after wounding at concentrations of 89-90µg/plant. When methyl salicylate and propyl gallate were mixed individually with MJ, neither inhibited MJ-induced nicotine synthesis, which suggests that the inhibitors block jasmonate synthesis or release from stored pools and not its effects. Linolenic acid or its methyl ester applied to undamaged plants or damaged plants (to either damaged or undamaged leaves) or to the roots of hydroponically growing plants did not induce nicotine accumulation or increase nicotine accumulation above levels found in damaged plants. Similarly, traumatic acid, linoleic acid, and abscisic acid did not induce nicotine accumulations. Methyl dihydrojasmonate, which is biosynthetically derived from linoleic acid, had 12-56% of the nicotine-inducing acitivity of MJ when added to the roots of hydroponically grown plants. The signal transduction pathway mediating wound-induced nicotine production therefore shares many features of the pathway eliciting wound-induced proteinase inhibitor production but differs in not being regulated at the lipase step in jasmonic acid production and not being responsive to abscisic acid.

Wound-induced changes in root and shoot jasmonic acid pools correlate with induced nicotine synthesis inNicotiana sylvestris spegazzini and comes

Leaf damage by herbivores inNicotiana sylvestris Spegazzini and Comes (Solanaceae) produces a damage signal that dramatically increasesde novo nicotine synthesis in the roots. The increased synthesis leads to increases in whole-plant nicotine pools, which in turn make plants more resistant to further herbivore attack. Because signal production and the response to the signal occur in widely separated tissues, the speed with which different damage signals exit a damaged leaf can be studied. We propose that electrical damage signals should exit a leaf faster (less than 60 min) than chemical damage signals. Excision of a leaf induces a smaller increase in nicotine production than does puncture damage, so we examined our proposition by excising previously punctured leaves at 1, 60, and 960 min after leaf puncture and quantifying the induced whole-plant nicotine pools six days later when the induced nicotine production had reached a maximum. Significant induced nicotine production occurred only if punctured leaves were excised more than 1 hr after puncture, which is consistent with the characteristics of a slow-moving chemical signal rather than a fast-moving electrical signal. We explore the nature of the chemical signal and demonstrate that additions of 90µg or more of methyl jasmonate (MJ) in an aqueous solution to the roots of hydroponically grown plants inducede novo nicotine synthesis from(15)NO3 in a manner similar to that induced by leaf damage. We examine the hypothesis that jasmonic acid (JA) functions in the transfer of the damage signal from shoot to root. Using GC-MS techniques to quantify whole-plant JA pools, we demonstrate that leaf damage rapidly (<0.5 hr) increases shoot JA pools and, more slowly (<2 hr), root JA pools. JA levels subsequently decay to levels found in undamaged plants within 24 hr and 10 hr for shoots and roots, respectively. The addition of sufficient quantities (186µg) of MJ in a lanolin paste to leaves from hydroponically grown plants significantly increased endogenous root JA pools and increasedde novo nicotine synthesis in these plants. However, the addition of 93µg or less of MJ did not significantly increase endogenous root JA pools and did not significantly affectde novo nicotine synthesis. We propose that wounding increases shoot JA pools, which either directly through transport or indirectly through a systemin-like signal increase root JA pools, which, in turn, stimulate root nicotine synthesis and increase whole-plant nicotine pools.

Damage-induced root nitrogen metabolism inNicotiana sylvestris: Testing C/N predictions for alkaloid production

"Nitrogen surplus" models for nicotine production induced by leaf damage predict that the observed increase in root nicotine synthesis after leaf damage results from "overflow" metabolism; reduced nitrogen existing in excess of growth requirements is shunted into nicotine biosynthesis. To test the nitrogen surplus model for induced nicotine production, we measured the concentrations of the majorN-containing metabolites exported from the roots and the nitrate reductase activity (NRA) of roots and shoots in damaged and undamagedNicotiana sylvestris plants. Leaf damage: (1) had no significant effect on root or shoot NRA, (2) increased nicotine export and decreased amino-acid and amide export from the roots of NO3-fertilized plants, and (3) had no significant effect on the export of anyN-containing metabolite from the roots of NH4-fertilized plants. These results are not consistent with the nitrogen surplus model and indicate that leaf damage has a direct influence on root alkaloid metabolism.

Auxin-induced Regulation of Amino Acid and Putrescine in the Free State and Nicotine Content in Cultured Tobacco Callus

Summary Callus tissues of Nicotiana tabacum cv. Burley 21 cultured on a low-auxin medium (1 μM NAA, optimal for nicotine synthesis) grew less and had a rather greater cytoplasm/vacuole ratio and consequently a lower water content than those cultured in a high-auxin medium (11.5 μM NAA, supraoptimal for nicotine synthesis). The former showed a marked increase of nicotine and putrescine levels, as well as ornithine decarboxylase (ODC) (EC 4.1.1.17) and arginine decarboxylase (ADC) (EC 4.1.1.19) activities, particularly ODC, but a decrease in the free amino acid level and arginase (ARG) (EC 3.5.3.7) activity compared with callus cultured at a high-auxin concentration. It can be deduced that ornithine and ODC were the chief amino acid and enzyme, respectively, involved in nicotine synthesis under our experimental conditions.

