Nitrogen-containing Secondary Metabolites Research Articles

Leaves and roots metabolomic signatures underlying rootstock-mediated water stress tolerance in grafted pepper plants

Grafting onto pepper rootstock NIBER® is an effective strategy to mitigate water stress effects on the grafted variety. In this work, we comparatively explored the metabolomic responses to water stress in the pepper variety “Maestral F1” (V) grafted onto NIBER® (V/N) and self-grafted (V/V) by untargeted metabolomics on leaves and roots. Leaf water status was also evaluated by relative water content (RWC) and gas exchange measurements. Under water stress, the V/N water use efficiency (WUE) and leaf RWC were higher than V/V, in agreement with major stomata closure and water retention in leaves. V/N showed a tolerance response, which was manifested in the untargeted metabolomic analysis. NIBER® modulated the grafted variety response to water stress as reflected in the differential metabolomic profiles in leaves and roots. The V/N-enriched metabolic pathways showed that the NIBER® response to water stress involved cutin and suberin biosynthesis, which act as protection layers, and jasmonic acid (JA) and jasmonates biosynthesis to favor signaling pathways. NIBER® did not induce flavonols and chlorophyll b synthesis, but likely promoted anthocyanins biosynthesis and maintained an undisturbed chlorophyll a:chlorophyll b ratio. Moreover, NIBER® increased vitamin B6, anthocyanins and stearic acid concentration in the variety leaves, whereas siroheme content rose in roots to improve nitrogen assimilation. Further studies are required to understand the contribution of secondary metabolites, such as phenylpropanoids, glycoalkaloids, and nitrogen-containing secondary metabolites, to NIBER® water stress tolerance.

Current Status and De Novo Synthesis of Anti-Tumor Alkaloids in Nicotiana

Alkaloids are the most diversified nitrogen-containing secondary metabolites, having antioxidant and antimicrobial properties, and are extensively used in pharmaceuticals to treat different types of cancer. Nicotiana serves as a reservoir of anti-cancer alkaloids and is also used as a model plant for the de novo synthesis of various anti-cancer molecules through genetic engineering. Up to 4% of the total dry weight of Nicotiana was found to be composed of alkaloids, where nicotine, nornicotine, anatabine, and anabasine are reported as the dominant alkaloids. Additionally, among the alkaloids present in Nicotiana, β-carboline (Harmane and Norharmane) and Kynurenines are found to show anti-tumor effects, especially in the cases of colon and breast cancers. Creating new or shunting of existing biosynthesis pathways in different species of Nicotiana resulted in de novo or increased synthesis of different anti-tumor molecules or their derivatives or precursors including Taxadiane (~22.5 µg/g), Artemisinin (~120 μg/g), Parthenolide (~2.05 ng/g), Costunolide (~60 ng/g), Etoposide (~1 mg/g), Crocin (~400 µg/g), Catharanthine (~60 ng/g), Tabersonine (~10 ng/g), Strictosidine (~0.23 mg/g), etc. Enriching the precursor pool, especially Dimethylallyl Diphosphate (DMAPP), down-regulating other bi-product pathways, compartmentalization or metabolic shunting, or organelle-specific reconstitution of the precursor pool, might trigger the enhanced accumulation of the targeted anti-cancer alkaloid in Nicotiana.

Nitrogen-Containing Secondary Metabolites from a Deep-Sea Fungus Aspergillus unguis and Their Anti-Inflammatory Activity.

