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Sample records for nicotiana tabacum altered

  1. Metabolite Fingerprinting in Transgenic Nicotiana tabacum Altered by the Escherichia coli Glutamate Dehydrogenase Gene

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    R. Mungur

    2005-01-01

    Full Text Available With about 200 000 phytochemicals in existence, identifying those of biomedical significance is a mammoth task. In the postgenomic era, relating metabolite fingerprints, abundances, and profiles to genotype is also a large task. Ion analysis using Fourier transformed ion cyclotron resonance mass spectrometry (FT-ICR-MS may provide a high-throughput approach to measure genotype dependency of the inferred metabolome if reproducible techniques can be established. Ion profile inferred metabolite fingerprints are coproducts. We used FT-ICR-MS-derived ion analysis to examine gdhA (glutamate dehydrogenase (GDH; EC 1.4.1.1 transgenic Nicotiana tabacum (tobacco carrying out altered glutamate, amino acid, and carbon metabolisms, that fundamentally alter plant productivity. Cause and effect between gdhA expression, glutamate metabolism, and plant phenotypes was analyzed by 13NH4+ labeling of amino acid fractions, and by FT-ICR-MS analysis of metabolites. The gdhA transgenic plants increased 13N labeling of glutamate and glutamine significantly. FT-ICR-MS detected 2 012 ions reproducible in 2 to 4 ionization protocols. There were 283 ions in roots and 98 ions in leaves that appeared to significantly change abundance due to the measured GDH activity. About 58% percent of ions could not be used to infer a corresponding metabolite. From the 42% of ions that inferred known metabolites we found that certain amino acids, organic acids, and sugars increased and some fatty acids decreased. The transgene caused increased ammonium assimilation and detectable ion variation. Thirty-two compounds with biomedical significance were altered in abundance by GDH including 9 known carcinogens and 14 potential drugs. Therefore, the GDH transgene may lead to new uses for crops like tobacco.

  2. Profiling of Altered Metabolomic States in Nicotiana tabacum Cells Induced by Priming Agents

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    Mhlongo, Msizi I.; Steenkamp, Paul A.; Piater, Lizelle A.; Madala, Ntakadzeni E.; Dubery, Ian A.

    2016-01-01

    Metabolomics has developed into a valuable tool for advancing our understanding of plant metabolism. Plant innate immune defenses can be activated and enhanced so that, subsequent to being pre-sensitized, plants are able to launch a stronger and faster defense response upon exposure to pathogenic microorganisms, a phenomenon known as priming. Here, three contrasting chemical activators, namely acibenzolar-S-methyl, azelaic acid and riboflavin, were used to induce a primed state in Nicotiana tabacum cells. Identified biomarkers were then compared to responses induced by three phytohormones—abscisic acid, methyljasmonate, and salicylic acid. Altered metabolomes were studied using a metabolite fingerprinting approach based on liquid chromatography and mass spectrometry. Multivariate data models indicated that these inducers cause time-dependent metabolic perturbations in the cultured cells and revealed biomarkers of which the levels are affected by these agents. A total of 34 metabolites were annotated from the mass spectral data and online databases. Venn diagrams were used to identify common biomarkers as well as those unique to a specific agent. Results implicate 20 cinnamic acid derivatives conjugated to (i) quinic acid (chlorogenic acids), (ii) tyramine, (iii) polyamines, or (iv) glucose as discriminatory biomarkers of priming in tobacco cells. Functional roles for most of these metabolites in plant defense responses could thus be proposed. Metabolites induced by the activators belong to the early phenylpropanoid pathway, which indicates that different stimuli can activate similar pathways but with different metabolite fingerprints. Possible linkages to phytohormone-dependent pathways at a metabolomic level were indicated in the case of cells treated with salicylic acid and methyljasmonate. The results contribute to a better understanding of the priming phenomenon and advance our knowledge of cinnamic acid derivatives as versatile defense metabolites. PMID

  3. Distribution of solanesol in Nicotiana tabacum

    Institute of Scientific and Technical Information of China (English)

    ZHAO Chun-jian; ZU Yuan-gang; LI Chun-ying; TIAN Cheng-yu

    2007-01-01

    Solanesol is an important secondary metabolite in Nicotiana tabacum. Distribution of solanesol in Nicotiana tabacum was investigated by High Performance Liquid Chromatography (HPLC) method. The quantitative distribution of solanesol in various organs and tissues of N. Tabacum showed that solanesol content, obviously different in all organs, was 6.8, 18.3, 27.5, 45.8, and 68.0 times higher in leaves than that in the stalks, flowers, seeds, fruits and roots, respectively. The contents of solanesol in various parts of leaf, stalk and flower were determined. The content of solanesol in top leaf, middle leaf and bottom leaf gradually decreased (6.124, 5.813 and 5.687 mg·g-1, respectively) and the content of solanesol in various leaf-parts (leaf apex, leaf middle and leaf base) also gradually decreased. The content of solanesol in top stalk was 1.19 times and 1.92 times higher than that in the middle stalk and the bottom stalk, respectively. The content of solanesol in various tissues of stalk (epidermis, cortex and stele) dramatically decreased. The sepal contained higher concentration of solanesol (1.192 mg·g-1) compared to any other parts in flower. The study will provide the base data for the regulation and control of solanesol,moreover, it will provide the scientific evidences for the rational development and utilization of N. Tabacum resources.

  4. Silicon delays Tobacco ringspot virus systemic symptoms in Nicotiana tabacum.

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    Zellner, Wendy; Frantz, Jonathan; Leisner, Scott

    2011-10-15

    Soluble silicon (Si) provides protection to plants against a variety of abiotic and biotic stress. However, the effects of Si on viral infections are largely unknown. To investigate the role of Si in viral infections, hydroponic studies were conducted in Nicotiana tabacum with two pathogens: Tobacco ringspot virus (TRSV) and Tobacco mosaic virus (TMV). Plants grown in elevated Si showed a delay in TRSV systemic symptom formation and a reduction in symptomatic leaf area, compared to the non-supplemented controls. TRSV-infected plants showed significantly higher levels of foliar Si compared to mock-inoculated plants. However, the Si effect appeared to be virus-specific, since the element did not alter TMV symptoms nor did infection by this virus alter foliar Si levels. Hence, increased foliar Si levels appear to correlate with Si-modulated protection against viral infection. This is all the more intriguing since N. tabacum is classified as a low Si accumulator.

  5. Ectopic expression of class 1 KNOX genes induce and adventitious shoot regeneration and alter growth and development of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L)

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    Transgenic plants of tobacco (Nicotiana tabacum L) and plum (Prunus domestica L) were produced by transforming with apple class 1 KNOX genes (MdKN1 and MdKN2) or corn KN1 gene. Transgenic tobacco plants were regenerated in vitro from transformed leaf discs cultured in a tissue medium lacking cytoki...

  6. Citroenzuur- en nicotinegehalte in enkele variëteiten van Nicotiana tabacum en Nicotiana rustica

    NARCIS (Netherlands)

    Fatton, Numa Edouard

    1939-01-01

    Van technisch rijpe bladen van verschillende varieteiten van Nicotiana rustica en Nicotiana Tabacum werden het citroenzuuren het nicotinegehalte bepaald, uitgedrukt in % op de droge stof. Hetzelfde werd gedaan van bladmoes en hoofdnerf afzonderlijk, verder van de wortel, de stengel, de bloemen en de

  7. Silicon delays tobacco Ringspot virus systemic symptoms in Nicotiana tabacum

    Science.gov (United States)

    Soluble silicon (Si) provides protection to plants against a variety of abiotic and biotic stress. However, the role of Si in viral infections has been elusive. To investigate the role of Si in viral infections, hydroponic studies were conducted in Nicotiana tabacum with two pathogens: Tobacco rings...

  8. Antinuclear human autoantibodies as markers in Nicotiana tabacum pollen tubes

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    C. Poggialini

    2014-02-01

    Full Text Available In the present paper we report on the use of antinuclear human autoantibodies as specific markers in Nicotiana tabacum pollen tubes. The antibodies have been tested by fluorescence techniques using a confocal laser scanning microscope. All the antibodies showed specifc labelling pattern and the results, although preliminary in nature, could open new perspectives of research.

  9. Manipulation of the napin primary structure alters its packaging and deposition in transgenic tobacco (Nicotiana tabacum L.) seeds.

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    Scarafoni, A; Carzaniga, R; Harris, N; Croy, R R

    2001-08-01

    Napin is a 2S storage protein found in the seeds of oilseed rape (Brassica napus L.) and related species. Using protein structural prediction programs we have identified a region in the napin protein sequence which forms a 'hydrophilic loop' composed of amino acid residues located at the protein surface. Targeting this region, we have constructed two napin chimeric genes containing the coding sequence for the peptide hormone leucine-enkephalin as a topological marker. One version has a single enkephalin sequence of 11 amino acids including linkers and the second contains a tandem repeat of this peptide comprising 22 amino acids, inserted into the napin large subunit. The inserted peptide sequences alter the balance of hydrophilic to hydrophobic amino acids and introduce flexibility into this region of the polypeptide chain. The chimeric genes have been expressed in tobacco plants under the control of the seed-specific napA gene promoter. Analyses indicate that the engineered napin proteins are expressed, transported, post-translationally modified and deposited inside the protein bodies of the transgenic seeds demonstrating that the altered napin proteins behave in a similar fashion to the authentic napin protein. Detailed immunolocalisation studies indicate that the insertion of the peptide sequences has a significant effect on the distribution of the napin proteins within the tobacco seed protein bodies.

  10. Ectopic expression of class 1 KNOX genes induce adventitious shoot regeneration and alter growth and development of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L).

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    Srinivasan, C; Liu, Zongrang; Scorza, Ralph

    2011-04-01

    Transgenic plants of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L) were produced by transforming with the apple class 1 KNOX genes (MdKN1 and MdKN2) or corn KNOX1 gene. Transgenic tobacco plants were regenerated in vitro from transformed leaf discs cultured in a medium lacking cytokinin. Ectopic expression of KNOX genes retarded shoot growth by suppressing elongation of internodes in transgenic tobacco plants. Expression of each of the three KNOX1 genes induced malformation and extensive lobbing in tobacco leaves. In situ regeneration of adventitious shoots was observed from leaves and roots of transgenic tobacco plants expressing each of the three KNOX genes. In vitro culture of leaf explants and internode sections excised from in vitro grown MdKN1 expressing tobacco shoots regenerated adventitious shoots on MS (Murashige and Skoog 1962) basal medium in the absence of exogenous cytokinin. Transgenic plum plants that expressed the MdKN2 or corn KNOX1 gene grew normally but MdKN1 caused a significant reduction in plant height, leaf shape and size and produced malformed curly leaves. A high frequency of adventitious shoot regeneration (96%) was observed in cultures of leaf explants excised from corn KNOX1-expressing transgenic plum shoots. In contrast to KNOX1-expressing tobacco, leaf and internode explants of corn KNOX1-expressing plum required synthetic cytokinin (thidiazuron) in the culture medium to induce adventitious shoot regeneration. The induction of high-frequency regeneration of adventitious shoots in vitro from leaves and stem internodal sections of plum through the ectopic expression of a KNOX1 gene is the first such report for a woody perennial fruit trees.

  11. Ectopic expression of Malus domestica class 1 knox genes altered growth and development of Nicotiana tabacum and Prunus domestica, and induced adventitious shoot regeneration from leaf explants without exogenous cytokinin

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    Transgenic tobacco (Nicotiana tabacum L) and plum (Prunus domestica L) plants were regenerated by transforming with apple class 1 KNOX genes (MdKNP1 and MdKNP2) or a corn KN1 (ZmKN1) gene. Transgenic tobacco plants were produced in vitro from transformed leaf discs in the absence of cytokinin in th...

  12. ANTIBACTERIAL ACTIVITY, PHYTOCHEMICAL SCREENING AND ANTIOXIDANT ACTIVITY OF STEM OF NICOTIANA TABACUM

    National Research Council Canada - National Science Library

    Y Sharma; D Dua; A Nagar; N S Srivastava

    2016-01-01

    .... The purpose of this present study is to investigate the antibacterial activity, phytochemical screening and the antioxidant activity of aqueous and alcoholic extracts of the stem of Nicotiana tabacum...

  13. Pollination triggers female gametophyte development in immature Nicotiana tabacum flowers

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    Brito, Michael S.; Bertolino, Lígia T.; Cossalter, Viviane; Quiapim, Andréa C.; DePaoli, Henrique C.; Goldman, Gustavo H.; Teixeira, Simone P.; Goldman, Maria H. S.

    2015-01-01

    In Nicotiana tabacum, female gametophytes are not fully developed at anthesis, but flower buds pollinated 12 h before anthesis produce mature embryo sacs. We investigated several pollination-associated parameters in N. tabacum flower buds to determine the developmental timing of important events in preparation for successful fertilization. First, we performed hand pollinations in flowers from stages 4 to 11 to study at which developmental stage pollination would produce fruits. A Peroxtesmo test was performed to correlate peroxidase activity on the stigma surface, indicative of stigma receptivity, with fruit set. Pollen tube growth and female gametophyte development were microscopically analyzed in pistils of different developmental stages. Fruits were obtained only after pollinations of flower buds at late stage 7 and older; fruit weight and seed germination capacity increased as the developmental stage of the pollinated flower approached anthesis. Despite positive peroxidase activity and pollen tube growth, pistils at stages 5 and 6 were unable to produce fruits. At late stage 7, female gametophytes were undergoing first mitotic division. After 24 h, female gametophytes of unpollinated pistils were still in the end of the first division, whereas those of pollinated pistils showed egg cells. RT-qPCR assay showed that the expression of the NtEC1 gene, a marker of egg cell development, is considerably higher in pollinated late stage 7 ovaries compared with unpollinated ovaries. To test whether ethylene is the signal eliciting female gametophyte maturation, the expression of ACC synthase was examined in unpollinated and pollinated stage 6 and late stage 7 stigmas/styles. Pollination induced NtACS expression in stage 6 pistils, which are unable to produce fruits. Our results show that pollination is a stimulus capable of triggering female gametophyte development in immature tobacco flowers and suggests the existence of a yet undefined signal sensed by the pistil. PMID

  14. Molecular genetics of alkaloid biosynthesis in Nicotiana tabacum.

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    Dewey, Ralph E; Xie, Jiahua

    2013-10-01

    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.

  15. CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum.

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    Gao, Junping; Wang, Genhong; Ma, Sanyuan; Xie, Xiaodong; Wu, Xiangwei; Zhang, Xingtan; Wu, Yuqian; Zhao, Ping; Xia, Qingyou

    2015-01-01

    Genome editing is one of the most powerful tools for revealing gene function and improving crop plants. Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been used as a powerful and efficient tool for genome editing in various organisms. Here, we report genome editing in tobacco (Nicotiana tabacum) mediated by the CRISPR/Cas9 system. Two genes, NtPDS and NtPDR6, were used for targeted mutagenesis. First, we examined the transient genome editing activity of this system in tobacco protoplasts, insertion and deletion (indel) mutations were observed with frequencies of 16.2-20.3% after transfecting guide RNA (gRNA) and the nuclease Cas9 in tobacco protoplasts. The two genes were also mutated using multiplexing gRNA at a time. Additionally, targeted deletions and inversions of a 1.8-kb fragment between two target sites in the NtPDS locus were demonstrated, while indel mutations were also detected at both the sites. Second, we obtained transgenic tobacco plants with NtPDS and NtPDR6 mutations induced by Cas9/gRNA. The mutation percentage was 81.8% for NtPDS gRNA4 and 87.5% for NtPDR6 gRNA2. Obvious phenotypes were observed, etiolated leaves for the psd mutant and more branches for the pdr6 mutant, indicating that highly efficient biallelic mutations occurred in both transgenic lines. No significant off-target mutations were obtained. Our results show that the CRISPR/Cas9 system is a useful tool for targeted mutagenesis of the tobacco genome.

  16. Nicotiana tabacum as model for ozone - plant surface reactions

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    Jud, Werner; Fischer, Lukas; Wohlfahrt, Georg; Tissier, Alain; Canaval, Eva; Hansel, Armin

    2015-04-01

    Elevated tropospheric ozone concentrations are considered a toxic threat to plants, responsible for global crop losses with associated economic costs of several billion dollars per year. The ensuing injuries have been related to the uptake of ozone through the stomatal pores and oxidative effects damaging the internal leaf tissue. A striking question of current research is the environment and plant specific partitioning of ozone loss between gas phase, stomatal or plant surface sink terms. Here we show results from ozone fumigation experiments using various Nicotiana Tabacum varieties, whose surfaces are covered with different amounts of unsaturated diterpenoids exuded by their glandular trichomes. Exposure to elevated ozone levels (50 to 150 ppbv) for 5 to 15 hours in an exceptionally clean cuvette system did neither result in a reduction of photosynthesis nor caused any visible leaf damage. Both these ozone induced stress effects have been observed previously in ozone fumigation experiments with the ozone sensitive tobacco line Bel-W3. In our case ozone fumigation was accompanied by a continuous release of oxygenated volatile organic compounds, which could be clearly associated to their condensed phase precursors for the first time. Gas phase reactions of ozone were avoided by choosing a high enough gas exchange rate of the plant cuvette system. In the case of the Ambalema variety, that is known to exude only the diterpenoid cis-abienol, ozone fumigation experiments yield the volatiles formaldehyde and methyl vinyl ketone (MVK). The latter could be unequivocally separated from isomeric methacrolein (MACR) by the aid of a Selective Reagent Ion Time-of-Flight Mass Spectrometer (SRI-ToF-MS), which was switched every six minutes from H3O+ to NO+ primary ion mode and vice versa. Consistent with the picture of an ozone protection mechanism caused by reactive diterpenoids at the leaf surface are the results from dark-light experiments. The ozone loss obtained from the

  17. Transcriptome profiling of male gametophyte development in Nicotiana tabacum

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    Pavel Bokvaj

    2015-03-01

    Full Text Available Pollen, an extremely reduced bicellular or tricellular male reproductive structure of flowering plants, serves as a model for numerous studies covering wide range of developmental and physiological processes. The pollen development represents a fragile and vital phase of plant ontogenesis and pollen was among the first singular plant tissues thoroughly characterized at the transcriptomic level (Honys and Twell [5]. Arabidopsis pollen developmental transcriptome has been published over a decade ago (Honys and Twell, 2004 and transcriptomes of developing pollen of other species have followed (Rice, Deveshwar et al. [2]; Triticeae, Tran et al. [11]; upland cotton, Ma et al. [8]. However, the transcriptomic data describing the development of tobacco pollen, a bicellular model for cell biology studies, have been missing. Here we provide the transcriptomic data covering three stages (Tupý et al., 1983 of wild type tobacco (Nicotiana tabacum, cv. Samsun pollen development: uninucleate microspores (UNM, stage 1, early bicellular pollen (eBCP, stage 3 and late bicellular pollen (lBCP, stage 5 as a supplement to the mature pollen (MP, 4 h-pollen tube (PT4, 24 h-pollen tubes (PT24, leaf (LF and root (RT transcriptomic data presented in our previous studies (Hafidh et al., 2012a; Hafidh et al., 2012b. We characterized these transcriptomes to refine the knowledge base of male gametophyte-enriched genes as well as genes expressed preferentially at the individual stages of pollen development. Alongside updating the list of tissue-specific genes, we have investigated differentially expressed genes with respect to early expressed genes. Pollen tube growth and competition of pollen tubes in female pistil can be viewed as a race of the fittest. Accordingly, there is an apparent evolutionary trend among higher plants to store significant material reserves and nutrients during pollen maturation. This supply ensures that after pollen germination, the pollen tube

  18. Agroinfiltration reduces ABA levels and suppresses Pseudomonas syringae-elicited salicylic acid production in Nicotiana tabacum.

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    Arantza Rico

    Full Text Available BACKGROUND: Agrobacterium tumefaciens strain GV3101 (pMP90 is widely used in transient gene expression assays, including assays to study pathogen effectors and plant disease resistance mechanisms. However, inoculation of A. tumefaciens GV3101 into Nicotiana tabacum (tobacco leaves prior to infiltration with pathogenic and non-host strains of Pseudomonas syringae results in suppression of macroscopic symptoms when compared with leaves pre-treated with a buffer control. METHODOLOGY/FINDINGS: To gain further insight into the mechanistic basis of symptom suppression by A. tumefaciens we examined the effect of pre-treatment with A. tumefaciens on the growth of P. syringae, the production of the plant signalling molecules salicylic acid (SA and abscisic acid (ABA, and the presence of callose deposits. Pre-treatment with A. tumefaciens reduced ABA levels, P. syringae multiplication and P. syringae-elicited SA and ABA production, but promoted increased callose deposition. However, pre-treatment with A. tumefaciens did not suppress necrosis or SA production in leaves inoculated with the elicitor HrpZ. CONCLUSIONS/SIGNIFICANCE: Collectively, these results show that inoculation of N. tabacum leaves with A. tumefaciens alters plant hormone levels and plant defence responses to P. syringae, and demonstrate that researchers should consider the impact of A. tumefaciens on plant signal transduction when using A. tumefaciens-mediated transient expression assays to investigate ABA-regulated processes or pathogenicity and plant defence mechanisms.

  19. GENE-REGULATION IN INTERTYPIC HETEROKARYONS OF SOLANUM-TUBEROSUM AND NICOTIANA-TABACUM TISSUE PROTOPLASTS

    NARCIS (Netherlands)

    VANKESTEREN, WJP; BIJMOLT, EW; TEMPELAAR, MJ

    1994-01-01

    Activities of the beta-glucuronidase (GUS) reporter enzyme were evaluated in transgenic plants, protoplasts, and intertypic heterokaryons of Solanum tuberosum and Nicotiana tabacum. With GUS under control of the promoter of the cauliflower-mosaicvirus 35S RNA gene (CaMV), activities of the enzyme we

  20. Variations in alkaloidal constituents of plant tissue cultures. [Nicotiana tabacum, Nicotiana rustica, Datura stramonium and Hyoscyamus niger

    Energy Technology Data Exchange (ETDEWEB)

    Essa, A.K.

    1987-01-01

    The accumulation of tobacco and Solanaceae-tropane alkaloids in calluses and suspensions of Nicotiana tabacum, Nicotiana rustica, Datura stramonium and Hyoscyamus niger was the main concern of this work. Nicotine, anabasine and anatabine had regularly been found in tobacco callus tissues grown for several passages. For the first time, N. tabacum suspensions were shown able to accumulate anabasine, anatabine, anatalline, myosmine and nicotelline whereas N. rustica calluses, N-methyl-, N,N-dimethyl- and N-methyl-N-nitroso anilines. The aim of these experiments was an attempt to affect the yields and types of alkaloids produced. The interrelationship between nicotine and protein contents in N. tabacum and N. rustica calluses was investigated. The possible role of urea and sodium propionate as precursors of nicotine in tobacco suspensions was checked by feeding the latter with carbon-14 radioactive substrates. The scope and quantum of the principle alkaloidal components present in the source N. tabacum plants and D. stramonium and H. niger seeds were investigated to stand as references against in vitro production. Identification of the products found was made by using gas chromatography-mass spectrometry (GC-MS) and/or MS.

  1. A red algal cyclophilin has an effect on development and growth in Nicotiana tabacum.

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    Cho, Eun Kyung; Kim, Mihyang

    2008-10-01

    In this study, an algal Cyp was introduced into plant to research the effect of the gene on growth and development. cDNA GjCyp-1 was isolated from the red alga (Griffithsia japonica), and a recombinant GjCyp-1 containing a CaMV35S promoter at the amino-terminus was constructed in Nicotiana tabacum. The altered GjCyp-1 levels in plants and the expression pattern of Cyp after hormone treatment were confirmed by RNA blotting. Transcript of GjCyp-1 was induced by plant hormones such as gibberellic acid (GA(3)), indoleacetic acid (IAA), and zeatin (ZA). Constitutive overexpression of GjCyp-1 appeared to be beneficial to seed germination. The ratio of emergence of cotyledon from seeds overexpressing GjCyp-1 was almost three times higher than that of the transgenic seeds carrying only the vector. In addition, it was found that most of the seedlings overexpressing GjCyp-1 were dwarfs with altered root systems. The ratio of leaf length and width and root length from transgenic seedlings overexpressing GjCyp-1 was almost 2 and 3.5 times lower than that of the transgenic seedlings carrying only the vector, respectively. The data in this study suggest that GjCyp-1 may affect development and growth in organisms.

  2. The haploid embryoids development in anther culture of transformed tobacco (Nicotiana tabacum L.)

    OpenAIRE

    Jaroslava Dubová

    2014-01-01

    Induction of pollen embryogenesis was used for the test of cytokinin influence on the development of pollen grain. Gene for maize cytokinin-specific β-glucosidase were introduced into tobacco genome together with the methotrexate resistance marker (Brzobohatý et al. 1994). Anther culture of T1 generation of transformed Nicotiana tabacum L. plants revealed that high activity of β-glucosidase caused a reduced regeneration of pollen embryoids. The stages of pollen embryoid development were compa...

  3. Pollination following grafting introduces efficiently Ocimum basilicum L. genes into Nicotiana tabacum L.

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    K. Q. Wei

    2013-09-01

    Full Text Available Tobacco is an important cash crop in the world. However, the genetic basis is comparatively narrow among the modern Nicotiana tabacum cultivars, limiting its potential for quality improvement. To introduce genes conferring desirable chemical constituents from medicinal plants, a distant hybridization test was conducted between N. tabacum and Ocimum basilicum L. Seedlings of wild type Nicotiana sylvestris and N. tabacum cultivar 78-04 respectively acted as rootstock and scion. During the flowering season, hand pollination between 78-04 as pistillate parent and O. basilicum as pollen parent was carried out under 22-25°C temperature and 70-80% of relative humidity in the greenhouse. Seed sets of 55% were obtained in 78-04, and about 400 seeds per capsule were produced. But both non-grafted and self-grafted 78-04 plants rarely resulted in fruits by hand pollination and those obtained were without seed. Similar results were obtained in different material combination. The interfamilial F1 hybrids acquired showed distinct variation with various morphological characteristics, and their hybrid nature was confirmed by isozyme and random amplified polymorphic DNA (RAPD analyses. This result indicated that pollination following grafting can facilitate gene exchange and recombination at the interfamilial level and efficiently overcome barriers of sexual incompatibility between N. tabacum and O. basilicum. Our research not only extends the genetic basis of tobacco but also will provide valuable germplasm for improvement of varieties.

  4. Sincronización de Células de Tabaco (Nicotiana tabacum NT-1 Synchronization of tobacco cells (Nicotiana tabacum NT-1

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    León F Ruiz

    2010-01-01

    Full Text Available Se ha evaluado la capacidad sincronizante de afidicolina e hidroxiurea en cultivos de células de tabaco (Nicotiana tabacum NT-1. Los cultivos sincronizados son poderosas herramientas en estudios moleculares y bioquímicos relacionados al ciclo celular y comúnmente se utilizan químicos para bloquear el ciclo celular. La línea celular de tabaco (Nicotiana tabacum NT-1 proviene de la línea celular TBY-2, caracterizándose NT-1 por su menor velocidad de crecimiento y tamaño celular heterogéneo. Los resultados mostraron una sincronía del 30 % de NT-1 con afidicolina 5 mg/ml, similar a la obtenida con TBY-2. Se probaron diferentes concentraciones de hidroxiurea, obteniéndose niveles óptimos de sincronización de hasta 31 % con 0.75 mM. Se muestra también que es posible sincronizar cultivos NT-1 en porcentajes similares a cultivos TBY-2 con afidicolina e hidroxiurea. Para este propósito, se recomienda la hidroxiurea por ser químicamente más estable y económica.The synchronizing capacity of aphidicolin and hydroxyurea on NT-1 cultures of tobacco cells (Nicotiana tabacum NT-1. Synchronized cell cultures are a powerful tool in biochemistry and molecular studies related to cell cycle and commonly chemicals are used to block the biochemical cycle. The tobacco cell line NT-1 comes from cell line TBY-2, the former being characterized with a slower growth rate and heterogeneous cell size. Results show 30 % synchrony with 5 mg/ml aphidicolin, similar to that obtained with TBY-2. Different concentrations of hydroxyurea were tested, obtaining an optimal synchrony of 31 % at 0.75 mM of concentration. It is also shown that it is possible to synchronize NT-1 cultures in similar percentages to those obtained with TBY-2, with either aphidicolin or hydroxyurea. For this purpose, hydroxyurea is recommended because of its lower cost and chemical stability.

  5. The haploid embryoids development in anther culture of transformed tobacco (Nicotiana tabacum L.

    Directory of Open Access Journals (Sweden)

    Jaroslava Dubová

    2014-01-01

    Full Text Available Induction of pollen embryogenesis was used for the test of cytokinin influence on the development of pollen grain. Gene for maize cytokinin-specific β-glucosidase were introduced into tobacco genome together with the methotrexate resistance marker (Brzobohatý et al. 1994. Anther culture of T1 generation of transformed Nicotiana tabacum L. plants revealed that high activity of β-glucosidase caused a reduced regeneration of pollen embryoids. The stages of pollen embryoid development were comparable with those of zygotic embryos. Optimal nutritive medium contained charcoal and chelated iron. The absence of iron in the initiation medium caused a distinct decrease of pollen embryoid regeneration.

  6. Satellite RNA-mediated Reduction of Cucumber Mosaic Virus Genomic RNAs Accumulation in Nicotiana tabacum

    Institute of Scientific and Technical Information of China (English)

    Qiansheng LIAO; Liping ZHU; Zhiyou DU; Rong ZENG; Junli FENG; Jishuang CHEN

    2007-01-01

    Satellite RNAs (satRNAs) are molecular parasites that interfere with the pathogenesis of the helper viruses.In this study,the relative accumulation of cucumber mosaic virus (CMV)-Fny genomic RNAs with or without satRNAs were quantitatively analyzed by real-time RT-PCR.The results showed that satRs apparently attenuated the symptoms of CMV-Fny on Nicotiana tabacum by depressing the accumulation of CMV-Fny genomic RNAs,tested as open reading frames.The accumulation of CMV-Fny la,2a,2b,3a,and CP genes was much higher than that of CMV-Fny with satRs added(CMV-Fsat),at different inoculation times.CMV-Fny△2b,in which the complete 2b gene and 41 amino acids at the C-terminal of the 2a gene were deleted,caused only a slight mosaic effect on N.tabacum seedlings,similar to that of CMVFsat,but the addition of satRs to CMV-Fny△2b showed further decrease in the accumulation of CMVFny△2b genomic RNAs.Our results indicated that the attenuation of CMV,by adding satRs or deleting the 2b gene,was due to the low accumulation of CMV genomic RNAs,and that satRNA-mediated reduction of CMV genomic RNAs accumulation in N.tabacum was possibly related to the 2b gene.

  7. Molecular characterization and expression pattern of tobacco (Nicotiana tabacum ascorbate oxidase gene

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    Zhihua Li

    2016-11-01

    Full Text Available The complete coding sequence of one tobacco (Nicotiana tabacum gene, ascorbate oxidase (AO, was isolated by the application of reverse transcription-polymerase chain reaction. The tobacco AO gene consists of a 1722-bp open reading frame and encodes a protein of 573 amino acids. Sequence comparison analysis revealed that the tobacco AO protein shares high homology with the AO proteins of Lycopersicon esculentum (89%, Populus trichocarpa (75%, soybean (74%, castor bean (73% and peach (73%. The prediction of transmembrane helices showed that tobacco AO might be a transmembrane protein. The expression profile was studied and the results indicated that the tobacco AO gene was diversely expressed in different tobacco tissues, including leaves, stem, roots and flowers. Our experiment laid the grounds for further research on this tobacco gene.

  8. [Induction of polyploid in hairy roots of Nicotiana tabacum and its plant regeneration].

    Science.gov (United States)

    Hou, Lili; Shi, Heping; Yu, Wu; Tsang, Po Keung Eric; Chow, Cheuk Fai Stephen

    2014-04-01

    By genetic transformation with Agrobacterum rhizogenes and artificial chromosome doubling techniques, we studied the induction of hairy roots and their polyploidization, and subsequent plant regeneration and nicotine determination to enhance the content of nicotine in Nicotiana tabacum. The results show that hairy roots could be induced from the basal surface of leaf explants of N. tabacum 8 days after inoculation with Agrobacterium rhizogenes ATCC15834. The percentage of the rooting leaf explants was 100% 15 days after inoculation. The hairy roots could grow rapidly and autonomously on solid or liquid phytohormones-free MS medium. The transformation was confirmed by PCR amplification of rol gene of Ri plasmid and paper electrophoresis of opines from N. tabacum hairy roots. The highest rate of polyploidy induction, more than 64.71%, was obtained after treatment of hairy roots with 0.1% colchicine for 36 h. The optimum medium for plant regeneration from polyploid hairy roots was MS+2.0 mg/L 6-BA +0.2 mg/L NAA. Compared with the control diploid plants, the hairy roots-regenerated plants had weak apical dominance, more axillary buds and more narrow leaves; whereas the polyploid hairy root-regenerated plants had thicker stems, shorter internodes and the colour, width and thickness of leaves were significantly higher than that of the control. Observation of the number of chromosomes in their root tip cells reveals that the obtained polyploid regenerated plants were tetraploidy, with 96 (4n = 96) chromosomes. Pot-grown experiments showed compared to the control, the flowering was delayed by 21 days in diploid hairy roots-regenerated plants and polyploid hairy root-regenerated plants. GC-MS detection shows that the content of nicotine in polyploid plants was about 6.90 and 4.57 times the control and the diploid hairy roots-regenerated plants, respectively.

  9. Deciphering the complex leaf transcriptome of the allotetraploid species Nicotiana tabacum: a phylogenomic perspective

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    Bombarely Aureliano

    2012-08-01

    Full Text Available Abstract Background Polyploidization is an important mechanism in plant evolution. By analyzing the leaf transcriptomes taken from the allotetraploid Nicotiana tabacum (tobacco and parental genome donors, N. sylvesteris (S-Genome and N. tomentosiformis (T-Genome, a phylogenomic approach was taken to map the fate of homeologous gene pairs in this plant. Results A comparison between the genes present in the leaf transcriptomes of N. tabacum and modern day representatives of its progenitor species demonstrated that only 33% of assembled transcripts could be distinguished based on their sequences. A large majority of the genes (83.6% of the non parent distinguishable and 87.2% of the phylogenetic topology analyzed clusters expressed above background level (more than 5 reads showed similar overall expression levels. Homeologous sequences could be identified for 968 gene clusters, and 90% (6% of all genes of the set maintained expression of only one of the tobacco homeologs. When both homeologs were expressed, only 15% (0.5% of the total showed evidence of differential expression, providing limited evidence of subfunctionalization. Comparing the rate of synonymous nucleotide substitution (Ks and non-synonymous nucleotide substitution (Kn provided limited evidence for positive selection during the evolution of tobacco since the polyploidization event took place. Conclusions Polyploidization is a powerful mechanism for plant speciation that can occur during one generation; however millions of generations may be necessary for duplicate genes to acquire a new function. Analysis of the tobacco leaf transcriptome reveals that polyploidization, even in a young tetraploid such as tobacco, can lead to complex changes in gene expression. Gene loss and gene silencing, or subfunctionalization may explain why both homeologs are not expressed by the associated genes. With Whole Genome Duplication (WGD events, polyploid genomes usually maintain a high percentage of

  10. Immobilization of Nicotiana tabacum plant cell suspensions within calcium alginate gel beads for the production of enhanced amounts of scopolin.

    Science.gov (United States)

    Gilleta; Roisin; Fliniaux; Jacquin-Dubreuil; Barbotin; Nava-Saucedo

    2000-02-01

    Scopolin-producing cells of Nicotiana tabacum were immobilized within Ca-alginate gel beads. Free cell suspensions accumulated scopolin within cytoplasmic compartments and cell disruption was necessary to recover scopolin. On the contrary, immobilized plant cells excreted considerable amounts of scopolin. Scopolin diffused throughout the gel matrix and reached the culture media. A large fraction of produced scopolin could then be recovered from the culture medium without disrupting cells. Immobilized N. tabacum cells produced more scopolin than free cell suspensions did (3.8 mg/g fresh weight biomass [into the culture media] versus 0.2 mg/g fresh weight biomass [intracellular]). Variation of the immobilization conditions revealed a marked influence on the behavior of N. tabacum plant cells: production of scopolin and enhanced excretion, cell growth, and morphological aspect of plant cell colonies. This excretion phenomenon could be used advantageously at an industrial production level.

  11. Optimisation of contained Nicotiana tabacum cultivation for the production of recombinant protein pharmaceuticals.

    Science.gov (United States)

    Colgan, Richard; Atkinson, Christopher J; Paul, Matthew; Hassan, Sally; Drake, Pascal M W; Sexton, Amy L; Santa-Cruz, Simon; James, David; Hamp, Keith; Gutteridge, Colin; Ma, Julian K-C

    2010-04-01

    Nicotiana tabacum is emerging as a crop of choice for production of recombinant protein pharmaceuticals. Although there is significant commercial expertise in tobacco farming, different cultivation practices are likely to be needed when the objective is to optimise protein expression, yield and extraction, rather than the traditional focus on biomass and alkaloid production. Moreover, pharmaceutical transgenic tobacco plants are likely to be grown initially within a controlled environment, the parameters for which have yet to be established. Here, the growth characteristics and functional recombinant protein yields for two separate transgenic tobacco plant lines were investigated. The impacts of temperature, day-length, compost nitrogen content, radiation and plant density were examined. Temperature was the only environmental variable to affect IgG concentration in the plants, with higher yields observed in plants grown at lower temperature. In contrast, temperature, supplementary radiation and plant density all affected the total soluble protein yield in the same plants. Transgenic plants expressing a second recombinant protein (cyanovirin-N) responded differently to IgG transgenic plants to elevated temperature, with an increase in cyanovirin-N concentration, although the effect of the environmental variables on total soluble protein yields was the same as the IgG plants. Planting density and radiation levels were important factors affecting variability of the two recombinant protein yields in transgenic plants. Phenotypic differences were observed between the two transgenic plant lines and non-transformed N. tabacum, but the effect of different growing conditions was consistent between the three lines. Temperature, day length, radiation intensity and planting density all had a significant impact on biomass production. Taken together, the data suggest that recombinant protein yield is not affected substantially by environmental factors other than growth

  12. Phytoaccumulation of lead by sunflower (Helianthus annuus), tobacco (Nicotiana tabacum), and vetiver (Vetiveria zizanioides).

    Science.gov (United States)

    Boonyapookana, Benjaporn; Parkpian, Preeda; Techapinyawat, Sombun; DeLaune, R D; Jugsujinda, Aroon

    2005-01-01

    The ability of three plant species: Helianthus annuus, Nicotiana tabacum, and Vetiveria zizanioides for phytoaccumulation of Pb was studied. Plants were grown in hydroponic solution containing Pb(NO3)2 at concentration of 0.25 and 2.5 mM Pb in the presence or absence of chelating agents (EDTA or DTPA). Lead (Pb) transport and localization within the tissues of the plant species was determined using scanning electron microscope equipped with energy dispersive X-ray spectrometers (SEM-EDS). The addition of chelators increased Pb uptake as compared to plants grown in solution containing Pb alone. Lead taken up by the plant species were concentrated in both leaf and stem at the region of vascular bundles with greater amounts in the leaf portion. Lead granules were also found in the H. annuus root tissue from the epidermis layer to the central axis. After four weeks of growth a 23-fold increase in shoot Pb content for H. annuus and N. tabacum and 17-fold increase in shoot Pb for V. zizanioides resulted from plants grown in the 2.5 mM Pb-EDTA treatment. The higher Pb treatment (2.5 mM Pb containing EDTA) resulted in higher concentrations of Pb in plant tissue at the fourth week of exposure as compared to Pb treatment containing DTPA. Overall, Pb accumulation potential of H. annuus was greater than that of N. tabacum and V. zizanioides as indicated by the bioconcentration factor (171, 70, and 88, respectively). The highest measured Pb concentrations were found in H. annuus roots, stems, and leaves (2668, 843, and 3611 microg/g DW, respectively) grown in the 2.5 mM Pb-EDTA treatment. The addition of chelators caused some reduction in plant growth and biomass. Results showed that the three plant species tested have potential for use in phytoaccumulation of Pb since the Pb was concentrated in leaf and stem as compared to control plants. H. annuus however best meet the prerequisites for a hyperaccumulator plant and would have the potential for use in the restoration of

  13. Assessment of {sup 210}Pb concentration in Nicotiana tabacum L., burley variety, cultivated in Brazil

    Energy Technology Data Exchange (ETDEWEB)

    Rocha, Rique J.F.X.; Silva, Carolina F.; Frujuele, Jonatan V.; Bovolini, Raquel R.; Damatto, Sandra R., E-mail: rjrocha@ipen.br, E-mail: cfsilva@ipen.br, E-mail: jonatanfrujuele@hotmail.com, E-mail: ra_bovolini@yahoo.com.br, E-mail: damatto@ipen.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil). Lab. de Radiometria Ambiental

    2013-07-01

    Tobacco products are extensively used throughout the world and the most consumed are cigarettes, cigars and narghile. The damaging effects that these products cause to human health are discussed worldwide and many researches are performed with the aim of relating the use of these products with various diseases. Brazil is the largest exporter and second largest producer of tobacco worldwide, according to the crop year 2009/2010 production. The tobacco plant (Nicotiana tabacum L.) is used to manufacture all derivatives and the chemical composition of the resulting tobacco varies with the type of tobacco leaves, how they are grown, the region where they are cultivated, the characteristics of preparation and the temperature variations resulting from the tobacco incomplete combustion. There is lack of information about the chemical and radiological characterization of the tobacco plant both in international and Brazilian literature. Thus a project was established with the objectives of characterizing chemically and radiologically the three varieties most cultivated in Brazil of Nicotiana tobacum L., Virginia, Burley and Common; this paper presents the preliminary results of {sup 210}Pb concentrations for the Burley variety. Plants from this variety were cultivated in pots with organic substrate and fertilizer and in a small farm in natural conditions. The entire plant was analyzed, the organic substrates, the fertilizers and the soil. The results obtained presented higher values for {sup 210}Pb in leaves when compared with the other parts of the plant. Comparing the three study areas the highest results of {sup 210}Pb concentration were obtained in the plants cultivated in the urban area probably due to its atmospheric deposition. (author)

  14. Transcriptome analysis of Nicotiana tabacum infected by Cucumber mosaic virus during systemic symptom development.

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    Jie Lu

    Full Text Available Virus infection of plants may induce a variety of disease symptoms. However, little is known about the molecular mechanism of systemic symptom development in infected plants. Here we performed the first next-generation sequencing study to identify gene expression changes associated with disease development in tobacco plants (Nicotiana tabacum cv. Xanthi nc induced by infection with the M strain of Cucumber mosaic virus (M-CMV. Analysis of the tobacco transcriptome by RNA-Seq identified 95,916 unigenes, 34,408 of which were new transcripts by database searches. Deep sequencing was subsequently used to compare the digital gene expression (DGE profiles of the healthy plants with the infected plants at six sequential disease development stages, including vein clearing, mosaic, severe chlorosis, partial and complete recovery, and secondary mosaic. Thousands of differentially expressed genes were identified, and KEGG pathway analysis of these genes suggested that many biological processes, such as photosynthesis, pigment metabolism and plant-pathogen interaction, were involved in systemic symptom development. Our systematic analysis provides comprehensive transcriptomic information regarding systemic symptom development in virus-infected plants. This information will help further our understanding of the detailed mechanisms of plant responses to viral infection.

  15. Plant coexistence can enhance phytoextraction of cadmium by tobacco (Nicotiana tabacum L.) in contaminated soil

    Institute of Scientific and Technical Information of China (English)

    Ling Liu; Yuefang Li; Jianjun Tang; Liangliang Hu; Xin Chen

    2011-01-01

    A mesocosm experiment was conducted to investigate whether plant coexistence affects cadmium (Cd) uptake by plant in contaminated soil. Tobacco (Nicotiana tabacum L. var. K326) and Japanese clover (Kummerowia striata (Thunb.) Schindl.) were used.Cadmium was applied as 3CdSO4.8H2O in solution at three levels (0, 1, and 3 mg/kg soil) to simulate an unpolluted soil and soils that were slightly and moderately polluted with Cd. Tobacco (crop), Japanese clover (non-crop), and their combination were grown under each Cd treatment. Compared to monoculture and under all Cd treatments, co-planting with Japanese clover did not affect tobacco biomass but significantly increased Cd concentration in all tobacco tissues and enhanced Cd accumulation in tobacco shoots and roots.Compared to monoculture, co-planting reduced soil pH and increased Cd bioavailability. For tobacco, co-planting with Japanese clover increased the Cd bioconcentration factor (BCF) in Cd contaminated soil. Japanese clover also accumulated substantial quantities of Cd in shoots and roots. Thus, total Cd uptake by the plants was much greater with co-planting than with monoculture. The results suggested that phytoextraction can be effectively increased through tobacco co-planting with Japanese clover in mildly Cd-contaminated soil.

  16. Mitochondrial alternative oxidase maintains respiration and preserves photosynthetic capacity during moderate drought in Nicotiana tabacum.

    Science.gov (United States)

    Dahal, Keshav; Wang, Jia; Martyn, Greg D; Rahimy, Farkhunda; Vanlerberghe, Greg C

    2014-11-01

    The mitochondrial electron transport chain includes an alternative oxidase (AOX) that is hypothesized to aid photosynthetic metabolism, perhaps by acting as an additional electron sink for photogenerated reductant or by dampening the generation of reactive oxygen species. Gas exchange, chlorophyll fluorescence, photosystem I (PSI) absorbance, and biochemical and protein analyses were used to compare respiration and photosynthesis of Nicotiana tabacum 'Petit Havana SR1' wild-type plants with that of transgenic AOX knockdown (RNA interference) and overexpression lines, under both well-watered and moderate drought-stressed conditions. During drought, AOX knockdown lines displayed a lower rate of respiration in the light than the wild type, as confirmed by two independent methods. Furthermore, CO2 and light response curves indicated a nonstomatal limitation of photosynthesis in the knockdowns during drought, relative to the wild type. Also relative to the wild type, the knockdowns under drought maintained PSI and PSII in a more reduced redox state, showed greater regulated nonphotochemical energy quenching by PSII, and displayed a higher relative rate of cyclic electron transport around PSI. The origin of these differences may lie in the chloroplast ATP synthase amount, which declined dramatically in the knockdowns in response to drought. None of these effects were seen in plants overexpressing AOX. The results show that AOX is necessary to maintain mitochondrial respiration during moderate drought. In its absence, respiration rate slows and the lack of this electron sink feeds back on the photosynthetic apparatus, resulting in a loss of chloroplast ATP synthase that then limits photosynthetic capacity.

  17. Short Exogenous Peptides Regulate Expression of CLE, KNOX1, and GRF Family Genes in Nicotiana tabacum.

    Science.gov (United States)

    Fedoreyeva, L I; Dilovarova, T A; Ashapkin, V V; Martirosyan, Yu Ts; Khavinson, V Kh; Kharchenko, P N; Vanyushin, B F

    2017-04-01

    Exogenous short biologically active peptides epitalon (Ala-Glu-Asp-Gly), bronchogen (Ala-Glu-Asp-Leu), and vilon (Lys-Glu) at concentrations 10(-7)-10(-9) M significantly influence growth, development, and differentiation of tobacco (Nicotiana tabacum) callus cultures. Epitalon and bronchogen, in particular, both increase growth of calluses and stimulate formation and growth of leaves in plant regenerants. Because the regulatory activity of the short peptides appears at low peptide concentrations, their action to some extent is like that of the activity of phytohormones, and it seems to have signaling character and epigenetic nature. The investigated peptides modulate in tobacco cells the expression of genes including genes responsible for tissue formation and cell differentiation. These peptides differently modulate expression of CLE family genes coding for known endogenous regulatory peptides, the KNOX1 genes (transcription factor genes) and GRF (growth regulatory factor) genes coding for respective DNA-binding proteins such as topoisomerases, nucleases, and others. Thus, at the level of transcription, plants have a system of short peptide regulation of formation of long-known peptide regulators of growth and development. The peptides studied here may be related to a new generation of plant growth regulators. They can be used in the experimental botany, plant molecular biology, biotechnology, and practical agronomy.

  18. Lysinibacillus tabacifolii sp. nov., a novel endophytic bacterium isolated from Nicotiana tabacum leaves.

    Science.gov (United States)

    Duan, Yan-Qing; He, Song-Tao; Li, Qing-Qing; Wang, Ming-Feng; Wang, Wen-Yuan; Zhe, Wei; Cao, Yong-Hong; Mo, Ming-He; Zhai, Yu-Long; Li, Wen-Jun

    2013-06-01

    A Gram-positive, catalase- and oxidase-positive, strictly aerobic, endospore-forming rod bacterium, designated K3514(T), was isolated from the leaves of Nicotiana tabacum. The strain was able to grow at temperatures of 8-40°C, pH 5.0-10.0 and NaCl concentrations of 0-7%. The predominant quinones (>30%) of this strain were MK-7(H2) and MK-7. Phylogenetic analysis of 16S rRNA gene sequence showed that strain K3514(T) was affiliated to the genus Lysinibacillus, with its closest relatives being Lysinibacillus mangiferihumi (98.3% sequence similarity), Lysinibacillus sphaericus (97.9% sequence similarity), Lysinibacillus fusiformis (97.4% sequence similarity), and Lysinibacillus xylanilyticus (97.3% sequence similarity). However, low levels of DNA-DNA relatedness values suggested that the isolate was distinct from the other closest Lysinibacillus species. Additionally, based on analysis of morphological, physiological, and biochemical characteristics, the isolate could be differentiated from the closest known relatives. Therefore, based on polyphasic taxonomic data, the novel isolate likely represents a novel species, for which the name Lysinibacillus tabacifolii sp. nov. and the type strain K3514(T) (=KCTC 33042(T) =CCTCC AB 2012050(T)) are proposed.

  19. Light and clomazone effects on tobacco (Nicotiana tabacum) callus and leaf discs.

    Science.gov (United States)

    Camper, N D; McDonald, S K; Burrows, P M

    2003-11-01

    The effects of clomazone on the growth of tobacco (Nicotiana tabacum L. 'NC2326') callus and leaf discs were studied under four light regimes. Callus cultures and leaf discs were grown on Murashige and Skoog medium supplemented with IAA and kinetin. Light regimes were: dark grown callus kept in the dark and also transferred to the light; light grown callus kept in the light and also transferred to the dark. Two-month-old callus (cultured for 2 months from initiation) grew more rapidly than twelve-month-old callus (cultured for 12 months from initiation) under all conditions tested. Callus transferred from light to dark, or from dark to light, increased in fresh weight slower than did the callus maintained totally in light or dark. Clomazone (2-[(2-chlorophenyl)methyl]-4,4-dimethyl-3-isoxazolidinone) at 140 mg l(-1) or more was lethal to both callus and leaf discs whereas 10 mg l(-1) was stimulatory to growth. Callus tissue responded to clomazone differently depending on the light regime under which it was grown. While clomazone may be affecting the isoprenoid pathway in the callus and leaf disks resulting in growth inhibition, it is possible that other target sites are also being affected and contribute to the reduced growth.

  20. Identification of donor lines containing favorable alleles for quantitative traits in Burley tobacco (Nicotiana tabacum L.

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    Dražić Slobodan

    2010-01-01

    Full Text Available Well-adapted local lines might be used as donors to improve existing commercial hybrids. The objective of this study was to identify the best burley tobacco (Nicotiana tabacum L. line among a group of four lines (SA 130, Bols 334, Barlej T and TN 90 from germplasm collection, for improving an elite single hybrid Bols 335 x TN 86. Values of three quantitative traits (number of leaves, total leaf area and leaf yield were measured in two-year field experiments. Evaluation of donor lines as sources of new favorable alleles not present in the elite hybrid parent lines was conducted. According to Dudley (1984, 1987 the most significant class for improving quantitative traits is locus class G. Donor line TN 90 had positive values of the parameter μG for number of leaves and for total leaf area. Studied donor lines could not be use directly as the source of new alleles for the improvement of leaf yield in elite hybrid. The improvement of leaf yield in tobacco hybrid Bols 335 x TN 86 would be conducted indirectly via back crossing (TN 86 x TN 90 x TN 86. Significance of GxE interaction for studied traits indicates the same breeding method would be effective in broad range of environmental conditions.

  1. Jasmonate mediates salt-induced nicotine biosynthesis in tobacco (Nicotiana tabacum L.

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    Xiaodong Chen

    2016-04-01

    Full Text Available Jasmonate (JA, as an important signal, plays a key role in multiple processes of plant growth, development and stress response. Nicotine and related pyridine alkaloids in tobacco (Nicotiana tabacum L. are essential secondary metabolites. Whether environmental factors control nicotine biosynthesis and the underlying mechanism remains previously unreported. Here, we applied physiological and biochemical approaches to investigate how salt stress affects nicotine biosynthesis in tobacco. We found that salt stress induced the biosynthesis of JA, which subsequently triggered the activation of JA-responsive gene expression and, ultimately, nicotine synthesis. Bioinformatics analysis revealed the existence of many NtMYC2a-recognized G-box motifs in the promoter regions of NtLOX, NtAOS, NtAOC and NtOPR genes. Applying exogenous JA increased nicotine content, while suppressing JA biosynthesis reduced nicotine biosynthesis. Salt treatment could not efficiently induce nicotine biosynthesis in transgenic anti-COI1 tobacco plants. These results demonstrate that JA acts as the essential signal which triggers nicotine biosynthesis in tobacco after salt stress.

  2. Differential patterns of dehydroabietic acid biotransformation by Nicotiana tabacum and Catharanthus roseus cells.

    Science.gov (United States)

    Häkkinen, Suvi T; Lackman, Petri; Nygrén, Heli; Oksman-Caldentey, Kirsi-Marja; Maaheimo, Hannu; Rischer, Heiko

    2012-01-20

    The aim of this study was to use whole cell catalysts as tools for modification of selected resin acids in order to obtain value-added functional derivatives. The enzymatic bioconversion capacities of two plant species were tested towards dehydroabietic acid. Dehydroabietic acid (DHA) is an abundant resin acid in conifers, representing a natural wood protectant. It is also one of the constituents found in by-products of the kraft chemical pulping industry. DHA was fed to tobacco (Nicotiana tabacum) and Madagascar periwinkle (Catharanthus roseus) plant cell and tissue cultures and bioconversion product formation was monitored using NMR analysis. Both plant species took up DHA from culture medium, and various types of typical detoxification processes occurred in both cultures. In addition, diverse responses to DHA treatment were observed, including differences in uptake kinetics, chemical modification of added substrate and changes in overall metabolism of the cells. Interestingly, Catharanthus roseus, a host species for pharmaceutically valuable terpenoid indole alkaloids, exhibited a very different bioconversion pattern for exogenously applied DHA than tobacco, which does not possess a terpenoid indole pathway. In tobacco, DHA is readily glycosylated in the carbonyl group, whereas in periwinkle it is proposed that a cytochrome P450-catalyzed enzymatic detoxification reaction takes place before the formation of glycosylated product.

  3. In vitro antioxidant properties of flavonoids and polysaccharides extract from tobacco (Nicotiana tabacum L.) leaves.

    Science.gov (United States)

    Ru, Qiao-Mei; Wang, Li-Juan; Li, Wei-Ming; Wang, Jing-Lu; Ding, Yu-Ting

    2012-09-21

    In the present study, antioxidant properties of flavonoids and polysaccharides from tobacco (Nicotiana tabacum L.) leaves were evaluated in several in vitro systems, e.g., scavenging activities on hydroxyl, superoxide anion, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radicals, and reducing power. Flavonoids showed much better activity than polysaccharides in scavenging activities on free radicals. When compared to the positive control, ascorbic acid, both showed weaker antioxidant potential. However, flavonoids possessed comparable superoxide anion, DPPH and ABTS radical scavenging abilities to ascorbic acid at high concentration (600 μg/mL). Meanwhile, it was found that flavonoids had prominent effects on the reducing power, which was equivalent to ascorbic acid, and was significantly higher than polysaccharides. These results clearly indicate that flavonoids are effective in scavenging free radicals and have the potential to be powerful antioxidants. Thus, tobacco leaves could be considered as a potential source of natural antioxidants for food, pharmaceutical, cosmetics or nutraceutical industries.

  4. The transmitting tissue of Nicotiana tabacum is not essential to pollen tube growth, and its ablation can reverse prezygotic interspecific barriers.

    Science.gov (United States)

    Smith, Alan G; Eberle, Carrie A; Moss, Nicole G; Anderson, Neil O; Clasen, Benjamin M; Hegeman, Adrian D

    2013-12-01

    The Nicotiana tabacum transmitting tissue is a highly specialized file of metabolically active cells that is the pathway for pollen tubes from the stigma to the ovules where fertilization occurs. It is thought to be essential to pollen tube growth because of the nutrients and guidance it provides to the pollen tubes. It also regulates gametophytic self-incompatibility in the style. To test the function of the transmitting tissue in pollen tube growth and to determine its role in regulating prezygotic interspecific incompatibility, genetic ablation was used to eliminate the mature transmitting tissue, producing a hollow style. Despite the absence of the mature transmitting tissue and greatly reduced transmitting-tissue-specific gene expression, self-pollen tubes had growth to the end of the style. Pollen tubes grew at a slower rate in the transmitting-tissue-ablated line during the first 24 h post-pollination. However, pollen tubes grew to a similar length 40 h post-pollination with and without a transmitting tissue. Ablation of the N. tabacum transmitting tissue significantly altered interspecific pollen tube growth. These results implicate the N. tabacum transmitting tissue in facilitating or inhibiting interspecific pollen tube growth in a species-dependent manner and in controlling prezygotic reproductive barriers.

  5. Glutathione transferase from Trichoderma virens enhances cadmium tolerance without enhancing its accumulation in transgenic Nicotiana tabacum.

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    Prachy Dixit

    Full Text Available BACKGROUND: Cadmium (Cd is a major heavy metal pollutant which is highly toxic to plants and animals. Vast agricultural areas worldwide are contaminated with Cd. Plants take up Cd and through the food chain it reaches humans and causes toxicity. It is ideal to develop plants tolerant to Cd, without enhanced accumulation in the edible parts for human consumption. Glutathione transferases (GST are a family of multifunctional enzymes known to have important roles in combating oxidative stresses induced by various heavy metals including Cd. Some GSTs are also known to function as glutathione peroxidases. Overexpression/heterologous expression of GSTs is expected to result in plants tolerant to heavy metals such as Cd. RESULTS: Here, we report cloning of a glutathione transferase gene from Trichoderma virens, a biocontrol fungus and introducing it into Nicotiana tabacum plants by Agrobacterium-mediated gene transfer. Transgenic nature of the plants was confirmed by Southern blot hybridization and expression by reverse transcription PCR. Transgene (TvGST showed single gene Mendelian inheritance. When transgenic plants expressing TvGST gene were exposed to different concentrations of Cd, they were found to be more tolerant compared to wild type plants, with transgenic plants showing lower levels of lipid peroxidation. Levels of different antioxidant enzymes such as glutathione transferase, superoxide dismutase, ascorbate peroxidase, guiacol peroxidase and catalase showed enhanced levels in transgenic plants expressing TvGST compared to control plants, when exposed to Cd. Cadmium accumulation in the plant biomass in transgenic plants were similar or lower than wild-type plants. CONCLUSION: The results of the present study suggest that transgenic tobacco plants expressing a Trichoderma virens GST are more tolerant to Cd, without enhancing its accumulation in the plant biomass. It should be possible to extend the present results to crop plants for

  6. Evaluation of Nicotiana tabacum plants transformed for the expression of verocytotoxic Escherichia coli antigens.

    Directory of Open Access Journals (Sweden)

    Angela Lombardi

    2015-07-01

    Full Text Available Two transgenic Nicotiana tabacum plants, carrying respectively the F18 adhesive fimbriae and the B subunit of verocytotoxin genes from O138 Verocytotoxic E.coli serotype were developed as a model of edible vaccine. Tobacco plants were transformed by agroinfection according to Rossi et al. (2013 stably.  The F18 adhesive fimbriae and VT2e B-subunit were placed under control of the GLOB promoter for the seed-specific protein expression. Agrobacterium tumefaciens binary vector system is an efficient tool to transform plant cells; however, the exogenous gene integrates at semi-random into the nuclear chromosome. PCR products, using specific oligonucleotides putatively encoding the B-subunit of VT2e-B and F18 fimbriae were identified on agarose gel (1.5% - 0.9% as bands with a length of 270 and 519 base pairs, respectively. We showed that the foreign VT2e-B and F18 fimbriae genes were stably integrated into the tobacco genome. Northern blot and Western blot analyses carried out respectively on total mRNA and total soluble protein extract obtained from seeds. For each line, the obtained amount of antigens is sufficient for subsequent oral immunization trials. Three lines of tobacco seeds (F18, VT2e-B, and WT were seeded in homogeneous conditions and were harvested simultaneously. Tobacco plants were analysed also by optical and electronic microscope in different phases of growth. Germination of transgenic seeds were delayed of three/five days compared to WT in two replicated experiments, suggesting that genetic manipulation may influenced mechanisms leading to germination. In conclusion the genes coding for VT2e-B and the F18 are stably maintained in the seeds and obtained tobacco seeds represent a valid strategy to ferry antigenic proteins to the gut and a promising non-invasive method of vaccination in pig industry.

  7. The sclerophyllous Eucalyptus camaldulensis and herbaceous Nicotiana tabacum have different mechanisms to maintain high rates of photosynthesis

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    Wei Huang

    2016-11-01

    Full Text Available It is believed that high levels of mesophyll conductance (gm largely contribute to the high rates of photosynthesis in herbaceous C3 plants. However, some sclerophyllous C3 plants that display low levels of gm have high rates of photosynthesis, and the underlying mechanisms responsible for high photosynthetic rates in sclerophyllous C3 plants are unclear. In the present study, we examined photosynthetic characteristics in two high-photosynthesis plants (the sclerophyllous Eucalyptus camaldulensis and the herbaceous Nicotiana tabacum using measurements of gas exchange and chlorophyll fluorescence. Under saturating light intensities, both species had similar rates of CO2 assimilation at 400 μmol mol-1 CO2 (A400. However, E. camaldulensis exhibited significantly lower gm and chloroplast CO2 concentration (Cc than N. tabacum. A quantitative analysis revealed that, in E. camaldulensis, the gm limitation was the most constraining factor for photosynthesis. By comparison, in N. tabacum, the biochemical limitation was the strongest, followed by gm and gs limitations. In conjunction with a lower Cc, E. camaldulensis up-regulated the capacities of photorespiratory pathway and alternative electron flow. Furthermore, the rate of alternative electron flow was positively correlated with the rates of photorespiration and ATP supply from other flexible mechanisms, suggesting the important roles of photorespiratory pathway and alternative electron flow in sustaining high rate of photosynthesis in E. camaldulensis. These results highlight the different mechanisms used to maintain high rates of photosynthesis in the sclerophyllous E. camaldulensis and the herbaceous N. tabacum.

  8. The Sclerophyllous Eucalyptus camaldulensis and Herbaceous Nicotiana tabacum Have Different Mechanisms to Maintain High Rates of Photosynthesis

    Science.gov (United States)

    Huang, Wei; Tong, You-Gui; Yu, Guo-Yun; Yang, Wei-Xian

    2016-01-01

    It is believed that high levels of mesophyll conductance (gm) largely contribute to the high rates of photosynthesis in herbaceous C3 plants. However, some sclerophyllous C3 plants that display low levels of gm have high rates of photosynthesis, and the underlying mechanisms responsible for high photosynthetic rates in sclerophyllous C3 plants are unclear. In the present study, we examined photosynthetic characteristics in two high-photosynthesis plants (the sclerophyllous Eucalyptus camaldulensis and the herbaceous Nicotiana tabacum) using measurements of gas exchange and chlorophyll fluorescence. Under saturating light intensities, both species had similar rates of CO2 assimilation at 400 μmol mol−1 CO2 (A400). However, E. camaldulensis exhibited significantly lower gm and chloroplast CO2 concentration (Cc) than N. tabacum. A quantitative analysis revealed that, in E. camaldulensis, the gm limitation was the most constraining factor for photosynthesis. By comparison, in N. tabacum, the biochemical limitation was the strongest, followed by gm and gs limitations. In conjunction with a lower Cc, E. camaldulensis up-regulated the capacities of photorespiratory pathway and alternative electron flow. Furthermore, the rate of alternative electron flow was positively correlated with the rates of photorespiration and ATP supply from other flexible mechanisms, suggesting the important roles of photorespiratory pathway, and alternative electron flow in sustaining high rate of photosynthesis in E. camaldulensis. These results highlight the different mechanisms used to maintain high rates of photosynthesis in the sclerophyllous E. camaldulensis and the herbaceous N. tabacum. PMID:27933083

  9. INFLUENCE OF STATIONARY MAGNETIC FIELD ON THE EARLY STAGES OF THE DEVELOPMENT OF TOBACCO SEEDS (NICOTIANA TABACUM L.

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    ANNA ALADJADJIYAN

    2003-10-01

    Full Text Available The influence of a stationary magnetic field with induction of 0,15 Т at expositions 10 min, 20 min и 30 min, on tobacco seeds (Nicotiana tabacum L., cv. Harmanly 11 has been investigated. The germination energy GE and the germination G have been determined with the aim of establishing the effect of the magnetic field on seed vitality. It has been found out that the magnetic field stimulates the development of the germ and leads to increasing the germination energy and germination. A hypothesis about the explanation of the results obtained has been proposed, especially about the stimulating effect of the magnetic field treatment.

  10. Biological and chemical induction of resistance to the Globodera tabacum solanacearum in oriental and flue-cured tobacco (Nicotiana tabacum L.).

    Science.gov (United States)

    Parkunan, Venkatesan; Johnson, Charles S; Eisenback, Jon D

    2009-09-01

    The effects of acibenzolar-S-methyl (ASM) and four combinations of plant growth-promoting rhizobacteria (PGPR) on the reproduction of a tobacco cyst nematode, Globodera tabacum solanacearum, and growth of Nicotiana tabacum (cv. K326 and Xanthi) were tested under greenhouse and field conditions. The PGPR included combinations of Bacillus subtilis A13 with B. pumilis INR7, B. pumilis SE34, B. licheniformis IN937b, or B. amyloliquefaciens IN937a, respectively. Among the four rhizobacterial combinations, IN937a + A13 exhibited the most consistent reduction in G. t. solanacearum cysts under greenhouse and field conditions. No undesirable effects of IN937a + A13 were observed on tobacco growth under greenhouse and field conditions. Use of INR7 + A13 reduced G. t. solanacearum reproduction on flue-cured tobacco cv. K326 but not on oriental tobacco cv. Xanthi. Application of ASM reduced final numbers of G. t. solanacearum cysts, but also resulted in phytotoxicity mainly under the greenhouse conditions. When oriental tobacco seedlings were pre-grown in a IN937a + A13-treated soil-less medium, a single application of ASM at 200 mg/L one week after transplanting significantly reduced G. t. solanacearum reproduction in the field.

  11. Elevated pCO(2 )favours nitrate reduction in the roots of wild-type tobacco (Nicotiana tabacum cv. Gat.) and significantly alters N-metabolism in transformants lacking functional nitrate reductase in the roots.

    Science.gov (United States)

    Kruse, Jörg; Hetzger, Ilka; Hänsch, Robert; Mendel, Ralf-R; Walch-Liu, Pia; Engels, Christof; Rennenberg, Heinz

    2002-12-01

    The impact of elevated pCO(2 )on N-metabolism of hydroponically grown wild-type and transformed tobacco plants lacking root nitrate reduction was studied in order to elucidate the effects on (i) nitrate uptake, (ii) long-distance transport of N, (iii) nitrate reduction with emphasis on root-NR, and (iv) the allocation of N between the root and shoot. The findings were related to alterations of growth rates. At elevated pCO(2 )the wild type exhibited higher growth rates, which were accompanied by an increase of NO(3)(-)-uptake per plant, due to a higher root:shoot ratio. Furthermore, elevated pCO(2 )enhanced nitrate reduction in the roots of the wild type, resulting in enhanced xylem-loading of organic N (amino-N) to supply the shoot with sufficient nitrogen, and decreased phloem-transport of organic N in a basipetal direction. Transformed tobacco plants lacking root nitrate reduction were smaller than the wild type and exhibited lower growth rates. Nitrate uptake per plant was decreased in transformed plants as a consequence of an impeded root growth and, thus, a significantly decreased root:shoot ratio. Surprisingly, transformed plants showed an altered allocation of amino-N between the root and the shoot, with an increase of amino-N in the root and a substantial decrease of amino-N in the shoot. In transformed plants, xylem-loading of nitrate was increased and the roots were supplied with organic N via phloem transport. Elevated pCO(2 )increased shoot-NR, but only slightly affected the growth rates of transformed plants, whereas carbohydrates accumulated at elevated pCO(2 )as indicated by a significant increase of the C/N ratio in the leaves of transformed plants. Unexpectedly, the C/N balance and the functional equilibrium between root and shoot growth was disturbed dramatically by the loss of nitrate reduction in the root.

  12. EFECTO DE LA MADUREZ EN LA CRIOCONSERVACIÓN DE SEMILLAS DE Nicotiana tabacum L.

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    Juan L. Pérez-Rodríguez

    2016-01-01

    Full Text Available La presente investigación tuvo como objetivo determinar el efecto de la madurez en la crioconservación de semillas de Nicotiana tabacum L. cv Sancti Spíritus 96 (SS-96. El estudio se realizó en la Estación experimental del tabaco de Cabaiguán, Sancti Spíritus, perteneciente al Instituto de Investigaciones del Tabaco, entre los años 2012 y 2014. Las semillas de SS-96 se colectaron a los 14, 21, 28, 35, 42, 49 y 56 días después de la antesis (DDA y se les determinó masa fresca y masa seca de 1000 semillas, porcentaje de humedad (base masa fresca y potencia germinativa (PG, además de evaluar su tolerancia a la desecación. Al mismo tiempo, semillas de cada día de colecta se conservaron a 5 ºC o en nitrógeno líquido y, transcurridos 30 días, les fue evaluada su PG y se sometieron a las pruebas de envejecimiento acelerado y pérdida de electrólitos. Durante el desarrollo de la investigación se determinó que la madurez fisiológica de las semillas de SS-96 se alcanza a los 29,6 DDA, con un contenido de agua de 30,6 % y masa seca de 1000 semillas de 81,2 mg. Se estableció 35 DDA como el momento idóneo para la colecta de semillas en este cultivar. Una colecta antes de esta fecha provoca una disminución en la tolerancia a la desecación producto de la inmadurez de las semillas; una recolección posterior conlleva a la pérdida de vigor debido a la mayor exposición de las semillas a las condiciones ambientales.

  13. Cloning of the Lycopene β-cyclase Gene in Nicotiana tabacum and Its Overexpression Confers Salt and Drought Tolerance

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    Yanmei Shi

    2015-12-01

    Full Text Available Carotenoids are important pigments in plants that play crucial roles in plant growth and in plant responses to environmental stress. Lycopene β cyclase (β-LCY functions at the branch point of the carotenoid biosynthesis pathway, catalyzing the cyclization of lycopene. Here, a β-LCY gene from Nicotiana tabacum, designated as Ntβ-LCY1, was cloned and functionally characterized. Robust expression of Ntβ-LCY1 was found in leaves, and Ntβ-LCY1 expression was obviously induced by salt, drought, and exogenous abscisic acid treatments. Strong accumulation of carotenoids and expression of carotenoid biosynthesis genes resulted from Ntβ-LCY1 overexpression. Additionally, compared to wild-type plants, transgenic plants with overexpression showed enhanced tolerance to salt and drought stress with higher abscisic acid levels and lower levels of malondialdehyde and reactive oxygen species. Conversely, transgenic RNA interference plants had a clear albino phenotype in leaves, and some plants did not survive beyond the early developmental stages. The suppression of Ntβ-LCY1 expression led to lower expression levels of genes in the carotenoid biosynthesis pathway and to reduced accumulation of carotenoids, chlorophyll, and abscisic acid. These results indicate that Ntβ-LCY1 is not only a likely cyclization enzyme involved in carotenoid accumulation but also confers salt and drought stress tolerance in Nicotiana tabacum.

  14. Experimental and computational studies of Nicotiana tabacum leaves extract as green corrosion inhibitor for mild steel in acidic medium

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    Jeetendra Bhawsar

    2015-09-01

    Full Text Available In the present work corrosion inhibition of mild steel in 2 M H2SO4 solution by Nicotiana tabacum extract was studied by weight loss method. It has been found that the extract acts as an effective corrosion inhibitor for mild steel in Sulfuric acid medium. The inhibition process is attributed to the formation of an adsorbed film of inhibitor on the metal surface which protects the metal against corrosion. The inhibition efficiency (%IE and surface coverage (θ of N. tabacum extract increased with increase in inhibitor concentration but decreased with increasing the temperature. The adsorption of extract on the mild steel surface was found to obey Langmuir’s adsorption isotherm. The free energy value (ΔGads indicated that the adsorption of inhibitor molecules was typical of physisorption. The results obtained show that N. tabacum Extract could serve as an excellent eco-friendly green corrosion inhibitor. Quantum chemical parameters such as highest occupied molecular orbital energy (EHOMO, lowest unoccupied molecular orbital energy (ELUMO, energy gap (ΔE, dipole moment (μ and Mulliken charges were calculated. Quantum chemical calculations also supported experimental data and the adsorption of inhibitor molecules onto the metal surface.

  15. Effects of aluminum oxide nanoparticles on the growth, development, and microRNA expression of tobacco (Nicotiana tabacum.

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    Caitlin E Burklew

    Full Text Available Nanoparticles are a class of newly emerging environmental pollutions. To date, few experiments have been conducted to investigate the effect nanoparticles may have on plant growth and development. It is important to study the effects nanoparticles have on plants because they are stationary organisms that cannot move away from environmental stresses like animals can, therefore they must overcome these stresses by molecular routes such as altering gene expression. microRNAs (miRNA are a newly discovered, endogenous class of post-transcriptional gene regulators that function to alter gene expression by either targeting mRNAs for degradation or inhibiting mRNAs translating into proteins. miRNAs have been shown to mediate abiotic stress responses such as drought and salinity in plants by altering gene expression, however no study has been performed on the effect of nanoparticles on the miRNA expression profile; therefore our aim in this study was to classify if certain miRNAs play a role in plant response to Al(2O(3 nanoparticle stress. In this study, we exposed tobacco (Nicotiana tabacum plants (an important cash crop as well as a model organism to 0%, 0.1%, 0.5%, and 1% Al(2O(3 nanoparticles and found that as exposure to the nanoparticles increased, the average root length, the average biomass, and the leaf count of the seedlings significantly decreased. We also found that miR395, miR397, miR398, and miR399 showed an extreme increase in expression during exposure to 1% Al(2O(3 nanoparticles as compared to the other treatments and the control, therefore these miRNAs may play a key role in mediating plant stress responses to nanoparticle stress in the environment. The results of this study show that Al(2O(3 nanoparticles have a negative effect on the growth and development of tobacco seedlings and that miRNAs may play a role in the ability of plants to withstand stress to Al(2O(3 nanoparticles in the environment.

  16. Jasmonate-Sensitivity-Assisted Screening and Characterization of Nicotine Synthetic Mutants from Activation-Tagged Population of Tobacco (Nicotiana tabacum L.)

    Science.gov (United States)

    Yin, Guoying; Wang, Wenjing; Niu, Haixia; Ding, Yongqiang; Zhang, Dingyu; Zhang, Jie; Liu, Guanshan; Wang, Sangen; Zhang, Hongbo

    2017-01-01

    Nicotine is a secondary metabolite that is important to the defense system and commercial quality of tobacco (Nicotiana tabacum L.). Jasmonate and its derivatives (JAs) are phytohormone regulators of nicotine formation; however, the underlying molecular mechanism of this process remains largely unclear. Owing to the amphitetraploid origin of N. tabacum, research on screening and identification of nicotine-synthetic mutants is relatively scarce. Here, we describe a method based on JA-sensitivity for screening nicotine mutants from an activation-tagged population of tobacco. In this approach, the mutants were first screened for abnormal JA responses in seed germination and root elongation, and then the levels of nicotine synthesis and expression of nicotine synthetic genes in the mutants with altered JA-response were measured to determine the nicotine-synthetic mutants. We successfully obtained five mutants that maintained stable nicotine contents and JA responses for three generations. This method is simple, effective and low-cost, and the finding of transcriptional changes of nicotine synthetic genes in the mutants shows potentials for identifying novel regulators involved in JA-regulated nicotine biosynthesis. PMID:28243248

  17. Spermine either delays or promotes cell death in Nicotiana tabacum L. corolla depending on the floral developmental stage and affects the distribution of transglutaminase.

    Science.gov (United States)

    Cai, Giampiero; Della Mea, Massimiliano; Faleri, Claudia; Fattorini, Laura; Aloisi, Iris; Serafini-Fracassini, Donatella; Del Duca, Stefano

    2015-12-01

    The role of spermine (SM) was studied to verify if SM supplied to Nicotiana tabacum flower can modulate programmed cell death (PCD) of the corolla. SM has strong effects on the development and senescence of excised flowers despite its low physiological levels. The timing and duration of SM treatment is a key factor; SM counteracts PCD (verified by morphological observations, pigment contents and DNA laddering) only in the narrow developmental window of corolla expansion. Before and after, SM promotes PCD. SM exerts its pro-survival role by delaying fresh weight loss, by inhibiting reduction of pigments and finally by preventing DNA degradation. Moreover, SM deeply alters the distribution of the PA-conjugating enzyme transglutaminase (TGase). TGase is present in the epidermis during development, but it sprays also in the cell walls of inner parenchyma at senescence. After SM treatment, parenchyma cells accumulate TGase, increase in size and their cell walls do not undergo stiffening contrarily to control cells. The subcellular localization of TGase has been validated by biolistic-transformation of onion epidermal cells. Results indicated that SM is a critical factor in the senescence of N. tabacum corolla by controlling biochemical and morphological parameters; the lasts are probably interconnected with the action of TGase.

  18. [Preferential localization of cadmium on the iterative DNA sequences from cultured tissues of the crown gall of tobacco (Nicotiana tabacum, var. Wisconsis 38)].

    Science.gov (United States)

    Sissoëff, I; Grisvard, J; Guillé, E; Laterjet, R

    1975-05-26

    Fractionation of total crown-gall tissue culture DNA from Nicotiana tabacum by Ag+-Cs2SO4 density gradient is described. Cadmium ions determination is performed in each fraction by anodic stripping voltammetry. The cadmium content of the DNA in the lightest density fractions is 100 to 1000 times higher than in the other fractions.

  19. ANÁLISIS COMPARATIVO DE INDICADORES QUÍMICOS DE LA HOJA Y DITERPENOS DE EXUDADOS FOLIARES DE Nicotiana tabacum L

    National Research Council Canada - National Science Library

    Yanelis Capdesuñer; Maribel Rivas; Janet Quiñones-Galvez; Madelin Gallo; Erinelvis Rodríguez; Juan L. Pérez; Ermis Yanes-Paz; Martha Hernández

    2016-01-01

    .... El objetivo de esta investigación es determinar la composición química de la hoja y de los extractos etanólicos obtenidos a partir de exudados foliares de diez accesiones no comerciales de Nicotiana tabacum L...

  20. Cloning and functional analysis of chloroplast division gene NtFtsZ2-1 in Nicotiana tabacum

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    FtsZ protein plays an important role in the division of chloroplasts. With the finding and functional analysis of higher plant FtsZ proteins, people have deepened the understanding in the molecular mechanism of chloroplast division. Multiple ftsZ genes are diversified into two families in higher plants, ftsZ1 and ftsZ2. On the basis of the research on ftsZ1 family, we analyzed the function of NtFtsZ2-1 gene in Nicotiana tabacum. Microscopic analysis of the sense and antisense NtFtsZ2-1 transgenic tobacco plants revealed that the chloroplasts were abnormal in size and also in number when compared with wild-type tobacco chloroplasts. Our investigations confirmed that the NtFtsZ2-1 gene is involved in plant chloroplast division.

  1. Study of photosynthesis process in the presence of low concentrations of clomazone herbicide in tobacco (Nicotiana tabacum

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    Darwish, Majd

    2013-02-01

    Full Text Available The effect of chemical residues of clomazone on photosynthetic processes has been studiedby using several low concentrations of the herbicide (0, 1, 0.1, 0.01, 0.001, 0.0001 and 0.00001 µM and seedlings of two varieties of tobacco (Nicotiana tabacum L. cv. Virginie vk51, Nicotiana tabacum, L. cv. Xanthi. The content of photosynthetic pigments, the parameters of the chlorophyll-a fluorescence and the JIP-test were performed on an adult leaf (AL and a young leaf (YL, that gave a complementary designto know the action's mode of clomazone on the plant physiological processes. Clomazone reducedthe total chlorophyll (a+b, carotenoids pigments (reduction in size antenna pigments judged by an increase in the chlorophyll a/b ratio in young leaves more than adults leaves. The maximal photochemical efficiency (Fv/Fm of photosystem II (PSII decreased significantly in youngleavescompared to adult leaves and in (Virginie variety than (Xanthi variety. Among the parameters calculated of the JIP-test most affected by the treatment, PIabs, 1-VJ, ABS/RC, DI0/RC, TR0/RC, ET0/RC, ET0/ABS, which indicated acomparable effects of clomazone(1μM, 0.1µM, 0.01µM between the two types of leaves and the varieties used. More, the results showed that the concentration ( 1μM was the most effective amongthe other low concentrations used and the (Virginie variety ismore sensitive than the (Xanthivariety. We conclude that clomazone has probably two combined functions (physiological, toxic judged by the different behavior of both types of leaves in the presence of the herbicide.

  2. A pleiotropic drug resistance transporter in Nicotiana tabacum is involved in defense against the herbivore Manduca sexta.