Correlation between K+ content, activities of arginine and ornithine decarboxylase, and levels of putrescine and nicotine in cultured tobacco callus

In callus cultures of Nicotiana tabacum L. cv. Burley 21 we have examined the effect of two auxin concentrations (1 and 11.5 μMα‐naphthaleneacetic acid) in the culture medium on K+, putrescine and nicotine levels and activities of putrescine‐biosyn‐thetic enzymes l‐arginine decarboxylase (EC 4.1.1.19) and l‐ornithine decarboxylase (EC 4.1.1.17). The calli grown on the low‐auxin medium (with optimal auxin concentration for nicotine synthesis) had significantly lower concentrations of K+ and higher concentrations of nicotine than those grown on the high‐auxin medium (with a supraoptimal auxin concentration). Furthermore, in the calli grown on both culture media, there was a positive correlation between the levels of HCIO4‐soluble free putrescine and nicotine, as well as a negative correlation between those of HCIO4‐soluble bound putrescine and the alkaloid. The results suggest that in tobacco callus K+ uptake, the accumulation of HCIO4‐soluble free putrescine and nicotine synthesis are related processes that depend upon the concentration of auxin in the culture medium; a concentration of 1 μM NAA would increase HCIO4‐soluble free putrescine level to a greater degree than that of 11,5 μM NAA, and consequently lead to a higher production of the alkaloid. Although both putrescine‐biosynthetic enzymes are active in our callus cultures, ornithine decarboxylase activity was considerably greater. This interpretation is supported by the enhancement of the 35.5 kDa band and 38.9 kDa band (detected by SDS‐PAGE) which showed ornithine and arginine decarboxylase activity, respectively.

The pyridine-nucleotide cycle in tobacco

In order to elucidate the NAD-recycling pathway the following enzyme activities have been characterized in different tobacco tissues and in tomato root: NAD pyrophosphatase, nicotinamide mononucleotide (NMN)/nicotinic acid mononucleotide (NaMN) glycohydrolases, nicotinamidase and nicotinic acid phosphoribosyltransferase. The investigations were performed with protein extracts purified by gel filtration and enzymatic activities were determined by high-performance liquid chromatography methods. The kinetic parameters of the different enzymes from tobacco root and their specificity are reported. The data are in favor of the so-called pyridine-nucleotide cycle VI (NAD→NMN→nicotinamide→nicotinic acid→NaMN→nicotinic acid adenine dinucleotide→NAD). In the nicotine-producing tobacco root a further direct route leading from NaMN to nicotinic acid is proposed. These data are reconciled with the assumption that it is nicotinic acid which is provided by the pyridine-nucleotide cycle for the synthesis of nicotine.

Regulation in tobacco callus of enzyme activities of the nicotine pathway

In tobacco callus, the induction of nicotine synthesis, which stimulates enzyme activities of the ornithine-methylpyrroline route (see the preceding paper), also leads to marked changes in the enzyme activities of the pyridine-nucleotide cycle. This cycle provides the metabolite (probably nicotinic acid) for condensation with methylpyrroline to produce nicotine. The activities of eight enzymes of the pyridine-nucleotide cycle and of quinolinic-acid phosphoribosyltransferase, the anaplerotic enzyme, were determined by high-performance liquid chromatography assays. The distinct changes of their activities upon induction of nicotine synthesis lead to the following conclusions: i) nicotinic acid is the relevant metabolite which is provided by the pyridine-nucleotide cycle and consumed for nicotine synthesis. ii) The enhancement of the nicotinic-acid pool arises in two ways, by synthesis of NAD and degradation via nicotinamide mononucleotide and by a direct route from nicotinic-acid mononucleotide (NaMN) which is degraded by a glycohydrolase with a rather high K m value. Such a K m value prevents the complete depletion of the NaMN pool.

Regulation in tobacco callus of enzyme activities of the nicotine pathway

Nicotine synthesis was stimulated by reduction of the medium auxin concentration (induction medium) in callus tissue originating from Nicotiana tabacum cv. Samsun. The enzyme activities of the route ornithine to methylpyrroline, which are those of ornithine decarboxylase, putrescine methyltransferase and methylputrescine oxidase, were determined during callus growth in the induction medium and as a control under non-nicotine-stimulating conditions (growth medium). The enzymes were assayed by high-performance liquid chromatography. Whereas the activities of ornithine decarboxylase were very similar under nicotine-stimulating and non-stimulating conditions, those of putrescine methyltransferase and methyl-putrescine oxidase increased strongly in the induction medium. In addition, the pools of putrescine and methylputrescine were determined throughout the callus growth cycle. Both sets of data strongly confirm the supposition that putrescine methyl-transferase is the enzyme under stringent control for nicotine biosynthesis, whereas the subsequent methylputrescine oxidase is co-regulated, although less stringently.