Aspergillus is well-known as the second-largest contributor of fungal natural products. Based on NMR guided isolation, three nitrogen-containing secondary metabolites, including two new compounds, variotin B (1) and coniosulfide E (2), together with a known compound, unguisin A (3), were isolated from the ethyl acetate (EtOAc) extract of the deep-sea fungus Aspergillus unguis IV17-109. The planar structures of 1 and 2 were elucidated by an extensive analysis of their spectroscopic data (HRESIMS, 1D and 2D NMR). The absolute configuration of 2 was determined by comparison of its optical rotation value with those of the synthesized analogs. Compound 2 is a rare, naturally occurring substance with an unusual cysteinol moiety. Furthermore, 1 showed moderate anti-inflammatory activity with an IC50 value of 20.0 µM. These results revealed that Aspergillus unguis could produce structurally diverse nitrogenous secondary metabolites, which can be used for further studies to find anti-inflammatory leads.

Genome-Wide Association Reveals Trait Loci for Seed Glucosinolate Accumulation in Indian Mustard (Brassica juncea L.).

Glucosinolates (GSLs) are sulphur- and nitrogen-containing secondary metabolites implicated in the fitness of Brassicaceae and appreciated for their pungency and health-conferring properties. In Indian mustard (Brassica juncea L.), GSL content and composition are seed-quality-determining traits affecting its economic value. Depending on the end use, i.e., condiment or oil, different GSL levels constitute breeding targets. The genetic control of GSL accumulation in Indian mustard, however, is poorly understood, and current knowledge of GSL biosynthesis and regulation is largely based on Arabidopsis thaliana. A genome-wide association study was carried out to dissect the genetic architecture of total GSL content and the content of two major GSLs, sinigrin and gluconapin, in a diverse panel of 158 Indian mustard lines, which broadly grouped into a South Asia cluster and outside-South-Asia cluster. Using 14,125 single-nucleotide polymorphisms (SNPs) as genotyping input, seven distinct significant associations were discovered for total GSL content, eight associations for sinigrin content and 19 for gluconapin. Close homologues of known GSL structural and regulatory genes were identified as candidate genes in proximity to peak SNPs. Our results provide a comprehensive map of the genetic control of GLS biosynthesis in Indian mustard, including priority targets for further investigation and molecular marker development.

A Bitter-Sweet Story: Unraveling the Genes Involved in Quinolizidine Alkaloid Synthesis in Lupinus albus.

Alkaloids are part of a structurally diverse group of over 21,000 cyclic nitrogen-containing secondary metabolites that are found in over 20% of plant species. Lupinus albus are naturally containing quinolizidine alkaloid (QA) legumes, with wild accessions containing up to 11% of QA in seeds. Notwithstanding their clear advantages as a natural protecting system, lupin-breeding programs have selected against QA content without proper understanding of quinolizidine alkaloid biosynthetic pathway. This review summarizes the current status in this field, with focus on the utilization of natural mutations such as the one contained in pauper locus, and more recently the development of molecular markers, which along with the advent of sequencing technology, have facilitated the identification of candidate genes located in the pauper region. New insights for future research are provided, including the utilization of differentially expressed genes located on the pauper locus, as candidates for genome editing. Identification of the main genes involved in the biosynthesis of QA will enable precision breeding of low-alkaloid, high nutrition white lupin. This is important as plant based high quality protein for food and feed is an essential for sustainable agricultural productivity.

Effects and mechanisms of nitrogen application on stress resistance of Chinese materia medica