    Science.gov (United States)

    Bienert, Manuela D; Siegmund, Stephanie E G; Drozak, Anna; Trombik, Tomasz; Bultreys, Alain; Baldwin, Ian T; Boutry, Marc

    2012-12-01

    Pleiotropic drug resistance (PDR) transporters are a group of membrane proteins belonging to the ABCG sub-family of ATP binding cassette (ABC) transporters. There is clear evidence for the involvement of plant ABC transporters in resistance to fungal and bacterial pathogens, but not in the biotic stress response to insect or herbivore attack. Here, we describe a PDR transporter, ABCG5/PDR5, from Nicotiana tabacum. GFP fusion and subcellular fractionation studies revealed that ABCG5/PDR5 is localized to the plasma membrane. Staining of transgenic plants expressing the GUS reporter gene under the control of the ABCG5/PDR5 transcription promoter and immunoblotting of wild-type plants showed that, under standard growth conditions, ABCG5/PDR5 is highly expressed in roots, stems and flowers, but is only expressed at marginal levels in leaves. Interestingly, ABCG5/PDR5 expression is induced in leaves by methyl jasmonate, wounding, pathogen infiltration, or herbivory by Manduca sexta. To address the physiological role of ABCG5/PDR5, N. tabacum plants silenced for the expression of ABCG5/PDR5 were obtained. No phenotypic modification was observed under standard conditions. However, a small increase in susceptibility to the fungus Fusarium oxysporum was observed. A stronger effect was observed in relation to herbivory: silenced plants allowed better growth and faster development of M. sexta larvae than wild-type plants, indicating an involvement of this PDR transporter in resistance to M. sexta herbivory.

  3. Heterologous expression of the BABY BOOM AP2/ERF transcription factor enhances the regeneration capacity of tobacco (Nicotiana tabacum L.).

    Science.gov (United States)

    Srinivasan, Chinnathambi; Liu, Zongrang; Heidmann, Iris; Supena, Ence Darmo Jaya; Fukuoka, Hiro; Joosen, Ronny; Lambalk, Joep; Angenent, Gerco; Scorza, Ralph; Custers, Jan B M; Boutilier, Kim

    2007-01-01

    Gain-of-function studies have shown that ectopic expression of the BABY BOOM (BBM) AP2/ERF domain transcription factor is sufficient to induce spontaneous somatic embryogenesis in Arabidopsis (Arabidopsis thaliana (L.) Heynh) and Brassica napus (B. napus L.) seedlings. Here we examined the effect of ectopic BBM expression on the development and regenerative capacity of tobacco (Nicotiana tabacum L.) through heterologous expression of Arabidopsis and B. napus BBM genes. 35S::BBM tobacco lines exhibited a number of the phenotypes previously observed in 35S::BBM Arabidopsis and B. napus transgenics, including callus formation, leaf rumpling, and sterility, but they did not undergo spontaneous somatic embryogenesis. 35S::BBM plants with severe ectopic expression phenotypes could not be assessed for enhanced regeneration at the seedling stage due to complete male and female sterility of the primary transformants, therefore fertile BBM ectopic expression lines with strong misexpression phenotypes were generated by expressing a steroid-inducible, post-translationally controlled BBM fusion protein (BBM:GR) under the control of a 35S promoter. These lines exhibited spontaneous shoot and root formation, while somatic embryogenesis could be induced from in-vitro germinated seedling hypocotyls cultured on media supplemented with cytokinin. Together these results suggest that ectopic BBM expression in transgenic tobacco also activates cell proliferation pathways, but differences exist between Arabidopsis/B. napus and N. tabacum with respect to their competence to respond to the BBM signalling molecule.

  4. Species origin of genomic factors in Nicotiana nudicaulis Watson controlling hybrid lethality in interspecific hybrids between N. nudicaulis Watson and N. tabacum L.

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    Hongshuo Liu

    Full Text Available Hybrid lethality is expressed at 28°C in the cross Nicotiana nudicaulis × N. tabacum. The S subgenome of N. tabacum has been identified as controlling this hybrid lethality. To clarify the responsible genomic factor(s of N. nudicaulis, we crossed N. trigonophylla (paternal progenitor of N. nudicaulis with N. tabacum, because hybrids between N. sylvestris (maternal progenitor of N. nudicaulis and N. tabacum are viable when grown in a greenhouse. In the cross N. trigonophylla×N. tabacum, approximately 50% of hybrids were vitrified, 20% were viable, and 20% were nonviable at 28°C. To reveal which subgenome of N. tabacum was responsible for these phenotypes, we crossed N. trigonophylla with two progenitors of N. tabacum, N. sylvestris (SS and N. tomentosiformis (TT. In the cross N. sylvestris × N. trigonophylla, we confirmed that over half of hybrids of N. sylvestris × N. trigonophylla were vitrified, and none of the hybrids of N. trigonophylla × N. tomentosiformis were. The results imply that the S subgenome, encoding a gene or genes inducing hybrid lethality in the cross between N. nudicaulis and N. tabacum, has one or more genomic factors that induce vitrification. Furthermore, in vitrified hybrids of N. trigonophylla × N. tabacum and N. sylvestris × N. trigonophylla, we found that nuclear fragmentation, which progresses during expression of hybrid lethality, was accompanied by vitrification. This observation suggests that vitrification has a relationship to hybrid lethality. Based on these results, we speculate that when N. nudicaulis was formed approximately 5 million years ago, several causative genomic factors determining phenotypes of hybrid seedlings were inherited from N. trigonophylla. Subsequently, genome downsizing and various recombination-based processes took place. Some of the causative genomic factors were lost and some became genomic factor(s controlling hybrid lethality in extant N. nudicaulis.

  5. Species origin of genomic factors in Nicotiana nudicaulis Watson controlling hybrid lethality in interspecific hybrids between N. nudicaulis Watson and N. tabacum L.

    Science.gov (United States)

    Liu, Hongshuo; Marubashi, Wataru

    2014-01-01

    Hybrid lethality is expressed at 28°C in the cross Nicotiana nudicaulis × N. tabacum. The S subgenome of N. tabacum has been identified as controlling this hybrid lethality. To clarify the responsible genomic factor(s) of N. nudicaulis, we crossed N. trigonophylla (paternal progenitor of N. nudicaulis) with N. tabacum, because hybrids between N. sylvestris (maternal progenitor of N. nudicaulis) and N. tabacum are viable when grown in a greenhouse. In the cross N. trigonophylla×N. tabacum, approximately 50% of hybrids were vitrified, 20% were viable, and 20% were nonviable at 28°C. To reveal which subgenome of N. tabacum was responsible for these phenotypes, we crossed N. trigonophylla with two progenitors of N. tabacum, N. sylvestris (SS) and N. tomentosiformis (TT). In the cross N. sylvestris × N. trigonophylla, we confirmed that over half of hybrids of N. sylvestris × N. trigonophylla were vitrified, and none of the hybrids of N. trigonophylla × N. tomentosiformis were. The results imply that the S subgenome, encoding a gene or genes inducing hybrid lethality in the cross between N. nudicaulis and N. tabacum, has one or more genomic factors that induce vitrification. Furthermore, in vitrified hybrids of N. trigonophylla × N. tabacum and N. sylvestris × N. trigonophylla, we found that nuclear fragmentation, which progresses during expression of hybrid lethality, was accompanied by vitrification. This observation suggests that vitrification has a relationship to hybrid lethality. Based on these results, we speculate that when N. nudicaulis was formed approximately 5 million years ago, several causative genomic factors determining phenotypes of hybrid seedlings were inherited from N. trigonophylla. Subsequently, genome downsizing and various recombination-based processes took place. Some of the causative genomic factors were lost and some became genomic factor(s) controlling hybrid lethality in extant N. nudicaulis.

  6. Improved phytoaccumulation of cadmium by genetically modified tobacco plants (Nicotiana tabacum L.). Physiological and biochemical response of the transformants to cadmium toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Gorinova, N. [AgroBioInstitute, 8 Dragan Tzankov Blvd., 1164 Sofia (Bulgaria)]. E-mail: noraig60@yahoo.co.uk; Nedkovska, M. [AgroBioInstitute, 8 Dragan Tzankov Blvd., 1164 Sofia (Bulgaria); Todorovska, E. [AgroBioInstitute, 8 Dragan Tzankov Blvd., 1164 Sofia (Bulgaria); Simova-Stoilova, L. [Institute of Plant Physiology, Bulgarian Academy of Sciences, 1113 Sofia (Bulgaria); Stoyanova, Z. [Institute of Plant Physiology, Bulgarian Academy of Sciences, 1113 Sofia (Bulgaria); Georgieva, K. [Institute of Plant Physiology, Bulgarian Academy of Sciences, 1113 Sofia (Bulgaria); Demirevska-Kepova, K. [Institute of Plant Physiology, Bulgarian Academy of Sciences, 1113 Sofia (Bulgaria); Atanassov, A. [AgroBioInstitute, 8 Dragan Tzankov Blvd., 1164 Sofia (Bulgaria); Herzig, R. [Phytotech-Foundation PT-F, Quartiergasse 12, CH 3013 Bern (Switzerland)

    2007-01-15

    The response of tobacco plants (Nicotiana tabacum L.)-non-transformed and transformed with a metallothionein gene MThis from Silene vulgaris L. - to increase cadmium supply in the nutrient solution was compared. The transgenic plants accumulated significantly more Cd both in the roots and the leaves. Visual toxicity symptoms and disturbance in water balance were correlated with Cd tissue content. Treatment with 300 {mu}M CdCl{sub 2} resulted in inhibition of photosynthesis and mobilization of the ascorbate-glutathione cycle. Treatment with 500 {mu}M CdCl{sub 2} led to irreversible damage of photosynthesis and oxidative stress. An appearance of a new peroxidase isoform and changes in the leaf polypeptide pattern were observed at the highest Cd concentration. The level of non-protein thiols gradually increased following the Cd treatment both in transgenic and non-transformed plants. - Genetic transformation of Nicotiana tabacum L. by metallothionein gene improved phytoaccumulation of cadmium.

  7. Coat protein gene and 3′ non-coding region of tobacco mosaic virus and tomato mosaic virus are associated with viral pathogenesis in Nicotiana tabacum

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The camellia isolate of tomato mosaic virus (ToMV-TL) can induce local necrotic lesions on the inoculated leaves in Nicotiana tabacum, whereas the broad bean isolate of tobacco mosaic virus (TMV-B) produces the mosaic symptom on systemic leaves. To examine viral determinant for differential infection phenotype in N. tabacum, the coat protein gene and the 3′ non-coding region of TMV was replaced with that of ToMV, the chimeric virus induced similar local necrotic lesions to that induced by ToMV. The results indicate that the coat protein gene and the 3′ non-coding region of TMV and ToMV influence the virus-induced pathogenesis in N. tabacum.

  8. Organ- and Growing Stage-Specific Expression of Solanesol Biosynthesis Genes in Nicotiana tabacum Reveals Their Association with Solanesol Content

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    Ning Yan

    2016-11-01

    Full Text Available Solanesol is a noncyclic terpene alcohol that is composed of nine isoprene units and mainly accumulates in solanaceous plants, especially tobacco (Nicotiana tabacum L.. In the present study, RNA-seq analyses of tobacco leaves, stems, and roots were used to identify putative solanesol biosynthesis genes. Six 1-deoxy-d-xylulose 5-phosphate synthase (DXS, two 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR, two 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (IspD, four 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (IspE, two 2-C-methyl-d-erythritol 2,4-cyclo-diphosphate synthase (IspF, four 1-hydroxy-2-methyl-2-(E-butenyl 4-diphosphate synthase (IspG, two 1-hydroxy-2-methyl-2-(E-butenyl 4-diphosphate reductase (IspH, six isopentenyl diphosphate isomerase (IPI, and two solanesyl diphosphate synthase (SPS candidate genes were identified in the solanesol biosynthetic pathway. Furthermore, the two N. tabacum SPS proteins (NtSPS1 and NtSPS2, which possessed two conserved aspartate-rich DDxxD domains, were highly homologous with SPS enzymes from other solanaceous plant species. In addition, the solanesol contents of three organs and of leaves from four growing stages of tobacco plants corresponded with the distribution of chlorophyll. Our findings provide a comprehensive evaluation of the correlation between the expression of different biosynthesis genes and the accumulation of solanesol, thus providing valuable insight into the regulation of solanesol biosynthesis in tobacco.

  9. Cadmium and zinc activate adaptive mechanisms in Nicotiana tabacum similar to those observed in metal tolerant plants.

    Science.gov (United States)

    Vera-Estrella, Rosario; Gómez-Méndez, María F; Amezcua-Romero, Julio C; Barkla, Bronwyn J; Rosas-Santiago, Paul; Pantoja, Omar

    2017-04-28

    Tobacco germinated and grew in the presence of high concentrations of cadmium and zinc without toxic symptoms. Evidence suggests that these ions are sequestered into the vacuole by heavy metal/H (+) exchanger mechanisms. Heavy metal hyperaccumulation and hypertolerance are traits shared by a small set of plants which show specialized physiological and molecular adaptations allowing them to accumulate and sequester toxic metal ions. Nicotiana tabacum was used to test its potential as a metal-accumulator in a glass house experiment. Seed germination was not affected in the presence of increasing concentrations of zinc and cadmium. Juvenile and adult plants could concentrate CdCl2 and ZnSO4 to levels exceeding those in the hydroponic growth medium and maintained or increased their leaf dry weight when treated with 0.5- or 1-mM CdCl2 or 1-mM ZnSO4 for 5 days. Accumulation of heavy metals did not affect the chlorophyll and carotenoid levels, while variable effects were observed in cell sap osmolarity. Heavy metal-dependent H(+) transport across the vacuole membrane was monitored using quinacrine fluorescence quenching. Cadmium- or zinc-dependent fluorescence recovery revealed that increasing concentrations of heavy metals stimulated the activities of the tonoplast Cd(2+) or Zn(2+)/H(+) exchangers. Immunodetection of the V-ATPase subunits showed that the increased proton transport by zinc was not due to changes in protein amount. MTP1 and MTP4 immunodetection and semiquantitative RT-PCR of NtMTP1, NtNRAMP1, and NtZIP1 helped to identify the genes that are likely involved in sequestration of cadmium and zinc in the leaf and root tissue. Finally, we demonstrated that cadmium and zinc treatments induced an accumulation of zinc in leaf tissues. This study shows that N. tabacum possesses a hyperaccumulation response, and thus could be used for phytoremediation purposes.

  10. Spectral reflectance, chlorophyll fluorescence and virological investigations of tobacco plants (Nicotiana tabacum L.) infected with Tobacco mosaic virus (TMV)

    Science.gov (United States)

    Krezhova, Dora; Hristova, Dimitrina; Iliev, Ilko; Yanev, Tony

    Application of multispectral remote sensing techniques to plant condition monitoring has been adopted for various purposes. Remote sensing is a reliable tool for detecting signs of vege-tation stress and diseases. Spectral reflectance and chlorophyll fluorescence are functions of tissue optical properties and biological status of the plants, and illumination conditions. The mean reflectance spectrum depends on the relative composition of all the pigments in the leaf including chlorophylls, carotenoids etc. Chlorophyll fluorescence results from the primary re-actions of photosynthesis and during the last decade it finds widening application as a means for revelation of stress and diseases. The changes in chlorophyll function take place before the alteration in chlorophyll content to occur so that changes in the fluorescence signal arise before any visible signs are apparent. The aim of our investigations was to study the development and spreading out of a viral infection on the leaves of two cultivars tobacco plants (Nicotiana tabacum L.) infected with Tobacco mosaic virus (TMV). We applied two remote sensing tech-niques (spectral reflectance and chlorophyll fluorescence measurements) for evaluation of the changes in the optical properties of the plants in accordance to their physiological status. The serological analyses via the Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) were made with appropriate kits (Leowe, Germany) for quantitative assessment of the concentration of viruses in the plants. The tobacco plants were grown in green house under controlled conditions. The first cultivar Nevrocop 1146 is known as resistive to the TMV, i.e. it shows hypersensitive response. The second cultivar named Krumovgrad is normally sen-sitive to the TMV. At growth stage 4-6 expanded leaf, up to one leaf from 20 plants for each cultivar were inoculated with TMV. The leaves opposite to the infected ones formed the group of control (untreated) leaves. The

  11. Cloning, expression analysis and recombinant expression of a gene encoding a polygalacturonase-inhibiting protein from tobacco, Nicotiana tabacum

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    Chengsheng Zhang

    2016-05-01

    Full Text Available Polygalacturonase inhibiting proteins (PGIPs are major defensive proteins produced by plant cell walls that play a crucial role in pathogen resistance by reducing polygalacturonase (PG activity. In the present study, a novel PGIP gene was isolated from tobacco (Nicotiana tabacum, hereafter referred as NtPGIP. A full-length NtPGIP cDNA of 1,412 bp with a 186 bp 5′-untranslated region (UTR, and 209 bp 3′-UTR was cloned from tobacco, NtPGIP is predicted to encode a protein of 338 amino acids. The NtPGIP sequence from genomic DNA showed no introns and sequence alignments of NtPGIP’s deduced amino acid sequence showed high homology with known PGIPs from other plant species. Moreover, the putative NtPGIP protein was closely clustered with several Solanaceae PGIPs. Further, the expression profile of NtPGIP was examined in tobacco leaves following stimulation with the oomycete Phytophthora nicotianae and other stressors, including salicylic acid (SA, abscisic acid (ABA, salt, and cold treatment. The results showed that all of the treatments up-regulated the expression of NtPGIP at different times. To understand the biochemical activity of NtPGIP gene, a full-length NtPGIP cDNA sequence was subcloned into a pET28a vector and transformed into E. coli BL21 (DE3. Recombinant proteins were successfully induced by 1.0 nmol/L IPTG and the purified proteins effectively inhibited Phytophthora capsici PG activity. The results of this study suggest that NtPGIP may be a new candidate gene with properties that could be exploited in plant breeding.

  12. Effects of down-regulating ornithine decarboxylase upon putrescine-associated metabolism and growth in Nicotiana tabacum L.

    Science.gov (United States)

    Dalton, Heidi L; Blomstedt, Cecilia K; Neale, Alan D; Gleadow, Ros; DeBoer, Kathleen D; Hamill, John D

    2016-05-01

    Transgenic plants of Nicotiana tabacum L. homozygous for an RNAi construct designed to silence ornithine decarboxylase (ODC) had significantly lower concentrations of nicotine and nornicotine, but significantly higher concentrations of anatabine, compared with vector-only controls. Silencing of ODC also led to significantly reduced concentrations of polyamines (putrescine, spermidine and spermine), tyramine and phenolamides (caffeoylputrescine and dicaffeoylspermidine) with concomitant increases in concentrations of amino acids ornithine, arginine, aspartate, glutamate and glutamine. Root transcript levels of S-adenosyl methionine decarboxylase, S-adenosyl methionine synthase and spermidine synthase (polyamine synthesis enzymes) were reduced compared with vector controls, whilst transcript levels of arginine decarboxylase (putrescine synthesis), putrescine methyltransferase (nicotine production) and multi-drug and toxic compound extrusion (alkaloid transport) proteins were elevated. In contrast, expression of two other key proteins required for alkaloid synthesis, quinolinic acid phosphoribosyltransferase (nicotinic acid production) and a PIP-family oxidoreductase (nicotinic acid condensation reactions), were diminished in roots of odc-RNAi plants relative to vector-only controls. Transcriptional and biochemical differences associated with polyamine and alkaloid metabolism were exacerbated in odc-RNAi plants in response to different forms of shoot damage. In general, apex removal had a greater effect than leaf wounding alone, with a combination of these injury treatments producing synergistic responses in some cases. Reduced expression of ODC appeared to have negative effects upon plant growth and vigour with some leaves of odc-RNAi lines being brittle and bleached compared with vector-only controls. Together, results of this study demonstrate that ornithine decarboxylase has important roles in facilitating both primary and secondary metabolism in Nicotiana.

  13. Gene-splitting technology: a novel approach for the containment of transgene flow in Nicotiana tabacum.

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    Xu-Jing Wang

    Full Text Available The potential impact of transgene escape on the environment and food safety is a major concern to the scientists and public. This work aimed to assess the effect of intein-mediated gene splitting on containment of transgene flow. Two fusion genes, EPSPSn-In and Ic-EPSPSc, were constructed and integrated into N. tabacum, using Agrobacterium tumefaciens-mediated transformation. EPSPSn-In encodes the first 295 aa of the herbicide resistance gene 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS fused with the first 123 aa of the Ssp DnaE intein (In, whereas Ic-EPSPSc encodes the 36 C-terminal aa of the Ssp DnaE intein (Ic fused to the rest of EPSPS C terminus peptide sequences. Both EPSPSn-In and Ic-EPSPSc constructs were introduced into the same N. tabacum genome by genetic crossing. Hybrids displayed resistance to the herbicide N-(phosphonomethyl-glycine (glyphosate. Western blot analysis of protein extracts from hybrid plants identified full-length EPSPS. Furthermore, all hybrid seeds germinated and grew normally on glyphosate selective medium. The 6-8 leaf hybrid plants showed tolerance of 2000 ppm glyphosate in field spraying. These results indicated that functional EPSPS protein was reassembled in vivo by intein-mediated trans-splicing in 100% of plants. In order to evaluate the effect of the gene splitting technique for containment of transgene flow, backcrossing experiments were carried out between hybrids, in which the foreign genes EPSPSn-In and Ic-EPSPSc were inserted into different chromosomes, and non-transgenic plants NC89. Among the 2812 backcrossing progeny, about 25% (664 plantlets displayed glyphosate resistance. These data indicated that transgene flow could be reduced by 75%. Overall, our findings provide a new and highly effective approach for biological containment of transgene flow.

  14. Cloning the bacterial bphC gene into Nicotiana tabacum to improve the efficiency of phytoremediation of polychlorinated biphenyls

    Science.gov (United States)

    Novakova, Martina; Mackova, Martina; Antosova, Zuzana; Viktorova, Jitka; Szekeres, Miklos; Demnerova, Katerina

    2010-01-01

    The aim of this work was to construct transgenic plants with increased capabilities to degrade organic pollutants, such as polychlorinated biphenyls. The environmentally important gene of bacterial dioxygenase, the bphC gene, was chosen to clone into a plant of Nicotiana tabacum. The chosen bphC gene encodes 2,3-dihydroxybiphenyl-1,2-dioxygenase, which cleaves the aromatic ring of dihydroxybiphenyl, and we cloned it in fusion with the gene for β-glucuronidase (GUS), luciferase (LUC) or with a histidine tail. Several genetic constructs were designed and prepared and the possible expression of desired proteins in tobacco plants was studied by transient expression. We used genetic constructs successfully expressing dioxygenase's genes we used for preparation of transgenic tobacco plants by agrobacterial infection. The presence of transgenic DNA , mRNA and protein was determined in parental and the first filial generation of transgenic plants with the bphC gene. Properties of prepared transgenic plants will be further studied. PMID:21468210

  15. Effects of selenium on the growth and photosynthetic characteristics of flue-cured tobacco (Nicotiana tabacum L.

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    Chaoqiang Jiang

    2015-03-01

    Full Text Available The objective of this study was to investigate the effect of Selenium (Se supply (0, 3, 6, 12, 24 mg kg−1 on the growth, photosynthetic characteristics, Se accumulation and distribution of flue-cured tobacco (Nicotiana tabacum L.. Results showed that low-dose Se treatments (≤6 mg kg−1 stimulated plant growth but high-dose Se treatments (≥12 mg kg−1 hindered plant growth. Optimal Se dose (6 mg kg−1 stimulated plant growth by reducing MDA content and improving photosynthetic capability. However, excess Se (24 mg kg−1 increased MDA content by 28%, decreased net photosynthetic rate and carboxylation efficiency by 34% and 39%, respectively. The Se concentration in the roots, stems, and leaves of the tobacco plants significantly increased with increasing Se application. A linear correlation (R = 0.95, P leaf > stem. The Se concentration in the roots was 3.17 and 7.57 times higher than that in the leaves and stems, respectively, after treatment with 24 mg kg−1 Se. In conclusion, the present study suggested that optimal Se dose (6 mg kg−1 improved the plant growth mainly by enhancing photosynthesis, stomatal conductance, carboxylation efficiency and Rubisco content in the flue-cured tobacco leaves. However, the inhibition of excess Se on tobacco growth might be due to high accumulation of Se in roots and the damage of photosynthesis in leaves.

  16. Pretreatment with alternation of light/dark periods improves the tolerance of tobacco (Nicotiana tabacum) to clomazone herbicide.

    Science.gov (United States)

    Darwish, Majd; Lopez-Lauri, Félicie; El Maataoui, Mohamed; Urban, Laurent; Sallanon, Huguette

    2014-05-01

    This work analyses the effects of alternation of light/dark periods pretreatment (AL) in tobacco plantlets (Nicotiana tabacum L. cv.Virginie vk51) growing in solution with low concentration of the clomazone herbicide. The experimentation has been carried out by exposing the plantlets to successive and regulated periods of light (16min light/8min dark cycles, PAR 50μmolm(-2)s(-1)) for three days. The photosynthesis efficiency was determined by mean of the chlorophyll fluorescence and JIP-test. The AL pretreatment improved the clomazone tolerance; this has been observed by the increase in the leaf area of the plant, the maximal photochemical quantum efficiency of PSII (Fv/Fm), the actual PSII efficiency (ФPSII), the performance index (PIabs), the electron flux beyond Quinone A (1-VJ), and also by the diminution of the energy dissipating into heat (DI0/RC). Furthermore, AL pretreatment led to low accumulation of hydrogen peroxide (H2O2) which proves that the scavenging enzymatic system have been activated before clomazone treatment. In the plantlets pretreated with AL, with regard to the ascorbate content, some of antioxidant enzyme whose function is associated with it have continued to scavenge reactive oxygen species (ROS) induced by clomazone, such as ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR). So, the observed photooxidative damages induced by clomazone herbicide were noticeably reduced.

  17. Behaviour of Potassium and Trace Elements in Rhizosphere of Flue—Cured Tobacco (Nicotiana Tabacum L.)

    Institute of Scientific and Technical Information of China (English)

    HUGUO-SONG; CAOZHI-HONG; 等

    1993-01-01

    The study on the behaviour of potassium,phosphate and trace elements,Fe,Mn,Cu and Zn,in the rhizosphere of different varieties of flue-cured tobacco (Nicotiana tabacum L.)with high and low potassium application rate with rhizobag technique showed that soil available K,soil available P,and slow available K was in depletion status,whereas DTPA extractable Fe,Mn,Zn and Cu accumulated obviously in rhizosphere.The depletion and accumulation rates of mineral nutrients differed in degree with K application rate,soil type,and tobacco variety.The content of available K in both rhizosphere and bulk soil and K concentration in tobacco leaf increased significantly,and the available P in rhizosphere dropped with more K application.The DTPA-Fe content of red soil much lower in pH was higher than that of calcareous soil in bulk soil.But the DTPA-Fe content of calcareous soil was much higher than that of red soil in rhizosphere,which was considered perhaps to be mainly related to releasing of Fe phytosiderophore.Nitrate coule increase depletion of a vailable K in rhizosphere and also soil pH in comparision with ammonium.

  18. ‘COROJO 2006’: NUEVA VARIEDAD CUBANA DE TABACO NEGRO (Nicotiana tabacum L.

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    Vivaldo García Morejón

    2013-01-01

    Full Text Available En la Estación Experimental del Tabaco, de San Juan y Martínez, Pinar del Río durante la campaña tabacalera 2001-2002, se realizó un cruzamiento sexual con el objetivo de obtener una variedad de tabaco negro con mayor rendimiento que la comercial ‘Criollo 98’, resistente al moho azul ( Peronospora hyocyami de Bary, a la pata prieta ( Phytophthora parasitica Dast. var . Nicotianae Breda de Haan, al virus del mosaico del tabaco (VMT y a la necrosis ambiental. Después de cinco generaciones de autofecundación y selección por el método genealógico se obtuvieron siete variedades resistentes. El cruce originó la nueva variedad `Corojo 2006 ́ que superó a la `Criollo 98 ́.

  19. Characterization of cDNA for PMT: a Partial Nicotine Biosynthesis-Related Gene Isolated from Indonesian Local Tobacco (Nicotiana tabacum cv. Sindoro1

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    Sesanti Basuki

    2013-12-01

    Full Text Available Nicotine is the major alkaloid compound in cultivated tobacco (Nicotiana tabacum that could potentially be converted into carcinogenic compound (nor-nicotine. The PMT gene encoding putrescine N-methyltransferase (PMT is one of the two key genes that play a prominent role in nicotine biosynthesis. The aimed of this study was to isolate and characterize the cDNA sequence originated from Indonesian local tobacco cv. Sindoro1 (Ntpmt_Sindoro1. The results showed that the Ntpmt_Sindoro1 was 1124 bp in length. This cDNA fragment encodes for 374 amino acid residues. The predicted polypeptide from the cDNA is a hidrophilic protein, and has a predicted molecular weight of 40.95 kD. The predicted amino acids sequence also showed high similarity to the PMT gene product Nicotiana sp. available in the GenBank data base. The amino acid sequences also exert conserved residues specifically exhibited only by PMT gene originated from N. tabacum. Clustering analysis revealed that Ntpmt_Sindoro1 belongs to the same clade as the PMT3 gene, a member of the N. tabacum PMT gene family. The Ntpmt_Sindoro1 cDNA sequence covering exon1-exon8 of the PMT gene fragment has been registered in the GenBank data base, under the accession number JX978277.

  20. Combination of Nicotiana tabacum and Azadirachta indica: A Novel Substitute to Contain Prevalence of Levamisole and Ivermectin-Resistant Haemonchus contortus in Ovine

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    Kareem Khoshnow Hamad

    2014-01-01

    Full Text Available The current study was aimed at exploring the activity of combined crude aqueous methanol extracts (CAMEs of Nicotiana (N. tabacum leaves and Azadirachta (A. indica seed kernels to control levamisole (LEV and ivermectin (IVM-resistant Haemonchus (H. contortus in sheep. The in vivo fecal egg count reduction test (FECRT demonstrated rampancy of resistance among H. contortus populations against LEV and IVM. There was a significant difference (P<0.05 between groups treated with the combined plant extracts compared to other groups treated with solitary N. tabacum and A. indica. The recorded FECR% at low (1+1g kg-1 BW and high (2+2g kg-1 BW doses was 52.71 and 94.59, respectively for the combined extracts, whilst for N. tabacum at low (2g kg-1 BW and high (4g kg-1 BW doses, the FECR% was 87.5 and 88.6, respectively. Moreover, the FECR% for A. indica was 45.62 and 85.14 at low (2g kg-1 BW and high (4g kg-1 BW doses, respectively. There was a dose and time dependent ovicidal and adulticidal activity of CAMEs with estimated LC50 values of 0.523, 0.566 and 1.169 μg ml-1 for the combined extracts, solitary N. tabacum and A. indica, respectively in egg hatch assay (in vitro test. The estimated LC50 values in adult motility test (in vitro test 10 hours post-exposure were 0.17, 0.20 and 0.80 mg ml-1 for the combined extracts, sole N. tabacum and A. indica, respectively. Accordingly, the aforementioned combined extracts at high dose were more efficacious than solitary N. tabacum and A. indica and, thus, can be used to restrict rampancy of LEV and IVM-resistant H. contortus populations parasitized in sheep.

  1. UJI TOLERANSI TANAMAN TEMBAKAU (Nicotiana tabacum L. TERHADAP CEKAMAN KADMIUM (Cd, TIMBAL (Pb, DAN TEMBAGA (Cu PADA KULTUR CAIR

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    S Rosidah

    2014-11-01

    Full Text Available Penelitian ini menyelidiki respon fisiologis, anatomis, dan morfologis tanaman tembakau (Nicotiana tabacum L. terhadap cekaman logam berat Cu, Cd, dan Pb. Sampel yang digunakan adalah tembakau umur 3-4 minggu yang dikecambahkan secara in vitro dan kemudian dipapar logam berat selama 14 hari. Desain penelitian yang digunakan yaitu rancangan acak lengkap dengan satu faktor, yaitu konsentrasi logam Cu (0 µM, 50 µM, 100 µM, 150 µM & 200 µM, Cd (0 µM, 50 µM, 100 µM, 200 µM & 300 µM, dan Pb (0 µM, 5 µM, 20 µM, 50 µM & 100 µM. Parameter yang digunakan: pertambahan panjang akar, pertambahan jumlah akar, akumulasi logam dalam akar, lokalisasi penimbunan dalam akar, dan warna daun. Bertambahnya konsentrasi logam menghambat pertumbuhan akar dan menyebabkan deposit logam pada jaringan akar dan gejala klorosis. Hasil uji Atomic Absorbtion Spectrophotometry (AAS menunjukkan semakin besar konsentrasi semakin banyak akumulasi logam pada jaringan akar. Akan tetapi, akumulasi Cd pada konsentrasi 200 µM lebih besar dibanding pada konsentrasi 300 µM. Analisis kualitatif membuktikan bahwa cekaman Cu tidak berpengaruh signifikan terhadap warna daun, sedangkan pada cekaman Cd (100, 150 dan 200 µM dan Pb (150 µM daun mengalami klorosis. Pada konsentrasi logam yang rendah seperti 50 µM Cu, 50 µM Cd, dan 5 µM Pb tidak berbeda nyata dengan kontrol. Dengan demikian disimpulkan bahwa tembakau mampu mentoleransi cekaman logam pada konsentrasi yang rendah.  This research investigated the physiological, anatomical, and morphological responses of tobacco (Nicotiana tabacum L. on stresses of heavy metals Cu, Cd, and Pb. The samples were 3- to 4-week tobacco plants germinated in vitro and then were exposed to heavy metals for 14 days. This study used a completed random design with single factor, i.e. the concentrations of Cu (0 µM, 50 µM, 100 µM, 150 µM & 200 µM, Cd (0 µM, 50 µM, 100 µM, 200 µM & 300 µM, and Pb (0 µM, 5 µM, 20 µM, 50 µM & 100

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

    Science.gov (United States)

    Lewis, Ramsey S; Bowen, Steven W; Keogh, Matthew R; Dewey, Ralph E

    2010-12-01

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

  3. Comparative study on macro- and micro-elements concentration in Nicotiana tabacum and Faba siliquis plants by ICP-MS

    Science.gov (United States)

    Balazs, Zoltan; Voica, Cezara; Dehelean, Adriana; Magdas, Dana Alina; Ristoiu, Dumitru

    2015-12-01

    Plants are important components of ecosystems as they transfer elements from abiotic into biotic environments. The concentration of macro and micro-elements in tobacco leaves (Nicotiana tabacum) and bean (Faba siliquis) was analyzed using ICP-MS technique. The results obtained indicated that the mean concentration of Mg, P, K and Ca in tobacco leaves was 0.965, 0.812, 4.412 and 2.694 g.kg-1, respectively, while in bean samples were 0.899, 2.024, 6.725 and 1.387 g.kg-1, respectively. Mn concentration ranged from 156.835 mg.kg-1 to 234.593 mg.kg-1 in tobacco leaves and from 116.174 mg.kg-1 to 440.423 mg.kg-1 in bean samples. The results for Cu and Zn were between 7.262 mg.kg-1 and 105.738 mg.kg-1, 68.549 mg.kg-1 and 113.720 mg.kg-1 (tobacco leaves); and 6.830 mg.kg-1 and 46.034 mg.kg-1, 50.166 mg.kg-1 and 77.242 mg.kg-1 (bean samples), respectively. In analyzed samples, Pb, Cd and As concentrations ranged between <0.001-0.717 mg.kg-1, 0.046 mg.kg-1 -6.218 mg.kg-1, <0.001-0.381 mg.kg-1. The paper discusses the transfer of metal ions (Mn, As, Cd, Cu, Pb and Zn, respectively) from soil to these plants in terms of transfer factors (TF).

  4. In vitro Anthelmintic Activity of Oily Extracts of Azadirachta indica and Aqueous Extracts of Nicotiana tabacum on Gastrointestinal Nematodes in Goats

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    Richard Zapata Salas

    2013-12-01

    Full Text Available Gastrointestinal nematodes are the most common parasites found in ruminants in the world. These parasites cause parasitic gastroenteritis and have a negative effect on productivity. Nematode control has been based on the use of anthelmintic chemicals—against which nematodes have developed a certain degree of resistance—which have been rated as residual substances in goat products that may cause adverse effects on the final consumer. As a result, the study of plant components has been proposed as a sustainable alternative to control nematodosis in goats. The anthelmintic potential of aqueous extracts of Nicotiana tabacum and oily extracts of Azadirachta indica on gastrointestinal nematodes affecting the goat chain was evaluated in vitro in this study. Nematicidal activity tests were performed on stool samples from goats with a high parasite load (trichostrongyles, by performing dose/ response curves. Percent inhibition in egg hatch for the aqueous extract of N. tabacum and the oily extract of A. indica was 99% and 80%, respectively. Extracts showed an effect on larva 3 (infective stage, with a mean lethal time of 8 ± 1 minutes for extracts of N. Tabacum, and of 8 ± 1 minutes for extract of A. indica. The in vitro results of the nematicidal activity show that N. tabacum and A. indica extracts can be a promising alternative for controlling nematodes in ruminants.

  5. Transfer of the cytochrome P450-dependent dhurrin pathway from Sorghum bicolor into Nicotiana tabacum chloroplasts for light-driven synthesis

    DEFF Research Database (Denmark)

    Gnanasekaran, Thiyagarajan; Karcher, Daniel; Nielsen, Agnieszka Janina Zygadlo;

    2016-01-01

    Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product....... For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble...... glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed...

  6. Effects of Initial Infestation Levels ofCallosobruchus maculatus(F.) (Coleoptera:Chrysomelidae) on Cowpea and Use ofNicotiana tabacum L. Aqueous Extract as Grain Protectant

    Institute of Scientific and Technical Information of China (English)

    Musa A K; Odunayo A; Adeyeye O E

    2015-01-01

    This study determined the effects of initial infestation of cowpea seeds (Ife brown variety) with different insect densities (0, 2, 4 and 6 pairs per 50 g seeds) ofCallosobruchus maculatus(F.) and evaluated the effects of aqueous leaf extract of Nicotiana tabacum L. onC. maculatus in the laboratory. It was observed that adult beetle population increased significantly (p<0.05) with increase in insect density. The increase in population of beetles and corresponding weight loss of the seeds in different levels of infestation showed that the cowpea variety was susceptible to beetle infestation, emergence and survival of progeny. Significantly more adults emerged on higher infestation compared to lower and no infestation. In Nigeria,Nicotiana tabacum L. is a locally available plant, with known insecticidal properties. The plant leaf extract was easily extracted with water and confirmed its effectiveness as a protective agent for stored cowpea seeds. Experiment was conducted to assess the effects of aqueous extracts of N. tabacumat 0, 0.1, 0.2 and 0.3 mL•50 g-1 cowpea seeds onC. maculatus. Data was recorded and showed varying levels of effectiveness againstC. maculatus. Result showed that seed appearance was dependent on levels of insect population, while N. tabacum aqueous extract exerted effects on survival ofC. maculatus. Aqueous leaf extract ofN. tabacum probably contained some insecticidal properties which might have significantly conferred beetle mortality and reduced beetle emergence leading to a decrease in seed weight loss.

  7. Cultivos celulares de nicotiana tabacum L.cv.BY-2 como sistema modelo en el estudio de la adaptación al estrés salino.

    OpenAIRE

    García de la Garma García, Jesús

    2013-01-01

    El objetivo general de esta tesis doctoral es ampliar los conocimientos sobre los mecanismos que permiten la adaptación de los cultivos celulares de Nicotiana tabacum cv. BY-2 al estrés abiótico, concretamente estrés salino. Para ello, hemos establecido como objetivos específicos los siguientes: 1. Estudiar diferentes mecanismos fisiológicos, bioquímicos y celulares de adaptación a salinidad. 2. Estudiar el papel que desempeñan las giberelinas (GAs) en el proceso de adaptación a salini...

  8. Assimilation of phytate-phosphorus by the extracellular phytase activity of tobacco (Nicotiana tabacum) is affected by the availability of soluble phytate

    OpenAIRE

    2006-01-01

    Phytate, the major organic phosphorus in soil, is not readily available to plants as a source of phosphorus (P). It is either complexed with cations or adsorbed to various soil components. The present study was carried out to investigate the extracellular phytase activities of tobacco (Nicotiana tabacum variety GeXin No.1) and its ability to assimilate external phytate-P. Whereas phytase activities in roots, shoots and growth media of P i-fed 14-day-old seedlings were only 1.3-4.9% of total a...

  9. "Nicotiana tabacum" L. cv Xanthi como sistema heterólogo para la producción de lactógeno placentario humano (hPL)

    OpenAIRE

    Urreta Gómez, Iratxe

    2012-01-01

    152 p. : il., col. El lactógeno placentario humano (hPL) es una hormona polipeptídica que forma parte de la pequeña lista de factores del crecimiento capaces de tratar la diabetes tipo 1 mediante el transplante de islotes humanos. Esta hormona tiene la capacidad de mejorar la supervivencia de los islotes humanos antes y/o después de su transplante. En este trabajo se aborda la producción de hPL en sistemas heterólogos vegetales basados en Nicotiana tabacum cv Xanthi. El cDNA del hPL ha sid...

  10. Transfer of the cytochrome P450-dependent dhurrin pathway from Sorghum bicolor into Nicotiana tabacum chloroplasts for light-driven synthesis

    DEFF Research Database (Denmark)

    Gnanasekaran, Thiyagarajan; Karcher, Daniel; Nielsen, Agnieszka Janina Zygadlo;

    2016-01-01

    . For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble...... compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons....

  11. Antigen production using heterologous expression of dengue virus-2 non-structural protein 1 (NS1) in Nicotiana tabacum (Havana) for immunodiagnostic purposes.

    Science.gov (United States)

    Amaro, Marilane O F; Xisto, Mariana F; Dias, Ana Carolina F; Versiani, Alice F; Cardoso, Silvia A; Otoni, Wagner C; da Silva, Cynthia C; De Paula, Sérgio O

    2015-06-01

    Expression of dengue-2 virus NS1 protein in Nicotiana tabacum plants for development of dengue immunodiagnostic kits. Dengue is one of the most important diseases caused by arboviruses in the world. A significant increase in its geographical distribution has been noticed over the last 20 years, with continuous transmission of several serotypes and emergence of the hemorrhagic fever in areas where the disease was previously not prevalent. Although the methodological processes for dengue diagnosis are in deep development and improvement, a limitation for the realization of dengue diagnostic tests is the difficulty of large-scale production of the antigen to be used in diagnostic tests. Due to this demand, the purpose of this study was to obtain the non-structural protein 1 (NS1) from dengue-2 serotype by heterologous expression in Nicotiana tabacum (Havana). After confirmation of the NS1 protein gene integration in the plant genome, the heterologous protein was characterized using SDS-PAGE and immunoblotting. In an immunoenzymatic test, the recombinant NS1 protein presents an antigen potential for development of dengue immunodiagnostic kits.

  12. Effectiveness of Biopesticide Derived fromCassia spectabilis and Nicotiana tabacum Leaves Against the Main Insect Pests of Coffee and Its Effect On Other Arthropods

    Directory of Open Access Journals (Sweden)

    Soekadar Wiryadiputra

    2006-05-01

    Full Text Available An experiment on the effectiveness of biopesticide made of ramayana (Cassia spectabilis and tobacco leaves (Nicotiana tabacum it called as Casnic, on Hypothenemus hampeiand Planococcus citriand its effect on the arthropods population fauna in coffee plantation had been conducted in Pest Laboratory of Indonesian Coffee and Cocoa Researh Institute and Bangelan Plantation in Malang. The treatment consisted of three level concentrations of botanical pesticide(15 ml, 30 ml, and 60 ml/lof water, Beauveria bassiana(at a dose 100 g spore per ha, metidation spraying (2 ml formulation per litre of water and two control treatments (with and without soap. The field experiment was arranged in randomized complete block design (RCBD with four replications. The results showed that botanical pesticide at the concentration of 30.0 ml stock solution per litre of water and applied four times with monthly interval was effective in controlling coffee berry borer (Hypothenemus hampei and coffee mealy bug (Planococcus citri. The effectiveness was not significantly different compared to metidation and Beauveria bassianaat the dose of 0.8 l and 100 g formulation per ha per application. Application of the botanical pesticide did not show negative effect on the usefull predatory insects and other arthropods population in coffee ecosystem. Key words : Botanical pesticide, Cassia spectabilis, Nicotiana tabacum, Coffee, Hypothenemus hampei, Planococcus citri, Arthropods.

  13. Isonitrosoacetophenone drives transcriptional reprogramming in Nicotiana tabacum cells in support of innate immunity and defense.

    Directory of Open Access Journals (Sweden)

    Arnaud T Djami-Tchatchou

    Full Text Available Plants respond to various stress stimuli by activating broad-spectrum defense responses both locally as well as systemically. As such, identification of expressed genes represents an important step towards understanding inducible defense responses and assists in designing appropriate intervention strategies for disease management. Genes differentially expressed in tobacco cell suspensions following elicitation with isonitrosoacetophenone (INAP were identified using mRNA differential display and pyro-sequencing. Sequencing data produced 14579 reads, which resulted in 198 contigs and 1758 singletons. Following BLAST analyses, several inducible plant defense genes of interest were identified and classified into functional categories including signal transduction, transcription activation, transcription and protein synthesis, protein degradation and ubiquitination, stress-responsive, defense-related, metabolism and energy, regulation, transportation, cytoskeleton and cell wall-related. Quantitative PCR was used to investigate the expression of 17 selected target genes within these categories. Results indicate that INAP has a sensitising or priming effect through activation of salicylic acid-, jasmonic acid- and ethylene pathways that result in an altered transcriptome, with the expression of genes involved in perception of pathogens and associated cellular re-programming in support of defense. Furthermore, infection assays with the pathogen Pseudomonas syringae pv. tabaci confirmed the establishment of a functional anti-microbial environment in planta.

  14. Expression of recombinant antibody (single chain antibody fragment) in transgenic plant Nicotiana tabacum cv. Xanthi.

    Science.gov (United States)

    Dobhal, S; Chaudhary, V K; Singh, A; Pandey, D; Kumar, A; Agrawal, S

    2013-12-01

    Plants offer an alternative inexpensive and convenient technology for large scale production of recombinant proteins especially recombinant antibodies (plantibodies). In this paper, we describe the expression of a model single chain antibody fragment (B6scFv) in transgenic tobacco. Four different gene constructs of B6scFv with different target signals for expression in different compartments of a tobacco plant cell with and without endoplasmic reticulum (ER) retention signal were used. Agrobacterium mediated plant transformation of B6scFv gene was performed with tobacco leaf explants and the gene in regenerated plants was detected using histochemical GUS assay and PCR. The expression of B6scFv gene was detected by western blotting and the recombinant protein was purified from putative transgenic tobacco plants using metal affinity chromatography. The expression level of recombinant protein was determined by indirect enzyme-linked immunosorbent assay. The highest accumulation of protein was found up to 3.28 % of the total soluble protein (TSP) in plants expressing B6scFv 1003 targeted to the ER, and subsequently expression of 2.9 % of TSP in plants expressing B6scFv 1004 (with target to apoplast with ER retention signal). In contrast, lower expression of 0.78 and 0.58 % of TSP was found in plants expressing antibody fragment in cytosol and apoplast, without ER retention signal. The described method/system could be used in the future for diverse applications including expression of other recombinant molecules in plants for immunomodulation, obtaining pathogen resistance against plant pathogens, altering metabolic pathways and also for the expression of different antibodies of therapeutic and diagnostic uses.

  15. Posttranslational processing of a new class of hydroxyproline-containing proteins. Prolyl hydroxylation and C-terminal cleavage of tobacco (Nicotiana tabacum) vacuolar chitinase.

    Science.gov (United States)

    Sticher, L; Hofsteenge, J; Neuhaus, J M; Boller, T; Meins, F

    1993-04-01

    The fungicidal class I chitinases (EC 3.2.1.14) are believed to be important in defending plants against microbial pathogens. The vacuolar isoforms of tobacco (Nicotiana tabacum), chitinases A and B, are the first examples of a new type of hydroxyproline-containing protein with intracellular location, enzymic activity, and a small number of hydroxyprolyl residues restricted to a single, short peptide sequence. We have investigated the posttranslational processing and intracellular transport of transgene-encoded chitinase A in callus cultures of Nicotiana tabacum L. cv Havana 425 and leaves of Nicotiana sylvestris Spegazzini and Comes. Pulse-chase experiments and cell fractionation show that chitinase A is processed in two distinct steps. In the first step, the nascent protein undergoes an increase in apparent M(r) of approximately 1500 detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experiments with the inhibitor of prolyl hydroxylation, alpha,alpha'-dipyridyl, and pulse-chase labeling of cells expressing recombinant forms of chitinase A indicate that the anomalous increase in M(r) is due to hydroxylation of prolyl residues. This step occurs in the endomembrane system before sorting for secretion and vacuolar transport and does not appear to be required for correct targeting of chitinase A to the vacuole. The second step is a proteolytic cleavage. Sequencing of tryptic peptides of the mature proteins indicates that during processing essentially all molecules of chitinase A and B lose a C-terminal heptapeptide, which has been shown to be a vacuolar targeting signal. This appears to occur primarily in the endomembrane system late in intracellular transport. A model for the posttranslational modification of chitinase A is proposed.

  16. Differences in the Detoxification Metabolism between Two clonal Lineages of the Aphid Myzus persicae (Sulzer (Hemiptera: Aphididae Reared on Tobacco (Nicotiana tabacum L. Diferencias en el Metabolismo de Detoxificación entre dos Linajes Clonales del Áfido Myzus persicae (Sulzer (Hemiptera: Aphididae creados sobre tabaco (Nicotiana tabacum L.

    Directory of Open Access Journals (Sweden)

    Marco A Cabrera-Brandt

    2010-12-01

    Full Text Available Myzus persicae (Sulzer is a highly polyphagous aphid species, with a subspecies (M. persicae nicotianae well adapted to tobacco (Nicotiana tabacum L.. We evaluated the effect of this host plant on the aphid performance and detoxification enzymes, in order to test the participation of xenobiotic metabolism on the ability of this aphid to overcome the tobacco chemical defences. Two genotypes, one corresponding to the only M. persicae nicotianae genotype reported in Chile on tobacco, and one genotype belonging to M. persicae sensu stricto were reared on tobacco and pepper (Capsicum annuum L., respectively. M. persicae nicotianae showed a significantly higher intrinsic rate of increase (r m on pepper than on tobacco, and M. persicae s.s. performed similarly, but with no reproduction at all on tobacco. In order to evaluate the effect of tobacco on detoxification enzymes, esterases, glutathione S-transferases (GST and cytochrome P-450 monooxygenases (MO were determined in both selected aphid genotypes after 12, 24, 36, 48 and 72 h of rearing on tobacco and pepper. M. persicae nicotianae exhibited the higher total esterase activities when reared on tobacco than on pepper after 48 h of rearing, while the activities of GST and MO did not show any significant difference between host-plants and duration of treatment. For M. persicae s.s., no significant differences were observed among host-plants for the studied enzymes. These results suggest a participation of the esterases, on the ability of this M. persicae nicotianae to overcome the tobacco defences.Myzus persicae (Sulzer es un áfido polífago que incluye a Myzus persicae nicotianae, una subespecie altamente adaptada sobre tabaco (Nicotiana tabacum L.. Evaluamos el efecto del tabaco sobre el desempeño biológico y sobre determinadas enzimas de detoxificación en áfidos, para estudiar su participación en la capacidad de M. persicae nicotianae de superar las defensas químicas del tabaco. Dos

  17. Molecular cloning and functional characterization of the lycopene ε-cyclase gene via virus-induced gene silencing and its expression pattern in Nicotiana tabacum.

    Science.gov (United States)

    Shi, Yanmei; Wang, Ran; Luo, Zhaopeng; Jin, Lifeng; Liu, Pingping; Chen, Qiansi; Li, Zefeng; Li, Feng; Wei, Chunyang; Wu, Mingzhu; Wei, Pan; Xie, He; Qu, Lingbo; Lin, Fucheng; Yang, Jun

    2014-08-22

    Lycopene ε-cyclase (ε-LCY) is a key enzyme that catalyzes the synthesis of α-branch carotenoids through the cyclization of lycopene. Two cDNA molecules encoding ε-LCY (designated Ntε-LCY1 and Ntε-LCY2) were cloned from Nicotiana tabacum. Ntε-LCY1 and Ntε-LCY2 are encoded by two distinct genes with different evolutionary origins, one originating from the tobacco progenitor, Nicotiana sylvestris, and the other originating from Nicotiana tomentosiformis. The two coding regions are 97% identical at the nucleotide level and 95% identical at the amino acid level. Transcripts of Ntε-LCY were detectable in both vegetative and reproductive organs, with a relatively higher level of expression in leaves than in other tissues. Subcellular localization experiments using an Ntε-LCY1-GFP fusion protein demonstrated that mature Ntε-LCY1 protein is localized within the chloroplast in Bright Yellow 2 suspension cells. Under low-temperature and low-irradiation stress, Ntε-LCY transcript levels substantially increased relative to control plants. Tobacco rattle virus (TRV)-mediated silencing of ε-LCY in Nicotiana benthamiana resulted in an increase of β-branch carotenoids and a reduction in the levels of α-branch carotenoids. Meanwhile, transcripts of related genes in the carotenoid biosynthetic pathway observably increased, with the exception of β-OHase in the TRV-ε-lcy line. Suppression of ε-LCY expression was also found to alleviate photoinhibition of Potosystem II in virus-induced gene silencing (VIGS) plants under low-temperature and low-irradiation stress. Our results provide insight into the regulatory role of ε-LCY in plant carotenoid biosynthesis and suggest a role for ε-LCY in positively modulating low temperature stress responses.

  18. Molecular Cloning and Functional Characterization of the Lycopene ε-Cyclase Gene via Virus-Induced Gene Silencing and Its Expression Pattern in Nicotiana tabacum

    Directory of Open Access Journals (Sweden)

    Yanmei Shi

    2014-08-01

    Full Text Available Lycopene ε-cyclase (ε-LCY is a key enzyme that catalyzes the synthesis of α-branch carotenoids through the cyclization of lycopene. Two cDNA molecules encoding ε-LCY (designated Ntε-LCY1 and Ntε-LCY2 were cloned from Nicotiana tabacum. Ntε-LCY1 and Ntε-LCY2 are encoded by two distinct genes with different evolutionary origins, one originating from the tobacco progenitor, Nicotiana sylvestris, and the other originating from Nicotiana tomentosiformis. The two coding regions are 97% identical at the nucleotide level and 95% identical at the amino acid level. Transcripts of Ntε-LCY were detectable in both vegetative and reproductive organs, with a relatively higher level of expression in leaves than in other tissues. Subcellular localization experiments using an Ntε-LCY1-GFP fusion protein demonstrated that mature Ntε-LCY1 protein is localized within the chloroplast in Bright Yellow 2 suspension cells. Under low-temperature and low-irradiation stress, Ntε-LCY transcript levels substantially increased relative to control plants. Tobacco rattle virus (TRV-mediated silencing of ε-LCY in Nicotiana benthamiana resulted in an increase of β-branch carotenoids and a reduction in the levels of α-branch carotenoids. Meanwhile, transcripts of related genes in the carotenoid biosynthetic pathway observably increased, with the exception of β-OHase in the TRV-ε-lcy line. Suppression of ε-LCY expression was also found to alleviate photoinhibition of Potosystem II in virus-induced gene silencing (VIGS plants under low-temperature and low-irradiation stress. Our results provide insight into the regulatory role of ε-LCY in plant carotenoid biosynthesis and suggest a role for ε-LCY in positively modulating low temperature stress responses.

  19. Synchronous high-resolution phenotyping of leaf and root growth in Nicotiana tabacum over 24-h periods with GROWMAP-plant

    Directory of Open Access Journals (Sweden)

    Ruts Tom

    2013-01-01

    Full Text Available Abstract Background Root growth is highly responsive to temporal changes in the environment. On the contrary, diel (24 h leaf expansion in dicot plants is governed by endogenous control and therefore its temporal pattern does not strictly follow diel changes in the environment. Nevertheless, root and shoot are connected with each other through resource partitioning and changing environments for one organ could affect growth of the other organ, and hence overall plant growth. Results We developed a new technique, GROWMAP-plant, to monitor growth processes synchronously in leaf and root of the same plant with a high resolution over the diel period. This allowed us to quantify treatment effects on the growth rates of the treated and non-treated organ and the possible interaction between them. We subjected the root system of Nicotiana tabacum seedlings to three different conditions: constant darkness at 22°C (control, constant darkness at 10°C (root cooling, and 12 h/12 h light–dark cycles at 22°C (root illumination. In all treatments the shoot was kept under the same 12 h/12 h light–dark cycles at 22°C. Root growth rates were found to be constant when the root-zone environment was kept constant, although the root cooling treatment significantly reduced root growth. Root velocity was decreased after light-on and light-off events of the root illumination treatment, resulting in diel root growth rhythmicity. Despite these changes in root growth, leaf growth was not affected substantially by the root-zone treatments, persistently showing up to three times higher nocturnal growth than diurnal growth. Conclusion GROWMAP-plant allows detailed synchronous growth phenotyping of leaf and root in the same plant. Root growth was very responsive to the root cooling and root illumination, while these treatments altered neither relative growth rate nor diel growth pattern in the seedling leaf. Our results that were obtained simultaneously in growing

  20. Alamethicin permeabilizes the plasma membrane and mitochondria but not the tonoplast in tobacco (Nicotiana tabacum L. cv Bright Yellow) suspension cells

    DEFF Research Database (Denmark)

    Matic, S.; Geisler, D.A.; Møller, I.M.

    2005-01-01

    The ion channel-forming peptide AlaM (alamethicin) is known to permeabilize isolated mitochondria as well as animal cells. When intact tobacco (Nicotiana tabacum L.) Bright Yellow-2 cells were treated with AlaM, the cells became permeable for low-molecular-mass molecules as shown by induced leakage...... remained intact, as indicated by an unaffected tonoplast proton gradient. Low-flux permeabilization of plasma membranes and mitochondria at moderate AlaM concentrations was reversible and did not affect cell vigour. Higher AlaM concentrations induced cell death. After the addition of catalase that removes...... of NAD(P)(+). After the addition of cofactors and substrates, activities of cytosolic as well as mitochondrial respiratory enzymes could be directly determined inside the permeabilized cells. However, at an AlaM concentration at which the cytoplasmic enzymes were maximally accessible, the vacuole...

  1. Uptake of NO, NO 2 and O 3 by sunflower ( Helianthus annuus L.) and tobacco plants ( Nicotiana tabacum L.): dependence on stomatal conductivity

    Science.gov (United States)

    Neubert, A.; Kley, D.; Wildt, J.; Segschneider, H. J.; Förstel, H.

    The uptake of NO, NO 2 and O 3 by sunflowers ( Helianthus annuus L. var. giganteus) and tobacco plants ( Nicotiana tabacum L. var. Bel W3), using concentrations representative for moderately polluted air, has been determined by gas exchange experiments. Conductivities for these trace gases were measured at different light fluxes ranging from 820 μEm -2s -1 to darkness. The conductivities to water vapor and the trace gases are highly correlated. It is concluded that the uptake of NO, NO 2 and O 3 by sunflowers and tobacco plants is linearly dependent on stomatal opening. While the uptake of NO is limited by the mesophyll resistance, the uptake of NO 2 is only by diffusion through the stomata. Loss processes by deposition to the leaf surfaces are more pronounced for O 3 than for NO and NO 2.

  2. Two aspartate residues at the putative p10 subunit of a type II metacaspase from Nicotiana tabacum L. may contribute to the substrate-binding pocket.

    Science.gov (United States)

    Acosta-Maspons, Alexis; Sepúlveda-García, Edgar; Sánchez-Baldoquín, Laura; Marrero-Gutiérrez, Junier; Pons, Tirso; Rocha-Sosa, Mario; González, Lien

    2014-01-01

    Metacaspases are cysteine proteases present in plants, fungi, prokaryotes, and early branching eukaryotes, although a detailed description of their cellular function remains unclear. Currently, three-dimensional (3D) structures are only available for two metacaspases: Trypanosoma brucei (MCA2) and Saccharomyces cerevisiae (Yca1). Furthermore, metacaspases diverged from animal caspases of known structure, which limits straightforward homology-based interpretation of functional data. We report for the first time the identification and initial characterization of a metacaspase of Nicotiana tabacum L., NtMC1. By combining domain search, multiple sequence alignment (MSA), and protein fold-recognition studies, we provide compelling evidences that NtMC1 is a plant metacaspase type II, and predict its 3D structure using the crystal structure of two type I metacaspases (MCA2 and Yca1) and Gsu0716 protein from Geobacter sulfurreducens as template. Analysis of the predicted 3D structure allows us to propose Asp353, at the putative p10 subunit, as a new member of the aspartic acid triad that coordinates the P1 arginine/lysine residue of the substrate. Nevertheless, site-directed mutagenesis and expression analysis in bacteria and Nicotiana benthamiana indicate the functionality of both Asp348 and Asp353. Through the co-expression of mutant and wild-type proteins by transient expression in N. benthamiana leaves we found that polypeptide processing seems to be intramolecular. Our results provide the first evidence in plant metacaspases concerning the functionality of the putative p10 subunit.

  3. Caracterización molecular de los genes pr1 y tpt1 en especies y variedades cubanas de tabaco (Nicotiana tabacum L.

    Directory of Open Access Journals (Sweden)

    Sandra Pérez Alvarez

    2012-04-01

    Full Text Available Normal 0 21 false false false ES-CO X-NONE X-NONE MicrosoftInternetExplorer4 Título en ingles: Molecular characterization of pr1 and tpt1 proteins in cuban tobacco  species and varieties (Nicoatiana tabacum L. Resumen: La identificación de genes nuevos relacionados con la respuesta de Nicotiana tabacum L. al estrés biótico y abiótico contribuye al mejoramiento genético del cultivo del tabaco en todo el mundo. El objetivo de este trabajo fue detectar la presencia de los genes tpt1 y pr1 en el genoma de algunas especies y variedades cubanas de tabaco. Se identificaron 265 etiquetas de secuencias expresadas (ESTs-expressed sequences tags que nunca se habían  informado con anterioridad en especies vegetales, y el gen que codifica para la proteína tumoral controlada durante la transcripción (Transcriptional Controlled Tumor Protein (tpt1 nunca se había informado en N. tabacum, pues los estudios del mismo se han centrado en su mayoría en animales y humanos. Los resultados del microarreglo se confirmaron utilizando la RT-PCR cuantitativa en tiempo real. Los genes tpt1 y proteína relacionada con la patogénesis (pr1 se utilizaron para diseñar cebadores para la caracterización molecular de algunas especies y variedades de tabaco cubano. El gen tpt1 solamente se expresó en dos especies (N. glutinosa y N. tomentosiformis y el pr1, después de la digestión, mostró diferentes bandas entre las variedades susceptibles y resistentes. La presencia de estos genes en una parte del germoplasma analizado constituye un paso inicial para la utilización de este conocimiento en el mejoramiento genético del tabaco. Palabras claves: etiquetas de secuencias expresadas (ESTs; microarreglo; caracterización molecular. Abstract: The identification of new genes related with Nicotiana tabacum L. response to biotic and abiotic stress contribute to the genetic improvement of tobacco plants around the world. The aim of this research was to identify tpt1 and pr

  4. Effet comparé des poudres de Nicotiana tabacum L, Cymbopogon citratus (D.C. Stapf et de l'huile de Ricinus communis L sur la conservation des graines de Vigna unguiculata (L Walp

    Directory of Open Access Journals (Sweden)

    Gakuru, S.

    1995-01-01

    Full Text Available Compared Effect of Nicotiana tabacum L, Cymbopogon citratus (D.C. Stapf Powders and Castor Oil Ricinus communis L. on Conservation of Cowpea Vigna Unguiculata (L. Walp Grains. The effect of powder of tobacco Nicotiana tabacum L. and citronella grass Cymbopogon citratus (D.C. Stapf and castor oil Ricinus communis L. on conservation of cowpea Vigna unguiculata (L. Walp. grains was investigated in Kisangani, Zaire. After 5 months of conservation, infestation rates by bean weevil Acanthoscelides obtectus Say were 72.5 %, 74.5 %, 49.5 % and 5 % respectively for the check, the samples treated by 1 % of citronella grass and tobacco powder and 1 % of castor oil. The powder dose of 7.5 % did not give more interesting results.

  5. Distribution and change patterns of free IAA, ABP 1 and PM H⁺-ATPase during ovary and ovule development of Nicotiana tabacum L.

    Science.gov (United States)

    Chen, Dan; Deng, Yingtian; Zhao, Jie

    2012-01-15

    Auxin plays key roles in flower induction, embryogenesis, seed formation and seedling development, but little is known about whether auxin regulates the development of ovaries and ovules before pollination. In the present report, we measured the content of free indole-3-acetic (IAA) in ovaries of Nicotiana tabacum L., and localized free IAA, auxin binding protein 1 (ABP1) and plasma membrane (PM) H⁺-ATPase in the ovaries and ovules. The level of free IAA in the developmental ovaries increased gradually from the stages of ovular primordium to the functional megaspore, but slightly decreased when the embryo sacs formed. Immunoenzyme labeling clearly showed that both IAA and ABP1 were distributed in the ovules, the edge of the placenta, vascular tissues and the ovary wall, while PM H⁺-ATPase was mainly localized in the ovules. By using immunogold labeling, the subcellular distributions of IAA, ABP1 and PM H⁺-ATPase in the ovules were also shown. The results suggest that IAA, ABP1 and PM H⁺-ATPase may play roles in the ovary and ovule initiation, formation and differentiation.

  6. Evaluation of tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots for the production of geraniol, the first committed step in terpenoid indole alkaloid pathway.

    Science.gov (United States)

    Ritala, Anneli; Dong, Lemeng; Imseng, Nicole; Seppänen-Laakso, Tuulikki; Vasilev, Nikolay; van der Krol, Sander; Rischer, Heiko; Maaheimo, Hannu; Virkki, Arho; Brändli, Johanna; Schillberg, Stefan; Eibl, Regine; Bouwmeester, Harro; Oksman-Caldentey, Kirsi-Marja

    2014-04-20

    The terpenoid indole alkaloids are one of the major classes of plant-derived natural products and are well known for their many applications in the pharmaceutical, fragrance and cosmetics industries. Hairy root cultures are useful for the production of plant secondary metabolites because of their genetic and biochemical stability and their rapid growth in hormone-free media. Tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots, which do not produce geraniol naturally, were engineered to express a plastid-targeted geraniol synthase gene originally isolated from Valeriana officinalis L. (VoGES). A SPME-GC-MS screening tool was developed for the rapid evaluation of production clones. The GC-MS analysis revealed that the free geraniol content in 20 hairy root clones expressing VoGES was an average of 13.7 μg/g dry weight (DW) and a maximum of 31.3 μg/g DW. More detailed metabolic analysis revealed that geraniol derivatives were present in six major glycoside forms, namely the hexose and/or pentose conjugates of geraniol and hydroxygeraniol, resulting in total geraniol levels of up to 204.3 μg/g DW following deglycosylation. A benchtop-scale process was developed in a 20-L wave-mixed bioreactor eventually yielding hundreds of grams of biomass and milligram quantities of geraniol per cultivation bag. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Salinity-induced accumulation of endogenous H2S and NO is associated with modulation of the antioxidant and redox defense systems in Nicotiana tabacum L. cv. Havana.

    Science.gov (United States)

    da Silva, Cristiane Jovelina; Batista Fontes, Elizabeth Pacheco; Modolo, Luzia Valentina

    2017-03-01

    Salinity is one of the abiotic factors that most affect crop growth and production. This study focused on the effect of high salinity on the endogenous levels of the signaling molecules hydrogen sulfite (H2S) and nitric oxide (NO) in Nicotiana tabacum leaves and the extent of these for the biochemically-driven plant tolerance to such abiotic stress. The NaCl treatment for 10days led to an expressive augment of H2S and NO levels. This increase was correlated with the raise of l-Cys and l-Arg and the induction of l-cysteine desulfhydrase, cyanoalanine synthase, cysteine synthase, nitrate reductase and arginase, enzymes known to be involved in the biosynthesis of H2S or NO. The enzymatic antioxidant system (superoxide dismutase and catalase activity) was boosted and the non-enzymatic antioxidant glutathione was intensively oxidized in leaves upon stress allowing plants to cope with oxidative stress. Lower stomatal conductance was observed in stressed plants in comparison with control ones. Moreover, the high activity of antioxidant enzymes and high rate of glutathione oxidation following salt stress were considerably decreased upon NO or H2S scavenging. Thus, increment in NO and H2S levels and their interplay, along with metabolic and physiological changes, contributed to tobacco survival to extreme salinity conditions. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  8. The Development of DNA Based Methods for the Reliable and Efficient Identification of Nicotiana tabacum in Tobacco and Its Derived Products

    Directory of Open Access Journals (Sweden)

    Sukumar Biswas

    2016-01-01

    Full Text Available Reliable methods are needed to detect the presence of tobacco components in tobacco products to effectively control smuggling and classify tariff and excise in tobacco industry to control illegal tobacco trade. In this study, two sensitive and specific DNA based methods, one quantitative real-time PCR (qPCR assay and the other loop-mediated isothermal amplification (LAMP assay, were developed for the reliable and efficient detection of the presence of tobacco (Nicotiana tabacum in various tobacco samples and commodities. Both assays targeted the same sequence of the uridine 5′-monophosphate synthase (UMPS, and their specificities and sensitivities were determined with various plant materials. Both qPCR and LAMP methods were reliable and accurate in the rapid detection of tobacco components in various practical samples, including customs samples, reconstituted tobacco samples, and locally purchased cigarettes, showing high potential for their application in tobacco identification, particularly in the special cases where the morphology or chemical compositions of tobacco have been disrupted. Therefore, combining both methods would facilitate not only the detection of tobacco smuggling control, but also the detection of tariff classification and of excise.

  9. iTRAQ-based quantitative proteomic analysis reveals proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum) in response to drought stress.

    Science.gov (United States)

    Xie, He; Yang, Da-Hai; Yao, Heng; Bai, Ge; Zhang, Yi-Han; Xiao, Bing-Guang

    2016-01-15

    Drought is one of the most severe forms of abiotic stresses that threaten the survival of plants, including crops. In turn, plants dramatically change their physiology to increase drought tolerance, including reconfiguration of proteomes. Here, we studied drought-induced proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum), a solanaceous plant, using the isobaric tags for relative and absolute quantitation (iTRAQ)-based protein labeling technology. Of identified 5570 proteins totally, drought treatment increased and decreased abundance of 260 and 206 proteins, respectively, compared with control condition. Most of these differentially regulated proteins are involved in photosynthesis, metabolism, and stress and defense. Although abscisic acid (ABA) levels greatly increased in drought-treated tobacco leaves, abundance of detected ABA biosynthetic enzymes showed no obvious changes. In contrast, heat shock proteins (HSPs), thioredoxins, ascorbate-, glutathione-, and hydrogen peroxide (H2O2)-related proteins were up- or down-regulated in drought-treated tobacco leaves, suggesting that chaperones and redox signaling are important for tobacco tolerance to drought, and it is likely that redox-induced posttranslational modifications play an important role in modulating protein activity. This study not only provides a comprehensive dataset on overall protein changes in drought-treated tobacco leaves, but also shed light on the mechanism by which solanaceous plants adapt to drought stress.

  10. Metabolism of methoxychlor by the P450-monooxygenase CYP6G1 involved in insecticide resistance of Drosophila melanogaster after expression in cell cultures of Nicotiana tabacum.

    Science.gov (United States)

    Joussen, Nicole; Schuphan, Ingolf; Schmidt, Burkhard

    2010-03-01

    Cytochrome P450 monooxygenase CYP6G1 of Drosophila melanogaster was heterologously expressed in a cell suspension culture of Nicotiana tabacum. This in vitro system was used to study the capability of CYP6G1 to metabolize the insecticide methoxychlor (=1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane, 1) against the background of endogenous enzymes of the corresponding non-transgenic culture. The Cyp6g1-transgenic cell culture metabolized 96% of applied methoxychlor (45.8 microg per assay) within 24 h by demethylation and hydroxylation mainly to trishydroxy and catechol methoxychlor (16 and 17%, resp.). About 34% of the metabolism and the distinct formation of trishydroxy and catechol methoxychlor were due to foreign enzyme CYP6G1. Furthermore, methoxychlor metabolism was inhibited by 43% after simultaneous addition of piperonyl butoxide (458 microg), whereas inhibition in the non-transgenic culture amounted to 92%. Additionally, the rate of glycosylation was reduced in both cultures. These results were supported by the inhibition of the metabolism of the insecticide imidacloprid (6; 20 microg, 24 h) in the Cyp6g1-transgenic culture by 82% in the presence of piperonyl butoxide (200 microg). Due to CYP6G1 being responsible for imidacloprid resistance of Drosophila or being involved in DDT resistance, it is likely that CYP6G1 conveys resistance to methoxychlor (1). Furthermore, treating Drosophila with piperonyl butoxide could weaken the observed resistance phenomena.

  11. The symbiosis between Nicotiana tabacum and the endomycorrhizal fungus Funneliformis mosseae increases the plant glutathione level and decreases leaf cadmium and root arsenic contents.

    Science.gov (United States)

    Degola, Francesca; Fattorini, Laura; Bona, Elisa; Sprimuto, Christian Triscari; Argese, Emanuele; Berta, Graziella; Sanità di Toppi, Luigi

    2015-07-01

    Over time, anthropogenic activities have led to severe cadmium (Cd) and arsenic (As) pollution in several environments. Plants inhabiting metal(loid)-contaminated areas should be able to sequester and detoxify these toxic elements as soon as they enter roots and leaves. We postulated here that an important role in protecting plants from excessive metal(loid) accumulation and toxicity might be played by arbuscular mycorrhizal (AM) fungi. In fact, human exploitation of plant material derived from Cd- and As-polluted environments may lead to a noxious intake of these toxic elements; in particular, a possible source of Cd and As for humans is given by cigarette and cigar smoke. We investigated the role of AM fungus Funneliformis mosseae (T.H. Nicolson & Gerd.) C. Walker & A. Schüßler in protecting Nicotiana tabacum L. (cv. Petit Havana) from the above-mentioned metal(loid) stress. Our findings proved that the AM symbiosis is effective in increasing the plant tissue content of the antioxidant glutathione (GSH), in influencing the amount of metal(loid)-induced chelators as phytochelatins, and in reducing the Cd and As content in leaves and roots of adult tobacco plants. These results might also prove useful in improving the quality of commercial tobacco, thus reducing the risks to human health due to inhalation of toxic elements contained in smoking products.

  12. Phenylpropanoid Defences in Nicotiana tabacum Cells: Overlapping Metabolomes Indicate Common Aspects to Priming Responses Induced by Lipopolysaccharides, Chitosan and Flagellin-22.

    Directory of Open Access Journals (Sweden)

    Msizi I Mhlongo

    Full Text Available Plants have evolved both constitutive and inducible defence strategies to cope with different biotic stimuli and stresses. Exposure of a plant to a challenging stress can lead to a primed state that allows it to launch a more rapid and stronger defence. Here we applied a metabolomic approach to study and compare the responses induced in Nicotiana tabacum cells by microbe-associated molecular pattern (MAMP molecules, namely lipopolysaccharides (LPS, chitosan (CHT and flagellin-22 (FLG22. Early response metabolites, extracted with methanol, were analysed by UHPLC-MS/MS. Using multivariate statistical tools the metabolic profiles induced by these elicitors were analysed. In the metabolic fingerprint of these agents a total of 19 cinnamic acid derivatives conjugated to quinic acids (chlorogenic acids, shikimic acid, tyramine, polyamines or glucose were found as discriminant biomarkers. In addition, treatment with the phytohormones salicylic acid (SA, methyljasmonic acid (MJ and abscisic acid (ABA resulted in differentially-induced phenylpropanoid pathway metabolites. The results indicate that the phenylpropanoid pathway is activated by these elicitors while hydroxycinnamic acid derivatives are commonly associated with the metabolic response to the MAMPs, and that the activated responses are modulated by both SA and MJ, with ABA not playing a role.

  13. In Vitro Antioxidant Properties of Flavonoids and Polysaccharides Extract from Tobacco (Nicotiana tabacum L. Leaves

    Directory of Open Access Journals (Sweden)

    Jing-Lu Wang

    2012-09-01

    Full Text Available In the present study, antioxidant properties of flavonoids and polysaccharides from tobacco (Nicotiana tabacum L. leaves were evaluated in several in vitro systems, e.g., scavenging activities on hydroxyl, superoxide anion, 1,1-diphenyl-2-picrylhydrazyl (DPPH and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS radicals, and reducing power. Flavonoids showed much better activity than polysaccharides in scavenging activities on free radicals. When compared to the positive control, ascorbic acid, both showed weaker antioxidant potential. However, flavonoids possessed comparable superoxide anion, DPPH and ABTS radical scavenging abilities to ascorbic acid at high concentration (600 μg/mL. Meanwhile, it was found that flavonoids had prominent effects on the reducing power, which was equivalent to ascorbic acid, and was significantly higher than polysaccharides. These results clearly indicate that flavonoids are effective in scavenging free radicals and have the potential to be powerful antioxidants. Thus, tobacco leaves could be considered as a potential source of natural antioxidants for food, pharmaceutical, cosmetics or nutraceutical industries.

  14. Tobacco plants transformed with the bean. alpha. ai gene express an inhibitor of insect. alpha. -amylase in their seeds. [Nicotiana tabacum; Tenebrio molitor

    Energy Technology Data Exchange (ETDEWEB)

    Altabella, T.; Chrispeels, M.J. (Univ. of California, San Diego, La Jolla (USA))

    1990-06-01

    Bean (Phaseolus vulgaris L.) seeds contain a putative plant defense protein that inhibits insect and mammalian but not plant {alpha}-amylases. We recently presented strong circumstantial evidence that this {alpha}-amylase inhibitor ({alpha}Al) is encoded by an already-identified lectin gene whose product is referred to as lectin-like-protein (LLP). We have now made a chimeric gene consisting of the coding sequence of the lectin gene that encodes LLP and the 5{prime} and 3{prime} flanking sequences of the lectin gene that encodes phytohemagglutinin-L. When this chimeric gene was expressed in transgenic tobacco (Nicotiana tabacum), we observed in the seeds a series of polypeptides (M{sub r} 10,000-18,000) that cross-react with antibodies to the bean {alpha}-amylase inhibitor. Most of these polypeptides bind to a pig pancreas {alpha}-amylase affinity column. An extract of the seeds of the transformed tobacco plants inhibits pig pancreas {alpha}-amylase activity as well as the {alpha}-amylase present in the midgut of Tenebrio molitor. We suggest that introduction of this lectin gene (to be called {alpha}ai) into other leguminous plants may be a strategy to protect the seeds from the seed-eating larvae of Coleoptera.

  15. The effect of methyl jasmonate and different chloride concentrations on photosynthetic pigments and proline content in Nicotiana tabacum L. cv. Cooker 347

    Directory of Open Access Journals (Sweden)

    Mahyar Moshtaghi

    2014-12-01

    Full Text Available Jasmonic acid and its methylated ester (methyl jasmonate, as natural plant growth regulators, are widely found in plants. In this study, the effect of methyl jasmonate (30 μM, in the early stages of Nicotiana tabacum L. cv. Cooker 347 growth, on photosynthetic pigments and proline was studied at high concentration of chloride. This experiment was performed in randomized complete block design with 4 replications, during 2011 crop year at Guilan Tobacco Research Center, outside greenhouse and under pot conditions. After transplanting similar seedlings to pots in rapid growth phase, the plants were irrigated with three levels chloride (50, 150 and 300 mg/L as CaCl2 both in presence and absence of methyl jasmonate for 4 weeks. Data analysis showed that by increasing Cl- concentrations up to 300 (mg/L, chloride and calcium accumulation of leaves increased, whilst photosynthetic pigments and proline decreased. Exogenous application of methyl jasmonate (30 μM significantly increased photosynthetic pigments in all treatments and proline in the 300 mg/L treatment. It seemed that the application of external methyl jasmonate (30 µM could improve tobacco plant injuries at high chloride concentration.

  16. Isolation and molecular characterization of an ethylene response factor NtERF1-1 in Nicotiana tabacum cv. Xanthi

    Indian Academy of Sciences (India)

    Jun-Shan Gao; Li Hu; Peng Xie; Yan Meng; Yong-Ping Cai; Yi Lin

    2014-12-01

    Apetala2/Ethylene Response Factors (AP2/ERF) play important roles in regulating gene expression under abiotic and biotic stress in the plant kingdom. Here, we isolated a member of the AP2/ERF transcription factors, NtERF1-1, from Nicotiana tabcum cv. Xanthi NN carrying the N gene, which is resistant to Tobacco mosaic virus (TMV). NtERF1-1 encoded a putative protein of 229 amino acids with a predicted molecular mass of 24.58 kDa. Nucleotide sequence analysis showed that NtERF1-1 contained a conserved DNA-binding domain at the N-terminal. Comparison of amino acid sequences revealed that NtERF1-1 possessed high similarities to ERFs from diverse plants. Semi-quantitative and real-time quantitative RT-PCR analyses indicated that NtERF1-1 was up-regulated following TMV infection. In addition, we speculated that NtERF1-1 might participate in the signal transduction pathway of defence response inducted by the interaction between the gene and TMV.

  17. Pneumatic hydrodynamics influence transplastomic protein yields and biological responses during in vitro shoot regeneration of Nicotiana tabacum callus: Implications for bioprocess routes to plant-made biopharmaceuticals.

    Science.gov (United States)

    Barretto, Sherwin S; Michoux, Franck; Hellgardt, Klaus; Nixon, Peter J

    2017-01-15

    Transplastomic plants are capable of high-yield production of recombinant biopharmaceutical proteins. Plant tissue culture combines advantages of agricultural cultivation with the bioprocess consistency associated with suspension culture. Overexpression of recombinant proteins through regeneration of transplastomic Nicotiana tabacum shoots from callus tissue in RITA(®) temporary immersion bioreactors has been previously demonstrated. In this study we investigated the hydrodynamics of periodic pneumatic suspension of liquid medium during temporary immersion culture (4 min aeration every 8 h), and the impact on biological responses and transplastomic expression of fragment C of tetanus toxin (TetC). Biomass was grown under a range of aeration rates for 3, 20 and 40-day durations. Growth, mitochondrial activity (a viability indicator) and TetC protein yields were correlated against the hydrodynamic parameters, shear rate and energy dissipation rate (per kg of medium). A critical aeration rate of 440 ml min(-1) was identified, corresponding to a shear rate of 96.7 s(-1), pneumatic power input of 8.8 mW kg(-1) and initial 20-day pneumatic energy dissipation of 127 J kg(-1), at which significant reductions in biomass accumulation and mitochondrial activity were observed. There was an exponential decline in TetC yields with increasing aeration rates at 40 days, across the entire range of conditions tested. These observations have important implications for the optimisation and scale-up of transplastomic plant tissue culture bioprocesses for biopharmaceutical production.

  18. Different blue-light requirement for the accumulation of transcripts from nuclear genes for thylakoid proteins in Nicotiana tabacum and Lycopersicon esculentum.