Effect of auxin on alkaloids, K+ and free amino acid content in cultured tobacco callus

Callus cultures derived from the petiole of Nicotiana tabacum L. cv. Burley 21 were grown at 25°C in the dark on two different basal media containing: (1) 11.5 μMα‐naphthaleneacetic acid and 1 μM kinetin, and (2) 1 μMα‐naphthaleneacetic acid and 1 μM kinetin. The contents of alkaloids, K+ and free amino acids of callus tissues were determined. The tissues were also examined microscopically for organization when organogenesis was not apparent. The first medium limited nicotine synthesis and stimulated its N‐demethylation to nornicotine. The second medium stimulated nicotine synthesis and limited tissue growth. Although significantly higher concentrations of K+ were observed in calli grown on the high‐auxin medium, both cultures were K+ deficient. The fact that the low‐auxin medium limited K+ uptake to a higher degree would account for the lower growth observed in calli cultured on this medium, and it is possible that the effect of auxin concentration on nicotine production may be mediated through its effects on K+ uptake by cells of the culture. The free amino acid concentration increased in the calli grown on the low‐auxin medium. Glutamic acid and proline, known as initial precursors of nicotine, increased significantly. Histological examination showed that the occurrence of meristematic areas in calli without organogenesis promoted nicotine synthesis. The relation between the accumulation of nicotine and formation of roots or shoot‐buds is discussed.

Interaction of nitrate and sulfate reduction in tobacco

Abstract A series of experiments was conducted to study the effects of apical meristem removal on sulfate and nitrate reduction in ‘Ky14’ tobacco plants (Nicotiana tabacum L.). Apical meristem removal is expected to modify N metabolism since it causes an increase in the synthesis of nicotine in the roots and its accumulation in shoots. There may not be any such complementary effect on S metabolism. Further, the experiments were designed to study the effects of apical meristem removal on the interaction of nitrate (NO3) and sulfate (SO4) reduction. Activities of ATP sulfurylase and NO3 reductase, the first steps of SO4 and NO3 reduction, respectively, have been considered indicators of the state of regulation of the assimilatory pathways of SO4 and NO3. The effect of apical meristem removal on the activities of the above enzymes in the leaf tissue and the interrelationship between them was examined. Removal of the apical meristem resulted in a large increase in nicotine concentration, a large increase in t...

Interaction of nitrate and sulfate reduction in tobacco

A series of experiments was conducted to study the effects of apical meristem removal on sulfate and nitrate reduction in ‘Ky14’ tobacco plants (Nicotiana tabacum L.). Apical meristem removal is expected to modify N metabolism since it causes an increase in the synthesis of nicotine in the roots and its accumulation in shoots. There may not be any such complementary effect on S metabolism. Further, the experiments were designed to study the effects of apical meristem removal on the interaction of nitrate (NO3) and sulfate (SO4) reduction. Activities of ATP sulfurylase and NO3 reductase, the first steps of SO4 and NO3 reduction, respectively, have been considered indicators of the state of regulation of the assimilatory pathways of SO4 and NO3. The effect of apical meristem removal on the activities of the above enzymes in the leaf tissue and the interrelationship between them was examined. Removal of the apical meristem resulted in a large increase in nicotine concentration, a large increase in t...

The pyridine-nucleotide cycle in tobacco Enzyme activities for the de-novo synthesis of NAD

The enzyme activities of the pyridine-nucleotide cycle, which transform nicotinic acid mononucleotide (NaMN) into NAD, have been characterized. The investigations were based on the extraction of protein, its purification on disposable gel-filtration columns, and determination of the enzymatic activities by high-performance liquid chromatography techniques. The latter technique avoided the synthesis and use of radioactive precursors. The NaMN-adenylyltransferase which converts NaMN into NaAD (nicotinic acid adenine dinucleotide) and NAD-synthetase which converts NaAD into NAD were characterized by their kinetic parameters and their specific activities in different tobacco tissues. This is the first report on NAD-synthetase from tissue of a higher plant. It was found that NAD-synthetase accepted both glutamine and asparagine for the amide transfer. Adenylyltransfer also occured with nicotinamide mononucleotide (NMN) which was transformed to NAD, whereas the glutamine-dependent amidation was only observed with NaAD. Thus, an additional route for the synthesis of NAD (NaMN→NMN→NAD) obviously does not exist. A comparison of the enzyme activities in tobacco tissues with different capacities for the synthesis of nicotine showed that, in contrast to quinolinic acid phosphoribosyltransferase whose activity was strictly correlated with the nicotine content, only NaMN-adenylyltransferase showed a smooth correlation, whereas NAD-synthetase was not affected at all.