Nitrogen fertilizers play an important role in the regulation of plant stress resistance. Impacts of nitrogen fertilizers on abiotic stress resistance and biotic stress resistance of Chinese materia medica(CMM) were summarized in this study. Adequate nitrogen application improves the abiotic stress resistance and weed resistance of CMM, however adverse effect appears when excess nitrogen is used. Generally, pest resistance decreases along with nitrogen deposition, while effects of nitrogen application on disease resistance vary with different diseases. Mechanisms underlying the impact of nitrogen fertilizers on plant stress resistance were also elucidated in this study from three aspects including physical defense mechanisms, biochemistry mechanisms and molecular defense mechanisms. Nitrogen availability modulates physical barrier of CMM like plant growth, formation of lignin and wax cuticle, and density of stomata. Growth of CMM promoted by nitrogen fertilizer may cause some decrease in pest resistance of CMM due to an increase in hiding places for pest along with plant growth. High ambient humidity caused by excessive plant growth facilitates the growth and development of CMM pathogen. Nitrogen application can also interfere with the accumulation of lignin in CMM which makes CMM more vulnerable to pest and pathogen attack. Stomatal closing delays due to nitrogen application is also a causal factor of increasing pathogen infection after nitrogen deposition. Biochemical defenses of plants are mainly achieved through nutrient elements, secondary metabolites, defense-related enzymes and proteins. Nutritional level of CMM and various antioxidant enzymes and resistance-related protein activities are elevated along with nitrogen deposition. These antioxidant enzymes can reduce the damage of reactive oxygen species content produced by plant in response to adversity and therefore enhance stress resistance of CMM. Researches showed that nitrogen application could also cause an increase in nitrogen-containing secondary metabolites content and a decrease in non-nitrogen-containing secondary metabolites content respectively. Nitrogen-mediated molecular defense mechanisms includes multiple plant hormones and nitric oxide signals. Plant hormones related to plant defense like salicylic acid, jasmonic acid and abscisic acid can be modulated by nitrogen application. Negative effect of nitrogen deposition was found on salicylic acid accumulation and the expression of related plant disease resistance genes. However, jasmonic acid level can be elevated by nitrogen. Nitric oxide signals constitute an important part of nitrogen mediated defense mechanisms. Nitric oxide signaling is related to many aspects of plant immunity. The roles of nitrogen fertilizers in CMM stress resistance are complex and may vary with different CMM varieties and environments. Further studies are urgently needed to provide a comprehensive understanding of how to improve stress resistance of CMM by using fertilizers.

Current situation of nitrogen application and its effects on yield and quality of Chinese materia medica

Nitrogen fertilizer has been the long-lasting crucial component in cultivation of Chinese materia medica(CMM) and crops for its profound effects on enhancing the productivity. In consideration of its role in better production, intensive and excessive application of N fertilizer is often found in CMM cultivation. Therefore, firstly, this review summarized various concentrations of N application with regards to different CMM and districts from the literatures published in the last two decades. The recommended concentration of nitrogen application of forty seven CMM species were covered in this review. We found that the optimum rates of nitrogen fertili-zer for different medicinal plants species were varied in the range between 0-1 035.55 kg·hm~(-2). Most of the optimum rates of nitrogen fertilizer for CMM in published researches fell between 100-199 kg·hm~(-2). The optimum rate of nitrogen fertilizer is not only related to amount of nitrogen required for different medicinal plants but also to soil fertilities of different fields. In addition, we outlined the diffe-rent effects of proper and excessive nitrogen deposition on yield of CMM. Proper nitrogen deposition benefits the yield of CMM, howe-ver, excessive nitrogen use accounts for a decrease in CMM yield. We elucidated that nutritional content, water use efficiencies, and photosynthesis capacity were major influencing factors. Researches showed that proper nitrogen fertilizer could promote the water use efficiencies of plants and boost photosynthesis. Consequently, the yield of CMM can be enhanced after nitrogen deposition. However, negative effects of nitrogen fertilizer were also found on plant including producing toxic substances to the soil and causing severe pest damages. Lastly we analyzed the impact of N fertilizer application on secondary metabolites which accounts for a large part of active pharmaceutical ingredients of CMM. It usually caused an increase in nitrogen-containing secondary metabolites content and a decrease in non-nitrogen-containing secondary metabolites content respectively. The potential underlying mechanisms are the different synthetic pathways of these metabolites and the plant nutritional status. Synthesis of non-nitrogen-containing secondary metabolites like phenols can be inhibited after nitrogen application because of the competition of the same precursor substances between metabolites synthesis and plant growth. To sum up, impacts and mechanisms of nitrogen fertilizer on yield and quality enhancement of CMM were discussed in this review. Negative effects of excessive nitrogen application on CMM should be paid special attention in CMM cultivation and prescription fertilization based on the field soil quality is strongly recommended. Overall, this review aims to provides insights on improving the proper application of N fertilizer in the cultivation of CMM.