    Science.gov (United States)

    Palomares, R; Herrmann, R G; Oelmüller, R

    1991-11-01

    We have isolated recombinant lambda gt11 phages which carry cDNA clones for the major light-harvesting chlorophyll a/b-binding proteins of photosystem I (LHCPI) and II (LHCPII), subunit II of photosystem I, a chlorophyll a/b-binding protein of photosystem II (CP24), the Rieske iron-sulphur protein of the cytochrome b6/f complex, and the 33, 23 and 16 kDa proteins of the water-oxidizing complex of photosystem II from Nicotiana tabacum. The nucleotide sequences of cDNA clones encoding the precursors for LHCPI and the FeS protein are presented. If tobacco or tomato seedlings, or seedlings of a phytochrome-deficient aurea mutant of tomato which lacks more than 95% of the phytochrome of the isogenic wild type, are kept in blue light, the transcript level of each of these genes is higher than in seedlings grown in red light suggesting the involvement of a blue-UVA-light photoreceptor. In the case of LHCPI, a 1 min blue-light pulse applied to red-light-grown seedlings is sufficient to increase the transcript levels to those present in blue-light-grown seedlings, whereas almost no increase is observed for transcripts encoding the FeS and 33 kDa proteins. If dark-grown tomato seedlings receive a single far-red-light pulse, significant stimulation is detected for LHCPI transcripts, whereas transcripts encoding the FeS and 33 kDa proteins are not stimulated. It is concluded that the lower light requirement for the increase in the LHCPI transcript level is not specific for one of the light-dependent signal transduction chains.

  19. Tolerance to clomazone herbicide is linked to the state of LHC, PQ-pool and ROS detoxification in tobacco (Nicotiana tabacum L.).

    Science.gov (United States)

    Darwish, Majd; Vidal, Véronique; Lopez-Lauri, Félicie; Alnaser, Osama; Junglee, Sanders; El Maataoui, Mohamed; Sallanon, Huguette

    2015-03-01

    In this study, plantlets of two tobacco (Nicotiana tabacum L.) varieties that are clomazone-tolerant (cv. Xanthi) and clomazone-sensitive (cv. Virginie vk51) were subjected to low concentration of clomazone herbicide. The oxygen-evolving rate of isolated chloroplasts, chlorophyll a fluorescence transients, JIP-test responses, hydrogen peroxide contents, antioxidant enzyme activities, cytohistological results and photosynthetic pigment contents were recorded. The results indicated that the carotenoid content was 2-fold higher in Virginie, which had greater clomazone sensitivity than Xanthi. Virginie exhibited noticeable decreases in the LHC content (Chl a/b ratio), the maximum photochemical quantum efficiency of PSII (Fv/Fm), the performance index on the absorption basis (PIabs), and the electron flux beyond the first PSII QA evaluated as (1-VJ) with VJ=(FJ-F0)/(Fm-F0) as well as increases in the rate of photon absorption (ABS/RC) and the energy dissipation as heat (DI0/RC). These results suggest that PSII photoinhibition occurred as a consequence of more reduced PQ-pool and accumulated QA(-). The oxygen evolution measurements indicate that PSI electron transport activity was not affected by clomazone. The more significant accumulation of H2O2 in Virginie compared to Xanthi was due to the absence of ROS-scavenging enzymes, and presumably induced programmed cell death (PCD). The symptoms of PCD were observed by cytohistological analysis, which also indicated that the leaf tissues of clomazone-treated Virginie exhibited significant starch accumulation compared to Xanthi. Taken together, these results indicate that the variable tolerance to clomazone observed between Virginie and Xanthi is independent of the carotenoid content and could be related to the state of the LHC, the redox state of the PQ-pool, and the activity of detoxification enzymes.

  20. PET imaging of thin objects: measuring the effects of positron range and partial-volume averaging in the leaf of Nicotiana tabacum

    Energy Technology Data Exchange (ETDEWEB)

    Alexoff, David L., E-mail: alexoff@bnl.gov; Dewey, Stephen L.; Vaska, Paul; Krishnamoorthy, Srilalan; Ferrieri, Richard; Schueller, Michael; Schlyer, David J.; Fowler, Joanna S.

    2011-02-15

    Introduction: PET imaging in plants is receiving increased interest as a new strategy to measure plant responses to environmental stimuli and as a tool for phenotyping genetically engineered plants. PET imaging in plants, however, poses new challenges. In particular, the leaves of most plants are so thin that a large fraction of positrons emitted from PET isotopes ({sup 18}F, {sup 11}C, {sup 13}N) escape while even state-of-the-art PET cameras have significant partial-volume errors for such thin objects. Although these limitations are acknowledged by researchers, little data have been published on them. Methods: Here we measured the magnitude and distribution of escaping positrons from the leaf of Nicotiana tabacum for the radionuclides {sup 18}F, {sup 11}C and {sup 13}N using a commercial small-animal PET scanner. Imaging results were compared to radionuclide concentrations measured from dissection and counting and to a Monte Carlo simulation using GATE (Geant4 Application for Tomographic Emission). Results: Simulated and experimentally determined escape fractions were consistent. The fractions of positrons (mean{+-}S.D.) escaping the leaf parenchyma were measured to be 59{+-}1.1%, 64{+-}4.4% and 67{+-}1.9% for {sup 18}F, {sup 11}C and {sup 13}N, respectively. Escape fractions were lower in thicker leaf areas like the midrib. Partial-volume averaging underestimated activity concentrations in the leaf blade by a factor of 10 to 15. Conclusions: The foregoing effects combine to yield PET images whose contrast does not reflect the actual activity concentrations. These errors can be largely corrected by integrating activity along the PET axis perpendicular to the leaf surface, including detection of escaped positrons, and calculating concentration using a measured leaf thickness.

  1. PET imaging of thin objects: measuring the effects of positron range and partial-volume averaging in the leag of Nicotiana Tabacum

    Energy Technology Data Exchange (ETDEWEB)

    Alexoff, D.L.; Alexoff, D.L.; Dewey, S.L.; Vaska, P.; Krishnamoorthy, S.; Ferrieri, R.; Schueller, M.; Schlyer, D.; Fowler, J.S.

    2011-03-01

    PET imaging in plants is receiving increased interest as a new strategy to measure plant responses to environmental stimuli and as a tool for phenotyping genetically engineered plants. PET imaging in plants, however, poses new challenges. In particular, the leaves of most plants are so thin that a large fraction of positrons emitted from PET isotopes ({sup 18}F, {sup 11}C, {sup 13}N) escape while even state-of-the-art PET cameras have significant partial-volume errors for such thin objects. Although these limitations are acknowledged by researchers, little data have been published on them. Here we measured the magnitude and distribution of escaping positrons from the leaf of Nicotiana tabacum for the radionuclides {sup 18}F, {sup 11}C and {sup 13}N using a commercial small-animal PET scanner. Imaging results were compared to radionuclide concentrations measured from dissection and counting and to a Monte Carlo simulation using GATE (Geant4 Application for Tomographic Emission). Simulated and experimentally determined escape fractions were consistent. The fractions of positrons (mean {+-} S.D.) escaping the leaf parenchyma were measured to be 59 {+-} 1.1%, 64 {+-} 4.4% and 67 {+-} 1.9% for {sup 18}F, {sup 11}C and {sup 13}N, respectively. Escape fractions were lower in thicker leaf areas like the midrib. Partial-volume averaging underestimated activity concentrations in the leaf blade by a factor of 10 to 15. The foregoing effects combine to yield PET images whose contrast does not reflect the actual activity concentrations. These errors can be largely corrected by integrating activity along the PET axis perpendicular to the leaf surface, including detection of escaped positrons, and calculating concentration using a measured leaf thickness.

  2. Actividad Fungicida e Insecticida de Emulsiones Agua/Aceite de Mezclas de Extractos de Nicotiana tabacum, Azadiractha indica y Eucalyptus tereticornis Fungicidal and Insecticide Activities of Water-in-Oil Emulsions of Mixtures from Extracts of Nicotiana taba-cum, Azadiractha indica and Eucalyptus tereticornis

    Directory of Open Access Journals (Sweden)

    Walter Murillo

    2012-01-01

    Full Text Available Se realizó un estudio sobre el potencial insecticida agudo y crónico sobre Drosophila melanogaster y antifúngico en Fusarium oxysporum de emulsiones aceite-en-agua de mezclas binarias y ternarias de extractos de Nicotiana tabacum, Azadiractha indica fneem, y aceite esencial de Eucalyptus tereticornis. Se construyeron curvas dosis/respuesta para el tiempo letal medio, relación pupa-huevo, adulto-pupa y porcentaje inhibitorio para la actividad antifúngica. Se observó alta actividad insecticida aguda del tabaco a 6 g/L (tiempo letal medio=2,3 ± 0,5 minutos, larvicida en el neem a 0,2 g/L (pupa-huevo= 0,05 y fungicida en el eucalipto a 3 g/L (porcentaje inhibitorio =100%. Las bioactividades se potenciaron en la mayoría de las mezclas binarias, exceptuando la actividad fungicida. La mezcla ternaria presentó actividad fungicida antagónica. Se concluye sobre la potencial aplicación de estos desarrollos para controlar plagas y enfermedades.A study on the acute and chronic insecticide potential on Drosophila melanogaster and antifungal on Fusarium oxysporum of oil-in-water emulsions of binary and ternary mixtures of extracts of Nicotiana tabacum and Azadiractha indica (neem and Eucalyptus tereticornis essential oil. Dose/response curves for the mean lethal time, pupae-egg ratio, adult-pupae and inhibitory rate for antifungal activity were constructed, compared with the bioactivity mixtures, measuring synergism, antagonism and joint activity. High insecticidal acute activity was observed in tobacco to 6 g/L (mean lethal time=2.3 ± 0.5 minutes, the neem larvicide to 0.2 g/L (pupae-egg ratio=0.05 and fungicide in the eucalyptus to 3 g/L (inhibitory rate=100%. The bioactivities were enhanced in almost all binary and ternary mixtures, except for the fungicidal activity. The ternary mixture exhibits fungicidal activity antagonistic. The study shows the potential application of these technologies to control pests and diseases.

  3. Profiling of altered metabolomic states in Nicotiana tabacum cells induced by priming agents

    CSIR Research Space (South Africa)

    Mhlongo, MI

    2016-10-01

    Full Text Available /h, m/z range: 100–1000, scan time: 0.15 s, interscan delay: 0.02 s, mode: centroid, lockmass: leucine enkephalin (556.3 ng/µL), lockmass flow rate: 0.4 mL/min, mass accuracy window: 0.5 mDa. Each sample originating from the three biological replicates...) ≥ 0.5 were chosen for metabolite identification using the m/z to generate elemental composition. XCMS-Online Data Analysis Like OPLS-DA, XCMS uses two pre-defined conditions to predict and analyze metabolome changes in the samples under study...

  4. Expression of a ferredoxin-dependent glutamate synthase gene in mesophyll and vascular cells and functions of the enzyme in ammonium assimilation in Nicotiana tabacum (L.).

    Science.gov (United States)

    Feraud, Magali; Masclaux-Daubresse, Céline; Ferrario-Méry, Sylvie; Pageau, Karine; Lelandais, Maud; Ziegler, Christine; Leboeuf, Edouard; Jouglet, Tiphaine; Viret, Lauriane; Spampinato, Axelle; Paganelli, Vanina; Hammouda, Mounir Ben; Suzuki, Akira

    2005-11-01

    GLU1 encodes the major ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) in Arabidopsis thaliana (ecotype Columbia). With the aim of providing clues on the role of Fd-GOGAT, we analyzed the expression of Fd-GOGAT in tobacco (Nicotiana tabacum L. cv. Xanthi). The 5' flanking element of GLU1 directed the expression of the uidA reporter gene in the palisade and spongy parenchyma of mesophyll, in the phloem cells of vascular tissue and in the roots of tobacco. White light, red light or sucrose induced GUS expression in the dark-grown seedlings in a pattern similar to the GLU1 mRNA accumulation in Arabidopsis. The levels of GLU2 mRNA encoding the second Fd-GOGAT and NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) were not affected by light. Both in the light and in darkness, (15)NH4(+) was incorporated into [5-(15)N]glutamine and [2-(15)N]glutamate by glutamine synthetase (GS, EC 6.3.1.2) and Fd-GOGAT in leaf disks of transgenic tobacco expressing antisense Fd-GOGAT mRNA and in wild-type tobacco. In the light, low level of Fd-glutamate synthase limited the [2-(15)N]glutamate synthesis in transgenic leaf disks. The efficient dark labeling of [2-(15)N]glutamate in the antisense transgenic tobacco leaves indicates that the remaining Fd-GOGAT (15-20% of the wild-type activity) was not the main limiting factor in the dark ammonium assimilation. The antisense tobacco under high CO2 contained glutamine, glutamate, asparagine and aspartate as the bulk of the nitrogen carriers in leaves (62.5%), roots (69.9%) and phloem exudates (53.2%). The levels of glutamate, asparagine and aspartate in the transgenic phloem exudates were similar to the wild-type levels while the glutamine level increased. The proportion of these amino acids remained unchanged in the roots of the transgenic plants. Expression of GLU1 in mesophyll cells implies that Fd-GOGAT assimilates photorespiratory and primary ammonium. GLU1 expression in vascular cells indicates that Fd-GOGAT provides

  5. EFECTO DE DOS OLIGOSACARINAS SOBRE LA EXPRESIÓN ISOENZIMÁTICA AL SER APLICADAS SOBRE DOS VARIEDADES DE TABACO (Nicotiana tabacum L.

    Directory of Open Access Journals (Sweden)

    A. Acosta

    2007-01-01

    Full Text Available En Cuba se trabaja en la búsqueda y producción de nuevas fitohormonas, entre las que se encuentran el Pectimorf y los xiloglucanos. El principio activo de estas es una mezcla de oligosacáridos, en el primer caso de origen péctico (Patente No. 1198/171 y en el segundo, de hemicelulosa. Se plantea que ambos son reguladores endógenos del desarrollo y la morfogénesis de las plantas y, además, inducen respuestas de defensa. Sin embargo, menos conocidos son los mecanismos moleculares que se activan, por lo que se continúan los estudios para evaluar el papel que juegan y las vías por las que actúan dichos compuestos en el desarrollo de las plantas. Para obtener una primera aproximación del mecanismo de acción de ambos compuestos sobre el genoma, se analizó la expresión de cuatro sistemas isoenzimáticos -Peroxidasas, Polifenoloxidasas, Anhidrasas Carbónicas y Esterasas- en dos variedades de tabaco (Nicotiana tabacum L., tratadas con los bioproductos. Los resultados se analizaron estadísticamente mediante el paquete de programas NTSYS-PC y se valoró su importancia sobre el desarrollo y la morfogénesis de las plantas. Como resultado se obtuvieron los zimotipos característicos para cada sistema isoenzimático de las dos variedades estudiadas (H-2000 y H-2.1.1. Además, se observó inhibición y/o inducción de isoformas en las plantas crecidas en medio suplementado con ambos biorreguladores, en comparación a los controles empleados. Estos resultados evidenciaron una modulación de la expresión de la información genética mediada por estos productos, al menos, para los cuatro sistemas analizados en el cultivo empleado.

  6. 烟草未授粉子房胚状体诱导的研究%Studies on the induction of embryoid from unpollinated ovaries of Nicotiana tabacum L.

    Institute of Scientific and Technical Information of China (English)

    潘莉; 杨铁钊

    2000-01-01

    Induction of embryoid from unpollinate ovaries of Nicotiana tabacum L.was investigated in this thesis.The results obtained were summarized as follows.Embryoid was originated from single cell,either megaspore or egg cell.Ovary inoculated at early stage produced embryoid from megaspore,while ovary inoculated at late stage produced embryoid from egg cell.When ovaries were plated at middle stage,embryoid originated more from megaspore than from egg cell.The ability of embryoid-production of various genotypes,various concentrations of sucrose,various light intensity changed greatly.%对烟草(Nicotiana tabacum L.)未授粉子房胚状体的诱导进行了研究,结果发现,胚状体是单细胞起源,起源于大孢子或卵细胞,接种发育早期的子房,胚状体起源于大孢子;接种发育晚期的子房,胚状体起源于卵细胞;接种发育中期的子房,胚状体 多起源于大孢子,少数起源于卵细胞。不同的基因型,蔗糖浓度及光照强度等对胚状体诱导 率的影响亦有不同

  7. 以单细胞压片技术观察烟草(Nicotiana tabacum L.)合子细胞器DNA的分布%A Single-Cell-Squashing Technique for Observation of Organelle DNA Distribution in Tobacco Zygotes

    Institute of Scientific and Technical Information of China (English)

    何玉池; 曲良焕; 孙蒙祥; 杨弘远

    2005-01-01

    通过实验建立了单个合子的压片技术,尝试了将染色与固定分开或同时进行的两种压片方法,两种方法均可以用于不同发育阶段合子细胞器DNA分布的检测,但二者各具特色.同时也利用共聚焦激光扫描显微镜观察了烟草(Nicotiana tabacum L.)合子细胞器的分布,并与单细胞压片技术进行了比较.实验表明它们各有其优越性.

  8. Assessment of natural radionuclides concentration from {sup 238}U and {sup 232}Th series in Virginia and Burley varieties of Nicotiana tabacum L; Avaliacao da concentracao dos radionuclideos naturais das series do {sup 238}U e {sup 232}Th nas variedades Burley e Virginia da Nicotiana tabacum L.

    Energy Technology Data Exchange (ETDEWEB)

    Silva, Carolina Fernanda da

    2015-07-01

    Brazil is the largest exporter and second largest producer of tobacco worldwide, according to the crop production of 2013/2014. The tobacco plant (Nicotiana tabacum L.) is used to manufacture all derivatives and the chemical composition of the resulting tobacco products varies with the type of tobacco leaves, how they are grown, the region where they are cultivated, the characteristics of preparation (compression, filter and paper) and the temperature variations resulting from the incomplete combustion of tobacco. Tobacco products are extensively used throughout the world, and the most consumed are cigarettes, cigars and narghile. The damaging effects that these products cause to human health are discussed globally, and many surveys are performed with the aim of relating the use of these products with various illnesses. There is a lack of information about the radiological characterization of the tobacco plant both in international and Brazilian literature. The objective of this study was to determine the concentration of radionuclides {sup 238}U, {sup 234}U, {sup 230}Th, {sup 22}'6Ra, {sup 210}Pb and {sup 210}Po, members from the {sup 238}U decay series, and the radionuclides {sup 232}Th and {sup 228}Ra members of the {sup 232}Th decay series in the varieties Burley and Virginia, which are the most cultivated in Brazil. Plants from these varieties were cultivated in pots with organic substrate and fertilizer and also acquired from the producers and analyzed by alpha spectrometry for U and Th isotopes and {sup 210}Po determination, and gross alpha and beta counting, {sup 228}Ra, {sup 226}Ra and {sup 210}Pb determination. The whole plant, from both places, was analyzed; root, stem, leaves, as well as the organic substrate, the fertilizers, and the soil. The results for U and Th isotopes presented values below the detection limits of the methods to the leaves and stems of all plants analyzed, with measurable results only in roots, soil, and substrate. The

  9. Inactivation of the β(1,2)-xylosyltransferase and the α(1,3)-fucosyltransferase genes in Nicotiana tabacum BY-2 Cells by a Multiplex CRISPR/Cas9 Strategy Results in Glycoproteins without Plant-Specific Glycans

    Science.gov (United States)

    Mercx, Sébastien; Smargiasso, Nicolas; Chaumont, François; De Pauw, Edwin; Boutry, Marc; Navarre, Catherine

    2017-01-01

    Plants or plant cells can be used to produce pharmacological glycoproteins such as antibodies or vaccines. However these proteins carry N-glycans with plant-typical residues [β(1,2)-xylose and core α(1,3)-fucose], which can greatly impact the immunogenicity, allergenicity, or activity of the protein. Two enzymes are responsible for the addition of plant-specific glycans: β(1,2)-xylosyltransferase (XylT) and α(1,3)-fucosyltransferase (FucT). Our aim consisted of knocking-out two XylT genes and four FucT genes (12 alleles altogether) in Nicotiana tabacum BY-2 suspension cells using CRISPR/Cas9. Three XylT and six FucT sgRNAs were designed to target conserved regions. After transformation of N. tabacum BY-2 cells with genes coding for sgRNAs, Cas9, and a selectable marker (bar), transgenic lines were obtained and their extracellular as well as intracellular protein complements were analyzed by Western blotting using antibodies recognizing β(1,2)-xylose and α(1,3)-fucose. Three lines showed a strong reduction of β(1,2)-xylose and α(1,3)-fucose, while two lines were completely devoid of them, indicating complete gene inactivation. The absence of these carbohydrates was confirmed by mass spectrometry analysis of the extracellular proteins. PCR amplification and sequencing of the targeted region indicated small INDEL and/or deletions between the target sites. The KO lines did not show any particular morphology and grew as the wild-type. One KO line was transformed with genes encoding a human IgG2 antibody. The IgG2 expression level was as high as in a control transformant which had not been glycoengineered. The IgG glycosylation profile determined by mass spectrometry confirmed that no β(1,2)-xylose or α(1,3)-fucose were present on the glycosylation moiety and that the dominant glycoform was the GnGn structure. These data represent an important step toward humanizing the glycosylation of pharmacological proteins expressed in N. tabacum BY-2 cells. PMID:28396675

  10. Over-expression of a scopoletin glucosyltransferase in Nicotiana tabacum leads to precocious lesion formation during the hypersensitive response to tobacco mosaic virus but does not affect virus resistance.

    Science.gov (United States)

    Gachon, Claire; Baltz, Rachel; Saindrenan, Patrick

    2004-01-01

    Nicotiana tabacum Togt encodes a scopoletin glucosyltransferase (UDPglucose:scopoletin O -beta-D-glucosyltrans- ferase, EC 2.4.1.128) known to act in vitro on many different substrates including the 6-methoxy-7-hydroxy- coumarin scopoletin. This phenolic compound accumulates in vast amounts, essentially in its glucosylated form scopolin, in tobacco during the hypersensitive response (HR) to tobacco mosaic virus (TMV). To identify the physiological role of this pathogen-inducible UDP-Glc glucosyltransferase (UGT), we generated TOGT over-expressing transgenic plants. Although no endogenous scopoletin or scopolin could be detected before infection, the accumulation of both the aglycone and the glucoside was found to be 2-fold higher in transgenic plants after inoculation with TMV than in wild-type plants. Scopoletin UGT activity in plants over-expressing Togt was significantly higher during the HR than in control plants. This up-regulated activity was associated with a strong increase of the bright blue fluorescence surrounding the HR-necrotic lesions under UV light, which is known to correlate with scopoletin and scopolin abundance. Necrosis appeared sooner in transgenic plants and lesions developed faster, suggesting an accelerated HR. Unexpectedly, the viral content in each lesion was not significantly different in transgenic and in wild-type plants. These results are discussed in relation to the role of TOGT as the major UDP-Glc: scopoletin glucosyltransferase and to the importance of scopoletin accumulation during the HR.

  11. Activity of the AtMRP3 promoter in transgenic Arabidopsis thaliana and Nicotiana tabacum plants is increased by cadmium, nickel, arsenic, cobalt and lead but not by zinc and iron.

    Science.gov (United States)

    Zientara, Katarzyna; Wawrzyńska, Anna; Lukomska, Jolanta; López-Moya, José Rafael; Liszewska, Frantz; Assunção, Ana G L; Aarts, Mark G M; Sirko, Agnieszka

    2009-02-05

    Characterization of the function, regulation and metal-specificity of metal transporters is one of the basic steps needed for the understanding of transport and accumulation of toxic metals and metalloids by plants. In this work GUS was used as a reporter for monitoring the activity of the promoter of the AtMRP3 gene from Arabidopsis thaliana, a gene encoding an ABC-transporter, expression of which is induced by heavy metals. The AtMRP3 promoter-GUS fusion expression cassette was introduced into the genome of two model plants, A. thaliana and Nicotiana tabacum. The promoter induces GUS activity in the roots as well as in the shoots upon metal exposure. Similar responses of the AtMRP3 promoter to the presence of the selected metals was observed in both plant species. Cadmium, nickel, arsenic, cobalt and lead strongly activated the transcription of the reporter gene, while zinc and iron had no impact. The AtMRP3 promoter thus seems to be a useful new tool in designing plants that can be used for biomonitoring of environmental contaminations.

  12. A 3D digital atlas of the Nicotiana tabacum root tip and its use to investigate changes in the root apical meristem induced by the Agrobacterium 6b oncogene.

    Science.gov (United States)

    Pasternak, Taras; Haser, Thomas; Falk, Thorsten; Ronneberger, Olaf; Palme, Klaus; Otten, Léon

    2017-10-01

    Using the intrinsic Root Coordinate System (iRoCS) Toolbox, a digital atlas at cellular resolution has been constructed for Nicotiana tabacum roots. Mitotic cells and cells labeled for DNA replication with 5-ethynyl-2'-deoxyuridine (EdU) were mapped. The results demonstrate that iRoCS analysis can be applied to roots that are thicker than those of Arabidopsis thaliana without histological sectioning. A three-dimensional (3-D) analysis of the root tip showed that tobacco roots undergo several irregular periclinal and tangential divisions. Irrespective of cell type, rapid cell elongation starts at the same distance from the quiescent center, however, boundaries between cell proliferation and transition domains are cell-type specific. The data support the existence of a transition domain in tobacco roots. Cell endoreduplication starts in the transition domain and continues into the elongation zone. The tobacco root map was subsequently used to analyse root organization changes caused by the inducible expression of the Agrobacterium 6b oncogene. In tobacco roots that express the 6b gene, the root apical meristem was shorter and radial cell growth was reduced, but the mitotic and DNA replication indexes were not affected. The epidermis of 6b-expressing roots produced less files and underwent abnormal periclinal divisions. The periclinal division leading to mature endodermis and cortex3 cell files was delayed. These findings define additional targets for future studies on the mode of action of the Agrobacterium 6b oncogene. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  13. Preliminary study of Lead (Pb) immobilization by meat and bone meal combustion residues (MBMCR) in soil: assessment of Pb toxicity (phytotoxicity and genotoxicity) using the tobacco model (Nicotiana tabacum var. xanthi Dulieu).

    Science.gov (United States)

    Mouchet, F; Cren, S; Deydier, E; Guilet, R; Gauthier, L

    2008-08-01

    Lead (Pb) is a major chemical pollutant in the environment. The present investigation evaluates the possible use of Meat and Bone Meal Combustion Residues (MBMCR), to sequester Pb from the soil compartment using the heterozygous tobacco model (Nicotiana tabacum var. xanthi Dulieu) characterized by the a1+ /a1 a2+ /a2 system. The toxic potential of Pb-contaminations (50, 100, 1,000, 2,000 and 10,000 mg Pb kg(-1)) as Pb(NO3) in standard soil was investigated in lab conditions according to three endpoints: (i) acute toxicity of plants (mortality, height and surface area parameters), (ii) Pb-accumulation in roots, stems and leaves, and (iii) genetic effects as the expression of reversion in the leaf of plants. Moreover, chemical investigations of Pb interactions with soil were realized to complete the toxicity evaluation. The results demonstrated that: (i) MBMCR were not acutely toxic or genotoxic to tobacco plants, (ii) Pb is acutely toxic to tobacco plants at 10,000 mg Pb kg(-1) of soil, (ii) but is not genotoxic, and (iii) Pb-bioaccumulation is significant in leaves, stems and roots (from 1,000, 2,000, and 50 mg Pb kg(-1) of soil, respectively). In contrast, in the presence of MBMCR, the toxic impacts of Pb were inhibited and Pb-accumulation in tobacco plants was reduced. In complement, chemical analyses highlighted the high capacity of the standard soil to immobilize Pb. The results suggest that even if Pb is bioavailable from soils to plants, complex mechanisms could occur in plants protecting them from the toxic impact of Pb.

  14. Alternative splicing of basic chitinase gene PR3b in the low-nicotine mutants of Nicotiana tabacum L. cv. Burley 21

    Science.gov (United States)

    Ma, Haoran; Wang, Feng; Wang, Wenjing; Yin, Guoying; Zhang, Dingyu; Ding, Yongqiang; Timko, Michael P.; Zhang, Hongbo

    2016-01-01

    Two unlinked semi-dominant loci, A (NIC1) and B (NIC2), control nicotine and related alkaloid biosynthesis in Burley tobaccos. Mutations in either or both loci (nic1 and nic2) lead to low nicotine phenotypes with altered environmental stress responses. Here we show that the transcripts derived from the pathogenesis-related (PR) protein gene PR3b are alternatively spliced to a greater extent in the nic1 and nic2 mutants of Burley 21 tobacco and the nic1nic2 double mutant. The alternative splicing results in a deletion of 65 nucleotides and introduces a premature stop codon into the coding region of PR3b that leads to a significant reduction of PR3b specific chitinase activity. Assays of PR3b splicing in F2 individuals derived from crosses between nic1 and nic2 mutants and wild-type plants showed that the splicing phenotype is controlled by the NIC1 and NIC2 loci, even though NIC1 and NIC2 are unlinked loci. Moreover, the transcriptional analyses showed that the splicing patterns of PR3b in the low-nicotine mutants were differentially regulated by jasmonate (JA) and ethylene (ET). These data suggest that the NIC1 and NIC2 loci display differential roles in regulating the alternative splicing of PR3b in Burley 21. The findings in this study have provided valuable information for extending our understanding of the broader effects of the low-nicotine mutants of Burley 21 and the mechanism by which JA and ET signalling pathways post-transcriptionally regulate the activity of PR3b protein. PMID:27664270

  15. [Construction and analysis of transgenic plants of Nicotiana tabacum L. expressing a bacterial gene for beta-1,3-glucanase. II. Transgenic tobacco plants expressing the bacterial beta-glucanase gene from Clostridium thermocellum,--a model for studying the differential expression of stress response-related genes].

    Science.gov (United States)

    Darbinian, N S; Popov, Iu G; Mochul'skiĭ, A V; Oming, D; Piruzian, E S; Vasilevko, V T

    1996-02-01

    The modified hybrid beta-1,3-glucanase gene (glc) of Clostridium thermocellum was expressed in tobacco Nicotiana tabacum. The glc gene was cloned into two plasmids, pC27-glc and pC29-glc, in which its expression was controlled by the TR2' promoter of the 2' gene of T-DNA and the rbcS promoter of Arabidopsis, respectively. These constructions were used for transformation of agrobacteria followed by transfer into plants. In transformed plants, each plasmid caused a high level of activity of thermostable bacterial glucanase not observed in reference plants. The plants obtained were used to study activation of some defense-related genes induced by their interaction with either tobacco mosaic virus (TMV) or a pathogenic fungus.

  16. Total Sugar,Reducing Sugar and Starch Content of Nicotiana tabacum L. CB-1 in Different Ecological Regions%翠碧1号烟叶总糖、还原糖和淀粉含量的区域分布特征

    Institute of Scientific and Technical Information of China (English)

    包可翔

    2011-01-01

    [ Objective ] The carbohydrates of Nicotiana tabacum L. CB-1 in different ecological regions were researched to provide a reference for the formulation of cigarettes. [ Method ] The total sugar,reducing sugar and starch content of Nicotiana tabacum L. CB-1 in 13 townships were analyzed by cluster and ANOVA method. [ Result ] The starch content of upper leaves in Yongding-Hulei and Shanghang-Rufeng were significantly higher than those in other locations. The upper leaves in Wuyi-Zheyang, Pucheng-Linjiang and Pucheng-Yongxing were classified to the sane class,in which the total sugar,reducing sugar and starch content of samples were significantly lower than those in other locations. The total sugar content of cutter leaves in Yongding-Hulei and Shanghang-Rufeng were significantly lower than those in other locations, but the starch content were significantly higher than those in other locations. The reducing sugar content of lower leaves in Yongding-Hulei was significantly lower than those in other locations,which in Wuyi-Zheyang and Pucheng-Zhongxing were significantly higher than those in other locations. The starch content of lower leaves in Yongding-Hulei was significantly higher than those in other locations. [Conclusion] The total sugar,reducing sugar and starch content of Nicotiana tabacum L. CB-1 in some townships had much in common,and those in some townships had significant difference. So it was necessary to classify 13 townships for better cigarettes formulation.%[目的]研究翠碧1号烟叶糖类物质在不同生态区域的分布特征,为卷烟配方提供参考依据.[方法]对13个乡镇翠碧1号烟叶样品的总糖、还原糖和淀粉含量进行聚类分析,并且进行类问方差分析.[结果]上部烟叶中永定湖雷和上杭庐丰样品的淀粉含量在0.05水平显著高于其他地点样品,武夷柘洋、浦城临江和浦城永兴聚为一类,其样品的总糖、还原糖和淀粉含量显著低于其他地点样品;中部烟叶

  17. 响应面法优化烟草花超临界二氧化碳萃取工艺及香气分析%Optimization of Supercritical CO2 Fluid Extraction of Flowers of Nicotiana tabacum by Response Surface Method and Analysis of Aroma Components

    Institute of Scientific and Technical Information of China (English)

    刘煜宇; 李仙; 念小魁; 普元柱; 郑琳

    2013-01-01

    Supercritical CO2 fluid extraction (SCFE) was optimized by response surface method (RSM) to study the utilization of flowers of Nicotiana tabacum in tobacco,spice and medicine,etc.Taking the extraction yield of flowers of Nicotiana tabacum as response value,the effects of technical conditions on the extraction yield were determined by designing experiments according to Box-Behnken Design (BBD).The volatile components in the extract were identified by GC/MS method.The results showed that:1) The optimal extraction conditions were:pressure 31.9 MPa,temperature 47.3 ℃,and time 108.5 mins.2) 28 components were identified,and their contents were determined with peak area normalization method,among them,the component with the highest content (43.81%) was 1,5,9-trimethyl-12-(1-methylethyl)-4,8,13-cyclotetradecatriene-1,3-diol.%为了研究烟草花在烟草、香料、医药等方面的应用,以响应面法对烟草花超临界CO2萃取工艺进行了优化.以烟草花萃取率为响应值,根据Box-Behnken Design(BBD)设计实验,确定各工艺条件对萃取率的影响;采用气相色谱-质谱法对萃取物挥发性成分进行分析鉴定.结果表明:①烟草花的最佳超临界CO2萃取条件为:萃取压力31.9 MPa,萃取温度47.3℃,萃取时间108.5 min.②烟草花超临界CO2提取物中共鉴定出28种化合物,其中,含量最高的是1,5,9-三甲基-12-(1-异丙基)-4,8,13-环十四碳三烯-1,3-二醇(43.81%).

  18. Ecotoxicity of natural insecticide based on tobacco plant extract and hematological effects on the Nile tilapia (Oreochromis niloticus. Ecotoxicity and hematological effects of a natural insecticide based on tobacco (Nicotiana tabacum extract on Nile tilapia (Oreochromis niloticus - doi: 10.4025/actascibiolsci.v35i2.14131

    Directory of Open Access Journals (Sweden)

    Marisa Narciso Fernandes

    2013-05-01

    Full Text Available Natural insecticides derived from plant extracts have been used as an alternative to synthetic products in order to reduce environmental contamination. The present study aimed to examine the effects of Fumydro®, a natural insecticide based in the tobacco plant Nicotiana tabacum, on the Nile tilapia (Oreochromis niloticus by determining the 48-h LC50 and evaluating their effects on hematological variables. Adult specimens of O. niloticus were exposed to four Fumydro® concentrations (200, 300, 400 and 500 μL L-1. The 48-h LC50 of Fumydro® was determined as 370 ± 50 μL L-1. Surviving fish showed increasing in the red blood cells, hemoglobin concentration, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration. The thrombocytes did not change but the percentage of neutrophils increased. These results indicated that the insecticide Fumydro® is toxic to Nile tilapia and the changes of the erythrocyte variables suggested hypoxemia induction with low effect on the immune system.Natural insecticides from plant extracts represent an alternative to the highly toxic synthetic products in order to reduce environmental contamination; however some might also be toxic for non-target organisms. The present study determined the 50% lethal concentration (48h; LC50 for adults Nile tilapia (Oreochromis niloticus exposed to the natural insecticide Fumydro®, based on the tobacco plant (Nicotiana tabacum, and evaluated its effect on hematological variables. After preliminary tests, adult specimens of O. niloticus were exposed to four Fumydro® concentrations (200, 300, 400 and 500 μL L-1. The 48h; LC50 of Fumydro® was determined at 370 ± 50 μL L-1. The surviving fish after exposure to Fumydro® showed an increase in the number of red blood cells, hemoglobin concentration, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration. The number of thrombocytes and leukocytes has not changed, unlike the differential leukocyte

  19. Cloning and Characterization of Diacylglycerol Acyltransferase Gene (NtDGAT2) from Tobacco (Nicotiana tabacum L.)%烟草二脂酰甘油酰基转移酶基因(NtDGAT2)的克隆与功能分析

    Institute of Scientific and Technical Information of China (English)

    阳天泉; 徐荣华; 刘爱忠

    2013-01-01

    Diacylglycerol acyltransferase (DGAT2), a key enzyme for lipid biosynthesis, plays a critical role in oil accumulation in oilseeds. In this study, using the silico cloning technique combined with RT-PCR method, a novel DGAT2 gene with the length of 999 bp encoding 332 amino acids was isolated from seed cDNAs of tobacco (Nicotiana tabacum), named NtDGAT2 (GenBank No JX843807). The sequence analyses showed that the amino acid sequence of NtDGAT2 with the typical functional motifs of DGAT2 was high similarity to those identified from other species. Based on Real-time quantitative PCR expression analysis, NtDGAT2 was expressed in all tissues such as root, stem, leaf, flower and developing seed; in particular, NtDGAT2 was highly expressed in flowers and developing seeds. Further, the function of NtDGAT2 was confirmed by heterologous transformation and the functional complementary experiments in yeast.%二脂酰甘油酰基转移酶(diacylglycerol acyltransferase,DGAT2)是植物储存油脂生物合成过程中的关键酶,对种子储存油脂累积具有重要的生理作用.本文采用电子克隆与实验相结合的方法,从烟草种子cDNA中克隆到DGAT2基因的开放阅读框序列,命名为NtDGAT2 (GenBank登录号JX843807),其序列长999 bp,编码332个氨基酸.多序列比对和进化分析表明该基因编码蛋白与其他植物DGAT2具有较高相似性和典型的DGAT2结构域.利用Real-time PCR定量表达分析显示Nt-DGA T2在烟草种子、花、茎、叶和根里面都有表达,且在发育中的种子和花的发育过程大量表达.酵母互补实验证实该基因编码蛋白具有DGAT酶活性.

  20. 转北美海蓬子胆碱单加氧酶基因(cmo)烟草的获得及耐盐性鉴定%Transformation of Salicornia bigelovii Choline Monooxygenase Gene (cmo) into Nicotiana tabacum and Salt Resistance Identification of Transgenic Plant

    Institute of Scientific and Technical Information of China (English)

    朱天艺; 龚一富; 刘增美; 王何瑜

    2015-01-01

    胆碱单加氧酶(choline monooxygenase,CMO)是植物甜菜碱合成过程中重要的限速酶.为探讨转北美海蓬子(Salicornia bigelovii)cmo基因烟草(Nicotiana tabacum)在耐盐基因工程中的应用,利用基础质粒pCAMBIA1300UR构建pCAMBIA 1300UR-cmo植物表达载体,通过电击法将重组质粒导入农杆菌(Agrobacterium tumefaciens)EHA105中,并采用农杆菌介导的叶盘转化法转入烟草,经抗生素筛选、PCR和qRT-PCR检测后,对阳性烟草植株进行耐盐性鉴定.结果表明,北美海蓬子cmo基因在烟草中具有80%的转化效率,在200 mmol/L盐胁迫下,转基因植株的超氧化物歧化酶(superoxide dismutase,SOD)、过氧化物酶(peroxidase,POD)、过氧化氢酶(catalase,CAT)及脯氨酸(proline,PRO)含量明显高于非转基因的烟草植株,而丙二醛(malondialdehyde,MDA)含量和过氧化氢(hydrogen peroxide,H2O2)含量则低于非转基因组.结果表明,cmo基因的转化提高了烟草的耐盐性,为进一步阐明cmo基因在植物体内的甜菜碱合成代谢途径、抗逆调控机制及培育耐盐植物新品种提供基因资源库和理论参考.

  1. Cloning and tissue expression of cysteine proteinase inhibitor (CPI) gene family inNicotiana tabacum L%烟草半胱氨酸蛋白酶抑制剂(CPI)基因家族的克隆及组织表达谱分析

    Institute of Scientific and Technical Information of China (English)

    林世锋; 元野; 任学良; 邹颉; 黎瑞源; 郭玉双; 赵杰宏; 王仁刚

    2014-01-01

    运用生物信息学方法,结合RT-PCR和SMART RACE技术从烟草(Nicotiana tabacum)中克隆了4个CPI基因的全长cDNA序列,分别命名为NtCPI1、NtCPI2、NtCPI3和NtCPI4, GenBank登陆号分别为KF057988、KF057989、KF057990和KF057991。基因序列分析表明4个基因分别编码98、98、120和123个氨基酸残基的蛋白质,都具有CPI反应位点的保守基序GG、QXVXQ和A/PW,同时具有植物CPI所特有的LARFAV基序,其中NtCPI3和NtCPI4的N端还包含一段27个氨基酸残基组成的信号肽。实时荧光定量PCR试验表明,4个基因的组织表达谱很广,在根、茎、叶和芽组织中都有表达。研究结果为进一步研究半胱氨酸蛋白酶抑制剂在植物中的生理功能奠定了基础。%Full-length cDNAs of fourCPI genes includingNtCPI1、NtCPI2、NtCPI3andNtCPI4were cloned fromNicotiana tabacum L. cv. K326 using RT-PCR and SMART RACE technique. Their sequences were deposited in GenBank with accession number KF057988, KF057989, KF057990 and KF057991. Sequence analysis showed that these four genes were predicted products of 98, 98, 120 and 123 amino acid residues, respectively. In addition to the typical inhibitory motifs, i.e. central signature motif QXVXG, a GG doublet in terminal region, and A/PW residues in C-terminal part. These deduced amino acid sequences contained PhyCys-specific LARFAV-like motif in the N-terminal region, of which a N-terminal signal peptide of 27 residues was found in both NtCPI3 and NtCPI4. Meanwhile, transcripts of these four genes were found in roots, stems, leaves and buds by real-time quantitative PCR, which indicated that they were broadly expressed in tobacco. This study laid foundation for further exploring physiological functions of these cysteine proteinase inhibitor genes in plants.

  2. Effect of cytokinins on shoot apical meristem in Nicotiana tabacum

    Directory of Open Access Journals (Sweden)

    Uzelac Branka

    2012-01-01

    Full Text Available Cytokinins are involved in plant cell proliferation leading to plant growth and morphogenesis. The size, activity and maintenance of the shoot apical meristem (SAM are defined by a balanced rate of mitotic cell divisions and functional cell differentiation that are controlled by cytokinins. In order to investigate the effect of exogenous cytokinin on SAM, morpho-anatomical changes in the shoot apices of tobacco treated with benzyladenine (BA were compared to those of untreated control plants.

  3. Management of broomrape (Orobanche cernua) in tobacco (Nicotiana tabacum)

    NARCIS (Netherlands)

    Dhanapal, G.N.

    1996-01-01


    Tobacco is an important commercial crop in India. India is the third largest tobacco producing country in the world. Tobacco is cultivated in an area of 0.428 million ha. Non- Virginia tobaccos such as bidi tobacco constitute about 65% of the total tobacco area in the

  4. Management of broomrape (Orobanche cernua) in tobacco (Nicotiana tabacum).

    NARCIS (Netherlands)

    Dhanapal, G.N.

    1996-01-01

    Tobacco is an important commercial crop in India. India is the third largest tobacco producing country in the world. Tobacco is cultivated in an area of 0.428 million ha. Non- Virginia tobaccos such as bidi tobacco constitute about 65% of the total tobacco area in the country.Broomrape (Orobanche ce

  5. Shifting Nicotiana attenuata’s diurnal rhythm does not alter its resistance to the specialist herbivore Manduca sexta

    Institute of Scientific and Technical Information of China (English)

    Jasmin Herden; Stefan Meldau; Sang-Gyu Kim; Grit Kunert; Youngsung Joo; Ian T Baldwin; Meredith C Schuman

    2016-01-01

    Arabidopsis thaliana plants are less resistant to attack by the generalist lepidopteran herbivore Trichoplusia ni when plants and herbivores are entrained to opposite, versus identical diurnal cycles and tested under constant conditions. This effect is associated with circadian fluctuations in levels of jasmonic acid, the transcription factor MYC2, and glucosino-late contents in leaves. We tested whether a similar effect could be observed in a different plant–herbivore system:the wild tobacco Nicotiana attenuata and its co-evolved specialist herbivore, Manduca sexta. We measured larval growth on plants under both constant and diurnal conditions following identical or opposite entrainment, profiled the metabolome of attacked leaf tissue, quantified specific metabolites known to reduce M. sexta growth, and monitored M. sexta feeding activity under all experimental conditions. Entrainment did not consistently affect M. sexta growth or plant defense induction. However, both were reduced under constant dark conditions, as was M. sexta feeding activity. Our data indicate that the response induced by M. sexta in N. attenuata is robust to diurnal cues and independent of plant or herbivore entrain-ment. We propose that while the patterns of constitutive or general damage-induced defense may undergo circadian fluctuation, the orchestration of specific induced responses is more complex.

  6. ANORMALIDADES EN FLORES DE NICOTIANA

    Directory of Open Access Journals (Sweden)

    Raúl Martínez Crovetto

    2010-11-01

    Full Text Available

    En este trabajo se describen las siguientes anormalidades florales: la adherencia de tallos florales, spirody, dialysepaly, dialypetaly, staminodiody, petalody de estambre y la supresión de estilo en Nicotiana rustica, y dialysepaly, dialypetaly, dialycarpely, supresión de estilo y staminody del sépalo en Nicotiana tabacum. Algunos de estos términos se proponen por primera vez por los fenómenos teratológicos.

  7. Cloning and expression analysis of osmotic stress tolerance gene NtP5CS in Nicotiana tabacum%普通烟草抗渗透胁迫基因NtP5CS的克隆与表达分析

    Institute of Scientific and Technical Information of China (English)

    焦蓉; 刘朝科; 冯祥国; 胡晓明; 刘贯山; 刘好宝; 王树林; 侯娜; 王全贞; 靳义荣; 白岩; 冯广林

    2012-01-01

    Sequence signature, evolutionary relationship, and expression profile of key enzyme of P5CS gene cloned from tobacco (Nicotiana tabacum) for Pro synthesis were analyzed to establish foundation for research in tobacco stress tolerance. Degenerate primers were designed to obtain segment of P5CS, and 5 ' and 3' ends of the cDNA were amplified by RACE. Bioinformatics was used to analyze sequence structure, coded protein's conserved domains, and basic characteristics. Expression pattern was studied by RT-PCR. The P5CS gene cloned from tobacco induced by drought stress, which was 2,584 bp in full length, were termed NtP5CS (GenBank accession HM854026). The ORF was 2160 bp in length, which encoded proteins of 719 amino acid residues. GenBank Blast analysis showed that the sequence is in high homologous with tomPR02 gene from tomato in level of nuleotide and amino acid. According to phylogenetic tree, NtP5CS and tomPR02 may be Ortholog gene. RT-PCR results showed that NtPSCS could be induced by drought, high salt, low temperature, ABA, and it had the highest expression in root and leaf of tobacco' s prosperous long-term and water stress condition. It was the. first time to obtain P5CS from tobacco. From the response to water stress, it may be inferred that it played a role in resistance to osmoticstress.%从普通烟草中克隆脯氨酸合成的关键酶基因P5CS,分析其序列特征、进化关系、表达模式,以期为烟草的抗逆研究奠定基础.设计兼并引物获得P5CS基因中间片段,利用RACE技术扩增其全长cDNA;采用生物信息学技术分析该基因的序列结构及其编码蛋白的保守域及基本特性;利用RT-PCR研究其表达模式.从受干旱胁迫诱导的普通烟草品种中烟14中克隆到全长为2584bp的烟草P5CS基因,命名为NtP5 CS,GenBank登录号为HM854026,开放读码框为2160 bp,编码719个氨基酸.经Blast同源性比对分析,该序列与番茄tomPRO2基因在核苷酸和氨基酸水平上高度同源.系

  8. Wild Nicotiana Species as a Source of Cytoplasmic Male Sterility in Nicotianatabacum

    Directory of Open Access Journals (Sweden)

    Nikova V

    2014-12-01

    Full Text Available The results of our experiments executed to obtain tobacco male sterile lines through interspecific hybridization are summarized. Ten wild species from the genus Nicotiana: N. excelsior (exc, N. amplexicaulis (amp, N. rustica (rus, Nicotianaglauca (gla, N. velutina (vel, N. benthamiana (ben, N. maritima (mar, N. paniculata (pan, N. longiflora (lon and N. africana (afr were used as cytoplasmic donors and N. tabacum, cv. HarmanliiskaBasma (HB as a donor of the nucleus. Genetic effects of cytoplasmic-nuclear interaction of the studied species are discussed. Our results suggested that cytoplasmic male sterility (CMS was expressed when the cytoplasms of the above mentioned wild Nicotiana species were combined with the nucleus of N. tabacum. The 10 sources of CMS obtained in tobacco were characterized by altered flower phenotypes. Flowers are classified into types according the stamen, pistil and corolla modification. All these CMS sources were backcrossed to Oriental tobaccos, cvs. Tekne, Nevrokop B-12, Kroumovgrad 90 and Djebel 576, to develop corresponding CMS lines. The investigated cytoplasms produced compete male sterility in all those cultivars. The CMS lines preserved flower types, specific for every “sterile” cytoplasm. The extent of male organ modifications varied from apparently normal (but pollenless stamens in CMS (pan, (afr, some plants of (vel (mar through different degrees of malformations (shriveled anther on shortened filaments (lon, pinnate-like anthers on filaments of normal length (amp, petal - (ben, pistil- or stigma-like structures (rus, (gla to lack of male reproductive organs in (exc and in some plants of (vel, (mar, (rus and (gla. Most of the above mentioned cytoplasms had normal female gametophyte and good seed productivity. Alterations of the pistils were observed in CMS (rus, (exc and (ben causing reduction of the seed set. Electrophoresis of seed proteins of the tobacco cultivars and their CMS lines also suggested that

  9. Genetic variation in alkaloid accumulation in leaves of Nicotiana

    Institute of Scientific and Technical Information of China (English)

    Bo SUN; Fen ZHANG; Guo-jun ZHOU; Guo-hai CHU; Fang-fang HUANG; Qiao-mei WANG; Li-feng JIN; Fu-cheng LIN; Jun YANG

    2013-01-01

    Alkaloids are plant secondary metabolites that are widely distributed in Nicotiana species and contribute greatly to the quality of tobacco leaves. Some alkaloids, such as nornicotine and myosmine, have adverse effects on human health. To reduce the content of harmful alkaloids in tobacco leaves through conventional breeding, a genetic study of the alkaloid variation among different genotypes is required. In this study, alkaloid profiles in leaves of five Nicotiana tabacum cultivars and Nicotiana tomentosiformis were investigated. Six alkaloids were identified from al six genotypes via gas chromatograph-mass spectrometry (GC-MS). Significant differences in alkaloid content were ob-served both among different leaf positions and among cultivars. The contents of nornicotine and myosmine were positively and significantly correlated (R2=0.881), and were also separated from those of other alkaloids by clustering. Thus, the genotype plays a major role in alkaloid accumulation, indicating a high potential for manipulation of alkaloid content through traditional breeding.

  10. Genetic variation in alkaloid accumulation in leaves of Nicotiana.

    Science.gov (United States)

    Sun, Bo; Zhang, Fen; Zhou, Guo-jun; Chu, Guo-hai; Huang, Fang-fang; Wang, Qiao-mei; Jin, Li-feng; Lin, Fu-cheng; Yang, Jun

    2013-12-01

    Alkaloids are plant secondary metabolites that are widely distributed in Nicotiana species and contribute greatly to the quality of tobacco leaves. Some alkaloids, such as nornicotine and myosmine, have adverse effects on human health. To reduce the content of harmful alkaloids in tobacco leaves through conventional breeding, a genetic study of the alkaloid variation among different genotypes is required. In this study, alkaloid profiles in leaves of five Nicotiana tabacum cultivars and Nicotiana tomentosiformis were investigated. Six alkaloids were identified from all six genotypes via gas chromatograph-mass spectrometry (GC-MS). Significant differences in alkaloid content were observed both among different leaf positions and among cultivars. The contents of nornicotine and myosmine were positively and significantly correlated (R(2)=0.881), and were also separated from those of other alkaloids by clustering. Thus, the genotype plays a major role in alkaloid accumulation, indicating a high potential for manipulation of alkaloid content through traditional breeding.

  11. Current status and prospects for the study of Nicotiana genomics, genetics, and nicotine biosynthesis genes.

    Science.gov (United States)

    Wang, Xuewen; Bennetzen, Jeffrey L

    2015-02-01

    Nicotiana, a member of the Solanaceae family, is one of the most important research model plants, and of high agricultural and economic value worldwide. To better understand the substantial and rapid research progress with Nicotiana in recent years, its genomics, genetics, and nicotine gene studies are summarized, with useful web links. Several important genetic maps, including a high-density map of N. tabacum consisting of ~2,000 markers published in 2012, provide tools for genetics research. Four whole genome sequences are from allotetraploid species, including N. benthamiana in 2012, and three N. tabacum cultivars (TN90, K326, and BX) in 2014. Three whole genome sequences are from diploids, including progenitors N. sylvestris and N. tomentosiformis in 2013 and N. otophora in 2014. These and additional studies provide numerous insights into genome evolution after polyploidization, including changes in gene composition and transcriptome expression in N. tabacum. The major genes involved in the nicotine biosynthetic pathway have been identified and the genetic basis of the differences in nicotine levels among Nicotiana species has been revealed. In addition, other progress on chloroplast, mitochondrial, and NCBI-registered projects on Nicotiana are discussed. The challenges and prospects for genomic, genetic and application research are addressed. Hence, this review provides important resources and guidance for current and future research and application in Nicotiana.

  12. Ectopic Terpene Synthase Expression Enhances Sesquiterpene Emission in Nicotiana attenuata without Altering Defense or Development of Transgenic Plants or Neighbors1[W

    Science.gov (United States)

    Schuman, Meredith C.; Palmer-Young, Evan C.; Schmidt, Axel; Gershenzon, Jonathan; Baldwin, Ian T.

    2014-01-01

    Sesquiterpenoids, with approximately 5,000 structures, are the most diverse class of plant volatiles with manifold hypothesized functions in defense, stress tolerance, and signaling between and within plants. These hypotheses have often been tested by transforming plants with sesquiterpene synthases expressed behind the constitutively active 35S promoter, which may have physiological costs measured as inhibited growth and reduced reproduction or may require augmentation of substrate pools to achieve enhanced emission, complicating the interpretation of data from affected transgenic lines. Here, we expressed maize (Zea mays) terpene synthase10 (ZmTPS10), which produces (E)-α-bergamotene and (E)-β-farnesene, or a point mutant ZmTPS10M, which produces primarily (E)-β-farnesene, under control of the 35S promoter in the ecological model plant Nicotiana attenuata. Transgenic N. attenuata plants had specifically enhanced emission of target sesquiterpene(s) with no changes detected in their emission of any other volatiles. Treatment with herbivore or jasmonate elicitors induces emission of (E)-α-bergamotene in wild-type plants and also tended to increase emission of (E)-α-bergamotene and (E)-β-farnesene in transgenics. However, transgenics did not differ from the wild type in defense signaling or chemistry and did not alter defense chemistry in neighboring wild-type plants. These data are inconsistent with within-plant and between-plant signaling functions of (E)-β-farnesene and (E)-α-bergamotene in N. attenuata. Ectopic sesquiterpene emission was apparently not costly for transgenics, which were similar to wild-type plants in their growth and reproduction, even when forced to compete for common resources. These transgenics would be well suited for field experiments to investigate indirect ecological effects of sesquiterpenes for a wild plant in its native habitat. PMID:25187528

  13. Putative Serine Protease Effectors of Clavibacter michiganensis Induce a Hypersensitive Response in the Apoplast of Nicotiana Species.

    Science.gov (United States)

    Lu, You; Hatsugai, Noriyuki; Katagiri, Fumiaki; Ishimaru, Carol A; Glazebrook, Jane

    2015-11-01

    Clavibacter michiganensis subspp. michiganensis and sepedonicus cause diseases on solanaceous crops. The genomes of both subspecies encode members of the pat-1 family of putative serine proteases known to function in virulence on host plants and induction of hypersensitive responses (HR) on nonhosts. One gene of this family in C. michiganensis subsp. sepedonicus, chp-7, is required for triggering HR in Nicotiana tabacum. Here, further investigation revealed that mutation of the putative catalytic serine residue at position 232 to threonine abolished the HR induction activity of Chp-7, suggesting that enzymatic activity is required. Purified Chp-7 triggered an HR in N. tabacum leaves in the absence of the pathogen, indicating Chp-7 itself is the HR elicitor from C. michiganensis subsp. sepedonicus. Ectopic expression of chp-7 constructs in N. tabacum leaves revealed that Chp-7 targeted to the apoplast triggered an HR while cytoplasmic Chp-7 did not, indicating that Chp-7 induces the HR in the apoplast of N. tabacum leaves. Chp-7 also induced HR in N. sylvestris, a progenitor of N. tabacum, but not in other Nicotiana species tested. ChpG, a related protein from C. michiganensis subsp. michiganensis, also triggered HR in N. tabacum and N. sylvestris. Unlike Chp-7, ChpG triggered HR in N. clevelandii and N. glutinosa.

  14. 侵染云南白肋烟的中国番茄黄化曲叶病毒及伴随卫星DNA分子的基因组特征%Genomic Characterization of DNA-A and Associated Satellite DNA Molecule of an Isolate of Tomato Yellow Leaf Curl China Virus Infecting Nicotiana tabacum White Burley in Yunnan

    Institute of Scientific and Technical Information of China (English)

    李桂新; 范三微; 李正和; 谢艳; 周雪平

    2003-01-01

    从中国云南省大理地区表现曲叶症状的白肋烟(Nicotina tabacum White Burley)上分离到病毒分离物Y43,该病毒可经烟粉虱(Bemisia tabaci)及嫁接传播.用15种粉虱传双生病毒的单抗对病样进行TAS-ELISA检测,结果表明,该病毒属菜豆金色花叶病毒属(Begomovirus)病毒.对DNA-A1.7kb基因组序列测定和分析表明,Y43与中国广西报道的中国番茄黄化曲叶病毒(Tomata yellow leaf curl China virus,TYLCCNV)同源性达89%,其中外壳蛋白(CP)氨基酸同源性达96%,因此Y43应为TYLCCNV的一个新分离物.进一步研究发现,Y43还伴随着一个长1 349 nt的卫星DNA分子(DNAβ).Y43DNAβ与AYVVDNAβ、BYVMVDNAβ和CLCuVDNAβ的同源性较低,而与中国分离的烟草曲叶病毒Y5 DNAβ和Y8 DNA β的亲缘关系较近.DNAβ可能编码7个分子量超过3.5kD的ORF,其中C1推测为有功能的ORF.

  15. [Construction and analysis of transgenic plants of Nicotiana tabacum L. expressing a bacterial gene for beta-1,3-glucanase. I. Construction of vector plasmids for transfer into plants and expression of a modified gene for beta-1,3-glucanase from Clostridium thermocellum in tobacco protoplasts].

    Science.gov (United States)

    Darbinian, N S; Popov, Iu G; Mochul'skiĭ, A V; Volkova, L V; Piruzian, E S; Vasilevko, V T

    1996-02-01

    We constructed two vectors, pC27-glc and pC29-glc, that allow expression of the beta-1,3-glucanase gene (glc) in plant cells. The glc gene was previously cloned from anaerobic thermophilous bacterium Clostridium thermocellum. To increase the efficiency of expression, the N-terminal fragment of the glc gene encoding bacterial transient peptide was deleted, and hybrid variants of lacZ-glc were obtained. Analysis of expression of the hybrid genes in Escherichia coli showed that deletion of the fragment corresponding to 31 amino acids (a.a.) of beta-glucanase affected neither activity nor thermostability of the enzyme. The modified gene was subcloned into two vectors, pC27 and pC29, in which its expression was controlled by the TR2' promoter of the 2' gene of T-DNA and the rbcS promoter from Arabidopsis, respectively. Each of the resulting plasmids, pC27-glc and pC29-glc, was transfected into protoplasts of Nicotiana plumbaginifolia. Both the plasmids were shown to allow a high level of activity of the thermostable beta-1,3-glucanase. We plan to use the vectors obtained for transformation of agrobacteria and construction of transgenic plants.

  16. A Distinct Endogenous Pararetrovirus Family in Nicotiana tomentosiformis, a Diploid Progenitor of Polyploid Tobacco1[w

    Science.gov (United States)

    Gregor, Wolfgang; Mette, M. Florian; Staginnus, Christina; Matzke, Marjori A.; Matzke, Antonius J.M.

    2004-01-01

    A distinct endogenous pararetrovirus (EPRV) family corresponding to a previously unknown virus has been identified in the genome of Nicotiana tomentosiformis, a diploid ancestor of allotetraploid tobacco (Nicotiana tabacum). The putative virus giving rise to N. tomentosiformis EPRVs (NtoEPRVs) is most similar to tobacco vein clearing virus, an episomal form of a normally silent EPRV family in Nicotiana glutinosa; it is also related to a putative virus giving rise to the NsEPRV family in Nicotiana sylvestris (the second diploid progenitor of tobacco) and in the N. sylvestris fraction of the tobacco genome. The copy number of NtoEPRVs is significantly higher in N. tomentosiformis than in tobacco. This suggests that after the polyploidization event, many copies were lost from the polyploid genome or were accumulated specifically in the diploid genome. By contrast, the copy number of NsEPRVs has remained constant in N. sylvestris and tobacco, indicating that changes have occurred preferentially in the NtoEPRV family during evolution of the three Nicotiana species. NtoEPRVs are often flanked by Gypsy retrotransposon-containing plant DNA. Although the mechanisms of NtoEPRV integration, accumulation, and/or elimination are unknown, these processes are possibly linked to retrotransposon activity. PMID:14988473

  17. PHANTASTICA regulates development of the adaxial mesophyll in Nicotiana leaves.

    Science.gov (United States)

    McHale, Neil A; Koning, Ross E

    2004-05-01

    Initiation and growth of leaf blades is oriented by an adaxial/abaxial axis aligned with the original axis of polarity in the leaf primordium. To investigate mechanisms regulating this process, we cloned the Nicotiana tabacum ortholog of PHANTASTICA (NTPHAN) and generated a series of antisense transgenics in N. sylvestris. We show that NSPHAN is expressed throughout emerging blade primordia in the wild type and becomes localized to the middle mesophyll in the expanding lamina. Antisense NSPHAN leaves show ectopic expression of NTH20, a class I KNOX gene. Juvenile transgenic leaves have normal adaxial/abaxial polarity and generate leaf blades in the normal position, but the adaxial mesophyll shows disorganized patterns of cell division, delayed maturation of palisade, and ectopic reinitiation of blade primordia along the midrib. Reversal of the phenotype with exogenous gibberellic acid suggests that NSPHAN, acting via KNOX repression, maintains determinacy in the expanding lamina and sustains the patterns of cell proliferation critical to palisade development.

  18. The Tomato spotted wilt virus cell-to-cell movement protein (NSM) triggers a hypersensitive response in Sw-5 containing resistant tomato lines and Nicotiana benthamiana transformed with the functional Sw-5b resistance gene copy.

    NARCIS (Netherlands)

    Hallwass, M.; Silva de Oliveira, A.; Dianese, E.C.; Lohuis, D.; Boiteux, L.S.; Inoue-Nagata, A.K.; Resende, de R.O.; Kormelink, R.J.M.

    2014-01-01

    Although the Sw-5 gene cluster has been cloned, and Sw-5b has been identified as the functional gene copy that confers resistance to Tomato spotted wilt virus (TSWV), its avirulence (Avr) determinant has not been identified to date. Nicotiana tabacum SR1 plants transformed with a copy of the Sw-5b

  19. Effects of air pollutants on Nicotiana cultivars and species used for virus studies

    Energy Technology Data Exchange (ETDEWEB)

    Grosso, J.J.; Menser, H.A.; Hodges, G.H.; McKinney, H.H.

    1971-08-01

    Three tobacco cultivars (Nicotiana tabacum) and two Nicotiana species, used for virus identification studies, were tested for susceptibility to air pollutants. The air pollutants used were (i) ozone; (ii) sulfur dioxide; and (iii) a mixture of both gases. Nicotiana rustica var. brasilia was the most susceptible cultivar to injury from individual gases, a gas mixture, and ambient polluted air. Nicotiana rustica var. brasilia displayed from 25 to 70% leaf injury when exposed to increasing ozone doses (10 to 30 pphm). Ozone injury to N. glutinosa fumigated at the same doses ranged from 10 to 60%. Samsun, Samsun (NN), and Xanthi cultivars were more resistant to ozone than N. rustica var. brasilia or N. glutinosa. Sulfur dioxide (ca. 45 pphm) added to an ozone (ca. 3.0 pphm) atmosphere induced a synergistic response evidenced as injury to all cultivars and species. Mixed gas injury was more acute on N. glutinosa and N. rustica var. brasilia than on Samsun, Samsun (NN), and Xanthi. Samsun and Samsun (NN) were more susceptible to sulfur dioxide (125 pphm) than N. glutinosa and Xanthi. Fumigations produced two injury syndromes: (i) light or dark punctate flecks, on upper leaf surfaces; or (ii) bifacial tissue collapse.

  20. (E)-β-farnesene synthase genes affect aphid (Myzus persicae) infestation in tobacco (Nicotiana tabacum).

    Science.gov (United States)

    Yu, Xiudao; Jones, Huw D; Ma, Youzhi; Wang, Genping; Xu, Zhaoshi; Zhang, Baoming; Zhang, Yongjun; Ren, Guangwei; Pickett, John A; Xia, Lanqin

    2012-03-01

    Aphids are major agricultural pests which cause significant yield losses of the crop plants each year. (E)-β-farnesene (EβF) is the alarm pheromone involved in the chemical communication between aphids and particularly in the avoidance of predation. In the present study, two EβF synthase genes were isolated from sweet wormwood and designated as AaβFS1 and AaβFS2, respectively. Overexpression of AaβFS1 or AaβFS2 in tobacco plants resulted in the emission of EβF ranging from 1.55 to 4.65 ng/day/g fresh tissues. Tritrophic interactions involving the peach aphids (Myzus persicae), predatory lacewings (Chrysopa septempunctata) demonstrated that the transgenic tobacco expressing AaβFS1 and AaβFS2 could repel peach aphids, but not as strongly as expected. However, AaβFS1 and AaβFS2 lines exhibited strong and statistically significant attraction to lacewings. Further experiments combining aphids and lacewing larvae in an octagon arrangement showed transgenic tobacco plants could repel aphids and attract lacewing larvae, thus minimizing aphid infestation. Therefore, we demonstrated a potentially valuable strategy of using EβF synthase genes from sweet wormwood for aphid control in tobacco or other economic important crops in an environmentally benign way.

  1. Multi-platform metabolomic analyses of ergosterol-induced dynamic changes in Nicotiana tabacum cells

    CSIR Research Space (South Africa)

    Tugizimana, F

    2014-01-01

    Full Text Available to variation in the biosynthesis of secondary metabolites. PCA scores plots revealed dose- and time-dependent metabolic variations, with optimal treatment conditions being found to be 300 nM ergosterol and an 18 h incubation period. The observed ergosterol...

  2. Pest insects at tobacco (Nicotiana tabacum L.) in Strumica region, Republic of Macedonia

    OpenAIRE

    Spasov, Dusan; Spasova, Dragica; Atanasova, Biljana; Serafimova, Mimoza

    2013-01-01

    The studies that were conducted in 2011 and 2012 about the pest insects at tobacco, at four localities in Strumica region, showed that the most important insect pests that cause damage to tobacco leaves are: the green peach aphid – Myzus persicae Sulz. (Homoptera: Aphididae), the onion trips – Thrips tabaci Lind. (Thysanoptera: Thripidae) and the tobacco flea beetle – Epitrix hirtipennis Melsh. (Coleoptera: Chrysomelidae). In both years of research the greatest number showed Myzus persica...

  3. Titanium dioxide nanoparticles affect the growth and microRNA expression of tobacco (Nicotiana tabacum).

    Science.gov (United States)

    Frazier, Taylor P; Burklew, Caitlin E; Zhang, Baohong

    2014-03-01

    Titanium dioxide (TiO(2)) is one of the most widely used pigments in the world. Due to its heavy use in industry and daily life, such as food additives, cosmetics, pharmaceuticals, and paints, many residues are released into the environment and currently TiO(2) nanoparticles are considered an emerging environmental contaminant. Although several studies have shown the effect of TiO(2) nanoparticles on a wide range of organisms including bacteria, algae, plankton, fish, mice, and rats, little research has been performed on land plants. In this study, we investigated the effect of TiO(2) nanoparticles on the growth, development, and gene expression of tobacco, an important economic and agricultural crop in the southeastern USA as well as around the world. We found that TiO(2) nanoparticles significantly inhibited the germination rates, root lengths, and biomasses of tobacco seedlings after 3 weeks of exposure to 0.1, 1, 2.5, and 5 % TiO(2) nanoparticles and that overall growth and development of the tobacco seedlings significantly decreased as TiO(2) nanoparticle concentrations increased. Overall, tobacco roots were the most sensitive to TiO(2) nanoparticle exposure. Nano-TiO(2) also significantly influenced the expression profiles of microRNAs (miRNAs), a recently discovered class of small endogenous noncoding RNAs (∼20-22 nt) that are considered important gene regulators and have been shown to play an important role in plant development as well as plant tolerance to abiotic stresses such as drought, salinity, cold, and heavy metal. Low concentrations (0.1 and 1 %) of TiO(2) nanoparticles dramatically induced miRNA expression in tobacco seedlings with miR395 and miR399 exhibiting the greatest fold changes of 285-fold and 143-fold, respectively. The results of this study show that TiO(2) nanoparticles have a negative impact on tobacco growth and development and that miRNAs may play an important role in tobacco response to heavy metals/nanoparticles by regulating gene expression.

  4. Fauna epigeica em sistemas de produção de Nicotiana tabacum L.

    Directory of Open Access Journals (Sweden)

    Marta Sandra Drescher

    2011-10-01

    Full Text Available O Rio Grande do Sul detém grande relevância na produção de tabaco. Adotam-se três sistemas de manejo: convencional, mínimo e plantio direto. Para analisar o impacto desses sistemas sobre a população da fauna do solo, efetuou-se uma amostragem da fauna epigeica. Foram avaliados preparo convencional (PC, cultivo mínimo (CM e plantio direto (PD, além de área de reconversão da fumicultura para a vitivinicultura (RV e de mata nativa (MN. Na amostragem, foram utilizadas dez armadilhas Provid em cada área. Os atributos avaliados foram: abundância de organismos, riqueza, índice de diversidade (H' e equitabilidade de Shannon (J. Collembola (Arthropoda: Hexapoda foi identificada até o nível de família, devido à sua sensibilidade às modificações do ambiente. Efetuaram-se a análise multivariada de Agrupamento Hierárquico e a Análise de Componentes Principais, e os índices H' e J foram comparados pelo teste t, de Student. O PD apresentou maior número total de organismos, isolando-se das demais áreas pela análise de agrupamento hierárquico. Os valores de riqueza foram muito semelhantes entre as áreas, destacando-se a aproximação entre RV e MN. Os tratamentos PC, PD e RV não diferiram estatisticamente quanto ao H', apresentando valores de 1,58; 1,60; e 1,52, respectivamente. CM apresentou menor valor de H' (1,18 e MN, o valor intermediário (1,33. O comportamento estatístico de J foi muito semelhante ao encontrado em H'. Em relação à Classe Collembola, foram identificadas as famílias Entomobrydae, Sminthuridae, Poduridae e Hipogasturidae. PD apresentou exemplares das quatro famílias identificadas, enquanto nas demais áreas foram identificadas apenas Entomobrydae e Sminthuridae. Pela Análise de Componentes Principais (PCA, as diferenças entre as áreas explicaram 32 % da variabilidade encontrada; desse percentual, 59,3 % foram explicados pelos eixos 1 e 2. A presença ou ausência de revolvimento do solo e a rotação com culturas de cobertura foram os fatores que mais influenciaram as populações de fauna epigeica.

  5. Multi-platform metabolomic analyses of ergosterol-induced dynamic changes in Nicotiana tabacum cells.

    Directory of Open Access Journals (Sweden)

    Fidele Tugizimana

    Full Text Available Metabolomics is providing new dimensions into understanding the intracellular adaptive responses in plants to external stimuli. In this study, a multi-technology-metabolomic approach was used to investigate the effect of the fungal sterol, ergosterol, on the metabolome of cultured tobacco cells. Cell suspensions were treated with different concentrations (0-1000 nM of ergosterol and incubated for different time periods (0-24 h. Intracellular metabolites were extracted with two methods: a selective dispersive liquid-liquid micro-extraction and a general methanol extraction. Chromatographic techniques (GC-FID, GC-MS, GC × GC-TOF-MS, UHPLC-MS and (1H NMR spectroscopy were used for quantitative and qualitative analyses. Multivariate data analyses (PCA and OPLS-DA models were used to extract interpretable information from the multidimensional data generated from the analytical techniques. The results showed that ergosterol triggered differential changes in the metabolome of the cells, leading to variation in the biosynthesis of secondary metabolites. PCA scores plots revealed dose- and time-dependent metabolic variations, with optimal treatment conditions being found to be 300 nM ergosterol and an 18 h incubation period. The observed ergosterol-induced metabolic changes were correlated with changes in defence-related metabolites. The 'defensome' involved increases in terpenoid metabolites with five antimicrobial compounds (the bicyclic sesquiterpenoid phytoalexins: phytuberin, solavetivone, capsidiol, lubimin and rishitin and other metabolites (abscisic acid and phytosterols putatively identified. In addition, various phenylpropanoid precursors, cinnamic acid derivatives and - conjugates, coumarins and lignin monomers were annotated. These annotated metabolites revealed a dynamic reprogramming of metabolic networks that are functionally correlated, with a high complexity in their regulation.

  6. The influence of light quality on the accumulation of flavonoids in tobacco (Nicotiana tabacum L.) leaves.

    Science.gov (United States)

    Fu, Bo; Ji, Xiaoming; Zhao, Mingqin; He, Fan; Wang, Xiaoli; Wang, Yiding; Liu, Pengfei; Niu, Lu

    2016-09-01

    Flavonoids are important secondary metabolites in plants regulated by the environment. To analyze the effect of light quality on the accumulation of flavonoids, we performed a rapid analysis of flavonoids in extracts of tobacco leaves using UHPLC-QTOF. A total of 12 flavonoids were detected and identified in tobacco leaves, which were classified into flavonoid methyl derivatives and flavonoid glycoside derivatives according to the groups linked to the flavonoid core. Correlation analysis was further conducted to investigate the effect of different wavelengths of light on their accumulation. The content of flavonoid methyl derivatives was positively correlated with the proportions of far-red light (FR; 716-810nm) and near-infrared light (NIR; 810-2200nm) in the sunlight spectrum and negatively correlated with the proportion of ultraviolet (UV-A; 350-400nm) and the red/far-red ratio (R/FR). By contrast, the content of flavonoid glycoside derivatives was positively correlated with the proportion of UV-A and the R/FR, and negatively correlated with FR and NIR. The results indicated that light quality with higher proportions of FR and NIR increases the activity of flavonoid methyltransferases but suppresses the activity of flavonoid glycoside transferases. While a high proportion of UV-A and a high R/FR can increase flavonoid glycoside transferase activity but suppress flavonoid methyltransferase activity.

  7. Inhibition of Trehalose Breakdown Increases New Carbon Partitioning into Cellulosic Biomass in Nicotiana tabacum

    Energy Technology Data Exchange (ETDEWEB)

    Best, F.M.; Ferrieri, R.; Best, F.M.; Koenig, K.; McDonald, K.; Schueller, M.J.; Rogers, A.; Ferrieri, R.A.

    2011-01-18

    Validamycin A was used to inhibit in vivo trehalase activity in tobacco enabling the study of subsequent changes in new C partitioning into cellulosic biomass and lignin precursors. After 12-h exposure to treatment, plants were pulse labeled using radioactive {sup 11}CO{sub 2}, and the partitioning of isotope was traced into [{sup 11}C]cellulose and [{sup 11}C]hemicellulose, as well as into [{sup 11}C]phenylalanine, the precursor for lignin. Over this time course of treatment, new carbon partitioning into hemicellulose and cellulose was increased, while new carbon partitioning into phenylalanine was decreased. This trend was accompanied by a decrease in phenylalanine ammonia-lyase activity. After 4 d of exposure to validamycin A, we also measured leaf protein content and key C and N metabolite pools. Extended treatment increased foliar cellulose and starch content, decreased sucrose, and total amino acid and nitrate content, and had no effect on total protein.

  8. Metabolomic analysis of isonitrosoacetophenone-induced perturbations in phenolic metabolism of Nicotiana tabacum cells

    CSIR Research Space (South Africa)

    Madala, NE

    2013-10-01

    Full Text Available -derived stress metabolite with anti-fungal and anti-oxidant properties, in order to investigate the effect thereof on cellular metabolism. Subsequent metabolomic-based analyses were employed to evaluate changes in the metabolome. UPLC-MS in conjunction...

  9. Characterisation of detergent-insoluble membranes in pollen tubes of Nicotiana tabacum (L.

    Directory of Open Access Journals (Sweden)

    Alessandra Moscatelli

    2015-02-01

    Full Text Available Pollen tubes are the vehicle for sperm cell delivery to the embryo sac during fertilisation of Angiosperms. They provide an intriguing model for unravelling mechanisms of growing to extremes. The asymmetric distribution of lipids and proteins in the pollen tube plasma membrane modulates ion fluxes and actin dynamics and is maintained by a delicate equilibrium between exocytosis and endocytosis. The structural constraints regulating polarised secretion and asymmetric protein distribution on the plasma membrane are mostly unknown. To address this problem, we investigated whether ordered membrane microdomains, namely membrane rafts, might contribute to sperm cell delivery. Detergent insoluble membranes, rich in sterols and sphingolipids, were isolated from tobacco pollen tubes. MALDI TOF/MS analysis revealed that actin, prohibitins and proteins involved in methylation reactions and in phosphoinositide pattern regulation are specifically present in pollen tube detergent insoluble membranes. Tubulins, voltage-dependent anion channels and proteins involved in membrane trafficking and signalling were also present. This paper reports the first evidence of membrane rafts in Angiosperm pollen tubes, opening new perspectives on the coordination of signal transduction, cytoskeleton dynamics and polarised secretion.

  10. Expression of a begomoviral DNAβ gene in transgenic Nicotiana plants induced abnormal cell division

    Institute of Scientific and Technical Information of China (English)

    CUI Xiao-feng; LI Yun-qin; HU Dong-wei; ZHOU Xue-ping

    2005-01-01

    An increasing number of monopartite begomoviruses are being identified that a satellite molecule (DNAβ) is required to induce typical symptoms in host plants. DNAβ encodes a single gene (termed βC1) encoded in the complementary-sense. We have produced transgenic Nicotiana benthamiana and N. tabacum plants expressing theβC1 gene of a DNAβ associated with Tomato yellow leaf curl China virus (TYLCCNV), under the control of the Cauliflower mosaic virus 35S promoter. Transgenic plants expressing βC1 showed severe developmental abnormalities in both species. Microscopic analysis of sections of both transgenic and non-transgenic N. tabacum leaves showed abnormal outgrowths of transgenic N. tabacum to be due to disorganized cell division (hyperplasia) of spongy and palisade parenchyma. Immuno-gold labeling of sections with a polyclonal antibody against the βC1 protein showed that the βC1 protein accumulated in the nuclei of cells. The possible biological function of the βC1 protein was discussed.

  11. Nicotiana small RNA sequences support a host genome origin of cucumber mosaic virus satellite RNA.

    Science.gov (United States)

    Zahid, Kiran; Zhao, Jian-Hua; Smith, Neil A; Schumann, Ulrike; Fang, Yuan-Yuan; Dennis, Elizabeth S; Zhang, Ren; Guo, Hui-Shan; Wang, Ming-Bo

    2015-01-01

    Satellite RNAs (satRNAs) are small noncoding subviral RNA pathogens in plants that depend on helper viruses for replication and spread. Despite many decades of research, the origin of satRNAs remains unknown. In this study we show that a β-glucuronidase (GUS) transgene fused with a Cucumber mosaic virus (CMV) Y satellite RNA (Y-Sat) sequence (35S-GUS:Sat) was transcriptionally repressed in N. tabacum in comparison to a 35S-GUS transgene that did not contain the Y-Sat sequence. This repression was not due to DNA methylation at the 35S promoter, but was associated with specific DNA methylation at the Y-Sat sequence. Both northern blot hybridization and small RNA deep sequencing detected 24-nt siRNAs in wild-type Nicotiana plants with sequence homology to Y-Sat, suggesting that the N. tabacum genome contains Y-Sat-like sequences that give rise to 24-nt sRNAs capable of guiding RNA-directed DNA methylation (RdDM) to the Y-Sat sequence in the 35S-GUS:Sat transgene. Consistent with this, Southern blot hybridization detected multiple DNA bands in Nicotiana plants that had sequence homology to Y-Sat, suggesting that Y-Sat-like sequences exist in the Nicotiana genome as repetitive DNA, a DNA feature associated with 24-nt sRNAs. Our results point to a host genome origin for CMV satRNAs, and suggest novel approach of using small RNA sequences for finding the origin of other satRNAs.

  12. Nicotiana small RNA sequences support a host genome origin of cucumber mosaic virus satellite RNA.

    Directory of Open Access Journals (Sweden)

    Kiran Zahid

    2015-01-01

    Full Text Available Satellite RNAs (satRNAs are small noncoding subviral RNA pathogens in plants that depend on helper viruses for replication and spread. Despite many decades of research, the origin of satRNAs remains unknown. In this study we show that a β-glucuronidase (GUS transgene fused with a Cucumber mosaic virus (CMV Y satellite RNA (Y-Sat sequence (35S-GUS:Sat was transcriptionally repressed in N. tabacum in comparison to a 35S-GUS transgene that did not contain the Y-Sat sequence. This repression was not due to DNA methylation at the 35S promoter, but was associated with specific DNA methylation at the Y-Sat sequence. Both northern blot hybridization and small RNA deep sequencing detected 24-nt siRNAs in wild-type Nicotiana plants with sequence homology to Y-Sat, suggesting that the N. tabacum genome contains Y-Sat-like sequences that give rise to 24-nt sRNAs capable of guiding RNA-directed DNA methylation (RdDM to the Y-Sat sequence in the 35S-GUS:Sat transgene. Consistent with this, Southern blot hybridization detected multiple DNA bands in Nicotiana plants that had sequence homology to Y-Sat, suggesting that Y-Sat-like sequences exist in the Nicotiana genome as repetitive DNA, a DNA feature associated with 24-nt sRNAs. Our results point to a host genome origin for CMV satRNAs, and suggest novel approach of using small RNA sequences for finding the origin of other satRNAs.

