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.

Alkaloids represent an extensive group of nitrogen-containing secondary metabolites that are widely distributed throughout the plant kingdom. The pyridine alkaloids of tobacco (Nicotiana tabacum L.) have been the subject of particularly intensive investigation, driven largely due to the widespread use of tobacco products by society and the role that nicotine (16) (see Fig. 1) plays as the primary compound responsible for making the consumption of these products both pleasurable and addictive. In a typical commercial tobacco plant, nicotine (16) comprises about 90% of the total alkaloid pool, with the alkaloids nornicotine (17) (a demethylated derivative of nicotine), anatabine (15) and anabasine (5) making up most of the remainder. Advances in molecular biology have led to the characterization of the majority of the genes encoding the enzymes directly responsible the biosynthesis of nicotine (16) and nornicotine (17), while notable gaps remain within the anatabine (15) and anabasine (5) biosynthetic pathways. Several of the genes involved in the transcriptional regulation and transport of nicotine (16) have also been elucidated. Investigations of the molecular genetics of tobacco alkaloids have not only provided plant biologists with insights into the mechanisms underlying the synthesis and accumulation of this important class of plant alkaloids, they have also yielded tools and strategies for modifying the tobacco alkaloid composition in a manner that can result in changing the levels of nicotine (16) within the leaf, or reducing the levels of a potent carcinogenic tobacco-specific nitrosamine (TSNA). This review summarizes recent advances in our understanding of the molecular genetics of alkaloid biosynthesis in tobacco, and discusses the potential for applying information accrued from these studies toward efforts designed to help mitigate some of the negative health consequences associated with the use of tobacco products. Copyright © 2013 Elsevier Ltd. All rights reserved.

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.

Nicotine alkaloids are synthesized in the root of Nicotiana species, and their synthesis increases after insect attack, wounding and jasmonate treatment of the leaf. Putrescine N-methyltransferase (PMT) catalyzes the first committed step in nicotine biosynthesis. The expression patterns of the three Nicotiana sylvestris PMT genes (NsPMT1, NsPMT2, and NsPMT3) are reported in this study. Transcripts of the NsPMT genes were detected only in the root, and were up-regulated by methyl jasmonate treatment. When the 5'-flanking regions of NsPMT1, NsPMT2, and NsPMT3 were fused independently to beta-glucuronidase reporter gene and introduced into N. sylvestris by Agrobacterium-mediated transformation, all introduced transgenes were expressed in the cortex, endodermis, and xylem in the root, as well as upregulated by methyl jasmonate treatment. These qualitatively similar patterns of expression for the NsPMT genes are achieved with only 0.25 kb of their conserved 5'-flanking regions, which contained no known jasmonate-responsive elements.

Nicotine has practical applications relating to smoking cessation devices and alternative nicotine products. Genetic manipulation for increasing nicotine content in cultivated tobacco (Nicotiana tabacum L.) may be of value for industrial purposes, including the possibility of enhancing the efficiency of nicotine extraction. Biotechnological approaches have been evaluated in connection with this objective, but field-based results are few. Here, we report characterization of two genes encoding basic-helix-loop-helix (bHLH) transcription factors (TFs), NtMYC2a and NtMYC2b from tobacco. Overexpression of NtMYC2a increased leaf nicotine levels in T-1 transgenic lines approximately 2.3-fold in greenhouse-grown plants of tobacco cultivar 'NC 95'. Subsequent field testing of T-2 and T-3 generations of transgenic NtMYC2a overexpression lines showed nicotine concentrations were 76% and 58% higher than control lines, respectively. These results demonstrated that the increased nicotine trait was stably inherited to the T-2 and T-3 generations, indicating the important role that NtMYC2a plays in regulating nicotine accumulation in N. tabacum and the great potential of NtMYC2a overexpression in tobacco plants for industrial nicotine production. Collected data in this study also indicated a negative feedback inhibition of nicotine biosynthesis. Further enhancement of nicotine accumulation in tobacco leaf may require modification of the processes of nicotine transport and deposition.