Three nitrogen-containing metabolites from an algicolous isolate of Trichoderma asperellum

As one of the most productive species, Trichoderma asperellum, especially isolated from marine algae, has contributed a number of new terpenes and nitrogen-bearing compounds, encouraging our continuous efforts to further explore them. Consequently, three new nitrogen-containing secondary metabolites, including cyclo(L-5-MeO-Pro-L-5-MeO-Pro) (1), 5′-acetoxy- deoxycyclonerin B (2), and 5′-acetoxy-deoxycyclonerin D (3) were isolated from the marine alga-derived endophytic fungus Trichoderma asperellum A-YMD-9-2. Their structures and relative configurations were fully elucidated through spectroscopic techniques, mainly including 1D/2D NMR and MS. The absolute configurations of 1 and 2 were ascertained on the basis of X-ray diffraction (Cu Kα radiation) and ECD data, respectively. The isolation of these new nitrogen-bearing compounds adds to the structural diversity of marine algicolous Trichoderma spp., and they display potent inhibition against some marine phytoplankton species.

Recent Discovery of Heterocyclic Alkaloids from Marine-Derived Aspergillus Species.

Nitrogen heterocycles have drawn considerable attention due to of their significant biological activities. The marine fungi residing in extreme environments are among the richest sources of these basic nitrogen-containing secondary metabolites. As one of the most well-known universal groups of filamentous fungi, marine-derived Aspergillus species produce a large number of structurally unique heterocyclic alkaloids. This review attempts to provide a comprehensive summary of the structural diversity and biological activities of heterocyclic alkaloids that are produced by marine-derived Aspergillus species. Herein, a total of 130 such structures that were reported from the beginning of 2014 through the end of 2018 are included, and 75 references are cited in this review, which will benefit future drug development and innovation.

Comparative analysis of glucosinolate production in hairy roots of green and red kale (Brassica oleracea var. acephala)

Glucosinolates (GSLs) are sulfur- and nitrogen-containing secondary metabolites that function in plant defense and provide benefits to human health. In this study, using Agrobacterium rhizogenes R1000, green and red kale hairy roots were established. The expression levels of GSLs biosynthesis genes and their accumulation in both kale hairy roots were analyzed by quantitative real-time PCR and HPLC. The results showed that the expression of most indolic GSLs biosynthesis genes was higher in the hairy roots of green kale than in that of red kale. In contrast, the expression of BoCYP83A1 and BoSUR1 encoding key enzymes aromatic GSL biosynthesis was significantly higher in red kale hairy root. The HPLC analysis identified six GSLs. The levels of 4-methoxyglucobrassicin, glucobrassicin, and 4-hydroxyglucobrassicin were 6.21, 5.98, and 2 times higher, respectively, in green kale than in red kale, whereas the levels of neoglucobrassicin and gluconasturtiin were 16.2 and 3.48 times higher, respectively, in red kale than in green kale. Our study provides insights into the underlying mechanisms of GSLs biosynthesis in kale hairy roots and can be potentially used as “biological factories” for producing bioactive substances such as GSLs.

A Complete Survey of Glycoalkaloids Using LC-FTICR-MS and IRMPD in a Commercial Variety and a Local Landrace of Eggplant (Solanum melongena L.) and their Anticholinesterase and Antioxidant Activities.