  13. Efficiency for Gene Silencing Induction in Nicotiana Species by a Viral Satellite DNA Vector

    Institute of Scientific and Technical Information of China (English)

    You-Ping Xu; Lu-Ping Zheng; Qiu-Fang Xu; Chang-Chun Wang; Xue-Ping Zhou; Zu-Jian Wu; Xin-Zhong Cai

    2007-01-01

    Virus-induced gene silencing (VIGS) is a useful technique for rapid plant gene function analysis.We recently reported a new VIGS vector modified from Tomato yellow leaf curl China virus (TYLCCNV) DNAβ (DNAm β).In this study we compared In detail DNAmβ-induced gene silencing in four Nicotiana species including N.benthamiana, N.glutinosa, N.tabacum and N.paniculata.We found that DNAmβ-induced gene silencing in the four species was distinct in developing dynamics, tissue specificity, efficiency, and constancy in the plant life span.It was most efficient in N.benthamiana, where development of VIGS was most rapid, without tissue specificity and nearly 100% efficient.DNAmβ-induced gene silencing in N.Glutinosa was also efficient despite being slightly less than in N.benthamiana.It initially occurred in veins, later was scattered to mesophyll, finally led to complete silencing in whole leaves.In both species, VIGS constantly expressed until the plants died.However, DNAmβ-mediated VIGS in the other two Nicotiana species, N.tabacum and N.paniculata, was significantly less efficient.It was strictly limited within the veins of the silenced leaves, and constantly occurred only over 3-4 weeks.The upper leaves that emerged later stopped showing the silencing phenotype, DNAm β-induced gene silencing in N.benthamiana and N.glutinosa was not significantly influenced by the growth stage when the plants were agro-inoculated,and was not sensitive to high growth temperature up to 32℃, Our results indicate that this system has great potential as a versatile VIGS system for routine functional analysis of genes in some Nicotiana species.

  14. Altered sucrose metabolism impacts plant biomass production and flower development.

    Science.gov (United States)

    Coleman, Heather D; Beamish, Leigh; Reid, Anya; Park, Ji-Young; Mansfield, Shawn D

    2010-04-01

    Nicotiana tabacum (tobacco) was transformed with three genes involved in sucrose metabolism, UDP-glucose pyrophosphorylase (UGPase, EC 2.7.7.9), sucrose synthase (SuSy, EC 2.4.1.13) and sucrose phosphate synthase (SPS, EC 2.4.1.14). Plants harbouring the single transgenes were subsequently crossed to produce double and triple transgenic lines, including: 2 x 35S::UGPase x SPS, 4CL::UGPase x SPS, 2 x 35S::SuSy x SPS, 4CL::SuSy x SPS, 2 x 35S::UGPase x SuSy x SPS, and 4CL::UGPase x SuSy x SPS. The ultimate aim of the study was to examine whether it is possible to alter cellulose production through the manipulation of sucrose metabolism genes. While altering sucrose metabolism using UGPase, SuSy and SPS does not have an end effect on cellulose production, their simultaneous overexpression resulted in enhanced primary growth as seen in an increase in height growth, in some cases over 50%. Furthermore, the pyramiding strategy of simultaneously altering the expression of multiple genes in combination resulted in increased time to reproductive bud formation as well as altered flower morphology and foliar stipule formation in 4CL lines. Upregulation of these sucrose metabolism genes appears to directly impact primary growth and therefore biomass production in tobacco.

  15. Recombinant protein expression in Nicotiana.

    Science.gov (United States)

    Matoba, Nobuyuki; Davis, Keith R; Palmer, Kenneth E

    2011-01-01

    Recombinant protein pharmaceuticals are now widely used in treatment of chronic diseases, and several recombinant protein subunit vaccines are approved for human and veterinary use. With growing demand for complex protein pharmaceuticals, such as monoclonal antibodies, manufacturing capacity is becoming limited. There is increasing need for safe, scalable, and economical alternatives to mammalian cell culture-based manufacturing systems, which require substantial capital investment for new manufacturing facilities. Since a seminal paper reporting immunoglobulin expression in transgenic plants was published in 1989, there have been many technological advances in plant expression systems to the present time where production of proteins in leaf tissues of nonfood crops such as Nicotiana species is considered a viable alternative. In particular, transient expression systems derived from recombinant plant viral vectors offer opportunities for rapid expression screening, construct optimization, and expression scale-up. Extraction of recombinant proteins from Nicotiana leaf tissues can be achieved by collection of secreted protein fractions, or from a total protein extract after grinding the leaves with buffer. After separation from solids, the major purification challenge is contamination with elements of the photosynthetic complex, which can be solved by application of a variety of facile and proven strategies. In conclusion, the technologies required for safe, efficient, scalable manufacture of recombinant proteins in Nicotiana leaf tissues have matured to the point where several products have already been tested in phase I clinical trials and will soon be followed by a rich pipeline of recombinant vaccines, microbicides, and therapeutic proteins.

  16. Demonstration of site-dependent dynamics of ozone effects in tobacco (Nicotina tabacum L. Bel W3) in Greater Rostock; Nachweis einer standoertlich differenzierten Dynamik der Ozonwirkung an Tabak (Nicotina tabacum L. Bel W3) im Grossraum Rostock

    Energy Technology Data Exchange (ETDEWEB)

    Odya, S.; Stuedemann, O.; Eckert, S. [Rostock Univ. (Germany). Inst. fuer Landschaftsplanung und Landschaftsoekologie

    1999-07-01

    The goal of the present study was to describe the genesis of a geographic pattern of phytotoxic ozone effects in the case of a mesoscale climate sequence. For this purpose the ''Applied Meteorology and Climatology'' working group carried out an active biomonitoring field trial with different bioindicator plants (Nicotina Tabacum L. Bel W3 and Bel B, bush bean, darnel, wheat and common nettle) over 4 vegetation periods. The trial was designed on the basis of existing knowledge on the spatially heterogeneous occurrence of ozone episodes and site-dependent phytotoxic ozone effects. [German] Das Ziel unserer Untersuchungen ist die Beschreibung der Genese des geographischen Musters phytotoxischer Ozonwirkungen im Bereich einer mesoskalen Klimasequenz in Nordost-Deutschland. Dazu wurde in Kenntnis des arealheterogenen Auftretens der Ozonepisoden und der standortabhaengigen phytotoxischen Ozonwirkung von der AG 'Angewandte Meteorologie und Klimatologie' ein aktives Biomonitoring mit verschiedenen Bioindikatorpflanzen (Nicotiana tabacum L. Bel W3 und Bel B, Buschbohne, Weidelgras, Weizen, Grosse Brennessel) ueber 4 Vegetationsperioden (1995-1998) im Freiland durchgefuehrt. (orig.)

  17. Taxonomy Icon Data: tobacco [Taxonomy Icon

    Lifescience Database Archive (English)

    Full Text Available tobacco Nicotiana tabacum Nicotiana_tabacum_L.png Nicotiana_tabacum_NL.png Nicotiana_tabacum_S.png Nico...tiana_tabacum_NS.png http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Nicotiana+tabacum&...t=L http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Nicotiana+tabacum&t=NL http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Nico...tiana+tabacum&t=S http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Nicotiana+tabacum&t=NS ...

  18. Over-expression of ascorbate oxidase in the apoplast of transgenic tobacco results in altered ascorbate and glutathione redox states and increased sensitivity to ozone

    DEFF Research Database (Denmark)

    Sanmartin, Maite; Drogoudi, Pavlina D.; Lyons, Tom

    2003-01-01

    Transgenic tobacco (Nicotiana tabacum L. cv. Xanthi) plants expressing cucumber ascorbate oxidase (EC.1.10.3.3) were used to examine the role of extracellular ascorbic acid in mediating tolerance to the ubiquitous air pollutant, ozone (O3). Three homozygous transgenic lines, chosen on the basis...

  19. Low abundant spacer 5S rRNA transcripts are frequently polyadenylated in Nicotiana.

    Science.gov (United States)

    Fulnecek, Jaroslav; Kovarik, Ales

    2007-11-01

    In plants, 5S rRNA genes (5S rDNA) encoding 120-nt structural RNA molecules of ribosomes are organized in tandem arrays comprising thousands of units. Failure to correctly terminate transcription would generate longer inaccurately processed transcripts interfering with ribosome biogenesis. Hence multiple termination signals occur immediately after the 5S rRNA coding sequence. To obtain information about the efficiency of termination of 5S rDNA transcription in plants we analyzed 5S rRNA pools in three Nicotiana species, N. sylvestris, N. tomentosiformis and N. tabacum. In addition to highly abundant 120-nt 5S rRNA transcripts, we also detected RNA species composed of a genic region and variable lengths of intergenic sequences. These genic-intergenic RNA molecules occur at a frequency severalfold lower than the mature 120-nt transcripts, and are posttranscriptionally modified by polyadenylation at their 3' end in contrast to 120-nt transcripts. An absence of 5S small RNAs (smRNA) argue against a dominant role for the smRNA biosynthesis pathway in the degradation of aberrant 5S rRNA in Nicotiana. This work is the first description of polyadenylated 5S rRNA species in higher eukaryotes originating from a read-through transcription into the intergenic spacer. We propose that polyadenylation may function in a "quality control" pathway ensuring that only correctly processed molecules enter the ribosome biogenesis.

  20. Nicotiana glauca poisoning in ostriches (Struthio camelus)

    CSIR Research Space (South Africa)

    Botha, CJ

    2011-01-01

    Full Text Available Putative Nicotiana glauca (wild tobacco) poisoning was diagnosed in a flock of ostriches near Oudtshoorn, South Africa. Post mortem examinations (n = 7) were performed on ostriches (Struthio camelus) that had died. Suspicious leaf remnants (weighing...

  1. Recognition of an Avr3a homologue plays a major role in mediating nonhost resistance to Phytophthora capsici in Nicotiana species.

    Science.gov (United States)

    Vega-Arreguín, Julio C; Jalloh, Abubakar; Bos, Jorunn I; Moffett, Peter

    2014-08-01

    Nonhost resistance is a commonly occurring phenomenon wherein all accessions or cultivars of a plant species are resistant to all strains of a pathogen species and is likely the manifestation of multiple molecular mechanisms. Phytophthora capsici is a soil-borne oomycete that causes Phytophthora blight disease in many solanaceous and cucurbitaceous plants worldwide. Interest in P. capsici has increased considerably with the sequencing of its genome and its increasing occurrence in multiple crops. However, molecular interactions between P. capsici and both its hosts and its nonhosts are poorly defined. We show here that tobacco (Nicotiana tabacum) acts like a nonhost for P. capsici and responds to P. capsici infection with a hypersensitive response (HR). Furthermore, we have found that a P. capsici Avr3a-like gene (PcAvr3a1) encoding a putative RXLR effector protein produces a HR upon transient expression in tobacco and several other Nicotiana species. This HR response correlated with resistance in 19 of 23 Nicotiana species and accessions tested, and knock-down of PcAvr3a1 expression by host-induced gene silencing allowed infection of resistant tobacco. Our results suggest that many Nicotiana species have the capacity to recognize PcAvr3a1 via the products of endogenous disease resistance (R) genes and that this R gene-mediated response is a major component of nonhost resistance to P. capsici.

  2. A survey of resistance to Tomato bushy stunt virus in the genus Nicotiana reveals that the hypersensitive response is triggered by one of three different viral proteins.

    Science.gov (United States)

    Angel, Carlos A; Schoelz, James E

    2013-02-01

    In this study, we screened 22 Nicotiana spp. for resistance to the tombusviruses Tomato bushy stunt virus (TBSV), Cucumber necrosis virus, and Cymbidium ringspot virus. Eighteen species were resistant, and resistance was manifested in at least two different categories. In all, 13 species responded with a hypersensitive response (HR)-type resistance, whereas another five were resistant but either had no visible response or responded with chlorotic lesions rather than necrotic lesions. Three different TBSV proteins were found to trigger HR in Nicotiana spp. in an agroinfiltration assay. The most common avirulence (avr) determinant was the TBSV coat protein P41, a protein that had not been previously recognized as an avr determinant. A mutational analysis confirmed that the coat protein rather than the viral RNA sequence was responsible for triggering HR, and it triggered HR in six species in the Alatae section. The TBSV P22 movement protein triggered HR in two species in section Undulatae (Nicotiana glutinosa and N. edwardsonii) and one species in section Alatae (N. forgetiana). The TBSV P19 RNA silencing suppressor protein triggered HR in sections Sylvestres (N. sylvestris), Nicotiana (N. tabacum), and Alatae (N. bonariensis). In general, Nicotiana spp. were capable of recognizing only one tombusvirus avirulence determinant, with the exceptions of N. bonariensis and N. forgetiana, which were each able to recognize P41, as well as P19 and P22, respectively. Agroinfiltration failed to detect the TBSV avr determinants responsible for triggering HR in N. arentsii, N. undulata, and N. rustica. This study illustrates the breadth and variety of resistance responses to tombusviruses that exists in the Nicotiana genus.

  3. Rational management of tobacco (Nicotiana tabacum L. crop residues to obtain organic compostManejo racional de resíduos da cultura do fumo (Nicotiana tabacum L. para obtenção de composto orgânico

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    Antonio Carlos Souza Borges Filho

    2011-10-01

    Full Text Available Crop residues may be used to produce organic composts for agricultural use. The aim of this study was to evaluate the effect of two inoculums on the decomposition speed of tobacco residues (stems. The experiment was carried out in a completely randomized design, with three treatments and six replications. The residue used in the study was passed through a forage chopper and mixed to the inoculums in a concrete truck mixer. The treatments evaluated were: TF + EB + RB - tobacco residue, bovine manure and bovine rumen; TF + EB + MP - tobacco residue, bovine manure and Microsept-Dust and TF + EB - tobacco residue and bovine manure. The temperature and humidity of the piles were monitored weekly. The piles were initially turned up side down every seven days, and then every fourteen days till closing 60 days. Samples for chemical analysis of the composted material were collected at 30, 60 and 90 days after the beginning of the composting process. The temperature of the piles stayed around 57 to 60 ºC in the period between the 20 to 40 days after the beginning of the composting process. The mix containing cattle rumen (RB presented the lowest temperature after forty days. At the end of the composting the mix containing Microsept-Dust (MP presented the lowest humidity. The C/N relation of the composts obtained was around 10/1 and 11/1. The three treatments presented pH values close to the neutrality in the mature stage. A period of sixty days seems to be sufficient to obtain a mature and stable biocompost of tobacco waste using manure independent of the addition of inoculums.Os resíduos vegetais podem ser utilizados na produção de adubo orgânico por meio da compostagem. O objetivo deste estudo foi avaliar o efeito de dois inoculantes sobre a velocidade de decomposição do resíduo da cultura do fumo. O experimento foi conduzido em delineamento inteiramente casualizado, com três tratamentos e seis repetições, sendo o resíduo picado em picadeira e misturado aos inoculantes em betoneira. Foram avaliados os tratamentos: TF + EB + RB - talo de fumo, esterco bovino e rúmen bovino; TF + EB + MP - talo de fumo, esterco bovino e microsept-pó e TF + EB - talo de fumo e esterco bovino. A temperatura e a umidade das pilhas foram monitoradas semanalmente. As pilhas foram revolvidas a cada 07 dias, inicialmente e a seguir a cada 14 dias até os 60 dias. Aos trinta, sessenta e noventa dias, foi feita a coleta, para análise química, do material em compostagem. Entre vinte e quarenta dias as pilhas mantiveram temperatura entre 57 e 60 ºC. A mistura contendo rúmen bovino (RB apresentou a menor temperatura após 40 dias. Ao final da compostagem a mistura contendo microsept-pó (MP foi a que apresentou menor umidade. A relação C/N, do composto obtido, ficou entre 10/1 e 11/1. Os três tratamentos apresentaram pH fracamente alcalino na fase de cura. Pode-se considerar que 60 dias de compostagem são suficientes para a obtenção de um composto estabilizado a partir da mistura resíduo de fumo usando esterco, com ou sem a adição de inoculantes.

  4. Influence of planting frame in the cultivar “Corojo 2006” of tobacco (Nicotiana tabacum L. on productive indicators

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    Luis Gustavo González Gómez

    2015-03-01

    Full Text Available The investigation was developed in the General CCS-F Victor Ramos Hernandez, Located in the area of the Oven, on a carbonated brown floor. The valued variety of tobacco was the Corojo 2006, with the objective of evaluating different plantation marks, for they were evaluated it four plantation distances that in turn were the valued treatments with a design of totally randomized in parcels of 20 m of long and wide The utilized materials they are characteristic of a field investigation and the cultural attentions were carried out by the Technical Instructive of the Tobacco (2011. the main indicators of the cultivation were evaluated like they were number of leaves, long and wide of the leaves, dry mass and yield, with its respective economic valuation. The data were processed with the statistical package it ESTATISTICA version 8 on Windows, through an analysis of simple variance and a test of multiple comparison for Tukey for 5% of probability of the error, The reached results indicate us that the best yields are obtained when the plants have a distance among them of 0.30 m and the lowest results when they are planted 0.25 m.

  5. Isolation and compositional analysis of a CP12-associated complex of calvin cycle enzymes from Nicotiana tabacum.

    Science.gov (United States)

    Carmo-Silva, A Elizabete; Marri, Lucia; Sparla, Francesca; Salvucci, Michael E

    2011-06-01

    Two Calvin Cycle enzymes, NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a multiprotein complex with CP12, a small intrinsically-unstructured protein. Under oxidizing conditions, association with CP12 confers redox-sensitivity to the otherwise redox-insensitive A isoform of GAPDH (GapA) and provides an additional level of down-regulation to the redox-regulated PRK. To determine if CP12-mediated regulation is specific for GAPDH and PRK in vivo, a high molecular weight complex containing CP12 was isolated from tobacco chloroplasts and leaves and its protein composition was characterized. Gel electrophoresis and immunoblot analyses after separation of stromal proteins by size fractionation verified that the GAPDH (both isoforms) and PRK co-migrated with CP12 in dark- but not light-adapted chloroplasts. Nano-liquid-chromatography-mass-spectrometry of the isolated complex identified only CP12, GAPDH and PRK. Since nearly all of the CP12 from darkened chloroplasts migrates with GADPH and PRK as a high molecular mass species, these data indicate that the tight association of tobacco CP12 with GAPDH and PRK is specific and involves no other Calvin Cycle enzymes.

  6. Arabidopsis LOS5/ABA3 overexpression in transgenic tobacco (Nicotiana tabacum cv. Xanthi-nc) results in enhanced drought tolerance.

    Science.gov (United States)

    Yue, Yuesen; Zhang, Mingcai; Zhang, Jiachang; Duan, Liusheng; Li, Zhaohu

    2011-10-01

    Drought is a major environmental stress factor that affects growth and development of plants. Abscisic acid (ABA), osmotically active compounds, and synthesis of specific proteins, such as proteins that scavenge oxygen radicals, are crucial for plants to adapt to water deficit. LOS5/ABA3 (LOS5) encodes molybdenum-cofactor sulfurase, which is a key regulator of ABA biosynthesis. We overexpressed LOS5 in tobacco using Agrobacterium-mediated transformation. Detached leaves of LOS5-overexpressing seedlings showed lower transpirational water loss than that of nontransgenic seedlings in the same period under normal conditions. When subjected to water-deficit stress, transgenic plants showed less wilting, maintained higher water content and better cellular membrane integrity, accumulated higher quantities of ABA and proline, and exhibited higher activities of antioxidant enzymes, i.e., superoxide dismutase, catalase, peroxidase and ascorbate peroxidase, as compared with control plants. Furthermore, LOS5-overexpressing plants treated with 30% polyethylene glycol showed similar performance in cellular membrane protection, ABA and proline accumulation, and activities of catalase and peroxidase to those under drought stress. Thus, overexpression of LOS5 in transgenic tobacco can enhance drought tolerance.

  7. Construction of phosphomannose isomerase (PMI) transformation vectors and evaluation of the effectiveness of vectors in tobacco (Nicotiana tabacum L).

    Science.gov (United States)

    Bahariah, Bohari; Parveez, Ghulam Kadir Ahmad; Masani, Mat Yunus Abdul; Khalid, Norzulaani

    2012-01-01

    Phosphomannose isomerase (pmi) gene isolated from Escherichia coli allows transgenic plants carrying it to convert mannose-6- phosphate (from mannose), a carbon source that could not be naturally utilized by plants into fructose-6-phosphate which can be utilized by plants as a carbon source. This conversion ability provides energy source to allow the transformed cells to survive on the medium containing mannose. In this study, four transformation vectors carrying the pmi gene alone or in combination with the β-glucuronidase (gusA) gene were constructed and driven by either the maize ubiquitin (Ubi1) or the cauliflower mosaic virus (CaMV35S) promoter. Restriction digestion, PCR amplification and sequencing were carried out to ensure sequence integrity and orientation. Tobacco was used as a model system to study the effectiveness of the constructs and selection system. PMI11G and pMI3G, which carry gusA gene, were used to study the gene transient expression in tobacco. PMI3 construct, which only carries the pmi gene driven by CaMV35S promoter, was stably transformed into tobacco using biolistics after selection on 30 g 1(-1) mannose without sucrose. Transgenic plants were verified using PCR analysis. PMI/pmi - Phosphomannose isomerase, Ubi1 - Maize ubiquitin promoter, CaMV35S - Cauliflower mosaic virus 35S promoter, gusA - β-glucuronidase GUS reporter gene.

  8. Induction of Callose Deposition in Tobacco (Nicotiana tabacum by Bacterial Lipopolysaccharide Pseudomonas syringae pv. tabaci and Pseudomonas syringae pv. glycinea

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    Pipit Marianingsih

    2014-12-01

    Full Text Available Lipopolysaccharide (LPS is a major component of outer-membrane gram-negative bacteria, and it can act as a Pathogen-Associated Molecular Pattern (PAMP for perception of pathogens by plants. LPS can be recognized by plants, triggering certain plant defense-related responses, including callose deposition. This study investigated induction of callose deposition by bacterial LPS in tobacco. Tobacco leaves were infiltrated with 400 μg/mL and 800 μg/mL LPS extracted from Pseudomonas syringae pv. tabaci (Pta and Pseudomonas syringae pv. glycinea (Pgl and incubated for 24 h or 48 h. To detect callose deposition, tobacco leaves were cleared in lactophenol solution, stained with aniline blue, and visualized by fluorescence microscopy. Results showed that LPS from Pgl induced more callose deposition in tobacco leaves than did that from Pta. In addition, a Pearson correlation test revealed that incubation period was the most significant factor in callose deposition, followed by the type of LPS bacteria. However, LPS concentration was not significantly corelated to callose deposition in tobacco leaves.

  9. Dynamic expression analysis of early response genes induced by potato virus Y in PVY-resistant Nicotiana tabacum.

    Science.gov (United States)

    Chen, Shuai; Li, Fengxia; Liu, Dan; Jiang, Caihong; Cui, Lijie; Shen, Lili; Liu, Guanshan; Yang, Aiguo

    2017-02-01

    Dynamic transcriptional changes of the host early responses genes were detected in PVY-resistant tobacco varieties infected with Potato virus Y; PVY resistance is a complex process that needs series of stress responses. Potato virus Y (PVY) causes a severe viral disease in cultivated crops, especially in Solanum plants. To understand the molecular basis of plant responses to the PVY stress, suppression subtractive hybridization (SSH) and microarray approaches were combined to identify the potentially important or novel genes that were involved in early stages (12 h, 1, 2, 3, 5, 8 days) of tobacco response to PVY infection. Dynamic changes of the host plant early responses to PVY infection on a transcriptional level were detected. In total, 167 different expressed ESTs were identified. The majority of genes involved in the metabolic process were found to be down-regulated at 12 h and 1 day, and then up-regulated at least one later period. Genes related to signaling and transcriptions were almost up-regulated at 12 h, 1 or 2 days, while stress response genes were almost up-regulated at a later stage. Genes involved in transcription, transport, cell wall, and several stress responses were found to have changed expression during the PVY infection stage, and numbers of these genes have not been previously reported to be associated with tobacco PVY infection. The diversity expression of these genes indicated that PVY resistance is a complex process that needs a series of stress responses. To resist the PVY infection, the tobacco plant has numerous active and silent responses.

  10. Using a Chlorophyll Meter to Evaluate the Nitrogen Leaf Content in Flue-Cured Tobacco (Nicotiana tabacum L.

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    Renato Contillo

    2009-06-01

    Full Text Available In flue-cured tobacco N fertilizer is commonly applied during pre-planting, and very often applied again later as a growth-starter. It is generally held that the efficiency of N-fertilizer use can be improved by evaluating the leaf Nstatus after transplanting and until flowering stage. N use efficiency in this context does not refer merely to the yield but also to the quality, in the meanwhile minimizing the negative effects on the environment. To investigate these aspects, we evaluated the capacity of a Minolta model SPAD-502 chlorophyll meter to estimate the N-status in flue-cured tobacco. The aims was to verify if a relationship exists between SPAD readings and leaf N content, and if a single leaf, in a well defined stalk position, could represent the nitrogen content of the whole plant. During the years 1995 and 1996, a pot experiment was conducted using two flue-cured tobacco varieties. SPAD values, total chlorophyll, total N contents and leaf area were measured throughout the growing season, on each odd leaf stalk position. SPAD values were well-correlated with both total chlorophyll and total N leaf concentration, and the regression coefficients were higher when relationships were calculated on a leaf-area basis. For both relationships, SPAD-total chlorophyll and SPAD-total N, the best fittings were obtained with quadratic equations. One leaf stalk position alone is able to monitor the N-status of the whole plant during the first six weeks after transplanting, without distinction of year and variety effects. The SPAD measurement of one leaf per plant, throughout the vegetative growing season, is therefore a valid tool to test the N-status of the crop in a period when a required N supply is still effective.

  11. Heterologous expression of the BABY BOOM AP2/ERF transcription factor enhances the regeneration capacity of tobacco (Nicotiana tabacum L.)

    NARCIS (Netherlands)

    Srinivasan, C.; Liu, Z.; Heidmann, I.; Supena, E.D.J.; Fukuoka, H.; Joosen, R.V.L.; Lambalk, J.; Angenent, G.C.; Scorza, R.; Custers, J.B.M.; Boutilier, K.A.

    2007-01-01

    Gain-of-function studies have shown that ectopic expression of the BABY BOOM (BBM) AP2/ERF domain transcription factor is sufficient to induce spontaneous somatic embryogenesis in Arabidopsis (Arabidopsis thaliana (L.) Heynh) and Brassica napus (B. napus L.) seedlings. Here we examined the effect of

  12. The Effect of Two Formulations of Sulfentrazone on Soil and Leaf Residues and Phytotoxicity in Tobacco (Nicotiana Tabacum L.

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    Bertha K. Mashayamombe

    2013-03-01

    Full Text Available Field studies were done to compare a wettable granular formulation (Authority 75 WG and a suspension concentrate formulation (Authority 48 SC of the herbicide sulfentrazone. A 6 x 2 factorial experiment was laid out in a split plot design to evaluate the effect of herbicide levels on phytotoxicity and residues in both leaves and the soil. One rate of the wettable granular formulation (0.225 kg/ha, four different rates of the suspension concentrate formulation (0.165 kg/ha, 0.205 kg/ha, 0.185 kg/ha, 0.225 kg/ha and the control (0.00 kg/ha were tested. Tobacco phytotoxicity at 27 days after transplanting (DAT and soil residues at 12 WAT was measured. There was significantly higher (P<0.05 phytotoxicity in all treatments where herbicides were applied compared to the control. The WG formulation showed significantly higher phytotoxicity than the SC formulation. Residues in both the soil and leaves were significantly higher in the herbicide applied plots as compared to the control for both formulations. The SC formulation of sulfentrazone at a rate of 0.225 kg/ha was significantly less phytotoxic than the same rate of the WG formulation at 27 DAT. The SC formulation of sulfentrazone can replace the WG formulation for broad spectrum weed control in tobacco in Zimbabwe at a recommended rate of 0.225 kg/ha.

  13. The role of gluconate production by Pseudomonas spp. in the mineralization and bioavailability of calcium-phytate to Nicotiana tabacum.

    Science.gov (United States)

    Giles, Courtney D; Hsu, Pei-Chun Lisa; Richardson, Alan E; Hurst, Mark R H; Hill, Jane E

    2015-12-01

    Organic phosphorus (P) is abundant in most soils but is largely unavailable to plants. Pseudomonas spp. can improve the availability of P to plants through the production of phytases and organic anions. Gluconate is a major component of Pseudomonas organic anion production and may therefore play an important role in the mineralization of insoluble organic P forms such as calcium-phytate (CaIHP). Organic anion and phytase production was characterized in 2 Pseudomonas spp. soil isolates (CCAR59, Ha200) and an isogenic mutant of strain Ha200, which lacked a functional glucose dehydrogenase (Gcd) gene (strain Ha200 gcd::Tn5B8). Wild-type and mutant strains of Pseudomonas spp. were evaluated for their ability to solubilize and hydrolyze CaIHP and to promote the growth and assimilation of P by tobacco plants. Gluconate, 2-keto-gluconate, pyruvate, ascorbate, acetate, and formate were detected in Pseudomonas spp. supernatants. Wild-type pseudomonads containing a functional gcd could produce gluconate and mineralize CaIHP, whereas the isogenic mutant could not. Inoculation with Pseudomonas improved the bioavailability of CaIHP to tobacco plants, but there was no difference in plant growth response due to Gcd function. Gcd function is required for the mineralization of CaIHP in vitro; however, further studies will be needed to quantify the relative contribution of specific organic anions such as gluconate to plant growth promotion by soil pseudomonads.

  14. Effect of calcium carbonate on cadmium and nutrients uptake in tobacco (Nicotiana tabacum L.) planted on contaminated soil.

    Science.gov (United States)

    Zeng, Wei-Ai; Li, Fan; Zhou, Hang; Qin, Xiao-Li; Zou, Zi-Jin; Tian, Tao; Zeng, Min; Liao, Bo-Han

    2016-01-01

    In the present study, calcium carbonate (CaCO3) was applied to Cd-contaminated soil at rates of 0, 0.5 and 1.0 g kg(-1). The effect of CaCO3 on soil pH, organic matter, available Cd, exchangeable Cd and level of major nutrients in a tobacco field and on accumulation of various elements in tobacco plants was determined. The results showed that CaCO3 application significantly increased the pH level, available P and exchangeable Ca but decreased organic matter, available Cd, exchangeable Cd, available heavy metals (Fe, Mn, Zn and Cu) and available K in soil. Additionally, CaCO3 application substantially reduced Cd accumulation in tobacco roots, stems, upper leaves, middle leaves and lower leaves, with maximum decrease of 22.3%, 32.1%, 24.5%, 22.0% and 18.2%, respectively. There were large increase in total Ca and slight increases in total N and K but decrease to varying degrees in total Fe, Cu and Zn due to CaCO3 application. CaCO3 had little effect on total P and Mn levels in tobacco leaves.

  15. Molecular diversity, population structure, and linkage disequilibrium in a worldwide collection of tobacco (Nicotiana tabacum L. germplasm

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    Fricano Agostino

    2012-03-01

    Full Text Available Abstract Background The goals of our study were to assess the phylogeny and the population structure of tobacco accessions representing a wide range of genetic diversity; identify a subset of accessions as a core collection capturing most of the existing genetic diversity; and estimate, in the tobacco core collection, the extent of linkage disequilibrium (LD in seven genomic regions using simple sequence repeat (SSR markers. To this end, a collection of accessions were genotyped with SSR markers. Molecular diversity was evaluated and LD was analyzed across seven regions of the genome. Results A genotyping database for 312 tobacco accessions was profiled with 49 SSR markers. Principal Coordinate Analysis (PCoA and Bayesian cluster analysis revealed structuring of the tobacco population with regard to commercial classes and six main clades were identified, which correspond to "Oriental", Flue-Cured", "Burley", "Dark", "Primitive", and "Other" classes. Pairwise kinship was calculated between accessions, and an overall low level of co-ancestry was observed. A set of 89 genotypes was identified that captured the whole genetic diversity detected at the 49 loci. LD was evaluated on these genotypes, using 422 SSR markers mapping on seven linkage groups. LD was estimated as squared correlation of allele frequencies (r2. The pattern of intrachromosomal LD revealed that in tobacco LD extended up to distances as great as 75 cM with r2 > 0.05 or up to 1 cM with r2 > 0.2. The pattern of LD was clearly dependent on the population structure. Conclusions A global population of tobacco is highly structured. Clustering highlights the accessions with the same market class. LD in tobacco extends up to 75 cM and is strongly dependent on the population structure.

  16. A membrane-bound matrix-metalloproteinase from Nicotiana tabacum cv. BY-2 is induced by bacterial pathogens

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    Wahner Verena

    2009-06-01

    Full Text Available Abstract Background Plant matrix metalloproteinases (MMP are conserved proteolytic enzymes found in a wide range of monocotyledonous and dicotyledonous plant species. Acting on the plant extracellular matrix, they play crucial roles in many aspects of plant physiology including growth, development and the response to stresses such as pathogen attack. Results We have identified the first tobacco MMP, designated NtMMP1, and have isolated the corresponding cDNA sequence from the tobacco suspension cell line BY-2. The overall domain structure of NtMMP1 is similar to known MMP sequences, although certain features suggest it may be constitutively active rather than dependent on proteolytic processing. The protein appears to be expressed in two forms with different molecular masses, both of which are enzymatically active as determined by casein zymography. Exchanging the catalytic domain of NtMMP1 with green fluorescent protein (GFP facilitated subcellular localization by confocal laser scanning microscopy, showing the protein is normally inserted into the plasma membrane. The NtMMP1 gene is expressed constitutively at a low level but can be induced by exposure to bacterial pathogens. Conclusion Our biochemical analysis of NtMMP1 together with bioinformatic data on the primary sequence indicate that NtMMP1 is a constitutively-active protease. Given its induction in response to bacterial pathogens and its localization in the plasma membrane, we propose a role in pathogen defense at the cell periphery.

  17. Electrochemical monitoring of phytochelatin accumulation in Nicotiana tabacum cells exposed to sub-cytotoxic and cytotoxic levels of cadmium

    Energy Technology Data Exchange (ETDEWEB)

    Fojta, Miroslav [Laboratory of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno (Czech Republic)]. E-mail: fojta@ibp.cz; Fojtova, Miloslava [Laboratory of Molecular Epigenetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno (Czech Republic); Havran, Ludek [Laboratory of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno (Czech Republic); Pivonkova, Hana [Laboratory of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno (Czech Republic); Dorcak, Vlastimil [Laboratory of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno (Czech Republic); Sestakova, Ivana [J. Heyrovsky Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Dolejskova 3, 182 23 Prague 8 (Czech Republic)

    2006-02-03

    Cadmium belongs to the most dangerous environmental pollutants among the toxic heavy metals seriously affecting vital functions in both animal and plant cells. It has been previously shown that cadmium ions at 50-100 {mu}M concentrations caused tobacco BY-2 (TBY-2) cells to enter apoptosis within several days of exposure. Phytochelatins (PCs), the 'plant metallothioneins', are cysteine-rich peptides involved in detoxification of heavy metals in plants. The PCs are synthesized in response to the heavy metal exposure. In this paper, we utilized electrochemical analysis to monitor accumulation of PCs in the TBY-2 cells exposed to cadmium ions. Measurements of a characteristic PC signal at mercury electrode in the presence of cobalt ions made it possible to detect changes in the cellular PC levels during the time of cultivation, starting from 30 min after exposure. Upon TBY-2 cultivation in the presence of cytotoxic cadmium concentrations, the PC levels remarkably increased during the pre-apoptotic phase and reached a limiting value at cultivation times coinciding with apoptosis trigger. The PC level observed for a sub-cytotoxic cadmium concentration (10 {mu}M) was about three-times lower than that observed for the 50 or 100 {mu}M cadmium ions after 5 days of exposure. We show that using a simple electrochemical analysis, synthesis of PCs in plant cells can be easily followed in parallel with other tests of the cellular response to the toxic heavy metal stress.

  18. Genetic characterization and molecular mapping of novel chlorophyll deficiency gene in air-cured tobacco (Nicotiana tabacum L.

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    Shahadati-Moghaddam Zeinalabedin

    2017-01-01

    Full Text Available This study was performed to genetical and morphological investigation of a novel chlorophyll deficiency gene in tobacco leaf. One low chlorophyll content (LCC variety (Urumieh 2 and high chlorophyll content (HCC variety (Burley Ree 103 from the Burley type was crossed and the F2 generation was grown on the field. One hundred plants were selected, contained low and high chlorophyll content. These plants were sampled and DNA was extracted. Sixty RAPD primers were tested on parents, LCC and HCC Bulks based on Bulk Segregant Analysis (BSA. Chi-square test confirmed the monogenic segregation. Regresion analysis showed that there was strong relationship between greenness degree and chlorophyll contents. Four primers (OPE17, OPC09, OPB08 and OPR02 showed polymorphism and after the test on 97 samples from the F2 generation two markers were selected (OPB08-1050 and OPC09-1900. That showed 15.9 and 10.8 CM distance from chlorophyll locus respectively.

  19. iTRAQ protein profile analysis provides integrated insight into mechanisms of tolerance to TMV in tobacco (Nicotiana tabacum).

    Science.gov (United States)

    Wang, Jing; Wang, Xiao-ran; Zhou, Qi; Yang, Jin-miao; Guo, Hong-xiang; Yang, Li-jun; Liu, Wei-qun

    2016-01-30

    To further investigate the mechanism of the plant tolerance to tobacco mosaic virus (TMV) infection, tobacco NC89 (N) hypersensitive to TMV and its natural mutant Yuyan8 (Y) with tolerance to TMV were employed for differential accumulation proteome analysis. There were 260 specifically accumulated proteins in Yuyan8 after 24 h inoculation (Yd), and the accumulations of 285 proteins inherent in Y have changed after TMV infection. Equally, there were 183 specifically accumulated proteins in NC89 after 24 h inoculation (Nd), and 132 proteins inherent in N have changed after TMV infection. These differential proteins were respectively enriched in two pathways, of which photosynthesis pathway was the common pathway in two varieties. In photoreaction system, the accumulations of differential proteins, especially D1 protein, were not decreased in Yd compared to Nd. The results indicated that maintaining the stability of D1 protein and reasonable utilization of the energy was the essential for tolerance to TMV infection. It was also revealed that 14-3-3 protein and PR4 was specific expressed, and the expression of LRR was enhanced in Yd, suggesting that regulation of defense protein mediated by 14-3-3 protein quickly activated resistance system and enhanced the plant tolerance to TMV infection. This is the first work that the molecular basis of tobacco tolerance was discussed basic on proteomic investigation performed on wild type and its natural mutant. Our results lay the foundation for development of molecular breeding and further proteome research in tobacco. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  20. RNA Sequencing Analysis Reveals Transcriptomic Variations in Tobacco (Nicotiana tabacum Leaves Affected by Climate, Soil, and Tillage Factors

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    Bo Lei

    2014-04-01

    Full Text Available The growth and development of plants are sensitive to their surroundings. Although numerous studies have analyzed plant transcriptomic variation, few have quantified the effect of combinations of factors or identified factor-specific effects. In this study, we performed RNA sequencing (RNA-seq analysis on tobacco leaves derived from 10 treatment combinations of three groups of ecological factors, i.e., climate factors (CFs, soil factors (SFs, and tillage factors (TFs. We detected 4980, 2916, and 1605 differentially expressed genes (DEGs that were affected by CFs, SFs, and TFs, which included 2703, 768, and 507 specific and 703 common DEGs (simultaneously regulated by CFs, SFs, and TFs, respectively. GO and KEGG enrichment analyses showed that genes involved in abiotic stress responses and secondary metabolic pathways were overrepresented in the common and CF-specific DEGs. In addition, we noted enrichment in CF-specific DEGs related to the circadian rhythm, SF-specific DEGs involved in mineral nutrient absorption and transport, and SF- and TF-specific DEGs associated with photosynthesis. Based on these results, we propose a model that explains how plants adapt to various ecological factors at the transcriptomic level. Additionally, the identified DEGs lay the foundation for future investigations of stress resistance, circadian rhythm and photosynthesis in tobacco.

  1. Non-host Plant Resistance against Phytophthora capsici Is Mediated in Part by Members of the I2 R Gene Family in Nicotiana spp.

    Science.gov (United States)

    Vega-Arreguín, Julio C.; Shimada-Beltrán, Harumi; Sevillano-Serrano, Jacobo; Moffett, Peter

    2017-01-01

    The identification of host genes associated with resistance to Phytophthora capsici is crucial to developing strategies of control against this oomycete pathogen. Since there are few sources of resistance to P. capsici in crop plants, non-host plants represent a promising source of resistance genes as well as excellent models to study P. capsici – plant interactions. We have previously shown that non-host resistance to P. capsici in Nicotiana spp. is mediated by the recognition of a specific P. capsici effector protein, PcAvr3a1 in a manner that suggests the involvement of a cognate disease resistance (R) genes. Here, we have used virus-induced gene silencing (VIGS) and transgenic tobacco plants expressing dsRNA in Nicotiana spp. to identify candidate R genes that mediate non-host resistance to P. capsici. Silencing of members of the I2 multigene family in the partially resistant plant N. edwardsonii and in the resistant N. tabacum resulted in compromised resistance to P. capsici. VIGS of two other components required for R gene-mediated resistance, EDS1 and SGT1, also enhanced susceptibility to P. capsici in N. edwardsonii, as well as in the susceptible plants N. benthamiana and N. clevelandii. The silencing of I2 family members in N. tabacum also compromised the recognition of PcAvr3a1. These results indicate that in this case, non-host resistance is mediated by the same components normally associated with race-specific resistance. PMID:28261255

  2. Quantitative Analysis of the Fate of Exogenous DNA in Nicotiana Protoplasts 1

    Science.gov (United States)

    Uchimiya, Hirofumi; Murashige, Toshio

    1977-01-01

    After a 5-hour incubation of protoplasts of Nicotiana tabacum L. `Xanthi' with 3H-DNA (7.26 μg/ml) from N. tabacum L. `Xanthi nc' 3.5% of the initial radioactivity was found in acid-insoluble substances of the protoplasts. The addition of DEAE-dextran and poly-l-lysine to the incubation medium nearly doubled radioactivity adsorption. The absorption was inhibited by 2,4-dinitrophenol, KCN, and low temperature (0 C); this inhibition could not be reversed by exogenous ATP. About 500 tobacco plants established from protoplasts of a normally tobacco-mosaic virus-susceptible cultivar that had been allowed to absorb DNA prepared from a resistant cultivar did not show transfer of the virus-resistant gene. A detailed analysis was performed of the disposition of exogenous DNA in plant protoplasts, by employing Escherichia coli3H-DNA and Nicotiana glutinosa protoplasts. In 5 to 20 hours, about 10% of the 3H-DNA entered the protoplasts. Competition experiments between the 3H-DNA and unlabeled DNA or thymidine showed that the entry occurred as undegraded 3H-DNA. Examination of intraprotoplast fractions revealed that 60 to 80% of the absorbed radioactivity resided in the “soluble” fraction of the cytoplasm and 20% in the nuclear fraction. The mitochondrion fraction also contained measurable radioactivity. Sizing on sucrose density gradients showed that the bulk of the absorbed E. coli DNA had been depolymerized. Of the incorporated radioactivity, 15% was accountable as DNA, exogenous as well as resynthesized, and 15% as RNA, protein, and other cell constituents. DNA/DNA hybridization test indicated that 17.6% of the re-extractable 3H-DNA retained homology with the E. coli DNA; this was equivalent to 2.6% of the absorbed radioactivity. Resynthesized receptor protoplast DNA was represented by a fraction at least 1.7% of the total absorbed radioactivity. The amount of bacterial DNA remaining in protoplasts suggests that each protoplast retained 2.3 × 10−15g donor DNA, or

  3. Plant oxidosqualene metabolism: cycloartenol synthase-dependent sterol biosynthesis in Nicotiana benthamiana.

    Science.gov (United States)

    Gas-Pascual, Elisabet; Berna, Anne; Bach, Thomas J; Schaller, Hubert

    2014-01-01

    The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans. The committed sterol precursor is the pentacyclic cycloartenol (9β,19-cyclolanost-24-en-3β-ol) and not lanosterol (lanosta-8,24-dien-3β-ol), as it was shown in the late sixties. However, plant genome mining over the last years revealed the general presence of lanosterol synthases encoding sequences (LAS1) in the oxidosqualene cyclase repertoire, in addition to cycloartenol synthases (CAS1) and to non-steroidal triterpene synthases that contribute to the metabolic diversity of C30H50O compounds on earth. Furthermore, plant LAS1 proteins have been unambiguously identified by peptidic signatures and by their capacity to complement the yeast lanosterol synthase deficiency. A dual pathway for the synthesis of sterols through lanosterol and cycloartenol was reported in the model Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ(5)-sterols was quite marginal (Ohyama et al. (2009) PNAS 106, 725). To investigate further the physiological relevance of CAS1 and LAS1 genes in plants, we have silenced their expression in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences from a Nicotiana tabacum CAS1 or derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis.

  4. Plant Oxidosqualene Metabolism: Cycloartenol Synthase–Dependent Sterol Biosynthesis in Nicotiana benthamiana

    Science.gov (United States)

    Gas-Pascual, Elisabet; Berna, Anne; Bach, Thomas J.; Schaller, Hubert

    2014-01-01

    The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans. The committed sterol precursor is the pentacyclic cycloartenol (9β,19-cyclolanost-24-en-3β-ol) and not lanosterol (lanosta-8,24-dien-3β-ol), as it was shown in the late sixties. However, plant genome mining over the last years revealed the general presence of lanosterol synthases encoding sequences (LAS1) in the oxidosqualene cyclase repertoire, in addition to cycloartenol synthases (CAS1) and to non-steroidal triterpene synthases that contribute to the metabolic diversity of C30H50O compounds on earth. Furthermore, plant LAS1 proteins have been unambiguously identified by peptidic signatures and by their capacity to complement the yeast lanosterol synthase deficiency. A dual pathway for the synthesis of sterols through lanosterol and cycloartenol was reported in the model Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ5-sterols was quite marginal (Ohyama et al. (2009) PNAS 106, 725). To investigate further the physiological relevance of CAS1 and LAS1 genes in plants, we have silenced their expression in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences from a Nicotiana tabacum CAS1 or derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis. PMID:25343375

  5. Two faces of Solanaceae telomeres: a comparison between Nicotiana and Cestrum telomeres and telomere-binding proteins.

    Science.gov (United States)

    Peska, V; Sýkorová, E; Fajkus, J

    2008-01-01

    While most Solanaceae genera (e.g.Solanum, Nicotiana) possess Arabidopsis-type telomeres of (TTTAGGG)n maintained by telomerase, the genera Cestrum, Vestia and Sessea (Cestrum group) lack these telomeres. Here we show that in the Cestrum-group the activity of telomerase has been lost. Nevertheless, proteins binding the single-stranded G-rich strand of the Arabidopsis-type and related human-type (TTAGGG)n telomeric sequences are present in nuclear extracts of both Nicotiana and Cestrum species. These proteins may have a role in telomere function or other cellular activities. In addition to characterizing DNA binding specificity and molecular weights of these proteins, we searched in both N. tabacum (tobacco) and C. parqui for the presence of POT1-like proteins, involved in telomere capping and telomerase regulation. Analysis of POT1-like proteins available on public databases and cloned by us from C. parqui, revealed the N-terminal OB folds typical for this protein family and a novel, plant-specific conserved C-terminal OB-fold domain (CTOB). We propose that CTOB is involved in protein-protein interactions.

  6. Digestive duet: Midgut digestive proteinases of Manduca sexta ingesting Nicotiana attenuata with manipulated trypsin proteinase inhibitor expression

    NARCIS (Netherlands)

    Zavala, J.A.; Giri, A.P.; Jongsma, M.A.; Baldwin, I.T.

    2008-01-01

    The defensive effect of endogenous trypsin proteinase inhibitors (NaTPIs) on the herbivore Manduca sexta was demonstrated by genetically altering NaTPI production in M. sexta's host plant, Nicotiana attenuata. To understand how this defense works, we studied the effects of NaTPI on M. sexta gut prot

  7. A glycoside of Nicotina tabacum affects mouse dopaminergic behavior.

    Science.gov (United States)

    Masuda, Y; Ohnuma, S; Kawagoe, M; Sugiyama, T

    2003-01-01

    Climbing in the forced swimming test is considered a dopaminergic-specific behavior. A substance of Nicotina tabacum affecting dopamine neuronal activity was investigated using the mouse behavioral system. The substance was found to be a glycoside with the peripheral sugar chain structures Fuc alpha 1-2Gal, Gal beta 1-4GlcNAc and GalNAc alpha 1-3GalNAc and with basic polymannoses. The glycoside dose-dependently increased behavior via D2 neuronal activity, but not D1 activity. This suggests that smoking can affect human brain function not only via the nicotinic cholinergic neuron, but also via the D2 neuron.

  8. Moss is a key nurse plant for reintroduction of the endangered herb, Primulina tabacum Hance.

    Science.gov (United States)

    Hai Ren; Guohua Ma; Qianmei Zhang; Qinfeng Guo; Jun Wang; Zhengfeng Wang

    2010-01-01

    The rare and endangered plant Primulina tabacum is a calciphilous perennial herb found only at the entrances of a small number of karst cave drainages in southern China. In a conservation effort, we identified potentially suitable habitats and reintroduced P. tabacum plantlets (propagated in vitro) to one historical and two new cave entrances. The transplanted...

  9. Sensitivity of Allium and Nicotiana in cellular and acellular comet assays to assess differential genotoxicity of direct and indirect acting mutagens.

    Science.gov (United States)

    Bandyopadhyay, Atrayee; Mukherjee, Anita

    2011-05-01

    We have evaluated the extent of DNA damage induced by direct and indirect mutagens by cellular and acellular comet assays in two plant systems, Nicotiana tabacum (wild type tobacco) and Allium cepa (common onion). The objectives of this study were: (1) to generate dose-response curves for DNA migration values from root and shoot nuclei of A. cepa and N. tabacum treated with the direct acting mutagens, ethyl methanesulphonate (EMS), hydrogen peroxide (H(2)O(2)) and the indirect acting mutagen, cadmium chloride (CdCl(2)), (2) to assess the differential response between isolated nuclei and nuclei of root and shoot and of both plants and (3) to examine the differences of sensitivity between direct and indirect acting mutagens by cellular and acellular comet assays. Similar sensitivities were evident in both plant systems to direct and indirect acting mutagens. The combination of cellular and acellular comet assays provided valuable insight to the mode of action of the genotoxicants used. The data obtained demonstrated the estimable capacity of the two plant systems to evaluate genotoxicity under different stress conditions and suggests Allium is a more desirable test system for rapid monitoring of genotoxicity.

  10. E-beam irradiation for the control of Phytophthora nicotianae var. nicotianae in stonewool cubes

    Directory of Open Access Journals (Sweden)

    Ptaszek Magdalena

    2015-09-01

    Full Text Available Effectiveness of electron beam irradiation was evaluated against Phytophthora nicotianae var. nicotianae, the causal agent of stem base and root rot of tomato. In laboratory trials, irradiation of 7-day-old Phytophthora cultures growing on potato-dextrose-agar (PDA medium with 1 kGy resulted in the disintegration of the pathogen’s hyphae. Increasing the irradiation dose to 3 kGy caused decay of the hyphae. Irradiation of infested stonewool with 5 kGy caused decrease of the pathogen population about 5 times. Application of 20 kGy completely eliminated the pathogen from stonewool. Irradiation of substratum resulted in significant increase of tomato seedlings healthiness, especially when the dose 20 kGy was applied.

  11. Expressing foreign genes in the pistil: a comparison of S-RNase constructs in different Nicotiana backgrounds.

    Science.gov (United States)

    Murfett, J; McClure, B A

    1998-06-01

    Transgenic plant experiments have great potential for extending our understanding of the role of specific genes in controlling pollination. Often, the intent of such experiments is to over-express a gene and test for effects on pollination. We have examined the efficiency of six different S-RNase constructs in Nicotiana species and hybrids. Each construct contained the coding region, intron, and downstream sequences from the Nicotiana alata S(A2)-RNase gene. Among the six expression constructs, two utilized the cauliflower mosaic virus (CaMV) 35S promoter with duplicated enhancer, and four utilized promoters from genes expressed primarily in pistils. The latter included promoters from the tomato Chi2;1 and 9612 genes, a promoter from the N. alata S(A2)-RNase gene, and a promoter from the Brassica SLG-13 gene. Some or all of the constructs were tested in N. tabacum, N. plumbaginifolia, N. plumbaginifolia x SI N. alata S(C10)S(c10) hybrids, N. langsdorffii, and N. langsdorffii x SC N. alata hybrids. Stylar specific RNase activities and S(A2)-RNase transcript levels were determined in transformed plants. Constructs including the tomato Chi2;1 gene promoter or the Brassica SLG-13 promoter provided the highest levels of S(A2)-RNase expression. Transgene expression patterns were tightly regulated, the highest level of expression was observed in post-anthesis styles. Expression levels of the S(A2)-RNase transgenes was dependent on the genetic background of the host. Higher levels of S(A2)-RNase expression were observed in N. plumbaginifolia x SC N. alata hybrids than in N. plumbaginifolia.

  12. Analysis of the photosynthetic apparatus in transgenic tobacco plants with altered endogenous cytokinin content: a proteomic study

    Directory of Open Access Journals (Sweden)

    Valcke Roland

    2011-06-01

    Full Text Available Abstract Background Cytokinin is a plant hormone that plays a crucial role in several processes of plant growth and development. In recent years, major breakthroughs have been achieved in the elucidation of the metabolism, the signal perception and transduction, as well as the biological functions of cytokinin. An important activity of cytokinin is the involvement in chloroplast development and function. Although this biological function has already been known for 50 years, the exact mechanisms remain elusive. Results To elucidate the effects of altered endogenous cytokinin content on the structure and function of the chloroplasts, chloroplast subfractions (stroma and thylakoids from transgenic Pssu-ipt and 35S:CKX1 tobacco (Nicotiana tabacum plants with, respectively, elevated and reduced endogenous cytokinin content were analysed using two different 2-DE approaches. Firstly, thykaloids were analysed by blue-native polyacrylamide gel electrophoresis followed by SDS-PAGE (BN/SDS-PAGE. Image analysis of the gel spot pattern thus obtained from thylakoids showed no substantial differences between wild-type and transgenic tobacco plants. Secondly, a quantitative DIGE analysis of CHAPS soluble proteins derived from chloroplast subfractions indicated significant gel spot abundance differences in the stroma fraction. Upon identification by MALDI-TOF/TOF mass spectrometry, these proteins could be assigned to the Calvin-Benson cycle and photoprotective mechanisms. Conclusion Taken together, presented proteomic data reveal that the constitutively altered cytokinin status of transgenic plants does not result in any qualitative changes in either stroma proteins or protein complexes of thylakoid membranes of fully developed chloroplasts, while few but significant quantitative differences are observed in stroma proteins.

  13. Pollen irradiation and possible gene transfer in Nicotiana species

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1985-01-01

    Progeny from crosses of Nicotiana langsdorffii with gamma irradiated pollen of Nicotiana alata ‘Crimson Bedder’ showed skewed segregation in the F2 favoring the maternal parent. This is probably not gene transfer in a strict sense, rather just an extreme case of reduced transmission of irradiated...... chromosomes, leading to massive overrepresentation of maternal genes. Gene transfer or mutational loss may explain some anomalous F1 plants. Segregation in the F2 progeny showed the presence of several genes from the irradiated pollen. Crosses of Nicotiana sylvestris, N. plumbaginifolia N. paniculata......, and Petunia parodii with irradiated pollen from N. alata and Petunia hybrida showed no evidence of gene transfer, nor did experiments with irradiated mentor pollen. This indicates that gene transfer with irradiated pollen between non-crossing species or between species giving sterile hybrids is probably...

  14. Molecular heterogeneity of photosystem I. psaD, psaE, psaF, psaH, and psaL are all present in isoforms in Nicotiana spp.

    Science.gov (United States)

    Obokata, J; Mikami, K; Hayashida, N; Nakamura, M; Sugiura, M

    1993-08-01

    The protein composition of photosystem I (PSI) was examined in Nicotiana spp. by high-resolution polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and immunoblot analysis. Five PSI proteins show polymorphism in an amphidiploid species, Nicotiana tabacum, but not in its ancestral diploid species, Nicotiana sylvestris and Nicotiana tomentosiformis. These Nicotiana spp. appear to have at least 18 PSI proteins per genome that range in molecular mass from 3 to 20 kD. They include the products of nuclear genes psaD, psaE, psaF, psaG, psaH, psaK, and psaL, the product of chloroplast gene psaC, N-terminally blocked proteins of 4.5 and 3.0 kD, and an unidentified protein of 12.5 kD. The psaD, psaF, psaH, and psaL products have two isoforms each that are distinguished by different mobilities in polyacrylamide gel electrophoresis, and the psaE product has four isoforms. The two isoforms of the psaD product have distinct amino acid sequences, indicating that they are encoded by different genes within the genome. Four isoforms of the psaE products can be classified into two groups by N-terminal amino acid sequence, indicating that at least two psaE genes are present in the genome. To examine whether the polymorphic nature of PSI is peculiar to Nicotiana spp., we carried out immunoblot analysis of the psaD and psaE products in isogenic lines of tomato (Lycopersicon esculentum), Arabidopsis thaliana, red bean (Vigna angularis), and corn (Zea mays). Two electrophoretically distinct isoforms were found for the psaD products of tomato, A. thaliana, and corn, and two isoforms of psaE products were detected in tomato, A. thaliana, and red bean. These results suggest that the nuclear-encoded subunits of PSI, except for the psaG and psaK products, generally have two isoforms.

  15. Hydrogen sulfide donor sodium hydrosulfide-induced heat tolerance in tobacco (Nicotiana tabacum L) suspension cultured cells and involvement of Ca(2+) and calmodulin.

    Science.gov (United States)

    Li, Zhong-Guang; Gong, Ming; Xie, Hong; Yang, Lan; Li, Jing

    2012-04-01

    Hydrogen sulfide (H(2)S) is considered as a new emerging cell signal in higher plants. Hydrogen sulfide donor, sodium hydrosulfide, pretreatment significantly increased survival percentage of tobacco suspension cultured cells under heat stress and regrowth ability after heat stress, and alleviated decrease in vitality of cells, increase in electrolyte leakage and accumulation of malondialdehyde (MDA). In addition, sodium hydrosulfide-induced heat tolerance was markedly strengthened by application of exogenous Ca(2+) and its ionophore A23187, respectively, while this heat tolerance was weakened by addition of Ca(2+) chelator ethylene glycol-bis(b-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), plasma membrane channel blocker La(3+), as well as calmodulin (CaM) antagonists chlorpromazine (CPZ) and trifluoperazine (TFP), respectively, but intracellular channel blocker ruthenium red (RR) did not. These results suggested that sodium hydrosulfide pretreatment could improve heat tolerance in tobacco suspension cultured cells and the acquisition of this heat tolerance requires the entry of extracellular Ca(2+) into cells across the plasma membrane and the mediation of intracellular CaM.

  16. Evaluation of tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots for the production of geraniol, the first committed step in terpenoid indole alkaloid pathway

    NARCIS (Netherlands)

    Ritala, A.; Dong, L.; Imseng, N.; Seppanen-Laakso, T.; Vasilev, N.; Krol, van der A.R.; Rischer, H.; Maaheimo, H.; Virkki, A.; Brandli, J.; Schillberg, S.; Eibl, R.; Bouwmeester, H.J.; Oksman-Caldentey, K.M.

    2014-01-01

    The terpenoid indole alkaloids are one of the major classes of plant-derived natural products and are well known for their many applications in the pharmaceutical, fragrance and cosmetics industries. Hairy root cultures are useful for the production of plant secondary metabolites because of their

  17. CDPK1 from ginger promotes salinity and drought stress tolerance without yield penalty by improving growth and photosynthesis in Nicotiana tabacum.

    Science.gov (United States)

    Vivek, Padmanabhan Jayanthi; Tuteja, Narendra; Soniya, Eppurathu Vasudevan

    2013-01-01

    In plants, transient changes in calcium concentrations of cytosol have been observed during stress conditions like high salt, drought, extreme temperature and mechanical disturbances. Calcium-dependent protein kinases (CDPKs) play important roles in relaying these calcium signatures into downstream effects. In this study, a stress-responsive CDPK gene, ZoCDPK1 was isolated from a stress cDNA generated from ginger using rapid amplification of cDNA ends (RLM-RACE) - PCR technique and characterized its role in stress tolerance. An important aspect seen during the analysis of the deduced protein is a rare coupling between the presence of a nuclear localization sequence in the junction domain and consensus sequence in the EF-hand loops of calmodulin-like domain. ZoCDPK1 is abundantly expressed in rhizome and is rapidly induced by high-salt stress, drought, and jasmonic acid treatment but not by low temperature stress or abscissic acid treatment. The sub-cellular localization of ZoCDPK1-GFP fusion protein was studied in transgenic tobacco epidermal cells using confocal laser scanning microscopy. Over-expression of ginger CDPK1 gene in tobacco conferred tolerance to salinity and drought stress as reflected by the high percentage of seed germination, higher relative water content, expression of stress responsive genes, higher leaf chlorophyll content, increased photosynthetic efficiency and other photosynthetic parameters. In addition, transgenic tobacco subjected to salinity/drought stress exhibited 50% more growth during stress conditions as compared to wild type plant during normal conditions. T3 transgenic plants are able to grow to maturity, flowers early and set viable seeds under continuous salinity or drought stress without yield penalty. The ZoCDPK1 up-regulated the expression levels of stress-related genes RD21A and ERD1 in tobacco plants. These results suggest that ZoCDPK1 functions in the positive regulation of the signaling pathways that are involved in the response to salinity and drought stress in ginger and it is likely operating in a DRE/CRT independent manner.

  18. Functional analysis of the Sesbania rostrata leghemoglobin glb3 gene 5'-upstream region in transgenic Lotus corniculatus and Nicotiana tabacum plants.

    Science.gov (United States)

    Szabados, L; Ratet, P; Grunenberg, B; de Bruijn, F J

    1990-10-01

    Expression of the Sesbania rostrata leghemoglobin glb3 gene was analyzed in transgenic Lotus corniculatus and tobacco plants harboring chimeric glb3-uidA (gus) gene fusions to identify cis-acting elements involved in nodule-specific gene expression and general transcriptional control. A 1.9-kilobase fragment of the glb3 5'-upstream region was found to direct a high level of nodule-specific beta-glucuronidase (GUS) activity in L. corniculatus, restricted to the Rhizobium-infected cells of the nodules. The same fragment directed a low level of GUS activity in tobacco, restricted primarily to the roots and to phloem cells of the stem and petiole vascular system. A deletion analysis revealed that the region between coordinates -429 and -48 relative to the ATG was sufficient for nodule-specific expression. Replacement of the -161 to -48 region, containing the glb3 CAAT and TATA boxes, with the heterologous truncated promoters delta-p35S and delta-pnos resulted in a loss of nodule specificity and reduction of GUS activity in L. corniculatus but a significant increase in tobacco, primarily in the roots. The same fragment could not direct nodule-specific expression when fused to a heterologous enhancer in cis. This region contains DNA sequences required, but not sufficient, for nodule-specific expression in L. corniculatus that function poorly or may be involved in promoter silencing in tobacco. By fusing further upstream fragments to the delta-p35S and delta-pnos promoters, two positive regulatory regions were delimited between coordinates -1601 and -670, as well as -429 and -162. The former region appears to function as a general enhancer because it significantly increased promoter activity in both orientations in L. corniculatus and tobacco. The latter region could enhance gene expression in both orientations in tobacco, but only in the correct orientation in L. corniculatus. These results show that efficient expression of the S. rostrata glb3 gene in nodules is mediated by an ATG-proximal, tissue-specific element, as well as further 5'-upstream positive elements; that the S. rostrata glb3 promoter is induced in a nodule-specific fashion in the heterologous legume L. corniculatus, suggesting a high degree of conservation of the relevant regulatory signals; and that the S. rostrata lb promoter is not silent in the nonlegume tobacco, but is expressed primarily in the roots.

  19. The Development of DNA Based Methods for the Reliable and Efficient Identification of Nicotiana tabacum in Tobacco and Its Derived Products

    NARCIS (Netherlands)

    Biswas, Sukumar; Fan, Wei; Li, Rong; Li, Sifan; Ping, Wenli; Li, Shujun; Naumova, Alexandra; Peelen, Tamara; Kok, Esther; Yuan, Zheng; Zhang, Dabing; Shi, Jianxin

    2016-01-01

    Reliable methods are needed to detect the presence of tobacco components in tobacco products to effectively control smuggling and classify tariff and excise in tobacco industry to control illegal tobacco trade. In this study, two sensitive and specific DNA based methods, one quantitative real-tim

  20. The Upregulation of NtAN2 Expression at Low Temperature is Required for Anthocyanin Accumulation in Juvenile Leaves of Lc-transgenic Tobacco (Nicotiana tabacum L.)

    Institute of Scientific and Technical Information of China (English)

    Zong-An Huang; Ting Zhao; Hua-Jie Fan; Ning Wang; Shu-Song Zheng; Hong-Qing Ling

    2012-01-01

    Anthocyanins often accumulate in plants subjected to environmental stress,including low temperature.However,the molecular regulatory mechanism of anthocyanin biosynthesis at low temperature is largely unknown.Here,tobacco was transformed with a maize anthocyanin regulatory gene Lc driven by AtSPX3 promoter to investigate the effect of Lc upon the anthocyanin-biosynthesis pathway.We found that the anthocyanin-biosynthesis pathway could not be activated in wild type,while Lc-transgenic tobacco lines exhibited purple pigmentation in juvenile leaves at low temperature.Accordingly,the total anthocyanin contents increased specifically in juvenilc leaves in Lc-transgenic lines.Transcriptional analysis showed that NtCHS and NtCHI were induced by low temperature in leaves of wild type and transgenic lines.NtDFR was uniquely expressed in Lc-transgenic lines,but its transcript was not detected in wild type,implying that NtDFR expression in tobacco leaves was dependent on Lc.Furthermore,the expression of NtAN2 (regulatory gene) and NtANS (anthocyanidin synthase gene) was coordinately upregulated in Lc-transgenic lines under low temperature,suggesting that both Lc and NtAN2 might activate the expression of NtANS,Based on our findings and previous reports,we postulated that Lc interacted with NtAN2 induced by low-temperature stress and consequently stimulated anthocyanin biosynthesis in juvenile leaves of Lc-transgenic tobacco lines.

  1. Influence of electrical fields (AC and DC) on phytoremediation of metal polluted soils with rapeseed (Brassica napus) and tobacco (Nicotiana tabacum).

    Science.gov (United States)

    Bi, Ran; Schlaak, Michael; Siefert, Eike; Lord, Richard; Connolly, Helen

    2011-04-01

    The combined use of electrokinetic remediation and phytoremediation to decontaminate soil polluted with heavy metals has been demonstrated in a laboratory-scale experiment. The plants species selected were rapeseed and tobacco. Three kinds of soil were used: un-contaminated soil from forest area (S1), artificially contaminated soil with 15mgkg(-1) Cd (S2) and multi-contaminated soil with Cd, Zn and Pb from an industrial area (S3). Three treatment conditions were applied to the plants growing in the experimental vessels: control (no electrical field), alternating current electrical field (AC, 1Vcm(-1)) and direct current electrical field (DC, 1Vcm(-1)) with switching polarity every 3h. The electrical fields were applied for 30d for rapeseed and 90d for tobacco, each experiment had three replicates. After a total of 90d growth for rapeseed and of 180d for tobacco, the plants were harvested. The pH variation from anode to cathode was eliminated by switching the polarity of the DC field. The plants reacted differently under the applied electrical field. Rapeseed biomass was enhanced under the AC field and no negative effect was found under DC field. However, no enhancement of the tobacco biomass under the AC treatment was found. The DC field had a negative influence on biomass production on tobacco plants. In general, Cd content was higher in both species growing in S2 treated with AC field compared to the control. Metal uptake (Cd, Cu, Zn and Pb) per rapeseed plant shoot was enhanced by the application of AC field in all soils. Copyright © 2010 Elsevier Ltd. All rights reserved.

  2. Evaluation of tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots for the production of geraniol, the first committed step in terpenoid indole alkaloid pathway

    NARCIS (Netherlands)

    Ritala, A.; Dong, L.; Imseng, N.; Seppanen-Laakso, T.; Vasilev, N.; Krol, van der A.R.; Rischer, H.; Maaheimo, H.; Virkki, A.; Brandli, J.; Schillberg, S.; Eibl, R.; Bouwmeester, H.J.; Oksman-Caldentey, K.M.

    2014-01-01

    The terpenoid indole alkaloids are one of the major classes of plant-derived natural products and are well known for their many applications in the pharmaceutical, fragrance and cosmetics industries. Hairy root cultures are useful for the production of plant secondary metabolites because of their ge

  3. VARIACIONES EN LA RESPUESTA DE INDICADORES DEFENSIVOS Y EN EL CONTENIDO DE COMPONENTES DEL METABOLISMO PRIMARIO EN PLÁNTULAS DE TABACO (Nicotiana tabacum, L.) APLICADAS CON QUITOSANA

    OpenAIRE

    Daimy Costales Menéndez; Trudy Ann Gordon; Alejandro B. Falcón Rodríguez

    2014-01-01

    Plántulas de tabaco de 25 días de sembradas (dds), variedad Criollo 98, fueron tratadas mediante adición a la rizosfera con un polímero de quitosana, a diferentes concentraciones: 0, 0,1, 1, 2,5 y 10 g.L-1. A los 30 dds se determinaron los contenidos de proteínas y carbohidratos reductores solubles y la actividad de indicadores defensivos (fenilalanina amonio liasa (PAL), EC 4.3.1.5 y β 1-3 glucanasa, EC 3.2.1.6) en hojas, mientras que la actividad de la enzima nitrato reductasa (EC 1.6.6....

  4. Transgenic Nicotiana tabacum cultivar Samsun plants carrying the wild sugar beet Hs1pro1 gene have resistance to root-knot nematodes

    OpenAIRE

    SÖNMEZ, Çağla; ELEKCİOĞLU, İbrahim Halil; YÜCEL, Ayşe Meral; ÖKTEM, Hüseyin Avni

    2014-01-01

    Nematodes are the principal animal parasites of plants, causing annual crop losses of more than US100 billion worldwide. Conventional control measures against nematode infection include toxic nematicide application to soil, crop rotation practices, and classical breeding approaches. However, due to the limitations of each technique, biotechnology presents itself as an effective alternative in nematode control. To date, several resistance genes against nematodes have been cloned. One such gene...

  5. Transformation of tobacco plant (Nicotiana tabacum L. with the recombinant hepatitis B virus genes 35SHBsAg and 35SHBsAgER

    Directory of Open Access Journals (Sweden)

    Juliana Martins Ribeiro

    2010-03-01

    Full Text Available The recombinant surface antigen of hepatitis B virus (HBsAg, purified from transgenic plants, proved to be efficient when utilized for raising anti-HB antibodies for the prevention of hepatitis B. Because of the important role of the HBsAg antigen in hepatitis B prevention, the coding sequence of HBsAg antigen, with or without the addition of the carboxi-terminus sequence for protein retention in the endoplasmatic reticulum, was linked to cauliflower mosaic virus 35S promoter, tobacco mosaic virus leader sequence Ω, and the transcription terminator sequence. The aim of this work was to clone the chimeric gene 35SHBsAgER in the plant expression vector pGPTV/Kan/Asc. The resulting plasmid, called pG35SHBsAgER, and another plasmid produced previously in our laboratory called pG35SHBsAg, were transferred to Agrobacterium tumefaciens, and tobacco leaves, of the SR1 cultivar were used as explants for genetic transformation. Twenty-one fully regenerated plants were obtained (10 for the pG35SHBsAg construction and 11 for the pG35SHBsAgER construction. The genomic DNA of all plants was analyzed by PCR, and the presence of the transgene was confirmed in all plants.

  6. Alternation of light/dark period priming enhances clomazone tolerance by increasing the levels of ascorbate and phenolic compounds and ROS detoxification in tobacco (Nicotiana tabacum L.) plantlets.

    Science.gov (United States)

    Darwish, Majd; Lopez-Lauri, Félicie; Vidal, Véronique; El Maâtaoui, Mohamed; Sallanon, Huguette

    2015-07-01

    The effect of the alternation of light/dark periods (AL) (16/8 min light/dark cycles and a photosynthetic photon flux density (PPFD) of 50 μmol photons m(-2) s(-1) for three days) to clarify the mechanisms involved in the clomazone tolerance of tobacco plantlets primed with AL was studied. Clomazone decreased PSII activity, the net photosynthetic rate (Pn), and the ascorbate and total polyphenol contents and increased H2O2 and starch grain accumulation and the number of the cells that underwent programmed cell death (PCD). The pretreatment with AL reduced the inhibitory effect of clomazone on the PSII activity and photosynthesis, as indicated by the decreases in the H2O2 and starch grain accumulation and the PCD levels, and increased the content of ascorbate and certain phenolic compounds, such as chlorogenic acid, neochlorogenic acid and rutin. The AL treatment could promote photorespiration via post-illumination burst (PIB) effects. This alternative photorespiratory electron pathway may reduce H2O2 generation via the consumption of photochemical energy, such as NADH+H(+). At 10 days (D10) of AL treatment, this process induced moderate stress which stimulates H2O2 detoxification systems by increasing the activity of antioxidant enzymes and the biosynthesis of antioxidant components. Therefore, the PCD levels provoked by clomazone were noticeably decreased.

  7. CDPK1 from ginger promotes salinity and drought stress tolerance without yield penalty by improving growth and photosynthesis in Nicotiana tabacum.

    Directory of Open Access Journals (Sweden)

    Padmanabhan Jayanthi Vivek

    Full Text Available In plants, transient changes in calcium concentrations of cytosol have been observed during stress conditions like high salt, drought, extreme temperature and mechanical disturbances. Calcium-dependent protein kinases (CDPKs play important roles in relaying these calcium signatures into downstream effects. In this study, a stress-responsive CDPK gene, ZoCDPK1 was isolated from a stress cDNA generated from ginger using rapid amplification of cDNA ends (RLM-RACE - PCR technique and characterized its role in stress tolerance. An important aspect seen during the analysis of the deduced protein is a rare coupling between the presence of a nuclear localization sequence in the junction domain and consensus sequence in the EF-hand loops of calmodulin-like domain. ZoCDPK1 is abundantly expressed in rhizome and is rapidly induced by high-salt stress, drought, and jasmonic acid treatment but not by low temperature stress or abscissic acid treatment. The sub-cellular localization of ZoCDPK1-GFP fusion protein was studied in transgenic tobacco epidermal cells using confocal laser scanning microscopy. Over-expression of ginger CDPK1 gene in tobacco conferred tolerance to salinity and drought stress as reflected by the high percentage of seed germination, higher relative water content, expression of stress responsive genes, higher leaf chlorophyll content, increased photosynthetic efficiency and other photosynthetic parameters. In addition, transgenic tobacco subjected to salinity/drought stress exhibited 50% more growth during stress conditions as compared to wild type plant during normal conditions. T3 transgenic plants are able to grow to maturity, flowers early and set viable seeds under continuous salinity or drought stress without yield penalty. The ZoCDPK1 up-regulated the expression levels of stress-related genes RD21A and ERD1 in tobacco plants. These results suggest that ZoCDPK1 functions in the positive regulation of the signaling pathways that are involved in the response to salinity and drought stress in ginger and it is likely operating in a DRE/CRT independent manner.

  8. Efectos de la deficiencia en boro sobre la asimilación de nitrato y algunos aspectos del metabolismo secundario en plantas de tabaco (Nicotiana tabacum L.)

    OpenAIRE

    Camacho Cristóbal, Juan José

    2003-01-01

    Aunque la deficiencia en boro (B) provoca alteraciones en muchos procesos fisiológicos, el papel de este micronutriente en la fisiología de las plantas vasculares no está bien establecido. Los principales objetivos de este trabajo han sido: 1, Evaluar si el boro desempeña algún papel sobre la asimilación de nitrato. 2, Estudio del metabolismo fenólico en condiciones de deficiencia en boro en plantas de tabaco. La deficiencia en B causó un drástico descenso en los contenidos foliares de nitrat...

  9. Isolation of beta-1, 4-endoglucanase genes from Globodera tabacum and their expression during parasitism

    NARCIS (Netherlands)

    Goellner, M.; Smant, G.; Boer, de J.M.; Baum, T.J.; Davis, E.L.

    2000-01-01

    Two beta-1,4-endoglucanase (EGase) cDNAs were isolated from Globodera tabacum, the tobacco cyst nematode, and have been designated as GT-eng-1 and GT-eng-2. GT-eng-1 and GT-eng-2 encode precursor proteins with a predicted secretion signal sequence, cellulolytic catalytic domain, and a linker domain.

  10. Effect of chitosan on tobacco mosaic virus (TMV) accumulation, hydrolase activity, and morphological abnormalities of the viral particles in leaves of N. tabacum L. cv. Samsun.

    Science.gov (United States)

    Nagorskaya, Vera; Reunov, Anatoliy; Lapshina, Larisa; Davydova, Viktoriya; Yermak, Irina

    2014-08-01

    The effect of chitosan on the development of infection caused by Tobacco mosaic virus (TMV) in leaves of Nicotiana tabacum L. cv. Samsun has been studied. It was shown that the infectivity and viral coat protein content in leaves inoculated with a mixture of TMV (2 μg/mL) and chitosan (1 mg/mL) were lower in the early period of infection (3 days after inoculation), by 63% and 66% respectively, than in leaves inoculated with TMV only. Treatment of leaves with chitosan 24 h before inoculation with TMV also caused the antiviral effects, but these were less apparent than when the virus and polysaccharide were applied simultaneously. The inhibitory effects of the agent decreased as the infection progressed. Inoculation of leaves with TMV together with chitosan considerably enhanced the activity of hydrolases (proteases, RNases) in the leaves, in comparison with leaves inoculated with TMV alone. Electron microscope assays of phosphotungstic acid (PTA)-stained suspensions from infected tobacco leaves showed that, in addition to the normal TMV particles (18 nm in diameter, 300 nm long), these suspensions contained abnormal (swollen, "thin" and "short") virions. The highest number of abnormal virions was found in suspensions from leaves inoculated with a mixture of TMV and chitosan. Immuno-electron microscopy showed that "thin" virus particles, in contrast to the particles of normal diameter, lost the ability to bind to specific antiserum. It seems that the chitosan-induced activation of hydrolases stimulates the intracellular degradation of TMV particles and hence hydrolase activation may be considered to be one of the polysaccharide-mediated cellular defense mechanisms that limit virus accumulation in cells.

  11. Overexpression of the Synthetic Chimeric Native-T-phylloplanin-GFP Genes Optimized for Monocot and Dicot Plants Renders Enhanced Resistance to Blue Mold Disease in Tobacco (N. tabacum L.

    Directory of Open Access Journals (Sweden)

    Dipak K. Sahoo

    2014-01-01

    Full Text Available To enhance the natural plant resistance and to evaluate the antimicrobial properties of phylloplanin against blue mold, we have expressed a synthetic chimeric native-phylloplanin-GFP protein fusion in transgenic Nicotiana tabacum cv. KY14, a cultivar that is highly susceptible to infection by Peronospora tabacina. The coding sequence of the tobacco phylloplanin gene along with its native signal peptide was fused with GFP at the carboxy terminus. The synthetic chimeric gene (native-phylloplanin-GFP was placed between the modified Mirabilis mosaic virus full-length transcript promoter with duplicated enhancer domains and the terminator sequence from the rbcSE9 gene. The chimeric gene, expressed in transgenic tobacco, was stably inherited in successive plant generations as shown by molecular characterization, GFP quantification, and confocal fluorescent microscopy. Transgenic plants were morphologically similar to wild-type plants and showed no deleterious effects due to transgene expression. Blue mold-sensitivity assays of tobacco lines were performed by applying P. tabacina sporangia to the upper leaf surface. Transgenic lines expressing the fused synthetic native-phyllopanin-GFP gene in the leaf apoplast showed resistance to infection. Our results demonstrate that in vivo expression of a synthetic fused native-phylloplanin-GFP gene in plants can potentially achieve natural protection against microbial plant pathogens, including P. tabacina in tobacco.

  12. Genetic variation in oriental tobacco (Nicotiana tabacum L. by agro-morphological traits and simple sequence repeat markers Variação genética em tabaco oriental (Nicotiana tabacum L. por marcadores agro-morfológicos e traços simples de repetição de sequência

    Directory of Open Access Journals (Sweden)

    Reza Darvishzadeh

    2013-06-01

    Full Text Available The objectives of this study were to assess genetic diversity and determine differences between several oriental tobacco genotypes by examining both agro-morphological traits and molecular markers. Simple lattice design with two replications was used to evaluate 100 oriental tobacco genotypes. Analysis of variance manifested that there is high level of genetic diversity in oriental-type tobaccos based on morphological traits including number of leaf, days to 50% flowering, leaf length, leaf width, leaf fresh weight, leaf dry weight, stem height and stem girth. Classification of genotypes using agro-morphological data by means of un-weighted pair-group method using arithmetic average (UPGMA algorithm based on squared standardized Euclidean distances resulted four distinguishable groups that pursuit own geographical distribution. In the molecular marker investigations, a total of 13 simple sequence repeats (SSR primer pairs were used to determine polymorphism of the test germplasm. Thirty five alleles were scored at 13 SSR loci. The average number of alleles per locus (na and the effective allele number (Ae were 2.69 and 2.34, respectively. By using SSR data, pair wise Jaccard's similarity coefficients were produced. Grouping of genotypes via Jaccard's similarity coefficients and using UPGMA clustering method lead to three groups that had not any accommodated with own origins. Results reveled that there is not completely agreement for classification based on agro-morphological and SSR loci in oriental-type tobaccos. Because of non influence of environmental effects on molecular marker, heterotic groups based on SSR markers could be closer to reality.Os objetivos deste estudo foram avaliar a diversidade genética e determinar as diferenças entre diversos genótipos de tabaco oriental através tratos morfológicos e marcadores moleculares. O delineamento utilizado foi o látice simples com duas repetições e foram avaliados 100 genótipos de tabaco simples. As análises de variância mostarram que há pouca alto nível de diversidade genética nos tabacos to tipo oriental baseado nos tratamentos morfológicos incluindo número de folhas, dias para os 50% do florescimento, comprimento da folha, largura da folha, peso fresco da folha, peso seco da folha e maior comprimento e espessura do caule. A classificação dos genótipos usando o método de dados morfológicos por médias de grupos pareados não ponderados usando média aritmética (UPGMA algoritmo baseado nas distâncias Euclidianas quadradas padronizadas resultou quatro grupos distintos de acordo co a própria distribuição geográfica. Nas investigações dos marcadores moleculares, um total de 13 repetições de sequências simples (RSS de iniciadores pares foram utilizados para determinar o polimorfismo do germoplasma de teste. Trinta e cinco alelos foram marcados em 13 locos RSS. O numero médio de alelos por loco (na e o número de alelo efetivo (Ae forma 2,69 e 2,34, respectivamente. Utilizando os dados RSS, coeficientes de similaridade de Jaccard foram produzidos. O agrupamento de genótipos através dos coeficientes de similaridade de Jaccard e usando o método de agrupamento UPGMA que considera três grupos que não tiveram algum alojamento com suas próprias origens. Os resultados revelaram que revelaram que não estão completamente de acordo para classificação baseada em agro-morfológicas por loco e RSS em tabacos do tipo oriental. Por causa da não influencia dos efeitos ambientais no marcador molecular, grupos heteróticos com base em marcadores SSR poderiam estar mais próximos da realidade.