Eggplant contains glycoalkaloids (GAs), a class of nitrogen-containing secondary metabolites of great structural variety that may have both adverse and beneficial biological effects. In this study, we performed a complete survey of GAs and their malonylated form, in two genotypes of eggplants: A commercial cultivated type, Mirabella (Mir), with purple peel and bitter taste and a local landrace, named Melanzana Bianca di Senise (Sen), characterized by white peel with purple strip and a typical sweet aroma. Besides the analysis of their morphological traits, nineteen glycoalkaloids were tentatively identified in eggplant berry extracts based upon LC-ESI-FTICR-MS analysis using retention times, elution orders, high-resolution mass spectra, as well as high-resolution fragmentation by IRMPD. The relative signal intensities (i.e., ion counts) of the GAs identified in Mir and Sen pulp extracts showed as solamargine, and its isomers are the most abundant. In addition, anticholinesterase and antioxidant activities of the extracts were evaluated. Pulp tissue was found to be more active in inhibiting acetylcholinesterase enzyme than peel showing an inhibitory effect higher than 20% for Mir pulp. The identification of new malonylated GAs in eggplant is proposed.

Identification of biphenyl 2, 3-dioxygenase and its catabolic role for phenazine degradation in Sphingobium yanoikuyae B1

Phenazines are important nitrogen-containing secondary metabolites that display a range of biological functionalities. However, these compounds have shown lethal effects on humans and, the fate of phenazine in the ecosystem remains uncertain. In this study, we investigated that Sphingobium yanoikuyae B1 could utilize phenazine as a sole carbon source for growth. Intermediate produced during phenazine degradation was purified and identified as 1, 2-dihydrogen 1, 2-dihydroxy phenazine. Biphenyl 2, 3-dioxygenase was determined to be the initial dioxygenase for phenazine degradation through gene cloning and whole cell transformation techniques. Phenazine was converted to 1, 2-dihydrogen 1, 2-dihydroxy phenazine through hydrogenation and hydroxylation, which then transformed to 2-hydroxy phenazine through spontaneous dehydration. ThebphA1fA2f, were evidenced to be the only genes encoding the initial dioxygenase for phenazine degradation. BphB (dihydrodiol dehydrogenase) and BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase) did not exhibit any 1, 2-dihydrogen 1, 2-dihydroxy phenazine and 1, 2-dihydroxy phenazine degradation capability, suggesting no contribution in phenazine degradation. Phylogenetic analysis of the dioxygenases demonstrated enormous biodegradation potential in strain B1. In conclusion, this study opens up new possibilities in better understanding the phenazine degradation in the environment.

The Interference of Selected Cytotoxic Alkaloids with the Cytoskeleton: An Insight into Their Modes of Action.

Alkaloids, the largest group among the nitrogen-containing secondary metabolites of plants, usually interact with several molecular targets. In this study, we provide evidence that six cytotoxic alkaloids (sanguinarine, chelerythrine, chelidonine, noscapine, protopine, homoharringtonine), which are known to affect neuroreceptors, protein biosynthesis and nucleic acids, also interact with the cellular cytoskeleton, such as microtubules and actin filaments, as well. Sanguinarine, chelerythrine and chelidonine depolymerized the microtubule network in living cancer cells (Hela cells and human osteosarcoma U2OS cells) and inhibited tubulin polymerization in vitro with IC50 values of 48.41 ± 3.73, 206.39 ± 4.20 and 34.51 ± 9.47 μM, respectively. However, sanguinarine and chelerythrine did not arrest the cell cycle while 2.5 μM chelidonine arrested the cell cycle in the G2/M phase with 88.27% ± 0.99% of the cells in this phase. Noscapine and protopine apparently affected microtubule structures in living cells without affecting tubulin polymerization in vitro, which led to cell cycle arrest in the G2/M phase, promoting this cell population to 73.42% ± 8.31% and 54.35% ± 11.26% at a concentration of 80 μM and 250.9 μM, respectively. Homoharringtonine did not show any effects on microtubules and cell cycle, while the known microtubule-stabilizing agent paclitaxel was found to inhibit tubulin polymerization in the presence of MAPs in vitro with an IC50 value of 38.19 ± 3.33 μM. Concerning actin filaments, sanguinarine, chelerythrine and chelidonine exhibited a certain effect on the cellular actin filament network by reducing the mass of actin filaments. The interactions of these cytotoxic alkaloids with microtubules and actin filaments present new insights into their molecular modes of action.

Alkaloid-Containing Plants Poisonous to Cattle and Horses in Europe

Alkaloids, nitrogen-containing secondary plant metabolites, are of major interest to veterinary toxicology because of their occurrence in plant species commonly involved in animal poisoning. Based on epidemiological data, the poisoning of cattle and horses by alkaloid-containing plants is a relatively common occurrence in Europe. Poisoning may occur when the plants contaminate hay or silage or when forage alternatives are unavailable. Cattle and horses are particularly at risk of poisoning by Colchicum autumnale (meadow saffron), Conium maculatum (poison hemlock), Datura stramonium (jimson weed), Equisetum palustre (marsh horsetail), Senecio spp. (ragwort and groundsel) and Taxus baccata (European yew). This review of poisonous alkaloid-containing plants describes the distribution of these plants, conditions under which poisoning occurs, active toxic principles involved and subsequent clinical signs observed.

Occurrence and De novo Biosynthesis of Caffeine and Theanine in Seedlings of Tea (Camellia sinensis)

Caffeine (1,3,7-trimethyl xanthine) and theanine (γ-glutamyl-L-ethylamide) are the major nitrogen-containing secondary metabolites in tea leaves. The aim of the present study was to elucidate the relative concentration and amounts of these compounds and the de novo biosynthetic activity in different parts of tea seedlings grown for 27-, 106- and 205 days. The results indicated that caffeine and its biosynthetic activity occur only in leaves and stems, while theanine is distributed in all organs, including roots. The concentration of caffeine and theanine in leaves ranged from 0.3-1.1 mg N/g and 0.1-0.5 mg N/g fresh weight, respectively. A higher concentration of theanine was found in roots (0.5-1.1 mg N). The total amounts of theanine expressed as g N/seedling were 1.1-1.5 times higher than that of caffeine. The high biosynthetic activity of caffeine from NH4+ was found in young leaves during the first 106 days after germination. Theanine biosynthetic activity probably occurs in roots, since higher 15N atom% excess was observed in roots during the first 27 days. Theanine may be synthesized mainly in roots and translocated to leaves. The de novo biosynthesis of caffeine and theanine in tea seedlings and their accumulation and translocation are discussed.

ChemInform Abstract: Chemistry and Anticarcinogenic Mechanisms of Glycoalkaloids Produced by Eggplants, Potatoes, and Tomatoes

AbstractReview: 141 refs.

Chemistry and anticarcinogenic mechanisms of glycoalkaloids produced by eggplants, potatoes, and tomatoes.

Inhibition of cancer can occur via apoptosis, a genetically directed process of cell self-destruction that involves numerous biomarkers and signaling pathways. Glycoalkaloids are nitrogen-containing secondary plant metabolites found in numerous Solanaceous plants including eggplants, potatoes, and tomatoes. Exposure of cancer cells to glycoalkaloids produced by eggplants (α-solamargine and α-solasonine), potatoes (α-chaconine and α-solanine), and tomatoes (α-tomatine) or their hydrolysis products (mono-, di-, and trisaccharide derivatives and the aglycones solasodine, solanidine, and tomatidine) inhibits the growth of the cells in culture (in vitro) as well as tumor growth in vivo. This overview comprehensively surveys and consolidates worldwide efforts to define the following aspects of these natural compounds: (a) their prevalence in the three foods; (b) their chemistry and structure-activity relationships; (c) the reported factors (biomarkers, signaling pathways) associated with apoptosis of bone, breast, cervical, colon, gastric, glioblastoma, leukemia, liver, lung, lymphoma, melanoma, pancreas, prostate, and squamous cell carcinoma cell lines in vitro and the in vivo inhibition of tumor formation and growth in fish and mice and in human skin cancers; and (d) future research needs. The described results may make it possible to better relate the structures of the active compounds to their health-promoting function, individually, in combination, and in food, and allow the consumer to select glycoalkaloid-containing food with the optimal content of nontoxic beneficial compounds. The described findings are expected to be a valuable record and resource for further investigation of the health benefits of food-related natural compounds.