  13. Genetic analysis of Phytophthora nicotianae populations from different hosts using microsatellite markers

    Science.gov (United States)

    Two hundred thirty-one isolates of P. nicotianae representing 14 populations from different host genera, including agricultural crops (Citrus, Nicotiana, and Lycopersicon), potted ornamental species in nurseries (Lavandula, Convolvulus, Myrtus, Correa and Ruta) and other plant genera of lesser econo...

  14. Conservation and possible reintroduction of an endangered plant based on an analysis of community ecology: a case study of Primulina tabacum Hance in China

    Science.gov (United States)

    Hai Ren; Qianmei Zhang; Zhengfeng Wang; Qinfeng Guo; June Wang; Nan Liu; Kaiming. Liang

    2010-01-01

    The distribution of the rare and endangered perennial herb Primulina tabacum Hance is restricted to eight karst caves in southern China. To conserve P. tabacum and to evaluate possible reintroduction, we studied its historical distribution and conducted field surveys of both its biotic and physical environment. We used detrended...

  15. NMR-based structural validation of therapeutic antibody produced in Nicotiana benthamiana.

    Science.gov (United States)

    Yagi, Hirokazu; Fukuzawa, Noriho; Tasaka, Yasushi; Matsuo, Kouki; Zhang, Ying; Yamaguchi, Takumi; Kondo, Sachiko; Nakazawa, Shiori; Hashii, Noritaka; Kawasaki, Nana; Matsumura, Takeshi; Kato, Koichi

    2015-06-01

    We successfully developed a method for metabolic isotope labeling of recombinant proteins produced in transgenic tobacco. This enabled assessment of structural integrity of plant-derived therapeutic antibodies by NMR analysis. A variety of expression vehicles have been developed for the production of promising biologics, including plants, fungi, bacteria, insects, and mammals. Glycoprotein biologics often experience altered folding and post-translational modifications that are typified by variant glycosylation patterns. These differences can dramatically affect their efficacy, as exemplified by therapeutic antibodies. However, it is generally difficult to validate the structural integrity of biologics produced using different expression vehicles. To address this issue, we have developed and applied a stable-isotope-assisted nuclear magnetic resonance (NMR) spectroscopy method for the conformational characterization of recombinant antibodies produced in plants. Nicotiana benthamiana used as a vehicle for the production of recombinant immunoglobulin G (IgG) was grown in a (15)N-enriched plant growth medium. The Fc fragment derived from the (15)N-labeled antibody thus prepared was subjected to heteronuclear two-dimensional (2D) NMR measurements. This approach enabled assessment of the structural integrity of the plant-derived therapeutic antibodies by comparing their NMR spectral properties with those of an authentic IgG-Fc derived from mammalian cells.

  16. Phytophthora nicotianae diseases worldwide: new knowledge of a long-recognised pathogen

    Directory of Open Access Journals (Sweden)

    Franck PANABIERES

    2016-05-01

    Full Text Available Phytophthora nicotianae was first isolated from tobacco at the end of the 19th century. This organism is now considered as one of the most devastating oomycete plant pathogens, with a recognized host range of more than 255 species over five continents and a wide diversity of climates. The economic losses caused by P. nicotianae are difficult to estimate, because of the diversity of its hosts and ecological niches. For these reasons, this pathogen represents a continuous challenge to plant disease management programmes, which frequently rely solely on the use of chemicals. Phytophthora nicotianae is better adapted than its competitors to abiotic stresses, especially to climate warming. As a result, its importance is increasing. This review illustrates, with some examples, how P. nicotianae currently impacts plant economies worldwide, and how it may constitute more severe threats to agriculture and natural ecosystems in the context of global climate change.

  17. Cell physiology of mortality and immortality in a Nicotiana interspecific F1 hybrid complies with the quantitative balance between reactive oxygen and nitric oxide.

    Science.gov (United States)

    Yamamoto, Takumi; Shomura, Sachiko; Mino, Masanobu

    2017-03-01

    The cultured cell line, GTH4, of an interspecific F1 hybrid between Nicotiana gossei Domin and N. tabacum L. died after a shift in temperature from 37°C to 26°C. Fluctuations in the cellular amounts of reactive oxygen species (ROS) and nitric oxide (NO) were detected in GTH4 after the temperature shift, but not in the mutant, GTH4S, which did not die at 26°C presumably due to the lack of genetic factors involved in cell death. The removal of ROS or NO suppressed cell death in GTH4, suggesting that ROS and NO both acted as mediators of cell death. However, excess amounts of the superoxide anion (O2(-)) or NO alleviated cell death. A series of experiments using generators and scavengers of ROS and NO showed that O2(-) affected the cellular levels of NO, and vice versa, indicating that a quantitative balance between O2(-) and NO was important for hybrid cell death. The combination of NO and hydrogen peroxide (H2O2) was necessary and sufficient to initiate cell death in GTH4 and GTH4S. Hypoxia, which suppressed cell death in GTH4 at 26°C, reduced the generation of H2O2 and NO, but allowed for the production of O2(-), which acted as a suppressor and/or modulator of cell death. The activation of MAPK was involved in the generation of H2O2 in GTG4 cells under normoxic conditions, but promoted O2(-) generation under hypoxic conditions. More protective cellular conditions against ROS, as estimated by the expression levels of genes for ROS-scavenging enzymes, may be involved in the mechanisms responsible for the low cell death rate of GTH4 under hypoxic conditions. Copyright © 2017 Elsevier GmbH. All rights reserved.

  18. Host-plant-mediated effects of Nadefensin on herbivore and pathogen resistance in Nicotiana attenuata

    Directory of Open Access Journals (Sweden)

    Baldwin Ian T

    2008-10-01

    Full Text Available Abstract Background The adage from Shakespeare, "troubles, not as single spies, but in battalions come," holds true for Nicotiana attenuata, which is commonly attacked by both pathogens (Pseudomonas spp. and herbivores (Manduca sexta in its native habitats. Defense responses targeted against the pathogens can directly or indirectly influence the responses against the herbivores. Nadefensin is an effective induced defense gene against the bacterial pathogen Pseudomonas syringae pv tomato (PST DC3000, which is also elicited by attack from M. sexta larvae, but whether this defense protein influences M. sexta's growth and whether M. sexta-induced Nadefensin directly or indirectly influences PST DC3000 resistance are unknown. Results M. sexta larvae consumed less on WT and on Nadefensin-silenced N. attenuata plants that had previously been infected with PST DC3000 than on uninfected plants. WT plants infected with PST DC3000 showed enhanced resistance to PST DC3000 and decreased leaf consumption by M. sexta larvae, but larval mass gain was unaffected. PST DC3000-infected Nadefensin-silenced plants were less resistant to subsequent PST DC3000 challenge, and on these plants, M. sexta larvae consumed less and gained less mass. WT and Nadefensin-silenced plants previously damaged by M. sexta larvae were better able to resist subsequent PST DC3000 challenges than were undamaged plants. Conclusion These results demonstrate that Na-defensin directly mediates defense against PST DC3000 and indirectly against M. sexta in N. attenuata. In plants that were previously infected with PST DC3000, the altered leaf chemistry in PST DC3000-resistant WT plants and PST DC3000-susceptible Nadefensin-silenced plants differentially reduced M. sexta's leaf consumption and mass gain. In plants that were previously damaged by M. sexta, the combined effect of the altered host plant chemistry and a broad spectrum of anti-herbivore induced metabolomic responses was more

  19. Effects of the suicide inhibitors of arginine and ornithine decarboxylase activities on organogenesis, growth, free polyamine and hydroxycinnamoyl putrescine levels in leaf explants of Nicotiana xanthi N.C. Cultivated in vitro in a medium producing callus formation.

    Science.gov (United States)

    Burtin, D; Martin-Tanguy, J; Paynot, M; Rossin, N

    1989-01-01

    We studied the effects of dl-alpha-difluoromethylarginine (DFMA) and dl-alpha-difluoromethylornithine (DFMO), specific, irreversible inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC), respectively, on organogenesis growth and titers of free polyamines and conjugated putrescines (hydroxycinnamoyl putrescines) in tobacco (Nicotiana tabacum cv Xanthi n.c.) calli. These results suggest that ADC and ODC regulate putrescine biosynthesis during early and later stages of tobacco callus development, respectively. ADC appears active in biosynthesis of large levels of free amines (agmatine and putrescine) while ODC appears active only in biosynthesis of large levels of putrescine conjugates (hydroxycinnamoyl putrescines). DFMA inhibits the fresh and dry weight increases of tobacco calli, whereas DFMO even promoted the fresh and dry weight increases, thus supporting the view that ADC is important for cell division and callus induction. Inhibition of ODC activity by DFMO resulting in an amide deficiency after 4 weeks of culture facilates the expression of differentiated cell functions. Formation of buds is associated with a significant decrease of hydroxycinnamoyl putrescines.

  20. Highly Oxygenated Flavonoids from the Leaves of Nicotiana plumbaginifolia (Solanaceae

    Directory of Open Access Journals (Sweden)

    Md. Shafiullah Shajib

    2017-11-01

    Full Text Available Nicotiana plumbaginifolia Viv. is an annual herb of the family Solanaceae, which grows abundantly in the weedy lands of Bangladesh . This plant possesses analgesic, antibacterial, anti-anxiety and hepatoprotective properties, and produces various phenolic compounds including flavonoids. The present study afforded determination of total phenolic and flavonoid contents, and for the first time, the isolation and characterization of highly oxygenated flavonoids, e.g., 3,3' ,5,6,7,8-hexamethoxy- 4',5'-methylenedioxyflavone (1, 3,3' ,4' ,5',5,6,7,8-octamethoxyflavone (2, exoticin, 6,7,4',5'-dimethylenedioxy-3,5,3'-trimethoxyflavone (3 and ( 3,3' ,4',5,5',8-hexamethoxy-6,7-methylenedioxyflavone (4 from the leaves of N. plumbaginifolia . All these flavonoids are rather rare natural products, and only found in a few genera, e.g.,Polygonum and Murraya. The structures of the isolated flavonoids were elucidated by comprehensive spectroscopic analyses, e.g., UV, 1H, 13C NMR, DEPT, HSQC, HMBC and MS.

  1. Allometric analysis of the induced flavonols on the leaf surface of wild tobacco (Nicotiana attenuata).

    Science.gov (United States)

    Roda, Amy L; Oldham, Neil J; Svatos, Ales; Baldwin, Ian T

    2003-02-01

    Trichomes excrete secondary metabolites that may alter the chemical composition of the leaf surface, reducing damage caused by herbivores, pathogens and abiotic stresses. We examined the surface exudates produced by Nicotiana attenuata Torr. Ex Wats., a plant known to contain and secrete a number of secondary metabolites that are toxic or a deterrent to herbivorous insects. Extractions specific to the leaf surface, the trichomes, and the laminar components demonstrated the localization of particular compounds. Diterpene glycosides occurred exclusively in leaf mesophyll, whereas nicotine was found in both the trichomes and mesophyll. Neither rutin nor nicotine was found on the leaf surface. Quercetin and 7 methylated derivatives were found in the glandular trichomes and appeared to be excreted onto the leaf surface. We examined the elicitation of these flavonols on the leaf surface with a surface-area allometric analysis, which measures changes in metabolites independent of the effects of leaf expansion. The flavonols responded differently to wounding, methyl jasmonate (MeJA), herbivore attack and UV-C radiation, and the response patterns corresponded to their compound-specific allometries. Finding greater amounts of quercetin on younger leaves and reduced amounts after herbivore feeding and MeJA treatment, we hypothesized that quercetin may function as an attractant, helping the insects locate a preferred feeding site. Consistent with this hypothesis, mirids (Tupiocoris notatus) were found more often on mature leaves sprayed with quercetin at a concentration typical of young leaves than on unsupplemented mature leaves. The composition of metabolites on the leaf surface of N. attenuata changes throughout leaf development and in response to herbivore attack or environmental stress, and these changes are mediated in part by responses of the glandular trichomes.

  2. Herbivore induction of jasmonic acid and chemical defences reduce photosynthesis in Nicotiana attenuata.

    Science.gov (United States)

    Nabity, Paul D; Zavala, Jorge A; DeLucia, Evan H

    2013-01-01

    Herbivory initiates a shift in plant metabolism from growth to defence that may reduce fitness in the absence of further herbivory. However, the defence-induced changes in carbon assimilation that precede this reallocation in resources remain largely undetermined. This study characterized the response of photosynthesis to herbivore induction of jasmonic acid (JA)-related defences in Nicotiana attenuata to increase understanding of these mechanisms. It was hypothesized that JA-induced defences would immediately reduce the component processes of photosynthesis upon attack and was predicted that wild-type plants would suffer greater reductions in photosynthesis than plants lacking JA-induced defences. Gas exchange, chlorophyll fluorescence, and thermal spatial patterns were measured together with the production of defence-related metabolites after attack and through recovery. Herbivore damage immediately reduced electron transport and gas exchange in wild-type plants, and gas exchange remained suppressed for several days after attack. The sustained reductions in gas exchange occurred concurrently with increased defence metabolites in wild-type plants, whereas plants lacking JA-induced defences suffered minimal suppression in photosynthesis and no increase in defence metabolite production. This suppression in photosynthesis occurred only after sustained defence signalling and defence chemical mobilization, whereas a short bout of feeding damage only transiently altered components of photosynthesis. It was identified that lipoxygenase signalling interacted with photosynthetic electron transport and that the resulting JA-related metabolites reduced photosynthesis. These data represent a metabolic cost to mounting a chemical defence against herbivory and link defence-signalling networks to the differential effects of herbivory on photosynthesis in remaining leaf tissues in a time-dependent manner.

  3. Efficacy of Chaetomium Species as Biological Control Agents against Phytophthora nicotianae Root Rot in Citrus.

    Science.gov (United States)

    Hung, Phung Manh; Wattanachai, Pongnak; Kasem, Soytong; Poeaim, Supattra

    2015-09-01

    Thailand is one of the largest citrus producers in Southeast Asia. Pathogenic infection by Phytophthora, however, has become one of major impediments to production. This study identified a pathogenic oomycete isolated from rotted roots of pomelo (Citrus maxima) in Thailand as Phytophthora nicotianae by the internal transcribed spacer ribosomal DNA sequence analysis. Then, we examined the in vitro and in vivo effects of Chaetomium globosum, Chaetomium lucknowense, Chaetomium cupreum and their crude extracts as biological control agents in controlling this P. nicotianae strain. Represent as antagonists in biculture test, the tested Chaetomium species inhibited mycelial growth by 50~56% and parasitized the hyphae, resulting in degradation of P. nicotianae mycelia after 30 days. The crude extracts of these Chaetomium species exhibited antifungal activities against mycelial growth of P. nicotianae, with effective doses of 2.6~101.4 µg/mL. Under greenhouse conditions, application of spores and methanol extracts of these Chaetomium species to pomelo seedlings inoculated with P. nicotianae reduced root rot by 66~71% and increased plant weight by 72~85% compared to that in the control. The method of application of antagonistic spores to control the disease was simple and economical, and it may thus be applicable for large-scale, highly effective biological control of this pathogen.

  4. Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana

    Science.gov (United States)

    Alkanaimsh, Salem; Karuppanan, Kalimuthu; Guerrero, Andrés; Tu, Aye M.; Hashimoto, Bryce; Hwang, Min Sook; Phu, My L.; Arzola, Lucas; Lebrilla, Carlito B.; Dandekar, Abhaya M.; Falk, Bryce W.; Nandi, Somen; Rodriguez, Raymond L.; McDonald, Karen A.

    2016-01-01

    To optimize the expression, extraction and purification of plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), we describe the development and use of plant viral amplicon-based gene expression system; Tobacco Mosaic Virus (TMV) RNA-based overexpression vector (TRBO) to express enzymatically active FLAG-tagged plant made recombinant butyrylcholinesterase (rBChE) in Nicotiana benthamiana leaves using transient agroinfiltration. Two gene expression cassettes were designed to express the recombinant protein in either the ER or to the apoplastic compartment. Leaf homogenization was used to isolate ER-retained recombinant butyrylcholinesterase (prBChE-ER) while apoplast-targeted rBChE was isolated by either leaf homogenization (prBChE) or vacuum-extraction of apoplastic wash fluid (prBChE-AWF). rBChE from apoplast wash fluid had a higher specific activity but lower enzyme yield than leaf homogenate. To optimize the isolation and purification of total recombinant protein from leaf homogenates, an acidic extraction buffer was used. The acidic extraction buffer yielded >95% enzymatically active tetrameric rBChE as verified by Coomassie stained and native gel electrophoresis. Furthermore, when compared to human butyrylcholinesterase, the prBChE was found to be similar in terms of tetramerization and enzyme kinetics. The N-linked glycan profile of purified prBChE-ER was found to be mostly high mannose structures while the N-linked glycans on prBChE-AWF were primarily complex. The glycan profile of the prBChE leaf homogenates showed a mixture of high mannose, complex and paucimannose type N-glycans. These findings demonstrate the ability of plants to produce rBChE that is enzymatically active and whose oligomeric state is comparable to mammalian butyrylcholinesterase. The process of plant made rBChE tetramerization and strategies for improving its pharmacokinetics properties are also discussed. PMID:27379103

  5. Nicotiana ovule extracts in duce nuclear reconstitution of demembranated Xenopus sperm in cell-free system

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    s Nicotiana tabaccum ovule extracts induced nuclear reconstitution of demembranated Xenopus leavis sperm in a ceil-free system. Demembranated Xenopus sperm began to swell after 15 rmin of incubation with Nicotiana ovulde extracts. Accompanying the process of incubation,Xenopus sperm decondensed and their shapes changed gradually from long and ellipse to round. The completely decondensed chromatin was surrounded with membrane structure, which was a mixture envelope of a double membrane and a single membrane. Nucleosome assembly was verified by means of micrococcal nuclease digestion to reconstituted nuclei and DNA electrophoresis. Nicotiana ovule extracts supplied one more experimental model and system.The new system could promote powerfully the research on mechanisms of cell division and cell cycle regulation.

  6. FIRST REPORT OF Phytophthora nicotianae CAUSING ROOT ROT OF SOURSOP IN NORTHEASTERN BRAZIL

    Directory of Open Access Journals (Sweden)

    JAQUELINE FIGUEREDO DE OLIVEIRA COSTA

    Full Text Available ABSTRACT In 2013, soursop trees showing symptoms of root rot were observed in a field in Maceió, state of Alagoas, Brazil. It was isolated Phytophthora sp. which pathogenicity was confirmed in the host seedlings. Morphological and physiological characteristics in carrot-agar modified medium were consistent with Phytophthora nicotianae description. The PCR sequences products obtained with ITS1/ITS4 primers were compared to sequences of ribosomal DNA of Phytophthora species from the GenBank database observing high identity with other P. nicotianae isolates. A phylogenetic tree was performed to compare the isolate with other sequences of P. nicotianae, which clustering has been verified with 99% of bootstrap, confirming the morphophysiological studies. This is the first report of this pathogen on annonaceous plants in the Northeastern Brazil.

  7. 不同镉积累基因型烟草CAX2基因克隆及序列分析%Cloning and sequence analysis of CAX2 from two nicotiana genotypes with different Cd accumulating pattern

    Institute of Scientific and Technical Information of China (English)

    郭园园; 陈江华; 周慧娜; 商慧文; 翟妞; 张艳玲

    2015-01-01

    Objective]The present study was conducted to clone and analyze the cation exchanger gene (CAX2) from two nicotiana genotypes in order to provide references for CAX2 function and mechanism in cadmium transportation of nicotiana . [Method]Based on CAX2 sequence of potato in NCBI and EST sequences of Nicotiana the primers CAX2-F and CAX2-R were designed to clone CAX2 gene from K326 (Nicotiana tabacum) and Nicotiana. rustica by PCR technique. The sequence of CAX2 was analyzed by bioinformatics methods. [Result]Both of CAX2 coding region in K326 and N. rustica (named as NtCAX2 and NrCAX2) were 1314 bp in length, their nucleotide sequences shared with 98.0% of homology. Their deduced amino acids were 437 aa long with only existing 5 residues differences. Nt-CAX2 and NrCAX2 proteins shared with 98.9% of homology, had two identical Na+-Ca2+ exchange protein domains in 102-249 aa and 293-426 aa regions, and were characterized by common structure characteristics of CAX family. The theoretical pI of NtCAX2 and NrCAX2 were 5.54 and 5.45, respectively. Both of them had 11 transmembrane spans with obvious hydrophobicity and two repeat regions of α-1 and α-2, and richened in alpha helix. Phylogenetic analysis results found that , NtCAX2 and NrCAX2 had the closest relationship to potato (StCAX2) , and their protein homolo-gies were higher than 92 . 0%. [Conclusion]The homology of CAX2 gene was very high in N. tabacum and N. rustica with different Cd accumulating pattern. The CAX2 gene was highly conserved in Solanaceae.%【目的】克隆不同镉积累基因型烟草中阳离子转运基因(CAX2),为研究烟草CAX2基因在镉转运过程中的作用机理提供参考。【方法】根据NCBI中马铃薯的CAX2序列,BLAST烟草EST数据库,设计引物克隆普通烟草(Nicotiana tabacum)K326和黄花烟草(N. rustica)的CAX2基因,使用生物信息学方法进行序列分析。【结果】K326和黄花烟草CAX2基因(分别命名为NtCAX2和NrCAX2

  8. Identification and validation of polymorphic microsatellite loci for the analysis of Phytophthora nicotianae populations

    Science.gov (United States)

    A large number of SSR loci were screened in the genomic assemblies of 14 different isolates of Phytophthora nicotianae and primers were developed for amplification of 17 markers distributed among different contigs. These loci were highly polymorphic and amplified from genetically distant isolates of...

  9. Product Variability of the ‘Cineole Cassette'Monoterpene Synthases of Related Nicotiana Species

    Institute of Scientific and Technical Information of China (English)

    Anke F(a)hnrich; Katrin Krause; Birgit Piechulla

    2011-01-01

    Nicotiana species of the section Alatae characteristically emit the floral scent compounds of the ‘cineole cassere' comprising 1,8-cineole,limonene,myrcene,α-pinene,β-pinene,sabinene,and α-terpineol.We successfully isolated genes of Nicotiana alata and Nicotiana langsdorfii that encoded enzymes,which produced the characteristic monoterpenes of this ‘cineole cassette' with α-terpineol being most abundant in the volatile spectra.The amino acid sequences of both terpineol synthases were 99% identical.The enzymes cluster in a monophyletic branch together with the closely related cineole synthase of Nicotiana suaveolens and monoterpene synthase 1 of Solanum lycopersicum.The cyclization reactions (α-terpineol to 1,8-cineole) of the terpineol synthases of N.alata and N.langsdorfii were less efficient compared to the ‘cineole cassette′ monoterpene synthases of Arabidopsis thaliana,N.suaveolens,Salvia fruticosa,Salvia officinalis,and Citrus unshiu.The terpineol synthases of N.alata and N.langsdorfii were localized in pistils and in the adaxial and abaxial epidermis of the petals.The enzyme activities reached their maxima at the second day after anthesis when flowers were fully opened and the enzyme activity in N.alata was highest at the transition from day to night (diurnal rhythm).

  10. Phenotypical and molecular characterization of the Tomato mottle Taino virus-Nicotiana megalosiphon interaction

    NARCIS (Netherlands)

    Collazo, C.; Ramos, P.L.; Chacón, O.; Borroto, C.J.; López, Y.; Pujol, M.; Thomma, B.P.H.J.; Hein, I.; Borrás-Hidalgo, O.

    2005-01-01

    Tomato mottle Taino virus (ToMoTV) infection causes significant yield losses in plants of various Solanaceous species. In this study, the interaction between Nicotiana megalosiphon and ToMoTV was characterized on a phenotypical and molecular basis. In order to isolate genes that are differentially e

  11. Genetic characterization of Phytophthora nicotianae by the analysis of polymorphic regions of the mitochondrial DNA.

    Science.gov (United States)

    A new method based on the analysis of mitochondrial intergenic regions characterized by intraspecific variation in DNA sequences was developed and applied to the study of the plant pathogen Phytophthora nicotianae. Two regions flanked by genes trny and rns and trnw and cox2 were identified by compa...

  12. Anaerobic soil disinfestation reduces survival and infectivity of Phytophthora nicotianae chlamydospores in pepper

    Science.gov (United States)

    Phytophthora nicotianae is the principal causal agent of root and crown rot disease of pepper plants in Extremadura (western Spain), a spring-summer crop in this region. Preplant soil treatment by anaerobic soil disinfestation (ASD) may effectively control plant pathogens in many crop production sys...

  13. In vitro grown pollen tubes of Nicotiana alata actively synthesise a fucosylated xyloglucan.

    Directory of Open Access Journals (Sweden)

    Edwin R Lampugnani

    Full Text Available Nicotiana alata pollen tubes are a widely used model for studies of polarized tip growth and cell wall synthesis in plants. To better understand these processes, RNA-Seq and de novo assembly methods were used to produce a transcriptome of N. alata pollen grains. Notable in the reconstructed transcriptome were sequences encoding proteins that are involved in the synthesis and remodelling of xyloglucan, a cell wall polysaccharide previously not thought to be deposited in Nicotiana pollen tube walls. Expression of several xyloglucan-related genes in actively growing pollen tubes was confirmed and xyloglucan epitopes were detected in the wall with carbohydrate-specific antibodies: the major xyloglucan oligosaccharides found in N. alata pollen grains and tubes were fucosylated, an unusual structure for the Solanaceae, the family to which Nicotiana belongs. Finally, carbohydrate linkages consistent with xyloglucan were identified chemically in the walls of N. alata pollen grains and pollen tubes grown in culture. The presence of a fucosylated xyloglucan in Nicotiana pollen tube walls was thus confirmed. The consequences of this discovery to models of pollen tube growth dynamics and more generally to polarised tip-growing cells in plants are discussed.

  14. Effect of light, temperature and phytohormone on seed germination and seedling growth of Nicotiana tabacum%光、温、激素对烟草种子萌发和幼苗生长的影响

    Institute of Scientific and Technical Information of China (English)

    招启柏; 宋平; 王广志; 吕冰; 曹显祖

    2001-01-01

    本文以4个烟草品种为材料,研究了激素、光、温对烟草种子萌发和幼苗生长的影响.光能明显加速烟草萌发,但对最终发芽率影响不大;红光、蓝光均能明显加速烟草萌发;光抑制烟草幼苗茎伸长,促进根伸长.烟草种子萌发的适宜温度为20~25℃;在10~30℃范围内,提高温度会促进幼苗生长.低浓度的茉莉酸甲酯(1×10-7mol/L~1×10-6mol/L)能明显加速烟草种子萌发,10-4mol/L以上浓度抑制种子萌发;适宜浓度的24-表油菜素内酯(0.01~0.1mg/L)也能促进烟草种子萌发;赤霉酸能明显促进烟草幼苗生长,其对茎伸长的促进效应明显强于对根伸长的促进效应,K346对GA3最敏感.

  15. Activity of the AtMRP3 promoter in transgenic Arabidopsis thaliana and Nicotiana tabacum plants is increased by cadmium, nickel, arsenic, cobalt and lead but not by zinc and iron

    NARCIS (Netherlands)

    Zientara, K.; Wawrzynska, A.; Lukomska, J.; Lopez Moya, J.R.; Liszewska, F.; Assuncao, A.G.L.; Aarts, M.G.M.; Sirko, A.

    2009-01-01

    Characterization of the function, regulation and metal-specificity of metal transporters is one of the basic steps needed for the understanding of transport and accumulation of toxic metals and metalloids by plants. In this work GUS was used as a reporter for monitoring the activity of the promoter

  16. Analysis of Root Differential Expression Proteins of Nicotiana Tabacum under Two Planting Systems%不同种植方式下烤烟根系差异表达蛋白质分析

    Institute of Scientific and Technical Information of China (English)

    尤垂淮; 陈冬梅; 黄锦文; 唐莉娜; 徐志兵; 张重义; 林文雄

    2014-01-01

    通过根系差异蛋白质组学探讨了种植方式(复种连作、复种轮作)对烤烟生长的影响。结果表明,共有15个蛋白质表达丰度发生变化,用 LC-MS/MS 鉴定了14个差异蛋白质点,经生物信息学分析,11个蛋白质的功能有注释,其中3个属于与香气形成有关的蛋白质,包括转酮醇酶、单脱氢抗坏血还原酶和 O-甲基转移酶,它们在复种轮作条件下均上调表达,此可能有助于烟叶品质提高,而复种连作处理下调表达,可能不利于品质提高。还鉴定到涉及能量、抗性、物质运输、核酸等功能的蛋白:磷酸甘油酸酯激酶、线粒体内膜移位酶亚基 Tim13、NBS-LRR、DnaJ、细胞内囊泡运输蛋白 Sly1、核糖体蛋白质,在复种轮作下也上调表达,此可能促进烤烟的生长,有利于提高烟草产量和品质且经济效益较好。而在复种连作处理下调表达,进而可能使得烟草生长变弱、产量降低、品质变差。%Root differential proteomics was employed in this study to explore the impact of different planting patterns (multiple cropping rotation (MR) and consecutive monoculture multiple cropping (MC) modes) on growth of flue-cured tobacco (Nicotina tobaccum L). The results showed that a total of 15 protein spots had significant expression difference; 14 protein spots were identified by LC-MS/MS analysis and database searching. Among which, the function of 11 proteins were identified, three proteins were related to the aroma formation, i.e. transketolase, monodehydroascorbate reductase(MDHA), O-methyltransferase-like protein were up-regulated under MR, it was helpful probably to enhance the tobacco quality. While under the MC treatment, they were down-regulated, and the tobacco quality could be worse. Phosphoglycerate kinase, mitochondrial import inner membrane translocase subunit Tim13, NBS-LRR(nucleotide binding site plus leucine-rich repeat), DnaJ protein, Vesicle trafficking protein Sly1, Ribosomal protein-like protein were involved in energy metabolism, plant antioxidation reaction, translocation of solute, metabolism of nucleic acid, respectively. They were very important for the growth development of tobacco and all up-regulated under MR treatments, probably increasing the yield and improving the quality of tobacco, and gaining better economic benefit. They were all down-regulated under MC, which may cause weak growth, low yield and poor quality of tobacco.

  17. 香蕉穿孔线虫对烟草和芒果的寄生性和致病性%Parasitism and Pathogenicity of Radopholus similis to Nicotiana tabacum and Mangifera indica

    Institute of Scientific and Technical Information of China (English)

    韩玉春; 谢辉; 秦丹; 黄春晓

    2009-01-01

    [目的]利用从进口观赏植物上截获,并培养保存在胡萝卜愈伤组织上的香蕉穿孔线虫10个种群,研究香蕉穿孔线虫对烟草和芒果的寄生和致病性.[方法]采用温室盆栽接种方法,进行寄生和致病性测定.[结果]香蕉穿孔线虫在烟草和芒果根际的繁殖率Rf>1,接种香蕉穿孔线虫的烟草和芒果植株生长缓慢、矮小,根系衰弱、变色腐烂,有明显受害症状.[结论]烟草和芒果是香蕉穿孔线虫新的适合寄主植物,香蕉穿孔线虫对这2种植物具有明显的致病性,不同种群的致病力有差异.

  18. Physiological and biochemical effects of morphactin IT 3233 on callus and tumour tissues of Nicotiana tabacum L. cultured in vitro III. Transamination processes catalysed by aminotransferase L-alanine: 2-oxoglutarate

    Directory of Open Access Journals (Sweden)

    Z. Chirek

    2015-01-01

    Full Text Available An active alanine transaminase was found both in callus and tumour tissues of tobacco. The enzyme is more active in the latter tissue, and the reaction balance is strongly shifted towards alanine production, while in callus tissue towards glutamic acid formation. Morphactin applied to the tissue cultures stimulates markedly the enzyme activity only in callus. A negative correlation was observed between the intensity of transamination processes and enhanced synthesis of proteins in the tissues studied. Morphactin disturbs nitrogen metabolism in the callus tissue. Tumour tissue is more resistant to the action of this substance. The different hormonal activities in these tissues may be the cause of the different effects of morphactin.

  19. Activity of the AtMRP3 promoter in transgenic Arabidopsis thaliana and Nicotiana tabacum plants is increased by cadmium, nickel, arsenic, cobalt and lead but not by zinc and iron

    NARCIS (Netherlands)

    Zientara, K.; Wawrzynska, A.; Lukomska, J.; Lopez Moya, J.R.; Liszewska, F.; Assuncao, A.G.L.; Aarts, M.G.M.; Sirko, A.

    2009-01-01

    Characterization of the function, regulation and metal-specificity of metal transporters is one of the basic steps needed for the understanding of transport and accumulation of toxic metals and metalloids by plants. In this work GUS was used as a reporter for monitoring the activity of the promoter

  20. Analyses of chlorogenic acids and related cinnamic acid derivatives from Nicotiana tabacum tissues with the aid of UPLC-QTOF-MS/MS based on the in-source collision-induced dissociation method

    CSIR Research Space (South Africa)

    Ncube, EN

    2014-11-01

    Full Text Available been attrib- uted to the different environmental conditions to which they are exposed to [19]. Furthermore, it has been re- ported that the biosynthesis of CGAs is highly dependenton the developmental stage of the tissues [20]. Cell type... and trans configurations) of CGAs. From our previous work [8], we could confidently conclude that the cis-5-CQA molecule could be a natural product of to- bacco plant systems, suggesting an interesting biochemical phenotype which is not fully explained...

  1. Cloning and Function Analysis of Transcription Factor Gene NtGRAS-R1 from Nicotiana tabacum L.%烟草转录因子基因NtGRAS-R1的克隆与功能分析

    Institute of Scientific and Technical Information of China (English)

    李富欣; 许芳芳; 李素敏; 肖万福; 孙艳敏; 刘卫群; 郭红祥

    2015-01-01

    为了分析NtGRAS-R1的生物学功能,在NCBI上BLAST植物的EST数据库拼接获得Nt-GRAS-R1的全长序列;根据该序列设计特异引物,利用PCR方法从烟草根系cDNA 中扩增Nt-GRAS-R1,将其连接到pS1300表达载体上,采用农杆菌介导的花序侵染法转化拟南芥,采用RT-PCR法检测转基因拟南芥植株;获得稳定转基因拟南芥后观察生长性状,并用qPCR方法检测At-CLV3基因的表达情况。结果显示,NtGRAS-R1基因属于HAM亚家族,编码508个氨基酸;观察发现,转基因拟南芥植株根长和根体积明显大于野生型;qPCR结果表明,转基因拟南芥AtCLV3的表达量明显低于野生型拟南芥。初步表明NtGRAS-R1参与根系生长发育调控过程。%The aim of this research is to explore the function of NtGRAS-R1 . The full-length sequence of NtGRAS-R1 was acquired with in silico cloning method. The ORF fragment of NtGRAS-R1 was obtained with PCR amplification method, and then pS1300-NtGRAS-R1 expression vector was constructed. Transgenic Arabidopsis with NtGRAS-R1 was obtained with Agrobacterium tumefaciens-mediated floral dip transformation method. RT-PCR was used to identify transgenic plants. The phenotypic of transgenic Arabidopsis was observed, and the expression of AtCLV3 was detected with qPCR method. The results showed that NtGRAS-R1 belonged to HAM subfamily,having an ORF of 1 527 bp to encode a protein of 508 amino acids. Compared to wild type plants, the root length and volume of transgenic Arabidopsis obviously increased. The expression of AtCLV3 in transgenic Arabidopsis was lower than wild type plant. In the present study, NtGRAS-R1 was successfully cloned, and the phenotypic analysis of transgenic Arabidopsis shows that NtGRAS-R1 has a role in the process of tobacco roots growth and development.

  2. 烟草小孢子母细胞减数分裂过程中微管分布变化%Microtubule Distribution and Change during Meiosis of Microspore Mother Cells of Nicotiana tabacum L.

    Institute of Scientific and Technical Information of China (English)

    王雅英; 张亚楠; 王宏毅; 林石明; 田惠桥

    2011-01-01

    The organization of microtubule cytoskeleton during tobacco microsporogenesis was observed.At the prophase of meiosis Ⅰ of microspore mother cell (MMC), the microtubules in the cell are short and dispersive in cytoplasm. At the metaphase of meiosis Ⅰ, the microtubules in the cell set up a spindle which makes chromosomes move at equatorial plate. When MMCs enter the anaphase of meiosis Ⅰ, the spindle microtubules break. At the metaphase of meiosis Ⅱ, the microtubules form two spindles in the same cytoplasm. In the telophase of meiosis Ⅱ,spindle microtubules depolymerize and change from short segment to tubulin. Then the MMC begins to cytokinesis by cytoplasmic constriction to form contractile furrows, which separate four nuclei into four microspores. There was not evidence to show that microtubules participate in the cytokinesis directly.%应用间接免疫荧光标记技术和激光共聚焦扫描显微镜成像技术观察了烟草小孢子母细胞减数分裂过程中微管的分布变化.在减数分裂前期,小孢子母细胞中的微管较短,随机分散在细胞质中.在减数分裂中期,细胞质中微管形成纺锤体,控制染色体的分布.进入减数分裂Ⅰ后期,部分纺锤体微管将两组染色体拉向两级.在减数分裂Ⅱ中期,细胞中的微管又形成两个纺锤体.在减数分裂Ⅱ后期,纺锤体微管解聚为微管蛋白分散在细胞质中.胞质分裂发生在四个细胞核形成之后.通过细胞核之间的质膜向内缢缩分隔四个细胞核,产生四个小孢子.

  3. Effects of Salicylic Acid to Early Growth and Quick Tillering of Transplanted Tobacco (Nicotiana tabacum L.) Plants%水杨酸对移栽后烟株早生快发的影响

    Institute of Scientific and Technical Information of China (English)

    赵静; 徐照丽; 段胜智; 王丽特; 杨利云; 盛业龙; 龚明

    2013-01-01

      为促进移栽后烟株的早生快发,以K326为试验材料,采用温室大棚漂浮育苗,在幼苗长至3~4叶1心时,叶面分别喷施0、0.5、1.0、1.5 mmol/L的水杨酸,成苗后移栽至大田。结果表明,水杨酸通过增强抗氧化系统的活性,及时有效地清除活性氧,为移栽后烟株快速适应变化的环境提供有利的条件,从而促进烟株根系的快速发育、增强根系活力和根冠比,最终缩短烟株的还苗期,促进早生快发,为后续的快速生长和干物质积累奠定基础。水杨酸浓度不同,作用效果不同,1.0、1.5 mmol/L浓度的作用效果好于0.5 mmol/L。%In order to accelerate early growth and quick tillering of transplanted tobacco plants, experimental material K326 seedlings were floated in greenhouse. Spraying 0, 0.5, 1.0, 1.5 mmol/L salicylic acid on foliar respectively when the seedlings with 3-4 leaves. Then the grown seedlings were transplanted into field. The results showed that transplanted tobacco plants could adapt to the changed environment quickly after treated by salicylic acid because it could increase the activity of antioxidant system and then remove reactive oxygen species ROS timely and effectively. So salicylic acid could promote the development of root system rapidly, strengthening the root vigor and increasing root-shoot ratio. The result was rosette stage was shorted; early growth and quick tillering were accelerated of transplanted tobacco plants. All of those were beneficial to tobacco plants subsequently grow and accumulate dry matter rapidly. Effects of 4 different salicylic acid concentrations were different. The effects of 1.0 and 1.5 mmol/L concentration were better than 0.5 mmol/L.

  4. 干旱和低温胁迫影响烟草幼苗海藻糖代谢的差异比较%Comparison of Metabolic Differences of Trehalose inNicotiana tabacum Seedlings Under Drought and Chilling Stress

    Institute of Scientific and Technical Information of China (English)

    张建波; 王莎莎; 郝大海; 杨慧芹; 马文广; 高雪; 崔明昆; 龚明

    2015-01-01

    In order to investigate differential response of trehalose metabolism to drought and low temperature stress, and roles of trehalose in the forming of drought and chilling resistance, tobacco seedlings(cv. Yunyan 203)were treated under drought and low temperature stresses. Then the changes of trehalose content, their activities of enzymes related to metabolism(TPS, TPP and THase)and gene expression were detected in the tobacco leaves during the drought and chilling stress at 4℃. The results indicated that trehalose content increased firstly, achieved the maximum level in 2 d, and the trehalose content in the leaves under drought stress always maintained higher level than that under the chilling stress. Similar to the change pattern of trehalose content, TPS, TPP activities were increased firstly and then decreased, and showed higher under the drought stress. On the other hand, THase activity demonstrated persistent increase under the drought and chilling stress, and was higher under chilling stress than that under drought stress. The expression level ofTPS,TPP, andTHasegenes was higher in the drought stress than the control at 0 d, but the expression level under chilling stress was lower than the control. Above results showed that drought and chilling stress induced the accumulation of trehalose, the drought stress led to relatively higher level of trehalose content, related enzyme activities and gene expression, implying that response of trehalose to drought stress was more sensitive than that to chilling stress.%为了明确海藻糖代谢在干旱和低温胁迫下的响应及其差异,以及海藻糖在烟草耐旱和耐冷性中的作用,选用烟草品种云烟203幼苗为实验材料,通过对烟草幼苗进行干旱和4℃低温胁迫处理,研究叶片中海藻糖含量及代谢相关酶(海藻糖-6-磷酸合成酶TPS、海藻糖-6-磷酸磷酸酶TPP、海藻糖酶THase)活性及基因表达变化情况。结果表明,在干旱和低温胁迫下,海藻糖含量表现出先升高后降低趋势,均在处理2 d时海藻糖积累达到最大,且干旱胁迫下海藻糖含量高于低温胁迫。TPS、TPP活性在干旱和低温胁迫下先升高后降低,且干旱高于低温;THase活性在干旱和低温胁迫下均不断升高,低温下的THase活性高于干旱。TPS、TPP、THase基因在干旱下的表达量均高于0 d对照,低温下低于0 d对照。以上结果表明,干旱和低温胁迫均能促使烟草体内海藻糖的积累,干旱更能诱导相关基因的表达、相关酶活性及海藻糖含量的升高,表明海藻糖对干旱响应更敏感。

  5. Effects of Bemisia tabaci (Homoptera: Aleyrodidae) Biotype B Infestation on Photosystem Ⅱ in Nicotiana tabacum%B型烟粉虱危害对烟草叶片光系统Ⅱ的影响

    Institute of Scientific and Technical Information of China (English)

    李庆亮; 谭伟; 薛明

    2012-01-01

    [目的]研究B型烟粉虱取食危害对烟草光系统Ⅱ的影响,为明确烟粉虱对寄主植物光合作用的影响机制提供依据.[方法]通过测定烟草叶片叶绿素荧光快速诱导曲线,使用JIP-test分析技术进行参数分析,研究B型烟粉虱危害烟草后对烟草的局部虫体叶和系统叶光系统Ⅱ (PSⅡ)的影响.[结果]B型烟粉虱危害烟草后虫体叶和系统叶上的最大光化学效率(φpo)和光化学性能指数(PIABS)与对照相比均明显升高(P<0.05),表明处理烟草植株的局部叶和系统叶的原初光化学反应受到较大的伤害.B型烟粉虱危害烟草后虫体叶和系统叶PSⅡ反应中心的电子传递均受阻,放氧复合体受到严重破坏.B型烟粉虱危害烟草后系统叶的单位反应中心吸收的能量(ABS/RC)和单位反应中心热耗散掉的能量(DIo/RC)增加,单位反应中心捕获的能量(TRo/RC)下降;B型烟粉虱危害的虫体叶中ABC/RC没有变化,DIo/RC增加,TRo/RC下降.B型烟粉虱危害烟草后虫体叶和系统叶的单位面积反应中心数量(RC/CS)均明显降低(P<0.05),而光系统PS Ⅱ反应中心的关闭程度(1-qP)却明显升高,分别较各自对照升高了69.83%和142.58%(P<0.05).[结论] B型烟粉虱的危害严重影响了烟草叶片的光系统Ⅱ(PSⅡ),主要是由于PSⅡ反应中心的失活和关闭以及对PSⅡ电子传递的抑制,电子传递中受抑制的位点包括放氧复合体和QA到Q8间的电子传递过程,同时B型烟粉虱危害影响了烟草叶片光系统的能量流动,且烟粉虱对烟草叶片PSⅡ的影响具有系统传导性.%[ Objective ] The objective of this study is to define the effects of Bemisia tabaci biotype B infestation on the photosystem II (PSII) in tobacco, and to reveal the mechanism of B. tabaci infestation affecting photosynthesis of host plant. [Method! Effects of B. tabaci infestation on the PSII performance in damaged leaves and systemic leaves of tobacco were investigated by measuring chlorophyll fluorescence transients and analyzing the related parameters using HP-test. [Result] B. tabaci infestation significantly increased the maximal photochemical efficiency of primary photochemistry (tppo) and performance index on absorption basis (PIabs) both in local and systemic leaves (P<0.05), indicating that the original photochemical reaction was greatly damaged. Photosynthetic electron transport was inhibited and oxygen-evolving complexes (OEC) were severely damaged. B. tabaci infestation increased the energy absorption per active reaction centers (ABS/RC) and the energy dissipation per active reaction centers (DIo/RC) increase while decreased energy trapping per active reaction centers (TRo/RC) in systemic leaves. In damaged leaves, DIo/RC increased and TRo/RC decreased, however, the ABC/RC was not affected. B. tabaci infestation reduced the density of active reaction centers per excited cross-section (RC/CS) both in damaged and systemic leaves (P<0.05) while increased the closure degree of the PS II reaction centers by 69.83% and 142.58% (P<0.05), respectively, in damaged and systemic leaves. [Conclusion] B. tabaci infestation damaged the PSII of tobacco leaves. The reaction centers were damaged and the electron transporting was inhibited. The inhibited points of the electron transporting included the OEC and the electron transporting from QA to QB. Energy flux of PSII was also affected by the B. tabaci infestation. The damage on PSII caused by B. tabaci in tobacco leaves was systematically conductive.

  6. Respuesta de algunos procesos metabólicos en plantas de tabaco (nicotiana tabacum l. var. tennesse 86) ante la aplicación conjunta de B y CA

    OpenAIRE

    Ruiz-Sáez, Juan Manuel

    2013-01-01

    La tesis doctoral defendida por D.Juan Manuel Ruiz Sáez se centra en el estudio de una de las relaciones menos estudiadas en la nutrición vegetal, como es la del B-Ca. Los resultados obtenidos muestran un efecto sinérgico del B sobre el Ca, mientras que el efecto del Ca sobre el B se defina como antagónica. Este tipo de relaciones obtenidas determina las acciones de estos nutrientes sobre los distintos procesos fisiológicos estudiados. En ...

  7. The dominant glutamic acid metabolic flux to produce γ-amino butyric acid over proline in Nicotiana tabacum leaves under water stress relates to its significant role in antioxidant activity.

    Science.gov (United States)

    Liu, Cuili; Zhao, Li; Yu, Guanghui

    2011-08-01

    γ-Amino butyric acid (GABA) and proline play a crucial role in protecting plants during various environmental stresses. Their synthesis is from the common precursor glutamic acid, which is catalyzed by glutamate decarboxylase and Δ(1) -pyrroline-5-carboxylate synthetase respectively. However, the dominant pathway under water stress has not yet been established. To explore this, excised tobacco leaves were used to simulate a water-stress condition. The results showed GABA content was much higher than that of proline in leaves under water-deficit and non-water-deficit conditions. Specifically, the amount of GABA significantly increased compared to proline under continuous water loss for 16 h, indicating that GABA biosynthesis is the dominant pathway from glutamic acid metabolism under these conditions. Quantitative reverse transcription polymerase chain reaction and protein Western gel-blot analysis further confirmed this. To explore the function of GABA accumulation, a system producing superoxide anion (O(2) (-) ), peroxide hydrogen (H(2) O(2) ), and singlet oxygen ((1) O(2) ) was employed to investigate the scavenging role on free-radical production. The results demonstrated that the scavenging ability of GABA for O(2) (-) , H(2) O(2) , and (1) O(2) was significantly higher than that of proline. This indicated that GABA acts as an effective osmolyte to reduce the production of reactive oxygen species under water stress.

  8. Over-expression of ascorbate oxidase in the apoplast of transgenic tobacco results in altered ascorbate and glutathione redox states and increased sensitivity to ozone

    DEFF Research Database (Denmark)

    Sanmartin, Maite; Drogoudi, Pavlina D.; Lyons, Tom

    2003-01-01

    Transgenic tobacco (Nicotiana tabacum L. cv. Xanthi) plants expressing cucumber ascorbate oxidase (EC.1.10.3.3) were used to examine the role of extracellular ascorbic acid in mediating tolerance to the ubiquitous air pollutant, ozone (O3). Three homozygous transgenic lines, chosen on the basis...... of a preliminary screen of AO activity in the leaves of 29 lines, revealed up to a 380-fold increase in AO activity, with expression predominantly associated with leaf cell walls. Overexpression of AO resulted in no change in the total ascorbate content recovered in apoplast washing fluid, but the redox state...... of ascorbate was reduced from 30% in wild-type leaves to below the threshold for detection in transgenic plants. Levels of ascorbic acid and glutathione in the symplast were not affected by AO overexpression, but the redox state of ascorbate was reduced, while that of glutathione was increased. AO...

  9. Genetic diversity of the endangered Chinese endemic herb Primulina tabacum (Gesneriaceae) revealed by amplified fragment length polymorphism (AFLP).

    Science.gov (United States)

    Ni, Xiaowei; Huang, Yelin; Wu, Lin; Zhou, Renchao; Deng, Shulin; Wu, Darong; Wang, Bosun; Su, Guohua; Tang, Tian; Shi, Suhua

    2006-05-01

    Primulina tabacum Hance, is a critically endangered perennial endemic to limestone area in South China. Genetic variability within and among four extant populations of this species was assessed using AFLP markers. We expected a low genetic diversity level of this narrowly distributed species, but our results revealed that a high level of genetic diversity remains, both at population level (55.5% of markers polymorphic, H (E) = 0.220, I (S) = 0.321), and at species level (P = 85.6% of markers polymorphic, H (E) = 0.339, I (S) = 0.495), probably resulting from its refugial history and/or breeding system. High levels of genetic differentiation among populations was apparent based on Nei's genetic diversity analysis (G (st)=0.350). The restricted gene flow between populations is a potential reason for the high genetic differentiation. The population genetic diversity of P. tabacum revealed here has clear implications for conservation and management. To maintain present levels of genetic diversity, in situ conservation of all populations is necessary.

  10. Silencing of host basal defense response-related gene expression increases susceptibility of Nicotiana benthamiana to Clavibacter michiganensis subsp. michiganensis.

    Science.gov (United States)

    Balaji, Vasudevan; Sessa, Guido; Smart, Christine D

    2011-03-01

    Clavibacter michiganensis subsp. michiganensis is an actinomycete, causing bacterial wilt and canker disease of tomato (Solanum lycopersicum). We used virus-induced gene silencing (VIGS) to identify genes playing a role in host basal defense response to C. michiganensis subsp. michiganensis infection using Nicotiana benthamiana as a model plant. A preliminary VIGS screen comprising 160 genes from tomato known to be involved in defense-related signaling identified a set of 14 genes whose suppression led to altered host-pathogen interactions. Expression of each of these genes and three additional targets was then suppressed in larger-scale VIGS experiments and the effect of silencing on development of wilt disease symptoms and bacterial growth during an N. benthamiana-C. michiganensis subsp. michiganensis compatible interaction was determined. Disease susceptibility and in planta bacterial population size were enhanced by silencing genes encoding N. benthamiana homologs of ubiquitin activating enzyme, snakin-2, extensin-like protein, divinyl ether synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase 2, and Pto-like kinase. The identification of genes having a role in the host basal defense-response to C. michiganensis subsp. michiganensis advances our understanding of the plant responses activated by C. michiganensis subsp. michiganensis and raises possibilities for devising novel and effective molecular strategies to control bacterial canker and wilt in tomato.

  11. Role of Rice stripe virus NSvc4 in cell-to-cell movement and symptom development in Nicotiana benthamiana

    Directory of Open Access Journals (Sweden)

    Yi eXu

    2012-12-01

    Full Text Available Our previous work has demonstrated that the NSvc4 protein of Rice stripe virus (RSV functions as a cell-to-cell movement protein. However, the mechanisms whereby RSV traffics through plasmodesmata (PD are unknown. Here we provide evidence that the NSvc4 moves on the actin filament and endoplasmic reticulum (ER network, but not microtubules, to reach cell wall PD. Disruption of cytoskeleton using different inhibitors altered NSvc4 localization to PD, thus impeding RSV infection of Nicotiana benthamiana. Sequence analyses and deletion mutagenesis experiment revealed that the N-terminal 125 amino acids (AAs of the NSvc4 determine PD targeting and that a transmembrane domain spanning AAs 106 to 125 is critical for PD localization. We also found that the NSvc4 protein can localize to chloroplasts in infected cells. Analyses using deletion mutants revealed that the N-terminal 73 AAs are essential for chloroplast localization. Furthermore, expression of NSvc4 from a Potato virus X (PVX vector resulted in more severe disease symptoms than PVX alone in systemically infected N. benthamiana leaves. Expression of NSvc4 in Spodoptera frugiperda 9 (Sf-9 cells did not elicit tubule formation, but instead resulted in punctate foci at the plasma membrane. These findings shed new light on our understanding of the movement mechanisms whereby RSV infects host plants.

  12. Screening micro-organisms for cadmium absorption from aqueous solution and cadmium absorption properties of Arthrobacter nicotianae.

    Science.gov (United States)

    Tsuruta, Takehiko; Umenai, Daishi; Hatano, Tomonobu; Hirajima, Tsuyoshi; Sasaki, Keiko

    2014-01-01

    To obtain basic information on how microbial cells absorb cadmium from aqueous solution, we examined cadmium absorption in various micro-organisms. Of 51 micro-organism strains tested, we found that some Gram-positive bacteria, such as, Arthrobacter nicotianae and Bacillus subtilis, and some actinomycetes, such as, Streptomyces flavoviridis and S. levoris were highly capable of absorbing cadmium from an aqueous solution. A. nicotianae absorbed the largest amount of cadmium, over 800 μmol cadmium per gram of dry wt. cells. However, cadmium absorption by A. nicotianae was affected by the solution pH, cadmium concentration, and cell density. The absorption of cadmium was very rapid. Some factors that affected cadmium absorption by A. nicotianae cells were also discussed.

  13. A Redox-Mediated Modulation of Stem Bolting in Transgenic Nicotiana sylvestris Differentially Expressing the External Mitochondrial NADPH Dehydrogenase

    National Research Council Canada - National Science Library

    Yun-Jun Liu; Adriano Nunes-Nesi; Sabá V. Wallström; Ida Lager; Agnieszka M. Michalecka; Fredrik E. B. Norberg; Susanne Widell; Kenneth M. Fredlund; Alisdair R. Fernie; Allan G. Rasmusson

    2009-01-01

    .... Nicotiana sylvestris plants overexpressing potato {Solarium tuberosum) NDB1 displayed early bolting, whereas sense suppression of the same gene led to delayed bolting, with consequential changes in flowering time...

  14. Nicotiana Occidentalis Chloroplast Ultrastructure imaged with Transmission Electron Microscopes Working at Different Accelerating Voltages

    OpenAIRE

    SVIDENSKÁ, Silvie

    2010-01-01

    The main goal of this thesis is to study and compare electron microscopy images of Nicotiana Occidentalis chloroplasts, obtained from two types of transmission electron microscopes,which work with different accelerating voltage of 80kV and 5kV. The two instruments, TEM JEOL 1010 and low voltage electron microscope LVEM5 are employed for experiments. In the first theoretical part, principle of electron microscopy and chloroplast morphology is described. In experimental part, electron microscop...

  15. 烟草5-磷酸脱氧木酮糖还原异构酶基因(dxr)的克隆和表达分析%Cloning and Expression Analysis of 1-deoxy-D-xylulose-5-phosphate Reductoisomerases Gene (dxr) in Nicotina tabacum

    Institute of Scientific and Technical Information of China (English)

    朱晓宇; 王景; 赵二卫; 姚姗姗; 崔红

    2011-01-01

    以烟草(Nicotiana tabacum)栽培品种K326为材料,采用RT-PCR技术,克隆了萜类代谢关键酶烟草5-磷酸脱氧木酮糖还原异构酶(dxr)的cDNA片段.该基因编码区长1422 bp,编码473个氨基酸残基.利用Clustal W(1.82)和Bioedit软件,对烟草与番茄(Lycopersicon esculentum)、长春花(Catharanthus roseus)、金鱼草(A ntirrhinum majus)、薄荷(Mentha piperita)、玉米(Zea mays)、拟南芥(A rabidopsis thaliana)、念珠藻(Nostoc sp.)等物种dar基因的同源性进行分析,其氨基酸同源性分别达到93.6%、87.9%、86.3%、84.6%、84.2%、82.9%和53.5%.原核表达结果证明,该基因编码蛋白的分子量约为50 kD,与氨基酸序列估算相符合.组织表达特异性分析表明,dxr基因在烟草组织中的表达强弱为花>叶>茎>腺毛>种子>根,在花和叶片中的表达量占优势.该结果对烟草萜类代谢的分子调控和品质改良具有重要的参考价值.%Isoprenoid biosynthesis via mvalonate-independent pathway is very important to tobacco resistance and leaf quality. 1-deoxy-D-Xylulose-5-phosphate Reductoisomerases (dxr) is a key enzyme in biosynthesis of isopentenyl diphosphate, which is the precusor for monoterpenoid, diterpenoid and tetratepenoid compounds. To regulate the terpenoid metabolism pathway for tobacco improvement, some important genes such as dxr should be studied firstly. In this paper, dxr gene was cloned successfully from tobacco (Nicotiana tabacum) cultivar K326 leaf by RT-PCR. The cDNA code region was 1 422 bp long and encoding 437 amino acids. Sequence analysis by Clustal W declared that this fragment was highly homologous to dxr gene of other species. It shared 93.6% amino acid homologous to Lycopersicon esculentum, 87.9% to Catharanthus roseus, 86.3% to Antirrhinum majus,84.6% to Mentha piperita, 84.2% to Zea mays, 82.9% to Arabidopsis thaliana, and 53.5% to Nostoc sp.PCC7120. The expression vector pET21b-dxr was constructed and expressed in

  16. Transcriptional and biochemical responses of monoacylglycerol acyltransferase-mediated oil synthesis and associated senescence-like responses in Nicotiana benthamiana.

    Directory of Open Access Journals (Sweden)

    Uday Kumar Divi

    2014-05-01

    Full Text Available Triacylglycerol (TAG accumulates in plant seeds as a major renewable source of carbon for food, fuel and industrial feedstock. Approaches to enhance TAG content by altering lipid pathways and genes in vegetative parts have gained significant attention for biofuel and other applications. However, consequences of these modifications are not always studied in detail. In an attempt to increase TAG levels in leaves we previously demonstrated that a novel substrate, monoacylglycerol (MAG, can be used for the biosynthesis of diacylglycerol (DAG and TAG. Transient expression of the Mus musculus monoacylglycerol acyltransferase MGAT1 and 2 in the model plant Nicotiana benthamiana increased TAG levels at 5 days post infiltration (dpi. Here we show that increased TAG and DAG levels can be achieved as early as 2 dpi. In addition, the MGAT1 infiltrated areas showed senescence-like symptoms from 3 dpi onwards. To unravel underlying molecular mechanisms, Illumina deep sequencing was carried out (a for de-novo assembling and annotation of N. benthamiana leaf transcripts and (b to characterize MGAT1-responsive transcriptome. We found that MGAT1-responsive genes are involved in several processes including TAG biosynthesis, photosynthesis, cell-wall, cutin, suberin, wax and mucilage biosynthesis, lipid and hormone metabolism. Comparative analysis with transcript profiles from other senescence studies identified characteristic gene expression changes involved in senescence induction. We confirmed that increased TAG and observed senescence-symptoms are due to the MAG depletion caused by MGAT1 activity and suggest a mechanism for MGAT1 induced TAG increase and senescence-like symptoms. The data generated will serve as a valuable resource for oil and senescence related studies and for future N. benthamiana transcriptome studies.

  17. Digestive duet: midgut digestive proteinases of Manduca sexta ingesting Nicotiana attenuata with manipulated trypsin proteinase inhibitor expression.

    Directory of Open Access Journals (Sweden)

    Jorge A Zavala

    Full Text Available BACKGROUND: The defensive effect of endogenous trypsin proteinase inhibitors (NaTPIs on the herbivore Manduca sexta was demonstrated by genetically altering NaTPI production in M. sexta's host plant, Nicotiana attenuata. To understand how this defense works, we studied the effects of NaTPI on M. sexta gut proteinase activity levels in different larval instars of caterpillars feeding freely on untransformed and transformed plants. METHODOLOGY/ PRINCIPAL FINDINGS: Second and third instars larvae that fed on NaTPI-producing (WT genotypes were lighter and had less gut proteinase activity compared to those that fed on genotypes with either little or no NaTPI activity. Unexpectedly, NaTPI activity in vitro assays not only inhibited the trypsin sensitive fraction of gut proteinase activity but also halved the NaTPI-insensitive fraction in third-instar larvae. Unable to degrade NaTPI, larvae apparently lacked the means to adapt to NaTPI in their diet. However, caterpillars recovered at least part of their gut proteinase activity when they were transferred from NaTPI-producing host plants to NaTPI-free host plants. In addition extracts of basal leaves inhibited more gut proteinase activity than did extracts of middle stem leaves with the same protein content. CONCLUSIONS/ SIGNIFICANCE: Although larvae can minimize the effects of high NaTPI levels by feeding on leaves with high protein and low NaTPI activity, the host plant's endogenous NaTPIs remain an effective defense against M. sexta, inhibiting gut proteinase and affecting larval performance.

  18. A Novel Isoenzyme of CuZn-superoxide Dismutase from Nicotiana tobacum

    Institute of Scientific and Technical Information of China (English)

    Liang Quan SHENG; Shao Min LIU; Hou Rong XIAO; Bing Le XIA; Qing Liang LIU

    2004-01-01

    An isoenzyme of CuZn-superoxide dismutase, denoted as CuZnSODⅢ, has been separated and purified from Nicotiana Tobacum (tobacco) leaves to apparent homogeneity. Its molecular mass is 22976.6Da. It is composed of one subunit, which is consisted of 187 amine acid residues and contains 1 copper and 0.5 zinc atom. The activation energy of the thermal denaturation process has been obtained as about 143.5kJmol-1. Meanwhile, some properties of spectra were investigated.

  19. Genetic Diversity and Phylogeny of Antagonistic Bacteria against Phytophthora nicotianae Isolated from Tobacco Rhizosphere

    OpenAIRE

    Jin, Fengli; Ding, Yanqin; Ding, Wei; Reddy, M. S.; Fernando, W. G. Dilantha; Du,Binghai

    2011-01-01

    The genetic diversity of antagonistic bacteria from the tobacco rhizosphere was examined by BOXAIR-PCR, 16S-RFLP, 16S rRNA sequence homology and phylogenetic analysis methods. These studies revealed that 4.01% of the 6652 tested had some inhibitory activity against Phytophthora nicotianae. BOXAIR-PCR analysis revealed 35 distinct amplimers aligning at a 91% similarity level, reflecting a high degree of genotypic diversity among the antagonistic bacteria. A total of 25 16S-RFLP patterns were i...

  20. L-Homoserylaminoethanol, a novel dipeptide alcohol inhibitor of eukaryotic DNA polymerase from a plant cultured cells, Nicotina tabacum L.

    Science.gov (United States)

    Kuriyama, Isoko; Asano, Naoki; Kato, Ikuo; Oshige, Masahiko; Sugino, Akio; Kadota, Yasuhiro; Kuchitsu, Kazuyuki; Yoshida, Hiromi; Sakaguchi, Kengo; Mizushina, Yoshiyuki

    2004-03-01

    We found a novel inhibitor specific to eukaryotic DNA polymerase epsilon(pol epsilon) from plant cultured cells, Nicotina tabacum L. The compound (compound 1) was a dipeptide alcohol, L-homoserylaminoethanol. The 50% inhibition of pol epsilon activity by the compound was 43.6 microg/mL, and it had almost no effect on the activities of the other eukaryotic DNA polymerases such as alpha, beta, gamma and delta, prokaryotic DNA polymerases, nor DNA metabolic enzymes such as human telomerase, human immunodeficiency virus type 1 reverse transcriptase, T7 RNA polymerase, human DNA topoisomerase I and II, T4 polynucleotide kinase and bovine deoxyribonuclease I. Kinetic studies showed that inhibition of pol epsilon by the compound was non-competitive with respect to both template-primer DNA and nucleotide substrate. We succeeded in chemically synthesizing the stereoisomers, L-homoserylaminoethanol and D-homoserylaminoethanol, and found both were effective to the same extent. The IC(50) values of L- and D-homoserylaminoethanols for pol epsilon were 42.0 and 41.5 microg/mL, respectively. This represents the second discovery of a pol epsilon-specific inhibitor, and the first report on a water-soluble peptide-like compound as the inhibitor, which is required in biochemical studies of pol epsilon.

  1. Overexpression of Citrus junos mitochondrial citrate synthase gene in Nicotiana benthamiana confers aluminum tolerance.

    Science.gov (United States)

    Deng, Wei; Luo, Keming; Li, Zhengguo; Yang, Yingwu; Hu, Nan; Wu, Yu

    2009-07-01

    Aluminum (Al) toxicity is one of the major factors that limit plant growth in acid soils. Al-induced release of organic acids into rhizosphere from the root apex has been identified as a major Al-tolerance mechanism in many plant species. In this study, Al tolerance of Yuzu (Citrus Junos Sieb. ex Tanaka) was tested on the basis of root elongation and the results demonstrated that Yuzu was Al tolerant compared with other plant species. Exposure to Al triggered the exudation of citrate from the Yuzu root. Thus, the mechanism of Al tolerance in Yuzu involved an Al-inducible increase in citrate release. Aluminum also elicited an increase of citrate content and increased the expression level of mitochondrial citrate synthase (CjCS) gene and enzyme activity in Yuzu. The CjCS gene was cloned from Yuzu and overexpressed in Nicotiana benthamiana using Agrobacterium tumefaciens-mediated methods. Increased expression level of the CjCS gene and enhanced enzyme activity were observed in transgenic plants compared with the wild-type plants. Root growth experiments showed that transgenic plants have enhanced levels of Al tolerance. The transgenic Nicotiana plants showed increased levels of citrate in roots compared to wild-type plants. The exudation of citrate from roots of the transgenic plants significantly increased when exposed to Al. The results with transgenic plants suggest that overexpression of mitochondrial CS can be a useful tool to achieve Al tolerance.

  2. Expression of a cucumber class III chitinase and Nicotiana plumbaginifolia class I glucanase genes in transgenic potato plants

    NARCIS (Netherlands)

    Moravcikova, J.; Matusikova, I.; Libantova, J.; Bauer, M.; Mlynarova, L.

    2004-01-01

    The genes encoding for a cucumber class III chitinase and Nicotiana plumbaginifolia class I glucanase were co-introduced into Slovak potato (Solanum tuberosum L.) breeding line 116/86 using Agrobacterium tumefaciens. For both transgenes the number of integrated copies and level of RNA expression wer

  3. Anti-sense expression of putrescine N-methyltransferase confirms defensive role of nicotine in Nicotiana sylvestris against Manduca sexta

    NARCIS (Netherlands)

    Voelckel, C.; Krugel, T.; Gase, K.; Heidrich, N.; Van Dam, N.M.; Winz, R.; Baldwin, I.T.

    2001-01-01

    Several lines of evidence support the defensive function of nicotine production in the Nicotiana genus against a range of herbivores, but the evidence is largely correlative. To suppress nicotine production in planta and to test its defensive function, we expressed DNA of putrescine N-methyl

  4. Anti-sense expression of putrescine N-methyltransferase confirms defensive role of nicotine in Nicotiana sylvestris against Manduca sexta

    NARCIS (Netherlands)

    Voelckel, C.; Krugel, T.; Gase, K.; Heidrich, N.; Van Dam, N.M.; Winz, R.; Baldwin, I.T.

    2001-01-01

    Several lines of evidence support the defensive function of nicotine production in the Nicotiana genus against a range of herbivores, but the evidence is largely correlative. To suppress nicotine production in planta and to test its defensive function, we expressed DNA of putrescine N-methyl transfe

  5. Changes in phenolic compound, TAL, PAL activity of Nicotiana rustica triggered by ethanolamine pretreatment under in vitro salt stress condition

    National Research Council Canada - National Science Library

    Ali Akbar Ehsanpour; Mohmmad Amin Toghyani; Somayeh Rajaeian

    2015-01-01

    .... For this purpose, 4-week-old Nicotiana rustica plants which had been kept in MS medium were pretreated with ethanolamine and 2 days later they were transferred to MS medium supplemented with 200 mM NaCl for 3 weeks...

  6. Expression of a cucumber class III chitinase and Nicotiana plumbaginifolia class I glucanase genes in transgenic potato plants

    NARCIS (Netherlands)

    Moravcikova, J.; Matusikova, I.; Libantova, J.; Bauer, M.; Mlynarova, L.

    2004-01-01

    The genes encoding for a cucumber class III chitinase and Nicotiana plumbaginifolia class I glucanase were co-introduced into Slovak potato (Solanum tuberosum L.) breeding line 116/86 using Agrobacterium tumefaciens. For both transgenes the number of integrated copies and level of RNA expression

  7. Spontaneous and induced loss of chromosomes in slow-growing somatic hybrid calli of Solanum tuberosum and Nicotiana plumbaginifolia

    NARCIS (Netherlands)

    Tempelaar, MJ; Drenth - Diephuis, L.J.; SAAT, TAWM; Jacobsen, E.

    1991-01-01

    Rate and extent of spontaneous and induced chromosome loss have been determined at the callus level of somatic hybrids of mutants of Solanum tuberosum and Nicotiana plumbaginifolia. AEC (amino ethyl cystein) resistance in potato and Nitrate-Reductase deficiency in N. plumbaginifolia have been used a

  8. [A hydroponic cultivation system for rapid high-yield transient protein expression in Nicotiana plants under laboratory conditions].

    Science.gov (United States)

    Mo, Qianzhen; Mai, Rongjia; Yang, Zhixiao; Chen, Minfang; Yang, Tiezhao; Lai, Huafang; Yang, Peiliang; Chen, Qiang; Zhou, Xiaohong

    2012-06-01

    To develop a hydroponic Nicotiana cultivation system for rapid and high-yield transient expression of recombinant proteins under laboratory conditions. To establish the hydroponic cultivation system, several parameters were examined to define the optimal conditions for the expression of recombinant proteins in plants. We used the green fluorescent protein (GFP) and the geminiviral plant transient expression vector as the model protein/expression vector. We examined the impact of Nicotiana species, the density and time of Agrobacterium infiltration, and the post-infiltration growth period on the accumulation of GFP. The expression levels of GFP in Nicotiana leaves were then examined by Western blotting and ELISA. Our data indicated that a hydroponic Nicotiana cultivation system with a light intensity of 9000 LX/layer, a light cycle of 16 h day/8 h night, a temperature regime of 28 degrees celsius; day/21 degrees celsius; night, and a relative humidity of 80% could support the optimal plant growth and protein expression. After agroinfiltration with pBYGFPDsRed.R/LBA4404, high levels of GFP expression were observed in both N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants cultured with this hydroponic cultivation system. An optimal GFP expression was achieved in both Nicotiana species leaves 4 days after infiltration by Agrobacterium with an OD(600) of 0.8. At a given time point, the average biomass of N. tobaccum (cv. Yuyan No.5) was significantly higher than that of N. benthamiana. The leaves from 6-week-old N. benthamiana plants and 5-week-old N. tobaccum (cv. Yuyan No.5) plants could be the optimal material for agroinfiltration. We have established a hydroponic cultivation system that allows robust growth of N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants and the optimal GFP expression in the artificial climate box.

  9. In vitro cytotoxicity of Nicotiana gossei leaves, used in the Australian Aboriginal smokeless tobacco known as pituri or mingkulpa.

    Science.gov (United States)

    Moghbel, Nahid; Ryu, BoMi; Cabot, Peter J; Steadman, Kathryn J

    2016-07-08

    The Aboriginal population of Central Australia use endemic Nicotiana species to make a smokeless tobacco product known usually as pituri or mingkulpa. Nicotiana leaves are masticated with wood ash into a 'quid' that is chewed/sucked for absorption of nicotine. In addition to nicotine, smokeless tobacco products contain a spectrum of biologically active compounds that may contribute to effects on health. The objective of this study was to quantify nicotine, and related alkaloids and tobacco specific nitrosamines (TSNAs), in Nicotiana leaves used in pituri, and compare in vitro toxicity of pure nicotine with Nicotiana leaf extract at the same concentration of nicotine. An aqueous extract of dry leaves of Nicotiana gossei and a reference smokeless tobacco (CORESTA CRP2) were quantified for major pyridine alkaloids and TSNAs using HPLC-UV and LC-MS/MS. A range of extract concentrations and corresponding concentrations of nicotine standard were tested using an MTS assay to measure human lung epithelium cell (A549) survival. Cells treated for 24h with the maximum concentration of 1.5mg/ml of nicotine resulted in 77% viability. In contrast, extracts from N. gossei leaves and CRP2 containing a similar concentration of nicotine (1.3mg/ml) resulted in remarkably lower viability of 1.5 and 6%, respectively. Comparison of cytotoxicity of pure nicotine with that of the extracts revealed that nicotine was not the source of their cytotoxicity. Other biologically active compounds such as the known carcinogens NNK and NNN, derived from nicotine and nornicotine and found to be present in the smokeless tobacco extracts, may be responsible.

  10. Optimization of engineered production of the glucoraphanin precursor dihomomethionine in Nicotiana benthamiana

    DEFF Research Database (Denmark)

    Crocoll, Christoph; Mirza, Nadia Muhammad Akram; Reichelt, Michael;

    2016-01-01

    Glucosinolates are natural products characteristic of the Brassicales order, which include vegetables such as cabbages and the model plant Arabidopsis thaliana. Glucoraphanin is the major glucosinolate in broccoli and associated with the health-promoting effects of broccoli consumption. Toward our...... goal of creating a rich source of glucoraphanin for dietary supplements, we have previously reported the feasibility of engineering glucoraphanin in Nicotiana benthamiana through transient expression of glucoraphanin biosynthetic genes from A. thaliana (Mikkelsen et al., 2010). As side-products, we...... to a ninefold increase compared to the highest production of this intermediate, as previously reported (Mikkelsen et al., 2010). The increased DHM production without increasing leucine-derived side-product levels provides new metabolic engineering strategies for improved glucoraphanin production...

  11. Partial Characterization of a Vicilin-Like Glycoprotein from Seeds of Flowering Tobacco (Nicotiana sylvestris

    Directory of Open Access Journals (Sweden)

    Jared Q. Gerlach

    2009-01-01

    Full Text Available A vicilin-like glycoprotein from the seeds of Nicotiana sylvestris, flowering tobacco, has been identified using nanoLC/ESI-MS/MS. Sequences from a fragment of protein demonstrated homology with vicilins from other members of the Solanaceae family, notably potato (Solanum demissum. Reducing and nonreducing SDS-PAGE analyses of the identified protein indicated that fragments resulting from in situ proteolytic processing are joined by intrachain disulphide bonds. Staining with Con A lectin was specifically inhibited by mannose suggested the presence of -linked glycosylation which was confirmed by carbohydrate compositional analysis of PVDF-bound protein subunits. HPAEC-PAD analysis of the monosaccharides released from the glycoprotein by acid hydrolysis revealed glucosamine and mannose. -acetylglucosamine termination of attached oligosaccharides was further verified by inhibitable WGA lectin staining. Immunostaining of PVDF-bound N. sylvestris proteins with antibodies against G. max total protein demonstrated cross-staining at masses corresponding to fragments from the proteolytically processed protein subunits.

  12. Protein-Protein Interaction Assays with Effector-GFP Fusions in Nicotiana benthamiana.

    Science.gov (United States)

    Petre, Benjamin; Win, Joe; Menke, Frank L H; Kamoun, Sophien

    2017-01-01

    Plant parasites secrete proteins known as effectors into host tissues to manipulate host cell structures and functions. One of the major goals in effector biology is to determine the host cell compartments and the protein complexes in which effectors accumulate. Here, we describe a five-step pipeline that we routinely use in our lab to achieve this goal, which consists of (1) Golden Gate assembly of pathogen effector-green fluorescent protein (GFP) fusions into binary vectors, (2) Agrobacterium-mediated heterologous protein expression in Nicotiana benthamiana leaf cells, (3) laser-scanning confocal microscopy assay, (4) anti-GFP coimmunoprecipitation-liquid chromatography-tandem mass spectrometry (coIP/MS) assay, and (5) anti-GFP western blotting. This pipeline is suitable for rapid, cost-effective, and medium-throughput screening of pathogen effectors in planta.

  13. Production of Recombinant Cholera Toxin B Subunit in Nicotiana benthamiana Using GENEWARE® Tobacco Mosaic Virus Vector.

    Science.gov (United States)

    Moore, Lauren; Hamorsky, Krystal; Matoba, Nobuyuki

    2016-01-01

    Here, we describe a method to produce a recombinant cholera toxin B subunit in Nicotiana benthamiana plants (CTBp) using the GENEWARE(®) tobacco mosaic virus vector system. Infectious transcripts of the vector RNA are generated in vitro and inoculated on N. benthamiana seedlings. After 11 days, CTBp is extracted in a simple tris buffer at room temperature. No protease inhibitor is required. The leaf homogenate is treated with mild heat and a pH shift to selectively precipitate host-derived proteins. CTBp is purified to >95 % homogeneity by two-step chromatography using immobilized metal affinity and ceramic hydroxyapatite resins. This procedure yields on average 400 mg of low-endotoxin CTBp from 1 kg of fresh leaf material.

  14. Wax esters of different compositions produced via engineering of leaf chloroplast metabolism in Nicotiana benthamiana.

    Science.gov (United States)

    Aslan, Selcuk; Sun, Chuanxin; Leonova, Svetlana; Dutta, Paresh; Dörmann, Peter; Domergue, Frédéric; Stymne, Sten; Hofvander, Per

    2014-09-01

    In a future bio-based economy, renewable sources for lipid compounds at attractive cost are needed for applications where today petrochemical derivatives are dominating. Wax esters and fatty alcohols provide diverse industrial uses, such as in lubricant and surfactant production. In this study, chloroplast metabolism was engineered to divert intermediates from de novo fatty acid biosynthesis to wax ester synthesis. To accomplish this, chloroplast targeted fatty acyl reductases (FAR) and wax ester synthases (WS) were transiently expressed in Nicotiana benthamiana leaves. Wax esters of different qualities and quantities were produced providing insights to the properties and interaction of the individual enzymes used. In particular, a phytyl ester synthase was found to be a premium candidate for medium chain wax ester synthesis. Catalytic activities of FAR and WS were also expressed as a fusion protein and determined functionally equivalent to the expression of individual enzymes for wax ester synthesis in chloroplasts.

  15. Polypeptide synthesis induced in Nicotiana clevelandii protoplasts by infection with raspberry ringspot nepovirus.

    Science.gov (United States)

    Acosta, O; Mayo, M A

    1993-01-01

    Infection of Nicotiana clevelandii protoplasts by raspberry ringspot nepovirus resulted in the accumulation of about 24 polypeptides that differed in M(r) and pI from polypeptides accumulating in mock-inoculated protoplasts. Similar polypeptides accumulated in protoplasts infected with the S and E strains of RRV but different infection-specific polypeptides were detected in protoplasts infected with tobacco ringspot nepovirus. The M(r) of RRV-specific polypeptides ranged from 210,000 to 18,000 and most are presumed to be derived from others by proteolytic cleavage. No evidence was found for marked changes in polypeptide abundance with time after inoculation or for any virus-specific polypeptide becoming disproportionately abundant in the medium during culture.

  16. Expression, Purification, and Biophysical Characterization of a Secreted Anthrax Decoy Fusion Protein in Nicotiana benthamiana

    Science.gov (United States)

    Karuppanan, Kalimuthu; Duhra-Gill, Sifti; Kailemia, Muchena J.; Phu, My L.; Lebrilla, Carlito B.; Dandekar, Abhaya M.; Rodriguez, Raymond L.; Nandi, Somen; McDonald, Karen A.

    2017-01-01

    Anthrax toxin receptor-mediated drug development for blocking anthrax toxin action has received much attention in recent decades. In this study, we produced a secreted anthrax decoy fusion protein comprised of a portion of the human capillary morphogenesis gene-2 (CMG2) protein fused via a linker to the fragment crystallizable (Fc) domain of human immunoglobulin G1 in Nicotiana benthamiana plants using a transient expression system. Using the Cauliflower Mosaic Virus (CaMV) 35S promoter and co-expression with the p19 gene silencing suppressor, we were able to achieve a high level of recombinant CMG2-Fc-Apo (rCMG2-Fc-Apo) protein accumulation. Production kinetics were observed up to eight days post-infiltration, and maximum production of 826 mg/kg fresh leaf weight was observed on day six. Protein A affinity chromatography purification of the rCMG2-Fc-Apo protein from whole leaf extract and apoplast wash fluid showed the homodimeric form under non-reducing gel electrophoresis and mass spectrometry analysis confirmed the molecular integrity of the secreted protein. The N-glycosylation pattern of purified rCMG2-Fc-Apo protein was analysed; the major portion of N-glycans consists of complex type structures in both protein samples. The most abundant (>50%) N-glycan structure was GlcNAc2(Xyl)Man3(Fuc)GlcNAc2 in rCMG2-Fc-Apo recovered from whole leaf extract and apoplast wash fluid. High mannose N-glycan structures were not detected in the apoplast wash fluid preparation, which confirmed the protein secretion. Altogether, these findings demonstrate that high-level production of rCMG2-Fc-Apo can be achieved by transient production in Nicotiana benthamiana plants with apoplast targeting. PMID:28054967

  17. Expression, Purification, and Biophysical Characterization of a Secreted Anthrax Decoy Fusion Protein in Nicotiana benthamiana.

    Science.gov (United States)

    Karuppanan, Kalimuthu; Duhra-Gill, Sifti; Kailemia, Muchena J; Phu, My L; Lebrilla, Carlito B; Dandekar, Abhaya M; Rodriguez, Raymond L; Nandi, Somen; McDonald, Karen A

    2017-01-04

    Anthrax toxin receptor-mediated drug development for blocking anthrax toxin action has received much attention in recent decades. In this study, we produced a secreted anthrax decoy fusion protein comprised of a portion of the human capillary morphogenesis gene-2 (CMG2) protein fused via a linker to the fragment crystallizable (Fc) domain of human immunoglobulin G1 in Nicotiana benthamiana plants using a transient expression system. Using the Cauliflower Mosaic Virus (CaMV) 35S promoter and co-expression with the p19 gene silencing suppressor, we were able to achieve a high level of recombinant CMG2-Fc-Apo (rCMG2-Fc-Apo) protein accumulation. Production kinetics were observed up to eight days post-infiltration, and maximum production of 826 mg/kg fresh leaf weight was observed on day six. Protein A affinity chromatography purification of the rCMG2-Fc-Apo protein from whole leaf extract and apoplast wash fluid showed the homodimeric form under non-reducing gel electrophoresis and mass spectrometry analysis confirmed the molecular integrity of the secreted protein. The N-glycosylation pattern of purified rCMG2-Fc-Apo protein was analysed; the major portion of N-glycans consists of complex type structures in both protein samples. The most abundant (>50%) N-glycan structure was GlcNAc₂(Xyl)Man₃(Fuc)GlcNAc₂ in rCMG2-Fc-Apo recovered from whole leaf extract and apoplast wash fluid. High mannose N-glycan structures were not detected in the apoplast wash fluid preparation, which confirmed the protein secretion. Altogether, these findings demonstrate that high-level production of rCMG2-Fc-Apo can be achieved by transient production in Nicotiana benthamiana plants with apoplast targeting.

  18. Membrane transporters in self resistance of Cercospora nicotianae to the photoactivated toxin cercosporin.

    Science.gov (United States)

    Beseli, Aydin; Amnuaykanjanasin, Alongkorn; Herrero, Sonia; Thomas, Elizabeth; Daub, Margaret E

    2015-11-01

    The goal of this work is to characterize membrane transporter genes in Cercospora fungi required for autoresistance to the photoactivated, active-oxygen-generating toxin cercosporin they produce for infection of host plants. Previous studies implicated a role for diverse membrane transporters in cercosporin resistance. In this study, transporters identified in a subtractive cDNA library between a Cercospora nicotianae wild type and a cercosporin-sensitive mutant were characterized, including two ABC transporters (CnATR2, CnATR3), an MFS transporter (CnMFS2), a uracil transporter, and a zinc transport protein. Phylogenetic analysis showed that only CnATR3 clustered with transporters previously characterized to be involved in cercosporin resistance. Quantitative RT-PCR analysis of gene expression under conditions of cercosporin toxicity, however, showed that only CnATR2 was upregulated, thus this gene was selected for further characterization. Transformation and expression of CnATR2 in the cercosporin-sensitive fungus Neurospora crassa significantly increased cercosporin resistance. Targeted gene disruption of CnATR2 in the wild type C. nicotianae, however, did not decrease resistance. Expression analysis of other transporters in the cnatr2 mutant under conditions of cercosporin toxicity showed significant upregulation of the cercosporin facilitator protein gene (CFP), encoding an MFS transporter previously characterized as playing an important role in cercosporin autoresistance in Cercospora species. We conclude that cercosporin autoresistance in Cercospora is mediated by multiple genes, and that the fungus compensates for mutations by up-regulation of other resistance genes. CnATR2 may be a useful gene, alone or in addition to other known resistance genes, for engineering Cercospora resistance in crop plants.

  19. High-level diterpene production by transient expression in Nicotiana benthamiana

    Science.gov (United States)

    2013-01-01

    Background Characterization of plant terpene synthases is typically done by production of recombinant enzymes in Escherichia coli. This is often difficult due to solubility and codon usage issues. Furthermore, plant terpene synthases which are targeted to the plastids, such as diterpene synthases, have to be shortened in a more or less empirical approach to improve expression. We report here an optimized Agrobacterium-mediated transient expression assay in Nicotiana benthamiana for plant diterpene synthase expression and product analysis. Results Agrobacterium-mediated transient expression of plant diterpene synthases in N. benthamiana led to the accumulation of diterpenes within 3 days of infiltration and with a maximum at 5 days. Over 50% of the products were exported onto the leaf surface, thus considerably facilitating the analysis by reducing the complexity of the extracts. The robustness of the method was tested by expressing three different plant enzymes, cembratrien-ol synthase from Nicotiana sylvestris, casbene synthase from Ricinus communis and levopimaradiene synthase from Gingko biloba. Furthermore, co-expression of a 1-deoxy-D-xylulose-5-phosphate synthase from tomato and a geranylgeranyl diphosphate synthase from tobacco led to a 3.5-fold increase in the amount of cembratrien-ol produced, with maximum yields reaching 2500 ng/cm2. Conclusion With this optimized method for diterpene synthase expression and product analysis, a single infiltrated leaf of N. benthamiana would be sufficient to produce quantities required for the structure elucidation of unknown diterpenes. The method will also be of general use for gene function discovery, pathway reconstitution and metabolic engineering of diterpenoid biosynthesis in plants. PMID:24330621

  20. A Redox-Mediated Modulation of Stem Bolting in Transgenic Nicotiana sylvestris Differentially Expressing the External Mitochondrial NADPH Dehydrogenase1[W][OA

    National Research Council Canada - National Science Library

    Yun-Jun Liu; Adriano Nunes-Nesi; Sabá V Wallström; Ida Lager; Agnieszka M Michalecka; Fredrik E B Norberg; Susanne Widell; Kenneth M Fredlund; Alisdair R Fernie; Allan G Rasmusson

    2009-01-01

    .... Nicotiana sylvestris plants overexpressing potato (Solanum tuberosum) NDB1 displayed early bolting, whereas sense suppression of the same gene led to delayed bolting, with consequential changes in flowering time...

  1. The influence of the manifestation resistance to pests and diseases varieties of nicotiana rustica on the yield performance of vegetative and generative mass

    National Research Council Canada - National Science Library

    Кароліна Артурівна Шейдик

    2015-01-01

    It is shown the phytosanitary state of Nicotiana rustica in Transcarpathian region and it is disclosed the development of harmful organisms, including requiring a detailed study of tomato spotted wilt...

  2. The influence of the manifestation resistance to pests and diseases varieties of nicotiana rustica on the yield performance of vegetative and generative mass

    Directory of Open Access Journals (Sweden)

    Кароліна Артурівна Шейдик

    2015-04-01

    Full Text Available It is shown the phytosanitary state of Nicotiana rustica in Transcarpathian region and it is disclosed the development of harmful organisms, including requiring a detailed study of tomato spotted wilt virus and tobacco stem in view of selection process on group stability. The result of research presented in this article is to assess the Nicotiana rustica varieties for resistance and seed productivity and identification of resistant varieties for implementation in production due to lack of varieties of domestic origin.

  3. Reduced content of homogalacturonan does not alter the ion-mediated increase in xylem hydraulic conductivity in tobacco.

    Science.gov (United States)

    Nardini, Andrea; Gascó, Antonio; Cervone, Felice; Salleo, Sebastiano

    2007-04-01

    Xylem hydraulic conductivity (K(s)) in stems of tobacco (Nicotiana tabacum) wild-type SR1 was compared to that of PG7 and PG16, two transgenic lines with increased levels of expression of the gene encoding the Aspergillus niger endopolygalacturonase (AnPGII). Activity of AnPGII removes in planta blocks of homogalacturonan (HG) with deesterified carboxyls, thus increasing the degree of neutrality of pectins. The effect of K+ was tested in increasing stem K(s) using model plants with more neutral polysaccharides in primary walls and, hence, in intervessel pit membranes. K(s) measured with deionized water was compared to that with KCl solutions at increasing concentrations (DeltaK(s), %). Plants transformed for HG degree of neutrality showed a dwarfed phenotype, but DeltaK(s) did not differ among the three experimental groups. The ion-mediated hydraulic effect saturated at a KCl concentration of 25 mm in SR1 plants. All the three tobacco lines showed DeltaK(s) of around +12.5% and +17.0% when perfused with 10 and 25 mm KCl, respectively. Because modification of HG content did not influence ion-mediated hydraulic enhancement, we suggest that pectin components other than HG, like rhamnogalacturonan-I and/or rhamnogalacturonan-II, might play important roles in the hydrogel behavior of pit membranes.

  4. 烟草黑胫病菌对农田草本植物的寄主范围%The host range of Phytophthora nicotianae var. nicotianae in field herbs

    Institute of Scientific and Technical Information of China (English)

    马国胜; 高智谋

    2011-01-01

    烟草黑胫病是烟草生产中的重要病害,对烟草的产量和质量均影响极大[1-2]。烟草黑胫病菌Phytophthora nicotianae var.nicotianae(Breda deHaan)Tucker是引起烟草黑胫病的唯一病原菌,以往的研究均认为,

  5. Simultaneous detection and quantification of Phytophthora nicotianae and P. cactorum, and distribution analyses in strawberry greenhouses by duplex real-time PCR.

    Science.gov (United States)

    Li, Mingzhu; Inada, Minoru; Watanabe, Hideki; Suga, Haruhisa; Kageyama, Koji

    2013-01-01

    Phytophthora nicotianae and P. cactorum cause Phytophthora rot of strawberry. A duplex real-time PCR technique for simultaneous detection and quantification of the two pathogens was developed. Species-specific primers for P. nicotianae and P. cactorum were designed based on the internal transcribed spacer regions (ITS) of rDNA and the ras-related protein gene Ypt1, respectively. TaqMan probes were labeled with FAM for P. nicotianae and HEX for P. cactorum. Specificities were demonstrated using 52 isolates, including various soil-borne pathogens. Sensitivities for P. nicotianae and P. cactorum DNAs were 10 fg and 1 pg, respectively. The technique was applied to naturally infested soil and root samples; the two pathogens were detected and the target DNA concentrations were quantified. Significant correlations of DNA quantities in roots and the surrounding soils were found. The minimum soil DNA concentration predicting the development of disease symptoms was estimated as 20 pg (g soil)(-1). In three strawberry greenhouses examined, the target DNA concentrations ranged from 1 to 1,655 pg (g soil)(-1) for P. nicotianae and from 13 to 233 pg (g soil)(-1) for P. cactorum. The method proved fast and reliable, and provides a useful tool to monitor P. nicotianae and P. cactorum in plants or soils.

  6. Enzymatic, expression and structural divergences among carboxyl O-methyltransferases after gene duplication and speciation in Nicotiana.

    Science.gov (United States)

    Hippauf, Frank; Michalsky, Elke; Huang, Ruiqi; Preissner, Robert; Barkman, Todd J; Piechulla, Birgit

    2010-02-01

    Methyl salicylate and methyl benzoate have important roles in a variety of processes including pollinator attraction and plant defence. These compounds are synthesized by salicylic acid, benzoic acid and benzoic acid/salicylic acid carboxyl methyltransferases (SAMT, BAMT and BSMT) which are members of the SABATH gene family. Both SAMT and BSMT were isolated from Nicotiana suaveolens, Nicotiana alata, and Nicotiana sylvestris allowing us to discern levels of enzyme divergence resulting from gene duplication in addition to species divergence. Phylogenetic analyses showed that Nicotiana SAMTs and BSMTs evolved in separate clades and the latter can be differentiated into the BSMT1 and the newly established BSMT2 branch. Although SAMT and BSMT orthologs showed minimal change coincident with species divergences, substantial evolutionary change of enzyme activity and expression patterns occurred following gene duplication. After duplication, the BSMT enzymes evolved higher preference for benzoic acid (BA) than salicylic acid (SA) whereas SAMTs maintained ancestral enzymatic preference for SA over BA. Expression patterns are largely complementary in that BSMT transcripts primarily accumulate in flowers, leaves and stems whereas SAMT is expressed mostly in roots. A novel enzyme, nicotinic acid carboxyl methyltransferase (NAMT), which displays a high degree of activity with nicotinic acid was discovered to have evolved in N. gossei from an ancestral BSMT. Furthermore a SAM-dependent synthesis of methyl anthranilate via BSMT2 is reported and contrasts with alternative biosynthetic routes previously proposed. While BSMT in flowers is clearly involved in methyl benzoate synthesis to attract pollinators, its function in other organs and tissues remains obscure.

  7. Use of Several Natural Products from Selected Nicotiana Species to Prevent Black Shank Disease in Tobacco

    Directory of Open Access Journals (Sweden)

    Kroumova Antoaneta B.

    2016-07-01

    Full Text Available Black shank is a major annual disease threat to all types of tobacco worldwide. It is caused by the fungus Phytophthora parasitica var. nicotianae (PPN. The major tobacco growing areas in US - Kentucky, Tennessee and North Carolina can experience devastating losses, reaching in some fields up to 100%. Thus far, the main approaches to control this disease have been creation of resistant varieties, fungicide treatments, and crop rotation. Some fungicides are reported to have negative effects on the environment. The goal of this work was to test the antifungal activity of several natural products that are synthesized by certain Nicotiana species, and secreted to the leaf surface. We hypothesized that phylloplanin, cis-abienol, labdenediol and sclareol can suppress PPN-race 0- and PPN-race 1-caused disease in Burley tobaccos KY 14 and MS KY 14 × L8LC in the greenhouse. We developed methods for leaf surface extraction, spore preparation and soil drench application of the natural compounds tested. Experiments were performed on 5–8 week-old greenhouse grown seedlings. cis-Abienol showed high inhibitory properties toward the disease. Race 0 infection was completely subdued in KY 14 while race 1 infection was reduced by 70–80%, and delayed by 6–10 days in KY 14 and MS KY14 × L8LC. Sclareol was very effective in inhibiting race 0-caused disease in both tobacco cultivars. In MS KY 14 × L8LC race 1 infection was inhibited while in KY 14 it was reduced by 85% and delayed by 6 days. Labdenediol reduced the disease by half in eight week-old KY 14 plants. Tobacco phylloplanin reduced plant infection by both races by 50–60% and delayed the disease by 6–10 days. Phylloplanin was least suppressive in both tobacco cultivars. We consider sclareol to be the best candidate for future studies due to its antifungal properties and availability. cis-Abienol, despite its good antifungal activity, is not feasible for large-scale use due to the production and

  8. Interaction of Se and S on uptake of Se by flue-cured tobacco (Nicotina tabacum L.)%硒硫相互作用对烤烟(Nicotina tabacum L.)吸收硒的影响

    Institute of Scientific and Technical Information of China (English)

    马友华; 丁瑞兴; 张继榛; 竺伟民

    2001-01-01

    采用硒硫二因素二次饱和D-最优设计进行烟草盆栽试验。结果表明,烟草各部位硒含量随施硒量的增加而增多,烟草生长前期根部硒含量高于地上部,成熟期烟叶和根部硒含量高于茎部,尤以上二棚烟叶硒含量最高。施硫对烟草硒的吸收和积累量的影响随烟草生育期、生长器官和硒硫浓度不同而异。前期烟草全株及地上部的硒积累量在低硫(<75 μg*g-1)条件下随施硫增加而增多,硒硫表现为协同作用;在高硫(>75 μg*g-1)条件下,烟草硒积累量则随施硫的增加而减少,表现为拮抗作用。不施硒时施硫会降低成熟烟叶硒含量,施硒时成熟烟叶硒含量则随施硫量的增加而增多,前者表现为硒硫拮抗作用,而后者则呈硒硫协同作用。%The Se and S pot experiment with flue-cured tobacco (Nicotina tabacum L.)was carried out using optimum design for two-factorial quadratic saturating D.The results showed that SeO2-3 application raised Se contents in different parts of tobacco.The contents of Se in tobacco roots at early growth were higher than those of Se in stalks and leaves,while the contents of Se in roots and leaves of tobacco at mature were higher than those of Se in stalks.The highest Se concentration appeared in upper leaves of tobacco.The infl uence of SO2-4 on the uptake and accumulation of SeO2-3 by to bacco changed with the growth period,organs of tobacco and the concentrations of added SeO2-3 and SO2-4.The Se accumulated amount of the stalks,leaves and total plant of tobacco at early growth stage raised with increasing of the SO2-4 application at lower S levels(<75 μg*g-1),sho wing the synergism interaction between Se and S;and while at higher S levels( >75 μg*g-1)the Se accumulated amount decreased with increasing of SO2-4,showing the ant agonistic interaction between Se and S.In the absence of added Se,SO2- 4 application reduced Se contents

  9. Functional characterization of a potassium transporter geneNrHAK1 in Nicotiana rustica

    Institute of Scientific and Technical Information of China (English)

    Zhao-kui GUO; Qian YANG; Xiu-qing WAN; Pei-qiang YAN

    2008-01-01

    The purpose of this study is to investigate the function of a novel potassium transporter gene(NrHAK1) isolated from Nicotiana rustica roots using yeast complement and real-time PCR technique. The complementary DNA (cDNA) of NrHAK1, 2 488 bp long, contains an open reading frame (ORF) of 2334 bp encoding a protein of 777 amino acids (87.6 kDa) with 12 predicted transmembrane domains. The NrHAK1 protein shows a high sequence similarity to those of high-affinity potassium transporters in Mesembryanthemum, Phytolacca acinosa, Arabidopsis thaliana, and so on. We found that the NrHAK1 gene could complement the yeast-mutant defect in K+ uptake. Among several tissues surveyed, the expression level of NrHAK1 was most abundant in the root tip and was up-regulated when exposed to potassium starvation. Moreover, the transcript accumulation was significantly reduced by adding 5 mmol/L NH4 to the solution. These results suggest that NrHAK1 plays an important role in potassium absorption in N. Rustica.

  10. Optimization of engineered production of the glucoraphanin precursor dihomo-methionine in Nicotiana benthamiana

    Directory of Open Access Journals (Sweden)

    Christoph eCrocoll

    2016-02-01

    Full Text Available Glucosinolates are natural products characteristic of the Brassicales order which include vegetables such as cabbages and the model plant Arabidopsis thaliana. Glucoraphanin is the major glucosinolate in broccoli and associated with the health-promoting effects of broccoli consumption. Towards our goal of creating a rich source of glucoraphanin for dietary supplements, we have previously reported the feasibility of engineering glucoraphanin in Nicotiana benthaminana through transient expression of glucoraphanin biosynthetic genes from Arabidopsis thaliana (Mikkelsen et al., 2010. As side-products, we obtained 5-8 fold higher levels of chain-elongated leucine-derived glucosinolates, not found in the native plant. Here, we investigated two different strategies to improve engineering of the methionine chain elongation part of the glucoraphanin pathway in N. benthamiana: 1 co-expression of the large subunit (LSU1 of the heterodimeric isopropylmalate isomerase, and 2 co-expression of BAT5 transporter for efficient transfer of intermediates across the chloroplast membrane. We succeeded in raising dihomo-methionine (DHM levels to a maximum of 432 nmol*g-1 fresh weight which is equivalent to a 9-fold increase compared to the highest production of this intermediate previously reported (Mikkelsen et al., 2010. The increased DHM production without increasing leucine-derived side-product levels provides new metabolic engineering strategies for improved glucoraphanin production in a heterologous host.

  11. Citrus leaf blotch virus invades meristematic regions in Nicotiana benthamiana and citrus.

    Science.gov (United States)

    Agüero, Jesús; Vives, María Carmen; Velázquez, Karelia; Ruiz-Ruiz, Susana; Juárez, Jose; Navarro, Luis; Moreno, Pedro; Guerri, José

    2013-08-01

    To invade systemically host plants, viruses need to replicate in the infected cells, spread to neighbouring cells through plasmodesmata and move to distal parts of the plant via sieve tubes to start new infection foci. To monitor the infection of Nicotiana benthamiana plants by Citrus leaf blotch virus (CLBV), leaves were agroinoculated with an infectious cDNA clone of the CLBV genomic RNA expressing green fluorescent protein (GFP) under the transcriptional control of a duplicate promoter of the coat protein subgenomic RNA. Fluorescent spots first appeared in agroinfiltrated leaves 11-12 days after infiltration, indicating CLBV replication. Then, after entering the phloem vascular system, CLBV was unloaded in the upper parts of the plant and invaded all tissues, including flower organs and meristems. GFP fluorescence was not visible in citrus plants infected with CLBV-GFP. Therefore, to detect CLBV in meristematic regions, Mexican lime (Citrus aurantifolia) plants were graft inoculated with CLBV, with Citrus tristeza virus (CTV), a virus readily eliminated by shoot-tip grafting in vitro, or with both simultaneously. Although CLBV was detected by hybridization and real-time reverse transcription-polymerase chain reaction (RT-PCR) in 0.2-mm shoot tips in all CLBV-inoculated plants, CTV was not detected. These results explain the difficulty in eliminating CLBV by shoot-tip grafting in vitro. © 2013 BSPP AND JOHN WILEY & SONS LTD.

  12. Cymbidium mosaic virus coat protein gene in antisense confers resistance to transgenic Nicotiana occidentalis.

    Science.gov (United States)

    Lim, S H; Ko, M K; Lee, S J; La, Y J; Kim, B D

    1999-12-31

    The nucleotide sequence of the 3'-terminal region of the Korean isolate of cymbidium mosaic virus (CyMV-Ca) from a naturally infected cattleya was determined. The sequence contains an open reading frame (ORF) coding for the viral coat protein (CP) at the 3'-end and three other ORFs (triple gene block or movement protein) of CyMV. The CP gene encodes a polypeptide chain of 220 amino acids with a molecular mass of 23,760 Da. The deduced CP sequence showed a strong homology with those of two CyMVs reported. A construct of the CyMV-Ca CP gene in the antisense orientation in the plant expression vector pMBP1 was transferred via Agrobacterium tumefaciens-mediated transformation into Nicotiana occidentalis which is a propagation host of CyMV. The T1 progeny of the transgenic plants were inoculated with CyMV and found to be highly resistant to CyMV infection.

  13. Identification of photoperiod-regulated gene in soybean and functional analysis in Nicotiana benthamiana

    Indian Academy of Sciences (India)

    Sha Ai-Hua; Chen Yin-Hua; Shan Zhi-Hui; Zhang Xiao-Juan; Wu Xue-Jun; Qiu De-Zheng; Zhou Xin-An

    2014-04-01

    Soybean (Glycine max) is a short-day crop and the photoperiod is a crucial factor regulating its flowering time. To investigate the molecular mechanism controlling the flowering time by photoperiod in soybean, cDNA-amplified fragment length polymorphism (cDNA-AFLP) was used to identify photoperiod-regulated genes in leaves of soybean growing under short-day length, neutral photoperiod and long-day length. A total of 36 transcript-derived fragments (TDFs) were identified to be regulated by photoperiod. Among them, 26 TDFs were homologues of genes with known function. These genes are involved in secondary metabolism, cellular metabolism, cell wall components metabolism, ion transport and hormone signalling. Silencing of the homologue genes in Nicotiana benthamiana for 14 TDFs was conducted by virus-induced gene silencing. The flowering time was delayed by silencing of the genes encoding rhodanese and 40S ribosomal protein S4 (RPS4). The results indicated that rhodanese and RPS4 probably play important roles in regulating flowering time.

  14. Detection of Nicotiana DNA in Tobacco Products Using a Novel Multiplex Real-Time PCR Assay.

    Science.gov (United States)

    Korchinski, Katie L; Land, Adrian D; Craft, David L; Brzezinski, Jennifer L

    2016-07-01

    Establishing that a product contains tobacco is a requirement for the U.S. Food and Drug Administration's regulation and/or prosecution of tobacco products. Therefore, a multiplex real-time PCR method was designed to determine if Nicotiana (tobacco) DNA is present in tobacco products. The PCR method simultaneously amplifies a 73 bp fragment of the cytochrome P450 monoxygenase CYP82E4 gene and 66 bp fragment in the nia-1 gene for nitrate reductase, which are detected using dual-labeled TaqMan probes. The assay is capable of detecting approximately 7.8 pg purified tobacco DNA, with a similar sensitivity for either gene target while incorporating an internal positive control (IPC). DNA was extracted from prepared tobacco products-including chewing tobacco, pipe tobacco, and snuff-or from the cut fill (no wrapper) of cigarettes and cigars. Of the 13 products analyzed, 12 were positive for both tobacco-specific markers and the IPC. DNA was also extracted from the fill of five varieties of herbal cigarettes, which were negative for both tobacco-specific gene targets and positive for the IPC. Our method expands on current assays by introducing a multiplex reaction, targeting two sequences in two different genes of interest, incorporating an IPC into the reaction, and lowering the LOD and LOQ while increasing the efficiency of the PCR.

  15. Techno-economic analysis of horseradish peroxidase production using a transient expression system in Nicotiana benthamiana.

    Science.gov (United States)

    Walwyn, David Richard; Huddy, Suzanne M; Rybicki, Edward P

    2015-01-01

    Despite the advantages of plant-based transient expression systems relative to microbial or mammalian cell systems, the commercial production of recombinant proteins using plants has not yet been achieved to any significant extent. One of the challenges has been the lack of published data on the costs of manufacture for products other than biopharmaceuticals. In this study, we report on the techno-economic analysis of the production of a standard commercial enzyme, namely, horseradish peroxidase (HRP), using a transient expression system in Nicotiana benthamiana. Based on the proven plant yield of 240 mg HRP/kg biomass, a biomass productivity of 15-kg biomass/m(2)/year and a process yield of 54 % (mg HRP product/mg HRP in biomass), it is apparent that HRP can be manufactured economically via transient expression in plants in a large-scale facility (>5 kg HRP/year). At this level, the process is competitive versus the existing technology (extraction of the enzyme from horseradish), and the product is of comparable or improved activity, containing only the preferred isoenzyme C. Production scale, protein yield and biomass productivity are found to be the most important determinants of overall viability.

  16. Salivary proteins of spider mites suppress defenses in Nicotiana benthamiana and promote mite reproduction.

    Science.gov (United States)

    Villarroel, Carlos A; Jonckheere, Wim; Alba, Juan M; Glas, Joris J; Dermauw, Wannes; Haring, Michel A; Van Leeuwen, Thomas; Schuurink, Robert C; Kant, Merijn R

    2016-04-01

    Spider mites (Tetranychidae sp.) are widely occurring arthropod pests on cultivated plants. Feeding by the two-spotted spider mite T. urticae, a generalist herbivore, induces a defense response in plants that mainly depends on the phytohormones jasmonic acid and salicylic acid (SA). On tomato (Solanum lycopersicum), however, certain genotypes of T. urticae and the specialist species T. evansi were found to suppress these defenses. This phenomenon occurs downstream of phytohormone accumulation via an unknown mechanism. We investigated if spider mites possess effector-like proteins in their saliva that can account for this defense suppression. First we performed an in silico prediction of the T. urticae and the T. evansi secretomes, and subsequently generated a short list of candidate effectors based on additional selection criteria such as life stage-specific expression and salivary gland expression via whole mount in situ hybridization. We picked the top five most promising protein families and then expressed representatives in Nicotiana benthamiana using Agrobacterium tumefaciens transient expression assays to assess their effect on plant defenses. Four proteins from two families suppressed defenses downstream of the phytohormone SA. Furthermore, T. urticae performance on N. benthamiana improved in response to transient expression of three of these proteins and this improvement was similar to that of mites feeding on the tomato SA accumulation mutant nahG. Our results suggest that both generalist and specialist plant-eating mite species are sensitive to SA defenses but secrete proteins via their saliva to reduce the negative effects of these defenses.

  17. Reduced Susceptibility to Xanthomonas citri in Transgenic Citrus Expressing the FLS2 Receptor From Nicotiana benthamiana.

    Science.gov (United States)

    Hao, Guixia; Pitino, Marco; Duan, Yongping; Stover, Ed

    2016-02-01

    Overexpression of plant pattern-recognition receptors by genetic engineering provides a novel approach to enhance plant immunity and broad-spectrum disease resistance. Citrus canker disease associated with Xanthomonas citri is one of the most important diseases damaging citrus production worldwide. In this study, we cloned the FLS2 gene from Nicotiana benthamiana cDNA and inserted it into the binary vector pBinPlus/ARS to transform Hamlin sweet orange and Carrizo citrange. Transgene presence was confirmed by polymerase chain reaction (PCR) and gene expression of NbFLS2 was compared by reverse transcription quantitative PCR. Reactive oxygen species (ROS) production in response to flg22Xcc was detected in transgenic Hamlin but not in nontransformed controls. Low or no ROS production was detected from nontransformed Hamlin seedlings challenged with flg22Xcc. Transgenic plants highly expressing NbFLS2 were selected and were evaluated for resistance to canker incited by X. citri 3213. Our results showed that the integration and expression of the NbFLS2 gene in citrus can increase canker resistance and defense-associated gene expression when challenged with X. citri. These results suggest that canker-susceptible Citrus genotypes lack strong basal defense induced by X. citri flagellin and the resistance of these genotypes can be enhanced by transgenic expression of the flagellin receptor from a resistant species.

  18. The Subcellular Localization and Functional Analysis of Fibrillarin2, a Nucleolar Protein in Nicotiana benthamiana

    Directory of Open Access Journals (Sweden)

    Luping Zheng

    2016-01-01

    Full Text Available Nucleolar proteins play important roles in plant cytology, growth, and development. Fibrillarin2 is a nucleolar protein of Nicotiana benthamiana (N. benthamiana. Its cDNA was amplified by RT-PCR and inserted into expression vector pEarley101 labeled with yellow fluorescent protein (YFP. The fusion protein was localized in the nucleolus and Cajal body of leaf epidermal cells of N. benthamiana. The N. benthamiana fibrillarin2 (NbFib2 protein has three functional domains (i.e., glycine and arginine rich domain, RNA-binding domain, and α-helical domain and a nuclear localization signal (NLS in C-terminal. The protein 3D structure analysis predicted that NbFib2 is an α/β protein. In addition, the virus induced gene silencing (VIGS approach was used to determine the function of NbFib2. Our results showed that symptoms including growth retardation, organ deformation, chlorosis, and necrosis appeared in NbFib2-silenced N. benthamiana.

  19. The effect of intermittent dosing of Nicotiana glauca on teratogenesis in goats.

    Science.gov (United States)

    Welch, K D; Panter, K E; Lee, S T; Gardner, D R

    2015-01-01

    Sustained inhibition of fetal movement in livestock species, induced by several poisonous plants, can result in numerous skeletal-contracture malformations. Lupines are responsible for a condition in cattle referred to as "crooked calf syndrome" that occurs when pregnant cattle graze teratogenic lupines. Similar malformations are also seen in animals poisoned by Conium maculatum (coniine) and Nicotiana glauca (anabasine). A proposed management strategy to limit these types of birth defects includes utilizing an intermittent grazing schedule to allow short durations of grazing lupine-infested areas interrupted by movement to a lupine-free pasture. The objective of this study was to use a goat model to determine if an intermittent schedule of five continuous days on treatment followed by two days off treatment would be sufficient to decrease, or prevent, the incidence of anabasine-induced malformations. The data from this study suggest that, for N. glauca in goats, the intermittent grazing program of five days exposure with two days of non-exposure is insufficient to prevent significant skeletal malformations from occurring. However, this study did demonstrate an inverse relationship between the amount of serum anabasine in the dam and the extent of fetal movement.

  20. Arabinogalactan-Proteins of the Female Sexual Tissue of Nicotiana alata

    Science.gov (United States)

    Gell, Andrew C.; Bacic, Antony; Clarke, Adrienne E.

    1986-01-01

    Arabinogalactan-proteins (AGPs), isolated from the pistils of Nicotiana alata, an ornamental tobacco, are developmentally regulated. Both the total amount and concentration of AGP in the stigma increase during flower development, reaching 10 micrograms AGP/stigma at maturity. In contrast, AGP concentration in the style remains constant throughout the maturation period reaching 12 micrograms AGP/style at maturity. The classes of AGP present in the stigma and style during flower development, separated according to their charge by crossed-electrophoresis, are different and change during development. Pollination of flowers of N. alata with compatible or incompatible pollen results in a significant and reproducible increase in the amount of AGPs in the stigma, but not the style, compared with control unpollinated pistils. Pollination with ethanol vapor inactivated pollen also results in an increase in the amount of AGP in the stigma, but this is less than half that observed following pollination with viable pollen. There are no significant differences in the classes of AGP, based on crossed-electrophoresis, present in the pistil following pollination. Images Fig. 1 Fig. 2 PMID:16665162

  1. Potato virus X movement in Nicotiana benthamiana: new details revealed by chimeric coat protein variants.

    Science.gov (United States)

    Betti, Camilla; Lico, Chiara; Maffi, Dario; D'Angeli, Simone; Altamura, Maria Maddalena; Benvenuto, Eugenio; Faoro, Franco; Baschieri, Selene

    2012-02-01

    Potato virus X coat protein is necessary for both cell-to-cell and phloem transfer, but it has not been clarified definitively whether it is needed in both movement phases solely as a component of the assembled particles or also of differently structured ribonucleoprotein complexes. To clarify this issue, we studied the infection progression of a mutant carrying an N-terminal deletion of the coat protein, which was used to construct chimeric virus particles displaying peptides selectively affecting phloem transfer or cell-to-cell movement. Nicotiana benthamiana plants inoculated with expression vectors encoding the wild-type, mutant and chimeric viral genomes were examined by microscopy techniques. These experiments showed that coat protein-peptide fusions promoting cell-to-cell transfer only were not competent for virion assembly, whereas long-distance movement was possible only for coat proteins compatible with virus particle formation. Moreover, the ability of the assembled PVX to enter and persist into developing xylem elements was revealed here for the first time.

  2. Effect of salicylic acid on tobacco (Nicotiana rustica plant under drought conditions

    Directory of Open Access Journals (Sweden)

    Ghader Habibi

    2015-09-01

    Full Text Available Drought stress impact photosynthesis and stomatal conductance, and may reduce the overall production capacity of plants. Since exogenous application of salicylic acid (SA can partially alleviatebe increased the negative effects tolerance of drought stress by improve the metabolism pathways and increase the net photosynthesison plant photosynthesis and metabolism, the main objective of this study was to clarify the roles of SA in enhancing 28 days tobacco (Nicotiana rustica Basmas tolerance to drought stress (50% FC. The results indicated that foliar application of SA (0.5 mM influenced negatively net CO2 assimilation rate and stomatal conductance and led to reduction of shoot and root dry masses. In contrast, the stress did not reduce significantly the maximal quantum yield of photosystem II (PSII. This that can be explained by enhancement of efficiency for dissipation of excess photon energy in the PSII antenna, determined as non-photochemical quenching, and consequently further protection of PSII from photodamage. Thus, under more drought stress, the reduction of photosynthesis of tobacco plants was due mainly to reduction of stomatal conductance. Under water-deficient conditions, plants showed an increase in chlorophyll a and amino acids concentrations in the leaves when treated with SA while this change for net photosynthesis was negligible. Our results indicated showed that the foliar application of SA had no ameliorative effect on tobacco growth under drought stress, because its effect on elevation of transpiration rate did not increase net photosynthesis under drought condition.

  3. Evaluation of hepatoprotective effect of Pistacia lentiscus, Phillyrea latifolia and Nicotiana glauca.

    Science.gov (United States)

    Janakat, Sana; Al-Merie, Hela

    2002-11-01

    The hepatoprotective effect of the boiled and non-boiled aqueous extracts of Pistacia lentiscus, Phillyrea latifolia, and Nicotiana glauca, that are alleged to be effective in the treatment of jaundice in Jordanian folk medicine, was evaluated in vivo using carbon tetrachloride (CCl(4)) intoxicated rats as an experimental model. Plant extracts were administrated orally at a dose of 4 ml/kg body weight, containing various amounts of solid matter. Only total serum bilirubin level was reduced by treatment with non-boiled aqueous extract of N. glauca leaves, while the boiled and non-boiled aqueous extracts of the N. glauca flowers were non effective. Bilirubin level and the activity of alkaline phosphatase (ALP) were both reduced upon treatment with boiled aqueous extract of P. latifolia without reducing the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Aqueous extract of P. lentiscus (both boiled and non-boiled) showed marked antihepatotoxic activity against CCl(4) by reducing the activity of the three enzymes and the level of bilirubin. The effect of the non-boiled aqueous extract was more pronounced than that of the boiled extract.

  4. The receptor-like kinase SERK3/BAK1 is required for basal resistance against the late blight pathogen phytophthora infestans in Nicotiana benthamiana.

    Directory of Open Access Journals (Sweden)

    Angela Chaparro-Garcia

    Full Text Available BACKGROUND: The filamentous oomycete plant pathogen Phytophthora infestans causes late blight, an economically important disease, on members of the nightshade family (Solanaceae, such as the crop plants potato and tomato. The related plant Nicotiana benthamiana is a model system to study plant-pathogen interactions, and the susceptibility of N. benthamiana to Phytophthora species varies from susceptible to resistant. Little is known about the extent to which plant basal immunity, mediated by membrane receptors that recognise conserved pathogen-associated molecular patterns (PAMPs, contributes to P. infestans resistance. PRINCIPAL FINDINGS: We found that different species of Phytophthora have varying degrees of virulence on N. benthamiana ranging from avirulence (incompatible interaction to moderate virulence through to full aggressiveness. The leucine-rich repeat receptor-like kinase (LRR-RLK BAK1/SERK3 is a major modulator of PAMP-triggered immunity (PTI in Arabidopsis thaliana and N. benthamiana. We cloned two NbSerk3 homologs, NbSerk3A and NbSerk3B, from N. benthamiana based on sequence similarity to the A. thaliana gene. N. benthamiana plants silenced for NbSerk3 showed markedly enhanced susceptibility to P. infestans infection but were not altered in resistance to Phytophthora mirabilis, a sister species of P. infestans that specializes on a different host plant. Furthermore, silencing of NbSerk3 reduced the cell death response triggered by the INF1, a secreted P. infestans protein with features of PAMPs. CONCLUSIONS/SIGNIFICANCE: We demonstrated that N. benthamiana NbSERK3 significantly contributes to resistance to P. infestans and regulates the immune responses triggered by the P. infestans PAMP protein INF1. In the future, the identification of novel surface receptors that associate with NbSERK3A and/or NbSERK3B should lead to the identification of new receptors that mediate recognition of oomycete PAMPs, such as INF1.

  5. Reação de híbridos somáticos de citros à infecção por Phytophthora nicotianae Reaction of citrus somatic hybrids to the infection by Phytophthora nicotianae

    Directory of Open Access Journals (Sweden)

    Meire Menezes Bassan

    2010-06-01

    Full Text Available Este trabalho avaliou a resistência à infecção de tronco e de raízes por Phytophthora nicotianae em híbridos somáticos de citros com potencial para serem utilizados como porta-enxertos. Os híbridos somáticos avaliados foram laranja 'Hamlin' (Citrus sinensis + toranja 'Indian Red' (Citrus grandis (plantas 1 e 2 e laranja 'Hamlin' (C. sinensis + toranja 'Singapura' (C. grandis. Plantas de limão 'Cravo' (Citrus limonia, laranja 'Caipira' (C. sinensis, laranja-azeda (C. aurantium e Poncirus trifoliata 'Davis A' (Poncirus trifoliata foram utilizadas como plantas-controle devido à reação conhecida à infecção pelo patógeno. Avaliações realizadas entre 30 e 60 dias após as inoculações com o patógeno incluíram o comprimento das lesões no tronco e a massa seca do sistema radicular nas plantas avaliadas. O híbrido somático laranja 'Hamlin' + toranja 'Indian Red' (planta 1 mostrou-se tolerante a P. nicotianae, indicando potencial para continuidade nas suas avaliações como porta-enxerto para citros.This study aimed to evaluate the resistance to trunk and root infection by Phytophthora nicotianae in citrus somatic hybrids with potential to be utilized as rootstocks. The somatic hybrids evaluated were 'Hamlin' sweet orange (Citrus sinensis + 'Indian Red' pummelo (Citrus grandis (plants 1 and 2, and 'Hamlin' sweet orange (C. sinensis + 'Singapura' pummelo (C. grandis. Plants of 'Rangpur' lime (Citrus limonia, 'Caipira' sweet orange (C. sinensis, sour orange (C. aurantium, and Poncirus trifoliata 'Davis A' (Poncirus trifoliata were used as control due to their known reaction to the pathogen. Evaluations performed between 30 and 60 days after pathogen inoculation included the length of trunk lesions, and root dry mass of the root system of evaluated plants. The somatic hybrid 'Hamlin' sweet orange + 'Indian Red' pummelo (plant 1 was tolerant to P. nicotianae, indicating potential to be further evaluated as a rootstock.

  6. Dicty_cDB: AFI857 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available he head. 52 7e-12 3 CV017773 |CV017773.1 tbt_009554 Normalized Nicotiana tabacum ...cDNA library Nicotiana tabacum cDNA clone tbt_009554 5', mRNA sequence. 66 1e-11 2 BQ489771 |BQ489771.1 14-E

  7. Dicty_cDB: AFJ139 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available expressed in the head. 52 7e-12 3 CV017773 |CV017773.1 tbt_009554 Normalized Nicotiana tabacum cDNA library ...Nicotiana tabacum cDNA clone tbt_009554 5', mRNA sequence. 66 1e-11 2 BQ489771 |B

  8. Dicty_cDB: AFI709 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available ne, complete cds. 38 9e-12 7 CV017773 |CV017773.1 tbt_009554 Normalized Nicotiana tabacum cDNA library Nicot...iana tabacum cDNA clone tbt_009554 5', mRNA sequence. 66 9e-12 2 BQ489771 |BQ489771.1 14-E9425-006-009-K03-T

  9. Dicty_cDB: CHD465 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available K-3-9, mRNA sequence. 50 6e-12 3 CV017773 |CV017773.1 tbt_009554 Normalized Nicot...iana tabacum cDNA library Nicotiana tabacum cDNA clone tbt_009554 5', mRNA sequence. 66 8e-12 2 AL408772 |AL

  10. Dicty_cDB: Contig-U10424-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available . 46 1.7 1 ( AM840768 ) Nicotiana tabacum EST, clone nt002017057. 46 1.7 1 ( CV021345 ) tbt..._000690 Normalized Nicotiana tabacum cDNA libr... 46 1.7 1 ( CV017227 ) tbt_000618 Normalized Nico

  11. Dicty_cDB: SLK619 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available nordered pieces. 44 0.23 3 BP128371 |BP128371.1 Nicotiana tabacum cDNA, clone: BY141, primer: M13 forward, s...equence 1. 34 1.1 2 BP128372 |BP128372.1 Nicotiana tabacum cDNA, clone: BY141, primer: M13 forward, sequence

  12. Multiplexed, targeted gene editing in Nicotiana benthamiana for glyco-engineering and monoclonal antibody production.

    Science.gov (United States)

    Li, Jin; Stoddard, Thomas J; Demorest, Zachary L; Lavoie, Pierre-Olivier; Luo, Song; Clasen, Benjamin M; Cedrone, Frederic; Ray, Erin E; Coffman, Andrew P; Daulhac, Aurelie; Yabandith, Ann; Retterath, Adam J; Mathis, Luc; Voytas, Daniel F; D'Aoust, Marc-André; Zhang, Feng

    2016-02-01

    Biopharmaceutical glycoproteins produced in plants carry N-glycans with plant-specific residues core α(1,3)-fucose and β(1,2)-xylose, which can significantly impact the activity, stability and immunogenicity of biopharmaceuticals. In this study, we have employed sequence-specific transcription activator-like effector nucleases (TALENs) to knock out two α(1,3)-fucosyltransferase (FucT) and the two β(1,2)-xylosyltransferase (XylT) genes within Nicotiana benthamiana to generate plants with improved capacity to produce glycoproteins devoid of plant-specific residues. Among plants regenerated from N. benthamiana protoplasts transformed with TALENs targeting either the FucT or XylT genes, 50% (80 of 160) and 73% (94 of 129) had mutations in at least one FucT or XylT allele, respectively. Among plants regenerated from protoplasts transformed with both TALEN pairs, 17% (18 of 105) had mutations in all four gene targets, and 3% (3 of 105) plants had mutations in all eight alleles comprising both gene families; these mutations were transmitted to the next generation. Endogenous proteins expressed in the complete knockout line had N-glycans that lacked β(1,2)-xylose and had a significant reduction in core α(1,3)-fucose levels (40% of wild type). A similar phenotype was observed in the N-glycans of a recombinant rituximab antibody transiently expressed in the homozygous mutant plants. More importantly, the most desirable glycoform, one lacking both core α(1,3)-fucose and β(1,2)-xylose residues, increased in the antibody from 2% when produced in the wild-type line to 55% in the mutant line. These results demonstrate the power of TALENs for multiplexed gene editing. Furthermore, the mutant N. benthamiana lines provide a valuable platform for producing highly potent biopharmaceutical products.

  13. Androgenesis Induced in Nicotiana alata and the Effect of Gamma Irradiation

    Directory of Open Access Journals (Sweden)

    Ayman EL-FIKI

    2015-03-01

    Full Text Available Nicotiana alata anthers cultured on different modified media based on MS, MT and N were used to obtain haploid plants through direct and indirect ways. The haploid plants resulting on MS medium ranged from 52% - 80%, on MT medium ranged from 32% - 52% and on N medium ranged from 28% - 44%. Accordingly, the best medium used for haploid induction was MS supplemented with 0.2 mgl-l NAA + 0.5 mgl-l KIN. On the other hand, MS medium supplemented with 0.4 mgl-l NAA + 0.5 mgl-l KIN or 1.0 mgl-l BAP + 0.5 mgl-l NAA were the best mediums for callus induction and plant regeneration, respectively. Morphologically, the leaf size, stem highest and diameter, flower size and diameter, anther length and number were about 67% of the diploid plants growth. Irradiated anthers with doses of 0, 2.5, 5, 7.5, 10, 15, 20 and 25 Gy caused reducing the number of haploid plants with increasing gamma radiation dose. For the haploid plants irradiated with same doses, the mortality percentage of bud survival was increasing with increasing gamma radiation dose. The irradiated callus with doses of 0, 5, 10, 15 and 20 Gy was affected negatively on growth rate and morphology. Proline content in irradiated plantlets increased with increasing gamma radiation dose. As well, total soluble protein content was increased with gamma irradiation up to 10 Gy. However, the higher doses caused a severe decrease of total soluble proteins. The production of proline and total soluble proteins in haploid plants were 48.6% and 69.5%, respectively comparing with diploid plants.

  14. HSPRO controls early Nicotiana attenuata seedling growth during interaction with the fungus Piriformospora indica.

    Science.gov (United States)

    Schuck, Stefan; Camehl, Iris; Gilardoni, Paola A; Oelmueller, Ralf; Baldwin, Ian T; Bonaventure, Gustavo

    2012-10-01

    In a previous study aimed at identifying regulators of Nicotiana attenuata responses against chewing insects, a 26-nucleotide tag matching the HSPRO (ORTHOLOG OF SUGAR BEET Hs1(pro)(-)(1)) gene was found to be strongly induced after simulated herbivory (Gilardoni et al., 2010). Here we characterized the function of HSPRO during biotic interactions in transgenic N. attenuata plants silenced in its expression (ir-hspro). In wild-type plants, HSPRO expression was not only induced during simulated herbivory but also when leaves were inoculated with Pseudomonas syringae pv tomato DC3000 and roots with the growth-promoting fungus Piriformospora indica. Reduced HSPRO expression did not affect the regulation of direct defenses against Manduca sexta herbivory or P. syringae pv tomato DC3000 infection rates. However, reduced HSPRO expression positively influenced early seedling growth during interaction with P. indica; fungus-colonized ir-hspro seedlings increased their fresh biomass by 30% compared with the wild type. Grafting experiments demonstrated that reduced HSPRO expression in roots was sufficient to induce differential growth promotion in both roots and shoots. This effect was accompanied by changes in the expression of 417 genes in colonized roots, most of which were metabolic genes. The lack of major differences in the metabolic profiles of ir-hspro and wild-type colonized roots (as analyzed by liquid chromatography time-of-flight mass spectrometry) suggested that accelerated metabolic rates were involved. We conclude that HSPRO participates in a whole-plant change in growth physiology when seedlings interact with P. indica.

  15. Expression of Aspergillus nidulans phy Gene in Nicotiana benthamiana Produces Active Phytase with Broad Specificities

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    Tae-Kyun Oh

    2014-09-01

    Full Text Available A full-length phytase gene (phy of Aspergillus nidulans was amplified from the cDNA library by polymerase chain reaction (PCR, and it was introduced into a bacterial expression vector, pET-28a. The recombinant protein (rPhy-E, 56 kDa was overexpressed in the insoluble fraction of Escherichia coli culture, purified by Ni-NTA resin under denaturing conditions and injected into rats as an immunogen. To express A. nidulans phytase in a plant, the full-length of phy was cloned into a plant expression binary vector, pPZP212. The resultant construct was tested for its transient expression by Agrobacterium-infiltration into Nicotiana benthamiana leaves. Compared with a control, the agro-infiltrated leaf tissues showed the presence of phy mRNA and its high expression level in N. benthamiana. The recombinant phytase (rPhy-P, 62 kDa was strongly reacted with the polyclonal antibody against the nonglycosylated rPhy-E. The rPhy-P showed glycosylation, two pH optima (pH 4.5 and pH 5.5, an optimum temperature at 45~55 °C, thermostability and broad substrate specificities. After deglycosylation by peptide-N-glycosidase F (PNGase-F, the rPhy-P significantly lost the phytase activity and retained 1/9 of the original activity after 10 min of incubation at 45 °C. Therefore, the deglycosylation caused a significant reduction in enzyme thermostability. In animal experiments, oral administration of the rPhy-P at 1500 U/kg body weight/day for seven days caused a significant reduction of phosphorus excretion by 16% in rat feces. Besides, the rPhy-P did not result in any toxicological changes and clinical signs.

  16. Expression of Aspergillus nidulans phy gene in Nicotiana benthamiana produces active phytase with broad specificities.

    Science.gov (United States)

    Oh, Tae-Kyun; Oh, Sung; Kim, Seongdae; Park, Jae Sung; Vinod, Nagarajan; Jang, Kyung Min; Kim, Sei Chang; Choi, Chang Won; Ko, Suk-Min; Jeong, Dong Kee; Udayakumar, Rajangam

    2014-09-03

    A full-length phytase gene (phy) of Aspergillus nidulans was amplified from the cDNA library by polymerase chain reaction (PCR), and it was introduced into a bacterial expression vector, pET-28a. The recombinant protein (rPhy-E, 56 kDa) was overexpressed in the insoluble fraction of Escherichia coli culture, purified by Ni-NTA resin under denaturing conditions and injected into rats as an immunogen. To express A. nidulans phytase in a plant, the full-length of phy was cloned into a plant expression binary vector, pPZP212. The resultant construct was tested for its transient expression by Agrobacterium-infiltration into Nicotiana benthamiana leaves. Compared with a control, the agro-infiltrated leaf tissues showed the presence of phy mRNA and its high expression level in N. benthamiana. The recombinant phytase (rPhy-P, 62 kDa) was strongly reacted with the polyclonal antibody against the nonglycosylated rPhy-E. The rPhy-P showed glycosylation, two pH optima (pH 4.5 and pH 5.5), an optimum temperature at 45~55 °C, thermostability and broad substrate specificities. After deglycosylation by peptide-N-glycosidase F (PNGase-F), the rPhy-P significantly lost the phytase activity and retained 1/9 of the original activity after 10 min of incubation at 45 °C. Therefore, the deglycosylation caused a significant reduction in enzyme thermostability. In animal experiments, oral administration of the rPhy-P at 1500 U/kg body weight/day for seven days caused a significant reduction of phosphorus excretion by 16% in rat feces. Besides, the rPhy-P did not result in any toxicological changes and clinical signs.

  17. Production of an active anti-CD20-hIL-2 immunocytokine in Nicotiana benthamiana.

    Science.gov (United States)

    Marusic, Carla; Novelli, Flavia; Salzano, Anna M; Scaloni, Andrea; Benvenuto, Eugenio; Pioli, Claudio; Donini, Marcello

    2016-01-01

    Anti-CD20 murine or chimeric antibodies (Abs) have been used to treat non-Hodgkin lymphomas (NHLs) and other diseases characterized by overactive or dysfunctional B cells. Anti-CD20 Abs demonstrated to be effective in inducing regression of B-cell lymphomas, although in many cases patients relapse following treatment. A promising approach to improve the outcome of mAb therapy is the use of anti-CD20 antibodies to deliver cytokines to the tumour microenvironment. In particular, IL-2-based immunocytokines have shown enhanced antitumour activity in several preclinical studies. Here, we report on the engineering of an anti-CD20-human interleukin-2 (hIL-2) immunocytokine (2B8-Fc-hIL2) based on the C2B8 mAb (Rituximab) and the resulting ectopic expression in Nicotiana benthamiana. The scFv-Fc-engineered immunocytokine is fully assembled in plants with minor degradation products as assessed by SDS-PAGE and gel filtration. Purification yields using protein-A affinity chromatography were in the range of 15-20 mg/kg of fresh leaf weight (FW). Glycopeptide analysis confirmed the presence of a highly homogeneous plant-type glycosylation. 2B8-Fc-hIL2 and the cognate 2B8-Fc antibody, devoid of hIL-2, were assayed by flow cytometry on Daudi cells revealing a CD20 binding activity comparable to that of Rituximab and were effective in eliciting antibody-dependent cell-mediated cytotoxicity of human PBMC versus Daudi cells, demonstrating their functional integrity. In 2B8-Fc-hIL2, IL-2 accessibility and biological activity were verified by flow cytometry and cell proliferation assay. To our knowledge, this is the first example of a recombinant immunocytokine based on the therapeutic Rituximab antibody scaffold, whose expression in plants may be a valuable tool for NHLs treatment.

  18. Altered apoplastic ascorbate redox state in tobacco plants via ascorbate oxidase overexpression results in delayed dark-induced senescence in detached leaves.

    Science.gov (United States)

    Fotopoulos, Vasileios; Kanellis, Angelos K

    2013-12-01

    Ascorbate oxidase (AO) is an apoplastic enzyme that uses oxygen to catalyse the oxidation of ascorbate (AA) to dehydroascorbate (DHA) via the unstable radical monodehydroascorbate (MDHA). Here, we report that transgenic tobacco plants (Nicotiana tabacum L. cv. Xanthi) with an in vivo lowered apoplastic AA redox state through increased AO expression demonstrate signs of delayed dark-induced senescence compared with wild-type plants, as shown by chlorophyll loss assay. In situ localization of hydrogen peroxide (H2O2) suggests that, although transgenic plants have higher constitutive levels of H2O2 under normal growth conditions, imposed dark-induced senescence results in smaller induction levels of H2O2, an observation which correlates with increased antioxidant enzyme activities and an induction in the expression of AA recycling genes compared with that in wild-type plants. Our current findings, combined with previous studies which showed the contribution of AO in the regulation of AA redox state, suggest that the reduction in AA redox state in the leaf apoplast of these transgenic plants results in an increase in the endogenous levels of H2O2, which provides a form of 'acquired tolerance' to oxidative stress imposed by dark-induced senescence.

  19. Coat protein-mediated resistance against an Indian isolate of the Cucumber mosaic virus subgroup IB in Nicotiana benthamiana

    Indian Academy of Sciences (India)

    A Srivastava; S K Raj

    2008-06-01

    Coat protein (CP)-mediated resistance against an Indian isolate of the Cucumber mosaic virus (CMV) subgroup IB was demonstrated in transgenic lines of Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transformation. Out of the fourteen independently transformed lines developed, two lines were tested for resistance against CMV by challenge inoculations. The transgenic lines exhibiting complete resistance remained symptomless throughout life and showed reduced or no virus accumulation in their systemic leaves after virus challenge. These lines also showed virus resistance against two closely related strains of CMV. This is the first report of CP-mediated transgenic resistance against a CMV subgroup IB member isolated from India.

  20. Virus-induced gene silencing reveals signal transduction components required for the Pvr9-mediated hypersensitive response in Nicotiana benthamiana.

    Science.gov (United States)

    Tran, Phu-Tri; Choi, Hoseong; Choi, Doil; Kim, Kook-Hyung

    2016-08-01

    Resistance to pathogens mediated by plant resistance (R) proteins requires different signaling transduction components and pathways. Our previous studies revealed that a potyvirus resistance gene in pepper, Pvr9, confers a hypersensitive response (HR) to pepper mottle virus in Nicotiana benthamiana. Our results show that the Pvr9-mediated HR against pepper mottle virus infection requires HSP90, SGT1, NDR1, but not EDS1. These results suggest that the Pvr9-mediated HR is possibly related to the SA pathway but not the ET, JA, ROS or NO pathways.

  1. Flower-bud formation in explants of photoperiodic and day-neutral Nicotiana biotypes and its bearing on the regulation of flower formation

    Energy Technology Data Exchange (ETDEWEB)

    Rajeevan, M.S.; Lang, A. (Michigan State Univ., East Lansing (United States))

    1993-05-15

    The capacity to form flower buds in thin-layer explants was studied in Nicotiana of several species, cultivars, and lines of differing in their response to photoperiod. This capacity was found in all biotypes examined and could extend into sepals and corolla. It varied depending on genotype, source tissue and its developmental state, and composition of the culture medium, particularly the levels of glucose, auxin, and cytokinin. It was greatest in the two day-neutral plants examined, Samsun tobacco and Nicotiana rustica, where it extended from the inflorescence region down the vegetative stem, in a basipetally decreasing gradient; it was least in the two qualitative photoperiodic plants studied, the long-day plant Nicotiana silvestris and the short-day plant Maryland Mammoth tobacco, the quantitative long-day plant Nicotiana alata and the quantitative short-day plant Nicotiana otophora line 38-G-81, where it was limited to the pedicels (and, in some cases, the sepals). Regardless of the photoperiodic response of the source plants, the response was the same in explants cultured under long and short days. The capacity to form flow buds in explants is present in all Nicotiana biotypes studied supports the idea that it is regulated by the same mechanism(s), regardless of the plant's photoperiodic character. However, flower formation in the explants is not identical with de novo flower formation in a hitherto vegetative plant: it is rather the expression of a floral state already established in the plant, although it can vary widely in extent and spatial distribution. Culture conditions that permit flower-bud formation in an explant are conditions that maintain the floral state and encourage its expression; conditions under which no flower buds are formed reduce this state and/or prevent its expression. 14 refs., 5 figs., 3 tabs.

  2. Exceptional inheritance of plastids via pollen in Nicotiana sylvestris with no detectable paternal mitochondrial DNA in the progeny.

    Science.gov (United States)

    Thyssen, Gregory; Svab, Zora; Maliga, Pal

    2012-10-01

    Plastids and mitochondria, the DNA-containing cytoplasmic organelles, are maternally inherited in the majority of angiosperm species. Even in plants with strict maternal inheritance, exceptional paternal transmission of plastids has been observed. Our objective was to detect rare leakage of plastids via pollen in Nicotiana sylvestris and to determine if pollen transmission of plastids results in co-transmission of paternal mitochondria. As father plants, we used N. sylvestris plants with transgenic, selectable plastids and wild-type mitochondria. As mother plants, we used N. sylvestris plants with Nicotiana undulata cytoplasm, including the CMS-92 mitochondria that cause cytoplasmic male sterility (CMS) by homeotic transformation of the stamens. We report here exceptional paternal plastid DNA in approximately 0.002% of N. sylvestris seedlings. However, we did not detect paternal mitochondrial DNA in any of the six plastid-transmission lines, suggesting independent transmission of the cytoplasmic organelles via pollen. When we used fertile N. sylvestris as mothers, we obtained eight fertile plastid transmission lines, which did not transmit their plastids via pollen at higher frequencies than their fathers. We discuss the implications for transgene containment and plant evolutionary histories inferred from cytoplasmic phylogenies.

  3. Spatial analysis of the incidence of Phytophthora infestans (Mont. De Bary and Phytophthora nicotianae Breda de Haan on potato

    Directory of Open Access Journals (Sweden)

    Leónides Castellanos González

    2016-03-01

    Full Text Available The objective of this research was to conduct a spatial analysis of the incidence of Phytophthora nicotianae Breda de Haan and Phytophthora infestans (Mont. De Bary, during five seasons of potato in Fields Company Miscellaneous Crops in Horquita, Cienfuegos (2003-2004 to 2007-2008. Information about pathogens, collected by the Plant Protection Station of Yaguaramas, was used in order to do so. An alpha numerical database was made, oriented to a group of areas under center pivot irrigation machines (Kubans and Bayamón and its quadrants. Parallel to this, mapping of center pivot machines and quadrants was generated in MapInfo GIS 8.5. Several automatic geo codifications were made in order to relate the alphanumeric database and the mapping, and thematic maps were generated in the presence or absence of pathogens. The two study agents have presented high spatial variability during the five planting seasons. Both have influenced the same geographical area with similar appearance date. P. infestans has not expressed a defined dispersion pattern and it has spread at random from the primary source of incidence, while Phytophthora nicotianae has become an endemic agent which shows a dispersion pattern towards neighboring areas or areas connected by the road network from the primary sources, which have been associated with poor land leveling and late or intermediate planting seasons.

  4. Pollination ecology of the invasive tree tobacco Nicotiana glauca: comparisons across native and non-native ranges

    Directory of Open Access Journals (Sweden)

    Jeff Ollerton

    2012-10-01

    Full Text Available Interactions with pollinators are thought to play a significant role in determining whether plant species become invasive, and ecologically generalised species are predicted to be more likely to invade than more specialised species. Using published and unpublished data we assessed the floral biology and pollination ecology of the South American native Nicotiana glauca (Solanaceae which has become a significant invasive of semi-arid parts of the world. In regions where specialised bird pollinators are available, for example hummingbirds in California and sunbirds in South Africa and Israel, N. glauca interacts with these local pollinators and sets seed by both out-crossing and selfing. In areas where there are no such birds, such as the Canary Islands and Greece, abundant viable seed is set by selfing, facilitated by the shorter stigma-anther distance compared to plants in native populations. Surprisingly, in these areas without pollinating birds, the considerable nectar resources are only rarely exploited by other flower visitors such as bees or butterflies, either legitimately or by nectar robbing. We conclude that Nicotiana glauca is a successful invasive species outside of its native range, despite its functionally specialised hummingbird pollination system, because it has evolved to become more frequently self pollinating in areas where it is introduced. Its invasion success is not predictable from what is known of its interactions with pollinators in its home range.

  5. A reversed-phase HPLC-UV method developed and validated for simultaneous quantification of six alkaloids from Nicotiana spp.

    Science.gov (United States)

    Moghbel, Nahid; Ryu, BoMi; Steadman, Kathryn J

    2015-08-01

    A reversed-phase HPLC-UV method was developed, optimized, and validated for the separation and quantitation of six target alkaloids from leaves of Nicotiana species (nicotine, nornicotine, anatabine, anabasine, myosmine, and cotinine). A bidentate reversed-phase C18 column was used as stationary phase and an alkaline ammonium formate buffer and acetonitrile as mobile phase. The alkaloids were well separated in a short run time of 13min with mobile phase pH 10.5 and a small gradient of 9-13% acetonitrile, and detected using UV at 260nm. Peak parameters were acceptable for all six closely related alkaloids. The proposed method has enough linearity with correlation coefficient >0.999 within the investigated range for all tested alkaloids. Satisfactory precision was achieved for both intra- and inter-day assay, with RSD less than 2% for all alkaloid standards. Reproducibility was also within the acceptable range of RSD alkaloids. The limit of quantification was 2.8 and 4.8μg/mL for nornicotine and nicotine respectively, and below 2μg/mL for all other alkaloids. The method was successfully applied for simultaneous analysis of alkaloids in leaves of Nicotiana benthamiana.

  6. The Cotton WRKY Gene GhWRKY41 Positively Regulates Salt and Drought Stress Tolerance in Transgenic Nicotiana benthamiana.

    Directory of Open Access Journals (Sweden)

    Xiaoqian Chu

    Full Text Available WRKY transcription factors constitute a very large family of proteins in plants and participate in modulating plant biological processes, such as growth, development and stress responses. However, the exact roles of WRKY proteins are unclear, particularly in non-model plants. In this study, Gossypium hirsutum WRKY41 (GhWRKY41 was isolated and transformed into Nicotiana benthamiana. Our results showed that overexpression of GhWRKY41 enhanced the drought and salt stress tolerance of transgenic Nicotiana benthamiana. The transgenic plants exhibited lower malondialdehyde content and higher antioxidant enzyme activity, and the expression of antioxidant genes was upregulated in transgenic plants exposed to osmotic stress. A β-glucuronidase (GUS staining assay showed that GhWRKY41 was highly expressed in the stomata when plants were exposed to osmotic stress, and plants overexpressing GhWRKY41 exhibited enhanced stomatal closure when they were exposed to osmotic stress. Taken together, our findings demonstrate that GhWRKY41 may enhance plant tolerance to stress by functioning as a positive regulator of stoma closure and by regulating reactive oxygen species (ROS scavenging and the expression of antioxidant genes.

  7. Changes in phenolic compound, TAL, PAL activity of Nicotiana rustica triggered by ethanolamine pretreatment under in vitro salt stress condition

    Directory of Open Access Journals (Sweden)

    Ali Akbar Ehsanpour

    2015-12-01

    Full Text Available Salt stress is one of the most important factors that endanger plant growth and production all around the world. Application of compounds which increase plant resistance to salt stress obviously has significant importance in reduction of economic losses. One of these compounds is the biological alcohol called ethanolamine, and the present study aimed to understand the way by which ethanolamine exerts its protecting effect against salt stress. For this purpose, 4-week-old Nicotiana rustica plants which had been kept in MS medium were pretreated with ethanolamine and 2 days later they were transferred to MS medium supplemented with 200 mM NaCl for 3 weeks. The our results revealed that Nicotiana rustica plants pretreated with exogenous ethanolamine showed elevated level of TAL activity and anthocyanin content whereas PAL activity was decreased, and reduced level of MDA, total flavonoid and phenol content in plants under salt stress condition. According to these results, it can be suggested that ethanolamine exerts its protective effect by anthocyanin content increase.

  8. Reversal by Calcium Ions of the Growth Inhibition of Debaryomyces nicotianae Caused by Antifungal Polyene Antibiotics1

    Science.gov (United States)

    Berdicevsky, Israela; Grossowicz, Nathan

    1972-01-01

    Only Debaryomyces nicotianae strain 77, of seven different yeast strains tested, was found to be resistant to heptamycin and other antifungal heptaenes when grown in a rich medium. This strain, however, like the other six, was completely susceptible to these antibiotics in a minimal medium. Addition of yeast extract to the minimal medium abolished the heptamycin effect; calcium ions fully duplicated the effect of yeast extract; Mg2+ and Mn2+ were also effective but less so than Ca2+. Ca2+ also counteracted the activity of the heptaenes ascosin and trichomycin. Complete reversal of the polyene inhibition by Ca2+ was obtained if the cation was added simultaneously with the antibiotic; addition of Ca2+ 2 hr after the polyene was without effect. Addition of Ca2+ in the absence of the polyene caused a slight, if any, growth stimulation of D. nicotianae 77. Cholesterol also counteracted polyene activity; this was due to the formation of a complex with the antibiotic which prevented the polyene from reaching the site of action—the cytoplasmic membrane. No evidence for complex formation between heptamycin and calcium was found. The importance of Ca2+ in membrane structure, as evidenced from heptaene studies, is discussed. PMID:4598328

  9. Ionomic profiling of Nicotiana langsdorffii wild-type and mutant genotypes exposed to abiotic stresses.

    Science.gov (United States)

    Ardini, Francisco; Soggia, Francesco; Abelmoschi, Maria Luisa; Magi, Emanuele; Grotti, Marco

    2013-01-01

    To provide a new insight into the response of plants to abiotic stresses, the ionomic profiles of Nicotiana langsdorffii specimens have been determined before and after exposure to toxic metals (chromium) or drought conditions. The plants were genetically transformed with the rat glucocorticoid receptor (GR) or the gene for Agrobacterium rhizogenes rolC, because these modifications are known to produce an imbalance in phytohormone equilibria and a significant change in the defence response of the plant. Elemental profiles were obtained by developing and applying analytical procedures based on inductively coupled plasma atomic emission and mass spectrometry (ICP-AES/MS). In particular, the removal of isobaric interferences affecting the determination of Cr and V by ICP-MS was accomplished by use of a dynamic reaction cell, after optimization of the relevant conditions. The combined use of ICP atomic emission and mass spectrometry enabled the determination of 29 major and trace elements (Ba, Bi, Ca, Cd, Co, Cr, Cu, Eu, Fe, Ga, K, Li, Mg, Mn, Mo, Na, P, Pb, Pt, Rb, S, Sb, Sn, Sr, Te, V, W, Y, and Zn) in different parts of the plants (roots, stems, and leaves), with high accuracy and precision. Multivariate data processing and study of element distribution patterns provided new information about the ionomic response of the target organism to chemical treatment or water stress. Genetic modification mainly affected the distribution of Bi, Cr, Mo, Na, and S, indicating that these elements were involved in biochemical processes controlled by the GR or rolC genes. Chemical stress strongly affected accumulation of several elements (Ba, Ca, Fe, Ga, K, Li, Mn, Mo, Na, P, Pb, Rb, S, Sn, Te, V, and Zn) in different ways; for Ca, Fe, K, Mn, Na, and P the effect was quite similar to that observed in other studies after treatment with other transition elements, for example Cu and Cd. The effect of water deficit was less evident, mainly consisting in a decrease of Ba, Cr, Na, and Sr

  10. Production of the main celiac disease autoantigen by transient expression in Nicotiana benthamiana

    Directory of Open Access Journals (Sweden)

    Vanesa Soledad Marin Viegas

    2015-12-01

    Full Text Available Celiac Disease (CD is a gluten sensitive enteropathy that remains widely undiagnosed and implementation of massive screening tests is needed to reduce the long term complications associated to untreated CD. The main CD autoantigen, human tissue transglutaminase (TG2, is a challenge for the different expression systems available since its cross-linking activity affects cellular processes. Plant-based transient expression systems can be an alternative for the production of this protein. In this work, a transient expression system for the production of human TG2 in Nicotiana benthamiana leaves was optimized and reactivity of plant-produced TG2 in CD screening test was evaluated. First, a subcellular targeting strategy was tested. Cytosolic, secretory, endoplasmic reticulum (C-terminal SEKDEL fusion and vacuolar (C-terminal KISIA fusion TG2 versions were transiently expressed in leaves and recombinant protein yields were measured. ER-TG2 and vac-TG2 levels were 9 to 16 fold higher than their cytosolic and secretory counterparts. As second strategy, TG2 variants were co-expressed with a hydrophobic elastin-like polymer (ELP construct encoding for 36 repeats of the pentapeptide VPGXG in which the guest residue X were V and F in ratio 8:1. Protein bodies (PB were induced by the ELP, with a consequent 2 fold-increase in accumulation of both ER-TG2 and vac-TG2. Subsequently, ER-TG2 and vac-TG2 were produced and purified using immobilized metal ion affinity chromatography. Plant purified ER-TG2 and vac-TG2 were recognized by three anti-TG2 monoclonal antibodies that bind different epitopes proving that plant-produced antigen has immunochemical characteristics similar to those of human TG2. Lastly, an ELISA was performed with sera of CD patients and healthy controls. Both vac-TG2 and ER-TG2 were positively recognized by IgA of CD patients while they were not recognized by serum from non-celiac controls. These results confirmed the usefulness of plant

  11. NmDef02, a novel antimicrobial gene isolated from Nicotiana megalosiphon confers high-level pathogen resistance under greenhouse and field conditions

    NARCIS (Netherlands)

    Portieles, R.; Ayra, C.; Gonzalez, E.; Gallo, A.; Rodriguez, R.; Chacón, O.; López, Y.; Rodriguez, M.; Castillo, J.; Pujol, M.; Enriquez, G.; Borroto, C.; Trujillo, L.; Thomma, B.P.H.J.; Borrás-Hidalgo, O.

    2010-01-01

    Plant defensins are small cysteine-rich peptides that inhibit the growth of a broad range of microbes. In this article, we describe NmDef02, a novel cDNA encoding a putative defensin isolated from Nicotiana megalosiphon upon inoculation with the tobacco blue mould pathogen Peronospora hyoscyami

  12. Back-transmission of a virus associated with apple stem pitting and pear vein yellows from Nicotiana occidentalis to apple and pear indicators

    NARCIS (Netherlands)

    Leone, G.; Lindner, J.L.; Jongedijk, G.; Meer, van der F.

    1995-01-01

    The successful back-transmission of the mechanically transmissible virus associated with apple stem pitting and pear vein yellows, from Nicotiana occidentalis to apple seedlings "Golden Delicious" under greenhouse conditions is reported. This result enabled a field experiment where isolates of apple

  13. Symptoms on apple and pear indicators after back-transmission from Nicotiana occidentalis confirm the identity of apple stem pitting virus with pear vein yellows virus

    NARCIS (Netherlands)

    Leone, G.; Lindner, J.L.; Meer, van der F.A.; Schoen, C.D.; Jongedijk, G.

    1998-01-01

    Isolates of apple stem pitting virus (ASPV) from diseased apple trees were maintained in Nicotiana occidentalis then back-transmitted mechanically from the herbaceous host to apple seedlings and indexed by double budding on apple and pear indicators for the following syndromes: apple stem pitting,

  14. Symptoms on apple and pear indicators after back-transmission from Nicotiana occidentalis confirm the identity of apple stem pitting virus with pear vein yellows virus

    NARCIS (Netherlands)

    Leone, G.; Lindner, J.L.; Meer, van der F.A.; Schoen, C.D.; Jongedijk, G.

    1998-01-01

    Isolates of apple stem pitting virus (ASPV) from diseased apple trees were maintained in Nicotiana occidentalis then back-transmitted mechanically from the herbaceous host to apple seedlings and indexed by double budding on apple and pear indicators for the following syndromes: apple stem pitting, p

  15. Differences in intensity and specificity of hypersensitive response induction in Nicotiana spp. by INF1, INF2A, and INF2B of Phytophthora infestans

    NARCIS (Netherlands)

    Huitema, E.; Vleeshouwers, V.G.A.A.; Cakir, C.; Kamoun, S.; Govers, F.

    2005-01-01

    Elicitins form a family of structurally related proteins that induce the hypersensitive response (HR) in plants, particularly Nicotiana spp. The elicitin family is composed of several classes. Most species of the plant-pathogenic oomycete genus Phytophthora produce the well-characterized 10-kDa

  16. NmDef02, a novel antimicrobial gene isolated from Nicotiana megalosiphon confers high-level pathogen resistance under greenhouse and field conditions

    NARCIS (Netherlands)

    Portieles, R.; Ayra, C.; Gonzalez, E.; Gallo, A.; Rodriguez, R.; Chacón, O.; López, Y.; Rodriguez, M.; Castillo, J.; Pujol, M.; Enriquez, G.; Borroto, C.; Trujillo, L.; Thomma, B.P.H.J.; Borrás-Hidalgo, O.

    2010-01-01

    Plant defensins are small cysteine-rich peptides that inhibit the growth of a broad range of microbes. In this article, we describe NmDef02, a novel cDNA encoding a putative defensin isolated from Nicotiana megalosiphon upon inoculation with the tobacco blue mould pathogen Peronospora hyoscyami f.sp

  17. Back-transmission of a virus associated with apple stem pitting and pear vein yellows from Nicotiana occidentalis to apple and pear indicators

    NARCIS (Netherlands)

    Leone, G.; Lindner, J.L.; Jongedijk, G.; Meer, van der F.

    1995-01-01

    The successful back-transmission of the mechanically transmissible virus associated with apple stem pitting and pear vein yellows, from Nicotiana occidentalis to apple seedlings "Golden Delicious" under greenhouse conditions is reported. This result enabled a field experiment where isolates of apple

  18. PENGENDALIAN PENYAKIT PATIK (Cercospora nicotianae PADA TEMBAKAU NA OOGST SECARA IN-VIVO DENGAN EKSTRAK DAUN GULMA KIPAHIT (Tithonia diversifolia

    Directory of Open Access Journals (Sweden)

    Aditya Reza Apriyadi

    2013-11-01

    Full Text Available [ENGLISH] Frog eyes diseases or leaf spot caused by Cercospora nicotinae that can reduce the quality of tobacco leaves, especially when used as a cigar deckblad. Affected leaves was easyly torn and the syhmptoms developed rapidly when processed in the storage. Therefore the alternative control of plant is by mexico sun flower leaf extracts. Mexico sunflower belonging to the broadleaf weeds. It suggested contains flavonoids, tannins, terpenoids, and saponins. The results showed mexico sunflower leaf extract concentration 50 g / L was effective to control this diseases, when compared mexico sunflower leaf extract concentration 25 g / L. However, there was no different between plant sprayed with mexico sun flower extract at concentration of 50 g/L and 75 g/L. Keywords: Mexico Sunflower leaf extract; Cercospora nicotianae; Tobacco. [INDONESIAN] Penyakit patik atau bercak daun Cercospora yang disebabkan oleh jamur Cercospora nicotinae dapat mengurangi mutu daun tembakau, terutama apabila digunakan sebagai daun pembalut cerutu. Biasanya penyakit ini dikendalikan dengan pestisida kimia tetapi karena ada batasan residu kimia maksimum sebesar 2,0 ppm, maka dicari alternatif pengendaliannya. Kipahit merupakan gulma berdaun lebar yang mempunyai potensi sebagai anti jamur patogen tanaman, karena mengandung senyawa flavonoid, tannin, terpenoid, dan saponin. Dengan penyemprotan empat kali dalam interval 15 hari, diketahui ekatrak daun kipahit dengan konsentrasi 50 g/l dan 75 g/L dapat menurunkan tingkat keparahan penyakit 1% sejak 60 hst sampai 70 hst. Akan tetapi, nilai insiden penyakit diketahui 100% pada semua perlakuan ekstrak daun kipahit. Ekstrak daun kipahit dengan konsentrasi 50 g/L sudah efektif mengendalikan penyakit patik jika dibandingkan dengan ekstrak daun kipahit dengan konsentrasi 25 g/L. Namun ekstrak daun kipahit dengan konsentrasi 75 g/L tidak berbeda efektifitasnya dengan ekstrak daun kipahit dengan konsentrasi 50 g/L. Kata Kunci: Ekstrak

  19. Tumores em certos híbridos do gênero nicotiana

    Directory of Open Access Journals (Sweden)

    F. G. Brieger

    1942-01-01

    Full Text Available The present paper gives some observations on the tumour formation and other abnormalities observed in the interspecific hybrids, Nicotiana glauca x N. Langsdorffii and N. glauca x N. Sanderae. Without entering into a detailed discussion of the literature and the various speculations proposed by Kostoff and Whitaker about the causes of tumour formations or on the relations between "spontaneous" and pathogenic tumours (Kostoff, Levine and others, two new observations are described. 1 The abnormalities in the two above mentioned hybrids are quite different. As already reported by other authors, the hybrids N. glauca x N. Langsdorffii are initially quite normal and vigorous, while tumours and other proliferations, on the stem, the stem base and the root appear more or less frequently in older plants. Tumours on leaf are rare and flowers are normal (fig. 1,2,7 and 10. The hybrids N. glauca x N. Sanderae are very different from the former. These remain either weak and rachitic, forming early large tumours at the stem base and on the root (fig. 5 and 6 right half. Numerous shoots with more or less normal leaves may arise from these tumours (fig. 11 and 12. Or the plants show an extremely vigorous and abnormal ramification with a number of serial axial shoots developing in each leaf axis (fig. 3 and 4. The leaves are frequently deformed and the flowers exhibit abnormally coloured patches. When crossing N. glauca with F1 (N. Langsdorffii x N. Sanderae the offspring could clearly be divided into two groups : plants exactly as described for N. glauca x N. Langsdorffii and others which correspond to the types of N. glauca x N. Sanderae. Thus it seems that we are dealing with a monogenic differences, each species, N. Langsdorffii and N. Sanderae, being homozygous for one allele each. These alleles have no phenotypic effect in either pure species, but only in hybrids when combined with the genom of N. glauca. The situation is somewhat similar to the sublethal

  20. Expression of the Minor Isoform Pea Ferredoxin in Tobacco Alters Photosynthetic Electron Partitioning and Enhances Cyclic Electron Flow1[W

    Science.gov (United States)

    Blanco, Nicolás E.; Ceccoli, Romina D.; Vía, María V. Dalla; Voss, Ingo; Segretin, María E.; Bravo-Almonacid, Fernando F.; Melzer, Michael; Hajirezaei, Mohammad-Reza; Scheibe, Renate; Hanke, Guy T.