Metabolomics‐assisted refinement of the pathways of steroidal glycoalkaloid biosynthesis in the tomato clade

Steroidal glycoalkaloids (SGAs) are nitrogen-containing secondary metabolites of the Solanum species, which are known to have large chemical and bioactive diversity in nature. While recent effort and development on LC/MS techniques for SGA profiling have elucidated the main pathways of SGA metabolism in tomato, the problem of peak annotation still remains due to the vast diversity of chemical structure and similar on overlapping of chemical formula. Here we provide a case study of peak classification and annotation approach by integration of species and tissue specificities of SGA accumulation for provision of comprehensive pathways of SGA biosynthesis. In order to elucidate natural diversity of SGA biosynthesis, a total of 169 putative SGAs found in eight tomato accessions (Solanum lycopersicum, S. pimpinellifolium, S. cheesmaniae, S. chmielewskii, S. neorickii, S. peruvianum, S. habrochaites, S. pennellii) and four tissue types were used for correlation analysis. The results obtained in this study contribute annotation and classification of SGAs as well as detecting putative novel biosynthetic branch points. As such this represents a novel strategy for peak annotation for plant secondary metabolites.

Molecular genetics of alkaloid biosynthesis in Nicotiana tabacum

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.

Detection of substrate binding motifs for morphine biosynthetic pathway intermediates in novel wound inducible (R,S)-reticuline 7-O-methyltransferase of Papaver somniferum

The benzylisoquinoline alkaloids (BIA) comprise a large and diverse group of nitrogen-containing secondary metabolites with about 2500 compounds identified in plants. BIA biosynthesis begins with the condensation of the tyrosine derived precursors dopamine and p-hydroxyphenylacetaldehyde to (S)-norcoclaurine. Subsequent regiospecific O- and N-methylations and aromatic ring hydroxylation lead to (S)-reticuline, which is the central intermediate for almost all BIAs. For morphinan alkaloid biosynthesis, (S)-reticuline undergoes an inversion of stereochemistry to (R)-reticuline, followed by C-C phenol coupling catalyzed by a unique cytochrome P450-dependent monooxygenase to yield salutaridine. The cDNA sequence of enzymes leading to (S)-reticuline, as well as those involved in the conversion of (R)-reticuline to salutaridine-7-O-acetate are already characterized. The inversion of (S)-reticuline to (R)-reticuline represent the important steps in morphine biosynthesis. Wound induced transcript accumulation in Papaver reveals a novel wound inducible EST (NCBI DbEST: GO238757) showing homology with (R,S)-reticuline 7-O-methyltransferase (ID: Q6WUC2) isolated from Papaver somniferum. We compare the substrate binding homology of this novel wound inducible (R,S)-reticuline 7-O-methyltransferase (7-OMT) using template of P. somniferum (Q6WUC2; gb∣AAQ01668) as experimental control. Homology modeling with 70% identity & 85% similarity with catalytic site of template protein i.e., (Q6WUC2) short chain dehydrogenase/reductase (SDR), showed docking energy -69.9 and -75.8 kcal/mol with (S)-Reticuline (CID:439653) and (R)-Reticuline (CID:440586) respectively, which are comparable with experimental control binding site interaction energies. Docking of S- & R-reticuline into the active site revealed eight (F(5), E(18), W(24), C(47), F(44), P(45), C(46) and I(47) amino acids presumably responsible for the high substrate specificity of (R,S)-reticuline 7-O-methyltransferase.