    2013-01-01

    Ferredoxins (Fds) are ferrosulfoproteins that function as low-potential electron carriers in plants. The Fd family is composed of several isoforms that share high sequence homology but differ in functional characteristics. In leaves, at least two isoforms conduct linear and cyclic photosynthetic electron transport around photosystem I, and mounting evidence suggests the existence of at least partial division of duties between these isoforms. To evaluate the contribution of different kinds of Fds to the control of electron fluxes along the photosynthetic electron transport chain, we overexpressed a minor pea (Pisum sativum) Fd isoform (PsFd1) in tobacco (Nicotiana tabacum) plants. The transplastomic OeFd1 plants exhibited variegated leaves and retarded growth and developmental rates. Photosynthetic studies of these plants indicated a reduction in carbon dioxide assimilation rates, photosystem II photochemistry, and linear electron flow. However, the plants showed an increase in nonphotochemical quenching, better control of excitation pressure at photosystem II, and no evidence of photoinhibition, implying a better dynamic regulation to remove excess energy from the photosynthetic electron transport chain. Finally, analysis of P700 redox status during illumination confirmed that the minor pea Fd isoform promotes enhanced cyclic flow around photosystem I. The two novel features of this work are: (1) that Fd levels achieved in transplastomic plants promote an alternative electron partitioning even under greenhouse light growth conditions, a situation that is exacerbated at higher light intensity measurements; and (2) that an alternative, minor Fd isoform has been overexpressed in plants, giving new evidence of labor division among Fd isoforms. PMID:23370717

  1. Identification of a mitochondrial external NADPH dehydrogenase by overexpression in transgenic ¤Nicotiana sylvestris¤

    DEFF Research Database (Denmark)

    Michalecka, A.M.; Agius, S.C.; Møller, I.M.;

    2004-01-01

    (P)H dehydrogenases, was introduced into Nicotiana sylvestris. Transgenic lines with high transcript and protein levels for St-NDB1 had up to threefold increased activity of external NADPH dehydrogenase in isolated mitochondria as compared to the wild type (WT). In two lines, the external NADPH dehydrogenase activity...... for NADPH and dependent on calcium for activity. Transgenic lines overexpressing St-ndb1 had specifically increased protein levels for alternative oxidase and uncoupling protein, as compared to the WT and one co-suppressing line. This indicates cross-talk in the expressional control, or metabolic conditions...... influencing it, for the different categories of energy-dissipating proteins that bypass oxidative phosphorylation. The potential effects of external NADPH oxidation on other cellular processes are discussed....

  2. Macrocyclic Trichothecenes from Myrothecium roridum Strain M10 with Motility Inhibitory and Zoosporicidal Activities against Phytophthora nicotianae.

    Science.gov (United States)

    Mondol, Muhammad Abdul Mojid; Surovy, Musrat Zahan; Islam, M Tofazzal; Schüffler, Anja; Laatsch, Hartmut

    2015-10-14

    The cytotoxicity of the extract obtained from Myrothecium roridum M10 and a characteristic (1)H signal at δH ∼8 led to the assumption that verrucarin/roridin-type compounds were present. Upscaling on rice medium led to the isolation of four new metabolites: verrucarins Y (1) and Z (6) (macrocyclic trichothecenes), bilain D (12) (a diketopiperazine derivative), and hamavellone C (14) (an unusual cyclopropyl diketone). In addition, nine known trichothecenes [verrucarin A (3), 16-hydroxyverrucarin A (5), verrucarin B (7), 16-hydroxyverrucarin B (8), verrucarin J (2), verrucarin X (4), roridin A (9), roridin L-2 (10), and trichoverritone (11)] and a bicyclic lactone [myrotheciumone A (15)] were identified. Their structures and configurations were determined by spectroscopic methods, published data, Mosher's method, and considering biosyntheses. Some trichothecenes showed motility inhibition followed by lysis of the zoospores against devastating Phytophthora nicotianae within 5 min. Compounds 2, 3, 7, and 9 also exhibited potent activities against Candida albicans and Mucor miehei.

  3. Cloning and characterization of a 9-lipoxygenase gene induced by pathogen attack from Nicotiana benthamiana for biotechnological application

    Directory of Open Access Journals (Sweden)

    Schwab Wilfried

    2011-03-01

    Full Text Available Abstract Background Plant lipoxygenases (LOXs have been proposed to form biologically active compounds both during normal developmental stages such as germination or growth as well as during responses to environmental stress such as wounding or pathogen attack. In our previous study, we found that enzyme activity of endogenous 9-LOX in Nicotiana benthamiana was highly induced by agroinfiltration using a tobacco mosaic virus (TMV based vector system. Results A LOX gene which is expressed after treatment of the viral vectors was isolated from Nicotiana benthamiana. As the encoded LOX has a high amino acid identity to other 9-LOX proteins, the gene was named as Nb-9-LOX. It was heterologously expressed in yeast cells and its enzymatic activity was characterized. The yeast cells expressed large quantities of stable 9-LOX (0.9 U ml-1 cell cultures which can oxygenate linoleic acid resulting in high yields (18 μmol ml-1 cell cultures of hydroperoxy fatty acid. The product specificity of Nb-9-LOX was examined by incubation of linoleic acid and Nb-9-LOX in combination with a 13-hydroperoxide lyase from watermelon (Cl-13-HPL or a 9/13-hydroperoxide lyase from melon (Cm-9/13-HPL and by LC-MS analysis. The result showed that Nb-9-LOX possesses both 9- and 13-LOX specificity, with high predominance for the 9-LOX function. The combination of recombinant Nb-9-LOX and recombinant Cm-9/13-HPL produced large amounts of C9-aldehydes (3.3 μmol mg-1 crude protein. The yield of C9-aldehydes from linoleic acid was 64%. Conclusion The yeast expressed Nb-9-LOX can be used to produce C9-aldehydes on a large scale in combination with a HPL gene with 9-HPL function, or to effectively produce 9-hydroxy-10(E,12(Z-octadecadienoic acid in a biocatalytic process in combination with cysteine as a mild reducing agent.

  4. Genetic Diversity in Haploid Nicotiana alata Induced by Gamma Irradiation, Salt Tolerance and Detection of These Differences by RAPD

    Directory of Open Access Journals (Sweden)

    Ayman EL-FIKI

    2016-03-01

    Full Text Available Haploid plants of Nicotiana alata were cultured in vitro on MS medium with IAA + KIN. The resulting plantlets were irradiated using gamma radiation doses of 10, 15, 20 and 25 Gy. Single node pieces were cut and transferred onto fresh MS medium. Gamma radiation doses caused the death of 9% and up to 28% of explants. NaCl concentrations caused the death of 8% up to 36% of explants, while the combined effect between gamma radiation doses and salinity had an impact suffused on the percentage of survival. The combined effect of gamma radiation doses 20 Gy and 25 Gy on NaCl concentrations of 100, 150 and 200 mM were deadly. Even more, the combined effect of gamma radiation doses and salinity had a severe negative impact on both the proline content and total soluble protein. Random amplified polymorphic DNA (RAPD analysis was used to determine the degree of genetic variation in treated haploid Nicotiana alata plants. Total genomic DNAs from different haploid plantlets treated were amplified using five arbitrary primers. Two hundred and seventy bands were detected from plantlets irradiated with doses of 15, 20 and 25 Gy, with polymorphic band number 226 (83.7%. The total number of bands resulted from plant grew on 150 mM and 200 mM NaCl were 260 bands with polymorphic bands 185 (85.6%. However, the total number of bands produced from combined effects between gamma rays and salinity (20 Gy X 50 mM NaCl, 20 Gy X 100 mM NaCl and 25 Gy X 50 mM NaCl were 270, with polymorphic band number 231 (85.5%. High similarity between treatments was revealed. Treatments relationships were estimated through cluster analysis (UPGMA based on RAPD data.

  5. Modulation of Quorum Sensing in Acylhomoserine Lactone-Producing or -Degrading Tobacco Plants Leads to Alteration of Induced Systemic Resistance Elicited by the Rhizobacterium Serratia marcescens 90-166.

    Science.gov (United States)

    Ryu, Choong-Min; Choi, Hye Kyung; Lee, Chi-Ho; Murphy, John F; Lee, Jung-Kee; Kloepper, Joseph W

    2013-06-01

    Numerous root-associated bacteria (rhizobacteria) are known to elicit induced systemic resistance (ISR) in plants. Bacterial cell-density-dependent quorum sensing (QS) is thought to be important for ISR. Here, we investigated the role of QS in the ISR elicited by the rhizobacterium, Serratia marcescens strain 90-166, in tobacco. Since S. marcescens 90-166 produces at least three QS signals, QS-mediated ISR in strain 90-166 has been difficult to understand. Therefore, we investigated the ISR capacity of two transgenic tobacco (Nicotiana tabacum) plants that contained either bacterial acylhomoserine lactone-producing (AHL) or -degrading (AiiA) genes in conjunction with S. marcescens 90-166 to induce resistance against bacterial and viral pathogens. Root application of S. marcescens 90-166 increased ISR to the bacterial pathogens, Pectobacterium carotovorum subsp. carotovorum and Pseudomonas syringae pv. tabaci, in AHL plants and decreased ISR in AiiA plants. In contrast, ISR to Cucumber mosaic virus was reduced in AHL plants treated with S. marcescens 90-166 but enhanced in AiiA plants. Taken together, these data indicate that QS-dependent ISR is elicited by S. marcescens 90-166 in a pathogen-dependent manner. This study provides insight into QS-dependent ISR in tobacco elicited by S. marcescens 90-166.

  6. Modulation of Quorum Sensing in Acylhomoserine Lactone-Producing or -Degrading Tobacco Plants Leads to Alteration of Induced Systemic Resistance Elicited by the Rhizobacterium Serratia marcescens 90-166

    Directory of Open Access Journals (Sweden)

    Choong-Min Ryu

    2013-06-01

    Full Text Available Numerous root-associated bacteria (rhizobacteria are known to elicit induced systemic resistance (ISR in plants. Bacterial cell-density-dependent quorum sensing (QS is thought to be important for ISR. Here, we investigated the role of QS in the ISR elicited by the rhizobacterium, Serratia marcescens strain 90–166, in tobacco. Since S. marcescens 90–166 produces at least three QS signals, QS-mediated ISR in strain 90–166 has been difficult to understand. Therefore, we investigated the ISR capacity of two transgenic tobacco (Nicotiana tabacum plants that contained either bacterial acylhomoserine lactone-producing (AHL or -degrading (AiiA genes in conjunction with S. marcescens 90–166 to induce resistance against bacterial and viral pathogens. Root application of S. marcescens 90–166 increased ISR to the bacterial pathogens, Pectobacterium carotovorum subsp. carotovorum and Pseudomonas syringae pv. tabaci, in AHL plants and decreased ISR in AiiA plants. In contrast, ISR to Cucumber mosaic virus was reduced in AHL plants treated with S. marcescens 90–166 but enhanced in AiiA plants. Taken together, these data indicate that QS-dependent ISR is elicited by S. marcescens 90–166 in a pathogen-dependent manner. This study provides insight into QS-dependent ISR in tobacco elicited by S. marcescens 90–166.

  7. Effect of carbon monoxide on respiration in higher plants. [Fagopyrum esculentum L. ; Delphinium Ajacis L. ; Raphanus sativus L. ; Pyrus Malus L. ; Beta vulgaris L. ; Phaseolus vulgaris L. ; Phaseolus limensis L. ; Medicago pratense L. ; Nicotiana Tabacum L. ; Cucumis sativus L. ; Helianthus annuus L

    Energy Technology Data Exchange (ETDEWEB)

    Webster, G.C.

    1954-01-01

    The effect of carbon monoxide and light on the respiration of a number of plant tissues were examined. The respiration of root or other tissue was measured at 25/sup 0/C by standard manometric techniques in a ratio of 95% CO and 5% O/sub 2/. The respiration of all eleven tissues studied was strongly inhibited by carbon monoxide. In ten of the eleven cases examined the inhibition was largely or completely eliminated by irradiation of the tissue with light. The evidence fairly well precludes the participation of a tyrosinase and definitely supports the participation of a cytochrome oxidase in respiration. 5 references, 1 table.

  8. Effects of Temperature and Light Intensity on Expression of Lycopene Cyclase Gene in Tobacco (Nicotiana tabacum L.)%温度与光照强度对烤烟番茄红素β-环化酶基因表达的影响

    Institute of Scientific and Technical Information of China (English)

    贾峰; 徐文; 刘卫群; 王彦伟

    2011-01-01

    选用水培烤烟叶圆片,在3种温度、3种光照及其交互作用的正交试验处理下进行试验,检测了Lyc-β基因表达的结果表明:光照是影响Lyc-β基因表达的主要因子,其次为温度,最后是温度和光强交互作用.光照时间试验结果表明,Lyc-β基因的表达在光照处理6 h时达到峰顶之后开始下调.%In this research, the tobacco leaf discs were treated with three different temperatures and three different light intensities using orthogonal experiment. The results of the expression of Lyc-β gene indicated that the key factor affecting the expression of Lyc-β was light, followed by temperature, and then interactions of temperature with light. Moreover, the results of light experiment showed that the expression of Lyc-β increased to the highest point after 6 hours' light-treatment, and then down-regulated.

  9. 小麦TaCRF2基因的克隆及其在烟草中的初步功能验证%TaCRF2 Gene Isolation from Triticum aestivum and Primary Function Validation in Tobacco (Nicotiana tabacum L.)

    Institute of Scientific and Technical Information of China (English)

    郭丽香; 高世庆; 唐益苗; 王永波; 刘美英; 张朝; 徐蓓; 赵昌平

    2011-01-01

    通过RT-PCR方法从小麦cDNA中扩增获得1个锌指蛋白基因TaCRF2,该基因的cDNA长度为843 bp,序列分析表明它编码1个含有280个氨基酸的蛋白质.在线软件预测该蛋白质的相对分子质量为30.97 kD,等电点为7.03,且在C-末端含有1个典型的RING-H2型锌指蛋白结构域,在N-末端含有2个跨膜结构域.氨基酸序列比对发现,TaCRF2与水稻中1个RING型锌指蛋白(ABF95226)的相似度为82%.亚细胞定位分析显示,该蛋白分布在细胞核和细胞膜上.real-time RT-PCR表达特性分析显示,TaCRF2基因的表达受干旱、高盐和外源ABA的强烈诱导,低温对该基因的表达量影响不明显.初步功能验证发现过表达TaCRF2基因增强了转基因烟草对干旱和高盐胁迫的耐性.%Wheat, like other crops, suffers from drought, salt, low-temperature and other abiotic stresses, seriously resulting in yield decline. It is an effective way for crop breeding to improve resistance via molecular biology techniques. Zinc finger protein, an important transcription factor commonly found in plant, can regulate the expression of multiple stress-inducible genes and enhance comprehensive resistances effectively. In this study, a RING-H2 zinc finger protein gene, designated as TaCRF2, was isolated from Triticum aestivum by RT-PCR. Its cDN A was 843 bp and encoded a putative protein of 280 amino acids with a predicted molecular mass of 30.97 kD and an isoelectric point (PI) of 7.03. A typical RING-H2 finger domain was found at the C-terminal region of TaCRF2 protein, and two transmembrane domains were found at the N-terminal region. Alignment of amino acid sequence showed that TaCRF2 was 82% identical to Oryza saliva putative RING zinc finger protein ABF95226. Subcellular localization analysis showed that the TaCRF2 was expressed in both nuclear and cytoplasm membrane, real-time PCR showed that the transcript of TaCRF2 was strongly induced by drought, salinity and cold to some extent. The TaCRF2 gene was transformed into tobacco cultivar Wisconsin 38 (W38) by Agrobacterium mediated under the control of the CaMV 35S promoter. Under drought, salt and low-temperature stresses, transgenic tobacco lines carrying TaCRF2 gene developed the strong primary root, more lateral root and smaller yellow leaves, and performed higher tolerance to these stresses than the wide-type tobaccos, The primary function verification showed that overexpression of TaCRF2 gene in tobaccos enhanced their tolerance to drought and salinity stresses. This study will help further research of wheat RING-H2 type zinc finger protein reactions in the role of stress resistance, and bring wheat resistance breeding excellent candidate genes.

  10. [Altered states of consciousness].

    Science.gov (United States)

    Gora, E P

    2005-01-01

    The review of modern ideas concerning the altered states of consciousness is presented in this article. Various methods of entry into the altered states of consciousness are looked over. It is shown that the altered states of consciousness are insufficiently known, but important aspects of human being existence. The role of investigation of the altered states of consciousness for the creation of integrative scientific conception base is discussed.

  11. NaStEP: a proteinase inhibitor essential to self-incompatibility and a positive regulator of HT-B stability in Nicotiana alata pollen tubes.

    Science.gov (United States)

    Jiménez-Durán, Karina; McClure, Bruce; García-Campusano, Florencia; Rodríguez-Sotres, Rogelio; Cisneros, Jesús; Busot, Grethel; Cruz-García, Felipe

    2013-01-01

    In Solanaceae, the self-incompatibility S-RNase and S-locus F-box interactions define self-pollen recognition and rejection in an S-specific manner. This interaction triggers a cascade of events involving other gene products unlinked to the S-locus that are crucial to the self-incompatibility response. To date, two essential pistil-modifier genes, 120K and High Top-Band (HT-B), have been identified in Nicotiana species. However, biochemistry and genetics indicate that additional modifier genes are required. We recently reported a Kunitz-type proteinase inhibitor, named NaStEP (for Nicotiana alata Stigma-Expressed Protein), that is highly expressed in the stigmas of self-incompatible Nicotiana species. Here, we report the proteinase inhibitor activity of NaStEP. NaStEP is taken up by both compatible and incompatible pollen tubes, but its suppression in Nicotiana spp. transgenic plants disrupts S-specific pollen rejection; therefore, NaStEP is a novel pistil-modifier gene. Furthermore, HT-B levels within the pollen tubes are reduced when NaStEP-suppressed pistils are pollinated with either compatible or incompatible pollen. In wild-type self-incompatible N. alata, in contrast, HT-B degradation occurs preferentially in compatible pollinations. Taken together, these data show that the presence of NaStEP is required for the stability of HT-B inside pollen tubes during the rejection response, but the underlying mechanism is currently unknown.

  12. N-Glycosylation of cholera toxin B subunit in Nicotiana benthamiana: impacts on host stress response, production yield and vaccine potential

    OpenAIRE

    Hamorsky, Krystal Teasley; Kouokam, J. Calvin; Jurkiewicz, Jessica M.; Nelson, Bailey; Moore, Lauren J.; Husk, Adam S.; Kajiura, Hiroyuki; Fujiyama, Kazuhito; Matoba, Nobuyuki

    2015-01-01

    Plant-based transient overexpression systems enable rapid and scalable production of subunit vaccines. Previously, we have shown that cholera toxin B subunit (CTB), an oral cholera vaccine antigen, is N-glycosylated upon expression in transgenic Nicotiana benthamiana. Here, we found that overexpression of aglycosylated CTB by agroinfiltration of a tobamoviral vector causes massive tissue necrosis and poor accumulation unless retained in the endoplasmic reticulum (ER). However, the re-introduc...

  13. C3HC4-type RING finger protein NbZFP1 is involved in growth and fruit development in Nicotiana benthamiana.

    Directory of Open Access Journals (Sweden)

    Wenxian Wu

    Full Text Available C3HC4-type RING finger proteins constitute a large family in the plant kingdom and play important roles in various physiological processes of plant life. In this study, a C3HC4-type zinc finger gene was isolated from Nicotiana benthamiana. Sequence analysis indicated that the gene encodes a 24-kDa protein with 191 amino acids containing one typical C3HC4-type zinc finger domain; this gene was named NbZFP1. Transient expression of pGDG-NbZFP1 demonstrated that NbZFP1 was localized to the chloroplast, especially in the chloroplasts of cells surrounding leaf stomata. Virus-induced gene silencing (VIGS analysis indicated that silencing of NbZFP1 hampered fruit development, although the height of the plants was normal. An overexpression construct was then designed and transferred into Nicotiana benthamiana, and PCR and Southern blot showed that the NbZFP1 gene was successfully integrated into the Nicotiana benthamiana genome. The transgenic lines showed typical compactness, with a short internode length and sturdy stems. This is the first report describing the function of a C3HC4-type RING finger protein in tobacco.

  14. [Effects of seven RNA silencing suppressors on heterologous expression of green fluorescence protein expression mediated by a plant virus-based system in Nicotiana benthamiana].

    Science.gov (United States)

    Wang, Sheng; Dong, Jie; Cao, Min; Mu, Hongzhen; Ding, Guoping; Zhang, Hong

    2012-11-01

    To test the effects of 7 virus-encoded RNA silencing suppressors (RSSs) for enhancement of a plant virus-based vector system-mediated heterologous expression of green fluorescence protein (GFP) in Nicotiana benthamiana. Seven transient expression vectors for the 7 RSSs were constructed and co-inoculated on the leaves of Nicotiana benthamiana with PVXdt-GFP vector, a novel Potato virus X-based plant expression vector, through agroinfiltration. The protein and mRNA expression levels of the reporter gene GFP in the co-inoculated Nicotiana leaves were examined by Western blotting, ELISA and RT-qPCR to assess the effect of the RSSs for GFP expression enhancement. The 7 RSSs differed in the degree and duration of enhancement of heterologous GFP expression, and the p19 protein of Tomato bushy stunt virus (TBSV) induced the highest expression of GFP. African cassava mosaic virus AC2 protein and Rice yellow mettle virus P1 protein produced no obvious enhancement GFP expression. Transient co-expression of RSSs suppresses host silencing response to allow high-level and long-term expression of heterologous genes in plant, but the optimal RSS has to be identified for each plant virus-based expression vector system.

  15. Study on the Cultural Characteristics of Phytophthora parasitica var. nicotianae%烟草黑胫病菌培养特性的研究

    Institute of Scientific and Technical Information of China (English)

    张文友; 刘铭; 尹福强; 赵云飞; 刘朝科

    2013-01-01

    研究了采自攀西地区的烟草黑胫病菌在不同培养基、不同温度及不同pH值等条件下培养特性。结果表明:烟草黑胫病菌最适合在燕麦培养基上生长,菌丝生长快。菌丝在20~36℃都能生长,28℃生长最佳。在不同pH值的培养基培养下,菌丝最适合在pH值为5.5的培养基上生长。%Phytophthora parasitica var.nicotianae was cultivated under the conditions of different cultural media ,pH value and temperature ,then observed its growth status and morphological characteristics.The results showed that oatmeal agar was most suitable for the growth of the strains of P. Nicotianae.The hyphae can grow at 20~36℃,the temperature of 28℃and the pH value of 5.5 were the best for the growth of the strains of P.Nicotianae.

  16. The ABC transporter ATR1 is necessary for efflux of the toxin cercosporin in the fungus Cercospora nicotianae.

    Science.gov (United States)

    Amnuaykanjanasin, Alongkorn; Daub, Margaret E

    2009-02-01

    The Cercospora nicotianae mutant deficient for the CRG1 transcription factor has marked reductions in both resistance and biosynthesis of the toxin cercosporin. We cloned and sequenced full-length copies of two genes, ATR1 and CnCFP, previously identified from a subtractive library between the wild type (WT) and a crg1 mutant. ATR1 is an ABC transporter gene and has an open reading frame (ORF) of 4368bp with one intron. CnCFP encodes a MFS transporter with homology to Cercospora kikuchii CFP, previously implicated in cercosporin export, and has an ORF of 1975bp with three introns. Disruption of ATR1 indicated atr1-null mutants had dramatic reductions in cercosporin production (25% and 20% of WT levels) in solid and liquid cultures, respectively. The ATR1 disruptants also showed moderately higher sensitivity to cercosporin. Constitutive expression of ATR1 in the crg1 mutant restored cercosporin biosynthesis and moderately increased resistance. In contrast, CnCFP overexpression in the mutant did not restore toxin production, however, it moderately enhanced toxin resistance. The results together indicate ATR1 acts as a cercosporin efflux pump in this fungus and plays a partial role in resistance.

  17. The dihydrolipoyl acyltransferase gene BCE2 participates in basal resistance against Phytophthora infestans in potato and Nicotiana benthamiana.

    Science.gov (United States)

    Wang, Hongyang; Sun, Chunlian; Jiang, Rui; He, Qin; Yang, Yu; Tian, Zhejuan; Tian, Zhendong; Xie, Conghua

    2014-07-01

    Dihydrolipoyl acyltransferase (EC 2.3.1.12), a branched-chain α-ketoacid dehydrogenase E2 subunit (BCE2), catalyzes the transfer of the acyl group from the lipoyl moiety to coenzyme A. However, the role of BCE2 responding to biotic stress in plant is not clear. In this study, we cloned and characterized a BCE2 gene from potato, namely StBCE2, which was previously suggested to be involved in Phytophthora infestans-potato interaction. We found that the expression of StBCE2 was strongly induced by both P. infestans isolate HB09-14-2 and salicylic acid. Besides, when the homolog of StBCE2 in Nicotiana benthamiana named NbBCE2 was silenced, plants showed increased susceptibility to P. infestans and reduced accumulation of hydrogen peroxide (H2O2). Furthermore, we found that a marker gene NbrbohB involved in the production of reactive oxygen species, was also suppressed in NbBCE2-silenced plants. However, silencing of NbBCE2 had no significant effect on the hypersensitive responses trigged by INF1, R3a-AVR3a(KI) pair or Rpi-vnt1.1-AVR-vnt1.1 pair. Our results suggest that BCE2 is associated with the basal resistance to P. infestans by regulating H2O2 production.

  18. Mechanisms of Optimal Defense Patterns in Nicotiana attenuata: Flowering Attenuates Herbivory-elicited Ethylene and Jasmonate Signaling

    Institute of Scientific and Technical Information of China (English)

    Celia Diezel; Silke AIImann; lan T. Baldwin

    2011-01-01

    To defend themselves against herbivore attack,plants produce secondary metabolites,which are variously inducible and constitutively deployed,presumably to optimize their fitness benefits in light of their fitness costs.Three phytohormones,jasmonates (JA) and their active forms,the JA-isoleucine (JA-Ile) and ethylene (ET),are known to play central roles in the elicitation of induced defenses,but little is known about how this mediation changes over ontogeny.The Optimal Defense Theory (ODT) predicts changes in the costs and benefits of the different types of defenses and has been usefully extrapolated to their modes of deployment.Here we studied whether the herbivore-induced accumulation of JA,JA-lle and ET changed over ontogeny in Nicotiana attenuata,a native tobacco in which inducible defenses are particularly well studied.Herbivore-elicited ET production changed dramatically during six developmental stages,from rosette through flowering,decreasing with the elongation of the first corollas during flower development.This decrease was largely recovered within a day after flower removal by decapitation.A similar pattern was found for the herbivore-induced accumulation of JA and JA-IIe.These results are consistent with ODT predictions and suggest that the last steps in floral development control the inducibility of at least three plant hormones,optimizing defense-growth tradeoffs.

  19. Rapid Transient Production of a Monoclonal Antibody Neutralizing the Porcine Epidemic Diarrhea Virus (PEDV) in Nicotiana benthamiana and Lactuca sativa.

    Science.gov (United States)

    Rattanapisit, Kaewta; Srijangwad, Anchalee; Chuanasa, Taksina; Sukrong, Suchada; Tantituvanont, Angkana; Mason, Hugh S; Nilubol, Dachrit; Phoolcharoen, Waranyoo

    2017-06-02

    Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration, weight loss, and high mortality rate in neonatal piglets. Porcine epidemic diarrhea (PED) has been reported in Europe, America, and Asia including Thailand. The disease causes substantial losses to the swine industry in many countries. Presently, there is no effective PEDV vaccine available. In this study, we developed a plant-produced monoclonal antibody (mAb) 2C10 as a prophylactic candidate to prevent the PEDV infection. Recently, plant expression systems have gained interest as an alternative for the production of antibodies because of many advantages, such as low production cost, lack of human and animal pathogen, large scalability, etc. The 2C10 mAb was transiently expressed in Nicotiana benthamiana and lettuce using geminiviral vector. After purification by protein A affinity chromatography, the antibody was tested for the binding and neutralizing activity against PEDV. Our result showed that the plant produced 2C10 mAb can bind to the virus and also inhibit PEDV infection in vitro. These results show excellent potential for a plant-expressed 2C10 as a PEDV prophylaxis and a diagnostic for PEDV infection. Georg Thieme Verlag KG Stuttgart · New York.

  20. Relationship of microbial communities and suppressiveness of Trichoderma fortified composts for pepper seedlings infected by Phytophthora nicotianae.

    Science.gov (United States)

    Ros, Margarita; Raut, Iulia; Santisima-Trinidad, Ana Belén; Pascual, Jose Antonio

    2017-01-01

    The understanding of the dynamic of soil-borne diseases is related to the microbial composition of the rhizosphere which is the key to progress in the field of biological control. Trichoderma spp. is commonly used as a biological control agent. The use of next generation sequencing approaches and quantitative PCR are two successful approaches to assess the effect of using compost as substrate fortified with two Trichoderma strains (Trichoderma harzianum or Trichoderma asperellum) on bacterial and fungal communities in pepper rhizosphere infected with Phytophthora nicotianae. The results showed changes in the bacterial rhizosphere community not attributed to the Trichoderma strain, but to the pathogen infection, while, fungi were not affected by pathogen infection and depended on the type of substrate. The Trichoderma asperellum fortified compost was the most effective combination against the pathogen. This could indicate that the effect of fortified composts is greater than compost itself and the biocontrol effect should be attributed to the Trichoderma strains rather than the compost microbiota, although some microorganisms could help with the biocontrol effect.

  1. Effects and Effectiveness of Two RNAi Constructs for Resistance to Pepper golden mosaic virus in Nicotiana benthamiana Plants

    Directory of Open Access Journals (Sweden)

    Diana Medina-Hernández

    2013-11-01

    Full Text Available ToChLPV and PepGMV are Begomoviruses that have adapted to a wide host range and are able to cause major diseases in agronomic crops. We analyzed the efficacy of induced resistance to PepGMV in Nicotiana benthamiana plants with two constructs: one construct with homologous sequences derived from PepGMV, and the other construct with heterologous sequences derived from ToChLPV. Plants protected with the heterologous construct showed an efficacy to decrease the severity of symptoms of 45%, while plants protected with the homologous construct showed an efficacy of 80%. Plants protected with the heterologous construct showed a reduction of incidence of 42.86%, while the reduction of incidence in plants protected with the homologous construct was 57.15%. The efficacy to decrease viral load was 95.6% in plants protected with the heterologous construct, and 99.56% in plants protected with the homologous construct. We found, in both constructs, up-regulated key components of the RNAi pathway. This demonstrates that the efficacy of the constructs was due to the activation of the gene silencing mechanism, and is reflected in the decrease of viral genome copies, as well as in recovery phenotype. We present evidence that both constructs are functional and can efficiently induce transient resistance against PepGMV infections. This observation guarantees a further exploration as a strategy to control complex Begomovirus diseases in the field.

  2. Mp10 and Mp42 from the aphid species Myzus persicae trigger plant defenses in Nicotiana benthamiana through different activities.

    Science.gov (United States)

    Rodriguez, Patricia A; Stam, Remco; Warbroek, Tim; Bos, Jorunn I B

    2014-01-01

    Aphids are phloem-feeding insects that, like other plant parasites, deliver effectors inside their host to manipulate host responses. The Myzus persicae (green peach aphid) candidate effectors Mp10 and Mp42 were previously found to reduce aphid fecundity upon intracellular transient overexpression in Nicotiana benthamiana. We performed functional analyses of these proteins to investigate whether they activate defenses through similar activities. We employed a range of functional characterization experiments based on intracellular transient overexpression in N. benthamiana to determine the subcellular localization of Mp10 and Mp42 and investigate their role in activating plant defense signaling. Mp10 and Mp42 showed distinct subcellular localization in planta, suggesting that they target different host compartments. Also, Mp10 reduced the levels of Agrobacterium-mediated overexpression of proteins. This reduction was not due to an effect on Agrobacterium viability. Transient overexpression of Mp10 but not Mp42 activated jasmonic acid and salicylic acid signaling pathways and decreased susceptibility to the hemibiotrophic plant pathogen Phytophthora capsici. We found that two candidate effectors from the broad-host-range aphid M. persicae can trigger aphid defenses through different mechanisms. Importantly, we found that some (candidate) effectors such as Mp10 interfere with Agrobacterium-based overexpression assays, an important tool to study effector activity and function.

  3. Dicer-Like 4 Is Involved in Restricting the Systemic Movement of Zucchini yellow mosaic virus in Nicotiana benthamiana.

    Science.gov (United States)

    Cordero, Teresa; Cerdán, Lidia; Carbonell, Alberto; Katsarou, Konstantina; Kalantidis, Kriton; Daròs, José-Antonio

    2017-01-01

    Zucchini yellow mosaic virus (ZYMV) induces serious diseases in cucurbits. To create a tool to screen for resistance genes, we cloned a wild ZYMV isolate and inserted the visual marker Rosea1 to obtain recombinant clone ZYMV-Ros1. While in some plant-virus combinations Rosea1 induces accumulation of anthocyanins in infected tissues, ZYMV-Ros1 infection of cucurbits did not lead to detectable anthocyanin accumulation. However, the recombinant virus did induce dark red pigmentation in infected tissues of the model plant Nicotiana benthamiana. In this species, ZYMV-Ros1 multiplied efficiently in local inoculated tissue but only a few progeny particles established infection foci in upper leaves. We used this system to analyze the roles of Dicer-like (DCL) genes, core components of plant antiviral RNA silencing pathways, in ZYMV infection. ZYMV-Ros1 local replication was not significantly affected in single DCL knockdown lines nor in double DCL2/4 and triple DCL2/3/4 knockdown lines. ZYMV-Ros1 systemic accumulation was not affected in knockdown lines DCL1, DCL2, and DCL3. However in DCL4 and also in DCL2/4 and DCL2/3/4 knockdown lines, ZYMV-Ros1 systemic accumulation dramatically increased, which highlights the key role of DCL4 in restricting virus systemic movement. The effect of DCL4 on ZYMV systemic movement was confirmed with a wild-type version of the virus.

  4. [The role of Cd-binding proteins and phytochelatins in the formation of cadmium resistance in Nicotiana plumbaginifolia cell lines].

    Science.gov (United States)

    Fenik, S I; Solodushko, V G; Kaliniak, T B; Blium, Ia B

    2007-01-01

    Nicotiana plumbaginifolia callus lines with the equal resistance to cadmium have been produced under different selective conditions--either without inhibition of the phytochelatin synthesis (line Cd-R) or in the presence of the inhibitor butionine sulfoximine (line Cd-Ri). The level of phytochelatin synthesis in the line Cd-R five-fold exceeded the control value and in the line Cd-Ri it was twice as much as in the control. It was shown that in the control line mainly three cadmium-binding proteins are expressed of the molecular weihgts 41, 34 and 19 kD. The common feature of the both resistant lines is the expression of the cadmium-binding proteins of 40, 37 and 19 kD. The resistant lines differ with respect to the synthesis of relatively low-molecular cadmium-binding proteins. The proteins of the molecular weights 12.5, 11.5 and 9 kD are expressed in the line Cd-R, while the proteins of 13 and 10 kD are expressed in the line Cd-Ri. It was supposed that both the phytochelatins and the Cd-binding proteins contribute to the resisitance of N. plumbaginifolia callus lines to cadmium and the lack of the phytochelatins can be equilibrated by the changes in the low-molecular Cd-binding protein synthesis.

  5. Theobroxide Treatment Inhibits Wild Fire Disease Occurrence in Nicotiana benthamiana by the Overexpression of Defense-related Genes

    Directory of Open Access Journals (Sweden)

    Soon Young Ahn

    2013-03-01

    Full Text Available Theobroxide, a novel compound isolated from a fungus Lasiodiplodia theobromae, stimulates potato tuber formation and induces flowering of morning glory by initiating the jasmonic acid synthesis pathway. To elucidate the effect of theobroxide on pathogen resistance in plants, Nicotiana benthamiana plants treated with theobroxide were immediately infiltrated with Pseudomonas syringae pv. tabaci. Exogenous application of theobroxide inhibited development of lesion symptoms, and growth of the bacterial cells was significantly retarded. Semi-quantitative RT-PCRs using the primers of 18 defense-related genes were performed to investigate the molecular mechanisms of resistance. Among the genes, the theobroxide treatment increased the expression of pathogenesis-related protein 1a (PR1a, pathogenesis-related protein 1b (PR1b, glutathione S-transferase (GST, allen oxide cyclase (AOC, and lipoxyganase (LOX. All these data strongly indicate that theobroxide treatment inhibits disease development by faster induction of defense responses, which can be possible by the induction of defense-related genes including PR1a, PR1b, and GST triggered by the elevated jasmonic acid.

  6. Narboh D, a Respiratory Burst Oxidase Homolog in Nicotiana attenuata, is Required for Late Defense Responses After Herbivore Attack

    Institute of Scientific and Technical Information of China (English)

    Jinsong Wu; Lei Wang; Hendrik Wünsche; Ian T.Baldwin

    2013-01-01

    The superoxide (O2-)-generating NADPH oxidases are crucial for the defense of plants against attack from pathogens; however,it remains unknown whether they also mediate responses against chewing insect herbivores.The transcripts of the respiratory burst NADPH oxidase homolog Narboh D in Nicotiana attenuate are rapidly and transiently elicited by wounding,and are amplified when Manduca sexta oral secretions (OS) are added to the wounds.The fatty-acid-amino-acid-conjugates (FACs),demonstrably the major elicitors in M.sexta OS,are responsible for the increase in Narboh D transcripts.Silencing Narboh D significantly reduced reactive oxygen species (ROS) levels after OS elicitation,but neither OS-elicited jasmonic acid (JA) or JA-isoleucine (JA-Ile) bursts,pivotal hormones that regulates plant resistance to herbivores,nor early transcripts of herbivore defense-related genes (NaJAR4 and NaPAL1),were influenced.However,late OS-elicited increases in trypsin proteinase inhibitors (TPIs),as well as the transcript levels of defense genes such as polyphenol oxidase,TPI and Thionin were significantly reduced.In addition,Narboh D-silenced plants were more vulnerable to insect herbivores,especially the larvae of the generalist Spodoptera littoralis.We thus conclude that Narboh D-based defenses play an important role in late herbivore-elicited responses.

  7. Norovirus Narita 104 Virus-Like Particles Expressed in Nicotiana benthamiana Induce Serum and Mucosal Immune Responses

    Directory of Open Access Journals (Sweden)

    Lolita George Mathew

    2014-01-01

    Full Text Available Narita 104 virus is a human pathogen belonging to the norovirus (family Caliciviridae genogroup II. Noroviruses cause epidemic gastroenteritis worldwide. To explore the potential of developing a plant-based vaccine, a plant optimized gene encoding Narita 104 virus capsid protein (NaVCP was expressed transiently in Nicotiana benthamiana using a tobacco mosaic virus expression system. NaVCP accumulated up to approximately 0.3 mg/g fresh weight of leaf at 4 days postinfection. Initiation of hypersensitive response-like symptoms followed by tissue necrosis necessitated a brief infection time and was a significant factor limiting expression. Transmission electron microscopy of plant-derived NaVCP confirmed the presence of fully assembled virus-like particles (VLPs. In this study, an optimized method to express and partially purify NaVCP is described. Further, partially purified NaVCP was used to immunize mice by intranasal delivery and generated significant mucosal and serum antibody responses. Thus, plant-derived Narita 104 VLPs have potential for use as a candidate subunit vaccine or as a component of a multivalent subunit vaccine, along with other genotype-specific plant-derived VLPs.

  8. Norovirus Narita 104 Virus-Like Particles Expressed in Nicotiana benthamiana Induce Serum and Mucosal Immune Responses

    Science.gov (United States)

    Mathew, Lolita George; Herbst-Kralovetz, Melissa M.; Mason, Hugh S.

    2014-01-01

    Narita 104 virus is a human pathogen belonging to the norovirus (family Caliciviridae) genogroup II. Noroviruses cause epidemic gastroenteritis worldwide. To explore the potential of developing a plant-based vaccine, a plant optimized gene encoding Narita 104 virus capsid protein (NaVCP) was expressed transiently in Nicotiana benthamiana using a tobacco mosaic virus expression system. NaVCP accumulated up to approximately 0.3 mg/g fresh weight of leaf at 4 days postinfection. Initiation of hypersensitive response-like symptoms followed by tissue necrosis necessitated a brief infection time and was a significant factor limiting expression. Transmission electron microscopy of plant-derived NaVCP confirmed the presence of fully assembled virus-like particles (VLPs). In this study, an optimized method to express and partially purify NaVCP is described. Further, partially purified NaVCP was used to immunize mice by intranasal delivery and generated significant mucosal and serum antibody responses. Thus, plant-derived Narita 104 VLPs have potential for use as a candidate subunit vaccine or as a component of a multivalent subunit vaccine, along with other genotype-specific plant-derived VLPs. PMID:24949472

  9. Deep sequencing analysis reveals a TMV mutant with a poly(A) tract reduces host defense responses in Nicotiana benthamiana.

    Science.gov (United States)

    Guo, Song; Wong, Sek-Man

    2017-07-15

    Tobacco mosaic virus (TMV) possesses an upstream pseudoknotted domain (UPD), which is important for replication. After substituting the UPD with an internal poly(A) tract (43 nt), a mutant TMV-43A was constructed. TMV-43A replicated slower than TMV and induced a non-lethal mosaic symptom in Nicotiana benthamiana. In this study, deep sequencing was performed to detect the differences of small RNA profiles between TMV- and TMV-43A-infected N. benthamiana. The results showed that TMV-43A produced lesser amount of virus-derived interfering RNAs (vsiRNAs) than that of TMV. However, the distributions of vsiRNAs generation hotspots between TMV and TMV-43A were similar. Expression of genes related to small RNA biogenesis in TMV-43A-infected N. benthamiana was significantly lower than that of TMV, which leads to generation of lesser vsiRNAs. The expressions of host defense response genes were up-regulated after TMV infection, as compared to TMV-43A-infected plants. Host defense response to TMV-43A infection was lower than that to TMV. The absence of UPD might contribute to the reduced host response to TMV-43A. Our study provides valuable information in the role of the UPD in eliciting host response genes after TMV infection in N. benthamiana. (187 words). Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Temporal and spatial resolution of activated plant defense responses in leaves of Nicotiana benthamiana infected with Dickeya dadantii

    Directory of Open Access Journals (Sweden)

    María Luisa ePérez-Bueno

    2016-01-01

    Full Text Available The necrotrophic bacteria Dickeya dadantii is the causal agent of soft-rot disease in a broad range of hosts. The model plant Nicotiana benthamiana, commonly used as experimental host for a very broad range of plant pathogens, is susceptible to infection by D. dadantii. The inoculation with D. dadantii at high dose seems to overcome the plant defense capacity, inducing maceration and death of the tissue, although restricted to the infiltrated area. By contrast, the output of the defense response to low dose inoculation is inhibition of maceration and limitation in the growth, or even eradication, of bacteria. Responses of tissue invaded by bacteria (neighbouring the infiltrated areas after 2-3 days post-inoculation included: i inhibition of photosynthesis in terms of photosystem II efficiency; ii activation of energy dissipation as non-photochemical quenching in photosystem II, which is related to the activation of plant defense mechanisms; and iii accumulation of secondary metabolites in cell walls of the epidermis (lignins and the apoplast of the mesophyll (phytoalexins. Infiltrated tissues showed an increase in the content of the main hormones regulating stress responses, including abscisic acid (ABA, jasmonic acid (JA and salicylic acid (SA. We propose a mechanism involving the three hormones by which N. benthamiana could activate an efficient defense response against D. dadantii.

  11. The combination of plant translational enhancers and terminator increase the expression of human glucocerebrosidase in Nicotiana benthamiana plants.

    Science.gov (United States)

    Limkul, Juthamard; Misaki, Ryo; Kato, Ko; Fujiyama, Kazuhito

    2015-11-01

    Gaucher's disease is a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCase). It is currently treated by enzyme replacement therapy using recombinant GCase expressed in mammalian cells. Plant production systems are among the most attractive alternatives for pharmaceutical protein production due to such advantages as low-cost, high-scalability, and safety from human pathogen contamination. Because of its high biomass yield, Nicotiana benthamiana could be an economical recombinant GCase production system. In this study, a translational enhancer and suitable terminator were utilized to obtain a powerful expression system for GCase production in N. benthamiana plants. Six plasmid constructs were used. The highest activity of 44.5units/mg protein (after subtraction of endogenous glucosidase activity of the wild-type plant) was observed in transgenic plants transformed with pAt-GC-HSP combined with a 5' untranslated region of the Arabidopsis alcohol dehydrogenase gene with the Arabidopsis heat shock protein terminator. These transgenic plant lines could pave the way to a stable plant-production system for low-cost, high-yield human GCase production.

  12. Phytophthora nicotianae var. nicotianae on tomatoes

    NARCIS (Netherlands)

    Weststeijn, G.

    1973-01-01

    Around 1960 some disorders which initially were considered to be of a physiological nature were found in tomato plants grown in glasshouses in the Netherlands. One complex of symptoms was a brown rot of the lateral roots and the tap root, often followed by decomposition of the stem base ti

  13. Phytophthora nicotianae var. nicotianae on tomatoes

    NARCIS (Netherlands)

    Weststeijn, G.

    1973-01-01

    Around 1960 some disorders which initially were considered to be of a physiological nature were found in tomato plants grown in glasshouses in the Netherlands. One complex of symptoms was a brown rot of the lateral roots and the tap root, often followed by decomposition of the stem base tissue and d

  14. Phytophthora nicotianae var. nicotianae on tomatoes

    NARCIS (Netherlands)

    Weststeijn, G.

    1973-01-01

    Around 1960 some disorders which initially were considered to be of a physiological nature were found in tomato plants grown in glasshouses in the Netherlands. One complex of symptoms was a brown rot of the lateral roots and the tap root, often followed by decomposition of the stem base

  15. Salicylic acid and jasmonic acid are essential for systemic resistance against tobacco mosaic virus in Nicotiana benthamiana.

    Science.gov (United States)

    Zhu, Feng; Xi, De-Hui; Yuan, Shu; Xu, Fei; Zhang, Da-Wei; Lin, Hong-Hui

    2014-06-01

    Systemic resistance is induced by pathogens and confers protection against a broad range of pathogens. Recent studies have indicated that salicylic acid (SA) derivative methyl salicylate (MeSA) serves as a long-distance phloem-mobile systemic resistance signal in tobacco, Arabidopsis, and potato. However, other experiments indicate that jasmonic acid (JA) is a critical mobile signal. Here, we present evidence suggesting both MeSA and methyl jasmonate (MeJA) are essential for systemic resistance against Tobacco mosaic virus (TMV), possibly acting as the initiating signals for systemic resistance. Foliar application of JA followed by SA triggered the strongest systemic resistance against TMV. Furthermore, we use a virus-induced gene-silencing-based genetics approach to investigate the function of JA and SA biosynthesis or signaling genes in systemic response against TMV infection. Silencing of SA or JA biosynthetic and signaling genes in Nicotiana benthamiana plants increased susceptibility to TMV. Genetic experiments also proved the irreplaceable roles of MeSA and MeJA in systemic resistance response. Systemic resistance was compromised when SA methyl transferase or JA carboxyl methyltransferase, which are required for MeSA and MeJA formation, respectively, were silenced. Moreover, high-performance liquid chromatography-mass spectrometry analysis indicated that JA and MeJA accumulated in phloem exudates of leaves at early stages and SA and MeSA accumulated at later stages, after TMV infection. Our data also indicated that JA and MeJA could regulate MeSA and SA production. Taken together, our results demonstrate that (Me)JA and (Me)SA are required for systemic resistance response against TMV.

  16. Silencing of DS2 aminoacylase-like genes confirms basal resistance to Phytophthora infestans in Nicotiana benthamiana.

    Science.gov (United States)

    Nakano, Masahito; Nishihara, Masahiro; Yoshioka, Hirofumi; Ohnishi, Kouhei; Hikichi, Yasufumi; Kiba, Akinori

    2014-01-01

    Nicotiana benthamiana is a potential host to several plant pathogens, and immature leaves of N. benthamiana are susceptible to Phytophthora infestans. In contrast, mature leaves of N. benthamiana are weakly susceptible and show basal resistance to P. infestans. We screened a gene-silenced mature plant showing high resistance to P. infestans, designated as DS2 (Disease suppression 2). The deduced amino acid sequence of cDNA responsible for DS2 encoded a putative aminoacylase. Growth of P. infestans decreased in DS2 plants. Trypan blue staining revealed inhibited hyphae growth of P. infestans with an increased number of dead cells under the penetration site in DS2 plants. Consistent with growth inhibition of P. infestans, defense responses such as reactive oxygen generation and expression of a salicylic acid-dependent PR-1a increased markedly in DS2 plants compared with that of control plants. DS2 phenotype was compromised in NahG plants, suggesting DS2 phenotype depends on the salicylic acid signaling pathway. Accelerated defense response was observed in DS2 plants elicited by INF1 elicitin as well as by NbMEK2(DD), which is the constitutive active form of NbMEK2, and act as a downstream regulator of INF1 perception. On the other hand, INF1- and NbMEK2(DD)-induced defense responses were prevented by DS2-overexpressing transgenic tobacco. These results suggest that DS2 negatively regulates plant defense responses against P. infestans via NbMEK2 and SA-dependent signaling pathway in N. benthamiana.

  17. Growth and stress response in Arabidopsis thaliana, Nicotiana benthamiana, Glycine max, Solanum tuberosum and Brassica napus cultivated under polychromatic LEDs.

    Science.gov (United States)

    Janda, Martin; Navrátil, Oldřich; Haisel, Daniel; Jindřichová, Barbora; Fousek, Jan; Burketová, Lenka; Čeřovská, Noemi; Moravec, Tomáš

    2015-01-01

    The use of light emitting diodes (LEDs) brings several key advantages over existing illumination technologies for indoor plant cultivation. Among these are that LEDs have predicted lifetimes from 50-100.000 hours without significant drops in efficiency and energy consumption is much lower compared to traditional fluorescent tubes. Recent advances allow LEDs to be used with customized wavelengths for plant growth. However, most of these LED growth systems use mixtures of chips emitting in several narrow wavelengths and frequently they are not compatible with existing infrastructures. This study tested the growth of five different plant species under phosphor coated LED-chips fitted into a tube with a standard G13 base that provide continuous visible light illumination with enhanced blue and red light. The LED system was characterized and compared with standard fluorescence tubes in the same cultivation room. Significant differences in heat generation between LEDs and fluorescent tubes were clearly demonstrated. Also, LED lights allowed for better control and stability of preset conditions. Physiological properties such as growth characteristics, biomass, and chlorophyll content were measured and the responses to pathogen assessed for five plant species (both the model plants Arabidopsis thaliana, Nicotiana bentamiana and crop species potato, oilseed rape and soybean) under the different illumination sources. We showed that polychromatic LEDs provide light of sufficient quality and intensity for plant growth using less than 40% of the electricity required by the standard fluorescent lighting under test. The tested type of LED installation provides a simple upgrade pathway for existing infrastructure for indoor plant growth. Interestingly, individual plant species responded differently to the LED lights so it would be reasonable to test their utility to any particular application.

  18. Elicitation of hypersensitive responses in Nicotiana glutinosa by the suppressor of RNA silencing protein P0 from poleroviruses.

    Science.gov (United States)

    Wang, Ken-Der; Empleo, Roman; Nguyen, Tan Tri V; Moffett, Peter; Sacco, Melanie Ann

    2015-06-01

    Plant disease resistance (R) proteins that confer resistance to viruses recognize viral gene products with diverse functions, including viral suppressors of RNA silencing (VSRs). The P0 protein from poleroviruses is a VSR that targets the ARGONAUTE1 (AGO1) protein for degradation, thereby disrupting RNA silencing and antiviral defences. Here, we report resistance against poleroviruses in Nicotiana glutinosa directed against Turnip yellows virus (TuYV) and Potato leafroll virus (PLRV). The P0 proteins from TuYV (P0(T) (u) ), PLRV (P0(PL) ) and Cucurbit aphid-borne yellows virus (P0(CA) ) were found to elicit a hypersensitive response (HR) in N. glutinosa accession TW59, whereas other accessions recognized P0(PL) only. Genetic analysis showed that recognition of P0(T) (u) by a resistance gene designated RPO1 (Resistance to POleroviruses 1) is inherited as a dominant allele. Expression of P0 from a Potato virus X (PVX) expression vector transferred recognition to the recombinant virus on plants expressing RPO1, supporting P0 as the unique Polerovirus factor eliciting resistance. The induction of HR required a functional P0 protein, as P0(T) (u) mutants with substitutions in the F-box motif that abolished VSR activity were unable to elicit HR. We surmised that the broad P0 recognition seen in TW59 and the requirement for the F-box protein motif could indicate detection of P0-induced AGO1 degradation and disruption of RNA silencing; however, other viral silencing suppressors, including the PVX P25 that also causes AGO1 degradation, failed to elicit HR in N. glutinosa. Investigation of P0 elicitation of RPO1 could provide insight into P0 activities within the cell that trigger resistance. © 2014 BSPP AND JOHN WILEY & SONS LTD.

  19. High levels of jasmonic acid antagonize the biosynthesis of gibberellins and inhibit the growth of Nicotiana attenuata stems.

    Science.gov (United States)

    Heinrich, Maria; Hettenhausen, Christian; Lange, Theo; Wünsche, Hendrik; Fang, Jingjing; Baldwin, Ian T; Wu, Jianqiang

    2013-02-01

    Hormones play pivotal roles in regulating plant development, growth, and stress responses, and cross-talk among different hormones fine-tunes various aspects of plant physiology. Jasmonic acid (JA) is important for plant defense against herbivores and necrotic fungi and also regulates flower development; in addition, Arabidopsis mutants over-producing JA usually have stunted stems and wound-induced jasmonates suppress Arabidopsis growth, suggesting that JA is also involved in stem elongation. Gibberellins (GAs) promote stem and leaf growth and modulate seed germination, flowering time, and the development of flowers, fruits, and seeds. However, little is known about the interaction between the JA and GA pathways. Two calcium-dependent protein kinases, CDPK4 and CDPK5, are important suppressors of JA accumulation in a wild tobacco species, Nicotiana attenuata. The stems of N. attenuata silenced in CDPK4 and CDPK5 (irCDPK4/5 plants) had dramatically increased levels of JA and exhibited stunted elongation and had very high contents of secondary metabolites. Genetic analysis indicated that the high JA levels in irCDPK4/5 stems accounted for the suppressed stem elongation and the accumulation of secondary metabolites. Supplementation of GA(3) to irCDPK4/5 plants largely restored normal stem growth to wild-type levels. Measures of GA levels indicated that over-accumulation of JA in irCDPK4/5 stems inhibited the biosynthesis of GAs. Finally, we show that JA antagonizes GA biosynthesis by strongly inhibiting the transcript accumulation of GA20ox and possibly GA13ox, the key genes in GA production, demonstrating that high JA levels antagonize GA biosynthesis in stems.

  20. Pithy protection: Nicotiana attenuata's jasmonic acid-mediated defenses are required to resist stem-boring weevil larvae.

    Science.gov (United States)

    Diezel, Celia; Kessler, Danny; Baldwin, Ian T

    2011-04-01

    Folivory is the best studied plant-herbivore interaction, but it is unclear whether the signaling and resistance traits important for the defense of leaves are also important for other plant parts. Larvae of the tobacco stem weevil, Trichobaris mucorea, burrow into stems of Nicotiana attenuata and feed on the pith. Transgenic N. attenuata lines silenced in signaling and foliar defense traits were evaluated in a 2-year field study for resistance against attack by naturally occurring T. mucorea larva. Plants silenced in early jasmonic acid (JA) biosynthesis (antisense [as]-lipoxygenase3 [lox3]; inverted repeat [ir]-allene oxide cyclase), JA perception (as-coronatine insensitive1), proteinase inhibitors (ir-pi), and nicotine (ir-putrescine methyl-transferase) direct defenses and lignin (ir-cad) biosynthesis were infested more frequently than wild-type plants. Plants unable to emit C(6) aldehydes (as-hpl) had lower infestation rates, while plants silenced in late steps in JA biosynthesis (ir-acyl-coenzyme A oxidase, ir-opr) and silenced in diterpene glycoside production (ir-geranylgeranyl pyrophosphate synthase) did not differ from wild type. Pith choice assays revealed that ir-putrescine methyl-transferase, ir-coronatine insensitive1, and ir-lox3 pith, which all had diminished nicotine levels, were preferred by larvae compared to wild-type pith. The lack of preference for ir-lox2 and ir-cad piths, suggest that oviposition attraction and vascular defense, rather than pith palatability accounts for the higher attack rates observed for these plants. We conclude that traits that influence a plant's apparency, stem hardness, and pith direct defenses all contribute to resistance against this herbivore whose attack can be devastating to N. attenuata's fitness.

  1. Differential RNAi responses of Nicotiana benthamiana individuals transformed with a hairpin-inducing construct during Plum pox virus challenge.

    Science.gov (United States)

    Montes, Christian; Castro, Álvaro; Barba, Paola; Rubio, Julia; Sánchez, Evelyn; Carvajal, Denisse; Aguirre, Carlos; Tapia, Eduardo; DelÍ Orto, Paola; Decroocq, Veronique; Prieto, Humberto

    2014-10-01

    Gene silencing and large-scale small RNA analysis can be used to develop RNA interference (RNAi)-based resistance strategies for Plum pox virus (PPV), a high impact disease of Prunus spp. In this study, a pPPViRNA hairpin-inducing vector harboring two silencing motif-rich regions of the PPV coat protein (CP) gene was evaluated in transgenic Nicotiana benthamiana (NB) plants. Wild-type NB plants infected with a chimeric PPV virus (PPV::GFP) exhibited affected leaves with mosaic chlorosis congruent to GFP fluorescence at 21 day post-inoculation; transgenic lines depicted a range of phenotypes from fully resistant to susceptible. ELISA values and GFP fluorescence intensities were used to select transgenic-resistant (TG-R) and transgenic-susceptible (TG-S) lines for further characterization of small interfering RNAs (siRNAs) by large-scale small RNA sequencing. In infected TG-S and untransformed (WT) plants, the observed siRNAs were nearly exclusively 21- and 22-nt siRNAs that targeted the whole PPV::GFP genome; 24-nt siRNAs were absent in these individuals. Challenged TG-R plants accumulated a full set of 21- to 24-nt siRNAs that were primarily associated with the selected motif-rich regions, indicating that a trans-acting siRNAs process prevented viral multiplication. BLAST analysis identified 13 common siRNA clusters targeting the CP gene. 21-nt siRNA sequences were associated with the 22-nt siRNAs and the scarce 23- and 24-nt molecules in TG-S plants and with most of the observed 22-, 23-, and 24-nt siRNAs in TG-R individuals. These results validate the use of a multi-hot spot silencing vector against PPV and elucidate the molecules by which hairpin-inducing vectors initiate RNAi in vivo.

  2. Genetic and molecular requirements for function of the Pto/Prf effector recognition complex in tomato and Nicotiana benthamiana.

    Science.gov (United States)

    Balmuth, Alexi; Rathjen, John P

    2007-09-01

    The Pto gene of tomato (Solanum lycopersicum) confers specific recognition of the unrelated bacterial effector proteins AvrPto and AvrPtoB. Pto resides in a constitutive molecular complex with the nucleotide binding site-leucine rich repeats protein Prf. Prf is absolutely required for specific recognition of both effectors. Here, using stable transgenic lines, we show that expression of Pto from its genomic promoter in susceptible tomatoes was sufficient to complement recognition of Pseudomonas syringae pv. tomato (Pst) bacteria expressing either avrPto or avrPtoB. Pto kinase activity was absolutely required for specific immunity. Expression of the Pto N-myristoylation mutant, pto(G2A), conferred recognition of Pst (avrPtoB), but not Pst (avrPto), although bacterial growth in these lines was intermediate between resistant and susceptible lines. Overexpression of pto(G2A) complemented recognition of avrPto. Transgenic tomato plants overexpressing wild-type Pto exhibited constitutive growth phenotypes, but these were absent in lines overexpressing pto(G2A). Therefore, Pto myristoylation is a quantitative factor for effector recognition in tomato, but is absolutely required for overexpression phenotypes. Native expression of Pto in the heterologous species Nicotiana benthamiana did not confer resistance to P. syringae pv. tabaci (Pta) expressing avrPto or avrPtoB, but recognition of both effectors was complemented by Prf co-expression. Thus, specific resistance conferred solely by Pto in N. benthamiana is an artefact of overexpression. Finally, pto(G2A) did not confer recognition of either avrPto or avrPtoB in N. benthamiana, regardless of the presence of Prf. Thus, co-expression of Prf in N. benthamiana complements many but not all aspects of normal Pto function.

  3. Genetic modification of alternative respiration in Nicotiana benthamiana affects basal and salicylic acid-induced resistance to potato virus X

    Directory of Open Access Journals (Sweden)

    Verchot-Lubicz Jeanmarie

    2011-02-01

    Full Text Available Abstract Background Salicylic acid (SA regulates multiple anti-viral mechanisms, including mechanism(s that may be negatively regulated by the mitochondrial enzyme, alternative oxidase (AOX, the sole component of the alternative respiratory pathway. However, studies of this mechanism can be confounded by SA-mediated induction of RNA-dependent RNA polymerase 1, a component of the antiviral RNA silencing pathway. We made transgenic Nicotiana benthamiana plants in which alternative respiratory pathway capacity was either increased by constitutive expression of AOX, or decreased by expression of a dominant-negative mutant protein (AOX-E. N. benthamiana was used because it is a natural mutant that does not express a functional RNA-dependent RNA polymerase 1. Results Antimycin A (an alternative respiratory pathway inducer and also an inducer of resistance to viruses and SA triggered resistance to tobacco mosaic virus (TMV. Resistance to TMV induced by antimycin A, but not by SA, was inhibited in Aox transgenic plants while SA-induced resistance to this virus appeared to be stronger in Aox-E transgenic plants. These effects, which were limited to directly inoculated leaves, were not affected by the presence or absence of a transgene constitutively expressing a functional RNA-dependent RNA polymerase (MtRDR1. Unexpectedly, Aox-transgenic plants infected with potato virus X (PVX showed markedly increased susceptibility to systemic disease induction and virus accumulation in inoculated and systemically infected leaves. SA-induced resistance to PVX was compromised in Aox-transgenic plants but plants expressing AOX-E exhibited enhanced SA-induced resistance to this virus. Conclusions We conclude that AOX-regulated mechanisms not only play a role in SA-induced resistance but also make an important contribution to basal resistance against certain viruses such as PVX.

  4. Plasticity in allocation of nicotine to reproductive parts inNicotiana attenuata.

    Science.gov (United States)

    Baldwin, I T; Karb, M J

    1995-07-01

    Although little is known about the patterns of chemical defense allocation in reproductive tissues, optimal defense theory predicts a high constitutive allocation due to the tissues' high fitness value. To examine this prediction, we quantified the short- and long-term changes in the nicotine pools of reproductive tissues in response to both floral and leaf damage. Recently opened flowers (stage 5 capsules) do not alter their nicotine pools within a day in response to herbivory byManduca sexta larvae or mechanical damage to the corolla. Similarly, leaf damage during both vegetative and reproductive growth does not influence the nicotine pools of the first three stage-5 capsules produced. However, the nicotine pools of capsules produced later in reproductive growth were significantly larger (1.2- to 1.9-fold) on plants with leaf damage. These differences in floral nicotine pools were a result of both increases in nicotine pools of capsules on damaged plants and decreases in the nicotine pools of capsules on undamaged plants during reproductive growth. Leaf damage did not affect the rate of capsule maturation or the mass of stage-5 capsules at any time during reproductive growth. An allometric analysis of nicotine pools and biomass of reproductive parts in all stages of development from damaged and undamaged plants demonstrates that damaged plants allocated a significantly larger quantity of nicotine to reproductive parts in all stages of development than did undamaged plants. Given that nicotine is thought to be synthesized in the roots and transported to leaves and reproductive parts, nicotine could be allocated to reproductive parts in proportion to the number of developing capsules on a plant. We excised the first 27 stage-5 capsules on plants with and without leaf damage, with the expectation that plants with fewer capsules would allocate a larger amount of nicotine to the remaining capsules. In contrast to the prediction of this passive allocation model, floral

  5. 烟草 NtWRKY40在植物应答病毒侵染过程中的作用%Roles of Nicotiana tobacum NtWRKY40 in Plant Responding to Virus Infection

    Institute of Scientific and Technical Information of China (English)

    刘晶晶; 程春玲; 席玉珍; 魏书

    2016-01-01

    tobacco than non-infected control. No significant change in NtWRKY40 transcription levels was noted in the NtWRKY40 over-expressed tobacco after virus infection. NtWRKY40 over-expressed tobaccos were more sensitive to TMV infection than non-transgenic wild type. The data suggested that NtWRKY40 was a negative regulator in responding to TMV. In addition,an artificial miRNA vector amiR167-PVY was constructed,for silencing the coat protein gene of Potato virus Y,using the miR167 precursor of Nicotiana tabacum as backbone and cauliflower mosaic virus 35S promoter. Over-expression of amiR167-PVY exhibited inhibitory effect on the virus. The results provide the basis for the further research on amiRNA-mediated virus resistance mechanism,and contributes to antiviral transgenic plants cultivation with the optimal target sequence.

  6. Nectar sugars and amino acids in day- and night-flowering Nicotiana species are more strongly shaped by pollinators’ preferences than organic acids and inorganic ions

    Science.gov (United States)

    Tiedge, Kira; Lohaus, Gertrud

    2017-01-01

    Floral nectar contains mainly sugars but also amino acids, organic acids, inorganic ions and secondary compounds to attract pollinators. The genus Nicotiana exhibits great diversity among species in floral morphology, flowering time, nectar compositions, and predominant pollinators. We studied nectar samples of 20 Nicotiana species, composed equally of day- and night-flowering plants and attracting different groups of pollinators (e.g. hummingbirds, moths or bats) to investigate whether sugars, amino acids, organic acids and inorganic ions are influenced by pollinator preferences. Glucose, fructose and sucrose were the only sugars found in the nectar of all examined species. Sugar concentration of the nectar of day-flowering species was 20% higher and amino acid concentration was 2-3-fold higher compared to the nectar of night-flowering species. The sucrose-to-hexose ratio was significantly higher in night-flowering species and the relative share of sucrose based on the total sugar correlated with the flower tube length in the nocturnal species. Flowers of different tobacco species contained varying volumes of nectar which led to about 150-fold higher amounts of total sugar per flower in bat- or sunbird-pollinated species than in bee-pollinated or autogamous species. This difference was even higher for total amino acids per flower (up to 1000-fold). As a consequence, some Nicotiana species invest large amounts of organic nitrogen for certain pollinators. Higher concentrations of inorganic ions, predominantly anions, were found in nectar of night-flowering species. Therefore, higher anion concentrations were also associated with pollinator types active at night. Malate, the main organic acid, was present in all nectar samples but the concentration was not correlated with pollinator type. In conclusion, statistical analyses revealed that pollinator types have a stronger effect on nectar composition than phylogenetic relations. In this context, nectar sugars and amino

  7. Nectar sugars and amino acids in day- and night-flowering Nicotiana species are more strongly shaped by pollinators' preferences than organic acids and inorganic ions.

    Science.gov (United States)

    Tiedge, Kira; Lohaus, Gertrud

    2017-01-01

    Floral nectar contains mainly sugars but also amino acids, organic acids, inorganic ions and secondary compounds to attract pollinators. The genus Nicotiana exhibits great diversity among species in floral morphology, flowering time, nectar compositions, and predominant pollinators. We studied nectar samples of 20 Nicotiana species, composed equally of day- and night-flowering plants and attracting different groups of pollinators (e.g. hummingbirds, moths or bats) to investigate whether sugars, amino acids, organic acids and inorganic ions are influenced by pollinator preferences. Glucose, fructose and sucrose were the only sugars found in the nectar of all examined species. Sugar concentration of the nectar of day-flowering species was 20% higher and amino acid concentration was 2-3-fold higher compared to the nectar of night-flowering species. The sucrose-to-hexose ratio was significantly higher in night-flowering species and the relative share of sucrose based on the total sugar correlated with the flower tube length in the nocturnal species. Flowers of different tobacco species contained varying volumes of nectar which led to about 150-fold higher amounts of total sugar per flower in bat- or sunbird-pollinated species than in bee-pollinated or autogamous species. This difference was even higher for total amino acids per flower (up to 1000-fold). As a consequence, some Nicotiana species invest large amounts of organic nitrogen for certain pollinators. Higher concentrations of inorganic ions, predominantly anions, were found in nectar of night-flowering species. Therefore, higher anion concentrations were also associated with pollinator types active at night. Malate, the main organic acid, was present in all nectar samples but the concentration was not correlated with pollinator type. In conclusion, statistical analyses revealed that pollinator types have a stronger effect on nectar composition than phylogenetic relations. In this context, nectar sugars and amino

  8. Non-host resistance induced by the Xanthomonas effector XopQ is widespread within the genus Nicotiana and functionally depends on EDS1

    Directory of Open Access Journals (Sweden)

    Norman Adlung

    2016-11-01

    Full Text Available Most Gram-negative plant pathogenic bacteria translocate effector proteins (T3Es directly into plant cells via a conserved type III secretion system, which is essential for pathogenicity in susceptible plants. In resistant plants, recognition of some T3Es is mediated by corresponding resistance (R genes or R proteins and induces effector triggered immunity (ETI that often results in programmed cell death reactions. The identification of R genes and understanding their evolution/distribution bears great potential for the generation of resistant crop plants. We focus on T3Es from Xanthomonas campestris pv. vesicatoria (Xcv, the causal agent of bacterial spot disease on pepper and tomato plants. Here, 86 Solanaceae lines mainly of the genus Nicotiana were screened for phenotypical reactions after Agrobacterium tumefaciens-mediated transient expression of 21 different Xcv effectors to (i identify new plant lines for T3E characterization, (ii analyze conservation/evolution of putative R genes and (iii identify promising plant lines as repertoire for R-gene isolation. The effectors provoked different reactions on closely related plant lines indicative of a high variability and evolution rate of potential R genes. In some cases, putative R genes were conserved within a plant species but not within superordinate phylogenetical units. Interestingly, the effector XopQ was recognized by several Nicotiana spp. lines, and Xcv infection assays revealed that XopQ is a host range determinant in many Nicotiana species. Non-host resistance against Xcv and XopQ recognition in N. benthamiana required EDS1, strongly suggesting the presence of a TIR domain-containing XopQ-specific R protein in these plant lines. XopQ is a conserved effector among most xanthomonads, pointing out the XopQ-recognizing RxopQ as candidate for targeted crop improvement.

  9. 烟草黑胫病菌产孢性状的研究%Study on the Producing Spores Properties of Phytophthora Parasitica var nicotianae

    Institute of Scientific and Technical Information of China (English)

    周志成

    2005-01-01

    研究了湖南烟区烟草黑胫病菌(Phytophthora parasitica var nicotianae)分离菌株的产孢性状.结果表明,Py96-1和Py96-2两菌株用燕麦片培养基,在25~28℃温度下,培养21~28 d,产孢菌丝生长最好,菌丝茂密,产生孢子囊多.

  10. The hrpZ Gene of Pseudomonas syringae pv. phaseolicola Enhances Resistance to Rhizomania Disease in Transgenic Nicotiana benthamiana and Sugar Beet

    OpenAIRE

    Ourania I Pavli; Kelaidi, Georgia I.; Tampakaki, Anastasia P.; Skaracis,George N.

    2011-01-01

    To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yellow vein virus (BNYVV), Nicotiana benthamiana plants were constructed to express the harpin of Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)). The HrpZ protein was expressed as an N-terminal fusion to the PR1 signal peptide (SP/HrpZ) to direct harpin accumulation to the plant apoplast. Transgene integration was verified by mPCR in all primary transformants (T0), while immunoblot analysis confir...

  11. Comparative in vitro inhibitory effects of cold extracts of some ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-09-17

    Sep 17, 2008 ... Cold extracts of five fungicidal plants (Aloe barbadensis, Azdrichta indica, Nicotiana tabacum, Tridax precubens and ... from Cam- wood has been used to control yam tuber rot caused by .... Seed yam production by minisett ...

  12. Main: TACBBFNTEAS4 [PLACE

    Lifescience Database Archive (English)

    Full Text Available ty; EAS is involved in sesquiterpene phytoalexin biosynthesis; Found between -245 and -232; Appears to function as a silence...cBBF; sesquiterpene; phytoalexin; silencer; repressor; tobacco (Nicotiana tabacum) ACTCTACAGTACTC ...

  13. Arabidopsis CDS blastp result: AK073850 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK073850 J033073F11 At4g35785.2 transformer serine/arginine-rich ribonucleoprotein, putative similar to tran...sformer-SR ribonucleoprotein [Nicotiana tabacum] gi|1781299|emb|CAA70700 4e-26 ...

  14. Arabidopsis CDS blastp result: AK103177 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103177 J033121H17 At4g35785.2 transformer serine/arginine-rich ribonucleoprotein, putative similar to tran...sformer-SR ribonucleoprotein [Nicotiana tabacum] gi|1781299|emb|CAA70700 1e-25 ...

  15. Main: -141NTG13 [PLACE

    Lifescience Database Archive (English)

    Full Text Available pression of GST isoenzymes, especially in root tip meristems; TGA1a; G13; ASF1; ASF-1; bZip; xenobiotic stress; root; meristem; tobacco (Nicotiana tabacum) GCTTTTGATGACTTCAAACAC ...

  16. Main: PASNTPARA [PLACE

    Lifescience Database Archive (English)

    Full Text Available PASNTPARA S000336 7-Sep-2000 (last modified) seki pas; as-1-related element found i...eness; Located between -68 and -49; parA; pas; cadmium; as-1; tobacco (Nicotiana tabacum) TTACGCAAGCAATGACATCT ...

  17. Cytosolic Proteins From Tobacco Pollen Tubes That Crosslink Microtubules and Actin Filaments In Vitro Are Metabolic Enzymes

    NARCIS (Netherlands)

    Romagnoli, Silvia; Faleri, Claudia; Bini, Luca; Baskin, Tobias I.; Cresti, Mauro

    2010-01-01

    In plant cells, many processes require cooperative action of both microtubules and actin filaments, but proteins mediating interactions between these cytoskeletal members are mostly undiscovered. Here, we attempt to identify such proteins by affinity purification. Cytosol from Nicotiana tabacum (tob

  18. A concise synthesis of optically active solanacol, the germination stimulant for seeds of root parasitic weeds.

    Science.gov (United States)

    Kumagai, Hiroshi; Fujiwara, Mami; Kuse, Masaki; Takikawa, Hirosato

    2015-01-01

    Solanacol, isolated from tobacco (Nicotiana tabacum L.), is a germination stimulant for seeds of root parasitic weeds. A concise synthesis of optically active solanacol has been achieved by employing enzymatic resolution as a key step.

  19. Ergosterol-induced sesquiterpenoid synthesis in tobacco Cells

    CSIR Research Space (South Africa)

    Tugizimana, F

    2012-02-01

    Full Text Available molecule to trigger defence mechanisms. Here we investigated the effect of ergosterol on the secondary metabolites in tobacco (Nicotiana tabacum) cells by profiling the induced sesquiterpenoids. Suspensions of tobacco cells were treated with different...

  20. Induction of systemic disease resistance in Nicotiana benthamiana by the cyclodipeptides cyclo (l-Pro-l-Pro) and cyclo (d-Pro-d-Pro).

    Science.gov (United States)

    Wu, Liming; Wu, Huijun; Chen, Lina; Zhang, Hongyue; Gao, Xuewen

    2017-01-01

    Cyclodipeptides, formed from two amino acids by cyclodehydration, are produced naturally by many organisms, and are known to possess a large number of biological activities. In this study, we found that cyclo (l-Pro-l-Pro) and cyclo (d-Pro-d-Pro) (where Pro is proline) could induce defence responses and systemic resistance in Nicotiana benthamiana. Treatment with the two cyclodipeptides led to a reduction in disease severity by Phytophthora nicotianae and Tobacco mosaic virus (TMV) infections compared with controls. Both cyclopeptides triggered stomatal closure, induced reactive oxygen species production and stimulated cytosolic calcium ion and nitric oxide production in guard cells. In addition, the application of cyclodipeptides significantly up-regulated the expression of the plant defence gene PR-1a and the PR-1a protein, and increased cellular salicylic acid (SA) levels. These results suggest that the SA-dependent defence pathway is involved in cyclodipeptide-mediated pathogen resistance in N. benthamiana. We report the systemic resistance induced by cyclodipeptides, which sheds light on the potential of cyclodipeptides for the control of plant diseases.

  1. MORFOMETRÍA COMPARADA DE SEMILLAS DE NICOTIANA (SOLANACEAE E IDENTIFICACIÓN DE SEMILLAS CARBONIZADAS PROVENIENTES DE UN SITIO ARQUEOLÓGICO EN CHILE CENTRAL

    Directory of Open Access Journals (Sweden)

    M. Teresa Planella

    2012-01-01

    Full Text Available La presencia frecuente de pipas para fumar en sitios arqueológicos del Período Alfarero Temprano deChile central y las evidencias en relación con la costumbre de fumar especies de Nicotiana halladas en sitios prehispánicos de otros lugares de las Américas, muestran la necesidad de contar con una metodología para identificar las especies de este género usadas en Chile. En este trabajo se ha realizado un estudio morfométrico en semillas de especies de Nicotiana que sirve de referencia para comparaciones con semillas de origen arqueológico. La forma y tamaño de la semilla, el patrón de ornamentación dado por las células epidérmicas y la ubicación del hilum resultaron ser caracteres relevantes para identificaciones confiables. Utilizando estos caracteres, se determinaron como N. corymbosa a las semillas recuperadas en el sitio arqueológico Las Morrenas 1, ubicado en Chile central.

  2. A genetic system for Citrus Tristeza Virus using the non-natural host Nicotiana benthamiana: an update

    Directory of Open Access Journals (Sweden)

    Silvia eAmbrós

    2013-07-01

    Full Text Available In nature Citrus tristeza virus (CTV, genus Closterovirus, infects only the phloem cells of species of Citrus and related genera. Finding that the CTV T36 strain replicated in Nicotiana benthamiana (NB protoplasts and produced normal virions allowed development of the first genetic system based on protoplast transfection with RNA transcribed from a full-genome cDNA clone, a laborious and uncertain system requiring several months for each experiment. We developed a more efficient system based on agroinfiltration of NB leaves with CTV-T36-based binary plasmids, which caused systemic infection in this non-natural host within a few weeks yielding in the upper leaves enough CTV virions to readily infect citrus by slash inoculation. Stem agroinoculation of citrus and NB plants with oncogenic strains of Agrobacterium tumefaciens carrying a CTV-T36 binary vector with a GUS marker, induced GUS positive galls in both species. However, while most NB tumours were CTV positive and many plants became systemically infected, no coat protein or viral RNA was detected in citrus tumours, even though CTV cDNA was readily detected by PCR in the same galls. This finding suggests i strong silencing or CTV RNA processing in transformed cells impairing infection progress, and ii the need for using NB as an intermediate host in the genetic system. To maintain CTV-T36 in NB or assay other CTV genotypes in this host, we also tried to graft-transmit the virus from infected to healthy NB, or to mechanically inoculate NB leaves with virion extracts. While these trials were mostly unsuccessful on non-treated NB plants, agroinfiltration with silencing suppressors enabled for the first time infecting NB plants by side-grafting and by mechanical inoculation with virions, indicating that previous failure to infect NB was likely due to virus silencing in early infection steps. Using NB as a CTV host provides new possibilities to study virus-host interactions with a simple and

  3. Molecular Characterization of Two Lysophospholipid:acyl-CoA Acyltransferases Belonging to the MBOAT Family in Nicotiana benthamiana.

    Directory of Open Access Journals (Sweden)

    Donghui Zhang

    Full Text Available In the remodeling pathway for the synthesis of phosphatidylcholine (PC, acyl-CoA-dependent lysophosphatidylcholine (lysoPC acyltransferase (LPCAT catalyzes the reacylation of lysoPC. A number of genes encoding LPCATs have been cloned and characterized from several plants in recent years. Using Arabidopsis and other plant LPCAT sequences to screen the genome database of Nicotiana benthamiana, we identified two cDNAs encoding the putative tobacco LPCATs (NbLPCAT1 and NbLPCAT2. Both of them were predicted to encode a protein of 463 amino acids with high similarity to LPCATs from other plants. Protein sequence features such as the presence of at least eight putative transmembrane regions, four highly conserved signature motifs and several invariant residues indicate that NbLPCATs belong to the membrane bound O-acyltransferase family. Lysophospholipid acyltransferase activity of NbLPCATs was confirmed by testing lyso-platelet-activating factor (lysoPAF sensitivity through heterologous expression of each full-length cDNA in a yeast mutant Y02431 (lca1△ disrupted in endogenous LPCAT enzyme activity. Analysis of fatty acid profiles of phospholipids from the NbLPCAT-expressing yeast mutant Y02431 cultures supplemented with polyunsaturated fatty acids suggested more incorporation of linoleic acid (18:2n6, LA and α-linolenic acid (18:3n3, ALA into PC compared to yeast mutant harbouring empty vector. In vitro enzymatic assay demonstrated that NbLPCAT1had high lysoPC acyltransferase activity with a clear preference for α-linolenoyl-CoA (18:3, while NbLPCAT2 showed a high lysophosphatidic acid (lysoPA acyltransferase activity towards α-linolenoyl-CoA and a weak lysoPC acyltransferase activity. Tissue-specific expression analysis showed a ubiquitous expression of NbLPCAT1 and NbLPCAT2 in roots, stems, leaves, flowers and seeds, and a strong expression in developing flowers. This is the first report on the cloning and characterization of lysophospholipid

  4. Rapid modification of the insect elicitor N-linolenoyl-glutamate via a lipoxygenase-mediated mechanism on Nicotiana attenuata leaves

    Directory of Open Access Journals (Sweden)

    VanDoorn Arjen

    2010-08-01

    Full Text Available Abstract Background Some plants distinguish mechanical wounding from herbivore attack by recognizing specific constituents of larval oral secretions (OS which are introduced into plant wounds during feeding. Fatty acid-amino acid conjugates (FACs are major constituents of Manduca sexta OS and strong elicitors of herbivore-induced defense responses in Nicotiana attenuata plants. Results The metabolism of one of the major FACs in M. sexta OS, N-linolenoyl-glutamic acid (18:3-Glu, was analyzed on N. attenuata wounded leaf surfaces. Between 50 to 70% of the 18:3-Glu in the OS or of synthetic 18:3-Glu were metabolized within 30 seconds of application to leaf wounds. This heat-labile process did not result in free α-linolenic acid (18:3 and glutamate but in the biogenesis of metabolites both more and less polar than 18:3-Glu. Identification of the major modified forms of this FAC showed that they corresponded to 13-hydroxy-18:3-Glu, 13-hydroperoxy-18:3-Glu and 13-oxo-13:2-Glu. The formation of these metabolites occurred on the wounded leaf surface and it was dependent on lipoxygenase (LOX activity; plants silenced in the expression of NaLOX2 and NaLOX3 genes showed more than 50% reduced rates of 18:3-Glu conversion and accumulated smaller amounts of the oxygenated derivatives compared to wild-type plants. Similar to 18:3-Glu, 13-oxo-13:2-Glu activated the enhanced accumulation of jasmonic acid (JA in N. attenuata leaves whereas 13-hydroxy-18:3-Glu did not. Moreover, compared to 18:3-Glu elicitation, 13-oxo-13:2-Glu induced the differential emission of two monoterpene volatiles (β-pinene and an unidentified monoterpene in irlox2 plants. Conclusions The metabolism of one of the major elicitors of herbivore-specific responses in N. attenuata plants, 18:3-Glu, results in the formation of oxidized forms of this FAC by a LOX-dependent mechanism. One of these derivatives, 13-oxo-13:2-Glu, is an active elicitor of JA biosynthesis and differential

  5. MAPK-dependent JA and SA signalling in Nicotiana attenuata affects plant growth and fitness during competition with conspecifics

    Directory of Open Access Journals (Sweden)

    Meldau Stefan

    2012-11-01

    Full Text Available Abstract Background Induced defense responses to herbivores are generally believed to have evolved as cost-saving strategies that defer the fitness costs of defense metabolism until these defenses are needed. The fitness costs of jasmonate (JA-mediated defenses have been well documented. Those of the early signaling units mediating induced resistance to herbivores have yet to be examined. Early signaling components that mediate herbivore-induced defense responses in Nicotiana attenuata, have been well characterized and here we examine their growth and fitness costs during competition with conspecifics. Two mitogen-activated protein kinases (MAPKs, salicylic acid (SA-induced protein kinase (SIPK and wound-induced protein kinase (WIPK are rapidly activated after perception of herbivory and both kinases regulate herbivory-induced JA levels and JA-mediated defense metabolite accumulations. Since JA-induced defenses result in resource-based trade-offs that compromise plant productivity, we evaluated if silencing SIPK (irSIPK and WIPK (irWIPK benefits the growth and fitness of plants competiting with wild type (WT plants, as has been shown for plants silenced in JA-signaling by the reduction of Lipoxygenase 3 (LOX3 levels. Results As expected, irWIPK and LOX3-silenced plants out-performed their competing WT plants. Surprisingly, irSIPK plants, which have the largest reductions in JA signaling, did not. Phytohormone profiling of leaves revealed that irSIPK plants accumulated higher levels of SA compared to WT. To test the hypothesis that these high levels of SA, and their presumed associated fitness costs of pathogen associated defenses in irSIPK plants had nullified the JA-deficiency-mediated growth benefits in these plants, we genetically reduced SA levels in irSIPK plants. Reducing SA levels partially recovered the biomass and fitness deficits of irSIPK plants. We also evaluated whether the increased fitness of plants with reduced SA or JA levels

  6. Music and Alterity Processes

    Directory of Open Access Journals (Sweden)

    Josep Martí

    2014-10-01

    Full Text Available The concept of alterity constitutes an important issue in anthropological research and, therefore, in the study of musical practices, as well. Without it, we could hardly understand other kinds of music situated in different spaces and time from the observer. In order to effectively approach these musical practices, we have to develop strategies to help us reduce as much as possible that which distorts the vision of the other. However, beyond the strictly epistemological and methodological issues, the study of music cannot ignore the ethical question related to the manner in which Western thought has understood and treated the other: through a hierarchical and stereotypical type of thinking based on the condition of otherness. Throughout the article, different alterity procedures are presented and discussed, such as synecdochization, exoticization, undervaluation, overvaluation, misunderstanding and exclusion. Taking these different alterity strategies into account may help us to better understand how the musical other is constructed, used and ultimately instrumentalized.

  7. Attention Alters Perceived Attractiveness.

    Science.gov (United States)

    Störmer, Viola S; Alvarez, George A

    2016-04-01

    Can attention alter the impression of a face? Previous studies showed that attention modulates the appearance of lower-level visual features. For instance, attention can make a simple stimulus appear to have higher contrast than it actually does. We tested whether attention can also alter the perception of a higher-order property-namely, facial attractiveness. We asked participants to judge the relative attractiveness of two faces after summoning their attention to one of the faces using a briefly presented visual cue. Across trials, participants judged the attended face to be more attractive than the same face when it was unattended. This effect was not due to decision or response biases, but rather was due to changes in perceptual processing of the faces. These results show that attention alters perceived facial attractiveness, and broadly demonstrate that attention can influence higher-level perception and may affect people's initial impressions of one another.

  8. The role of Nicotiana gluca Graham (paraguayan herbs as an adjuvant in immunomodulation of Newcastle disease vaccine for broilers Estudo da ação de Nicotiana glauca Graham (erva paraguaia como coadjuvante em vacina contra a doença de Newcastle em frangos de corte

    Directory of Open Access Journals (Sweden)

    Fabiane Pereira Gentilini

    2008-07-01

    Full Text Available The Nicotiana glauca is a native plant species from Argentina, but found all over South América, being used against headaches, rheumatism, injuries, ulcers, and so on. Researchers have considered it as having immunomodulation effect. This study was conducted to investigate the use of a aqueous extract of Nicotiana glauca Graham as an immunomodulator (adjuvant of a Newcastle disease vaccine.. A total of 56 broilers were distributed into 4 experimental groups. Each one of them received 3 dosages of this vaccine with or without the addition of different concentrations of the extract Using hemmoaglutination inhibition techniques , the results have shown differences (P<0.05 in the third sera collection. An increase in the antibody titer with the inclusion of 5 mg/dosage of the extract (Treatment 3 as compared to 1 mg/dosage (Treatment 2 and 10 mg/dosage of the extract (Treatment 4 was observed, However, birds from Treatment 3 did not differ (P> 0.05 from Treatment 1. These results indicated that further investigations are required, including the use of cytotoxicity tests in vitro, to evaluate the immunomodulation effect of this extract.

     

    KEY WORDS: Immunomodulation effect, Nicotiana glauca Graham, vaccine.

    A Nicotiana glauca Graham é uma espécie nativa da Argentina, bem distribuída na América do Sul, sendo empregada, popularmente, contra dores de cabeça, dores reumáticas, cicatrização de feridas e úlceras, entre outros. Pesquisas têm avaliado a sua ação na potencialização da resposta imune. Assim, com este estudo, buscou-se avaliar a ação de um extrato aquoso de Nicotiana glauca Graham como coadjuvante imunológico em uma vacina contra a doença de Newcastle (DNC. Utilizaram-se 56 frangos de corte, distribuídos em quatro grupos experimentais, que receberam tr

  9. Ressourcenorientierte Diagnostik im Alter

    OpenAIRE

    Forstmeier, Simon; Maercker, Andreas

    2008-01-01

    Trotz der im Alter zunehmenden körperlichen, kognitiven und sozialen Verlusten bleibt das subjektive Wohlbefinden relativ stabil. Dies weist auf die vielen Ressourcen älterer Menschen hin. Dieser Artikel stellt für die klinische Ressourcendiagnostik relevante Verfahren vor und erläutert die zugrunde liegenden Konzepte. Berücksichtigt werden Aktivitäten und Erlebnisse als Ressourcen, emotionale Ressourcen (positiver Affekt, Lebenszufriedenheit, Selbstwerterleben, Lebensqualität), motivationale...

  10. Comparison of phenotypes produced in response to transient expression of genes encoded by four distinct begomoviruses in Nicotiana benthamiana and their correlation with the levels of developmental miRNAs

    Directory of Open Access Journals (Sweden)

    Amin Imran

    2011-05-01

    Full Text Available Abstract Background Whitefly-transmitted geminiviruses (begomoviruses are a major limiting factor for the production of numerous dicotyledonous crops throughout the world. Begomoviruses differ in the number of components that make up their genomes and association with satellites, and yet they cause strikingly similar phenotypes, such as leaf curling, chlorosis and stunted plant growth. MicroRNAs (miRNAs are small endogenous RNAs that regulate plant growth and development. The study described here was aimed at investigating the effects of each virus encoded gene on the levels of developmental miRNAs to identify common trends between distinct begomoviruses. Results All genes encoded by four distinct begomoviruses (African cassava mosaic virus [ACMV], Cabbage leaf curl virus [CbLCuV], Tomato yellow leaf curl virus [TYLCV] and Cotton leaf curl virus/Cotton leaf curl betasatellite [CLCuV/CLCuMB] were expressed from a Potato virus X (PVX vector in Nicotiana benthamiana. Changes in the levels of ten miRNAs in response to the virus genes were determined by northern blotting using specific miRNA probes. For the monopartite begomoviruses (TYLCV and CLCuMV the V2 gene product was identified as the major symptom determinant while for bipartite begomoviruses (ACMV and CbLCuV more than one gene appears to contribute to symptoms and this is reflected in changes in miRNA levels. The phenotype induced by expression of the βC1 gene of the betasatellite CLCuMB was the most distinct and consisted of leaf curling, vein swelling, thick green veins and enations and the pattern of changes in miRNA levels was the most distinct. Conclusions Our results have identified symptom determinants encoded by begomoviruses and show that developmental abnormalities caused by transient expression of begomovirus genes correlates with altered levels of developmental miRNAs. Additionally, all begomovirus genes were shown to modulate miRNA levels, the first time this has been shown to

  11. Heterologous expression of the isopimaric acid pathway in Nicotiana benthamiana and the effect of N-terminal modifications of the involved cytochrome P450 enzyme

    DEFF Research Database (Denmark)

    Gnanasekaran, Thiyagarajan; Vavitsas, Konstantinos; Andersen-Ranberg, Johan;

    2015-01-01

    in the chloroplast and subsequently oxidized by a cytochrome P450, CYP720B4. RESULTS: We transiently expressed the isopimaric acid pathway in Nicotiana benthamiana leaves and enhanced its productivity by the expression of two rate-limiting steps in the pathway (providing the general precursor of diterpenes). This co...... enzymes. CONCLUSIONS: It is possible to localize a diterpenoid pathway from spruce fully within the chloroplast of N. benthamiana and a few modifications of the N-terminal sequences of the CYP720B4 can facilitate the expression of plant P450s in the plastids. The coupling of terpene biosynthesis closer......BACKGROUND: Plant terpenoids are known for their diversity, stereochemical complexity, and their commercial interest as pharmaceuticals, food additives, and cosmetics. Developing biotechnology approaches for the production of these compounds in heterologous hosts can increase their market...

  12. A new virus-induced gene silencing vector based on Euphorbia mosaic virus-Yucatan peninsula for NPR1 silencing in Nicotiana benthamiana and Capsicum annuum var. Anaheim.

    Science.gov (United States)

    Villanueva-Alonzo, Hernan J; Us-Camas, Rosa Y; López-Ochoa, Luisa A; Robertson, Dominique; Guerra-Peraza, Orlene; Minero-García, Yereni; Moreno-Valenzuela, Oscar A

    2013-05-01

    Virus-induced gene silencing is based on the sequence-specific degradation of RNA. Here, a gene silencing vector derived from EuMV-YP, named pEuMV-YP:ΔAV1, was used to silence ChlI and NPR1 genes in Nicotiana benthamiana. The silencing of the ChlI transcripts was efficient in the stems, petioles and leaves as reflected in tissue bleaching and reduced transcript levels. The silencing was stable, reaching the flowers and fruits, and was observed throughout the life cycle of the plants. Additionally, the silencing of the NPR1 gene was efficient in both N. benthamiana and Capsicum annuum. After silencing, the plants' viral symptoms increased to levels similar to those seen in wild-type plants. These results suggest that NPR1 plays a role in the compatible interactions of EuMV-YP N. benthamiana and EuMV-C. annum var. anaheim.

  13. Heterologous expression of the isopimaric acid pathway in Nicotiana benthamiana and the effect of N-terminal modifications of the involved cytochrome P450 enzyme

    DEFF Research Database (Denmark)

    Gnanasekaran, Thiyagarajan; Vavitsas, Konstantinos; Andersen-Ranberg, Johan

    2015-01-01

    in the chloroplast and subsequently oxidized by a cytochrome P450, CYP720B4. RESULTS: We transiently expressed the isopimaric acid pathway in Nicotiana benthamiana leaves and enhanced its productivity by the expression of two rate-limiting steps in the pathway (providing the general precursor of diterpenes). This co...... enzymes. CONCLUSIONS: It is possible to localize a diterpenoid pathway from spruce fully within the chloroplast of N. benthamiana and a few modifications of the N-terminal sequences of the CYP720B4 can facilitate the expression of plant P450s in the plastids. The coupling of terpene biosynthesis closer......BACKGROUND: Plant terpenoids are known for their diversity, stereochemical complexity, and their commercial interest as pharmaceuticals, food additives, and cosmetics. Developing biotechnology approaches for the production of these compounds in heterologous hosts can increase their market...

  14. The Agrobacterium tumefaciens Ti Plasmid Virulence Gene virE2 Reduces Sri Lankan Cassava Mosaic Virus Infection in Transgenic Nicotiana benthamiana Plants

    Directory of Open Access Journals (Sweden)

    Thulasi Raveendrannair Resmi

    2015-05-01

    Full Text Available Cassava mosaic disease is a major constraint to cassava cultivation worldwide. In India, the disease is caused by Indian cassava mosaic virus (ICMV and Sri Lankan cassava mosaic virus (SLCMV. The Agrobacterium Ti plasmid virulence gene virE2, encoding a nuclear-localized, single-stranded DNA binding protein, was introduced into Nicotiana benthamiana to develop tolerance against SLCMV. Leaf discs of transgenic N. benthamiana plants, harboring the virE2 gene, complemented a virE2 mutation in A. tumefaciens and produced tumours. Three tested virE2 transgenic plants displayed reduction in disease symptoms upon agroinoculation with SLCMV DNA A and DNA B partial dimers. A pronounced reduction in viral DNA accumulation was observed in all three virE2 transgenic plants. Thus, virE2 is an effective candidate gene to develop tolerance against the cassava mosaic disease and possibly other DNA virus diseases.

  15. Molecular and functional analysis of phosphomannomutase (PMM) from higher plants and genetic evidence for the involvement of PMM in ascorbic acid biosynthesis in Arabidopsis and Nicotiana benthamiana

    DEFF Research Database (Denmark)

    Qian, W; Yu, C; Qin, H

    2007-01-01

    Phosphomannomutase (PMM) catalyzes the interconversion of mannose-6-phosphate and mannose-1-phosphate. However, systematic molecular and functional investigations on PMM from higher plants have hitherto not been reported. In this work, PMM cDNAs were isolated from Arabidopsis, Nicotiana benthamiana...... was constitutively expressed in both vegetative and reproductive organs. Reducing the PMM expression level through virus-induced gene silencing caused a substantial decrease in ascorbic acid (AsA) content in N. benthamiana leaves. Conversely, raising the PMM expression level in N. benthamiana using viral-vector......-mediated ectopic expression led to a 20-50% increase in AsA content. Consistent with this finding, transgenic expression of an AtPMM-GFP fusion protein in Arabidopsis also increased AsA content by 25-33%. Collectively, this study improves our understanding on the molecular and functional properties of plant PMM...

  16. 烟草疫霉分离及生长培养基的选择%Selection of Isolating and Culturing Medium for Phytophthora nicotianae

    Institute of Scientific and Technical Information of China (English)

    苏凯; 桑维钧; 张新强; 王慧

    2013-01-01

    Different base medium adding different combination of antibiotics were applied for isolation and culture of Phytophthora nicotianae.The results showed that selective medium with Vs base medium (100mL/L V8juice+0.02 g/L CaCO3+2 g/Lagar) adding 10 mg/mL ampicillin +5 mg/mL nysfungin+5 mg/mL carbendazim had the best isolation effect as the successful isolation rate was 100%,and the hypha grew well on this medium.After separation,the hypha could be cultured in Vs base medium as the hypha was white,tight and strong with big bacteria clone and strong growth potential.%采用不同的基本培养基添加抗生素组合对烟草疫霉(Phytophthora nicotianae)进行分离培养.结果表明,以V8培养基(100 mL/L Vs汁+0.02 g/L CaCO3+2 g/L洋菜)为基本培养基附加10 mg/mL氨苄青霉素+5 mg/mL制霉素+5 mg/mL多菌灵的选择培养基分离烟草疫霉的效果较好,分离成功率达100%,菌丝的生长状态也较好.菌丝分离后在V8基本培养基中培养,菌丝紧密、浓白、粗壮,菌落大、长势旺.

  17. New microsome-associated HT-family proteins from Nicotiana respond to pollination and define an HT/NOD-24 protein family

    Institute of Scientific and Technical Information of China (English)

    Katsuhiko Kondo; Bruce McClure

    2008-01-01

    HT-family proteins have been identified in Nicotiana, Solanum,and Petunia.HT-B-type proteins are implicated in S-RNase-based self-incompatibility,but the functions of other family members are unknown.Screening for cDNA sequences with an expression pattern similar to HT-B in Nicotiana alata revealed a new group of small HT-family proteins.designated HT-M.HT-M proteins resemble HT-B in several respects:their pistil-specific expression pattern iS indistinguish-able from HT-B,they pellet with a microsome fraction,and their abundance decreases after pollination.Unlike HT-B,there iS no S-specificity to this response,and RNAi experiments show that HT-M proteins are not necessary for self-incompatibility.Identification of a third group of pistil-specific HT-family proteins helps better define the characteristics of the family and allowed identification of putative new family members.By searching the databases with only the most conserved HT-family sequence elements,the signal sequence and cysteine motifs,we identified nodulin-24-1ike proteins and several small glycine-rich proteins as putative HT-family members.Like HT-M and HT-B,nodulin-24 iS membrane associated.We propose that the conserved features in HT-family proteins are important for targeting or modification and refer to the broader family that includes both HT-and nodulin-24-like proteins as the HT/NOD-24-family.

  18. A polygalacturonase-inhibiting protein from grapevine reduces the symptoms of the endopolygalacturonase BcPG2 from Botrytis cinerea in Nicotiana benthamiana leaves without any evidence for in vitro interaction

    NARCIS (Netherlands)

    Joubert, D.A.; Kars, I.; Wagemakers, L.; Bergmann, C.; Kemp, G.; Vivier, M.A.; Kan, van J.A.L.

    2007-01-01

    Six endopolygalacturonases from Botrytis cinerea (BcPG1 to BcPG6) as well as mutated forms of BcPG1 and BcPG2 were expressed transiently in leaves of Nicotiana benthamiana using agroinfiltration. Expression of BcPG1, BcPG2, BcPG4, BcPG5, and mutant BcPG1-D203A caused symptoms, whereas BcPG3, BcPG6,

  19. Genetic Alterations in Glioma

    Energy Technology Data Exchange (ETDEWEB)

    Bralten, Linda B. C.; French, Pim J., E-mail: p.french@erasmusmc.nl [Department of Neurology, Erasmus University Medical Center, Erasmus University Rotterdam, Dr Molewaterplein 50, 3000 CA, Rotterdam (Netherlands)

    2011-03-07

    Gliomas are the most common type of primary brain tumor and have a dismal prognosis. Understanding the genetic alterations that drive glioma formation and progression may help improve patient prognosis by identification of novel treatment targets. Recently, two major studies have performed in-depth mutation analysis of glioblastomas (the most common and aggressive subtype of glioma). This systematic approach revealed three major pathways that are affected in glioblastomas: The receptor tyrosine kinase signaling pathway, the TP53 pathway and the pRB pathway. Apart from frequent mutations in the IDH1/2 gene, much less is known about the causal genetic changes of grade II and III (anaplastic) gliomas. Exceptions include TP53 mutations and fusion genes involving the BRAF gene in astrocytic and pilocytic glioma subtypes, respectively. In this review, we provide an update on all common events involved in the initiation and/or progression across the different subtypes of glioma and provide future directions for research into the genetic changes.

  20. Music alters visual perception.

    Directory of Open Access Journals (Sweden)

    Jacob Jolij

    Full Text Available BACKGROUND: Visual perception is not a passive process: in order to efficiently process visual input, the brain actively uses previous knowledge (e.g., memory and expectations about what the world should look like. However, perception is not only influenced by previous knowledge. Especially the perception of emotional stimuli is influenced by the emotional state of the observer. In other words, how we perceive the world does not only depend on what we know of the world, but also by how we feel. In this study, we further investigated the relation between mood and perception. METHODS AND FINDINGS: We let observers do a difficult stimulus detection task, in which they had to detect schematic happy and sad faces embedded in noise. Mood was manipulated by means of music. We found that observers were more accurate in detecting faces congruent with their mood, corroborating earlier research. However, in trials in which no actual face was presented, observers made a significant number of false alarms. The content of these false alarms, or illusory percepts, was strongly influenced by the observers' mood. CONCLUSIONS: As illusory percepts are believed to reflect the content of internal representations that are employed by the brain during top-down processing of visual input, we conclude that top-down modulation of visual processing is not purely predictive in nature: mood, in this case manipulated by music, may also directly alter the way we perceive the world.

  1. Altered Perspectives: Immersive Environments

    Science.gov (United States)

    Shipman, J. S.; Webley, P. W.

    2016-12-01

    Immersive environments provide an exciting experiential technology to visualize the natural world. Given the increasing accessibility of 360o cameras and virtual reality headsets we are now able to visualize artistic principles and scientific concepts in a fully immersive environment. The technology has become popular for photographers as well as designers, industry, educational groups, and museums. Here we show a sci-art perspective on the use of optics and light in the capture and manipulation of 360o images and video of geologic phenomena and cultural heritage sites in Alaska, England, and France. Additionally, we will generate intentionally altered perspectives to lend a surrealistic quality to the landscapes. Locations include the Catacombs of Paris, the Palace of Versailles, and the Northern Lights over Fairbanks, Alaska. Some 360o view cameras now use small portable dual lens technology extending beyond the 180o fish eye lens previously used, providing better coverage and image quality. Virtual reality headsets range in level of sophistication and cost, with the most affordable versions using smart phones and Google Cardboard viewers. The equipment used in this presentation includes a Ricoh Theta S spherical imaging camera. Here we will demonstrate the use of 360o imaging with attendees being able to be part of the immersive environment and experience our locations as if they were visiting themselves.

  2. Virus-Induced Gene Silencing Using Tobacco Rattle Virus as a Tool to Study the Interaction between Nicotiana attenuata and Rhizophagus irregularis.

    Directory of Open Access Journals (Sweden)

    Karin Groten

    Full Text Available Most land plants live in a symbiotic association with arbuscular mycorrhizal fungi (AMF that belong to the phylum Glomeromycota. Although a number of plant genes involved in the plant-AMF interactions have been identified by analyzing mutants, the ability to rapidly manipulate gene expression to study the potential functions of new candidate genes remains unrealized. We analyzed changes in gene expression of wild tobacco roots (Nicotiana attenuata after infection with mycorrhizal fungi (Rhizophagus irregularis by serial analysis of gene expression (SuperSAGE combined with next generation sequencing, and established a virus-induced gene-silencing protocol to study the function of candidate genes in the interaction. From 92,434 SuperSAGE Tag sequences, 32,808 (35% matched with our in-house Nicotiana attenuata transcriptome database and 3,698 (4% matched to Rhizophagus genes. In total, 11,194 Tags showed a significant change in expression (p2-fold change after infection. When comparing the functions of highly up-regulated annotated Tags in this study with those of two previous large-scale gene expression studies, 18 gene functions were found to be up-regulated in all three studies mainly playing roles related to phytohormone metabolism, catabolism and defense. To validate the function of identified candidate genes, we used the technique of virus-induced gene silencing (VIGS to silence the expression of three putative N. attenuata genes: germin-like protein, indole-3-acetic acid-amido synthetase GH3.9 and, as a proof-of-principle, calcium and calmodulin-dependent protein kinase (CCaMK. The silencing of the three plant genes in roots was successful, but only CCaMK silencing had a significant effect on the interaction with R. irregularis. Interestingly, when a highly activated inoculum was used for plant inoculation, the effect of CCaMK silencing on fungal colonization was masked, probably due to trans-complementation. This study demonstrates that

  3. Virus-Induced Gene Silencing Using Tobacco Rattle Virus as a Tool to Study the Interaction between Nicotiana attenuata and Rhizophagus irregularis.

    Science.gov (United States)

    Groten, Karin; Pahari, Nabin T; Xu, Shuqing; Miloradovic van Doorn, Maja; Baldwin, Ian T

    2015-01-01

    Most land plants live in a symbiotic association with arbuscular mycorrhizal fungi (AMF) that belong to the phylum Glomeromycota. Although a number of plant genes involved in the plant-AMF interactions have been identified by analyzing mutants, the ability to rapidly manipulate gene expression to study the potential functions of new candidate genes remains unrealized. We analyzed changes in gene expression of wild tobacco roots (Nicotiana attenuata) after infection with mycorrhizal fungi (Rhizophagus irregularis) by serial analysis of gene expression (SuperSAGE) combined with next generation sequencing, and established a virus-induced gene-silencing protocol to study the function of candidate genes in the interaction. From 92,434 SuperSAGE Tag sequences, 32,808 (35%) matched with our in-house Nicotiana attenuata transcriptome database and 3,698 (4%) matched to Rhizophagus genes. In total, 11,194 Tags showed a significant change in expression (p2-fold change) after infection. When comparing the functions of highly up-regulated annotated Tags in this study with those of two previous large-scale gene expression studies, 18 gene functions were found to be up-regulated in all three studies mainly playing roles related to phytohormone metabolism, catabolism and defense. To validate the function of identified candidate genes, we used the technique of virus-induced gene silencing (VIGS) to silence the expression of three putative N. attenuata genes: germin-like protein, indole-3-acetic acid-amido synthetase GH3.9 and, as a proof-of-principle, calcium and calmodulin-dependent protein kinase (CCaMK). The silencing of the three plant genes in roots was successful, but only CCaMK silencing had a significant effect on the interaction with R. irregularis. Interestingly, when a highly activated inoculum was used for plant inoculation, the effect of CCaMK silencing on fungal colonization was masked, probably due to trans-complementation. This study demonstrates that large

  4. 广东连州上柏场报春苣苔种群及其生境特征%Habital characteristics of Primulina tabacum populations in a cave at Shangbaichang, Lianzhou, Guangdong

    Institute of Scientific and Technical Information of China (English)

    缪绅裕; 唐志信; 邓冬梅; 周健桃; 潘丽君; 王厚麟; 李春波; 黄福长

    2013-01-01

      2012年用自动记录仪对已知最大规模的广东连州上柏场报春苣苔Primulina tabacum种群所处生境的气温、相对湿度进行为期1年的监测,每季度监测植物叶片绿色度和种群数量;测定土壤、岩石、水体和植物体多种元素的含量,计算植物的吸收系数,以期为种群的保护和发展提供基础资料。结果表明:岩洞的气温变幅为1.262~24.847℃,平均气温和温差均为洞内>洞中>洞口;相对湿度为洞口>洞中>洞内,洞内湿度的变幅最大。报春苣苔能较好地适应4000 lx以下的光强,低于洞外最强光的10%。所测定的元素含量,以Ca最高,且岩石>土壤>植物体,以N最低,且植物体≈土壤>岩石。地面植株对元素的吸收系数顺序为H>N>C>P>K>Na>Ca>Mg>Al,侧壁植株的为H>C>K>Mg>N>Ca>Na>P>Al。植株叶片绿色度为洞口>洞中>洞内,但差异不显著。种群数量波动较大,夏季和秋季多,冬季和早春少,与气温和相对湿度有较明显相关性。结果提示,报春苣苔虽要求石灰岩生境,但对不同生态因子仍具一定适应性,为迁地保护提供了理论依据。%Using the sensor of an automatic onset HOBO mini-weather station, the air temperature, air relative humidity were monitored for one year (2012) in a lime cave located at Shanbaochang, Lianzhou, Guangdong where the national Grade I key protected plant species Primulina tabacum grows. The leaf SPAD values and the population sizes were measured seasonally. The elemental contents of soil, rock, underground water and plant were also measured, and the assimilative coefficient by plant were calculated, in order to provide basic data for protecting and enhancing the plant population. Results showed that the air temperature ranged from 1.622 ℃ to 24.847 ℃. Mean air temperature and the range of temperature were in the following order inner> middle> entrance of the cave. The order of range in air

  5. Silencing ribulose-1,5-bisphosphate carboxylase/oxygenase expression does not disrupt nitrogen allocation to defense after simulated herbivory in Nicotiana attenuata.

    Science.gov (United States)

    Stanton, Mariana A; Ullmann-Zeunert, Lynn; Wielsch, Natalie; Bartram, Stefan; Svatoš, Aleš; Baldwin, Ian T; Groten, Karin

    2013-01-01

    Ribulose-1,5-bisphosphate carboxylase/ oxygenase (RuBisCO) is the most abundant protein on the planet and in addition to its central role in photosynthesis it is thought to function as a nitrogen (N)-storage protein and a potential source of N for defense biosynthesis in plants. In a recent study in the wild tobacco Nicotiana attenuata, we showed that the decrease in absolute N invested in soluble proteins and RuBisCO elicited by simulated herbivory was much larger than the N-requirements of nicotine and phenolamide biosynthesis; (15)N flux studies revealed that N for defensive phenolamide synthesis originates from recently assimilated N rather than from RuBisCO turnover. Here we show that a transgenic line of N. attenuata silenced in the expression of RuBisCO (asRUB) invests similar or even larger amounts of N into phenolamide biosynthesis compared with wild type plants, consistent with our previous conclusion that recently assimilated N is channeled into phenolamide synthesis after elicitation. We suggest that the decrease in leaf proteins after simulated herbivory is a tolerance mechanism, rather than a consequence of N-demand for defense biosynthesis.

  6. Recombinant jacalin-like plant lectins are produced at high levels in Nicotiana benthamiana and retain agglutination activity and sugar specificity.

    Science.gov (United States)

    Fernandez-del-Carmen, Asun; Juárez, Paloma; Presa, Silvia; Granell, Antonio; Orzáez, Diego

    2013-02-20

    The plant kingdom is an underexplored source of valuable proteins which, like plant lectins, display unique interacting specificities. Furthermore, plant protein diversity remains under-exploited due to the low availability and heterogeneity of native sources. All these hurdles could be overcome with recombinant production. A narrow phylogenetic gap between the native source and the recombinant platform is likely to facilitate proper protein processing and stability; therefore, the plant cell chassis should be specially suited for the recombinant production of many plant native proteins. This is illustrated herein with the recombinant production of two representatives of the plant jacalin-related lectin (JRLs) protein family in Nicotiana benthamiana using state-of-the-art magnICON technology. Mannose-specific Banlec JRL was produced at very high levels in leaves, reaching 1.0mg of purified protein per gram of fresh weight and showing strong agglutination activity. Galactose-specific jacalin JRL, with its complicated processing requirements, was also successfully produced in N. benthamiana at levels of 0.25 mg of purified protein per gram of fresh weight. Recombinant Jacalin (rJacalin) proved efficient in the purification of human IgA1, and was able to discriminate between plant-made and native IgA1 due to their differential glycosylation status. Together, these results show that the plant cell factory should be considered a primary option in the recombinant production of valuable plant proteins.

  7. Influence of retinoblastoma-related gene silencing on the initiation of DNA replication by African cassava mosaic virus Rep in cells of mature leaves in Nicotiana benthamiana plants

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    Bruce Gareth

    2011-12-01

    Full Text Available Abstract Background Geminiviruses mainly infect terminally differentiated tissues and cells in plants. They need to reprogramme host cellular machinery for DNA replication. This process is thought to be mediated by inactivation of cell-cycle repressor proteins and by induction of host DNA synthesis protein expression through actions of the geminviral replication initiator protein (Rep. Findings Exploiting a Nicotiana benthamiana pOri2 line, which is transformed with a transgene consisting of a direct repeat of the African cassava mosaic virus (ACMV-replication origin (Ori flanking a non-viral DNA region, and virus-induced RNA silencing (VIGS, the impact of host gene expression on replication of the ACMV-derived replicon was investigated. The ACMV Rep trans-replicated the viral episomal replicon in leaves of young but not older pOri2 plants. Upon VIGS-mediated down-regulation of N. benthamiana NbRBR1, the retinoblastoma-related protein gene coding for a negative cell-cycle suppressor, recovered the ability of ACMV Rep for trans DNA replication, whereas the silencing of NbPCNA coding for the sliding clamp of DNA polymerase had no effect. Conclusions These results suggest that the cellular machinery for DNA replication in differentiated tissues of older leaves cannot be reprogrammed by Rep alone but may need other uncharacterised viral and plant factors.

  8. Accumulation of human EGF in nectar of transformed plants of Nicotiana langsdorffii x N. sanderae and transfer to honey by bees.

    Science.gov (United States)

    Helsper, J P F G; Ruyter-Spira, C P; Kwakman, P H S; Bleeker, W K; Keizer, L C P; Bade, J B; Te Velde, A A; Zaat, S A J; Verbeek, M; Creemers-Molenaar, J

    2011-09-01

    Honey has been used successfully in wound healing for thousands of years. The peptide hormone human epidermal growth factor (hEGF) is also known to have a beneficial effect in various wound healing processes via mechanisms that differ from those for honey. In this study, we show that hEGF can be incorporated into honey via nectar. Plants of Nicotiana langsdorffii x N. sanderae were transformed with the gene for hEGF, equipped with a nectary-targeted promoter and a signal sequence for secretion to nectar. These plants accumulated hEGF in the nectar. The maximum hEGF concentration recorded with ELISA in these plants is 2.5 ng·ml⁻¹. There is a significant linear relationship (Pplants did not allow production of honey from their nectar, we used feeding solutions, spiked with synthetic hEGF, to study transfer of this peptide into honey through bee activity. Transfer of hEGF from a feeding solution to honey by bees occurred with retention of the hEGF concentration and the capacity to induce hEGF-receptor phosphorylation. These observations indicate that plants can function as a production platform for honey containing biologically active peptides, which may enhance wound healing and other biological processes.

  9. Expression of a Recombinant Anti-HIV and Anti-Tumor Protein, MAP30, in Nicotiana tobacum Hairy Roots: A pH-Stable and Thermophilic Antimicrobial Protein.

    Science.gov (United States)

    Moghadam, Ali; Niazi, Ali; Afsharifar, Alireza; Taghavi, Seyed Mohsen

    2016-01-01

    In contrast to conventional antibiotics, which microorganisms can readily evade, it is nearly impossible for a microbial strain that is sensitive to antimicrobial proteins to convert to a resistant strain. Therefore, antimicrobial proteins and peptides that are promising alternative candidates for the control of bacterial infections are under investigation. The MAP30 protein of Momordica charantia is a valuable type I ribosome-inactivating protein (RIP) with anti-HIV and anti-tumor activities. Whereas the antimicrobial activity of some type I RIPs has been confirmed, less attention has been paid to the antimicrobial activity of MAP30 produced in a stable, easily handled, and extremely cost-effective protein-expression system. rMAP30-KDEL was expressed in Nicotiana tobacum hairy roots, and its effect on different microorganisms was investigated. Analysis of the extracted total proteins of transgenic hairy roots showed that rMAP30-KDEL was expressed effectively and that this protein exhibited significant antibacterial activity in a dose-dependent manner. rMAP30-KDEL also possessed thermal and pH stability. Bioinformatic analysis of MAP30 and other RIPs regarding their conserved motifs, amino-acid contents, charge, aliphatic index, GRAVY value, and secondary structures demonstrated that these factors accounted for their thermophilicity. Therefore, RIPs such as MAP30 and its derived peptides might have promising applications as food preservatives, and their analysis might provide useful insights into designing clinically applicable antibiotic agents.

  10. Expression of a Recombinant Anti-HIV and Anti-Tumor Protein, MAP30, in Nicotiana tobacum Hairy Roots: A pH-Stable and Thermophilic Antimicrobial Protein.

    Directory of Open Access Journals (Sweden)

    Ali Moghadam

    Full Text Available In contrast to conventional antibiotics, which microorganisms can readily evade, it is nearly impossible for a microbial strain that is sensitive to antimicrobial proteins to convert to a resistant strain. Therefore, antimicrobial proteins and peptides that are promising alternative candidates for the control of bacterial infections are under investigation. The MAP30 protein of Momordica charantia is a valuable type I ribosome-inactivating protein (RIP with anti-HIV and anti-tumor activities. Whereas the antimicrobial activity of some type I RIPs has been confirmed, less attention has been paid to the antimicrobial activity of MAP30 produced in a stable, easily handled, and extremely cost-effective protein-expression system. rMAP30-KDEL was expressed in Nicotiana tobacum hairy roots, and its effect on different microorganisms was investigated. Analysis of the extracted total proteins of transgenic hairy roots showed that rMAP30-KDEL was expressed effectively and that this protein exhibited significant antibacterial activity in a dose-dependent manner. rMAP30-KDEL also possessed thermal and pH stability. Bioinformatic analysis of MAP30 and other RIPs regarding their conserved motifs, amino-acid contents, charge, aliphatic index, GRAVY value, and secondary structures demonstrated that these factors accounted for their thermophilicity. Therefore, RIPs such as MAP30 and its derived peptides might have promising applications as food preservatives, and their analysis might provide useful insights into designing clinically applicable antibiotic agents.

  11. Ectopic expression of Capsicum-specific cell wall protein Capsicum annuum senescence-delaying 1 (CaSD1) delays senescence and induces trichome formation in Nicotiana benthamiana.

    Science.gov (United States)

    Seo, Eunyoung; Yeom, Seon-In; Jo, Sunghwan; Jeong, Heejin; Kang, Byoung-Cheorl; Choi, Doil

    2012-04-01

    Secreted proteins are known to have multiple roles in plant development, metabolism, and stress response. In a previous study to understand the roles of secreted proteins, Capsicum annuum secreted proteins (CaS) were isolated by yeast secretion trap. Among the secreted proteins, we further characterized Capsicum annuum senescence-delaying 1 (CaSD1), a gene encoding a novel secreted protein that is present only in the genus Capsicum. The deduced CaSD1 contains multiple repeats of the amino acid sequence KPPIHNHKPTDYDRS. Interestingly, the number of repeats varied among cultivars and species in the Capsicum genus. CaSD1 is constitutively expressed in roots, and Agrobacterium-mediated transient overexpression of CaSD1 in Nicotiana benthamiana leaves resulted in delayed senescence with a dramatically increased number of trichomes and enlarged epidermal cells. Furthermore, senescence- and cell division-related genes were differentially regulated by CaSD1-overexpressing plants. These observations imply that the pepper-specific cell wall protein CaSD1 plays roles in plant growth and development by regulating cell division and differentiation.

  12. A three-dimensional RNA motif in Potato spindle tuber viroid mediates trafficking from palisade mesophyll to spongy mesophyll in Nicotiana benthamiana.

    Science.gov (United States)

    Takeda, Ryuta; Petrov, Anton I; Leontis, Neocles B; Ding, Biao

    2011-01-01

    Cell-to-cell trafficking of RNA is an emerging biological principle that integrates systemic gene regulation, viral infection, antiviral response, and cell-to-cell communication. A key mechanistic question is how an RNA is specifically selected for trafficking from one type of cell into another type. Here, we report the identification of an RNA motif in Potato spindle tuber viroid (PSTVd) required for trafficking from palisade mesophyll to spongy mesophyll in Nicotiana benthamiana leaves. This motif, called loop 6, has the sequence 5'-CGA-3'...5'-GAC-3' flanked on both sides by cis Watson-Crick G/C and G/U wobble base pairs. We present a three-dimensional (3D) structural model of loop 6 that specifies all non-Watson-Crick base pair interactions, derived by isostericity-based sequence comparisons with 3D RNA motifs from the RNA x-ray crystal structure database. The model is supported by available chemical modification patterns, natural sequence conservation/variations in PSTVd isolates and related species, and functional characterization of all possible mutants for each of the loop 6 base pairs. Our findings and approaches have broad implications for studying the 3D RNA structural motifs mediating trafficking of diverse RNA species across specific cellular boundaries and for studying the structure-function relationships of RNA motifs in other biological processes.

  13. Simultaneous determination of shikimic acid, salicylic acid and jasmonic acid in wild and transgenic Nicotiana langsdorffii plants exposed to abiotic stresses.

    Science.gov (United States)

    Scalabrin, Elisa; Radaelli, Marta; Capodaglio, Gabriele

    2016-06-01

    The presence and relative concentration of phytohormones may be regarded as a good indicator of an organism's physiological state. The integration of the rolC gene from Agrobacterium rhizogenes and of the rat glucocorticoid receptor (gr) in Nicotiana langsdorffii Weinmann plants has shown to determine various physiological and metabolic effects. The analysis of wild and transgenic N. langsdorffii plants, exposed to different abiotic stresses (high temperature, water deficit, and high chromium concentrations) was conducted, in order to investigate the metabolic effects of the inserted genes in response to the applied stresses. The development of a new analytical procedure was necessary, in order to assure the simultaneous determination of analytes and to obtain an adequately low limit of quantification. For the first time, a sensitive HPLC-HRMS quantitative method for the simultaneous determination of salicylic acid, jasmonic acid and shikimic acid was developed and validated. The method was applied to 80 plant samples, permitting the evaluation of plant stress responses and highlighting some metabolic mechanisms. Salicylic, jasmonic and shikimic acids proved to be suitable for the comprehension of plant stress responses. Chemical and heat stresses showed to induce the highest changes in plant hormonal status, differently affecting plant response. The potential of each genetic modification toward the applied stresses was marked and particularly the resistance of the gr modified plants was evidenced. This work provides new information in the study of N. langsdorffii and transgenic organisms, which could be useful for the further application of these transgenes.

  14. Ser/Thr kinase-like protein of Nicotiana benthamiana is involved in the cell-to-cell movement of Bamboo mosaic virus.

    Directory of Open Access Journals (Sweden)

    Shun-Fang Cheng

    Full Text Available To investigate the plant genes affected by Bamboo mosaic virus (BaMV infection, we applied a cDNA-amplified fragment length polymorphism technique to screen genes with differential expression. A serine/threonine kinase-like (NbSTKL gene of Nicotiana benthamiana is upregulated after BaMV infection. NbSTKL contains the homologous domain of Ser/Thr kinase. Knocking down the expression of NbSTKL by virus-induced gene silencing reduced the accumulation of BaMV in the inoculated leaves but not in the protoplasts. The spread of GFP-expressing BaMV in the inoculated leaves is also impeded by a reduced expression of NbSTKL. These data imply that NbSTKL facilitates the cell-to-cell movement of BaMV. The subcellular localization of NbSTKL is mainly on the cell membrane, which has been confirmed by mutagenesis and fractionation experiments. Combined with the results showing that active site mutation of NbSTKL does not change its subcellular localization but significantly affects BaMV accumulation, we conclude that NbSTKL may regulate BaMV movement on the cell membrane by its kinase-like activity. Moreover, the transient expression of NbSTKL does not significantly affect the accumulation of Cucumber mosaic virus (CMV and Potato virus X (PVX; thus, NbSTKL might be a specific protein facilitating BaMV movement.

  15. Enhancement of RNA-directed DNA methylation of a transgene by simultaneously downregulating a ROS1 orthologue using a virus vector in Nicotiana benthamiana

    Directory of Open Access Journals (Sweden)

    Shungo eOtagaki

    2013-04-01

    Full Text Available Cytosine methylation can be induced by double-stranded RNAs through the RNA-directed DNA methylation (RdDM pathway. A DNA glycosylase REPRESSOR OF SILENCING 1 (ROS1 participates in DNA demethylation in Arabidopsis and may possibly counteract RdDM. Here, we isolated an orthologue of ROS1 (NbROS1 from Nicotiana benthamiana and examined the antagonistic activity of NbROS1 against virus-induced RdDM by simultaneously inducing RdDM and NbROS1 knockdown using a vector based on Cucumber mosaic virus. Plants were inoculated with a virus that contained a portion of the CaMV 35S promoter, which induced RdDM of the promoter integrated in the plant genome and transcriptional silencing of the green fluorescent protein gene driven by the promoter. Plants were also inoculated with a virus that contained a portion of NbROS1, which induced downregulation of NbROS1. Simultaneous induction of RdDM and NbROS1 knockdown resulted in an increase in the level of cytosine methylation of the target promoter. These results provide evidence for the presence of antagonistic activity of NbROS1 against virus-induced RdDM and suggest that the simultaneous induction of promoter-targeting RdDM and NbROS1 knockdown by a virus vector is useful as a tool to enhance targeted DNA methylation.

  16. Hydrophobin fusion of an influenza virus hemagglutinin allows high transient expression in Nicotiana benthamiana, easy purification and immune response with neutralizing activity.

    Directory of Open Access Journals (Sweden)

    Nicolas Jacquet

    Full Text Available The expression of recombinant hemagglutinin in plants is a promising alternative to the current egg-based production system for the influenza vaccines. Protein-stabilizing fusion partners have been developed to overcome the low production yields and the high downstream process costs associated with the plant expression system. In this context, we tested the fusion of hydrophobin I to the hemagglutinin ectodomain of the influenza A (H1N1pdm09 virus controlled by the hybrid En2PMA4 transcriptional promoter to rapidly produce high levels of recombinant antigen by transient expression in agro-infiltrated Nicotiana benthamiana leaves. The fusion increased the expression level by a factor of ∼ 2.5 compared to the unfused protein allowing a high accumulation level of 8.6% of the total soluble proteins. Hemagglutinin was located in ER-derived protein bodies and was successfully purified by combining an aqueous-two phase partition system and a salting out step. Hydrophobin interactions allowed the formation of high molecular weight hemagglutinin structures, while unfused proteins were produced as monomers. Purified protein was shown to be biologically active and to induce neutralizing antibodies after mice immunization. Hydrophobin fusion to influenza hemagglutinin might therefore be a promising approach for rapid, easy, and low cost production of seasonal or pandemic influenza vaccines in plants.

  17. Overexpression and self-assembly of virus-like particles in Nicotiana benthamiana by a single-vector DNA replicon system.

    Science.gov (United States)

    Moon, Ki-Beom; Lee, Jisu; Kang, Sebyung; Kim, Moonil; Mason, Hugh S; Jeon, Jae-Heung; Kim, Hyun-Soon

    2014-10-01

    Based on recent developments, virus-like particles (VLPs) are considered to be perfect candidates as nanoplatforms for applications in materials science and medicine. To succeed, mass production of VLPs and self-assembly into a correct form in plant systems are key factors. Here, we report expression of synthesized coat proteins of the three viruses, Brome mosaic virus, Cucumber mosaic virus, and Maize rayado fino virus, in Nicotiana benthamiana and production of self-assembled VLPs by transient expression system using agroinfiltration. Each coat protein was synthesized and cloned into a pBYR2fp single replicon vector. Target protein expression in cells containing p19 was fourfold higher than that of cells lacking p19. After agroinfiltration, protein expression was analyzed by SDS-PAGE and quantitative image analyzer. Quantitative analysis showed that BMVCP, CMVCP, and MRFVCP concentrations were 0.5, 1.0, and 0.8 mg · g(-1) leaf fresh weight, respectively. VLPs were purified by sucrose cushion ultracentrifugation and then analyzed by transmission electron microscopy. Our results suggested that BMVCP and CMVCP proteins expressed in N. benthamiana leaves were able to correctly self-assemble into particles. Moreover, we evaluated internal cavity accessibility of VLPs to load foreign molecules. Finally, plant growth conditions after agroinfiltration are critical for increasing heterologous protein expression levels in a transient expression system.

  18. A chimeric and truncated mitochondrial atpA gene is transcribed in alloplasmic cytoplasmic male-sterile tobacco with Nicotiana bigelovii mitochondria.

    Science.gov (United States)

    Bergman, P; Kofer, W; Håkansson, G; Glimelius, K

    1995-09-01

    Protoplast fusions were performed between two sexually produced alloplasmic male-sterile tobacco cultivars, with cytoplasms from Nicotiana bigelovii [Nta (big)S] and N. undulata[Nta(und)S], both of which exhibit homeotic-like phenotypes affecting the petal and stamen whorls. Among the fusion products obtained, both novel male-sterile and pollen-producing cybrid plants were identified. Of the pollen-producing cybrid plants, all of which were indehiscent, some had flowers with stamens that appeared normal when compared to male-fertile tobacco plants. Other hybrid plants were incompletely restored as they exhibited petaloid structures on the anther-bearing pollen-producing stamens. In this study, gel-blot analyses with mitochondrial geneprobes were conducted comparing the mitochondrial DNA of cybrids and male-sterile parents. It was found that the flower morphology typical of the Nta(big)S parental plants, as well as of the novel male-sterile cybrids, coincided with the presence of a chimeric atpA gene copy where an open reading frame of unknown origin was found to be linked in-frame to the 3'-end of a truncated atpA gene. RNA gel-blot hybridizations revealed the presence of atpA transcripts in the malesterile parent Nta(big)S and novel male-sterile cybrids, but which were absent in cybrids capable of pollen production.

  19. Downregulation of the NbNACa1 gene encoding a movement-protein-interacting protein reduces cell-to-cell movement of Brome mosaic virus in Nicotiana benthamiana.

    Science.gov (United States)

    Kaido, Masanori; Inoue, Yosuke; Takeda, Yoshika; Sugiyama, Kazuhiko; Takeda, Atsushi; Mori, Masashi; Tamai, Atsushi; Meshi, Tetsuo; Okuno, Tetsuro; Mise, Kazuyuki

    2007-06-01

    The 3a movement protein (MP) plays a central role in the movement of the RNA plant virus, Brome mosaic virus (BMV). To identify host factor genes involved in viral movement, a cDNA library of Nicotiana benthamiana, a systemic host for BMV, was screened with far-Western blotting using a recombinant BMV MP as probe. One positive clone encoded a protein with sequence similarity to the alpha chain of nascent-polypeptide-associated complex from various organisms, which is proposed to contribute to the fidelity of translocation of newly synthesized proteins. The orthologous gene from N. benthamiana was designated NbNACa1. The binding of NbNACa1 to BMV MP was confirmed in vivo with an agroinfiltration-immunoprecipitation assay. To investigate the involvement of NbNACa1 in BMV multiplication, NbNACa1-silenced (GSNAC) transgenic N. benthamiana plants were produced. Downregulation of NbNACa1 expression reduced virus accumulation in inoculated leaves but not in protoplasts. A microprojectile bombardment assay to monitor BMV-MP-assisted viral movement demonstrated reduced virus spread in GSNAC plants. The localization to the cell wall of BMV MP fused to green fluorescent protein was delayed in GSNAC plants. From these results, we propose that NbNACa1 is involved in BMV cell-to-cell movement through the regulation of BMV MP localization to the plasmodesmata.

  20. Expression of a Recombinant Anti-HIV and Anti-Tumor Protein, MAP30, in Nicotiana tobacum Hairy Roots: A pH-Stable and Thermophilic Antimicrobial Protein

    Science.gov (United States)

    Moghadam, Ali; Niazi, Ali; Afsharifar, Alireza; Taghavi, Seyed Mohsen

    2016-01-01

    In contrast to conventional antibiotics, which microorganisms can readily evade, it is nearly impossible for a microbial strain that is sensitive to antimicrobial proteins to convert to a resistant strain. Therefore, antimicrobial proteins and peptides that are promising alternative candidates for the control of bacterial infections are under investigation. The MAP30 protein of Momordica charantia is a valuable type I ribosome-inactivating protein (RIP) with anti-HIV and anti-tumor activities. Whereas the antimicrobial activity of some type I RIPs has been confirmed, less attention has been paid to the antimicrobial activity of MAP30 produced in a stable, easily handled, and extremely cost-effective protein-expression system. rMAP30-KDEL was expressed in Nicotiana tobacum hairy roots, and its effect on different microorganisms was investigated. Analysis of the extracted total proteins of transgenic hairy roots showed that rMAP30-KDEL was expressed effectively and that this protein exhibited significant antibacterial activity in a dose-dependent manner. rMAP30-KDEL also possessed thermal and pH stability. Bioinformatic analysis of MAP30 and other RIPs regarding their conserved motifs, amino-acid contents, charge, aliphatic index, GRAVY value, and secondary structures demonstrated that these factors accounted for their thermophilicity. Therefore, RIPs such as MAP30 and its derived peptides might have promising applications as food preservatives, and their analysis might provide useful insights into designing clinically applicable antibiotic agents. PMID:27459300

  1. The Sesquiterpenes(E-ß-Farnesene and (E-α-Bergamotene Quench Ozone but Fail to Protect the Wild Tobacco Nicotiana attenuata from Ozone, UVB, and Drought Stresses.

    Directory of Open Access Journals (Sweden)

    Evan C Palmer-Young

    Full Text Available Among the terpenes, isoprene (C5 and monoterpene hydrocarbons (C10 have been shown to ameliorate abiotic stress in a number of plant species via two proposed mechanisms: membrane stabilization and direct antioxidant effects. Sesquiterpene hydrocarbons (C15 not only share the structural properties thought to lend protective qualities to isoprene and monoterpene hydrocarbons, but also react rapidly with ozone, suggesting that sesquiterpenes may similarly enhance tolerance of abiotic stresses. To test whether sesquiterpenes protect plants against ozone, UVB light, or drought, we used transgenic lines of the wild tobacco Nicotiana attenuata. The transgenic plants expressed a maize terpene synthase gene (ZmTPS10 which produced a blend of (E-ß-farnesene and (E-α-bergamotene, or a point mutant of the same gene (ZmTPS10M which produced (E-ß-farnesene alone,. (E-ß-farnesene exerted a local, external, and transient ozone-quenching effect in ozone-fumigated chambers, but we found no evidence that enhanced sesquiterpene production by the plant inhibited oxidative damage, or maintained photosynthetic function or plant fitness under acute or chronic stress. Although the sesquiterpenes (E-ß-farnesene and (E-α-bergamotene might confer benefits under intermittent heat stress, which was not tested, any roles in relieving abiotic stress may be secondary to their previously demonstrated functions in biotic interactions.

  2. Isolation and characterization of an RNA-dependent RNA polymerase from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus.

    Science.gov (United States)

    Bates, H J; Farjah, M; Osman, T A; Buck, K W

    1995-06-01

    A template-bound RNA polymerase was isolated from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus (RCNMV) by differential centrifugation, solubilization with dodecyl beta-D-maltopyranoside, and chromatography on columns of Sephacryl S-400 and Q-Sepharose. Analysis of the purified polymerase by SDS-polyacrylamide gel electrophoresis, followed by silver staining or immunoblotting, showed that it contained virus-encoded proteins of molecular masses 27 kDa and 88 kDa together with several minor proteins possibly of host origin. After removal of endogenous RNA with micrococcal nuclease, the polymerase became template-dependent. It was also template-specific, being able to utilize as templates RNA of two strains of RCNMV, but not RNAs of three viruses in different taxonomic groups, namely cucumber mosaic cucumovirus, tomato bushy stunt tombusvirus and tomato mosaic tobamovirus. The products of RNA polymerase reactions were double-stranded RNAs corresponding to RCNMV RNAs 1 and 2. The ability of the template-dependent RNA polymerase to synthesize RNA was completely inhibited by antibodies to a peptide containing the GDD motif, whereas the activity of the template-bound enzyme was unaffected by these antibodies.

  3. Transplastomic Nicotiana benthamiana plants expressing multiple defence genes encoding protease inhibitors and chitinase display broad-spectrum resistance against insects, pathogens and abiotic stresses.

    Science.gov (United States)

    Chen, Peng-Jen; Senthilkumar, Rajendran; Jane, Wann-Neng; He, Yong; Tian, Zhihong; Yeh, Kai-Wun

    2014-05-01

    Plastid engineering provides several advantages for the next generation of transgenic technology, including the convenient use of transgene stacking and the generation of high expression levels of foreign proteins. With the goal of generating transplastomic plants with multiresistance against both phytopathogens and insects, a construct containing a monocistronic patterned gene stack was transformed into Nicotiana benthamiana plastids harbouring sweet potato sporamin, taro cystatin and chitinase from Paecilomyces javanicus. Transplastomic lines were screened and characterized by Southern/Northern/Western blot analysis for the confirmation of transgene integration and respective expression level. Immunogold localization analyses confirmed the high level of accumulation proteins that were specifically expressed in leaf and root plastids. Subsequent functional bioassays confirmed that the gene stacks conferred a high level of resistance against both insects and phytopathogens. Specifically, larva of Spodoptera litura and Spodoptera exigua either died or exhibited growth retardation after ingesting transplastomic plant leaves. In addition, the inhibitory effects on both leaf spot diseases caused by Alternaria alternata and soft rot disease caused by Pectobacterium carotovorum subsp. carotovorum were markedly observed. Moreover, tolerance to abiotic stresses such as salt/osmotic stress was highly enhanced. The results confirmed that the simultaneous expression of sporamin, cystatin and chitinase conferred a broad spectrum of resistance. Conversely, the expression of single transgenes was not capable of conferring such resistance. To the best of our knowledge, this is the first study to demonstrate an efficacious stacked combination of plastid-expressed defence genes which resulted in an engineered tolerance to various abiotic and biotic stresses.

  4. A calmodulin-like protein suppresses RNA silencing and promotes geminivirus infection by degrading SGS3 via the autophagy pathway in Nicotiana benthamiana

    Science.gov (United States)

    Li, Fangfang; Zhao, Nan; Xu, Xiongbiao; Wang, Yaqin; Yang, Xiuling; Liu, Shu-Sheng; Wang, Aiming; Zhou, Xueping

    2017-01-01

    A recently characterized calmodulin-like protein is an endogenous RNA silencing suppressor that suppresses sense-RNA induced post-transcriptional gene silencing (S-PTGS) and enhances virus infection, but the mechanism underlying calmodulin-like protein-mediated S-PTGS suppression is obscure. Here, we show that a calmodulin-like protein from Nicotiana benthamiana (NbCaM) interacts with Suppressor of Gene Silencing 3 (NbSGS3). Deletion analyses showed that domains essential for the interaction between NbSGS3 and NbCaM are also required for the subcellular localization of NbSGS3 and NbCaM suppressor activity. Overexpression of NbCaM reduced the number of NbSGS3-associated granules by degrading NbSGS3 protein accumulation in the cytoplasm. This NbCaM-mediated NbSGS3 degradation was sensitive to the autophagy inhibitors 3-methyladenine and E64d, and was compromised when key autophagy genes of the phosphatidylinositol 3-kinase (PI3K) complex were knocked down. Meanwhile, silencing of key autophagy genes within the PI3K complex inhibited geminivirus infection. Taken together these data suggest that NbCaM acts as a suppressor of RNA silencing by degrading NbSGS3 through the autophagy pathway. PMID:28212430

  5. N-glycosylation of cholera toxin B subunit in Nicotiana benthamiana: impacts on host stress response, production yield and vaccine potential.

    Science.gov (United States)

    Hamorsky, Krystal Teasley; Kouokam, J Calvin; Jurkiewicz, Jessica M; Nelson, Bailey; Moore, Lauren J; Husk, Adam S; Kajiura, Hiroyuki; Fujiyama, Kazuhito; Matoba, Nobuyuki

    2015-01-23

    Plant-based transient overexpression systems enable rapid and scalable production of subunit vaccines. Previously, we have shown that cholera toxin B subunit (CTB), an oral cholera vaccine antigen, is N-glycosylated upon expression in transgenic Nicotiana benthamiana. Here, we found that overexpression of aglycosylated CTB by agroinfiltration of a tobamoviral vector causes massive tissue necrosis and poor accumulation unless retained in the endoplasmic reticulum (ER). However, the re-introduction of N-glycosylation to its original or an alternative site significantly relieved the necrosis and provided a high CTB yield without ER retention. Quantitative gene expression analysis of PDI, BiP, bZIP60, SKP1, 26Sα proteasome and PR1a, and the detection of ubiquitinated CTB polypeptides revealed that N-glycosylation significantly relieved ER stress and hypersensitive response, and facilitated the folding/assembly of CTB. The glycosylated CTB (gCTB) was characterized for potential vaccine use. Glycan profiling revealed that gCTB contained approximately 38% plant-specific glycans. gCTB retained nanomolar affinity to GM1-ganglioside with only marginal reduction of physicochemical stability and induced an anti-cholera holotoxin antibody response comparable to native CTB in a mouse oral immunization study. These findings demonstrated gCTB's potential as an oral immunogen and point to a potential role of N-glycosylation in increasing recombinant protein yields in plants.

  6. Effects of sublethal concentrations of tobacco (Nicotiana tobaccum leaf dust on some biochemical parameters of Hybrid catfish (Clarias gariepinus and Heterobranchus bidorsalis

    Directory of Open Access Journals (Sweden)

    Kabir Mohammed Adamu

    2011-02-01

    Full Text Available The aim of this work was to study the effect of tobacco (Nicotiana tobaccum leaf dust at sublethal concentrations (375.6, 187.8, 93.9 0.0mg/L on biochemical parameters of Hybrid catfish in a static semi-renewable bioassay system with the aim to ascertain its effect on the test fish after 14 days exposure period. The effects of the plant dust on the test fish was dose-dependent, revealing significant difference in albumin, glucose, triglyceride, cholesterol, urea, and uric acid of the serum, liver and kidney; total protein and globulin of serum and kidney and creatinine and calcium of the serum while insignificant difference were observed for calcium and creatinine in liver and kidney and total protein and globulin in liver compared to the control. The effect of the plant dust was most pronounced in the serum, less in the kidney and least in the liver. However, the monitored water quality parameters revealed that the plant dust had effects on primary productivity, thus the biodiversity of organisms.

  7. Transcriptomic responses of the aphid Myzus persicae nicotianae Blackman (Hemiptera: Aphididae to insecticides: Analyses in the single Chilean clone of the tobacco aphid

    Directory of Open Access Journals (Sweden)

    Marco Cabrera-Brandt

    2014-04-01

    Full Text Available The tobacco aphid Myzus persicae nicotianae Blackman is a subspecies of the highly polyphagous and agricultural pest Myzus persicae (Sulzer. For its control, insecticide applications are widely used, but resistance to numerous molecules has been reported, displaying at least three insecticide resistance mechanisms, including: (i elevated carboxylesterases (E-Carb, (ii modification of the acetylcholinesterase (MACE, and (iii kdr and super-kdr insensitivity mutations. In Chile, populations of the tobacco aphid are characterized by the presence of a single predominant clone, which is also present in high proportions in other countries of the Americas. This aphid clone exhibits low levels of carboxylesterase activity and is kdr susceptible, but the MACE mechanism of insecticide resistance has not been studied. In order to characterize the tobacco aphid in terms of the MACE mechanism and to identify a preliminary group of aphid genes putatively involved in insecticide resistance, a cDNA microarray was used to study the transcriptomic responses when aphids are sprayed with a carbamate insecticide. The single Chilean clone of the tobacco aphid was characterized as MACE susceptible, but we found 38 transcripts significantly regulated by insecticide exposure (13 up- and 25 down-regulated genes. The expression of six of them was validated by qRT-PCR experiments at several time points (6, 12, 18, 24, 30, 36, and 42 h after insecticide application. This mutational and transcriptomic characterization of the tobacco aphid responding to insecticide spray opens new hypotheses in the understanding of the molecular mechanisms underlying insecticide resistance.

  8. An Accessory Protease Inhibitor to Increase the Yield and Quality of a Tumour-Targeting mAb in Nicotiana benthamiana Leaves.

    Science.gov (United States)

    Jutras, Philippe V; Marusic, Carla; Lonoce, Chiara; Deflers, Carole; Goulet, Marie-Claire; Benvenuto, Eugenio; Michaud, Dominique; Donini, Marcello

    2016-01-01

    The overall quality of recombinant IgG antibodies in plants is dramatically compromised by host endogenous proteases. Different approaches have been developed to reduce the impact of endogenous proteolysis on IgGs, notably involving site-directed mutagenesis to eliminate protease-susceptible sites or the in situ mitigation of host protease activities to minimize antibody processing in the cell secretory pathway. We here characterized the degradation profile of H10, a human tumour-targeting monoclonal IgG, in leaves of Nicotiana benthamiana also expressing the human serine protease inhibitor α1-antichymotrypsin or the cysteine protease inhibitor tomato cystatin SlCYS8. Leaf extracts revealed consistent fragmentation patterns for the recombinant antibody regardless of leaf age and a strong protective effect of SlCYS8 in specific regions of the heavy chain domains. As shown using an antigen-binding ELISA and LC-MS/MS analysis of antibody fragments, SlCYS8 had positive effects on both the amount of fully-assembled antibody purified from leaf tissue and the stability of biologically active antibody fragments containing the heavy chain Fc domain. Our data confirm the potential of Cys protease inhibitors as convenient antibody-stabilizing expression partners to increase the quality of therapeutic antibodies in plant protein biofactories.

  9. SGS3 Cooperates with RDR6 in Triggering Geminivirus-Induced Gene Silencing and in Suppressing Geminivirus Infection in Nicotiana Benthamiana

    Directory of Open Access Journals (Sweden)

    Fangfang Li

    2017-09-01

    Full Text Available RNA silencing has an important role in defending against virus infection in plants. Plants with the deficiency of RNA silencing components often show enhanced susceptibility to viral infections. RNA-dependent RNA polymerase (RDRs mediated-antiviral defense has a pivotal role in resistance to many plant viruses. In RDR6-mediated defense against viral infection, a plant-specific RNA binding protein, Suppressor of Gene Silencing 3 (SGS3, was also found to fight against some viruses in Arabidopsis. In this study, we showed that SGS3 from Nicotiana benthamiana (NbSGS3 is required for sense-RNA induced post-transcriptional gene silencing (S-PTGS and initiating sense-RNA-triggered systemic silencing. Further, the deficiency of NbSGS3 inhibited geminivirus-induced endogenous gene silencing (GIEGS and promoted geminivirus infection. During TRV-mediated NbSGS3 or N. benthamiana RDR6 (NbRDR6 silencing process, we found that their expression can be effectively fine-tuned. Plants with the knock-down of both NbSGS3 and NbRDR6 almost totally blocked GIEGS, and were more susceptible to geminivirus infection. These data suggest that NbSGS3 cooperates with NbRDR6 against GIEGS and geminivirus infection in N. benthamiana, which provides valuable information for breeding geminivirus-resistant plants.

  10. NaJAZh Regulates a Subset of Defense Responses against Herbivores and Spontaneous Leaf Necrosis in Nicotiana attenuata Plants[C][W][OA

    Science.gov (United States)

    Oh, Youngjoo; Baldwin, Ian T.; Gális, Ivan

    2012-01-01

    The JASMONATE ZIM DOMAIN (JAZ) proteins function as negative regulators of jasmonic acid signaling in plants. We cloned 12 JAZ genes from native tobacco (Nicotiana attenuata), including nine novel JAZs in tobacco, and examined their expression in plants that had leaves elicited by wounding or simulated herbivory. Most JAZ genes showed strong expression in the elicited leaves, but NaJAZg was mainly expressed in roots. Another novel herbivory-elicited gene, NaJAZh, was analyzed in detail. RNA interference suppression of this gene in inverted-repeat (ir)JAZh plants deregulated a specific branch of jasmonic acid-dependent direct and indirect defenses: irJAZh plants showed greater trypsin protease inhibitor activity, 17-hydroxygeranyllinalool diterpene glycosides accumulation, and emission of volatile organic compounds from leaves. Silencing of NaJAZh also revealed a novel cross talk in JAZ-regulated secondary metabolism, as irJAZh plants had significantly reduced nicotine levels. In addition, irJAZh spontaneously developed leaf necrosis during the transition to flowering. Because the lesions closely correlated with the elevated expression of programmed cell death genes and the accumulations of salicylic acid and hydrogen peroxide in the leaves, we propose a novel role of the NaJAZh protein as a repressor of necrosis and/or programmed cell death during plant development. PMID:22496510

  11. Multivariate optimization of a headspace solid-phase microextraction method followed by gas chromatography with mass spectrometry for the determination of terpenes in Nicotiana langsdorffii.

    Science.gov (United States)

    Ardini, Francisco; Carro, Marina Di; Abelmoschi, Maria Luisa; Grotti, Marco; Magi, Emanuele

    2014-07-01

    A simple and sensitive procedure based on headspace solid-phase microextraction and gas chromatography with mass spectrometry was developed for the determination of five terpenes (α-pinene, limonene, linalool, α-terpineol, and geraniol) in the leaves of Nicotiana langsdorffii. The microextraction conditions (extraction temperature, equilibration time, and extraction time) were optimized by means of a Doehlert design. The experimental design showed that, for α-pinene and limonene, a low temperature and a long extraction time were needed for optimal extraction, while linalool, α-terpineol, and geraniol required a high temperature and a long extraction time. The chosen compromise conditions were temperature 60°C, equilibration time 15 min and extraction time 50 min. The main analytical figures of the optimized method were evaluated; LODs ranged from 0.07 ng/g (α-pinene) to 8.0 ng/g (geraniol), while intraday and interday repeatability were in the range 10-17% and 9-13%, respectively. Finally, the procedure was applied to in vitro wild-type and transgenic specimens of N. langsdorffii subjected to abiotic stresses (chemical and heat stress). With the exception of geraniol (75-374 ng/g), low concentration levels of terpenes were measured (ng/g level or lower); some interesting variations in terpene concentration induced by abiotic stress were observed.

  12. Heterologous Expression Screens in Nicotiana benthamiana Identify a Candidate Effector of the Wheat Yellow Rust Pathogen that Associates with Processing Bodies

    Science.gov (United States)

    Petre, Benjamin; Saunders, Diane G. O.; Sklenar, Jan; Lorrain, Cécile; Krasileva, Ksenia V.; Win, Joe; Duplessis, Sébastien; Kamoun, Sophien

    2016-01-01

    Rust fungal pathogens of wheat (Triticum spp.) affect crop yields worldwide. The molecular mechanisms underlying the virulence of these pathogens remain elusive, due to the limited availability of suitable molecular genetic research tools. Notably, the inability to perform high-throughput analyses of candidate virulence proteins (also known as effectors) impairs progress. We previously established a pipeline for the fast-forward screens of rust fungal candidate effectors in the model plant Nicotiana benthamiana. This pipeline involves selecting candidate effectors in silico and performing cell biology and protein-protein interaction assays in planta to gain insight into the putative functions of candidate effectors. In this study, we used this pipeline to identify and characterize sixteen candidate effectors from the wheat yellow rust fungal pathogen Puccinia striiformis f sp tritici. Nine candidate effectors targeted a specific plant subcellular compartment or protein complex, providing valuable information on their putative functions in plant cells. One candidate effector, PST02549, accumulated in processing bodies (P-bodies), protein complexes involved in mRNA decapping, degradation, and storage. PST02549 also associates with the P-body-resident ENHANCER OF mRNA DECAPPING PROTEIN 4 (EDC4) from N. benthamiana and wheat. We propose that P-bodies are a novel plant cell compartment targeted by pathogen effectors. PMID:26863009

  13. Heterologous Expression Screens in Nicotiana benthamiana Identify a Candidate Effector of the Wheat Yellow Rust Pathogen that Associates with Processing Bodies.

    Directory of Open Access Journals (Sweden)

    Benjamin Petre

    Full Text Available Rust fungal pathogens of wheat (Triticum spp. affect crop yields worldwide. The molecular mechanisms underlying the virulence of these pathogens remain elusive, due to the limited availability of suitable molecular genetic research tools. Notably, the inability to perform high-throughput analyses of candidate virulence proteins (also known as effectors impairs progress. We previously established a pipeline for the fast-forward screens of rust fungal candidate effectors in the model plant Nicotiana benthamiana. This pipeline involves selecting candidate effectors in silico and performing cell biology and protein-protein interaction assays in planta to gain insight into the putative functions of candidate effectors. In this study, we used this pipeline to identify and characterize sixteen candidate effectors from the wheat yellow rust fungal pathogen Puccinia striiformis f sp tritici. Nine candidate effectors targeted a specific plant subcellular compartment or protein complex, providing valuable information on their putative functions in plant cells. One candidate effector, PST02549, accumulated in processing bodies (P-bodies, protein complexes involved in mRNA decapping, degradation, and storage. PST02549 also associates with the P-body-resident ENHANCER OF mRNA DECAPPING PROTEIN 4 (EDC4 from N. benthamiana and wheat. We propose that P-bodies are a novel plant cell compartment targeted by pathogen effectors.

  14. Genetic alteration in hepatocellular carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Yoo Chul; Kang, Tae Woong; Lee, Jin Oh [Korea Cancer Center Hospital of Korea Atomic Energy Research Institute, Seoul (Korea, Republic of)

    1994-12-01

    Cancer of stomach, colon and liver are a group of the most common cancer in Korea. However, results with current therapeutic modalities are still unsatisfactory. The intensive efforts have been made to understand basic pathogenesis and to find better therapeutic tools for the treatment of this miserable disease. We studied the alteration of tumor suppressor genes and oncogenes in hepatocellular carcinoma in Korea. We found that alteration of Rb gene, APC were 33 %, 13 % respectively. But alterations of oncogenes such as myc, ras and mdm2 were rarely found. Our results suggests that HBV may act as oncogenic role in hepatocarcinogenesis instead of oncogenes. 6 figs, 2 tabs. (Author).

  15. Epigenetic alterations in gastric carcinogenesis

    Institute of Scientific and Technical Information of China (English)

    In-Seon CHOI; Tsung-Teh WU

    2005-01-01

    Gastric cancer is believed to result in part from the accumulation of multiple genetic alterations leading to oncogene overexpression and tumor suppressor loss. Epigenetic alterations as a distinct and crucial mechanism to silence a variety of methylated tissue-specific and imprinted genes, have been extensively studied in gastric carcinoma and play important roles in gastric carcinogenesis. This review will briefly discuss the basic aspects of DNA methylation and CpG island methylation, in particular the epigenetic alterations of certain critical genes implicated in gastric carcinogenesis and its relevance of clinical implications.

  16. Complement Activation Alters Platelet Function

    Science.gov (United States)

    2015-12-01

    Award Number: W81XWH-12-1-0523 TITLE: Complement Activation Alters Platelet Function PRINCIPAL INVESTIGATOR: George Tsokos, M.D. CONTRACTING...Activation Alters Platelet Function 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-12-1-0523 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) George Tsokos, M.D...a decreased level of disease. Further studies will expand upon these observations better outlining the function of platelets in the injury associated

  17. The coat protein of Alternanthera mosaic virus is the elicitor of a temperature-sensitive systemic necrosis in Nicotiana benthamiana, and interacts with a host boron transporter protein

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Hyoun-Sub, E-mail: hyounlim@cnu.ac.kr [Department of Applied Biology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Nam, Jiryun, E-mail: jilyoon@naver.com [Department of Applied Biology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Seo, Eun-Young, E-mail: sey22@cnu.ac.kr [Department of Applied Biology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Nam, Moon, E-mail: moonlit51@cnu.ac.kr [Department of Applied Biology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Vaira, Anna Maria, E-mail: a.vaira@ivv.cnr.it [Floral and Nursery Plants Research Unit, US National Arboretum, USDA-ARS, 10300 Baltimore Avenue B-010A, Beltsville, MD 20705 (United States); Istituto di Virologia Vegetale, CNR, Strada delle Cacce 73, Torino 10135 (Italy); Bae, Hanhong, E-mail: hanhongbae@ynu.ac.kr [School of Biotechnology, Yeungnam University, Geongsan 712-749 (Korea, Republic of); Jang, Chan-Yong, E-mail: sunbispirit@gmail.com [Department of Applied Biology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Lee, Cheol Ho, E-mail: chlee1219@hanmail.net [Department of Chemical and Biological Engineering, Seokyoung University, Seoul 136-704 (Korea, Republic of); Kim, Hong Gi, E-mail: hgkim@cnu.ac.kr [Department of Applied Biology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Roh, Mark, E-mail: marksroh@gmail.com [Floral and Nursery Plants Research Unit, US National Arboretum, USDA-ARS, 10300 Baltimore Avenue B-010A, Beltsville, MD 20705 (United States); Laboratory of Floriculture and Plant Physiology, School of Bio-Resource Science, Dankook University, Cheonan, Chungnam 330-714 (Korea, Republic of); Hammond, John, E-mail: john.hammond@ars.usda.gov [Floral and Nursery Plants Research Unit, US National Arboretum, USDA-ARS, 10300 Baltimore Avenue B-010A, Beltsville, MD 20705 (United States)

    2014-03-15

    Different isolates of Alternanthera mosaic virus (AltMV; Potexvirus), including four infectious clones derived from AltMV-SP, induce distinct systemic symptoms in Nicotiana benthamiana. Virus accumulation was enhanced at 15 °C compared to 25 °C; severe clone AltMV 3-7 induced systemic necrosis (SN) and plant death at 15 °C. No interaction with potexvirus resistance gene Rx was detected, although SN was ablated by silencing of SGT1, as for other cases of potexvirus-induced necrosis. Substitution of AltMV 3-7 coat protein (CP{sub SP}) with that from AltMV-Po (CP{sub Po}) eliminated SN at 15 °C, and ameliorated symptoms in Alternanthera dentata and soybean. Substitution of only two residues from CP{sub Po} [either MN(13,14)ID or LA(76,77)IS] efficiently ablated SN in N. benthamiana. CP{sub SP} but not CP{sub Po} interacted with Arabidopsis boron transporter protein AtBOR1 by yeast two-hybrid assay; N. benthamiana homolog NbBOR1 interacted more strongly with CP{sub SP} than CP{sub Po} in bimolecular fluorescence complementation, and may affect recognition of CP as an elicitor of SN. - Highlights: • Alternanthera mosaic virus CP is an elicitor of systemic necrosis in N. benthamiana. • Virus-induced systemic necrosis is enhanced at 15 °C compared to 25 °C. • Induction of systemic necrosis is dependent on as few as two CP amino acid residues. • These residues are at subunit interfaces within the same turn of the virion helix. • Inducer/non-inducer CPs interact differentially with a boron transporter protein.

  18. Nicotiana benthamiana calreticulin 3a is required for the ethylene-mediated production of phytoalexins and disease resistance against oomycete pathogen Phytophthora infestans.

    Science.gov (United States)

    Matsukawa, Mizuki; Shibata, Yusuke; Ohtsu, Mina; Mizutani, Aki; Mori, Hitoshi; Wang, Ping; Ojika, Makoto; Kawakita, Kazuhito; Takemoto, Daigo

    2013-08-01

    Mature Nicotiana benthamiana shows strong resistance to the potato late blight pathogen Phytophthora infestans. By screening using virus-induced random gene silencing, we isolated a gene for plant-specific calreticulin NbCRT3a as a required gene for resistance of N. benthamiana against P. infestans. NbCRT3a encodes an endoplasmic reticulum quality-control (ERQC) chaperone for the maturation of glycoproteins, including glycosylated cell-surface receptors. NbCRT3a-silenced plants showed no detectable growth defects but resistance to P. infestans was significantly compromised. Defense responses induced by the treatment with INF1 (a secretory protein of P. infestans), such as production of reactive oxygen species and accumulation of phytoalexins, were suppressed in NbCRT3a-silenced N. benthamiana. Expression of an ethylene-regulated gene for phytoalexin biosynthesis, NbEAS, was reduced in NbCRT3a-silenced plants, whereas the expression of salicylic acid-regulated NbPR-1a was not affected. Consistently, induction of ethylene production by INF1 was suppressed in NbCRT3a-silenced plants. Resistance reactions induced by a hyphal wall components elicitor prepared from P. infestans were also impaired in NbCRT3a-silenced plants. However, cell death induced by active mitogen-activated protein kinase kinase (NbMEK2(DD)) was not affected by the silencing of NbCRT3a. Thus, NbCRT3a is required for the initiation of resistance reactions of N. benthamiana in response to elicitor molecules derived from P. infestans.

  19. The glutathione transferase of Nicotiana benthamiana NbGSTU4 plays a role in regulating the early replication of Bamboo mosaic virus.

    Science.gov (United States)

    Chen, I-Hsuan; Chiu, Meng-Hsuen; Cheng, Shun-Fang; Hsu, Yau-Heiu; Tsai, Ching-Hsiu

    2013-08-01

    Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus. One of the plant glutathione S-transferase (GST) genes, NbGSTU4, responds as an upregulated gene in Nicotiana benthamiana post BaMV infection. In order to identify the role of NbGSTU4 in BaMV infection, the expression of NbGSTU4 was knocked down using a virus-induced gene silencing technique or was transiently expressed in N. benthamiana in BaMV inoculation. The results show a significant decrease in BaMV RNA accumulation when the expression level of NbGSTU4 is reduced; whereas the viral RNA accumulation increases when NbGSTU4 is transiently expressed. Furthermore, this study identified that the involvement of NbGSTU4 in viral RNA accumulation occurs by its participation in the viral early replication step. The findings show that the NbGSTU4 protein expressed from Escherichia coli can interact with the 3' untranslated region (UTR) of the BaMV RNA in vitro in the presence of glutathione (GSH). The addition of GSH in the in vitro replication assay shows an enhancement of minus-strand but not plus-strand RNA synthesis. The results suggest that the plant GST protein plays a role in binding viral RNA and delivering GSH to the replication complex to create a reduced condition for BaMV minus-strand RNA synthesis. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  20. Influenza virus-like particles produced by transient expression in Nicotiana benthamiana induce a protective immune response against a lethal viral challenge in mice.

    Science.gov (United States)

    D'Aoust, Marc-André; Lavoie, Pierre-Olivier; Couture, Manon M-J; Trépanier, Sonia; Guay, Jean-Martin; Dargis, Michèle; Mongrand, Sébastien; Landry, Nathalie; Ward, Brian J; Vézina, Louis-P

    2008-12-01

    A strain-specific vaccine represents the best possible response to the threat of an influenza pandemic. Rapid delivery of such a vaccine to the world's population before the peak of the first infection wave seems to be an unattainable goal with the current influenza vaccine manufacturing capacity. Plant-based transient expression is one of the few production systems that can meet the anticipated surge requirement. To assess the capability of plant agroinfiltration to produce an influenza vaccine, we expressed haemagglutinin (HA) from strains A/Indonesia/5/05 (H5N1) and A/New Caledonia/20/99 (H1N1) by agroinfiltration of Nicotiana benthamiana plants. Size distribution analysis of protein content in infiltrated leaves revealed that HA was predominantly assembled into high-molecular-weight structures. H5-containing structures were purified and examination by transmission electron microscopy confirmed virus-like particle (VLP) assembly. High-performance thin layer chromatography analysis of VLP lipid composition highlighted polar and neutral lipid contents comparable with those of purified plasma membranes from tobacco plants. Electron microscopy of VLP-producing cells in N. benthamiana leaves confirmed that VLPs accumulated in apoplastic indentations of the plasma membrane. Finally, immunization of mice with two doses of as little as 0.1 microg of purified influenza H5-VLPs triggered a strong immune response against the homologous virus, whereas two doses of 0.5 microg of H5-VLPs conferred complete protection against a lethal challenge with the heterologous A/Vietnam/1194/04 (H5N1) strain. These results show, for the first time, that plants are capable of producing enveloped influenza VLPs budding from the plasma membrane; such VLPs represent very promising candidates for vaccination against influenza pandemic strains.

  1. Removal of bacterial suspension water occupying the intercellular space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a Nicotiana benthamiana transient gene expression system.

    Science.gov (United States)

    Fujiuchi, Naomichi; Matsuda, Ryo; Matoba, Nobuyuki; Fujiwara, Kazuhiro

    2016-04-01

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of bacterial suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow bacterial suspension water occupying intercellular space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g(-1) relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield