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Sample records for transgenic tobacco nicotiana

  1. [Induced expression of Serratia marcescens ribonuclease III gene in transgenic Nicotiana tabacum L. cv. SR1 tobacco plants].

    Science.gov (United States)

    Zhirnov, I V; Trifonova, E A; Romanova, A V; Filipenko, E A; Sapotsky, M V; Malinovsky, V I; Kochetov, A V; Shumny, V K

    2016-11-01

    Transgenic Nicotiana tabacum L. cv. SR1 plants, characterized by an increase in the level of dsRNA-specific hydrolytic activity after induction by wounding, were obtained. The Solanum lycopersicum anionic peroxidase gene promoter (new for plant genetic engineering) was for the first time used for the induced expression of the target Serratia marcescens RNase III gene. Upon infection with the tobacco mosaic virus (TMV), the transgenic plants of the obtained lines did not differ significantly from the control group in the level of TMV capsid protein accumulation. In general, no delay in the development of the infection symptoms was observed in transgenic plants as compared with the control group. The obtained transgenic plants represent a new model for the study of the biological role of endoribonucleases from the RNase III family, including in molecular mechanisms of resistance to pathogens.

  2. Developmental and environmental regulation of the Nicotiana plumbaginifolia cytosolic Cu/Zn-superoxide dismutase promoter in transgenic tobacco.

    Science.gov (United States)

    Hérouart, D; Van Montagu, M; Inzé, D

    1994-03-01

    Superoxide dismutases (SODs) play a key role in the cellular defense against reactive oxygen species. To study the transcriptional regulation at the cellular level, the promoter of the Nicotiana plumbaginifolia cytosolic gene encoding Cu/ZnSOD (SODCc) was fused to the beta-glucuronidase (GUS) reporter gene (gusA) and analyzed in transgenic tobacco plants. The promoter was highly active in vascular bundles of leaves and stems, where it is confined to phloem cells. In flowers, GUS activity was detected in ovules and pollen grains, in pigmented tissues of petals, and in vascular tissue of ovaries and anthers. In response to treatment with the superoxide-generating herbicide paraquat, very strong GUS staining was observed in photosynthetically active cells of leaves and in some epidermal root cells of seedlings. The expression of the SODCc-gusA was also induced in seedlings after heat shock and chilling and after treatment with sulfhydryl antioxidants such as reduced glutathione and cysteine. It is postulated that SODCc expression is directly linked to a cell-specific production of excess superoxide radicals in the cytosol.

  3. Truncated presequences of mitochondrial F1-ATPase beta subunit from Nicotiana plumbaginifolia transport CAT and GUS proteins into mitochondria of transgenic tobacco.

    Science.gov (United States)

    Chaumont, F; Silva Filho, M de C; Thomas, D; Leterme, S; Boutry, M

    1994-02-01

    The mitochondrial F1-ATPase beta subunit (ATPase-beta) of Nicotiana plumbaginifolia is nucleus-encoded as a precursor containing an NH2-terminal extension. By sequencing the mature N. tabacum ATPase-beta, we determined the length of the presequence, viz. 54 residues. To define the essential regions of this presequence, we produced a series of 3' deletions in the sequence coding for the 90 NH2-terminal residues of ATPase-beta. The truncated sequences were fused with the chloramphenicol acetyl transferase (cat) and beta-glucuronidase (gus) genes and introduced into tobacco plants. From the observed distribution of CAT and GUS activity in the plant cells, we conclude that the first 23 amino-acid residues of ATPase-beta remain capable of specifically targeting reporter proteins into mitochondria. Immunodetection in transgenic plants and in vitro import experiments with various CAT fusion proteins show that the precursors are processed at the expected cleavage site but also at a cryptic site located in the linker region between the presequence and the first methionine of native CAT.

  4. Tapetum development in transgenic tobacco (Nicotiana tabacum L. plants with modlfied level of histone H1 variants

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    Joanna Ślusarczyk

    2011-01-01

    Full Text Available The phenomenon of male sterility has often been observed in investigations on the role of histone H1 in regulation of morphogenetic and cytological processes in transgenic tobacco plants. These changes were accumulated by disturbances in flower development, consisting in lengthening of the pistil style in relation to stamen heads. This prevented pollination and production of seeds. As similar abnormalities occurred also in the present investigations (depending on combination, the sterility% was 84.4 to 19.9, at only 8.1 in the control, the main problem of our investigations was an attempt to explain their reasons. It is commonly known that one of the conditions for formation of fertile pollen is the properly functioning tapetum. Here, we carried out observations of ultrastructure of anther tapetum control cells in respect of abnormalities which occurred during microsporogenesis of transgenic plants with inactivated expression of two major (A, B and two minor (C, D histone H1 variants. The investigations were carried out on the following groups of plants: (1 control group with a full set of histone variants (K, (2 with inactivated A and B variants (-AB; (3 with inactivated A, B, C and D variants (-ABCD, (4 with inactivated C and D variants (-CD. It was found that tapetal development was normal in all the investigated groups of plants, and the sequence of changes was similar as in the control. However, certain ultrastructural differences appeared when tapetum functioned as secretory tissue, and in the degeneration phase. In tapetal cell cytoplasm, with participation of rER, lipid bodies were formed, which, having penetrated to the cell surface and to locules, took part in formation of pollen grain sporoderm. Both in the control and in the remaining combination, excluding -ABCD, these bodies looked similar: they were grey, homogenous and surrounded by black jagged deposits. In -ABCD plants, these bodies were more translucent, slightly rarefied, and

  5. Biotechnological Reduction of Tobacco (Nicotiana Tabacum L. Toxicity

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    Samane Sattar

    2012-11-01

    Full Text Available Background: Nicotiana tobacco contains large amounts of alkaloid nicotine. Tobacco plant is used for smoking and causes many health problems since it is high in nicotine which is one of the widely-recognized toxic compounds with serious side effects for different body organs. Reducing nicotine content of this plant is a way to reduce its health hazards in cigarette smokers. Utilizing the new methods of genetic engineering can modify nicotine levels in the plant. In this study, through transferring the blocking gene, the pathway of nicotine biosynthesis was blocked to produce transgenic tobacco with low levels of nicotine. Methods: Transgenic plants carrying T DNA, and non-transgenic plants were grown on MS medium. Then their leaves were dried and powdered. The plants were extracted with alkali solution. Eventually, the nicotine content of the extract were analyzed using GC. Results: The analysis of extracts showed a reduction in the nicotine content of the transgenic plant (contain T-DNA in comparison with non-transgenic plants. Conclusion: Tobacco with lower nicotine reduction can reduce the toxic effects of smoking on smokers and can facilitate withdrawal from it.

  6. 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...

  7. Acute toxicity of tobacco ( Nicotiana tobaccum ) leaf dust on ...

    African Journals Online (AJOL)

    Experiments were conducted using dry tobacco (Nicotiana tobaccum) leaves aqueous extract to determine the acute toxicity and sub lethal effects on some haematological indices of Oreochromis niloticus using static renewable bioassay method. The extract was found to be toxic with a 48-h LC50 value of 109.6 mg/l.

  8. Glutathione Transferase from Trichoderma virens Enhances Cadmium Tolerance without Enhancing Its Accumulation in Transgenic Nicotiana tabacum

    Science.gov (United States)

    Dixit, Prachy; Mukherjee, Prasun K.; Ramachandran, V.; Eapen, Susan

    2011-01-01

    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 developing Cd tolerance and

  9. 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

  10. Nitric oxide enhances osmoregulation of tobacco ( Nicotiana ...

    African Journals Online (AJOL)

    This study was carried out to investigate the effect of the intracellular signaling molecule nitric oxide (NO) on osmoregulation of tobacco cells under osmotic stress caused by phenylethanoid glycosides 6000 (PEG 6000). The results show that the PEG stress induced a specific pattern of endogenous NO production with two ...

  11. 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.

  12. Accumulation of nickel in transgenic tobacco

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    Sidik, Nik Marzuki; Othman, Noor Farhan

    2013-11-01

    The accumulation of heavy metal Ni in the roots and leaves of four T1 transgenic lines of tobacco (T(1)20E, T(1)24C, T(1)18B1 and T(1)20B) expressing eiMT1 from E.indica was assessed. The aim of the study was to investigate the level of Ni accumulation in the leaves and roots of each transgenic lines and to evaluate the eligibility of the plants to be classified as a phytoremediation agent. All of the transgenic lines showed different ability in accumulating different metals and has translocation factor (TF) less than 1 (TFtransgenic lines, transgenic line T(1)24C showed the highest accumulation of Ni (251.9 ± 0.014 mg/kg) and the lowest TF value (TFT(1)24C=0.0875) at 60 ppm Ni.

  13. Population structure and genetic diversity of Phytophthora nicotianae from tobacco in Georgia

    Science.gov (United States)

    Black shank caused by Phytophthora nicotianae occurs worldwide and is responsible for significant yield loss in tobacco production in Georgia. Management of the disease has primarily relied on utilization of tobacco cultivars with resistance to race 0 of the pathogen and application of the fungicide...

  14. 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

  15. The grapevine VvWRKY2 gene enhances salt and osmotic stress tolerance in transgenic Nicotiana tabacum.

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    Mzid, Rim; Zorrig, Walid; Ben Ayed, Rayda; Ben Hamed, Karim; Ayadi, Mariem; Damak, Yosra; Lauvergeat, Virginie; Hanana, Mohsen

    2018-06-01

    Our study aims to assess the implication of WRKY transcription factor in the molecular mechanisms of grapevine adaptation to salt and water stresses. In this respect, a full-length VvWRKY2 cDNA, isolated from a Vitis vinifera grape berry cDNA library, was constitutively over-expressed in Nicotiana tabacum seedlings. Our results showed that transgenic tobacco plants exhibited higher seed germination rates and better growth, under both salt and osmotic stress treatments, when compared to wild type plants. Furthermore, our analyses demonstrated that, under stress conditions, transgenic plants accumulated more osmolytes, such as soluble sugars and free proline, while no changes were observed regarding electrolyte leakage, H 2 O 2 , and malondialdehyde contents. The improvement of osmotic adjustment may be an important mechanism underlying the role of VvWRKY 2 in promoting tolerance and adaptation to abiotic stresses. Principal component analysis of our results highlighted a clear partition of plant response to stress. On the other hand, we observed a significant adaptation behaviour response for transgenic lines under stress. Taken together, all our findings suggest that over-expression of VvWRKY2 gene has a compelling role in abiotic stress tolerance and, therefore, would provide a useful strategy to promote abiotic stress tolerance in grape via molecular-assisted breeding and/or new biotechnology tools.

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

    International Nuclear Information System (INIS)

    Gorinova, N.; Nedkovska, M.; Todorovska, E.; Simova-Stoilova, L.; Stoyanova, Z.; Georgieva, K.; Demirevska-Kepova, K.; Atanassov, A.; Herzig, R.

    2007-01-01

    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 μM CdCl 2 resulted in inhibition of photosynthesis and mobilization of the ascorbate-glutathione cycle. Treatment with 500 μM CdCl 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

  17. Effect of lethal and sub-lethal concentrations of tobacco (Nicotiana ...

    African Journals Online (AJOL)

    Lethal and sub-lethal bioassays on Clarias gariepinus were conducted to evaluate the toxicity of tobacco (Nicotiana tobaccum) leaf dust on weight gain and haematological indices of Clarias gariepinus (mean weight 10.5±0.70g) in glass aquaria with aeration system. The concentrations used during the lethal exposure are: ...

  18. Expression and Chloroplast Targeting of Cholesterol Oxidase in Transgenic Tobacco Plants

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    Corbin, David R.; Grebenok, Robert J.; Ohnmeiss, Thomas E.; Greenplate, John T.; Purcell, John P.

    2001-01-01

    Cholesterol oxidase represents a novel type of insecticidal protein with potent activity against the cotton boll weevil (Anthonomus grandis grandis Boheman). We transformed tobacco (Nicotiana tabacum) plants with the cholesterol oxidase choM gene and expressed cytosolic and chloroplast-targeted versions of the ChoM protein. Transgenic leaf tissues expressing cholesterol oxidase exerted insecticidal activity against boll weevil larvae. Our results indicate that cholesterol oxidase can metabolize phytosterols in vivo when produced cytosolically or when targeted to chloroplasts. The transgenic plants exhibiting cytosolic expression accumulated low levels of saturated sterols known as stanols, and displayed severe developmental aberrations. In contrast, the transgenic plants expressing chloroplast-targeted cholesterol oxidase maintained a greater accumulation of stanols, and appeared phenotypically and developmentally normal. These results are discussed within the context of plant sterol distribution and metabolism. PMID:11457962

  19. Analysis of DNA methylation variation in sibling tobacco ( Nicotiana ...

    African Journals Online (AJOL)

    Amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP) analysis were used to investigate the genome of two sibling tobacco cultivars, Yunyan85 and Yunyan87, their parent K326 and the other tobacco cultivar NC89. AFLP analysis indicated that, the genome primary ...

  20. Cloning of transgenic tobacco BY-2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression.

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    Nocarova, Eva; Fischer, Lukas

    2009-04-22

    Phenotypic characterization of transgenic cell lines, frequently used in plant biology studies, is complicated because transgene expression in individual cells is often heterogeneous and unstable. To identify the sources and to reduce this heterogeneity, we transformed tobacco (Nicotiana tabacum L.) BY-2 cells with a gene encoding green fluorescent protein (GFP) using Agrobacterium tumefaciens, and then introduced a simple cloning procedure to generate cell lines derived from the individual transformed cells. Expression of the transgene was monitored by analysing GFP fluorescence in the cloned lines and also in lines obtained directly after transformation. The majority ( approximately 90%) of suspension culture lines derived from calli that were obtained directly from transformation consisted of cells with various levels of GFP fluorescence. In contrast, nearly 50% of lines generated by cloning cells from the primary heterogeneous suspensions consisted of cells with homogenous GFP fluorescence. The rest of the lines exhibited "permanent heterogeneity" that could not be resolved by cloning. The extent of fluorescence heterogeneity often varied, even among genetically identical clones derived from the primary transformed lines. In contrast, the offspring of subsequent cloning of the cloned lines was uniform, showing GFP fluorescence intensity and heterogeneity that corresponded to the original clone. The results demonstrate that, besides genetic heterogeneity detected in some lines, the primary lines often contained a mixture of epigenetically different cells that could be separated by cloning. This indicates that a single integration event frequently results in various heritable expression patterns, which are probably accidental and become stabilized in the offspring of the primary transformed cells early after the integration event. Because heterogeneity in transgene expression has proven to be a serious problem, it is highly advisable to use transgenes tagged with

  1. Co-expression of peppermint geranyl diphosphate synthase small subunit enhances monoterpene production in transgenic tobacco plants.

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    Yin, Jun-Lin; Wong, Woon-Seng; Jang, In-Cheol; Chua, Nam-Hai

    2017-02-01

    Monoterpenes are important for plant survival and useful to humans. In addition to their function in plant defense, monoterpenes are also used as flavors, fragrances and medicines. Several metabolic engineering strategies have been explored to produce monoterpene in tobacco but only trace amounts of monoterpenes have been detected. We investigated the effects of Solanum lycopersicum 1-deoxy-d-xylulose-5-phosphate synthase (SlDXS), Arabidopsis thaliana geranyl diphosphate synthase 1 (AtGPS) and Mentha × piperita geranyl diphosphate synthase small subunit (MpGPS.SSU) on production of monoterpene and geranylgeranyl diphosphate (GGPP) diversities, and plant morphology by transient expression in Nicotiana benthamiana and overexpression in transgenic Nicotiana tabacum. We showed that MpGPS.SSU could enhance the production of various monoterpenes such as (-)-limonene, (-)-linalool, (-)-α-pinene/β-pinene or myrcene, in transgenic tobacco by elevating geranyl diphosphate synthase (GPS) activity. In addition, overexpression of MpGPS.SSU in tobacco caused early flowering phenotype and increased shoot branching by elevating contents of GA 3 and cytokinins due to upregulated transcript levels of several plastidic 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway genes, geranylgeranyl diphosphate synthases 3 (GGPPS3) and GGPPS4. Our method would allow the identification of new monoterpene synthase genes using transient expression in N. benthamiana and the improvement of monoterpene production in transgenic tobacco plants. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  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

  3. In vitro and in vivo activities of eugenol against tobacco black shank caused by Phytophthora nicotianae.

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    Jing, Changliang; Gou, Jianyu; Han, Xiaobin; Wu, Qian; Zhang, Chengsheng

    2017-10-01

    Phytophthora nicotianae causes serious black shank disease in tobacco. Syringa oblata essential oil and its main components were evaluated to develop an effective and environmentally friendly biocontrol agent. Eugenol, which exhibited the strongest activity, was intensively investigated in vitro and in vivo. The mycelial growth of P. nicotianae was inhibited by eugenol at a minimum inhibitory concentration of 200μgmL -1 , and inhibition occurred in a dose-dependent manner. Extracellular pH and extracellular conductivity results indicated that eugenol increased membrane permeability. Flow cytometry and fluorescent staining results further showed that eugenol disrupted mycelial membranes but did not affect spore membrane integrity. The in vivo results confirmed that treatment of tobacco with various concentrations of eugenol formulations reduced disease incidence and better controlled against the disease. Our results suggested that the ability of eugenol to control tobacco black shank depended on its ability to damage mycelial membranes and that eugenol formulations have potential as an eco-friendly antifungal agent for controlling tobacco blank shank. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Characterization of transgenic tobacco plants containing bacterial bphC gene and study of their phytoremediation ability.

    Science.gov (United States)

    Viktorovtá, Jitka; Novakova, Martina; Trbolova, Ladislava; Vrchotova, Blanka; Lovecka, Petra; Mackova, Martina; Macek, Tomas

    2014-01-01

    Genetically modified plants can serve as an efficient tool for remediation of diverse dangerous pollutants of the environment such as pesticides, heavy metals, explosives and persistent organic compounds. Transgenic lines of Nicotiana tabacum containing bacterial bphC gene from the degradation pathway of polychlorinated biphenyls (PCBs) were tested. The product of the bphC gene - enzyme 2,3-dihydroxybiphenyl-1,2-dioxygenase is responsible for cleaving of the biphenyl ring. The presence of bphC gene in transgenic plants was detected on DNA, RNA and protein level. The expression of the bphC/His gene was verified afterpurification of the enzyme from plants by affinity chromatography followed by a Western blot and immunochemical assay. The enzyme activity of isolated protein was detected. Efficient transformation of 2,3-DHB by transgenic plants was achieved and the lines also exhibited high production of biomass. The transgenic plants were more tolerant to the commercial PCBs mixture Delor 103 than non-transgenic tobacco. And finally, the higher decrease of total PCB content and especially congener 28 in real contaminated soil from a dumpsite was determined after cultivation of transgenic plant in comparison with nontransgenic tobacco. The substrate specificity of transgenic plants was the same as substrate specificity of BphC enzyme.

  5. Sequencing and phylogenetic analysis of tobacco virus 2, a polerovirus from Nicotiana tabacum.

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    Zhou, Benguo; Wang, Fang; Zhang, Xuesong; Zhang, Lina; Lin, Huafeng

    2017-07-01

    The complete genome sequence of a new virus, provisionally named tobacco virus 2 (TV2), was determined and identified from leaves of tobacco (Nicotiana tabacum) exhibiting leaf mosaic, yellowing, and deformity, in Anhui Province, China. The genome sequence of TV2 comprises 5,979 nucleotides, with 87% nucleotide sequence identity to potato leafroll virus (PLRV). Its genome organization is similar to that of PLRV, containing six open reading frames (ORFs) that potentially encode proteins with putative functions in cell-to-cell movement and suppression of RNA silencing. Phylogenetic analysis of the nucleotide sequence placed TV2 alongside members of the genus Polerovirus in the family Luteoviridae. To the best our knowledge, this study is the first report of a complete genome sequence of a new polerovirus identified in tobacco.

  6. Processing, Targeting, and Antifungal Activity of Stinging Nettle Agglutinin in Transgenic Tobacco

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    Does, Mirjam P.; Houterman, Petra M.; Dekker, Henk L.; Cornelissen, Ben J.C.

    1999-01-01

    The gene encoding the precursor to stinging nettle (Urtica dioica L.) isolectin I was introduced into tobacco (Nicotiana tabacum). In transgenic plants this precursor was processed to mature-sized lectin. The mature isolectin is deposited intracellularly, most likely in the vacuoles. A gene construct lacking the C-terminal 25 amino acids was also introduced in tobacco to study the role of the C terminus in subcellular trafficking. In tobacco plants that expressed this construct, the mutant precursor was correctly processed and the mature isolectin was targeted to the intercellular space. These results indicate the presence of a C-terminal signal for intracellular retention of stinging nettle lectin and most likely for sorting of the lectin to the vacuoles. In addition, correct processing of this lectin did not depend on vacuolar deposition. Isolectin I purified from tobacco displayed identical biological activities as isolectin I isolated from stinging nettle. In vitro antifungal assays on germinated spores of the fungi Botrytis cinerea, Trichoderma viride, and Colletotrichum lindemuthianum revealed that growth inhibition by stinging nettle isolectin I occurs at a specific phase of fungal growth and is temporal, suggesting that the fungi had an adaptation mechanism. PMID:10364393

  7. Overexpression of a tea flavanone 3-hydroxylase gene confers tolerance to salt stress and Alternaria solani in transgenic tobacco.

    Science.gov (United States)

    Mahajan, Monika; Yadav, Sudesh Kumar

    2014-08-01

    Flavan-3-ols are the major flavonoids present in tea (Camellia sinensis) leaves. These are known to have antioxidant and free radical scavenging properties in vitro. Flavanone 3-hydroxylase is considered to be an important enzyme of flavonoid pathway leading to accumulation of flavan-3-ols in tea. Expression analysis revealed the upregulation in transcript levels of C. sinensis flavanone 3-hydroxylase (CsF3H) encoding gene under salt stress. In this study, the biotechnological potential of CsF3H was evaluated by gene overexpression in tobacco (Nicotiana tabacum cv. Xanthi). Overexpression of CsF3H cDNA increased the content of flavan-3-ols in tobacco and conferred tolerance to salt stress and fungus Alternaria solani infection. Transgenic tobaccos were observed for increase in primary root length, number of lateral roots, chlorophyll content, antioxidant enzyme expression and their activities. Also, they showed lesser malondialdehyde content and electrolyte leakage compared to control tobacco plants. Further, transgenic plants produced higher degree of pectin methyl esterification via decreasing pectin methyl esterase (PME) activity in roots and leaves under unstressed and salt stressed conditions. The effect of flavan-3-ols on pectin methyl esterification under salt stressed conditions was further validated through in vitro experiments in which non-transgenic (wild) tobacco seedlings were exposed to salt stress in presence of flavan-3-ols, epicatechin and epigallocatechin. The in vitro exposed seedlings showed similar trend of increase in pectin methyl esterification through decreasing PME activity as observed in CsF3H transgenic lines. Taken together, overexpression of CsF3H provided tolerance to salt stress and fungus A. solani infection to transgenic tobacco through improved antioxidant system and enhanced pectin methyl esterification.

  8. Dehydrins Impart Protection against Oxidative Stress in Transgenic Tobacco Plants.

    Science.gov (United States)

    Halder, Tanmoy; Upadhyaya, Gouranga; Basak, Chandra; Das, Arup; Chakraborty, Chandrima; Ray, Sudipta

    2018-01-01

    Environmental stresses generate reactive oxygen species (ROS) which might be detrimental to the plants when produced in an uncontrolled way. However, the plants ameliorate such stresses by synthesizing antioxidants and enzymes responsible for the dismutation of ROS. Additionally, the dehydrins were also able to protect the inactivation of the enzyme lactate dehydrogenase against hydroxyl radicals (OH ⋅ ) generated during Fenton's reaction. SbDhn1 and SbDhn2 overexpressing transgenic tobacco plants were able to protect against oxidative damage. Transgenic tobacco lines showed better photosynthetic efficiency along with high chlorophyll content, soluble sugar and proline. However, the malonyl dialdehyde (MDA) content was significantly lower in transgenic lines. Experimental evidence demonstrates the protective effect of dehydrins on electron transport chain in isolated chloroplast upon methyl viologen (MV) treatment. The transgenic tobacco plants showed significantly lower superoxide radical generation () upon MV treatment. The accumulation of the H 2 O 2 was also lower in the transgenic plants. Furthermore, in the transgenic plants the expression of ROS scavenging enzymes was higher compared to non-transformed (NT) or vector transformed (VT) plants. Taken together these data, during oxidative stress dehydrins function by scavenging the () directly and also by rendering protection to the enzymes responsible for the dismutation of () thereby significantly reducing the amount of hydrogen peroxides formed. Increase in proline content along with other antioxidants might also play a significant role in stress amelioration. Dehydrins thus function co-operatively with other protective mechanisms under oxidative stress conditions rendering protection in stress environment.

  9. Cloning and functional analysis in transgenic tobacco of a tapetum ...

    African Journals Online (AJOL)

    The 5'-flanking region of 1174 bp upstream of the translation start point (TSP) of a reported Arabidopsis anther-specific gene, Anther7 gene (ATA7), which putatively encodes a protein related to lipid transfer protein, was cloned and functionally analyzed in transgenic tobacco after been fused with β- glucuronidase (GUS) ...

  10. Expression of chimeric HCV peptide in transgenic tobacco plants ...

    African Journals Online (AJOL)

    Expression of chimeric HCV peptide in transgenic tobacco plants infected with recombinant alfalfa mosaic virus for development of a plant-derived vaccine against HCV. AK El Attar, AM Shamloul, AA Shalaby, BY Riad, A Saad, HM Mazyad, JM Keith ...

  11. Coniferyl alcohol hinders the growth of tobacco BY-2 cells and Nicotiana benthamiana seedlings.

    Science.gov (United States)

    Väisänen, Enni E; Smeds, Annika I; Fagerstedt, Kurt V; Teeri, Teemu H; Willför, Stefan M; Kärkönen, Anna

    2015-09-01

    Externally added coniferyl alcohol at high concentrations reduces the growth of Nicotiana cells and seedlings. Coniferyl alcohol is metabolized by BY-2 cells to several compounds. Coniferyl alcohol (CA) is a common monolignol and a building block of lignin. The toxicity of monolignol alcohols has been stated in the literature, but there are only few studies suggesting that this is true. We investigated the physiological effects of CA on living plant cells in more detail. Tobacco (Nicotiana tabacum) Bright yellow-2 cells (BY-2) and Nicotiana benthamiana seedlings both showed concentration-dependent growth retardation in response to 0.5-5 mM CA treatment. In some cases, CA addition caused cell death in BY-2 cultures, but this response was dependent on the growth stage of the cells. Based on LC-MS/MS analysis, BY-2 cells did not accumulate the externally supplemented CA, but metabolized it to ferulic acid, ferulic acid glycoside, coniferin, and to some other phenolic compounds. In addition to growth inhibition, CA caused the formation of a lignin-like compound detected by phloroglucinol staining in N. benthamiana roots and occasionally in BY-2 cells. To prevent this, we added potassium iodide (KI, at 5 mM) to overcome the peroxidase-mediated CA polymerization to lignin. KI had, however, toxic effects on its own: in N. benthamiana seedlings, it caused reduction in growth; in BY-2 cells, reduction in growth and cell viability. Surprisingly, CA restored the growth of KI-treated BY-2 cells and N. benthamiana seedlings. Our results suggest that CA at high concentrations is toxic to plant cells.

  12. 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

    overexpressing plants exposed to 100 nmol mol-1 ozone for 7 h day-1 exhibited a substantial increase in foliar injury, and a greater pollutant-induced reduction in both the light-saturated rate of CO2 assimilation and the maximum in vivo rate of ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation......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...

  13. Uptake and translocation of 109Cd and stable Cd within tobacco plants (Nicotiana sylvestris)

    International Nuclear Information System (INIS)

    Rosén, K.; Eriksson, J.; Vinichuk, M.

    2012-01-01

    The availability, uptake, and translocation of recently added ( 109 Cd) and naturally occurring (stable) soil Cd within tobacco plants were compared. 109 Cd was added to soil in two treatments, A (0.25 MBq kg soil −1 DW) and B (eight-fold dose): stable Cd was measured in both treatments. Both the added and the stable Cd were higher in leaves and reproductive structures of the plant than in stalks and roots. The uptake of 109 Cd was 5.3 kBq plant −1 for treatment A and 36.7 kBq plant −1 for treatment B, and about 26 μg plant −1 for stable Cd. Leaves of the tobacco plants accumulated 40–45% of the total 109 Cd and about 50% of total stable Cd taken up by the plant. Cadmium concentration in the plant was three times higher than in roots and two times higher than the concentration in soil: the concentration in roots was lower than in the soil. - Capsule: The availability, uptake, and translocation of recently added ( 109 Cd) and naturally occurring (stable) soil Cd within tobacco plants (Nicotiana sylvestris) were investigated. - Highlights: ► We compared uptake recently added and naturally occurring soil Cd by tobacco plant. ► Both added and stable Cd display similar uptake and translocation within the plant. ► Leaves of tobacco plants accumulate half of the total Cd taken up by the plant. ► Recently added 109 Cd to soil is more available than naturally occurring cadmium.

  14. 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.

  15. Phytoremediation of arsenic from the contaminated soil using transgenic tobacco plants expressing ACR2 gene of Arabidopsis thaliana.

    Science.gov (United States)

    Nahar, Noor; Rahman, Aminur; Nawani, Neelu N; Ghosh, Sibdas; Mandal, Abul

    2017-11-01

    We have cloned, characterized and transformed the AtACR2 gene (arsenic reductase 2) of Arabidopsis thaliana into the genome of tobacco (Nicotiana tabacum, var Sumsun). Our results revealed that the transgenic tobacco plants are more tolerant to arsenic than the wild type ones. These plants can grow on culture medium containing 200μM arsenate, whereas the wild type can barely survive under this condition. Furthermore, when exposed to 100μM arsenate for 35days the amount of arsenic accumulated in the shoots of transgenic plants was significantly lower (28μg/g d wt.) than that found in the shoots of non-transgenic controls (40μg/g d wt.). However, the arsenic content in the roots of transgenic plants was significantly higher (2400μg/g d. wt.) than that (2100μg/g d. wt.) observed in roots of wild type plants. We have demonstrated that Arabidopsis thaliana AtACR2 gene is a potential candidate for genetic engineering of plants to develop new crop cultivars that can be grown on arsenic contaminated fields to reduce arsenic content of the soil and can become a source of food containing no arsenic or exhibiting substantially reduced amount of this metalloid. Copyright © 2017 Elsevier GmbH. All rights reserved.

  16. Split-Cre complementation restores combination activity on transgene excision in hair roots of transgenic tobacco.

    Directory of Open Access Journals (Sweden)

    Mengling Wen

    Full Text Available The Cre/loxP system is increasingly exploited for genetic manipulation of DNA in vitro and in vivo. It was previously reported that inactive ''split-Cre'' fragments could restore Cre activity in transgenic mice when overlapping co-expression was controlled by two different promoters. In this study, we analyzed recombination activities of split-Cre proteins, and found that no recombinase activity was detected in the in vitro recombination reaction in which only the N-terminal domain (NCre of split-Cre protein was expressed, whereas recombination activity was obtained when the C-terminal (CCre or both NCre and CCre fragments were supplied. We have also determined the recombination efficiency of split-Cre proteins which were co-expressed in hair roots of transgenic tobacco. No Cre recombination event was observed in hair roots of transgenic tobacco when the NCre or CCre genes were expressed alone. In contrast, an efficient recombination event was found in transgenic hairy roots co-expressing both inactive split-Cre genes. Moreover, the restored recombination efficiency of split-Cre proteins fused with the nuclear localization sequence (NLS was higher than that of intact Cre in transgenic lines. Thus, DNA recombination mediated by split-Cre proteins provides an alternative method for spatial and temporal regulation of gene expression in transgenic plants.

  17. Transgenic tobacco plants having a higher level of methionine are more sensitive to oxidative stress.

    Science.gov (United States)

    Hacham, Yael; Matityahu, Ifat; Amir, Rachel

    2017-07-01

    Methionine is an essential amino acid the low level of which limits the nutritional quality of plants. We formerly produced transgenic tobacco (Nicotiana tabacum) plants overexpressing CYSTATHIONE γ-SYNTHASE (CGS) (FA plants), methionine's main regulatory enzyme. These plants accumulate significantly higher levels of methionine compared with wild-type (WT) plants. The aim of this study was to gain more knowledge about the effect of higher methionine content on the metabolic profile of vegetative tissue and on the morphological and physiological phenotypes. FA plants exhibit slightly reduced growth, and metabolic profiling analysis shows that they have higher contents of stress-related metabolites. Despite this, FA plants were more sensitive to short- and long-term oxidative stresses. In addition, compared with WT plants and transgenic plants expressing an empty vector, the primary metabolic profile of FA was altered less during oxidative stress. Based on morphological and metabolic phenotypes, we strongly proposed that FA plants having higher levels of methionine suffer from stress under non-stress conditions. This might be one of the reasons for their lesser ability to cope with oxidative stress when it appeared. The observation that their metabolic profiling is much less responsive to stress compared with control plants indicates that the delta changes in metabolite contents between non-stress and stress conditions is important for enabling the plants to cope with stress conditions. © 2017 Scandinavian Plant Physiology Society.

  18. Characterization of secretory phospholipase A₂ with phospholipase A₁ activity in tobacco, Nicotiana tabacum (L.).

    Science.gov (United States)

    Fujikawa, Yukichi; Fujikawa, Ritsuko; Iijima, Noriaki; Esaka, Muneharu

    2012-03-01

    A cDNA encoding protein with homology to plant secretory phospholipase A₂ (sPLA₂), denoted as Nt1 PLA₂, was isolated from tobacco (Nicotiana tabacum). The cDNA encodes a mature protein of 118 amino acid residues with a putative signal peptide of 29 residues. The mature form of Nt1 PLA₂ has 12 cysteines, Ca²⁺ binding loop and catalytic site domain that are commonly conserved in plant sPLA₂s. The recombinant Nt1 PLA₂ was expressed as a fusion protein with thioredoxin in E. coli BL21 cells and was purified by an ion exchange chromatography after digestion of the fusion proteins by Factor Xa protease to obtain the mature form. Interestingly, Nt1 PLA₂ could hydrolyze the ester bond at the sn-1 position of glycerophospholipids as well as at the sn-2 position, when the activities were determined using mixed-micellar phospholipids with sodium cholate. Both activities for the sn-1 and -2 positions of glycerophospholipids required Ca²⁺ essentially, and maximal activities were found in an alkaline region when phosphatidylcholine, phosphatidylglycerol or phosphatidylethanolamine was used as a substrate. The level of Nt1 PLA₂ mRNA was detected at a higher level in tobacco flowers than stem, leaves and roots, and was induced by salicylic acid.

  19. Co-expression of NCED and ALO improves vitamin C level and tolerance to drought and chilling in transgenic tobacco and stylo plants.

    Science.gov (United States)

    Bao, Gegen; Zhuo, Chunliu; Qian, Chunmei; Xiao, Ting; Guo, Zhenfei; Lu, Shaoyun

    2016-01-01

    Abscisic acid (ABA) regulates plant adaptive responses to various environmental stresses, while L-ascorbic acid (AsA) that is also named vitamin C is an important antioxidant and involves in plant stress tolerance and the immune system in domestic animals. Transgenic tobacco (Nicotiana tabacum L.) and stylo [Stylosanthes guianensis (Aublet) Swartz], a forage legume, plants co-expressing stylo 9-cis-epoxycarotenoid dioxygenase (SgNCED1) and yeast D-arabinono-1,4-lactone oxidase (ALO) genes were generated in this study, and tolerance to drought and chilling was analysed in comparison with transgenic tobacco overexpressing SgNCED1 or ALO and the wild-type plants. Compared to the SgNCED1 or ALO transgenic plants, in which only ABA or AsA levels were increased, both ABA and AsA levels were increased in transgenic tobacco and stylo plants co-expressing SgNCED1 and ALO genes. Compared to the wild type, an enhanced drought tolerance was observed in SgNCED1 transgenic tobacco plants with induced expression of drought-responsive genes, but not in ALO plants, while an enhanced chilling tolerance was observed in ALO transgenic tobaccos with induced expression of cold-responsive genes, but not in SgNCED1 plants. Co-expression of SgNCED1 and ALO genes resulted in elevated tolerance to both drought and chilling in transgenic tobacco and stylo plants with induced expression of both drought and cold-responsive genes. Our result suggests that co-expression of SgNCED1 and ALO genes is an effective way for use in forage plant improvement for increased tolerance to drought and chilling and nutrition quality. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  20. 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.

  1. 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.

  2. Assessment of cultivation factors that affect biomass and geraniol production in transgenic tobacco cell suspension cultures.

    Directory of Open Access Journals (Sweden)

    Nikolay Vasilev

    Full Text Available A large-scale statistical experimental design was used to determine essential cultivation parameters that affect biomass accumulation and geraniol production in transgenic tobacco (Nicotiana tabacum cv. Samsun NN cell suspension cultures. The carbohydrate source played a major role in determining the geraniol yield and factors such as filling volume, inoculum size and light were less important. Sucrose, filling volume and inoculum size had a positive effect on geraniol yield by boosting growth of plant cell cultures whereas illumination of the cultures stimulated the geraniol biosynthesis. We also found that the carbohydrates sucrose and mannitol showed polarizing effects on biomass and geraniol accumulation. Factors such as shaking frequency, the presence of conditioned medium and solubilizers had minor influence on both plant cell growth and geraniol content. When cells were cultivated under the screened conditions for all the investigated factors, the cultures produced ∼ 5.2 mg/l geraniol after 12 days of cultivation in shaking flasks which is comparable to the yield obtained in microbial expression systems. Our data suggest that industrial experimental designs based on orthogonal arrays are suitable for the selection of initial cultivation parameters prior to the essential medium optimization steps. Such designs are particularly beneficial in the early optimization steps when many factors must be screened, increasing the statistical power of the experiments without increasing the demand on time and resources.

  3. Characterization and subcellular compartmentation of recombinant 4-hydroxyphenylpyruvate dioxygenase from Arabidopsis in transgenic tobacco.

    Science.gov (United States)

    Garcia, I; Rodgers, M; Pepin, R; Hsieh, T F; Matringe, M

    1999-04-01

    4-Hydroxyphenylpyruvate dioxygenase (4HPPD) catalyzes the formation of homogentisate (2,5-dihydroxyphenylacetate) from p-hydroxyphenylpyruvate and molecular oxygen. In plants this enzyme activity is involved in two distinct metabolic processes, the biosynthesis of prenylquinones and the catabolism of tyrosine. We report here the molecular and biochemical characterization of an Arabidopsis 4HPPD and the compartmentation of the recombinant protein in chlorophyllous tissues. We isolated a 1508-bp cDNA with one large open reading frame of 1338 bp. Southern analysis strongly suggested that this Arabidopsis 4HPPD is encoded by a single-copy gene. We investigated the biochemical characteristics of this 4HPPD by overproducing the recombinant protein in Escherichia coli JM105. The subcellular localization of the recombinant 4HPPD in chlorophyllous tissues was examined by overexpressing its complete coding sequence in transgenic tobacco (Nicotiana tabacum), using Agrobacterium tumefaciens transformation. We performed western analyses for the immunodetection of protein extracts from purified chloroplasts and total leaf extracts and for the immunocytochemistry on tissue sections. These analyses clearly revealed that 4HPPD was confined to the cytosol compartment, not targeted to the chloroplast. Western analyses confirmed the presence of a cytosolic form of 4HPPD in cultured green Arabidopsis cells.

  4. Effects of biotic stress caused by Potato virus Y on photosynthesis in ipt transgenic and control Nicotiana tabacum L

    Czech Academy of Sciences Publication Activity Database

    Synková, Helena; Semorádová, Šárka; Schnablová, Renáta; Muller, K.; Pospíšilová, Jana; Ryšlavá, H.; Malbeck, Jiří; Čeřovská, Noemi

    2006-01-01

    Roč. 171, - (2006), s. 607-616 ISSN 0168-9452 R&D Projects: GA ČR GA206/03/0310 Grant - others:Grantová agentura University Karlovy GAUK428/2004/B-Ch/PrF Institutional research plan: CEZ:AV0Z50380511 Keywords : cytokinins * ipt * transgenic tobacco * photosynthesis * Potato virus Y Subject RIV: EF - Botanics Impact factor: 1.631, year: 2006

  5. Heterogenous expression of Pyrus pyrifolia PpCAD2 and PpEXP2 in tobacco impacts lignin accumulation in transgenic plants.

    Science.gov (United States)

    Wang, Yuling; Zhang, Xinfu; Yang, Shaolan; Wang, Caihong; Lu, Guilong; Wang, Ran; Yang, Yingjie; Li, Dingli

    2017-12-30

    Lignin, a natural macromolecular compound, plays an important role in the texture and taste of fruit. Hard end is a physiological disorder of pear fruit, in which the level of lignification in fruit tissues is dramatically elevated. Cinnamyl alcohol dehydrogenase and expansin genes (PpCAD2 and PpEXP2, respectively) exhibit higher levels of expression in 'Whangkeumbae' (Pyrus pyrifolia) pear fruit exhibiting this physiological disorder, relative to control fruit without symptoms. These genes were isolated from pear fruit and subsequently expressed in tobacco (Nicotiana tabacum) to investigate their function. Histochemical staining for lignin revealed that the degree of lignification in leaf veins and stem tissues increased in plants transformed with sense constructs and decreased in plants transformed with antisense constructs of PpCAD2. The expression of native NtCADs was also inhibited in the antisense PpCAD2 transgenic tobacco. Sense and antisense PpCAD2 transgenic tobacco exhibited an 86.7% increase and a 60% decrease in CAD activity, respectively, accompanied by a complementary response in lignin content in root tissues. The basal portion of the stem in PpEXP2 transgenic tobacco was bent and highly lignified. Additionally, the level of cellulose also increased in the stem of PpEXP2 transgenic tobacco. Collectively, these results suggested that PpCAD2 and PpEXP2 genes play a significant role in lignin accumulation in transgenic tobacco plants, and it is inferred that these two genes may also participate in the increased lignification observed in hard end pear fruit. Copyright © 2017. Published by Elsevier B.V.

  6. Transgenic tobacco expressing a modified spider peptide inhibits the growth of plant pathogens and insect larvae

    Science.gov (United States)

    The gene encoding lycotoxin I, an amphipathic pore-forming peptide, was modified to increase oral toxicity to insects. One of the most active modified genes was then constitutively expressed in tobacco (Nicotiana tabacum) and transformants were evaluated for insect and disease resistance. Pathogenic...

  7. An active role for endogenous beta-1,3-glucanase genes in transgene-mediated co-suppression in tobacco.

    Science.gov (United States)

    Sanders, Matthew; Maddelein, Wendy; Depicker, Anna; Van Montagu, Marc; Cornelissen, Marc; Jacobs, John

    2002-11-01

    Post-transcriptional gene silencing (PTGS) is characterized by the accumulation of short interfering RNAs that are proposed to mediate sequence-specific degradation of cognate and secondary target mRNAs. In plants, it is unclear to what extent endogenous genes contribute to this process. Here, we address the role of the endogenous target genes in transgene-mediated PTGS of beta-1,3-glucanases in tobacco. We found that mRNA sequences of the endogenous glucanase glb gene with varying degrees of homology to the Nicotiana plumbaginifolia gn1 transgene are targeted by the silencing machinery, although less efficiently than corresponding transgene regions. Importantly, we show that endogene-specific nucleotides in the glb sequence provide specificity to the silencing process. Consistent with this finding, small sense and antisense 21- to 23-nucleotide RNAs homologous to the endogenous glb gene were detected. Combined, these data demonstrate that a co-suppressed endogenous glucan ase gene is involved in signal amplification and selection of homologous targets, and show that endogenous genes can actively participate in PTGS in plants. The findings are introduced as a further sophistication of the post-transciptional silencing model.

  8. The signal peptide-like segment of hpaXm is required for its association to the cell wall in transgenic tobacco plants.

    Science.gov (United States)

    Li, Le; Miao, Weiguo; Liu, Wenbo; Zhang, Shujian

    2017-01-01

    Harpins, encoded by hrp (hypersensitive response and pathogenicity) genes of Gram-negative plant pathogens, are elicitors of hypersensitive response (HR). HpaXm is a novel harpin-like protein described from cotton leaf blight bacteria, Xanthomonas citri subsp. malvacearum-a synonym of X. campestris pv. malvacearum (Smith 1901-1978). A putative signal peptide (1-MNSLNTQIGANSSFL-15) of hpaXm was predicted in the nitroxyl-terminal (N-terminal)by SignalP (SignalP 3.0 server). Here, we explored the function of the N-terminal leader peptide like segment of hpaXm using transgenic tobacco (Nicotiana tabacum cv. Xanthi nc.). Transgenic tobacco lines expressing the full-length hpaXm and the signal peptide-like segment-deleted mutant hpaXmΔLP were developed using transformation mediated by Agrobacterium tumefaciens. The target genes were confirmed integrated into the tobacco genomes and expressed normally. Using immune colloidal-gold detection technique, hpaXm protein was found to be transferred to the cytoplasm, the cell membrane, and organelles such as chloroplasts, mitochondria, and nucleus, as well as the cell wall. However, the deletion mutant hpaXmΔLP expressed in transgenic tobacco was found unable to cross the membrane to reach the cell wall. Additionally, soluble proteins extracted from plants transformed with hpaXm and hpaXmΔLP were bio-active. Defensive micro-HR induced by the transgene expression of hpaXm and hpaXmΔLP were observed on transgenic tobacco leaves. Disease resistance bioassays to tobacco mosaic virus (TMV) showed that tobacco plants transformed with hpaXm and with hpaXmΔLP exhibited enhanced resistance to TMV. In summary, the N-terminal signal peptide-like segment (1-45 bp) in hpaXm sequence is not necessary for transgene expression, bioactivity of hpaXm and resistance to TMV in transgenic tobacco, but is required for the protein to be translocated to the cell wall.

  9. Jasmonoyl-l-Isoleucine Coordinates Metabolic Networks Required for Anthesis and Floral Attractant Emission in Wild Tobacco (Nicotiana attenuata)[C][W][OPEN

    Science.gov (United States)

    Stitz, Michael; Hartl, Markus; Baldwin, Ian T.; Gaquerel, Emmanuel

    2014-01-01

    Jasmonic acid and its derivatives (jasmonates [JAs]) play central roles in floral development and maturation. The binding of jasmonoyl-l-isoleucine (JA-Ile) to the F-box of CORONATINE INSENSITIVE1 (COI1) is required for many JA-dependent physiological responses, but its role in anthesis and pollinator attraction traits remains largely unexplored. Here, we used the wild tobacco Nicotiana attenuata, which develops sympetalous flowers with complex pollination biology, to examine the coordinating function of JA homeostasis in the distinct metabolic processes that underlie flower maturation, opening, and advertisement to pollinators. From combined transcriptomic, targeted metabolic, and allometric analyses of transgenic N. attenuata plants for which signaling deficiencies were complemented with methyl jasmonate, JA-Ile, and its functional homolog, coronatine (COR), we demonstrate that (1) JA-Ile/COR-based signaling regulates corolla limb opening and a JA-negative feedback loop; (2) production of floral volatiles (night emissions of benzylacetone) and nectar requires JA-Ile/COR perception through COI1; and (3) limb expansion involves JA-Ile-induced changes in limb fresh mass and carbohydrate metabolism. These findings demonstrate a master regulatory function of the JA-Ile/COI1 duet for the main function of a sympetalous corolla, that of advertising for and rewarding pollinator services. Flower opening, by contrast, requires JA-Ile signaling-dependent changes in primary metabolism, which are not compromised in the COI1-silenced RNA interference line used in this study. PMID:25326292

  10. Molecular cloning of a catalase cDNA from Nicotiana glutinosa L. and its repression by tobacco mosaic virus infection.

    Science.gov (United States)

    Yi, S Y; Yu, S H; Choi, D

    1999-06-30

    Recent reports revealed that catalase has a role in the plant defense mechanism against a broad range of pathogens through being inhibited by salicylic acid (SA). During an effort to clone disease resistance-responsive genes, a cDNA encoding catalase (Ngcat1; Nicotiana glutinosa cat1) was isolated from a tobacco cDNA library. In N. glutinosa, catalase is encoded by a small gene family. The deduced amino acid sequence of the Ngcat1 cDNA has 98% homology with the cat1 gene of N. plumbaginifolia. The Ngcat1 expression is controlled by the circadian clock, and its mRNA level is the most abundant in leaves. Both the expression of Ngcat1 mRNA and its enzyme activity in the tobacco plant undergoing a hypersensitive response (HR) to TMV infection were repressed. The repression of the mRNA level was also observed following treatment with SA. These results imply that SA may act as an inhibitor of catalase transcription during the HR of tobacco. Cloning and expression of the Ngcat1 in tobacco following pathogen infection and SA treatment are presented.

  11. GhWRKY68 reduces resistance to salt and drought in transgenic Nicotiana benthamiana.

    Directory of Open Access Journals (Sweden)

    Haihong Jia

    Full Text Available The WRKY transcription factors modulate numerous physiological processes, including plant growth, development and responses to various environmental stresses. Currently, our understanding of the functions of the majority of the WRKY family members and their possible roles in signalling crosstalk is limited. In particular, very few WRKYs have been identified and characterised from an economically important crop, cotton. In this study, we characterised a novel group IIc WRKY gene, GhWRKY68, which is induced by different abiotic stresses and multiple defence-related signalling molecules. The β-glucuronidase activity driven by the GhWRKY68 promoter was enhanced after exposure to drought, salt, abscisic acid (ABA and H2O2. The overexpression of GhWRKY68 in Nicotiana benthamiana reduced resistance to drought and salt and affected several physiological indices. GhWRKY68 may mediate salt and drought responses by modulating ABA content and enhancing the transcript levels of ABA-responsive genes. GhWRKY68-overexpressing plants exhibited reduced tolerance to oxidative stress after drought and salt stress treatments, which correlated with the accumulation of reactive oxygen species (ROS, reduced enzyme activities, elevated malondialdehyde (MDA content and altered ROS-related gene expression. These results indicate that GhWRKY68 is a transcription factor that responds to drought and salt stresses by regulating ABA signalling and modulating cellular ROS.

  12. An overexpression of chalcone reductase of Pueraria montana var. lobata alters biosynthesis of anthocyanin and 5'-deoxyflavonoids in transgenic tobacco.

    Science.gov (United States)

    Joung, Jae-youl; Kasthuri, G Mangai; Park, Ji-young; Kang, Won-jin; Kim, Hyun-soon; Yoon, Bong-sik; Joung, Hyouk; Jeon, Jae-heung

    2003-03-28

    We isolated the chalcone reductase (pl-chr) gene of Pueraria montana var. lobata by using a PCR strategy from cDNA pools of storage roots. A high level of expression of RNA was found in both stems and roots. The genomic Southern blot result suggests that pl-chr exists as a member of a small gene family. By introducing a pl-chr gene under the control of the 35S CaMV promoter into the pink-flowering Xanthi line of Nicotiana tabacum, the flower color was changed from pink to white-to-pink. The contents of anthocyanin in the flowers of the transgenic lines were dramatically decreased by 40%, but the total UV absorption compounds remained unchanged. The production of liquiritigenin in pl-chr overexpressed transgenic tobacco lines was confirmed by HPLC and MS analysis. The introduction of pl-chr gene provides a method to redirect the flavonoid pathway into 5'-deoxyflavonoid production in non-legume crops, in order to manipulate the phenylpropanoid pathway for isoflavonoid production.

  13. A wheat WRKY transcription factor TaWRKY10 confers tolerance to multiple abiotic stresses in transgenic tobacco.

    Directory of Open Access Journals (Sweden)

    Chen Wang

    Full Text Available WRKY transcription factors are reported to be involved in defense regulation, stress response and plant growth and development. However, the precise role of WRKY transcription factors in abiotic stress tolerance is not completely understood, especially in crops. In this study, we identified and cloned 10 WRKY genes from genome of wheat (Triticum aestivum L.. TaWRKY10, a gene induced by multiple stresses, was selected for further investigation. TaWRKY10 was upregulated by treatment with polyethylene glycol, NaCl, cold and H2O2. Result of Southern blot indicates that the wheat genome contains three copies of TaWRKY10. The TaWRKY10 protein is localized in the nucleus and functions as a transcriptional activator. Overexpression of TaWRKY10 in tobacco (Nicotiana tabacum L. resulted in enhanced drought and salt stress tolerance, mainly demonstrated by the transgenic plants exhibiting of increased germination rate, root length, survival rate, and relative water content under these stress conditions. Further investigation showed that transgenic plants also retained higher proline and soluble sugar contents, and lower reactive oxygen species and malonaldehyde contents. Moreover, overexpression of the TaWRKY10 regulated the expression of a series of stress related genes. Taken together, our results indicate that TaWRKY10 functions as a positive factor under drought and salt stresses by regulating the osmotic balance, ROS scavenging and transcription of stress related genes.

  14. Overexpression of CsANR increased flavan-3-ols and decreased anthocyanins in transgenic tobacco.

    Science.gov (United States)

    Kumar, Vinay; Yadav, Sudesh Kumar

    2013-06-01

    Anthocyanins and flavan-3-ols are distributed widely in plants and synthesized by a common biosynthetic pathway. Anthocyanin reductase (ANR) represents branching-point enzyme of this pathway converting anthocyanidins to flavan-3-ols. Since tea contains highest amount of flavonoids, a cDNA encoding anthocyanin reductase from tea (CsANR) was overexpressed in transgenic tobacco to check the influence on anthocyanin and flavan-3-ols. The transgenic tobacco was confirmed by genomic PCR and expression of transgene was analyzed through semiquantitative PCR. Interestingly flowers of transgenic tobacco were light pink/white in color instead of dark pink in wild tobacco, documenting the decrease in anthocyanins content. Upon measurement, flower anthocyanin content was found to be lesser. While flavan-3-ols (epicatechin and epigallocatechin) contents were increased in leaf tissue of transgenic lines. The expressions of other endogenous flavonoid biosynthetic pathway genes in different floral parts (sepal, petal, stamen, and carpel) of CsANR overexpressing tobacco as well as wild tobacco were analyzed. The transcript levels of PAL and CHI genes were downregulated, while transcript levels of F3H, FLS, CHS, ANR1, and ANR2 genes were upregulated in all floral parts of CsANR transgenic plants compared to wild tobacco. The expressions of DFR and ANS genes were also spatially modulated in different floral parts due to overexpression of CsANR. Thus, CsANR overexpression increased flavan-3-ols and decreased anthocyanin content by modulating the expressions of various flavonoid biosynthetic pathway genes in flower of tobacco. These changes might be responsible for the observed pollen tube in the pollens of CsANR overexpressing transgenic tobacco when they were still in the anther before pollination.

  15. Development of a transgenic tobacco plant for phytoremediation of methylmercury pollution.

    Science.gov (United States)

    Nagata, Takeshi; Morita, Hirofumi; Akizawa, Toshifumi; Pan-Hou, Hidemitsu

    2010-06-01

    To develop the potential of plant for phytoremediation of methylmercury pollution, a genetically engineered tobacco plant that coexpresses organomercurial lyase (MerB) with the ppk-specified polyphosphate (polyP) and merT-encoding mercury transporter was constructed by integrating a bacterial merB gene into ppk/merT-transgenic tobacco. A large number of independent transgenic tobaccos was obtained, in some of which the merB gene was stably integrated in the plant genome and substantially translated to the expected MerB enzyme in the transgenic tobacco. The ppk/merT/merB-transgenic tobacco callus showed more resistance to methylmercury (CH3Hg+) and accumulated more mercury from CH3Hg+-containing medium than the ppk/merT-transgenic and wild-type progenitors. These results suggest that the MerB enzyme encoded by merB degraded the incorporated CH3Hg+ to Hg2+, which then accumulated as a less toxic Hg-polyP complex in the tobacco cells. Phytoremediation of CH3Hg+ and Hg2+ in the environment with this engineered ppk/merT/merB-transgenic plant, which prevents the release mercury vapor (Hg0) into the atmosphere in addition to generating potentially recyclable mercury-rich plant residues, is believed to be more acceptable to the public than other competing technologies, including phytovolatilization.

  16. Spicing Up the N Gene: F. O. Holmes and Tobacco mosaic virus Resistance in Capsicum and Nicotiana Plants.

    Science.gov (United States)

    Scholthof, Karen-Beth G

    2017-02-01

    One of the seminal events in plant pathology was the discovery by Francis O. Holmes that necrotic local lesions induced on certain species of Nicotiana following rub-inoculation of Tobacco mosaic virus (TMV) was due to a specific interaction involving a dominant host gene (N). From this, Holmes had an idea that if the N gene from N. glutinosa was introgressed into susceptible tobacco, the greatly reduced titer of TMV would, by extension, prevent subsequent infection of tomato and pepper plants by field workers whose hands were contaminated with TMV from their use of chewing and smoking tobacco. The ultimate outcome has many surprising twists and turns, including Holmes' failure to obtain fertile crosses of N. glutinosa × N. tabacum after 3 years of intensive work. Progress was made with N. digluta, a rare amphidiploid that was readily crossed with N. tabacum. And, importantly, the first demonstration by Holmes of the utility of interspecies hybridization for virus resistance was made with Capsicum (pepper) species with the identification of the L gene in Tabasco pepper, that he introgressed into commercial bell pepper varieties. Holmes' findings are important as they predate Flor's gene-for-gene hypothesis, show the use of interspecies hybridization for control of plant pathogens, and the use of the local lesion as a bioassay to monitor resistance events in crop plants.

  17. An N-terminal peptide extension results in efficient expression, but not secretion, of a synthetic horseradish peroxidase gene in transgenic tobacco.

    Science.gov (United States)

    Kis, Mihaly; Burbridge, Emma; Brock, Ian W; Heggie, Laura; Dix, Philip J; Kavanagh, Tony A

    2004-03-01

    Native horseradish (Armoracia rusticana) peroxidase, HRP (EC 1.11.1.7), isoenzyme C is synthesized with N-terminal and C-terminal peptide extensions, believed to be associated with protein targeting. This study aimed to explore the specific functions of these extensions, and to generate transgenic plants with expression patterns suitable for exploring the role of peroxidase in plant development and defence. Transgenic Nicotiana tabacum (tobacco) plants expressing different versions of a synthetic horseradish peroxidase, HRP, isoenzyme C gene were constructed. The gene was engineered to include additional sequences coding for either the natural N-terminal or the C-terminal extension or both. These constructs were placed under the control of a constitutive promoter (CaMV-35S) or the tobacco RUBISCO-SSU light inducible promoter (SSU) and introduced into tobacco using Agrobacterium-mediated transformation. To study the effects of the N- and C-terminal extensions, the localization of recombinant peroxidase was determined using biochemical and molecular techniques. Transgenic tobacco plants can exhibit a ten-fold increase in peroxidase activity compared with wild-type tobacco levels, and the majority of this activity is located in the symplast. The N-terminal extension is essential for the production of high levels of recombinant protein, while the C-terminal extension has little effect. Differences in levels of enzyme activity and recombinant protein are reflected in transcript levels. There is no evidence to support either preferential secretion or vacuolar targeting of recombinant peroxidase in this heterologous expression system. This leads us to question the postulated targeting roles of these peptide extensions. The N-terminal extension is essential for high level expression and appears to influence transcript stability or translational efficiency. Plants have been generated with greatly elevated cytosolic peroxidase activity, and smaller increases in apoplastic

  18. Suppressed phenylalanine ammonia-lyase activity after heat shock in transgenic Nicotiana plumbaginifolia containing an Arabidopsis HSP18.2-parsley PAL2 chimera gene.

    Science.gov (United States)

    Moriwaki, M; Yamakawa, T; Washino, T; Kodama, T; Igarashi, Y

    1999-01-01

    The activity of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) after heat shock (HS) in leaves and buds of transgenic Nicotiana plumbaginifolia containing an Arabidopsis HSP18.2 promoter-parsley phenylalanine ammonia-lyase 2 (HSP18.2-PAL2) chimera gene was examined. Immediately after HS treatment at 44 degrees C for 5 h, the PAL activity in both transgenic and normal (untransformed) plants was 35-38% lower than that before HS. At normal temperature (25-26 degrees C), the PAL activity recovered within 5 h of ending the HS treatment in normal plants, but not until 12-24 h in transgenic plants containing the HSP18.2-PAL2 gene. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed the presence of parsley PAL2 mRNA in transgenic plants, which remained for 8-12 h following 5-h HS at 44 degrees C; the mRNA was not observed before HS. The content of chlorogenic acid (CGA; 3-caffeoylquinic acid) decreased drastically 8-12 h after HS in transgenic plants, but only slightly in normal plants. Thus, the decrease in PAL activity accompanied by expression of the parsley PAL2 gene after HS treatment corresponded to the decrease in CGA synthesis. These results might be attributed to post-transcriptional degradation of endogenous PAL mRNA triggered by transcription of the transgene.

  19. Expression of a defence-related intercellular barley peroxidase in transgenic tobacco

    DEFF Research Database (Denmark)

    Kristensen, B.K.; Brandt, J.; Bojsen, K.

    1997-01-01

    genetically, phenotypically and biochemically. The T-DNA was steadily inherited through three generations. The barley peroxidase is expressed and sorted to the intercellular space in the transgenic tobacco plants. The peroxidase can be extracted from the intercellular space in two molecular forms from both...... barley and transgenic tobacco. The tobacco expressed forms are indistinguishable from the barley expressed forms as determined by analytical isoelectric focusing (pI 8.5) and Western-blotting. Staining for N-glycosylation showed that one form only was glycosylated. The N-terminus of purified Prx8 from...... transgenic tobacco was blocked by pyroglutamate, after the removal of which, N-terminal sequencing verified the transit signal-peptide cleavage site deduced from the cDNA sequence. Phenotype comparisons show that the constitutive expression of Prx8 lead to growth retardation. However, an infection assay...

  20. Functional analysis of multiple carotenogenic genes from Lycium barbarum and Gentiana lutea L. for their effects on beta-carotene production in transgenic tobacco.

    Science.gov (United States)

    Ji, Jing; Wang, Gang; Wang, Jiehua; Wang, Ping

    2009-02-01

    Carotenoids are red, yellow and orange pigments, which are widely distributed in nature and are especially abundant in yellow-orange fruits and vegetables and dark green leafy vegetables. Carotenoids are essential for photosynthesis and photoprotection in plant life and also have different beneficial effects in humans and animals (van den Berg et al. 2000). For example, beta-carotene plays an essential role as the main dietary source of vitamin A. To obtain further insight into beta-carotene biosynthesis in two important economic plant species, Lycium barbarum and Gentiana lutea L., and to investigate and prioritize potential genetic engineering targets in the pathway, the effects of five carotenogenic genes from these two species, encoding proteins including geranylgeranyl diphosphate synthase, phytoene synthase and delta-carotene desaturase gene, lycopene beta-cyclase, lycopene epsilon-cyclase were functionally analyzed in transgenic tobacco (Nicotiana tabacum) plants. All transgenic tobacco plants constitutively expressing these genes showed enhanced beta-carotene contents in their leaves and flowers to different extents. The addictive effects of co-ordinate expression of double transgenes have also been investigated.

  1. Impact of herbicides on some agronomic and chemical characteristics of flue-cured virginia (FCV tobacco (Nicotiana tabacum L.

    Directory of Open Access Journals (Sweden)

    Muhammad Azim Khan

    2006-09-01

    Full Text Available A field experiment was carried out at Tobacco Research Station Khan Ghari, Mardan, (NWFP- Pakistan during spring 2003 to study the impact of herbicides on some agronomic and chemical characteristics of flue-cured virginia (FCV tobacco (Nicotiana tabacum L.. The experiment was laid out in RCB design, replicated four times with ten treatments, comprising hand weeding, weedy check, pre-transplanting herbicides; S-metalocholar @ 1.92, pendimethalin (EC @ 1.00, pendimethalin (CS @ 1.00, and butralin @ 1.44 kg a.i ha-1 and the post-transplanting herbicides include; clodinafop @ 0.04, fenoxaprop-p-ethyl @ 1.00, acetochlor @ 0.125 and glyphosate @ 0.95 kg a.i ha-1. None of the herbicides except S-metalocholar had a phytotoxic effect on tobacco. All the parameters except the number of leaves plant-1 were significantly affected by different treatments. The highest (228.3 weeds density m-2 was observed in weedy check while minimum (69 was recorded in pendimethalin (EC treatment. The maximum grade index of 74.60% was recorded in acetochlor and minimum grade index of 53.88% was recorded in S-metalocholar treatments. Nicotine (% was higher in pendimethalin (EC treated plots with 2.362%; however it was comparable to all other treatments. The maximum percent reducing sugar of 18.22% was recorded in pendimethalin (CS treatment, while minimum of 12.42% reducing sugar was recorded in weedy check. Similarly maximum yield of 2465 kg ha-1 was recorded in pendimethalin (EC treatment and minimum yield of 1703 kg ha-1 was recorded in weedy check (control treatment. Thus it can be concluded from the experiment that herbicides proved effective against weeds and their growth and promoted tobacco quality and yield. Hence the use of herbicides not only increases the net income of the farmers but also will make the weed seed bank poorer.

  2. MsZEP, a novel zeaxanthin epoxidase gene from alfalfa (Medicago sativa), confers drought and salt tolerance in transgenic tobacco.

    Science.gov (United States)

    Zhang, Zhiqiang; Wang, Yafang; Chang, Leqin; Zhang, Tong; An, Jie; Liu, Yushi; Cao, Yuman; Zhao, Xia; Sha, Xuyang; Hu, Tianming; Yang, Peizhi

    2016-02-01

    The zeaxanthin epoxidase gene ( MsZEP ) was cloned and characterized from alfalfa and validated for its function of tolerance toward drought and salt stresses by heterologous expression in Nicotiana tabacum. Zeaxanthin epoxidase (ZEP) plays important roles in plant response to various environment stresses due to its functions in ABA biosynthetic and the xanthophyll cycle. To understand the expression characteristics and the biological functions of ZEP in alfalfa (Medicago sativa), a novel gene, designated as MsZEP (KM044311), was cloned, characterized and overexpressed in Nicotiana tabacum. The open reading frame of MsZEP contains 1992 bp nucleotides and encodes a 663-amino acid polypeptide. Amino acid sequence alignment indicated that deduced MsZEP protein was highly homologous to other plant ZEP sequences. Phylogenetic analysis showed that MsZEP was grouped into a branch with other legume plants. Real-time quantitative PCR revealed that MsZEP gene expression was clearly tissue-specific, and the expression levels were higher in green tissues (leaves and stems) than in roots. MsZEP expression decreased in shoots under drought, cold, heat and ABA treatment, while the expression levels in roots showed different trends. Besides, the results showed that nodules could up-regulate the MsZEP expression under non-stressful conditions and in the earlier stage of different abiotic stress. Heterologous expression of the MsZEP gene in N. tabacum could confer tolerance to drought and salt stress by affecting various physiological pathways, ABA levels and stress-responsive genes expression. Taken together, these results suggested that the MsZEP gene may be involved in alfalfa responses to different abiotic stresses and nodules, and could enhance drought and salt tolerance of transgenic tobacco by heterologous expression.

  3. 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.

  4. 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.

  5. PDH45 overexpressing transgenic tobacco and rice plants provide salinity stress tolerance via less sodium accumulation.

    Science.gov (United States)

    Nath, Manoj; Garg, Bharti; Sahoo, Ranjan Kumar; Tuteja, Narendra

    2015-01-01

    Salinity stress negatively affects the crop productivity worldwide, including that of rice. Coping with these losses is a major concern for all countries. The pea DNA helicase, PDH45 is a unique member of helicase family involved in the salinity stress tolerance. However, the exact mechanism of the PDH45 in salinity stress tolerance is yet to be established. Therefore, the present study was conducted to investigate the mechanism of PDH45-mediated salinity stress tolerance in transgenic tobacco and rice lines along with wild type (WT) plants using CoroNa Green dye based sodium localization in root and shoot sections. The results showed that under salinity stress root and shoot of PDH45 overexpressing transgenic tobacco and rice accumulated less sodium (Na(+)) as compared to their respective WT. The present study also reports salinity tolerant (FL478) and salinity susceptible (Pusa-44) varieties of rice accumulated lowest and highest Na(+) level, respectively. All the varieties and transgenic lines of rice accumulate differential Na(+) ions in root and shoot. However, roots accumulate high Na(+) as compared to the shoots in both tobacco and rice transgenic lines suggesting that the Na(+) transport in shoot is somehow inhibited. It is proposed that the PDH45 is probably involved in the deposition of apoplastic hydrophobic barriers and consequently inhibit Na(+) transport to shoot and therefore confers salinity stress tolerance to PDH45 overexpressing transgenic lines. This study concludes that tobacco (dicot) and rice (monocot) transgenic plants probably share common salinity tolerance mechanism mediated by PDH45 gene.

  6. Overexpression of a Plasma Membrane-Localized SbSRP-Like Protein Enhances Salinity and Osmotic Stress Tolerance in Transgenic Tobacco

    Directory of Open Access Journals (Sweden)

    Avinash Mishra

    2017-04-01

    Full Text Available An obligate halophyte, Salicornia brachiata grows in salt marshes and is considered to be a potential resource of salt- and drought-responsive genes. It is important to develop an understanding of the mechanisms behind enhanced salt tolerance. To increase this understanding, a novel SbSRP gene was cloned, characterized, over-expressed, and functionally validated in the model plant Nicotiana tabacum. The genome of the halophyte S. brachiata contains two homologs of an intronless SbSRP gene of 1,262 bp in length that encodes for a stress-related protein. An in vivo localization study confirmed that SbSRP is localized on the plasma membrane. Transgenic tobacco plants (T1 that constitutively over-express the SbSRP gene showed improved salinity and osmotic stress tolerance. In comparison to Wild Type (WT and Vector Control (VC plants, transgenic lines showed elevated relative water and chlorophyll content, lower malondialdehyde content, lower electrolyte leakage and higher accumulation of proline, free amino acids, sugars, polyphenols, and starch under abiotic stress treatments. Furthermore, a lower build-up of H2O2 content and superoxide-radicals was found in transgenic lines compared to WT and VC plants under stress conditions. Transcript expression of Nt-APX (ascorbate peroxidase, Nt-CAT (catalase, Nt-SOD (superoxide dismutase, Nt-DREB (dehydration responsive element binding factor, and Nt-AP2 (apetala2 genes was higher in transgenic lines under stress compared to WT and VC plants. The results suggested that overexpression of membrane-localized SbSRP mitigates salt and osmotic stress in the transgenic tobacco plant. It was hypothesized that SbSRP can be a transporter protein to transmit the environmental stimuli downward through the plasma membrane. However, a detailed study is required to ascertain its exact role in the abiotic stress tolerance mechanism. Overall, SbSRP is a potential candidate to be used for engineering salt and osmotic

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

    OpenAIRE

    León F Ruiz; Ana E Higareda; Marco A Pardo

    2010-01-01

    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. L...

  8. Analysis of methylated patterns and quality-related genes in tobacco (Nicotiana tabacum) cultivars.

    Science.gov (United States)

    Jiao, Junna; Jia, Yanlong; Lv, Zhuangwei; Sun, Chuanfei; Gao, Lijie; Yan, Xiaoxiao; Cui, Liusu; Tang, Zongxiang; Yan, Benju

    2014-08-01

    Methylation-sensitive amplified polymorphism was used in this study to investigate epigenetic information of four tobacco cultivars: Yunyan 85, NC89, K326, and Yunyan 87. The DNA fragments with methylated information were cloned by reamplified PCR and sequenced. The results of Blast alignments showed that the genes with methylation information included chitinase, nitrate reductase, chloroplast DNA, mitochondrial DNA, ornithine decarboxylase, ribulose carboxylase, and promoter sequences. Homologous comparison in three cloned gene sequences (nitrate reductase, ornithine decarboxylase, and ribulose decarboxylase) indicated that geographic factors had significant influence on the whole genome methylation. Introns also contained different information in different tobacco cultivars. These findings suggest that synthetic mechanisms for tobacco aromatic components could be affected by different environmental factors leading to variation of noncoding regions in the genome, which finally results in different fragrance and taste in different tobacco cultivars.

  9. RNAi-mediated resistance to SMV and BYMV in transgenic tobacco

    Directory of Open Access Journals (Sweden)

    Lo Thi Mai Thu

    2016-09-01

    Full Text Available Soybean mosaic virus (SMV and bean yellow mosaic virus (BYMV are two typical types of viruses that cause mosaic in soybean plants. Multiple viral infections at the same site can lead to 66% to 80% yield reduction. We have aimed to improve SMV and BYMV resistance in Vietnamese soybeans using gene transfer techniques under the mechanism of RNAi. In this study, we present newly generated transgenic tobacco plants carrying RNAi [CPi (SMV-BYMV] resistance to the two types of viruses; 73.08% of transgenic tobacco lines proved to be fully resistant to SMV and BYMV. In addition, the number of virus copies in transgenic tobacco plants was reduced on average by more than 51% compared to the control plants (wild type. This promising result shows the potential of transerring the CPi (SMV-BYMV structure in soybean to increase resistance of soybean to SMV and BYMV and advance the aims of antiviral soybean breeding in Vietnam.

  10. Acidic α-galactosidase is the most abundant nectarin in floral nectar of common tobacco (Nicotiana tabacum)

    Science.gov (United States)

    Zha, Hong-Guang; Flowers, V. Lynn; Yang, Min; Chen, Ling-Yang; Sun, Hang

    2012-01-01

    Background and Aims To date, most floral nectarins (nectar proteins) are reported to function in nectar defence, particularly for insect-pollinated outcrossing species. We compared nectarin composition and abundance in selfing common tobacco (Nicotiana tobaccum) with outcrossing ornamental tobacco plants to elucidate the functional difference of nectarins in different reproductive systems. Methods Common tobacco (CT) nectarins were separated by SDS-PAGE and the N terminus of the most abundant nectarin was sequenced via Edman degradation. The full-length nectarin gene was amplified and cloned from genomic DNA and mRNA with hiTail-PCR and RACE (rapid amplification of cDNA ends), and expression patterns were then investigated in different tissues using semi-quantitative reverse transcriptase PCR. Additionally, high-performance liquid chromatography and enzymatic analyses of nectar sugar composition, and other biochemical traits and functions of the novel nectarin were studied. Key Results The most abundant nectarin in CT nectar is an acidic α-galactosidase, here designated NTα-Gal. This compound has a molecular mass of 40 013 Da and a theoretical pI of 5·33. NTα-Gal has a conserved α-Gal characteristic signature, encodes a mature protein of 364 amino acids and is expressed in different organs. Compared with 27 other melliferous plant species from different families, CT floral nectar demonstrated the highest α-Gal activity, which is inhibited by d-galactose. Raffinose family oligosaccharides were not detected in CT nectar, indicating that NTα-Gal does not function in post-secretory hydrolysis. Moreover, tobacco plant fruits did not develop intact skin with galactose inhibition of NTα-Gal activity in nectar, suggesting that NTα-Gal induces cell-wall surface restructuring during the initial stages of fruit development. Conclusions α-Gal was the most abundant nectarin in selfing CT plants, but was not detected in the nectar of strictly outcrossing sister tobacco

  11. Column chromatography isolation of nicotine from tobacco leaf extract (Nicotiana tabaccum L.)

    Science.gov (United States)

    Fathi, Raden Muhammad; Fauzantoro, Ahmad; Rahman, Siti Fauziyah; Gozan, Misri

    2018-02-01

    Restrictions on the use of dried tobacco leaf for cigarette production must be accompanied by the development of non-cigarette alternative products that are made from tobacco leaves. One of the alternative that can be done is to use the nicotine compound in tobacco leaf extract as medical product, such as Parkinson's medication or to be used as active substance in biopesticide. Nicotine was isolated using column chromatography method with the variation of mobile phase mixture ratio (petroleum ether and ethanol), started from 8:2, 6:4, 4:6, 2:8, to 0:10. All of the chromatographic fraction from each mobile phase's ratio was then tested qualitatively using thin layer chromatography (TLC) and also quantitatively using HPLC instrument. The column chromatography process could isolate 4.006% of nicotine compound from 4.19% tobacco leaf extract's nicotine. It is also known that ethanol is a good solution to be used as chromatography's mobile phase for nicotine isolation from tobacco leaf extract.

  12. Effects of a petunia scaffold/matrix attachment region on copy number dependency and stability of transgene expression in Nicotiana tabacum.

    Science.gov (United States)

    Dietz-Pfeilstetter, Antje; Arndt, Nicola; Manske, Ulrike

    2016-04-01

    Transgenes in genetically modified plants are often not reliably expressed during development or in subsequent generations. Transcriptional gene silencing (TGS) as well as post-transcriptional gene silencing (PTGS) have been shown to occur in transgenic plants depending on integration pattern, copy number and integration site. In an effort to reduce position effects, to prevent read-through transcription and to provide a more accessible chromatin structure, a P35S-ß-glucuronidase (P35S-gus) transgene flanked by a scaffold/matrix attachment region from petunia (Petun-SAR), was introduced in Nicotiana tabacum plants by Agrobacterium tumefaciens mediated transformation. It was found that Petun-SAR mediates enhanced expression and copy number dependency up to 2 gene copies, but did not prevent gene silencing in transformants with multiple and rearranged gene copies. However, in contrast to the non-SAR transformants where silencing was irreversible and proceeded during long-term vegetative propagation and in progeny plants, gus expression in Petun-SAR plants was re-established in the course of development. Gene silencing was not necessarily accompanied by DNA methylation, while the gus transgene could still be expressed despite considerable CG methylation within the coding region.

  13. Regulatory approval and a first-in-human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants.

    Science.gov (United States)

    Ma, Julian K-C; Drossard, Jürgen; Lewis, David; Altmann, Friedrich; Boyle, Julia; Christou, Paul; Cole, Tom; Dale, Philip; van Dolleweerd, Craig J; Isitt, Valerie; Katinger, Dietmar; Lobedan, Martin; Mertens, Hubert; Paul, Mathew J; Rademacher, Thomas; Sack, Markus; Hundleby, Penelope A C; Stiegler, Gabriela; Stoger, Eva; Twyman, Richard M; Vcelar, Brigitta; Fischer, Rainer

    2015-10-01

    Although plant biotechnology has been widely investigated for the production of clinical-grade monoclonal antibodies, no antibody products derived from transgenic plants have yet been approved by pharmaceutical regulators for clinical testing. In the Pharma-Planta project, the HIV-neutralizing human monoclonal antibody 2G12 was expressed in transgenic tobacco (Nicotiana tabacum). The scientific, technical and regulatory demands of good manufacturing practice (GMP) were addressed by comprehensive molecular characterization of the transgene locus, confirmation of genetic and phenotypic stability over several generations of transgenic plants, and by establishing standard operating procedures for the creation of a master seed bank, plant cultivation, harvest, initial processing, downstream processing and purification. The project developed specifications for the plant-derived antibody (P2G12) as an active pharmaceutical ingredient (API) based on (i) the guidelines for the manufacture of monoclonal antibodies in cell culture systems; (ii) the draft European Medicines Agency Points to Consider document on quality requirements for APIs produced in transgenic plants; and (iii) de novo guidelines developed with European national regulators. From the resulting process, a GMP manufacturing authorization was issued by the competent authority in Germany for transgenic plant-derived monoclonal antibodies for use in a phase I clinical evaluation. Following preclinical evaluation and ethical approval, a clinical trial application was accepted by the UK national pharmaceutical regulator. A first-in-human, double-blind, placebo-controlled, randomized, dose-escalation phase I safety study of a single vaginal administration of P2G12 was carried out in healthy female subjects. The successful completion of the clinical trial marks a significant milestone in the commercial development of plant-derived pharmaceutical proteins. © 2015 Society for Experimental Biology, Association of

  14. Comparison of leaf smearing and wick feeding techniques for root distribution studies of tobacco (Nicotiana tabacum)

    International Nuclear Information System (INIS)

    Nagaraj, G.; Hanumantha Rao, A.; Gopalachari, N.C.

    1976-01-01

    Wick feeding and leaf smearing methods have been compared for their relative efficiencies for root distribution studies with tobacco plant. The applied radioactivity gets equilibrated within 3 days in the tobacco plant. Root sections of the plants fed through the wick contained higher quantity for the radioactivity over those of the leaf smeared ones. Because of the case of application and better translocation of applied radioactivity the wick-feeding method appears to have good utility for root distribution studies with hard stemmed plants. (author)

  15. A dominant-negative mutation within AtMYB90 blocks flower pigment production in transgenic tobacco.

    Science.gov (United States)

    During de novo shoot induction in cultured transgenic tobacco callus a spontaneous mutation within the coding region of a AtMYB90 transgene produced a plant line in which the original transgene-induced over-pigmented phenotype (dark red/purple from anthocyanin overproduction in most tissues) was los...

  16. Immediate effects of nectar robbing by Palestine sunbirds (Nectarinia osea) on nectar alkaloid concentrations in tree tobacco (Nicotiana glauca).

    Science.gov (United States)

    Kaczorowski, Rainee L; Koplovich, Avi; Sporer, Frank; Wink, Michael; Markman, Shai

    2014-04-01

    Plant secondary metabolites (PSMs), such as alkaloids, are often found in many parts of a plant, including flowers, providing protection to the plant from various types of herbivores or microbes. PSMs are also present in the floral nectar of many species, but typically at lower concentrations than in other parts of the plant. Nectar robbers often damage floral tissue to access the nectar. By doing so, these nectar robbers may initiate an increase of PSMs in the floral nectar. It is often assumed that it takes at least a few hours before the plant demonstrates an increase in PSMs. Here, we addressed the question of whether PSMs in the floral tissue are immediately being released into the floral nectar following nectar robbing. To address this research question, we investigated whether there was an immediate effect of nectar robbing by the Palestine Sunbird (Nectarinia osea) on the concentration of nectar alkaloids, nicotine and anabasine, in Tree Tobacco (Nicotiana glauca). We found that the concentration of anabasine, but not nicotine, significantly increased in floral nectar immediately following simulated nectar robbing. These findings suggest that nectar robbers could be ingesting greater amounts of PSMs than they would if they visit flowers legitimately. As a consequence, increased consumption of neurotoxic nectar alkaloids or other PSMs could have negative effects on the nectar robber.

  17. 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.

  18. 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

  19. Increased production of wax esters in transgenic tobacco plants by expression of a fatty acid reductase:wax synthase gene fusion.

    Science.gov (United States)

    Aslan, Selcuk; Hofvander, Per; Dutta, Paresh; Sun, Chuanxin; Sitbon, Folke

    2015-12-01

    Wax esters are hydrophobic lipids consisting of a fatty acid moiety linked to a fatty alcohol with an ester bond. Plant-derived wax esters are today of particular concern for their potential as cost-effective and sustainable sources of lubricants. However, this aspect is hampered by the fact that the level of wax esters in plants generally is too low to allow commercial exploitation. To investigate whether wax ester biosynthesis can be increased in plants using transgenic approaches, we have here exploited a fusion between two bacterial genes together encoding a single wax ester-forming enzyme, and targeted the resulting protein to chloroplasts in stably transformed tobacco (Nicotiana benthamiana) plants. Compared to wild-type controls, transgenic plants showed both in leaves and stems a significant increase in the total level of wax esters, being eight-fold at the whole plant level. The profiles of fatty acid methyl ester and fatty alcohol in wax esters were related, and C16 and C18 molecules constituted predominant forms. Strong transformants displayed certain developmental aberrations, such as stunted growth and chlorotic leaves and stems. These negative effects were associated with an accumulation of fatty alcohols, suggesting that an adequate balance between formation and esterification of fatty alcohols is crucial for a high wax ester production. The results show that wax ester engineering in transgenic plants is feasible, and suggest that higher yields may become achieved in the near future.

  20. Transgenic ipt tobacco overproducing cytokinins overaccumulates phenolic compounds during in vitro growth

    Czech Academy of Sciences Publication Activity Database

    Schnablová, Renáta; Synková, Helena; Vičánková, Anna; Burketová, Lenka; Eder, Josef; Cvikrová, Milena

    2006-01-01

    Roč. 44, - (2006), s. 526-534 ISSN 0981-9428 R&D Projects: GA ČR GA206/03/0310 Institutional research plan: CEZ:AV0Z50380511 Keywords : cytokinins * ipt * transgenic tobacco * in vitro cultivation Subject RIV: EF - Botanics Impact factor: 1.847, year: 2006

  1. Potential of MuS1 Transgenic Tobacco for Phytoremediation of the Urban Soils Contaminated with Cadmium

    Science.gov (United States)

    Kim, K. H.; Kim, Y. N.; Kim, S. H.

    2010-05-01

    Urban soils are prone to contamination by trace elements such as Cd, Cu, Pb and Zn. Phytoremediation is one of the attractive remediation methods for soils contaminated with trace elements due to its non-destructive and environmentally-friendly characteristic. Scientists have tried to find hyper-accumulator plants in nature or to develop transgenic plant through genetic engineering. This study was carried out to identify a potential of MuS1 transgenic tobacco for phytoremediation of the urban soils contaminated with Cd. MuS1 is known as a multiple stress related gene with several lines. The previous study using RT-PCR showed that the expression of MuS1 gene in tobacco plant induced tolerance to Cd stress. For this study, MuS1 transgenic tobacco and wild-type tobacco (control) were cultivated in a hydroponic system treated with Cd (0, 50, 100 and 200μM Cd) for 3 weeks. At harvest, both tobacco and nutrient solution were collected and were analyzed for Cd. Effect of Cd treatment on morphological change of the tobacco leaves was also observed by variable-pressure scanning electron microscopy (VP-SEM). The tolerance of MuS1 transgenic tobacco to Cd stress was better than that of wild-type tobacco at all Cd levels. Especially, wild-type tobacco showed chlorosis and withering with 200μM Cd treatment, whereas MuS1 transgenic tobacco gradually recovered from Cd damage. Wild-type tobacco accumulated more Cd (4.65mg per plant) than MuS1 transgenic tobacco (2.37mg per plant) with 200μM Cd treatment. Cd translocation rate from root to leaves was 81.8 % for wild-type tobacco compared to 37.1 % for MuS1 transgenic tobacco. Result of VP-SEM showed that the number of trichome in the leaves for wild-type tobacco increased in comparison with that for untreated samples after 3 weeks, while that for MuS1 transgenic tobacco was not changed by Cd treatment. Results showed that the mechanism of the recovery of the MuS1 tobacco plant was not by high level of Cd uptake and accumulation

  2. Overexpression of monoubiquitin improves photosynthesis in transgenic tobacco plants following high temperature stress.

    Science.gov (United States)

    Tian, Fengxia; Gong, Jiangfeng; Zhang, Jin; Feng, Yanan; Wang, Guokun; Guo, Qifang; Wang, Wei

    2014-09-01

    The ubiquitin/26S proteasome system (Ub/26S) is implicated in abiotic stress responses in plants. In this paper, transgenic tobacco plants overexpressing Ta-Ub2 from wheat were used to study the functions of Ub in the improvement of photosynthesis under high temperature (45°C) stress. We observed higher levels of Ub conjugates in transgenic plants under high temperature stress conditions compared to wild type (WT) as a result of the constitutive overexpression of Ta-Ub2, suggesting increased protein degradation by the 26S proteasome system under high temperature stress. Overexpressing Ub increased the photosynthetic rate (Pn) of transgenic tobacco plants, consistent with the improved ATPase activity in the thylakoid membrane and enhanced efficiency of PSII photochemistry. The higher D1 protein levels following high temperature stress in transgenic plants than WT were also observed. These findings imply that Ub may be involved in tolerance of photosynthesis to high temperature stress in plants. Compared with WT, the transgenic plants showed lower protein carbonylation and malondialdehyde (MDA) levels, less reactive oxygen species (ROS) accumulation, but higher antioxidant enzyme activity under high temperature stress. These findings suggest that the improved antioxidant capacity of transgenic plants may be one of the most important mechanisms underlying Ub-regulated high temperature tolerance. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  3. Enhanced tolerance and remediation of anthracene by transgenic tobacco plants expressing a fungal glutathione transferase gene

    Energy Technology Data Exchange (ETDEWEB)

    Dixit, Prachy; Mukherjee, Prasun K.; Sherkhane, Pramod D.; Kale, Sharad P. [Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Eapen, Susan, E-mail: eapenhome@yahoo.com [Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai 400085 (India)

    2011-08-15

    Highlights: {yields} Transgenic plants expressing a TvGST gene were tested for tolerance, uptake and degradation of anthracene. {yields} Transgenic plants were more tolerant to anthracene and take up more anthracene from soil and solutions compared to control plants. {yields} Using in vitro T{sub 1} seedlings, we showed that anthracene-a three fused benzene ring compound was phytodegraded to naphthalene derivatives, having two benzene rings. {yields} This is the first time that a transgenic plant was shown to have the potential to phytodegrade anthracene. - Abstract: Plants can be used for remediation of polyaromatic hydrocarbons, which are known to be a major concern for human health. Metabolism of xenobiotic compounds in plants occurs in three phases and glutathione transferases (GST) mediate phase II of xenobiotic transformation. Plants, although have GSTs, they are not very efficient for degradation of exogenous recalcitrant xenobiotics including polyaromatic hydrocarbons. Hence, heterologous expression of efficient GSTs in plants may improve their remediation and degradation potential of xenobiotics. In the present study, we investigated the potential of transgenic tobacco plants expressing a Trichoderma virens GST for tolerance, remediation and degradation of anthracene-a recalcitrant polyaromatic hydrocarbon. Transgenic plants with fungal GST showed enhanced tolerance to anthracene compared to control plants. Remediation of {sup 14}C uniformly labeled anthracene from solutions and soil by transgenic tobacco plants was higher compared to wild-type plants. Transgenic plants (T{sub 0} and T{sub 1}) degraded anthracene to naphthalene derivatives, while no such degradation was observed in wild-type plants. The present work has shown that in planta expression of a fungal GST in tobacco imparted enhanced tolerance as well as higher remediation potential of anthracene compared to wild-type plants.

  4. Enhanced tolerance and remediation of anthracene by transgenic tobacco plants expressing a fungal glutathione transferase gene

    International Nuclear Information System (INIS)

    Dixit, Prachy; Mukherjee, Prasun K.; Sherkhane, Pramod D.; Kale, Sharad P.; Eapen, Susan

    2011-01-01

    Highlights: → Transgenic plants expressing a TvGST gene were tested for tolerance, uptake and degradation of anthracene. → Transgenic plants were more tolerant to anthracene and take up more anthracene from soil and solutions compared to control plants. → Using in vitro T 1 seedlings, we showed that anthracene-a three fused benzene ring compound was phytodegraded to naphthalene derivatives, having two benzene rings. → This is the first time that a transgenic plant was shown to have the potential to phytodegrade anthracene. - Abstract: Plants can be used for remediation of polyaromatic hydrocarbons, which are known to be a major concern for human health. Metabolism of xenobiotic compounds in plants occurs in three phases and glutathione transferases (GST) mediate phase II of xenobiotic transformation. Plants, although have GSTs, they are not very efficient for degradation of exogenous recalcitrant xenobiotics including polyaromatic hydrocarbons. Hence, heterologous expression of efficient GSTs in plants may improve their remediation and degradation potential of xenobiotics. In the present study, we investigated the potential of transgenic tobacco plants expressing a Trichoderma virens GST for tolerance, remediation and degradation of anthracene-a recalcitrant polyaromatic hydrocarbon. Transgenic plants with fungal GST showed enhanced tolerance to anthracene compared to control plants. Remediation of 14 C uniformly labeled anthracene from solutions and soil by transgenic tobacco plants was higher compared to wild-type plants. Transgenic plants (T 0 and T 1 ) degraded anthracene to naphthalene derivatives, while no such degradation was observed in wild-type plants. The present work has shown that in planta expression of a fungal GST in tobacco imparted enhanced tolerance as well as higher remediation potential of anthracene compared to wild-type plants.

  5. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Response morphology and anatomy of tobacco (Nicotiana tabacum L.) plant on waterlogging

    Science.gov (United States)

    Nurhidayati, Tutik; Wardhani, Selfrina Puri; Purnobasuki, Hery; Hariyanto, Sucipto; Jadid, Nurul; Nurcahyani, Desy Dwi

    2017-11-01

    This study has conducted research on morphological and anatomical responses of some varieties of tobacco plants to waterlogging stress. Parameters measured were morphology, anatomy, and plants sensitivity index. Results were analyzed using two-way ANOVA followed by the Tukey test. The results show that waterlogging stress can reduce the growth of tobacco plants, including a decrease in plant height with the lowest value of 15.6 cm, root length reduction to the lowest value of 4.6 cm and plant dry weight reduction to the lowest value of 0.26 gr. But waterlogging stress can increase the number of adventitious roots with the highest value of 18.33. In addition, waterlogging stress can lead to the formation of aerenchyma tissue. The sensitivity index showed that plant varieties that are resistant to waterlogging stress are the varieties Kemloko 3 (index value of 0.03), varieties of Paiton 2 (index value of 0.18), and the varieties Kemloko 2 (index value of 0.42).

  7. Heterologous production of a ginsenoside saponin (compound K) and its precursors in transgenic tobacco impairs the vegetative and reproductive growth.

    Science.gov (United States)

    Gwak, Yu Shin; Han, Jung Yeon; Adhikari, Prakash Babu; Ahn, Chang Ho; Choi, Yong Eui

    2017-06-01

    Production of compound K (a ginsenoside saponin) and its precursors in transgenic tobacco resulted in stunted growth and seed set failure, which may be caused by strong autotoxicity of heterologously produced phytochemicals against the tobacco itself. Panax ginseng roots contain various saponins (ginsenosides), which are major bioactive compounds. A monoglucosylated saponin, compound K (20-O-(β-D-glucopyranosyl)-20(S)-protopanaxadiol), has high medicinal and cosmetic values but is present in undetectable amounts in naturally grown ginseng roots. The production of compound K (CK) requires complicated deglycosylation of ginsenosides using physicochemical and/or enzymatic degradation. In this work, we report the production of CK in transgenic tobacco by co-overexpressing three genes (PgDDS, CYP716A47 and UGT71A28) isolated from P. ginseng. Introduction and expression of the transgenes in tobacco lines were confirmed by genomic PCR and RT-PCR. All the lines of transgenic tobacco produced CK including its precursors, protopanaxadiol and dammarenediol-II (DD). The concentrations of CK in the leaves ranged from 1.55 to 2.64 µg/g dry weight, depending on the transgenic line. Interestingly, production of CK in tobacco brought stunted plant growth and gave rise to seed set failure. This seed set failure was caused by both long-styled flowers and abnormal pollen development in transgenic tobacco. Both CK and DD treatments highly suppressed in vitro germination and tube growth in wild-type pollens. Based on these results, metabolic engineering for CK production in transgenic tobacco was successfully achieved, but the production of CK and its precursors in tobacco severely affects vegetative and reproductive growth due to the cytotoxicity of phytochemicals that are heterologously produced in transgenic tobacco.

  8. 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

  9. Biosynthesis of a new tobacco alkaloid, hydroxy-N-acylnornicotine in the trichomes of Nicotiana stocktonii

    International Nuclear Information System (INIS)

    Zador, E.; Jones, D.

    1986-01-01

    A new tobacco alkaloid from section Repandae is highly toxic to an insect (Manduca sexta) unsusceptible to previously described nicotine alkaloids (1). They have localized the alkaloid, HO-N-acylnornicotine (HO-NAN) nearly entirely to the exudate secreted by the epidermal trichomes of N. stocktonii. Only the nicotine and nornicotine were found in abundance inside the trichomes, while primarily nicotine was present inside the aerial vegetative parts and root. These results suggest that the HO-NAN is synthesized by the trichomes. When unlabelled nicotine was fed to isolated leaves there was an increase in internal nicotine, nornicotine and secretion of HO-NAN. Feeding leaves with 2'-C 14 nicotine resulted in labelling of both nornicotine and HO-NAN. These data strongly suggest synthesis of HO-NAN from nicotine via nornicotine in the trichomes, followed by rapid secretion. The possible evolutionary significance of this pathway of synthesis and secretion is discussed

  10. Optimization of non-catalytic transesterification of tobacco (Nicotiana tabacum) seed oil using supercritical methanol to biodiesel production

    International Nuclear Information System (INIS)

    García-Martínez, Nuria; Andreo-Martínez, Pedro; Quesada-Medina, Joaquín; Pérez de los Ríos, Antonia Pérez; Chica, Antonio; Beneito-Ruiz, Rubén; Carratalá-Abril, Juan

    2017-01-01

    Highlights: • Biodiesel from tobacco oil was produced by non-catalytic supercritical methanolysis. • Maximum experimental yield of FAMEs (92.8%) was reached at 300 °C and 90 min. • Optimal conditions by RSM (303.4 °C and 90 min) predicted a maximum FAME yield of 91.1%. • Thermal decomposition of biodiesel was observed above 325 °C and 60 min of reaction. • Glycerol generated at 300 °C and 90 min was degraded and incorporated to the biodiesel. - Abstract: The biodiesel production from non-edible oils has high potential as renewable and ecological fuel. Few researches have been conducted to date on the production of biodiesel from tobacco (Nicotiana tabacum) seed oil. The aim of this study was to optimize the biodiesel production from this crude oil by non-catalytic supercritical methanolysis using response surface methodology (RSM). Triglyceride conversion, total and individual FAME yield, monoglyceride and diglyceride yield, and thermal decomposition degree of biodiesel were determined in the temperature and reaction time ranges of 250–350 °C (12–43 MPa) and 15–90 min, respectively, at a fixed methanol-to-oil molar ratio of 43:1. According to the RSM, the optimal conditions were 303.4 °C and 90 min, reaching a predicted maximum FAME yield of 91.1 ± 3.2 mol%. This maximum was very close to that obtained experimentally (92.8 ± 2.1 mol%) at 300 °C and 90 min. Decomposition of biodiesel became evident at 325 °C and 60 min of reaction due to the thermal instability of unsaturated methyl esters (methyl linoleate and oleate). The biodiesel obtained in the best experimental reaction conditions (300 °C and 90 min), where no thermal decomposition of FAMEs was observed, contained most of the byproduct glycerol generated, which was degraded and incorporated to the product. This biodiesel basically failed to meet the content of FAMEs as required by the standard EN 14214, the content of monoglycerides and total glycerol, and the acid value, being a

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    Burklew, Caitlin E; Ashlock, Jordan; Winfrey, William B; Zhang, Baohong

    2012-01-01

    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(2)O(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(2)O(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(2)O(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(2)O(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(2)O(3) nanoparticles in the environment.

  13. 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. Copyright © 2015 Yunnan Academy of Tobacco Agricultural Sciences. Published by Elsevier Inc. All rights reserved.

  14. An N‐terminal Peptide Extension Results in Efficient Expression, but not Secretion, of a Synthetic Horseradish Peroxidase Gene in Transgenic Tobacco

    Science.gov (United States)

    KIS, MIHALY; BURBRIDGE, EMMA; BROCK, IAN W.; HEGGIE, LAURA; DIX, PHILIP J.; KAVANAGH, TONY A.

    2004-01-01

    • Background and Aims Native horseradish (Armoracia rusticana) peroxidase, HRP (EC 1.11.1.7), isoenzyme C is synthesized with N‐terminal and C‐terminal peptide extensions, believed to be associated with protein targeting. This study aimed to explore the specific functions of these extensions, and to generate transgenic plants with expression patterns suitable for exploring the role of peroxidase in plant development and defence. • Methods Transgenic Nicotiana tabacum (tobacco) plants expressing different versions of a synthetic horseradish peroxidase, HRP, isoenzyme C gene were constructed. The gene was engineered to include additional sequences coding for either the natural N‐terminal or the C‐terminal extension or both. These constructs were placed under the control of a constitutive promoter (CaMV‐35S) or the tobacco RUBISCO‐SSU light inducible promoter (SSU) and introduced into tobacco using Agrobacterium‐mediated transformation. To study the effects of the N‐ and C‐terminal extensions, the localization of recombinant peroxidase was determined using biochemical and molecular techniques. • Key Results Transgenic tobacco plants can exhibit a ten‐fold increase in peroxidase activity compared with wild‐type tobacco levels, and the majority of this activity is located in the symplast. The N‐terminal extension is essential for the production of high levels of recombinant protein, while the C‐terminal extension has little effect. Differences in levels of enzyme activity and recombinant protein are reflected in transcript levels. • Conclusions There is no evidence to support either preferential secretion or vacuolar targeting of recombinant peroxidase in this heterologous expression system. This leads us to question the postulated targeting roles of these peptide extensions. The N‐terminal extension is essential for high level expression and appears to influence transcript stability or translational efficiency. Plants have been

  15. Increased yield of heterologous viral glycoprotein in the seeds of homozygous transgenic tobacco plants cultivated underground.

    Science.gov (United States)

    Tackaberry, Eilleen S; Prior, Fiona; Bell, Margaret; Tocchi, Monika; Porter, Suzanne; Mehic, Jelica; Ganz, Peter R; Sardana, Ravinder; Altosaar, Illimar; Dudani, Anil

    2003-06-01

    The use of transgenic plants in the production of recombinant proteins for human therapy, including subunit vaccines, is being investigated to evaluate the efficacy and safety of these emerging biopharmaceutical products. We have previously shown that synthesis of recombinant glycoprotein B (gB) of human cytomegalovirus can be targeted to seeds of transgenic tobacco when directed by the rice glutelin 3 promoter, with gB retaining critical features of immunological reactivity (E.S. Tackaberry et al. 1999. Vaccine, 17: 3020-3029). Here, we report development of second generation transgenic plant lines (T1) homozygous for the transgene. Twenty progeny plants from two lines (A23T(1)-2 and A24T(1)-3) were grown underground in an environmentally contained mine shaft. Based on yields of gB in their seeds, the A23T(1)-2 line was then selected for scale-up in the same facility. Analyses of mature seeds by ELISA showedthat gB specific activity in A23T(1)-2 seeds was over 30-fold greater than the best T0 plants from the same transformation series, representing 1.07% total seed protein. These data demonstrate stable inheritance, an absence of transgene inactivation, and enhanced levels of gB expression in a homozygous second generation plant line. They also provide evidence for the suitability of using this environmentally secure facility to grow transgenic plants producing therapeutic biopharmaceuticals.

  16. Cell culture-induced gradual and frequent epigenetic reprogramming of invertedly repeated tobacco transgene epialleles

    Czech Academy of Sciences Publication Activity Database

    Křížová, Kateřina; Fojtová, Miloslava; Depicker, A.; Kovařík, Aleš

    2009-01-01

    Roč. 149, č. 3 (2009), s. 1493-1504 ISSN 0032-0889 R&D Projects: GA AV ČR(CZ) IAA600040611; GA ČR(CZ) GD204/05/H505; GA ČR(CZ) GA521/07/0116 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : tobacco * cell culture * transgene silencing Subject RIV: BO - Biophysics Impact factor: 6.235, year: 2009

  17. Ubiquitin fusion expression and tissue-dependent targeting of hG-CSF in transgenic tobacco

    Science.gov (United States)

    2011-01-01

    Background Human granulocyte colony-stimulating factor (hG-CSF) is an important human cytokine which has been widely used in oncology and infection protection. To satisfy clinical needs, expression of recombinant hG-CSF has been studied in several organisms, including rice cell suspension culture and transient expression in tobacco leaves, but there was no published report on its expression in stably transformed plants which can serve as a more economical expression platform with potential industrial application. Results In this study, hG-CSF expression was investigated in transgenic tobacco leaves and seeds in which the accumulation of hG-CSF could be enhanced through fusion with ubiquitin by up to 7 fold in leaves and 2 fold in seeds, leading to an accumulation level of 2.5 mg/g total soluble protein (TSP) in leaves and 1.3 mg/g TSP in seeds, relative to hG-CSF expressed without a fusion partner. Immunoblot analysis showed that ubiquitin was processed from the final protein product, and ubiquitination was up-regulated in all transgenic plants analyzed. Driven by CaMV 35S promoter and phaseolin signal peptide, hG-CSF was observed to be secreted into apoplast in leaves but deposited in protein storage vacuole (PSV) in seeds, indicating that targeting of the hG-CSF was tissue-dependent in transgenic tobacco. Bioactivity assay showed that hG-CSF expressed in both seeds and leaves was bioactive to support the proliferation of NFS-60 cells. Conclusions In this study, the expression of bioactive hG-CSF in transgenic plants was improved through ubiquitin fusion strategy, demonstrating that protein expression can be enhanced in both plant leaves and seeds through fusion with ubiquitin and providing a typical case of tissue-dependent expression of recombinant protein in transgenic plants. PMID:21985646

  18. Long-chain bases and their phosphorylated derivatives differentially regulate cryptogein-induced production of reactive oxygen species in tobacco (Nicotiana tabacum) BY-2 cells.

    Science.gov (United States)

    Coursol, Sylvie; Fromentin, Jérôme; Noirot, Elodie; Brière, Christian; Robert, Franck; Morel, Johanne; Liang, Yun-Kuan; Lherminier, Jeannine; Simon-Plas, Françoise

    2015-02-01

    The proteinaceous elicitor cryptogein triggers defence reactions in Nicotiana tabacum (tobacco) through a signalling cascade, including the early production of reactive oxygen species (ROS) by the plasma membrane (PM)-located tobacco respiratory burst oxidase homologue D (NtRbohD). Sphingolipid long-chain bases (LCBs) are emerging as potent positive regulators of plant defence-related mechanisms. This led us to question whether both LCBs and their phosphorylated derivatives (LCB-Ps) are involved in the early signalling process triggered by cryptogein in tobacco BY-2 cells. Here, we showed that cryptogein-induced ROS production was inhibited by LCB kinase (LCBK) inhibitors. Additionally, Arabidopsis thaliana sphingosine kinase 1 and exogenously supplied LCB-Ps increased cryptogein-induced ROS production, whereas exogenously supplied LCBs had a strong opposite effect, which was not driven by a reduction in cellular viability. Immunogold-electron microscopy assay also revealed that LCB-Ps are present in the PM, which fits well with the presence of a high LCBK activity associated with this fraction. Our data demonstrate that LCBs and LCB-Ps differentially regulate cryptogein-induced ROS production in tobacco BY-2 cells, and support a model in which a cooperative synergism between LCBK/LCB-Ps and NtRbohD/ROS in the cryptogein signalling pathway is likely at the PM in tobacco BY-2 cells. © 2014 INRA New Phytologist © 2014 New Phytologist Trust.

  19. 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. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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 kDa. 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.

  1. The role of mesophyll conductance during water stress and recovery in tobacco (Nicotiana sylvestris): acclimation or limitation?

    Science.gov (United States)

    Galle, Alexander; Florez-Sarasa, Igor; Tomas, Magdalena; Pou, Alicia; Medrano, Hipolito; Ribas-Carbo, Miquel; Flexas, Jaume

    2009-01-01

    While the responses of photosynthesis to water stress have been widely studied, acclimation to sustained water stress and recovery after re-watering is poorly understood. In particular, the factors limiting photosynthesis under these conditions, and their possible interactions with other environmental conditions, are unknown. To assess these issues, changes of photosynthetic CO(2) assimilation (A(N)) and its underlying limitations were followed during prolonged water stress and subsequent re-watering in tobacco (Nicotiana sylvestris) plants growing under three different climatic conditions: outdoors in summer, outdoors in spring, and indoors in a growth chamber. In particular, the regulation of stomatal conductance (g(s)), mesophyll conductance to CO(2) (g(m)), leaf photochemistry (chlorophyll fluorescence), and biochemistry (V(c,max)) were assessed. Leaf gas exchange and chlorophyll fluorescence data revealed that water stress induced a similar degree of stomatal closure and decreased A(N) under all three conditions, while V(c,max) was unaffected. However, the behaviour of g(m) differed depending on the climatic conditions. In outdoor plants, g(m) strongly declined with water stress, but it recovered rapidly (1-2 d) after re-watering in spring while it remained low many days after re-watering in summer. In indoor plants, g(m) initially declined with water stress, but then recovered to control values during the acclimation period. These differences were reflected in different velocities of recovery of A(N) after re-watering, being the slowest in outdoor summer plants and the fastest in indoor plants. It is suggested that these differences among the experiments are related to the prevailing climatic conditions, i.e. to the fact that stress factors other than water stress have been superimposed (e.g. excessive light and elevated temperature). In conclusion, besides g(s), g(m) contributes greatly to the limitation of photosynthesis during water stress and during

  2. Amyloid-like protein inclusions in tobacco transgenic plants.

    Directory of Open Access Journals (Sweden)

    Anna Villar-Piqué

    Full Text Available The formation of insoluble protein deposits in human tissues is linked to the onset of more than 40 different disorders, ranging from dementia to diabetes. In these diseases, the proteins usually self-assemble into ordered β-sheet enriched aggregates known as amyloid fibrils. Here we study the structure of the inclusions formed by maize transglutaminase (TGZ in the chloroplasts of tobacco transplastomic plants and demonstrate that they have an amyloid-like nature. Together with the evidence of amyloid structures in bacteria and fungi our data argue that amyloid formation is likely a ubiquitous process occurring across the different kingdoms of life. The discovery of amyloid conformations inside inclusions of genetically modified plants might have implications regarding their use for human applications.

  3. Phenotypic Changes in Transgenic Tobacco Plants Overexpressing Vacuole-Targeted Thermotoga maritima BglB Related to Elevated Levels of Liberated Hormones

    Science.gov (United States)

    Nguyen, Quynh Anh; Lee, Dae-Seok; Jung, Jakyun; Bae, Hyeun-Jong

    2015-01-01

    The hyperthermostable β-glucosidase BglB of Thermotoga maritima was modified by adding a short C-terminal tetrapeptide (AFVY, which transports phaseolin to the vacuole, to its C-terminal sequence). The modified β-glucosidase BglB was transformed into tobacco (Nicotiana tabacum L.) plants. We observed a range of significant phenotypic changes in the transgenic plants compared to the wild-type (WT) plants. The transgenic plants had faster stem growth, earlier flowering, enhanced root systems development, an increased biomass biosynthesis rate, and higher salt stress tolerance in young plants compared to WT. In addition, programed cell death was enhanced in mature plants. Furthermore, the C-terminal AFVY tetrapeptide efficiently sorted T. maritima BglB into the vacuole, which was maintained in an active form and could perform its glycoside hydrolysis function on hormone conjugates, leading to elevated hormone [abscisic acid (ABA), indole 3-acetic acid (IAA), and cytokinin] levels that likely contributed to the phenotypic changes in the transgenic plants. The elevation of cytokinin led to upregulation of the transcription factor WUSCHELL, a homeodomain factor that regulates the development, division, and reproduction of stem cells in the shoot apical meristems. Elevation of IAA led to enhanced root development, and the elevation of ABA contributed to enhanced tolerance to salt stress and programed cell death. These results suggest that overexpressing vacuole-targeted T. maritima BglB may have several advantages for molecular farming technology to improve multiple targets, including enhanced production of the β-glucosidase BglB, increased biomass, and shortened developmental stages, that could play pivotal roles in bioenergy and biofuel production. PMID:26618153

  4. Expression of kenaf mitochondrial chimeric genes HM184 causes male sterility in transgenic tobacco plants.

    Science.gov (United States)

    Zhao, Yanhong; Liao, Xiaofang; Huang, Zhipeng; Chen, Peng; Zhou, Bujin; Liu, Dongmei; Kong, Xiangjun; Zhou, Ruiyang

    2015-08-01

    Chimeric genes resulting from the rearrangement of a mitochondrial genome were generally thought to be a causal factor in the occurrence of cytoplasmic male sterility (CMS). In the study, earlier we reported that identifying a 47 bp deletion at 3'- flanking of atp9 that was linked to male sterile cytoplasm in kenaf. The truncated fragment was fused with atp9, a mitochondrial transit signal (MTS) and/or GFP, comprised two chimeric genes MTS-HM184-GFP and MTS-HM184. The plant expression vector pBI121 containing chimeric genes were then introduced to tobacco plants by Agrobacterium-mediated T-DNA transformation. The result showed that certain transgenic plants were male sterility or semi-sterility, while some were not. The expression analysis further demonstrated that higher level of expression were showed in the sterility plants, while no expression or less expression in fertility plants, the levels of expression of semi-sterility were in between. And the sterile plant (containing MTS-HM184-GFP) had abnormal anther produced malformed/shriveled pollen grains stained negative that failed to germinate (0%), the corresponding fruits was shrunken, the semi-sterile plants having normal anther shape produced about 10-50% normal pollen grains, the corresponding fruits were not full, and the germination rate was 58%. Meanwhile these transgenic plants which altered on fertility were further analyzed in phenotype. As a result, the metamorphosis leaves were observed in the seedling stage, the plant height of transgenic plants was shorter than wild type. The growth duration of transgenic tobacco was delayed 30-45 days compared to the wild type. The copy numbers of target genes of transgenic tobacco were analyzed using the real-time quantitative method. The results showed that these transgenic plants targeting-expression in mitochondrial containing MTS-HM184-GFP had 1 copy and 2 copies, the other two plants containing MTS-HM184 both had 3 copies, but 0 copy in wild type. In

  5. GhWRKY25, a group I WRKY gene from cotton, confers differential tolerance to abiotic and biotic stresses in transgenic Nicotiana benthamiana.

    Science.gov (United States)

    Liu, Xiufang; Song, Yunzhi; Xing, Fangyu; Wang, Ning; Wen, Fujiang; Zhu, Changxiang

    2016-09-01

    WRKY transcription factors are involved in various processes, ranging from plant growth to abiotic and biotic stress responses. Group I WRKY members have been rarely reported compared with group II or III members, particularly in cotton (Gossypium hirsutum). In this study, a group I WRKY gene, namely, GhWRKY25, was cloned from cotton and characterized. Expression analysis revealed that GhWRKY25 can be induced or deduced by the treatments of abiotic stresses and multiple defense-related signaling molecules. Overexpression of GhWRKY25 in Nicotiana benthamiana reduced plant tolerance to drought stress but enhanced tolerance to salt stress. Moreover, more MDA and ROS accumulated in transgenic plants after drought treatment with lower activities of SOD, POD, and CAT. Our study further demonstrated that GhWRKY25 overexpression in plants enhanced sensitivity to the fungal pathogen Botrytis cinerea by reducing the expression of SA or ET signaling related genes and inducing the expression of genes involved in the JA signaling pathway. These results indicated that GhWRKY25 plays negative or positive roles in response to abiotic stresses, and the reduced pathogen resistance may be related to the crosstalk of the SA and JA/ET signaling pathways.

  6. Overexpression of the wheat aquaporin gene, TaAQP7, enhances drought tolerance in transgenic tobacco.

    Directory of Open Access Journals (Sweden)

    Shiyi Zhou

    Full Text Available Aquaporin (AQP proteins have been shown to transport water and other small molecules through biological membranes, which is crucial for plants to combat stress caused by drought. However, the precise role of AQPs in drought stress response is not completely understood in plants. In this study, a PIP2 subgroup gene AQP, designated as TaAQP7, was cloned and characterized from wheat. Expression of TaAQP7-GFP fusion protein revealed its localization in the plasma membrane. TaAQP7 exhibited high water channel activity in Xenopus laevis oocytes and TaAQP7 transcript was induced by dehydration, and treatments with polyethylene glycol (PEG, abscisic acid (ABA and H(2O(2. Further, TaAQP7 was upregulated after PEG treatment and was blocked by inhibitors of ABA biosynthesis, implying that ABA signaling was involved in the upregulation of TaAQP7 after PEG treatment. Overexpression of TaAQP7 increased drought tolerance in tobacco. The transgenic tobacco lines had lower levels of malondialdehyde (MDA and H(2O(2, and less ion leakage (IL, but higher relative water content (RWC and superoxide dismutase (SOD and catalase (CAT activities when compared with the wild type (WT under drought stress. Taken together, our results show that TaAQP7 confers drought stress tolerance in transgenic tobacco by increasing the ability to retain water, reduce ROS accumulation and membrane damage, and enhance the activities of antioxidants.

  7. Overexpression of MfPIP2-7 from Medicago falcata promotes cold tolerance and growth under NO3 (-) deficiency in transgenic tobacco plants.

    Science.gov (United States)

    Zhuo, Chunliu; Wang, Ting; Guo, Zhenfei; Lu, Shaoyun

    2016-06-14

    Plasma membrane intrinsic proteins (PIPs), which belong to aquaporins (AQPs) superfamily, are subdivided into two groups, PIP1 and PIP2, based on sequence similarity. Several PIP2s function as water channels, while PIP1s have low or no water channel activity, but have a role in water permeability through interacting with PIP2. A cold responsive PIP2 named as MfPIP2-7 was isolated from Medicago falcata (hereafter falcata), a forage legume with great cold tolerance, and transgenic tobacco plants overexpressing MfPIP2-7 were analyzed in tolerance to multiple stresses including freezing, chilling, and nitrate reduction in this study. MfPIP2-7 transcript was induced by 4 to 12 h of cold treatment and 2 h of abscisic acid (ABA) treatment. Pretreatment with inhibitor of ABA synthesis blocked the cold induced MfPIP2-7 transcript, indicating that ABA was involved in cold induced transcription of MfPIP2-7 in falcata. Overexpression of MfPIP2-7 resulted in enhanced tolerance to freezing, chilling and NO3 (-) deficiency in transgenic tobacco (Nicotiana tabacum L.) plants as compared with the wild type. Moreover, MfPIP2-7 was demonstrated to facilitate H2O2 diffusion in yeast. Higher transcript levels of several stress responsive genes, such as NtERD10B, NtERD10C, NtDREB1, and 2, and nitrate reductase (NR) encoding genes (NtNIA1, and NtNIA2) were observed in transgenic plants as compared with the wild type with dependence upon H2O2. In addition, NR activity was increased in transgenic plants, which led to alterations in free amino acid components and concentrations. The results suggest that MfPIP2-7 plays an important role in plant tolerance to freezing, chilling, and NO3 (-) deficiency by promoted H2O2 diffusion that in turn up-regulates expression of NIAs and multiple stress responsive genes.

  8. Protection of the photosynthetic apparatus from extreme dehydration and oxidative stress in seedlings of transgenic tobacco.

    Directory of Open Access Journals (Sweden)

    Concepción Almoguera

    Full Text Available A genetic program that in sunflower seeds is activated by Heat Shock transcription Factor A9 (HaHSFA9 has been analyzed in transgenic tobacco seedlings. The ectopic overexpression of the HSFA9 program protected photosynthetic membranes, which resisted extreme dehydration and oxidative stress conditions. In contrast, heat acclimation of seedlings induced thermotolerance but not resistance to the harsh stress conditions employed. The HSFA9 program was found to include the expression of plastidial small Heat Shock Proteins that accumulate only at lower abundance in heat-stressed vegetative organs. Photosystem II (PSII maximum quantum yield was higher for transgenic seedlings than for non-transgenic seedlings, after either stress treatment. Furthermore, protection of both PSII and Photosystem I (PSI membrane protein complexes was observed in the transgenic seedlings, leading to their survival after the stress treatments. It was also shown that the plastidial D1 protein, a labile component of the PSII reaction center, and the PSI core protein PsaB were shielded from oxidative damage and degradation. We infer that natural expression of the HSFA9 program during embryogenesis may protect seed pro-plastids from developmental desiccation.

  9. Transgenic tobacco plants constitutively expressing peanut BTF3 exhibit increased growth and tolerance to abiotic stresses.

    Science.gov (United States)

    Pruthvi, V; Rama, N; Parvathi, M S; Nataraja, K N

    2017-05-01

    Abiotic stresses limit crop growth and productivity worldwide. Cellular tolerance, an important abiotic stress adaptive trait, involves coordinated activities of multiple proteins linked to signalling cascades, transcriptional regulation and other diverse processes. Basal transcriptional machinery is considered to be critical for maintaining transcription under stressful conditions. From this context, discovery of novel basal transcription regulators from stress adapted crops like peanut would be useful for improving tolerance of sensitive plant types. In this study, we prospected a basal transcription factor, BTF3 from peanut (Arachis hypogaea L) and studied its relevance in stress acclimation by over expression in tobacco. AhBTF3 was induced under PEG-, NaCl-, and methyl viologen-induced stresses in peanut. The constitutive expression of AhBTF3 in tobacco increased plant growth under non stress condition. The transgenic plants exhibited superior phenotype compared to wild type under mannitol- and NaCl-induced stresses at seedling level. The enhanced cellular tolerance of transgenic plants was evidenced by higher cell membrane stability, reactive oxygen species (ROS) scavenging activity, seedling survival and vigour than wild type. The transgenic lines showed better in vitro regeneration capacity on growth media supplemented with NaCl than wild type. Superior phenotype of transgenic plants under osmotic and salinity stresses seems to be due to constitutive activation of genes of multiple pathways linked to growth and stress adaptation. The study demonstrated that AhBTF3 is a positive regulator of growth and stress acclimation and hence can be considered as a potential candidate gene for crop improvement towards stress adaptation. © 2016 German Botanical Society and The Royal Botanical Society of the Netherlands.

  10. 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

    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...... concentrations. Possible uses and limitations of this method for plant cell research are discussed.......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...

  11. Light-grown plants of transgenic tobacco expressing an introduced oat phytochrome A gene under the control of a constitutive viral promoter exhibit persistent growth inhibition by far-red light

    International Nuclear Information System (INIS)

    McCormac, A.; Whitelam, G.; Smith, H.

    1992-01-01

    A comparison of the photoregulation of development has been made for etiolated and light-grown plants of wild-type (WT) tobacco (Nicotiana tabacun L.) and an isogenic transgenic line which expresses an introduced oat phytochrome gene (phyA) under the control of a constitutive viral promoter. Etiolated seedlings of both the WT and transgenic line showed irradiance-dependent inhibition of hypocotyl growth under continuous far-red (FR) light; transgenic seedlings showed a greater level of inhibition under a given fluence rate and this is considered to be the result of the heterologous phytochrome protein (PhyA) functioning in a compatible manner with the native etiolated phytochrome. Deetiolation of WT seedlings resulted in a loss of responsiveness to prolonged FR. Light-grown transgenic seedlings, however, continued to respond in an irradiance-dependent manner to prolonged FR and it is proposed that this is a specific function of the constitutive PhyA. Mature green plants of the WT and transgenic lines showed a qualitatively similar growth promotion to a brief end-of-day FR-treatment but this response was abolished in the transgenic plants under prolonged irradiation by this same FR source. Growth inhibition (McCormac et al. 1991, Planta 185, 162-170) and enhanced levels of nitrate-reductase activity under irradiance of low red:far-red ratio, as achieved by the FR-supplementation of white light, emphasised that the introduced PhyA was eliciting an aberrant mode of photoresponse compared with the normal phytochrome population of light-grown plants. Total levels of the oat-encoded phytochrome in the etiolated transgenic tobacco were shown to be influenced by the wavelength of continuous irradiation in a manner which was qualitatively similar to that seen for the native, etiolated tobacco phytochrome, and distinct from that seen in etiolated oat tissues. These results are discussed in terms of the proposal that the constitutive oat-PhyA pool in the transgenic plants

  12. Enhanced whitefly resistance in transgenic tobacco plants expressing double stranded RNA of v-ATPase A gene.

    Science.gov (United States)

    Thakur, Nidhi; Upadhyay, Santosh Kumar; Verma, Praveen C; Chandrashekar, Krishnappa; Tuli, Rakesh; Singh, Pradhyumna K

    2014-01-01

    Expression of double strand RNA (dsRNA) designed against important insect genes in transgenic plants have been shown to give protection against pests through RNA interference (RNAi), thus opening the way for a new generation of insect-resistant crops. We have earlier compared the efficacy of dsRNAs/siRNAs, against a number of target genes, for interference in growth of whitefly (Bemisia tabaci) upon oral feeding. The v-ATPase subunit A (v-ATPaseA) coding gene was identified as a crucial target. We now report the effectiveness of transgenic tobacco plants expressing siRNA to silence v-ATPaseA gene expression for the control of whitefly infestation. Transgenic tobacco lines were developed for the expression of long dsRNA precursor to make siRNA and knock down the v-ATPaseA mRNA in whitefly. Molecular analysis and insecticidal properties of the transgenic plants established the formation of siRNA targeting the whitefly v-ATPaseA, in the leaves. The transcript level of v-ATPaseA in whiteflies was reduced up to 62% after feeding on the transgenic plants. Heavy infestation of whiteflies on the control plants caused significant loss of sugar content which led to the drooping of leaves. The transgenic plants did not show drooping effect. Host plant derived pest resistance was achieved against whiteflies by genetic transformation of tobacco which generated siRNA against the whitefly v-ATPaseA gene. Transgenic tobacco lines expressing dsRNA of v-ATPaseA, delivered sufficient siRNA to whiteflies feeding on them, mounting a significant silencing response, leading to their mortality. The transcript level of the target gene was reduced in whiteflies feeding on transgenic plants. The strategy can be taken up for genetic engineering of plants to control whiteflies in field crops.

  13. Enhanced whitefly resistance in transgenic tobacco plants expressing double stranded RNA of v-ATPase A gene.

    Directory of Open Access Journals (Sweden)

    Nidhi Thakur

    Full Text Available BACKGROUND: Expression of double strand RNA (dsRNA designed against important insect genes in transgenic plants have been shown to give protection against pests through RNA interference (RNAi, thus opening the way for a new generation of insect-resistant crops. We have earlier compared the efficacy of dsRNAs/siRNAs, against a number of target genes, for interference in growth of whitefly (Bemisia tabaci upon oral feeding. The v-ATPase subunit A (v-ATPaseA coding gene was identified as a crucial target. We now report the effectiveness of transgenic tobacco plants expressing siRNA to silence v-ATPaseA gene expression for the control of whitefly infestation. METHODOLOGY/PRINCIPAL FINDINGS: Transgenic tobacco lines were developed for the expression of long dsRNA precursor to make siRNA and knock down the v-ATPaseA mRNA in whitefly. Molecular analysis and insecticidal properties of the transgenic plants established the formation of siRNA targeting the whitefly v-ATPaseA, in the leaves. The transcript level of v-ATPaseA in whiteflies was reduced up to 62% after feeding on the transgenic plants. Heavy infestation of whiteflies on the control plants caused significant loss of sugar content which led to the drooping of leaves. The transgenic plants did not show drooping effect. CONCLUSIONS/SIGNIFICANCE: Host plant derived pest resistance was achieved against whiteflies by genetic transformation of tobacco which generated siRNA against the whitefly v-ATPaseA gene. Transgenic tobacco lines expressing dsRNA of v-ATPaseA, delivered sufficient siRNA to whiteflies feeding on them, mounting a significant silencing response, leading to their mortality. The transcript level of the target gene was reduced in whiteflies feeding on transgenic plants. The strategy can be taken up for genetic engineering of plants to control whiteflies in field crops.

  14. Expression of Finger Millet EcDehydrin7 in Transgenic Tobacco Confers Tolerance to Drought Stress.

    Science.gov (United States)

    Singh, Rajiv Kumar; Singh, Vivek Kumar; Raghavendrarao, Sanagala; Phanindra, Mullapudi Lakshmi Venkata; Venkat Raman, K; Solanke, Amolkumar U; Kumar, Polumetla Ananda; Sharma, Tilak Raj

    2015-09-01

    One of the critical alarming constraints for agriculture is water scarcity. In the current scenario, global warming due to climate change and unpredictable rainfall, drought is going to be a master player and possess a big threat to stagnating gene pool of staple food crops. So it is necessary to understand the mechanisms that enable the plants to cope with drought stress. In this study, effort was made to prospect the role of EcDehydrin7 protein from normalized cDNA library of drought tolerance finger millet in transgenic tobacco. Biochemical and molecular analyses of T0 transgenic plants were done for stress tolerance. Leaf disc assay, seed germination test, dehydration assay, and chlorophyll estimation showed EcDehydrin7 protein directly link to drought tolerance. Northern and qRT PCR analyses shows relatively high expression of EcDehydrin7 protein compare to wild type. T0 transgenic lines EcDehydrin7(11) and EcDehydrin7(15) shows superior expression among all lines under study. In summary, all results suggest that EcDehydrin7 protein has a remarkable role in drought tolerance and may be used for sustainable crop breeding program in other food crops.

  15. Potential Impacts of Pharmaceutical Uses of Transgenic Tobacco: The Case of Human Serum Albumin and Gaucher's Disease Treatment

    OpenAIRE

    Kostandini, Gentian

    2004-01-01

    This thesis examines the size and distribution of benefits from the use of transgenic tobacco as a production vehicle for pharmaceutical proteins. Ex-ante welfare benefits are estimated for the introduction of two biotech innovations. In both cases economic surplus model with imperfect competition is employed to assess the size and distribution of benefits from these alternative uses of tobacco. An introductory chapter presents an overview of the topic followed by chapters 2 and 3 which conta...

  16. Enhanced poly(3-hydroxybutyrate) production in transgenic tobacco BY-2 cells using engineered acetoacetyl-CoA reductase.

    Science.gov (United States)

    Yokoo, Toshinori; Matsumoto, Ken'ichiro; Ooba, Takashi; Morimoto, Kenjiro; Taguchi, Seiichi

    2015-01-01

    Highly active mutant of NADPH-dependent acetoacetyl-CoA reductase (PhaB) was expressed in Nicotiana tabacum cv. Bright Yellow-2 cultured cells to produce poly(3-hydroxybutyrate) [P(3HB)]. The mutated PhaB increased P(3HB) content by three-fold over the control, indicating that the mutant was a versatile tool for P(3HB) production. Additionally, the PhaB-catalyzed reaction was suggested to be a rate-limiting step of P(3HB) biosynthesis in tobacco BY-2 cells.

  17. The influence of EDDS and EDTA on the uptake of heavy metals of Cd and Cu from soil with tobacco Nicotiana tabacum.

    Science.gov (United States)

    Evangelou, Michael W H; Bauer, Uwe; Ebel, Mathias; Schaeffer, Andreas

    2007-06-01

    Phytoextraction, the use of plants to extract contaminants from soils and groundwater, is a promising approach for cleaning up soils contaminated with heavy metals. In order to enhance phytoextraction the use of chelating agents has been proposed. This study aims to assess whether ethylene diamine disuccinate (EDDS), a biodegradable chelator, can be used for enhanced phytoextraction purposed, as an alternative to ethylene diamine tetraacetate (EDTA). EDDS revealed a higher toxicity to tobacco (Nicotiana tabacum) in comparison to EDTA, but no toxicity to microorganisms. The uptake of Cu was increased by the addition of EDTA and EDDS, while no increase was observed in the uptake of Cd. Both chelating agents showed a very low root to shoot translocation capability and the translocation factor was lower than the one of the control. Heavy metals where significantly more phytoavailable than in the control, even after harvesting, resulting in a high heavy metal leaching possibility, probably owing to a low biodegradation rate of EDDS. New seedlings which were transplanted into the EDDS treated pots 7d after the phytoextraction experiment, showed signs of necrosis and chlorosis, which resulted in a significantly lower biomass in comparison to the control. The seedlings on the EDTA treated pots showed no toxicity signs. Contrary to previous opinions the results of this study revealed the chelating agents EDTA and EDDS as unsuitable for enhanced phytoextraction using tobacco.

  18. Protective effect of oral administration of transgenic tobacco seeds against verocytotoxic Escherichia coli strain in piglets.

    Science.gov (United States)

    Rossi, Luciana; Dell'Orto, Vittorio; Vagni, Simona; Sala, Vittorio; Reggi, Serena; Baldi, Antonella

    2014-03-01

    The use of transgenic plants as delivery system for antigenic proteins is attractive for its simplicity and increases likelihood for local immune response at sites of infection. The aim of this study was to evaluate the protective effect of oral administration of tobacco seeds, expressing the FedA, the major protein of the F18 adhesive fimbriae, and B subunit of verocytotoxin, against verocytotoxin-producing E. coli (VTEC) strain in piglets. Forty-three early weaned piglets, were randomly divided into 4 experimental groups: 3 test groups and a control. Treatment groups orally received a bolus, with different dose of tobacco seeds on 0, 1, 2, 14 days post primary administration. After challenge, with 1*10(10) CFU of O138 Escherichia coli strain, piglets showed clinical scores significantly higher in the control group compared to orally immunized groups (P administration of recombinant tobacco seeds expressing antigenic proteins against VTEC strains can induce a protective effect against challenger strain in piglets.

  19. 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.

  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. Inheritance and effectiveness of two transgenes determining PVY resistance in progeny from crossing independently transformed tobacco lines.

    Science.gov (United States)

    Czubacka, Anna; Sacco, Ermanno; Olszak-Przybyś, Hanna; Doroszewska, Teresa

    2017-05-01

    Genetic transformation of plants allows us to obtain improved genotypes enriched with the desired traits. However, if transgenic lines were to be used in breeding programs the stability of inserted transgenes is essential. In the present study, we followed the inheritance of transgenes in hybrids originated from crossing two transgenic tobacco lines resistant to Potato virus Y (PVY): MN 944 LMV with the transgene containing Lettuce mosaic virus coat protein gene (LMV CP) and AC Gayed ROKY2 with PVY replicase gene (ROKY2). Progeny populations generated by successive self-pollination were analyzed with respect to the transgene segregation ratio and resistance to Potato virus Y in tests carried out under greenhouse conditions. The presence of the virus in inoculated plants was detected by DAS-ELISA method. The results demonstrated the Mendelian fashion of inheritance of transgenes which were segregated independently and stably. As a result, we obtained T 4 generation of hybrid with both transgenes stacked and which was highly resistant to PVY.

  2. Genetically pyramiding protease-inhibitor genes for dual broad-spectrum resistance against insect and phytopathogens in transgenic tobacco.

    Science.gov (United States)

    Senthilkumar, Rajendran; Cheng, Chiu-Ping; Yeh, Kai-Wun

    2010-01-01

    Protease inhibitors provide a promising means of engineering plant resistance against attack by insects and pathogens. Sporamin (trypsin inhibitor) from sweet potato and CeCPI (phytocystatin) from taro were stacked in a binary vector, using pMSPOA (a modified sporamin promoter) to drive both genes. Transgenic tobacco lines of T0 and T1 generation with varied inhibitory activity against trypsin and papain showed resistance to both insects and phytopathogens. Larvae of Helicoverpa armigera that ingested tobacco leaves either died or showed delayed growth and development relative to control larvae. Transgenic tobacco-overexpressing the stacked genes also exhibited strong resistance against bacterial soft rot disease caused by Erwinia carotovora and damping-off disease caused by Pythium aphanidermatum. Thus, stacking protease-inhibitor genes, driven by the wound and pathogen responsive pMSPOA promoter, is an effective strategy for engineering crops to resistance against insects and phytopathogens.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Overexpression of the Wheat Expansin Gene TaEXPA2 Improved Seed Production and Drought Tolerance in Transgenic Tobacco Plants.

    Science.gov (United States)

    Chen, Yanhui; Han, Yangyang; Zhang, Meng; Zhou, Shan; Kong, Xiangzhu; Wang, Wei

    2016-01-01

    Expansins are cell wall proteins that are grouped into two main families, α-expansins and β-expansins, and they are implicated in the control of cell extension via the disruption of hydrogen bonds between cellulose and matrix glucans. TaEXPA2 is an α-expansin gene identified in wheat. Based on putative cis-regulatory elements in the TaEXPA2 promoter sequence and the expression pattern induced when polyethylene glycol (PEG) is used to mimic water stress, we hypothesized that TaEXPA2 is involved in plant drought tolerance and plant development. Through transient expression of 35S::TaEXPA2-GFP in onion epidermal cells, TaEXPA2 was localized to the cell wall. Constitutive expression of TaEXPA2 in tobacco improved seed production by increasing capsule number, not seed size, without having any effect on plant growth patterns. The transgenic tobacco exhibited a significantly greater tolerance to water-deficiency stress than did wild-type (WT) plants. We found that under drought stress, the transgenic plants maintained a better water status. The accumulated content of osmotic adjustment substances, such as proline, in TaEXPA2 transgenic plants was greater than that in WT plants. Transgenic plants also displayed greater antioxidative competence as indicated by their lower malondialdehyde (MDA) content, relative electrical conductivity, and reactive oxygen species (ROS) accumulation than did WT plants. This result suggests that the transgenic plants suffer less damage from ROS under drought conditions. The activities of some antioxidant enzymes as well as expression levels of several genes encoding key antioxidant enzymes were higher in the transgenic plants than in the WT plants under drought stress. Collectively, our results suggest that ectopic expression of the wheat expansin gene TaEXPA2 improves seed production and drought tolerance in transgenic tobacco plants.

  5. Ectopic Expression of JcWRKY Confers Enhanced Resistance in Transgenic Tobacco Against Macrophomina phaseolina.

    Science.gov (United States)

    Agarwal, Parinita; Patel, Khantika; Agarwal, Pradeep K

    2018-04-01

    Plants possess an innate immune system comprising of a complex network of closely regulated defense responses involving differential gene expression mediated by transcription factors (TFs). The WRKYs comprise of an important plant-specific TF family, which is involved in regulation of biotic and abiotic defenses. The overexpression of JcWRKY resulted in improved resistance in transgenic tobacco against Macrophomina phaseolina. The production of reactive oxygen species (ROS) and its detoxification through antioxidative system in the transgenics facilitates defense against Macrophomina. The enhanced catalase activity on Macrophomina infection limits the spread of infection. The transcript expression of antioxidative enzymes gene (CAT and SOD) and salicylic acid (SA) biosynthetic gene ICS1 showed upregulation during Macrophomina infection and combinatorial stress. The enhanced transcript of pathogenesis-related genes PR-1 indicates the accumulation of SA during different stresses. The PR-2 and PR-5 highlight the activation of defense responses comprising of activation of hydrolytic cleavage of glucanases and thaumatin-like proteins causing disruption of fungal cells. The ROS homeostasis in coordination with signaling molecules regulate the defense responses and inhibit fungal growth.

  6. Wheat bran soil inoculant of sumateran nematode-trapping fungi as biocontrol agents of the root-knot nematode meloidogyne incognita on deli tobacco (nicotiana tabaccum l) cv. deli 4

    Science.gov (United States)

    Dwi Sri Hastuti, Liana; Faull, Jane

    2018-03-01

    A pot experiment was carried out to test the effectiveness of nematode-trapping fungi (NTF) isolated from Sumatera for controlling infection by the root-knot nematode (RKN) on Deli tobacco plant. Wheat bran soil containing 109 conidia of Arthrobotrys. oligospora, Candellabrella musiformis and Dactylella eudermata was added to the soil as a dry inoculum. Carbofuran was also applied as chemical agent and comparison treatment. Seedling tobacco (Nicotiana tabacum L.) cv. Deli 4 was inoculated with root knot (Meloidogyne incognita Chitwood.) seven days after the plant were transplanted to the pots. A. oligospora, C. musiformis and D. eudermata were found to be reliable as biocontrol agents, reducing the number of vermiform nematodes, swollen root, sausage shaped and galls in tobacco plant after 7, 15 and 30 days of infection with M. incognita. Treatment with NTF produced results that were comparable with Carbofuran® as a control agent in the reduction of the number of infections in tobacco plant caused by M. incognita in Nicotiana tabacum var. Deli 4. They also optimize the growth of the tobacco plants especially up to 15 days after infection.

  7. 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.

  8. Utilization of a tobacco rattle virus vector to clone an Nicotiana benthamiana cDNA library for VIGS

    Science.gov (United States)

    Virus-induced gene silencing (VIGS) is an efficient and rapid method to identify plant gene functions. One of the most widely used VIGS vectors is Tobacco rattle virus (TRV) which has been used successfully for RNA interference (RNAi) in N. benthamiana and tomato. We previously modified a TRV VIGS v...

  9. Transient Expression of Lumbrokinase (PI239 in Tobacco (Nicotiana tabacum Using a Geminivirus-Based Single Replicon System Dissolves Fibrin and Blood Clots

    Directory of Open Access Journals (Sweden)

    Alexia Dickey

    2017-01-01

    Full Text Available Lumbrokinases, a group of fibrinolytic enzymes extracted from earthworm, have been widely used to prevent and treat various cardiovascular diseases. They specifically target fibrin to effectively degrade thrombi without major side effects. Plant expression systems are becoming potential alternative expression platforms for producing pharmaceutical proteins. In this work, a lumbrokinase (PI239 was produced from a plant system. Both wild-type (WT and plant codon-optimized (OP PI239 gene sequences were synthesized and cloned into a geminivirus-based single-vector DNA replicon system. Both vectors were independently expressed in tobacco (Nicotiana tabacum leaves transiently by agroinfiltration. Overexpressed PI239 resulted in sudden tissue necrosis 3 days after infiltration. Remaining proteins were purified through His-tag affinity chromatography and analyzed with SDS-PAGE and Western blot methods. Purified PI239 successfully degraded artificial fibrin with relative activity of 13,400 U/mg when compared with commercial lumbrokinase product. In vitro tests demonstrated that plant-derived PI239 dissolved human blood clots and that the plant expression system is capable of producing functional PI239.

  10. Effect of vermicompost on some physiological attributes involved in carbon and nitrogen metabolism as well as nutrient status in leaves of tobacco (nicotiana tabacum L.)

    International Nuclear Information System (INIS)

    Qin, C.; Zheng, P.; Akram, N.A.

    2016-01-01

    A pot experiment was carried out to examine the influence of vermicompost application on some key enzymes and metabolites involved in carbon (C) and nitrogen (N) metabolism as well as nutrient status in the leaves of tobacco (Nicotiana tabacum L.). Two types of vermicompost with two application rates were used in this study. Regardless of application rate, both types of vermicompost significantly increased total N, phosphorus (P) and potassium (K) contents in the leaves. They also caused enhancements in contents of total soluble carbohydrates, reducing sugars, starch and total organic C as well as amylase and invertase activities involved in C metabolism, contents of soluble protein and nicotine in N metabolism in the leaves. With an increase in application rate, each vermicompost type had an increasing effect on almost all measured parameters except nitrate reductase activity. Regardless of vermicompost type, the high rate (50%) of application showed the best effects compared with controls. The effects of V1 type vermicompost were superior to those of V2 at the same application rate. Therefore, the above effects might appear to be dependent on both type and dose. Vermicompost could be considered as an effective organic matter for attaining improved plant nutrition as well as C and N metabolism. (author)

  11. Genome-wide identification, subcellular localization and gene expression analysis of the members of CESA gene family in common tobacco (Nicotiana tabacum L.).

    Science.gov (United States)

    Xu, Zong-Chang; Kong, Yingzhen

    2017-06-20

    Cellulose-synthase proteins (CESAs) are membrane localized proteins and they form protein complexes to produce cellulose in the plasma membrane. CESA proteins play very important roles in cell wall construction during plant growth and development. In this study, a total of 21 NtCESA gene sequences were identified by using PF03552 conserved protein sequence and 10 AtCESA protein sequences of Arabidopsis thaliana to blast against the common tobacco (Nicotiana tabacum L.) genome database with TBLASTN protocol. We analyzed the physical and chemical properties of protein sequences based on some software or on-line analysis tools. The results showed that there were no significant variances in terms of the physical and chemical properties of the 21 NtCESA proteins. First, phylogenetic tree analysis showed that 21 NtCESA genes and 10 AtCESA genes were clustered into five groups, and the gene structures were similar among the genes that are clustered into the same group. Second, in all of the 21 NtCESA proteins the conserved zinc finger domain was identified in the N-terminus, transmembrane domains were identified in the C-terminus and the DDD-QXXRW conserved domains were also identified. Third, gene expression analysis results indicated that most NtCESA genes were expressed in roots and leaves of seedling or mature tissues of tobacco, seeds and callus tissues. The genes that clustered into the same group share similar expression patterns. Importantly, NtCESA proteins that are involved in secondary cell wall cellulose synthesis have two extra transmembrane domains compared with that involved in primary cell wall cellulose biosynthesis. In addition, subcellular localization results showed that NtCESA9 and NtCESA14 were two plasma membrane anchored proteins. This study will lay a foundation for further functional characterization of these NtCESA genes.

  12. Over-expression of SlJA2 decreased heat tolerance of transgenic tobacco plants via salicylic acid pathway.

    Science.gov (United States)

    Liu, Zhong-Ming; Yue, Meng-Meng; Yang, Dong-Yue; Zhu, Shao-Bo; Ma, Na-Na; Meng, Qing-Wei

    2017-04-01

    Over-expression of SlJA2 decreased the accumulation of SA, which resulted in significant physiological and gene expression changes in transgenic tobacco plants, leading to the decreased heat tolerance of transgenic tobacco. NAC family, the largest transcription factors in plants, responses to different environmental stimuli. Here, we isolated a typical NAC transcription factor (SlJA2) from tomato and got transgenic tobacco with SlJA2 over-expression. Expression of SlJA2 was induced by heat stress (42 °C), chilling stress (4 °C), drought stress, osmotic stress, abscisic acid, and salicylic acid. Over-expression of SlJA2 decreased the accumulation of salicylic acid by regulating expression of salicylic acid degradation gene under heat stress. Compared to WT plants, stomatal apertures and water loss increased in transgenic plants, and the damage of photosynthetic apparatus and chlorophyll breakdown were more serious in transgenic plants under heat stress. Meanwhile, more H 2 O 2 and O 2 ·- were accumulated transgenic plants and proline synthesis was restricted, which resulted in more serious oxidative damage compared to WT. qRT-PCR analysis showed that over-expression of SlJA2 could down-regulate genes involved in reactive oxygen species scavenging, proline biosynthesis, and response to heat stress. All the above results indicated that SlJA2 may be a negative regulator responded to plant's heat tolerance. Thus, this study provides new insight into roles of NAC family member in plant response to abiotic stress.

  13. Membrane topology of Golgi-localized probable S-adenosylmethionine-dependent methyltransferase in tobacco (Nicotiana tabacum) BY-2 cells.

    Science.gov (United States)

    Liu, Jianping; Hayashi, Kyoko; Matsuoka, Ken

    2015-01-01

    S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) transfer methyl groups to substrates. In this study, a novel putative tobacco SAM-MTase termed Golgi-localized methyl transferase 1 (GLMT1) has been characterized. GLMT1 is comprised of 611 amino acids with short N-terminal region, putative transmembrane region, and C-terminal SAM-MTase domain. Expression of monomeric red fluorescence protein (mRFP)-tagged protein in tobacco BY-2 cell indicated that GLMT1 is a Golgi-localized protein. Analysis of the membrane topology by protease digestion suggested that both C-terminal catalytic region and N-terminal region seem to be located to the cytosolic side of the Golgi apparatus. Therefore, GLMT1 might have a different function than the previously studied SAM-MTases in plants.

  14. Degradation of Red Ferralitic (Rhodic Ferralsol soils grown with tobacco (Nicotiana tabacum L. in the Artemisa province, Cuba

    Directory of Open Access Journals (Sweden)

    Óscar Ricote Jorge

    2017-01-01

    Full Text Available “Partido” is a tobacco growing area which extends for some 3000 hectares among the municipalities of San Antonio de los Baños, Güira de Melena and Alquízar in the Cuban province of Artemisa. Predominant soils are Red Ferralitic (Rhodic Ferralsol according to the World Reference Base, with a strong tendency to alkalinization which has a negative impact on the quality of their agricultural use. The aim of this research was to quantify the geographical extension of the degradation process, to determine how deep it happens along the soil profile and to establish its possible relationship with the quality and quantity of water applied to tobacco fields. The chemical, physical and mineralogical analyses of two test pits carried out in the area were compared: one profile without agricultural use with one characteristic soil profile under continuous production. After being subjected to the same irrigation regime in laboratory conditions, it was concluded that degradation affects to 89.56% of the area of tobacco soils evaluated. This phenomenon occurs very deeply along the soil profile and happens downwards, causing the accumulation of calcium and the loss of sodium and potassium in the superficial horizon, what is shown in pH rises. Such processes, associated to irrigation water and to insufficient rainfall regime which are traditional in the territory, have led to changes in the mineralogical composition of these tobacco soils appearance of minerals such as gibbsite which was absent in uncultivated Red Ferralitic soils, which involve the modification of soil classification at gender level.

  15. Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation

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    Burgos Lorenzo

    2010-07-01

    Full Text Available Abstract Background The routine generation of transgenic plants involves analysis of transgene integration into the host genome by means of Southern blotting. However, this technique cannot distinguish between uniformly transformed tissues and the presence of a mixture of transgenic and non-transgenic cells in the same tissue. On the other hand, the use of reporter genes often fails to accurately detect chimerical tissues because their expression can be affected by several factors, including gene silencing and plant development. So, new approaches based on the quantification of the amount of the transgene are needed urgently. Results We show here that chimeras are a very frequent phenomenon observed after regenerating transgenic plants. Spatial and temporal analyses of transformed tobacco and apricot plants with a quantitative, real-time PCR amplification of the neomycin phosphotransferase (nptII transgene as well as of an internal control (β-actin, used to normalise the amount of target DNA at each reaction, allowed detection of chimeras at unexpected rates. The amount of the nptII transgene differed greatly along with the sub-cultivation period of these plants and was dependent on the localisation of the analysed leaves; being higher in roots and basal leaves, while in the apical leaves it remained at lower levels. These data demonstrate that, unlike the use of the gus marker gene, real-time PCR is a powerful tool for detection of chimeras. Although some authors have proposed a consistent, positive Southern analysis as an alternative methodology for monitoring the dissociation of chimeras, our data show that it does not provide enough proof of uniform transformation. In this work, however, real-time PCR was applied successfully to monitor the dissociation of chimeras in tobacco plants and apricot callus. Conclusions We have developed a rapid and reliable method to detect and estimate the level of chimeras in transgenic tobacco and apricot

  16. Generation of Resistance to the Diphenyl Ether Herbicide, Oxyfluorfen, via Expression of the Bacillus subtilis Protoporphyrinogen Oxidase Gene in Transgenic Tobacco Plants.

    Science.gov (United States)

    Choi, K W; Han, O; Lee, H J; Yun, Y C; Moon, Y H; Kim, M; Kuk, Y I; Han, S U; Guh, J O

    1998-01-01

    In an effort to develop transgenic plants resistant to diphenyl ether herbicides, we introduced the protoporphyrinogen oxidase (EC 1.3.3.4) gene of Bacillus subtilis into tobacco plants. The results from a Northern analysis and leaf disc assay indicate that the expression of the B. subtilis protoporphyrinogen oxidase gene under the cauliflower mosaic virus 35S promoter generated resistance to the diphenyl ether herbicide, oxyfluorfen, in transgenic tobacco plants.

  17. An Alcohol Dehydrogenase Gene from Synechocystis sp. Confers Salt Tolerance in Transgenic Tobacco

    Directory of Open Access Journals (Sweden)

    So Young Yi

    2017-11-01

    Full Text Available Synechocystis salt-responsive gene 1 (sysr1 was engineered for expression in higher plants, and gene construction was stably incorporated into tobacco plants. We investigated the role of Sysr1 [a member of the alcohol dehydrogenase (ADH superfamily] by examining the salt tolerance of sysr1-overexpressing (sysr1-OX tobacco plants using quantitative real-time polymerase chain reactions, gas chromatography-mass spectrometry, and bioassays. The sysr1-OX plants exhibited considerably increased ADH activity and tolerance to salt stress conditions. Additionally, the expression levels of several stress-responsive genes were upregulated. Moreover, airborne signals from salt-stressed sysr1-OX plants triggered salinity tolerance in neighboring wild-type (WT plants. Therefore, Sysr1 enhanced the interconversion of aldehydes to alcohols, and this occurrence might affect the quality of green leaf volatiles (GLVs in sysr1-OX plants. Actually, the Z-3-hexenol level was approximately twofold higher in sysr1-OX plants than in WT plants within 1–2 h of wounding. Furthermore, analyses of WT plants treated with vaporized GLVs indicated that Z-3-hexenol was a stronger inducer of stress-related gene expression and salt tolerance than E-2-hexenal. The results of the study suggested that increased C6 alcohol (Z-3-hexenol induced the expression of resistance genes, thereby enhancing salt tolerance of transgenic plants. Our results revealed a role for ADH in salinity stress responses, and the results provided a genetic engineering strategy that could improve the salt tolerance of crops.

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

    Directory of Open Access Journals (Sweden)

    Fabio Castelli

    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.

  19. Constitutive expression of nitrate reductase allows normal growth and development of Nicotiana plumbaginifolia plants.

    Science.gov (United States)

    Vincentz, M; Caboche, M

    1991-01-01

    A nitrate reductase (NR) deficient mutant of Nicotiana plumbaginifolia totally impaired in the production of NR transcript and protein was restored for NR activity by transformation with a chimaeric NR gene. This gene was composed of a full-length tobacco NR cDNA fused to the CaMV 35S promoter and to termination signals from the tobacco NR gene. The transgenic plants we obtained were viable and fertile and expressed from one-fifth to three times the wild-type NR activity in their leaves. The analysis of chimeric NR gene expression in these plants showed, by comparison with wild-type plants, that the regulation of NR gene expression by light, nitrate and circadian rhythm takes place at the transcriptional level. However, unlike nitrate, light was required for the accumulation of NR protein in transgenic plants, suggesting that NR expression is also controlled at the translational and/or post-translational level. Images PMID:2022181

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

    Directory of Open Access Journals (Sweden)

    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. Expression of a pathogen-induced cysteine protease (AdCP) in tapetum results in male sterility in transgenic tobacco.

    Science.gov (United States)

    Shukla, Pawan; Singh, Naveen Kumar; Kumar, Dilip; Vijayan, Sambasivam; Ahmed, Israr; Kirti, Pulugurtha Bharadwaja

    2014-06-01

    Usable male sterility systems have immense potential in developing hybrid varieties in crop plants, which can also be used as a biological safety containment to prevent horizontal transgene flow. Barnase-Barstar system developed earlier was the first approach to engineer male sterility in plants. In an analogous situation, we have evolved a system of inducing pollen abortion and male sterility in transgenic tobacco by expressing a plant gene coding for a protein with known developmental function in contrast to the Barnase-Barstar system, which deploys genes of prokaryotic origin, i.e., from Bacillus amyloliquefaciens. We have used a plant pathogen-induced gene, cysteine protease for inducing male sterility. This gene was identified in the wild peanut, Arachis diogoi differentially expressed when it was challenged with the late leaf spot pathogen, Phaeoisariopsis personata. Arachis diogoi cysteine protease (AdCP) was expressed under the strong tapetum-specific promoter (TA29) and tobacco transformants were generated. Morphological and histological analysis of AdCP transgenic plants showed ablated tapetum and complete pollen abortion in three transgenic lines. Furthermore, transcript analysis displayed the expression of cysteine protease in these male sterile lines and the expression of the protein was identified in western blot analysis using its polyclonal antibody raised in the rabbit system.

  2. Expression of the Native Cholera Toxin B Subunit Gene and Assembly as Functional Oligomers in Transgenic Tobacco Chloroplasts

    Science.gov (United States)

    Daniell, Henry; Lee, Seung-Bum; Panchal, Tanvi; Wiebe, Peter O.

    2012-01-01

    The B subunits of enterotoxigenic Escherichia coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are candidate vaccine antigens. Integration of an unmodified CTB-coding sequence into chloroplast genomes (up to 10,000 copies per cell), resulted in the accumulation of up to 4.1% of total soluble tobacco leaf protein as functional oligomers (410-fold higher expression levels than that of the unmodified LTB gene expressed via the nuclear genome). However, expresssion levels reported are an underestimation of actual accumulation of CTB in transgenic chloroplasts, due to aggregation of the oligomeric forms in unboiled samples similar to the aggregation observed for purified bacterial antigen. PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome. Western blot analysis showed that the chloroplast-synthesized CTB assembled into oligomers and were antigenically identical with purified native CTB. Also, binding assays confirmed that chloroplast- synthesized CTB binds to the intestinal membrane GM1-ganglioside receptor, indicating correct folding and disulfide bond formation of CTB pentamers within transgenic chloroplasts. In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed in chloroplasts. Introduced genes were inherited stably in subsequent generations, as confirmed by PCR and Southern blot analyses. Increased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant-based oral vaccines and fusion proteins with CTB needing oral administration commercially feasible. Successful expression of foreign genes in transgenic chromoplasts and availability of marker-free chloroplast transformation techniques augurs well for development of vaccines in edible parts of transgenic plants. Furthermore, since the quaternary structure of

  3. The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco.

    Science.gov (United States)

    Aharoni, A; De Vos, C H; Wein, M; Sun, Z; Greco, R; Kroon, A; Mol, J N; O'Connell, A P

    2001-11-01

    Fruit ripening is characterized by dramatic changes in gene expression, enzymatic activities and metabolism. Although the process of ripening has been studied extensively, we still lack valuable information on how the numerous metabolic pathways are regulated and co-ordinated. In this paper we describe the characterization of FaMYB1, a ripening regulated strawberry gene member of the MYB family of transcription factors. Flowers of transgenic tobacco lines overexpressing FaMYB1 showed a severe reduction in pigmentation. A reduction in the level of cyanidin 3-rutinoside (an anthocyanin) and of quercetin-glycosides (flavonols) was observed. Expression of late flavonoid biosynthesis genes and their enzyme activities were adversely affected by FaMYB1 overexpression. Two-hybrid assays in yeast showed that FaMYB1 could interact with other known anthocyanin regulators, but it does not act as a transcriptional activator. Interestingly, the C-terminus of FaMYB1 contains the motif pdLNL(D)/(E)Lxi(G)/S. This motif is contained in a region recently proposed to be involved in the repression of transcription by AtMYB4, an Arabidopsis MYB protein. Our results suggest that FaMYB1 may play a key role in regulating the biosynthesis of anthocyanins and flavonols in strawberry. It may act to repress transcription in order to balance the levels of anthocyanin pigments produced at the latter stages of strawberry fruit maturation, and/or to regulate metabolite levels in various branches of the flavonoid biosynthetic pathway.

  4. Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco

    DEFF Research Database (Denmark)

    Jach, G; Görnhardt, B; Mundy, J

    1995-01-01

    cDNAs encoding three proteins from barley (Hordeum vulgare), a class-II chitinase (CHI), a class-II beta-1,3-glucanase (GLU) and a Type-I ribosome-inactivating protein (RIP) were expressed in tobacco plants under the control of the CaMV 35S-promoter. High-level expression of the transferred genes...... was detected in the transgenic plants by Northern and Western blot analysis. The leader peptides in CHI and GLU led to accumulation of these proteins in the intercellular space of tobacco leaves. RIP, which is naturally deposited in the cytosol of barley endosperm cells, was expressed either in its original...... cytosolic form or fused to a plant secretion peptide (spRIP). Fungal infection assays revealed that expression of the individual genes in each case resulted in an increased protection against the soilborne fungal pathogen Rhizoctonia solani, which infects a range of plant species including tobacco...

  5. Cloning of Ammopiptanthus mongolicus C-repeat-binding factor gene and its cold-induced tolerance in transgenic tobacco

    Directory of Open Access Journals (Sweden)

    Lijiang Gu

    2013-12-01

    Full Text Available C-repeat-binding factors (CBFs are a type of important regulon in stress-related signal transduction pathways that control plant tolerance of abiotic stress. Ammopiptanthus mongolicus is the only evergreen broadleaf shrub in the northwest desert of China. The species shows strong resistance to environmental stress, especially to cold stress. An A. mongolicus CBF1 gene (AmCBF1 was cloned and transformed into tobacco. Expression of AmCBF1 could be detected in A. mongolicus shortly after exposure to low temperature of 4°C. Analysis on ratio of electrolytic leakage, soluble sugar content, free proline content, malondialdehyde (MDA content and peroxidase (POD activity before and after cold treatment (4°C for 24 h indicated AmCBF1 conferred higher cold tolerance to AmCBF1 transgenic tobacco compared with the wild type and empty vector transformed tobacco.

  6. High-efficiency Agrobacterium rhizogenes-mediated transformation of heat inducible sHSP18.2-GUS in Nicotiana tabacum.

    Science.gov (United States)

    Chen, Shih-Cheng; Liu, Hui-Wen; Lee, Kung-Ta; Yamakawa, Takashi

    2007-01-01

    The chimerical gene, Arabidopsis thaliana sHSP18.2 promoter fused to E. coli gusA gene, was Agrobacterium rhizogenes-mediated transformed into Nicotiana tabacum as a heat-regulatable model, and the thermo-inducible expression of GUS activity in N. tabacum transgenic hairy roots was profiled. An activation of A. rhizogenes with acetosyringone (AS) before cocultured with tobacco's leaf disc strongly promoted transgenic hairy roots formation. Transgenic hairy roots formation efficiency of A. rhizogenes precultured with 200 microM AS supplementation was 3.1-fold and 7.5-fold, respectively, compared to the formation efficiency obtained with and without AS supplementation in coculture. Transgenic hairy roots transformed with different AS concentration exhibited a similar pattern of thermo-inducibility after 10 min to 3 h heat treatments detected by GUS expression. The peak of expressed GUS specific activity, 399,530 pmol MUG per mg total protein per min, of the transgenic hairy roots was observed at 48 h after 3 h of 42 degrees C heat treatment, and the expressed GUS specific activity was 7-26 times more than that reported in A. thaliana, tobacco BY-2 cells and Nicotiana plumbaginifolia. Interference caused by AS supplementation on the growth of transgenic hairy roots, time-course of GUS expression and its expression level were not observed.

  7. Hyperactive mutant of a wheat plasma membrane Na+/H+ antiporter improves the growth and salt tolerance of transgenic tobacco.

    Science.gov (United States)

    Zhou, Yang; Lai, Zesen; Yin, Xiaochang; Yu, Shan; Xu, Yuanyuan; Wang, Xiaoxiao; Cong, Xinli; Luo, Yuehua; Xu, Haixia; Jiang, Xingyu

    2016-12-01

    Wheat SOS1 (TaSOS1) activity could be relieved upon deletion of the C-terminal 168 residues (the auto-inhibitory domain). This truncated form of wheat SOS1 (TaSOS1-974) was shown to increase compensation (compared to wild-type TaSOS1) for the salt sensitivity of a yeast mutant strain, AXT3K, via increased Na + transportation out of cells during salinity stress. Expression of the plasma membrane proteins TaSOS1-974 or TaSOS1 improved the growth of transgenic tobacco plants compared with wild-type plants under normal conditions. However, plants expressing TaSOS1-974 grew better than TaSOS1-transformed plants. Upon salinity stress, Na + efflux and K + influx rates in the roots of transgenic plants expressing TaSOS1-974 or TaSOS1 were greater than those of wild-type plants. Furthermore, compared to TaSOS1-transgenic plants, TaSOS1-974-expressing roots showed faster Na + efflux and K + influx, resulting in less Na + and more K + accumulation in TaSOS1-974-transgenic plants compared to TaSOS1-transgenic and wild-type plants. TaSOS1-974-expressing plants had the lowest MDA content and electrolyte leakage among all tested plants, indicating that TaSOS1-974 might protect the plasma membrane against oxidative damage generated by salt stress. Overall, TaSOS1-974 conferred higher salt tolerance in transgenic plants compared to TaSOS1. Consistent with this result, transgenic plants expressing TaSOS1-974 showed a better growth performance than TaSOS1-expressing and wild-type plants under saline conditions. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  8. GhZFP1, a novel CCCH-type zinc finger protein from cotton, enhances salt stress tolerance and fungal disease resistance in transgenic tobacco by interacting with GZIRD21A and GZIPR5.

    Science.gov (United States)

    Guo, Ying-Hui; Yu, Yue-Ping; Wang, Dong; Wu, Chang-Ai; Yang, Guo-Dong; Huang, Jin-Guang; Zheng, Cheng-Chao

    2009-01-01

    * Zinc finger proteins are a superfamily involved in many aspects of plant growth and development. However, CCCH-type zinc finger proteins involved in plant stress tolerance are poorly understood. * A cDNA clone designated Gossypium hirsutum zinc finger protein 1 (GhZFP1), which encodes a novel CCCH-type zinc finger protein, was isolated from a salt-induced cotton (G. hirsutum) cDNA library using differential hybridization screening and further studied in transgenic tobacco Nicotiana tabacum cv. NC89. Using yeast two-hybrid screening (Y2H), proteins GZIRD21A (GhZFP1 interacting and responsive to dehydration protein 21A) and GZIPR5 (GhZFP1 interacting and pathogenesis-related protein 5), which interacted with GhZFP1, were isolated. * GhZFP1 contains two typical zinc finger motifs (Cx8Cx5Cx3H and Cx5Cx4Cx3H), a putative nuclear export sequence (NES) and a potential nuclear localization signal (NLS). Transient expression analysis using a GhZFP1::GFP fusion gene in onion epidermal cells indicated a nuclear localization for GhZFP1. RNA blot analysis showed that the GhZFP1 transcript was induced by salt (NaCl), drought and salicylic acid (SA). The regions in GhZFP1 that interact with GZIRD21A and GZIPR5 were identified using truncation mutations. * Overexpression of GhZFP1 in transgenic tobacco enhanced tolerance to salt stress and resistance to Rhizoctonia solani. Therefore, it appears that GhZFP1 might be involved as an important regulator in plant responses to abiotic and biotic stresses.

  9. Transgenic tobacco plants carrying the non-structural P3 gene of potato virus A

    Czech Academy of Sciences Publication Activity Database

    Nováková, S.; Mazúrová, Ľ.; Čeřovská, Noemi; šubr, Z. W.

    2005-01-01

    Roč. 49, č. 4 (2005), s. 593-598 ISSN 0006-3134 Institutional research plan: CEZ:AV0Z5038910 Keywords : potyvirus * Agrobacterium tumefaciens * Nicotiana tabacum Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.792, year: 2005

  10. Determination of content of metallothionein and low molecular mass stress peptides in transgenic tobacco plants

    Czech Academy of Sciences Publication Activity Database

    Diopan, V.; Shestivska, V.; Adam, V.; Macek, Tomáš; Macková, M.; Havel, L.; Kizek, R.

    2008-01-01

    Roč. 94, č. 3 (2008), s. 291-298 ISSN 0167-6857 R&D Projects: GA MŠk 1M06030 Institutional research plan: CEZ:AV0Z40550506 Keywords : metallothionein * Nicotiana tabacum * thiols * phytoremediation Subject RIV: EI - Biotechnology ; Bionics Impact factor: 1.017, year: 2008

  11. Effect of potassium application in drought-stressed tobacco (Nicotiana rustica L. plants: Comparison of root with foliar application

    Directory of Open Access Journals (Sweden)

    Sara Bahrami-Rad

    2017-12-01

    Full Text Available Effect of potassium (K application through leaves (LA or roots (RA was studied in tobacco plants grown under K deficiency and drought stress conditions. Application of K was effective in improving the shoot growth only under drought conditions, whereas root biomass and length responded under both watering regimes. Under drought conditions, photosynthesis and transpiration activities increased upon K application leading to a reduced water use efficiency. Both RA and LA increased the leaf water potential, relative water content and turgor under both well-watered and drought conditions; RA was more effective than LA in the recovery of leaf turgor. Analyses of water relation parameters in different aged leaves showed lower susceptibility of the middle-aged leaves to both K deficiency and drought stresses than the upper and lower leaves; this phenomenon was accompanied by a more conservative control of water loss in the middle-aged leaves. In contrast, proline was accumulated in the young leaves, and K application increased it further. Although various organic osmolytes were accumulated under the combinative effect of K deficiency and drought stress, they did not exceed the amounts found in the control (well-watered +K plants and were merely a result of the concentration effect. Collectively, our results revealed that the majority of leaf biochemical responses to drought stress are developmentally regulated processes. In addition, the alleviating effect of both RA and LA despite higher water loss indicated that an improved stomatal function upon K application allowed carbohydrates synthesis, thus, enhancing plant growth under water stress.

  12. Over-expression of a Rab family GTPase from phreatophyte Prosopis juliflora confers tolerance to salt stress on transgenic tobacco.

    Science.gov (United States)

    George, Suja; Parida, Ajay

    2011-03-01

    Plant growth and productivity are adversely affected by various abiotic and biotic stress factors. In our previous study, we used Prosopis juliflora, an abiotic stress tolerant tree species of Fabaceae, as a model plant system for isolating genes functioning in abiotic stress tolerance. Here we report the isolation and characterization of a Rab family GTPase from P. juliflora (Pj Rab7) and the ability of this gene to confer salt stress tolerance in transgenic tobacco. Northern analysis for Pj Rab7 in P. juliflora leaf tissue revealed up-regulation of this gene under salt stress under the concentrations and time points analyzed. Pj Rab7 transgenic tobacco lines survived better under conditions of 150 mM NaCl stress compared to control un-transformed plants. Pj Rab7 transgenic plants were found to accumulate more sodium than control plants during salt stress. The results of our studies could be used as a starting point for generation of crop plants tolerant to abiotic stress.

  13. Cytoprotective effect of recombinant human erythropoietin produced in transgenic tobacco plants.

    Directory of Open Access Journals (Sweden)

    Farooqahmed S Kittur

    Full Text Available Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPO(M by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and β1,4-galactosyltransferase (GalT genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene (GapC promoters, respectively. Plant-produced asialo-rhuEPO (asialo-rhuEPO(P was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS/MS revealed that asialo-rhuEPO(P bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPO(P (20 U/ml provides 2-fold better cytoprotection (44% to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPO(M (21%. The cytoprotective effect of the asialo-rhuEPO(P was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 (JAK2 and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production.

  14. Simultaneous Expression of PDH45 with EPSPS Gene Improves Salinity and Herbicide Tolerance in Transgenic Tobacco Plants.

    Science.gov (United States)

    Garg, Bharti; Gill, Sarvajeet S; Biswas, Dipul K; Sahoo, Ranjan K; Kunchge, Nandkumar S; Tuteja, Renu; Tuteja, Narendra

    2017-01-01

    To cope with the problem of salinity- and weed-induced crop losses, a multi-stress tolerant trait is need of the hour but a combinatorial view of such traits is not yet explored. The overexpression of PDH45 (pea DNA helicase 45) and EPSPS (5-enoylpruvyl shikimate-3-phosphate synthase) genes have been reported to impart salinity and herbicide tolerance. Further, the understanding of mechanism and pathways utilized by PDH45 and EPSPS for salinity and herbicide tolerance will help to improve the crops of economical importance. In the present study, we have performed a comparative analysis of salinity and herbicide tolerance to check the biochemical parameters and antioxidant status of tobacco transgenic plants. Collectively, the results showed that PDH45 overexpressing transgenic lines display efficient tolerance to salinity stress, while PDH45+EPSPS transgenics showed tolerance to both the salinity and herbicide as compared to the control [wild type (WT) and vector control (VC)] plants. The activities of the components of enzymatic antioxidant machinery were observed to be higher in the transgenic plants indicating the presence of an efficient antioxidant defense system which helps to cope with the stress-induced oxidative-damages. Photosynthetic parameters also showed significant increase in PDH45 and PDH45+EPSPS overexpressing transgenic plants in comparison to WT, VC and EPSPS transgenic plants under salinity stress. Furthermore, PDH45 and PDH45+EPSPS synergistically modulate the jasmonic acid and salicylic acid mediated signaling pathways for combating salinity stress. The findings of our study suggest that pyramiding of the PDH45 gene with EPSPS gene renders host plants tolerant to salinity and herbicide by enhancing the antioxidant machinery thus photosynthesis.

  15. Ectopic expression of wheat expansin gene TaEXPA2 improved the salt tolerance of transgenic tobacco by regulating Na+ /K+ and antioxidant competence.

    Science.gov (United States)

    Chen, Yanhui; Han, Yangyang; Kong, Xiangzhu; Kang, Hanhan; Ren, Yuanqing; Wang, Wei

    2017-02-01

    High salinity is one of the most serious environmental stresses that limit crop growth. Expansins are cell wall proteins that regulate plant development and abiotic stress tolerance by mediating cell wall expansion. We studied the function of a wheat expansin gene, TaEXPA2, in salt stress tolerance by overexpressing it in tobacco. Overexpression of TaEXPA2 enhanced the salt stress tolerance of transgenic tobacco plants as indicated by the presence of higher germination rates, longer root length, more lateral roots, higher survival rates and more green leaves under salt stress than in the wild type (WT). Further, when leaf disks of WT plants were incubated in cell wall protein extracts from the transgenic tobacco plants, their chlorophyll content was higher under salt stress, and this improvement from TaEXPA2 overexpression in transgenic tobacco was inhibited by TaEXPA2 protein antibody. The water status of transgenic tobacco plants was improved, perhaps by the accumulation of osmolytes such as proline and soluble sugar. TaEXPA2-overexpressing tobacco lines exhibited lower Na + but higher K + accumulation than WT plants. Antioxidant competence increased in the transgenic plants because of the increased activity of antioxidant enzymes. TaEXPA2 protein abundance in wheat was induced by NaCl, and ABA signaling was involved. Gene expression regulation was involved in the enhanced salt stress tolerance of the TaEXPA2 transgenic plants. Our results suggest that TaEXPA2 overexpression confers salt stress tolerance on the transgenic plants, and this is associated with improved water status, Na + /K + homeostasis, and antioxidant competence. ABA signaling participates in TaEXPA2-regulated salt stress tolerance. © 2016 Scandinavian Plant Physiology Society.

  16. TaCIPK29, a CBL-interacting protein kinase gene from wheat, confers salt stress tolerance in transgenic tobacco.

    Directory of Open Access Journals (Sweden)

    Xiaomin Deng

    Full Text Available Calcineurin B-like protein-interacting protein kinases (CIPKs have been found to be responsive to abiotic stress. However, their precise functions and the related molecular mechanisms in abiotic stress tolerance are not completely understood, especially in wheat. In the present study, TaCIPK29 was identified as a new member of CIPK gene family in wheat. TaCIPK29 transcript increased after NaCl, cold, methyl viologen (MV, abscisic acid (ABA and ethylene treatments. Over-expression of TaCIPK29 in tobacco resulted in increased salt tolerance, which was demonstrated by higher germination rates, longer root lengths and better growth status of transgenic tobacco plants compared to controls when both were treated with salt stress. Physiological measurements indicated that transgenic tobacco seedlings retained high K(+/Na(+ ratios and Ca(2+ content by up-regulating some transporter genes expression and also possessed lower H2O2 levels and reduced membrane injury by increasing the expression and activities of catalase (CAT and peroxidase (POD under salt stress. Moreover, transgenic lines conferred tolerance to oxidative stress by increasing the activity and expression of CAT. Finally, TaCIPK29 was located throughout cells and it preferentially interacted with TaCBL2, TaCBL3, NtCBL2, NtCBL3 and NtCAT1. Taken together, our results showed that TaCIPK29 functions as a positive factor under salt stress and is involved in regulating cations and reactive oxygen species (ROS homeostasis.

  17. The SbMT-2 gene from a halophyte confers abiotic stress tolerance and modulates ROS scavenging in transgenic tobacco.

    Directory of Open Access Journals (Sweden)

    Amit Kumar Chaturvedi

    Full Text Available Heavy metals are common pollutants of the coastal saline area and Salicornia brachiata an extreme halophyte is frequently exposed to various abiotic stresses including heavy metals. The SbMT-2 gene was cloned and transformed to tobacco for the functional validation. Transgenic tobacco lines (L2, L4, L6 and L13 showed significantly enhanced salt (NaCl, osmotic (PEG and metals (Zn++, Cu++ and Cd++ tolerance compared to WT plants. Transgenic lines did not show any morphological variation and had enhanced growth parameters viz. shoot length, root length, fresh weight and dry weight. High seed germination percentage, chlorophyll content, relative water content, electrolytic leakage and membrane stability index confirmed that transgenic lines performed better under salt (NaCl, osmotic (PEG and metals (Zn++, Cu++ and Cd++ stress conditions compared to WT plants. Proline, H2O2 and lipid peroxidation (MDA analyses suggested the role of SbMT-2 in cellular homeostasis and H2O2 detoxification. Furthermore in vivo localization of H2O2 and O2-; and elevated expression of key antioxidant enzyme encoding genes, SOD, POD and APX evident the possible role of SbMT-2 in ROS scavenging/detoxification mechanism. Transgenic lines showed accumulation of Cu++ and Cd++ in root while Zn++ in stem under stress condition. Under control (unstressed condition, Zn++ was accumulated more in root but accumulation of Zn++ in stem under stress condition suggested that SbMT-2 may involve in the selective translocation of Zn++ from root to stem. This observation was further supported by the up-regulation of zinc transporter encoding genes NtZIP1 and NtHMA-A under metal ion stress condition. The study suggested that SbMT-2 modulates ROS scavenging and is a potential candidate to be used for phytoremediation and imparting stress tolerance.

  18. Overexpression of the Maize Sulfite Oxidase Increases Sulfate and GSH Levels and Enhances Drought Tolerance in Transgenic Tobacco

    Directory of Open Access Journals (Sweden)

    Zongliang Xia

    2018-03-01

    Full Text Available Sulfite oxidase (SO plays a pivotal role in sulfite metabolism. In our previous study, sulfite-oxidizing function of the SO from Zea mays (ZmSO was characterized. To date, the knowledge of ZmSO’s involvement in abiotic stress response is scarce. In this study, we aimed to investigate the role of ZmSO in drought stress. The transcript levels of ZmSO were relatively high in leaves and immature embryos of maize plants, and were up-regulated markedly by PEG-induced water stress. Overexpression of ZmSO improved drought tolerance in tobacco. ZmSO-overexpressing transgenic plants showed higher sulfate and glutathione (GSH levels but lower hydrogen peroxide (H2O2 and malondialdehyde (MDA contents under drought stress, indicating that ZmSO confers drought tolerance by enhancing GSH-dependent antioxidant system that scavenged ROS and reduced membrane injury. In addition, the transgenic plants exhibited more increased stomatal response than the wild-type (WT to water deficit. Interestingly, application of exogenous GSH effectively alleviated growth inhibition in both WT and transgenic plants under drought conditions. qPCR analysis revealed that the expression of several sulfur metabolism-related genes was significantly elevated in the ZmSO-overexpressing lines. Taken together, these results imply that ZmSO confers enhanced drought tolerance in transgenic tobacco plants possibly through affecting stomatal regulation, GSH-dependent antioxidant system, and sulfur metabolism-related gene expression. ZmSO could be exploited for developing drought-tolerant maize varieties in molecular breeding.

  19. The SbMT-2 gene from a halophyte confers abiotic stress tolerance and modulates ROS scavenging in transgenic tobacco.

    Science.gov (United States)

    Chaturvedi, Amit Kumar; Patel, Manish Kumar; Mishra, Avinash; Tiwari, Vivekanand; Jha, Bhavanath

    2014-01-01

    Heavy metals are common pollutants of the coastal saline area and Salicornia brachiata an extreme halophyte is frequently exposed to various abiotic stresses including heavy metals. The SbMT-2 gene was cloned and transformed to tobacco for the functional validation. Transgenic tobacco lines (L2, L4, L6 and L13) showed significantly enhanced salt (NaCl), osmotic (PEG) and metals (Zn++, Cu++ and Cd++) tolerance compared to WT plants. Transgenic lines did not show any morphological variation and had enhanced growth parameters viz. shoot length, root length, fresh weight and dry weight. High seed germination percentage, chlorophyll content, relative water content, electrolytic leakage and membrane stability index confirmed that transgenic lines performed better under salt (NaCl), osmotic (PEG) and metals (Zn++, Cu++ and Cd++) stress conditions compared to WT plants. Proline, H2O2 and lipid peroxidation (MDA) analyses suggested the role of SbMT-2 in cellular homeostasis and H2O2 detoxification. Furthermore in vivo localization of H2O2 and O2-; and elevated expression of key antioxidant enzyme encoding genes, SOD, POD and APX evident the possible role of SbMT-2 in ROS scavenging/detoxification mechanism. Transgenic lines showed accumulation of Cu++ and Cd++ in root while Zn++ in stem under stress condition. Under control (unstressed) condition, Zn++ was accumulated more in root but accumulation of Zn++ in stem under stress condition suggested that SbMT-2 may involve in the selective translocation of Zn++ from root to stem. This observation was further supported by the up-regulation of zinc transporter encoding genes NtZIP1 and NtHMA-A under metal ion stress condition. The study suggested that SbMT-2 modulates ROS scavenging and is a potential candidate to be used for phytoremediation and imparting stress tolerance.

  20. Daya Insektisida Alami Kombinasi Perasan Umbi Gadung (Dioscorea Hispida Dennst ) Dan Ekstrak Tembakau ( Nicotiana Tabacum L) (the Natural Insecticide Capacity of Squeeze Combination of Cassava (Dioscoreahispida Dennst) and Tobacco's Extract (Nicotiana T)

    OpenAIRE

    Hasanah, Misroul; Tangkas, I Made; Sakung, Jamaluddin

    2012-01-01

    The research regarding the natural insecticide capacity of squeeze combination of cassava (dioscoreahispida Dennst) and tobacco's extract has been done. The purposes are to measure the capacity of squeeze combination of cassava and tobacco's extract as a natural insecticide and to determine the most effective combination as natural insecticide. The method was the laboratory experiments by using squeeze cassava and tobacco's extract with the comparison ratio (in mL) as follow; 0:100, 25:75, 50...

  1. Transgenic tobacco overexpressing Brassica juncea HMG-CoA synthase 1 shows increased plant growth, pod size and seed yield.

    Directory of Open Access Journals (Sweden)

    Pan Liao

    Full Text Available Seeds are very important not only in the life cycle of the plant but they represent food sources for man and animals. We report herein a mutant of 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS, the second enzyme in the mevalonate (MVA pathway that can improve seed yield when overexpressed in a phylogenetically distant species. In Brassica juncea, the characterisation of four isogenes encoding HMGS has been previously reported. Enzyme kinetics on recombinant wild-type (wt and mutant BjHMGS1 had revealed that S359A displayed a 10-fold higher enzyme activity. The overexpression of wt and mutant (S359A BjHMGS1 in Arabidopsis had up-regulated several genes in sterol biosynthesis, increasing sterol content. To quickly assess the effects of BjHMGS1 overexpression in a phylogenetically more distant species beyond the Brassicaceae, wt and mutant (S359A BjHMGS1 were expressed in tobacco (Nicotiana tabacum L. cv. Xanthi of the family Solanaceae. New observations on tobacco OEs not previously reported for Arabidopsis OEs included: (i phenotypic changes in enhanced plant growth, pod size and seed yield (more significant in OE-S359A than OE-wtBjHMGS1 in comparison to vector-transformed tobacco, (ii higher NtSQS expression and sterol content in OE-S359A than OE-wtBjHMGS1 corresponding to greater increase in growth and seed yield, and (iii induction of NtIPPI2 and NtGGPPS2 and downregulation of NtIPPI1, NtGGPPS1, NtGGPPS3 and NtGGPPS4. Resembling Arabidopsis HMGS-OEs, tobacco HMGS-OEs displayed an enhanced expression of NtHMGR1, NtSMT1-2, NtSMT2-1, NtSMT2-2 and NtCYP85A1. Overall, increased growth, pod size and seed yield in tobacco HMGS-OEs were attributed to the up-regulation of native NtHMGR1, NtIPPI2, NtSQS, NtSMT1-2, NtSMT2-1, NtSMT2-2 and NtCYP85A1. Hence, S359A has potential in agriculture not only in improving phytosterol content but also seed yield, which may be desirable in food crops. This work further demonstrates HMGS function in plant

  2. The SbASR-1 gene cloned from an extreme halophyte Salicornia brachiata enhances salt tolerance in transgenic tobacco.

    Science.gov (United States)

    Jha, Bhavanath; Lal, Sanjay; Tiwari, Vivekanand; Yadav, Sweta Kumari; Agarwal, Pradeep K

    2012-12-01

    Salinity severely affects plant growth and development. Plants evolved various mechanisms to cope up stress both at molecular and cellular levels. Halophytes have developed better mechanism to alleviate the salt stress than glycophytes, and therefore, it is advantageous to study the role of different genes from halophytes. Salicornia brachiata is an extreme halophyte, which grows luxuriantly in the salty marshes in the coastal areas. Earlier, we have isolated SbASR-1 (abscisic acid stress ripening-1) gene from S. brachiata using cDNA subtractive hybridisation library. ASR-1 genes are abscisic acid (ABA) responsive, whose expression level increases under abiotic stresses, injury, during fruit ripening and in pollen grains. The SbASR-1 transcript showed up-regulation under salt stress conditions. The SbASR-1 protein contains 202 amino acids of 21.01-kDa molecular mass and has 79 amino acid long signatures of ABA/WDS gene family. It has a maximum identity (73 %) with Solanum chilense ASR-1 protein. The SbASR-1 has a large number of disorder-promoting amino acids, which make it an intrinsically disordered protein. The SbASR-1 gene was over-expressed under CaMV 35S promoter in tobacco plant to study its physiological functions under salt stress. T(0) transgenic tobacco seeds showed better germination and seedling growth as compared to wild type (Wt) in a salt stress condition. In the leaf tissues of transgenic lines, Na(+) and proline contents were significantly lower, as compared to Wt plant, under salt treatment, suggesting that transgenic plants are better adapted to salt stress.

  3. Insect-resistance and high-yield transgenic tobacco obtained by ...

    African Journals Online (AJOL)

    The modified synthesized VHb gene and insectidal gene (GFMcryIA) were transferred to tobacco plants by Agrobacterium-mediated transformation. The bivalent genes were inserted successfully into the tobacco genome and detected by PCR amplification. Southern blot and Western blot analyses showed that VHb gene ...

  4. Expression of TaWRKY44, a wheat WRKY gene, in transgenic tobacco confers multiple abiotic stress tolerances

    Directory of Open Access Journals (Sweden)

    Xiatian eWang

    2015-08-01

    Full Text Available The WRKY transcription factors have been reported to be involved in various plant physiological and biochemical processes. In this study, we successfully assembled ten unigenes from expressed sequence tags (ESTs of wheat and designated them as TaWRKY44–TaWRKY53, respectively. Among these genes, a subgroup I gene, TaWRKY44, was found to be upregulated by treatments with PEG6000, NaCl, 4°C, abscisic acid (ABA, H2O2 and gibberellin (GA. The TaWRKY44-GFP fusion protein was localized to the nucleus of onion epidermal cells, and TaWRKY44 was able to bind to the core DNA sequences of TTGACC and TTAACC in yeast. The N-terminal of TaWRKY44 showed transcriptional activation activity. Expression of TaWRKY44 in tobacco plants conferred drought and salt tolerance and transgenic tobacco exhibited a higher survival rate, relative water content (RWC, soluble sugar, proline and superoxide dismutase (SOD content, as well as higher activities of catalase (CAT and peroxidase (POD, but less ion leakage (IL, lower contents of malondialdehyde (MDA, and H2O2. In addition, expression of TaWRKY44 also increased the seed germination rate in the transgenic lines under osmotic stress conditions while exhibiting a lower H2O2 content and higher SOD, CAT and POD activities. Expression of TaWRKY44 upregulated the expression of some reactive oxygen species (ROS-related genes and stress-responsive genes in tobacco under osmotic stresses. These data demonstrate that TaWRKY44 may act as a positive regulator in drought/salt/osmotic stress responses by either efficient ROS elimination through direct or indirect activation of the cellular antioxidant systems or activation of stress-associated gene expression.

  5. Overexpression of snapdragon Delila (Del) gene in tobacco enhances anthocyanin accumulation and abiotic stress tolerance

    OpenAIRE

    Naing, Aung Htay; Park, Kyeung Il; Ai, Trinh Ngoc; Chung, Mi Young; Han, Jeung Sul; Kang, Young-Wha; Lim, Ki Byung; Kim, Chang Kil

    2017-01-01

    Background Rosea1 (Ros1) and Delila (Del) co-expression controls anthocyanin accumulation in snapdragon flowers, while their overexpression in tomato strongly induces anthocyanin accumulation. However, little data exist on how Del expression alone influences anthocyanin accumulation. Results In tobacco (Nicotiana tabacum ?Xanthi?), Del expression enhanced leaf and flower anthocyanin production through regulating NtCHS, NtCHI, NtF3H, NtDFR, and NtANS transcript levels. Transgenic lines display...

  6. Cre/lox system to develop selectable marker free transgenic tobacco plants conferring resistance against sap sucking homopteran insect.

    Science.gov (United States)

    Chakraborti, Dipankar; Sarkar, Anindya; Mondal, Hossain A; Schuermann, David; Hohn, Barbara; Sarmah, Bidyut K; Das, Sampa

    2008-10-01

    A binary expression vector was constructed containing the insecticidal gene Allium sativum leaf agglutinin (ASAL), and a selectable nptII marker gene cassette, flanked by lox sites. Similarly, another binary vector was developed with the chimeric cre gene construct. Transformed tobacco plants were generated with these two independent vectors. Each of the T(0) lox plants was crossed with T(0) Cre plants. PCR analyses followed by the sequencing of the target T-DNA part of the hybrid T(1) plants demonstrated the excision of the nptII gene in highly precised manner in certain percentage of the T(1) hybrid lines. The frequency of such marker gene excision was calculated to be 19.2% in the hybrids. Marker free plants were able to express ASAL efficiently and reduce the survivability of Myzus persiceae, the deadly pest of tobacco significantly, compared to the control tobacco plants. Results of PCR and Southern blot analyses of some of the T(2) plants detected the absence of cre as well as nptII genes. Thus, the crossing strategy involving Cre/lox system for the excision of marker genes appears to be very effective and easy to execute. Documentation of such marker excision phenomenon in the transgenic plants expressing the important insecticidal protein for the first time has a great significance from agricultural and biotechnological points of view.

  7. A spontaneous dominant-negative mutation within a 35S::AtMYB90 transgene inhibits flower pigment production in tobacco.

    Science.gov (United States)

    Velten, Jeff; Cakir, Cahid; Cazzonelli, Christopher I

    2010-03-29

    In part due to the ease of visual detection of phenotypic changes, anthocyanin pigment production has long been the target of genetic and molecular research in plants. Specific members of the large family of plant myb transcription factors have been found to play critical roles in regulating expression of anthocyanin biosynthetic genes and these genes continue to serve as important tools in dissecting the molecular mechanisms of plant gene regulation. A spontaneous mutation within the coding region of an Arabidopsis 35S::AtMYB90 transgene converted the activator of plant-wide anthocyanin production to a dominant-negative allele (PG-1) that inhibits normal pigment production within tobacco petals. Sequence analysis identified a single base change that created a premature nonsense codon, truncating the encoded myb protein. The resulting mutant protein lacks 78 amino acids from the wild type C-terminus and was confirmed as the source of the white-flower phenotype. A putative tobacco homolog of AtMYB90 (NtAN2) was isolated and found to be expressed in flower petals but not leaves of all tobacco plants tested. Using transgenic tobacco constitutively expressing the NtAN2 gene confirmed the NtAN2 protein as the likely target of PG-1-based inhibition of tobacco pigment production. Messenger RNA and anthocyanin analysis of PG-1Sh transgenic lines (and PG-1Sh x purple 35S::NtAN2 seedlings) support a model in which the mutant myb transgene product acts as a competitive inhibitor of the native tobacco NtAN2 protein. This finding is important to researchers in the field of plant transcription factor analysis, representing a potential outcome for experiments analyzing in vivo protein function in test transgenic systems that over-express or mutate plant transcription factors.

  8. Assessing the bioconfinement potential of a Nicotiana hybrid platform for use in plant molecular farming applications.

    Science.gov (United States)

    Rice, J Hollis; Mundell, Richard E; Millwood, Reginald J; Chambers, Orlando D; Stewart, C Neal; Davies, H Maelor

    2013-08-06

    The introduction of pharmaceutical traits in tobacco for commercial production could benefit from the utilization of a transgene bioconfinement system. It has been observed that interspecific F1Nicotiana hybrids (Nicotiana tabacum × Nicotiana glauca) are sterile and thus proposed that hybrids could be suitable bioconfined hosts for biomanufacturing. We genetically tagged hybrids with green fluorescent protein (GFP), which was used as a visual marker to enable gene flow tracking and quantification for field and greenhouse studies. GFP was used as a useful proxy for pharmaceutical transgenes. Analysis of DNA content revealed significant genomic downsizing of the hybrid relative to that of N. tabacum. Hybrid pollen was capable of germination in vitro, albeit with a very low frequency and with significant differences between plants. In two field experiments, one each in Tennessee and Kentucky, we detected outcrossing at only one location (Tennessee) at 1.4%. Additionally, from 50 hybrid plants at each field site, formation of 84 and 16 seed was observed, respectively. Similar conclusions about hybrid fertility were drawn from greenhouse crosses. In terms of above-ground biomass, the hybrid yield was not significantly different than that of N. tabacum in the field. N. tabacum × N. glauca hybrids show potential to contribute to a bioconfinement- and biomanufacturing host system. Hybrids exhibit extremely low fertility with no difference of green biomass yields relative to N. tabacum. In addition, hybrids are morphologically distinguishable from tobacco allowing for identity preservation. This hybrid system for biomanufacturing would optimally be used where N. glauca is not present and in physical isolation of N. tabacum production to provide total bioconfinement.

  9. Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp.

    Directory of Open Access Journals (Sweden)

    Sandra Fresquet-Corrales

    Full Text Available Proanthocyanidins (PAs, or condensed tannins, are powerful antioxidants that remove harmful free oxygen radicals from cells. To engineer the anthocyanin and proanthocyanidin biosynthetic pathways to de novo produce PAs in two Nicotiana species, we incorporated four transgenes to the plant chassis. We opted to perform a simultaneous transformation of the genes linked in a multigenic construct rather than classical breeding or retransformation approaches. We generated a GoldenBraid 2.0 multigenic construct containing two Antirrhinum majus transcription factors (AmRosea1 and AmDelila to upregulate the anthocyanin pathway in combination with two Medicago truncatula genes (MtLAR and MtANR to produce the enzymes that will derivate the biosynthetic pathway to PAs production. Transient and stable transformation of Nicotiana benthamiana and Nicotiana tabacum with the multigenic construct were respectively performed. Transient expression experiments in N. benthamiana showed the activation of the anthocyanin pathway producing a purple color in the agroinfiltrated leaves and also the effective production of 208.5 nmol (- catechin/g FW and 228.5 nmol (- epicatechin/g FW measured by the p-dimethylaminocinnamaldehyde (DMACA method. The integration capacity of the four transgenes, their respective expression levels and their heritability in the second generation were analyzed in stably transformed N. tabacum plants. DMACA and phoroglucinolysis/HPLC-MS analyses corroborated the activation of both pathways and the effective production of PAs in T0 and T1 transgenic tobacco plants up to a maximum of 3.48 mg/g DW. The possible biotechnological applications of the GB2.0 multigenic approach in forage legumes to produce "bloat-safe" plants and to improve the efficiency of conversion of plant protein into animal protein (ruminal protein bypass are discussed.

  10. A CBL-Interacting Protein Kinase TaCIPK2 Confers Drought Tolerance in Transgenic Tobacco Plants through Regulating the Stomatal Movement.

    Science.gov (United States)

    Wang, Yan; Sun, Tao; Li, Tingting; Wang, Meng; Yang, Guangxiao; He, Guangyuan

    2016-01-01

    In plants, the CBL-CIPK signaling pathways play key roles in the response to abiotic stresses. However, functional studies of CIPKs in the important staple crop wheat are very rare. In this study, we identified a CIPK gene from wheat, designated TaCIPK2. Expression analysis results showed that TaCIPK2 could be up-regulated in wheat leaves by polyethylene glycol, abscisic acid and H2O2 treatments. Subcellular localization analyses revealed that TaCIPK2 was present in whole wheat epidermal cells. A yeast two-hybrid assay indicated that TaCIPK2 interacted with TaCBL1, 2, 3 and 4 in vitro. Transgenic tobacco plants over-expressing TaCIPK2 exhibited increased drought tolerance, indicated by a larger proportion of green cotyledons and higher survival rates under the osmotic and drought stress conditions compared with control plants. Additionally, physiological index analyses revealed that the transgenic tobacco plants had lower water loss rates and ion leakage, accumulated less malondialdehyde and H2O2, and had higher catalase and superoxide dismutase activities than the control plants. The transgenic plants also exhibited faster stomatal closure following exposure to osmotic stress conditions. The seed germination rates and stomatal aperture of TaCIPK2-overexpressing tobacco plants decreased after exogenous abscisic acid treatment was applied, implying that the transgenic tobacco plants were more sensitive to exogenous abscisic acid than the control plants. Our results indicate that TaCIPK2 plays a positive regulatory role in drought stress responses in transgenic tobacco plants.

  11. In trans silencing properties of a tobacco transgene locus depend on its epigenetic state

    Czech Academy of Sciences Publication Activity Database

    Fojtová, Miloslava; Van Houdt, H.; Depicker, A.; Kovařík, Aleš

    2004-01-01

    Roč. 26, č. 3 (2004), s. 147 ISSN 0137-5881. [Congress of the Federation of European Societies of Plant Biology /14./. 23.08.2004-27.08.2004, Cracow] Keywords : plant transgenes * gene expression * silencing Subject RIV: BO - Biophysics

  12. Gene dosage induction of silencing directed against an Arabidopsis Myb transgene in tobacco

    Science.gov (United States)

    An unexpected reduction in petal pigmentation on petunia plants genetically engineered for enhanced flower color was one of the first experimental demonstrations of the natural process of RNA-associated gene silencing. The obvious visual nature of such alterations to pigment patterns of transgenic ...

  13. Overexpression of a New Chitinase Gene EuCHIT2 Enhances Resistance to Erysiphe cichoracearum DC. in Tobacco Plants

    Directory of Open Access Journals (Sweden)

    Xuan Dong

    2017-11-01

    Full Text Available In this study, we cloned a new chitinase gene, EuCHIT2, from Eucommia ulmoides Oliver (E. ulmoides using rapid amplification of cDNA ends (RACE technology and constructed an overexpression vector, pSH-35S-EuCHIT2, to introduce it into tobacco (Nicotiana tabacum cv. Xanthi. Resistance to Erysiphe cichoracearum de Candolle (E.cichoracearum DC and molecular mechanisms in the transgenic tobacco were determined by drop inoculation, spore counting, determination of physicochemical indicators, and analysis of gene expression. The chitinase activity and resistance to E. cichoracearum DC were significantly higher in the transgenic tobacco than in wild-type tobacco (p < 0.05. The activities of peroxidase (POD and catalase (CAT, after inoculation with E. cichoracearum DC, were higher in the transgenic tobacco than in the wild-type. Conversely, the malondialdehyde (MDA content was significantly lower in the transgenic tobacco than the wild-type before and after inoculation. In addition, our study also indicated that the resistance to E. cichoracearum DC might involve the salicylic acid (SA and jasmonic acid (JA pathways, because the expression levels of pathogenesis-related gene 1 (PR-1a and coronatine-insensitive 1 (COI1 were significantly increased and decreased, respectively, after inoculation with E. cichoracearum DC. The present study supports the notion that PR-1a and POD participate in resistance to E. cichoracearum DC in the transgenic tobacco plants.

  14. Transgenic tobacco plants expressing BoRS1 gene from Brassica ...

    Indian Academy of Sciences (India)

    Water stress is by far the leading environmental stress limiting crop yields worldwide. Genetic engineering techniques hold great promise for developing crop cultivars with high tolerance to water stress. In this study, the Brassica oleracea var. acephala BoRS1 gene was transferred into tobacco through ...

  15. Expression of Hemagglutinin–Neuraminidase and fusion epitopes of Newcastle Disease Virus in transgenic tobacco

    Directory of Open Access Journals (Sweden)

    Amir Ghaffar Shahriari

    2016-07-01

    Conclusion: Developments in genetic engineering have led to plant-based systems for recombinant vaccine production. In this research, tobacco plant was used to express F and HN epitopes of NDV. Our results indicate that for the production of recombinant vaccine, it is a novel strategy to use concatenated epitopes without their genetic fusion onto larger scaffold structure such as viral coat protein.

  16. Paramutation of tobacco transgenes by small RNA-mediated transcriptional gene silencing

    Czech Academy of Sciences Publication Activity Database

    Crhák Khaitová, Lucie; Fojtová, M.; Křížová, Kateřina; Lunerová Bedřichová, Jana; Fulneček, Jaroslav; Depicker, A.; Kovařík, Aleš

    2011-01-01

    Roč. 6, č. 5 (2011), s. 650-660 ISSN 1559-2294 R&D Projects: GA ČR(CZ) GD204/09/H002; GA MŠk(CZ) LC06004 Grant - others:GA ČR(CZ) GPP501/11/P667 Program:GP Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : transcriptional gene silencing * transgene epialleles * DNA methylation Subject RIV: BO - Biophysics Impact factor: 4.318, year: 2011

  17. Rapid transient expression of human granulocyte-macrophage colony-stimulating factor in two industrial cultivars of tobacco (Nicotiana tabacum L. by agroinfiltration

    Directory of Open Access Journals (Sweden)

    Lea Vojta

    2015-09-01

    Full Text Available We report the production of hGM-CSF cytokine in leaves of industrial tobacco cultivars DH-17 and DH-27 by using Agrobacterium-mediated transient expression. We prove the concept that very high biomass industrial tobacco plants are suitable platforms for rapid, low cost production of foreign proteins. Successful transient expression of the GM-CSF was achieved in less than three months, opening the possibility for future applications of this approach in rapid response production of various proteins of non-plant origin in industrial tobacco.

  18. Tobacco

    Science.gov (United States)

    ... Second-hand smoke is the smoke that fills restaurants, offices or other enclosed spaces when people burn ... as smuggling, illicit manufacturing and counterfeiting. The tobacco industry and others often argue that high tobacco product ...

  19. Increased tolerance to two oomycete pathogens in transgenic tobacco expressing pathogenesis-related protein 1a.

    OpenAIRE

    Alexander, D; Goodman, R M; Gut-Rella, M; Glascock, C; Weymann, K; Friedrich, L; Maddox, D; Ahl-Goy, P; Luntz, T; Ward, E

    1993-01-01

    Expression of pathogenesis-related protein 1a (PR-1a), a protein of unknown biochemical function, is induced to high levels in tobacco in response to pathogen infection. The induction of PR-1a expression is tightly correlated with the onset of systemic acquired resistance (SAR), a defense response effective against a variety of fungal, viral, and bacterial pathogens. While PR-1a has been postulated to be involved in SAR, and is the most highly expressed of the PR proteins, evidence for its ro...

  20. Expression of chickpea CIPK25 enhances root growth and tolerance to dehydration and salt stress in transgenic tobacco

    Directory of Open Access Journals (Sweden)

    Mukesh Kumar Meena

    2015-09-01

    Full Text Available Calcium signaling plays an important role in adaptation and developmental processes in plants and animals. A class of calcium sensors, known as Calcineurin B-like (CBL proteins sense specific temporal changes in cytosolic Ca2+ concentration and regulate activities of a group of ser/thr protein kinases called CBL-interacting protein kinases (CIPKs. Although a number of CIPKs have been shown to play crucial roles in the regulation of stress signaling, no study on the function of CIPK25 or its orthologues has been reported so far. In the present study, an orthologue of Arabidopsis CIPK25 was cloned from chickpea (Cicer arietinum. CaCIPK25 gene expression in chickpea increased upon salt, dehydration, and different hormonal treatments. CaCIPK25 gene showed differential tissue-specific expression. 5'-upstream activation sequence (5'-UAS of the gene and its different truncated versions were fused to a reporter gene and studied in Arabidopsis to identify promoter regions directing its tissue-specific expression. Replacement of a conserved threonine residue with an aspartic acid at its catalytic site increased the kinase activity of CaCIPK25 by 2.5-fold. Transgenic tobacco plants overexpressing full-length and the high active versions of CaCIPK25 displayed a differential germination period and longer root length in comparison to the control plants. Expression of CaCIPK25 and its high active form differentially increased salt and water-deficit tolerance demonstrated by improved growth and reduced leaf chlorosis suggesting that the kinase activity of CaCIPK25 was required for these functions. Expressions of the abiotic stress marker genes were enhanced in the CaCIPK25-expressing tobacco plants. Our results suggested that CaCIPK25 functions in root development and abiotic stress tolerance.

  1. Enhanced cadmium accumulation and tolerance in transgenic tobacco overexpressing rice metal tolerance protein gene OsMTP1 is promising for phytoremediation.

    Science.gov (United States)

    Das, Natasha; Bhattacharya, Surajit; Maiti, Mrinal K

    2016-08-01

    One of the most grievous heavy metal pollutants in the environment is cadmium (Cd), which is not only responsible for the crop yield loss owing to its phytotoxicity, but also for the human health hazards as the toxic elements usually accumulate in the consumable parts of crop plants. In the present study, we aimed to isolate and functionally characterize the OsMTP1 gene from indica rice (Oryza sativa L. cv. IR64) to study its potential application for efficient phytoremediation of Cd. The 1257 bp coding DNA sequence (CDS) of OsMTP1 encodes a ∼46 kDa protein belonging to the cation diffusion facilitator (CDF) or metal tolerance/transport protein (MTP) family. The OsMTP1 transcript in rice plant was found to respond during external Cd stress. Heterologous expression of OsMTP1 in tobacco resulted in the reduction of Cd stress-induced phytotoxic effects, including growth inhibition, lipid peroxidation, and cell death. Compared to untransformed control, the transgenic tobacco plants showed enhanced vacuolar thiol content, indicating vacuolar localization of the sequestered Cd. The transgenic tobacco plants exhibited significantly higher biomass growth (2.2-2.8-folds) and hyperaccumulation of Cd (1.96-2.22-folds) compared to untransformed control under Cd exposure. The transgenic plants also showed moderate tolerance and accumulation of arsenic (As) upon exogenous As stress, signifying broad substrate specificity of OsMTP1. Together, findings of our research suggest that the transgenic tobacco plants overexpressing OsMTP1 with its hyperaccumulating activity and increased growth rate could be useful for future phytoremediation applications to clean up the Cd-contaminated soil. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  2. Analysis of the function of the photoreceptors phytochrome B and phytochrome D in Nicotiana plumbaginifolia and Arabidopsis thaliana.

    Science.gov (United States)

    Fernández, Aurora Piñas; Gil, Patricia; Valkai, Ildiko; Nagy, Ferenc; Schäfer, Eberhard

    2005-05-01

    To investigate the mechanism of phytochrome action in vivo, NtPHYB, AtPHYB and phyD:green fluorescent protein (GFP) were overexpressed in Nicotiana plumbaginifolia and Arabidopsis thaliana. The expression of 35S:NtPHYB:GFP and 35S:AtPHYB:GFP complemented the tobacco hgl2 and Arabidopsis phyB-9 mutations, whereas the 35S:AtPHYD:GFP only rescued the hgl2 mutant. All three fusion proteins are transported into the nucleus in all genetic backgrounds. These data indicate that AtPHYD:GFP is biologically active and functions as the main red light receptor in transgenic tobacco, and establish an experimental system for the functional analysis of this elusive photoreceptor in vivo.

  3. A Wheat R2R3-type MYB Transcription Factor TaODORANT1 Positively Regulates Drought and Salt Stress Responses in Transgenic Tobacco Plants

    Directory of Open Access Journals (Sweden)

    Qiuhui Wei

    2017-08-01

    Full Text Available MYB transcription factors play important roles in plant responses to biotic and abiotic stress. In this study, TaODORANT1, a R2R3-MYB gene, was cloned from wheat (Triticum aestivum L.. TaODORANT1 was localized in the nucleus and functioned as a transcriptional activator. TaODORANT1 was up-regulated in wheat under PEG6000, NaCl, ABA, and H2O2 treatments. TaODORANT1-overexpressing transgenic tobacco plants exhibited higher relative water content and lower water loss rate under drought stress, as well as lower Na+ accumulation in leaves under salt stress. The transgenic plants showed higher CAT activity but lower ion leakage, H2O2 and malondialdehyde contents under drought and salt stresses. Besides, the transgenic plants also exhibited higher SOD activity under drought stress. Our results also revealed that TaODORANT1 overexpression up-regulated the expression of several ROS- and stress-related genes in response to both drought and salt stresses, thus enhancing transgenic tobacco plants tolerance. Our studies demonstrate that TaODORANT1 positively regulates plant tolerance to drought and salt stresses.

  4. Multiple different defense mechanisms are activated in the young transgenic tobacco plants which express the full length genome of the Tobacco mosaic virus, and are resistant against this virus.

    Science.gov (United States)

    Jada, Balaji; Soitamo, Arto J; Siddiqui, Shahid Aslam; Murukesan, Gayatri; Aro, Eva-Mari; Salakoski, Tapio; Lehto, Kirsi

    2014-01-01

    Previously described transgenic tobacco lines express the full length infectious Tobacco mosaic virus (TMV) genome under the 35S promoter (Siddiqui et al., 2007. Mol Plant Microbe Interact, 20: 1489-1494). Through their young stages these plants exhibit strong resistance against both the endogenously expressed and exogenously inoculated TMV, but at the age of about 7-8 weeks they break into TMV infection, with typical severe virus symptoms. Infections with some other viruses (Potato viruses Y, A, and X) induce the breaking of the TMV resistance and lead to synergistic proliferation of both viruses. To deduce the gene functions related to this early resistance, we have performed microarray analysis of the transgenic plants during the early resistant stage, and after the resistance break, and also of TMV-infected wild type tobacco plants. Comparison of these transcriptomes to those of corresponding wild type healthy plants indicated that 1362, 1150 and 550 transcripts were up-regulated in the transgenic plants before and after the resistance break, and in the TMV-infected wild type tobacco plants, respectively, and 1422, 1200 and 480 transcripts were down-regulated in these plants, respectively. These transcriptome alterations were distinctly different between the three types of plants, and it appears that several different mechanisms, such as the enhanced expression of the defense, hormone signaling and protein degradation pathways contributed to the TMV-resistance in the young transgenic plants. In addition to these alterations, we also observed a distinct and unique gene expression alteration in these plants, which was the strong suppression of the translational machinery. This may also contribute to the resistance by slowing down the synthesis of viral proteins. Viral replication potential may also be suppressed, to some extent, by the reduction of the translation initiation and elongation factors eIF-3 and eEF1A and B, which are required for the TMV replication

  5. Functional components of the bacterial CzcCBA efflux system reduce cadmium uptake and accumulation in transgenic tobacco plants.

    Science.gov (United States)

    Nesler, Andrea; DalCorso, Giovanni; Fasani, Elisa; Manara, Anna; Di Sansebastiano, Gian Pietro; Argese, Emanuele; Furini, Antonella

    2017-03-25

    Cadmium (Cd) is a toxic trace element released into the environment by industrial and agricultural practices, threatening the health of plants and contaminating the food/feed chain. Biotechnology can be used to develop plant varieties with a higher capacity for Cd accumulation (for use in phytoremediation programs) or a lower capacity for Cd accumulation (to reduce Cd levels in food and feed). Here we generated transgenic tobacco plants expressing components of the Pseudomonas putida CzcCBA efflux system. Plants were transformed with combinations of the CzcC, CzcB and CzcA genes, and the impact on Cd mobilization was analysed. Plants expressing PpCzcC showed no differences in Cd accumulation, whereas those expressing PpCzcB or PpCzcA accumulated less Cd in the shoots, but more Cd in the roots. Plants expressing both PpCzcB and PpCzcA accumulated less Cd in the shoots and roots compared to controls, whereas plants expressing all three genes showed a significant reduction in Cd levels only in shoots. These results show that components of the CzcCBA system can be expressed in plants and may be useful for developing plants with a reduced capacity to accumulate Cd in the shoots, potentially reducing the toxicity of food/feed crops cultivated in Cd-contaminated soils. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. DNA damage induced by indirect and direct acting mutagens in catalase-deficient transgenic tobacco Cellular and acellular Comet assays

    Czech Academy of Sciences Publication Activity Database

    Gichner, Tomáš

    2003-01-01

    Roč. 535, - (2003), s. 187-193 ISSN 1383-5718 R&D Projects: GA ČR GA521/02/0400 Institutional research plan: CEZ:AV0Z5038910 Keywords : Hydrogen peroxide * Single-cell gel electrophoresis * Nicotiana tabacum Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.748, year: 2003

  7. Overexpression of SoCYP85A1, a Spinach Cytochrome p450 Gene in Transgenic Tobacco Enhances Root Development and Drought Stress Tolerance

    Directory of Open Access Journals (Sweden)

    Fangmeng Duan

    2017-11-01

    Full Text Available Brassinosteroids (BRs play an essential role in plant growth, development, and responses to diverse abiotic stresses. However, previous studies mainly analyzed how exogenous BRs influenced plant physiological reactions to drought stress, therefore, genetic evidences for the endogenous BRs-mediated regulation of plant responses still remain elusive. In this study, a key BRs biosynthetic gene, SoCYP85A1 was cloned from Spinacia oleracea, which has a complete open reading frame of 1,392 bp encoding a 464 amino acid peptide and shares high sequence similarities with CYP85A1 from other plants. The expression of SoCYP85A1 which was higher in leaf compared with root and stem, was induced by treatments of PEG6000, abscisic acid (ABA, low temperature and high salt. Increases in both SoCYP85A1 transcripts and endogenous BRs in transgenic tobacco which resulted in longer primary root and more lateral roots enhanced drought tolerance compared with wild types. The transgenic tobacco accumulated much lower levels of reactive oxygen species and malondialdehyde (MDA than wild types did, accompanied by significantly higher content of proline and notably enhanced activities of antioxidant enzymes. Besides, transcriptional expressions of six stress-responsive genes were regulated to higher levels in transgenic lines under drought stress. Taken together, our results demonstrated that SoCYP85A1 involves in response to drought stress by promoting root development, scavenging ROS, and regulating expressions of stress-responsive genes.

  8. The Metallothionein Gene, TaMT3, from Tamarix androssowii Confers Cd2+ Tolerance in Tobacco

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    Boru Zhou

    2014-06-01

    Full Text Available Cadmium (Cd is a nonessential microelement and low concentration Cd2+ has strong toxicity to plant growth. Plant metallothioneins, a class of low molecular, cystein(Cys-rich and heavy-metal binding proteins, play an important role in both metal chaperoning and scavenging of reactive oxygen species (ROS with their large number of cysteine residues and therefore, protect plants from oxidative damage. In this study, a metallothionein gene, TaMT3, isolated from Tamarix androssowii was transformed into tobacco (Nicotiana tobacum through Agrobacterium-mediated leaf disc method, and correctly expressed under the control of 35S promoter. Under Cd2+ stress, the transgenic tobacco showed significant increases of superoxide dismutase (SOD activity and chlorophyll concentration, but decreases of peroxidase (POD activity and malondialdehyde (MDA accumulation when compared to the non-transgenic tobacco. Vigorous growth of transgenic tobacco was observed at the early development stages, resulting in plant height and fresh weight were significantly larger than those of the non-transgenic tobacco under Cd2+ stress. These results demonstrated that the expression of the exogenous TaMT3 gene increased the ability of ROS cleaning-up, indicating a stronger tolerance to Cd2+ stress.

  9. The metallothionein gene, TaMT3, from Tamarix androssowii confers Cd2+ tolerance in tobacco.

    Science.gov (United States)

    Zhou, Boru; Yao, Wenjing; Wang, Shengji; Wang, Xinwang; Jiang, Tingbo

    2014-06-10

    Cadmium (Cd) is a nonessential microelement and low concentration Cd2+ has strong toxicity to plant growth. Plant metallothioneins, a class of low molecular, cystein(Cys)-rich and heavy-metal binding proteins, play an important role in both metal chaperoning and scavenging of reactive oxygen species (ROS) with their large number of cysteine residues and therefore, protect plants from oxidative damage. In this study, a metallothionein gene, TaMT3, isolated from Tamarix androssowii was transformed into tobacco (Nicotiana tobacum) through Agrobacterium-mediated leaf disc method, and correctly expressed under the control of 35S promoter. Under Cd2+ stress, the transgenic tobacco showed significant increases of superoxide dismutase (SOD) activity and chlorophyll concentration, but decreases of peroxidase (POD) activity and malondialdehyde (MDA) accumulation when compared to the non-transgenic tobacco. Vigorous growth of transgenic tobacco was observed at the early development stages, resulting in plant height and fresh weight were significantly larger than those of the non-transgenic tobacco under Cd2+ stress. These results demonstrated that the expression of the exogenous TaMT3 gene increased the ability of ROS cleaning-up, indicating a stronger tolerance to Cd2+ stress.

  10. Sequential path analysis for determining interrelationships between yield and related traits in tobacco (Nicotiana tabacum L. under normal and abiotic stress conditions

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    Bayat Mahdi

    2014-01-01

    Full Text Available In the present work the relationships between yield and its related traits were investigated in tobacco genotypes under normal and abiotic stress conditions (Orobanche aegyptiaca weed at Urmia Tobacco Research Centre, Iran, during 2006-2009 cropping seasons. The experimental design was a randomized complete block design (RCBD with three replications in each condition every year. Analysis of variance revealed extent genetic variability among the genotypes for most of the traits studied. In comparison with normal condition, the mean value of studied traits decreased in stress condition. LAI and FD showed the maximum and minimum diminution in the mean values under stress condition compared to normal one so known as more sensitive and more tolerant traits, respectively. Based on CV values, the traits FD and DLYP showed the minimum and maximum variation among traits in both normal and stress conditions. Correlation analysis revealed significant and positive correlations between DLYP with all studied traits in both normal and stress conditions. Path analysis detected the traits including biomass, APDW and DWR as the first-order variables at normal condition and biomass, APDW, DWR and harvest index as the first-order variables under abiotic stress condition. Based on results, the traits such as biomass, APDW, DWR detected as more important factors in both conditions can be used in tobacco breeding programs for increasing yield. Abbreviation: aerial part fresh weight without leaves weight (APFW, aerial part dry weight without leaves weight (APDW, biomass (BIO, coefficient of variation (CV, dry weight of root (DWR, flowering date (FD, fresh weight of leaf (FWL, fresh weight of root (FWR, harvest index (HI, leaf area index (LAI, dry leaf yield per plant (DLYP, number of leaf (NL, plant height (PH, randomized complete block design (RCBD, standard deviation (Std.

  11. Tobacco

    Science.gov (United States)

    ... 1 in 3 countries, representing 39% of the world's population, monitors tobacco use by repeating nationally representative youth ... 1.4 billion people, or 20% of the world's population, are protected by comprehensive national smoke-free laws. ...

  12. Transgenic tobacco plants with improved cyanobacterial Rubisco expression but no extra assembly factors grow at near wild-type rates if provided with elevated CO2.

    Science.gov (United States)

    Occhialini, Alessandro; Lin, Myat T; Andralojc, P John; Hanson, Maureen R; Parry, Martin A J

    2016-01-01

    Introducing a carbon-concentrating mechanism and a faster Rubisco enzyme from cyanobacteria into higher plant chloroplasts may improve photosynthetic performance by increasing the rate of CO2 fixation while decreasing losses caused by photorespiration. We previously demonstrated that tobacco plants grow photoautotrophically using Rubisco from Synechococcus elongatus, although the plants exhibited considerably slower growth than wild-type and required supplementary CO2 . Because of concerns that vascular plant assembly factors may not be adequate for assembly of a cyanobacterial Rubisco, prior transgenic plants included the cyanobacterial chaperone RbcX or the carboxysomal protein CcmM35. Here we show that neither RbcX nor CcmM35 is needed for assembly of active cyanobacterial Rubisco. Furthermore, by altering the gene regulatory sequences on the Rubisco transgenes, cyanobacterial Rubisco expression was enhanced and the transgenic plants grew at near wild-type growth rates, although still requiring elevated CO2 . We performed detailed kinetic characterization of the enzymes produced with and without the RbcX and CcmM35 cyanobacterial proteins. These transgenic plants exhibit photosynthetic characteristics that confirm the predicted benefits of introduction of non-native forms of Rubisco with higher carboxylation rate constants in vascular plants and the potential nitrogen-use efficiency that may be achieved provided that adequate CO2 is available near the enzyme. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  13. A cold-induced myo-inositol transporter-like gene confers tolerance to multiple abiotic stresses in transgenic tobacco plants.

    Science.gov (United States)

    Sambe, Mame Abdou Nahr; He, Xueying; Tu, Qinghua; Guo, Zhenfei

    2015-03-01

    A full length cDNA encoding a myo-inositol transporter-like protein, named as MfINT-like, was cloned from Medicago sativa subsp. falcata (herein falcata), a species with greater cold tolerance than alfalfa (M. sativa subsp. sativa). MfINT-like is located on plasma membranes. MfINT-like transcript was induced 2-4 h after exogenous myo-inositol treatment, 24-96 h with cold, and 96 h by salinity. Given that myo-inositol accumulates higher in falcata after 24 h of cold treatment, myo-inositol is proposed to be involved in cold-induced expression of MfINT-like. Higher levels of myo-inositol was observed in leaves of transgenic tobacco plants overexpressing MfINT-like than the wild-type but not in the roots of plants grown on myo-inositol containing medium, suggesting that transgenic plants had higher myo-inositol transport activity than the wild-type. Transgenic plants survived better to freezing temperature, and had lower ion leakage and higher maximal photochemical efficiency of photosystem II (Fv /Fm ) after chilling treatment. In addition, greater plant fresh weight was observed in transgenic plants as compared with the wild-type when plants were grown under drought or salinity stress. The results suggest that MfINT-like mediated transport of myo-inositol is associated with plant tolerance to abiotic stresses. © 2014 Scandinavian Plant Physiology Society.

  14. Overexpression of a specific soybean GmGSTU4 isoenzyme improves diphenyl ether and chloroacetanilide herbicide tolerance of transgenic tobacco plants.

    Science.gov (United States)

    Benekos, Kostantinos; Kissoudis, Christos; Nianiou-Obeidat, Irini; Labrou, Nikolaos; Madesis, Panagiotis; Kalamaki, Mary; Makris, Antonis; Tsaftaris, Athanasios

    2010-10-01

    Plant glutathione transferases (GSTs) superfamily consists of multifunctional enzymes and forms a major part of the plants herbicide detoxification enzyme network. The tau class GST isoenzyme GmGSTU4 from soybean, exhibits catalytic activity towards the diphenyl ether herbicide fluorodifen and is active as glutathione-dependent peroxidase (GPOX). Transgenic tobacco plants of Basmas cultivar were generated via Agrobacterium transformation. The aim was to evaluate in planta, GmGSTU4's role in detoxifying the diphenyl ether herbicides fluorodifen and oxyfluorfen and the chloroacetanilides alachlor and metolachlor. Transgenic tobacco plants were verified by PCR and Southern blot hybridization and expression of GmGSTU4 was determined by RT-PCR. Leaf extracts from transgenic plants showed moderate increase in GST activity towards CDNB and a significant increase towards fluorodifen and alachlor, and at the same time an increased GPOX activity towards cumene hydroperoxide. GmGSTU4 overexpressing plants when treated with 200 μM fluorodifen or oxyfluorfen exhibited reduced relative electrolyte leakage compared to wild type plants. Moreover all GmGSTU4 overexpressing lines exhibited significantly increased tolerance towards alachlor when grown in vitro at 7.5 mg/L alachlor compared to wild type plants. No significant increased tolerance was observed to metolachlor. These results confirm the contribution of this particular GmGSTU4 isoenzyme from soybean in the detoxification of fluorodifen and alachlor, and provide the basis towards the development of transgenic plants with improved phytoremediation capabilities for future use in environmental cleanup of herbicides. Copyright © 2010 Elsevier B.V. All rights reserved.

  15. Increased and Altered Fragrance of Tobacco Plants after Metabolic Engineering Using Three Monoterpene Synthases from Lemon

    Science.gov (United States)

    Lücker, Joost; Schwab, Wilfried; van Hautum, Bianca; Blaas, Jan; van der Plas, Linus H. W.; Bouwmeester, Harro J.; Verhoeven, Harrie A.

    2004-01-01

    Wild-type tobacco (Nicotiana tabacum) plants emit low levels of terpenoids, particularly from the flowers. By genetic modification of tobacco cv Petit Havana SR1 using three different monoterpene synthases from lemon (Citrus limon L. Burm. f.) and the subsequent combination of these three into one plant by crossings, we show that it is possible to increase the amount and alter the composition of the blend of monoterpenoids produced in tobacco plants. The transgenic tobacco plant line with the three introduced monoterpene synthases is emitting β-pinene, limonene, and γ-terpinene and a number of side products of the introduced monoterpene synthases, from its leaves and flowers, in addition to the terpenoids emitted by wild-type plants. The results show that there is a sufficiently high level of substrate accessible for the introduced enzymes. PMID:14718674

  16. Recombinant Promoter (MUASCsV8CP) Driven Totiviral Killer Protein 4 (KP4) Imparts Resistance Against Fungal Pathogens in Transgenic Tobacco

    Science.gov (United States)

    Deb, Debasish; Shrestha, Ankita; Maiti, Indu B.; Dey, Nrisingha

    2018-01-01

    Development of disease-resistant plant varieties achieved by engineering anti-microbial transgenes under the control of strong promoters can suffice the inhibition of pathogen growth and simultaneously ensure enhanced crop production. For evaluating the prospect of such strong promoters, we comprehensively characterized the full-length transcript promoter of Cassava Vein Mosaic Virus (CsVMV; -565 to +166) and identified CsVMV8 (-215 to +166) as the highest expressing fragment in both transient and transgenic assays. Further, we designed a new chimeric promoter ‘MUASCsV8CP’ through inter-molecular hybridization among the upstream activation sequence (UAS) of Mirabilis Mosaic Virus (MMV; -297 to -38) and CsVMV8, as the core promoter (CP). The MUASCsV8CP was found to be ∼2.2 and ∼2.4 times stronger than the CsVMV8 and CaMV35S promoters, respectively, while its activity was found to be equivalent to that of the CaMV35S2 promoter. Furthermore, we generated transgenic tobacco plants expressing the totiviral ‘Killer protein KP4’ (KP4) under the control of the MUASCsV8CP promoter. Recombinant KP4 was found to accumulate both in the cytoplasm and apoplast of plant cells. The agar-based killing zone assays revealed enhanced resistance of plant-derived KP4 against two deuteromycetous foliar pathogenic fungi viz. Alternaria alternata and Phoma exigua var. exigua. Also, transgenic plants expressing KP4 inhibited the growth progression of these fungi and conferred significant fungal resistance in detached-leaf and whole plant assays. Taken together, we establish the potential of engineering “in-built” fungal stress-tolerance in plants by expressing KP4 under a novel chimeric caulimoviral promoter in a transgenic approach. PMID:29556246

  17. TaPP2C1, a Group F2 Protein Phosphatase 2C Gene, Confers Resistance to Salt Stress in Transgenic Tobacco.

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    Wei Hu

    Full Text Available Group A protein phosphatases 2Cs (PP2Cs are essential components of abscisic acid (ABA signaling in Arabidopsis; however, the function of group F2 subfamily PP2Cs is currently less known. In this study, TaPP2C1 which belongs to group F2 was isolated and characterized from wheat. Expression of the TaPP2C1-GFP fusion protein suggested its ubiquitous localization within a cell. TaPP2C1 expression was downregulated by abscisic acid (ABA and NaCl treatments, but upregulated by H2O2 treatment. Overexpression of TaPP2C1 in tobacco resulted in reduced ABA sensitivity and increased salt resistance of transgenic seedlings. Additionally, physiological analyses showed that improved resistance to salt stress conferred by TaPP2C1 is due to the reduced reactive oxygen species (ROS accumulation, the improved antioxidant system, and the increased transcription of genes in the ABA-independent pathway. Finally, transgenic tobacco showed increased resistance to oxidative stress by maintaining a more effective antioxidant system. Taken together, these results demonstrated that TaPP2C1 negatively regulates ABA signaling, but positively regulates salt resistance. TaPP2C1 confers salt resistance through activating the antioxidant system and ABA-independent gene transcription process.

  18. The SbSOS1 gene from the extreme halophyte Salicornia brachiata enhances Na+ loading in xylem and confers salt tolerance in transgenic tobacco

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    Yadav Narendra

    2012-10-01

    Full Text Available Abstract Background Soil salinity adversely affects plant growth and development and disturbs intracellular ion homeostasis resulting cellular toxicity. The Salt Overly Sensitive 1 (SOS1 gene encodes a plasma membrane Na+/H+ antiporter that plays an important role in imparting salt stress tolerance to plants. Here, we report the cloning and characterisation of the SbSOS1 gene from Salicornia brachiata, an extreme halophyte. Results The SbSOS1 gene is 3774 bp long and encodes a protein of 1159 amino acids. SbSOS1 exhibited a greater level of constitutive expression in roots than in shoots and was further increased by salt stress. Overexpressing the S. brachiata SbSOS1 gene in tobacco conferred high salt tolerance, promoted seed germination and increased root length, shoot length, leaf area, fresh weight, dry weight, relative water content (RWC, chlorophyll, K+/Na+ ratio, membrane stability index, soluble sugar, proline and amino acid content relative to wild type (WT plants. Transgenic plants exhibited reductions in electrolyte leakage, reactive oxygen species (ROS and MDA content in response to salt stress, which probably occurred because of reduced cytosolic Na+ content and oxidative damage. At higher salt stress, transgenic tobacco plants exhibited reduced Na+ content in root and leaf and higher concentrations in stem and xylem sap relative to WT, which suggests a role of SbSOS1 in Na+ loading to xylem from root and leaf tissues. Transgenic lines also showed increased K+ and Ca2+ content in root tissue compared to WT, which reflect that SbSOS1 indirectly affects the other transporters activity. Conclusions Overexpression of SbSOS1 in tobacco conferred a high degree of salt tolerance, enhanced plant growth and altered physiological and biochemical parameters in response to salt stress. In addition to Na+ efflux outside the plasma membrane, SbSOS1 also helps to maintain variable Na+ content in different organs and also affect the other

  19. The SbSOS1 gene from the extreme halophyte Salicornia brachiata enhances Na(+) loading in xylem and confers salt tolerance in transgenic tobacco.

    Science.gov (United States)

    Yadav, Narendra Singh; Shukla, Pushp Sheel; Jha, Anupama; Agarwal, Pradeep K; Jha, Bhavanath

    2012-10-11

    Soil salinity adversely affects plant growth and development and disturbs intracellular ion homeostasis resulting cellular toxicity. The Salt Overly Sensitive 1 (SOS1) gene encodes a plasma membrane Na(+)/H(+) antiporter that plays an important role in imparting salt stress tolerance to plants. Here, we report the cloning and characterisation of the SbSOS1 gene from Salicornia brachiata, an extreme halophyte. The SbSOS1 gene is 3774 bp long and encodes a protein of 1159 amino acids. SbSOS1 exhibited a greater level of constitutive expression in roots than in shoots and was further increased by salt stress. Overexpressing the S. brachiata SbSOS1 gene in tobacco conferred high salt tolerance, promoted seed germination and increased root length, shoot length, leaf area, fresh weight, dry weight, relative water content (RWC), chlorophyll, K(+)/Na(+) ratio, membrane stability index, soluble sugar, proline and amino acid content relative to wild type (WT) plants. Transgenic plants exhibited reductions in electrolyte leakage, reactive oxygen species (ROS) and MDA content in response to salt stress, which probably occurred because of reduced cytosolic Na(+) content and oxidative damage. At higher salt stress, transgenic tobacco plants exhibited reduced Na(+) content in root and leaf and higher concentrations in stem and xylem sap relative to WT, which suggests a role of SbSOS1 in Na(+) loading to xylem from root and leaf tissues. Transgenic lines also showed increased K(+) and Ca(2+) content in root tissue compared to WT, which reflect that SbSOS1 indirectly affects the other transporters activity. Overexpression of SbSOS1 in tobacco conferred a high degree of salt tolerance, enhanced plant growth and altered physiological and biochemical parameters in response to salt stress. In addition to Na(+) efflux outside the plasma membrane, SbSOS1 also helps to maintain variable Na(+) content in different organs and also affect the other transporters activity indirectly. These

  20. The SbSOS1 gene from the extreme halophyte Salicornia brachiata enhances Na+ loading in xylem and confers salt tolerance in transgenic tobacco

    Science.gov (United States)

    2012-01-01

    Background Soil salinity adversely affects plant growth and development and disturbs intracellular ion homeostasis resulting cellular toxicity. The Salt Overly Sensitive 1 (SOS1) gene encodes a plasma membrane Na+/H+ antiporter that plays an important role in imparting salt stress tolerance to plants. Here, we report the cloning and characterisation of the SbSOS1 gene from Salicornia brachiata, an extreme halophyte. Results The SbSOS1 gene is 3774 bp long and encodes a protein of 1159 amino acids. SbSOS1 exhibited a greater level of constitutive expression in roots than in shoots and was further increased by salt stress. Overexpressing the S. brachiata SbSOS1 gene in tobacco conferred high salt tolerance, promoted seed germination and increased root length, shoot length, leaf area, fresh weight, dry weight, relative water content (RWC), chlorophyll, K+/Na+ ratio, membrane stability index, soluble sugar, proline and amino acid content relative to wild type (WT) plants. Transgenic plants exhibited reductions in electrolyte leakage, reactive oxygen species (ROS) and MDA content in response to salt stress, which probably occurred because of reduced cytosolic Na+ content and oxidative damage. At higher salt stress, transgenic tobacco plants exhibited reduced Na+ content in root and leaf and higher concentrations in stem and xylem sap relative to WT, which suggests a role of SbSOS1 in Na+ loading to xylem from root and leaf tissues. Transgenic lines also showed increased K+ and Ca2+ content in root tissue compared to WT, which reflect that SbSOS1 indirectly affects the other transporters activity. Conclusions Overexpression of SbSOS1 in tobacco conferred a high degree of salt tolerance, enhanced plant growth and altered physiological and biochemical parameters in response to salt stress. In addition to Na+ efflux outside the plasma membrane, SbSOS1 also helps to maintain variable Na+ content in different organs and also affect the other transporters activity indirectly

  1. A Survey of Fertility Program Responses of Kentucky Dark Fire-Cured Tobacco (Nicotiana tabacum L. Yield and Quality for Cigars Manufacture in the Benevento Province (Southern Italy

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    Sifola Maria Isabella

    2018-04-01

    Full Text Available Nitrogen (N fertilization of Kentucky dark fire-cured tobacco can be used to increase weight of high quality cured leaves for cigar manufacture. We conducted field experiments at 11 different locations in the province of Benevento (Southern Italy where the following four N treatments were compared: 1 unfertilized control (N0; 2 a site-specific N rate, calculated by a N fertilization plan (NFP based on physical and chemical soil characteristics, which ranged between 113 and 145 kg N ha−1; 3 200 kg N ha−1 (rate commonly used by farmers, N200; 4 100 kg N ha−1 (half of the rate commonly used by farmers, N100. Yields of the following five commercial quality categories of cured leaves were measured: i wrappers, ii heavy filler (Fh, iii light filler (Fl, iv heavy shredded (Sh and v light shredded (Sl. Fh cured products of B1, B4, B6 and B10 locations were analyzed for: total alkaloids, reducing sugars, chlorides, total N (Kjeldahl, ammonium-N (NH4-N, nitrate-N (NO3-N, and tobacco specific nitrosamines (TSNA. Color parameters: Lightness (L, Chroma (C and Hue (H were determined on five cured leaves / plot of both Fh and Fl types at B1, B2, B3, B6, B8 and B10. A blind evaluation of cured leaves collected across locations was conducted by a panel test who considered the main basic characteristics of cured leaves (stalk position, leaf structure, texture, etc.. The total yield of cured products increased with fertilization across locations, up to NFP treatment, without any statistically significant increase at N200 treatment. Fertilization increased yield of wrappers at B1 up to NFP treatment (113.5 kg N ha−1, without any significant increase at N200 treatment. Yield of light filler product was positively influenced by fertilization up to the maximum dose only in 5 out of 11 locations. Total alkaloids significantly increased with increasing fertilization up to 100 kg N ha−1 without any significant changes at higher N rate. Fertilization hardly

  2. Ectopic Expression of Xylella fastidiosa rpfF Conferring Production of Diffusible Signal Factor in Transgenic Tobacco and Citrus Alters Pathogen Behavior and Reduces Disease Severity.

    Science.gov (United States)

    Caserta, R; Souza-Neto, R R; Takita, M A; Lindow, S E; De Souza, A A

    2017-11-01

    The pathogenicity of Xylella fastidiosa is associated with its ability to colonize the xylem of host plants. Expression of genes contributing to xylem colonization are suppressed, while those necessary for insect vector acquisition are increased with increasing concentrations of diffusible signal factor (DSF), whose production is dependent on RpfF. We previously demonstrated that transgenic citrus plants ectopically expressing rpfF from a citrus strain of X. fastidiosa subsp. pauca exhibited less susceptibility to Xanthomonas citri subsp. citri, another pathogen whose virulence is modulated by DSF accumulation. Here, we demonstrate that ectopic expression of rpfF in both transgenic tobacco and sweet orange also confers a reduction in disease severity incited by X. fastidiosa and reduces its colonization of those plants. Decreased disease severity in the transgenic plants was generally associated with increased expression of genes conferring adhesiveness to the pathogen and decreased expression of genes necessary for active motility, accounting for the reduced population sizes achieved in the plants, apparently by limiting pathogen dispersal through the plant. Plant-derived DSF signal molecules in a host plant can, therefore, be exploited to interfere with more than one pathogen whose virulence is controlled by DSF signaling.

  3. Cloning of two individual cDNAS encoding 9-cis-epoxycarotenoid dioxygenase from Gentiana lutea, their tissue-specific expression and physiological effect in transgenic tobacco.

    Science.gov (United States)

    Zhu, Changfu; Kauder, Friedrich; Römer, Susanne; Sandmann, Gerhard

    2007-02-01

    Two 9-cis-epoxycarotenoid dioxygenase (NCED) cDNAs have been cloned from a petal library of Gentiana lutea. Both cDNAs carry a putative transit sequence for chloroplast import and differ mainly in their length and the 5'-flanking regions. GlNCED1 was evolutionary closely related to Arabidopsis thaliana NCED6 whereas GlNCED2 showed highest homology to tomato NCED1 and A. thaliana NCED3. The amounts of GlNCED2 transcript were below Northern detection in G. lutea. In contrast, GlNCED1 was specifically expressed at higher levels in developing flowers when petals start appearing. By genetic engineering of tobacco with coding regions of either gene under a constitutive promoter, their function was further analyzed. Although mRNA of both genes was detectable in the corresponding transgenic plants, a physiological effect was only found for GlNCED1 but not for GlNCED2. In germination experiments of GlNCED1 transgenic lines, delayed radicle formation and cotyledon appearance were observed. However, the transformants exhibited no improved tolerance against desiccation stress. In contrast to other plants with over-expressed NCEDs, prolonged delay of seed germination is the only abscisic-acid-related phenotypic effect in the GlNCED1 transgenic lines.

  4. 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

  5. Stress-inducible expression of an F-box gene TaFBA1 from wheat enhanced the drought tolerance in transgenic tobacco plants without impacting growth and development

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    Xiangzhu Kong

    2016-09-01

    Full Text Available E3 ligase plays an important role in the response to many environment stresses in plants. In our previous study, constitutive overexpression of an F-box protein gene TaFBA1 driven by 35S promoter improved the drought tolerance in transgenic tobacco plants, but the growth and development in transgenic plants was altered in normal conditions. In this study, we used stress-inducible promoter RD29A instead of 35S promoter, as a results, the stress-inducible transgenic tobacco plants exhibit a similar phenotype with WT plants. However, the drought tolerance of the transgenic plants with stress-inducible expressed TaFBA1 was enhanced. The improved drought tolerance of transgenic plants was indicated by their higher seed germination rate and survival rate, greater biomass and photosynthesis than those of WT under water stress, which may be related to their greater water retention capability and osmotic adjustment. Moreover, the transgenic plants accumulated less reactive oxygen species (ROS, kept lower MDA content and membrane leakage under water stress, which may be related to their higher levels of antioxidant enzyme activity and upregulated gene expression of some antioxidant enzymes. These results suggest that stress induced expression of TaFBA1 confers drought tolerance via the improved water retention and antioxidative compete abilibty. Meanwhile, this stress-inducible expression strategy by RD29A promoter can minimize the unexpectable effects by 35S constitutive promoter on phenotypes of the transgenic plants.

  6. Stress-Inducible Expression of an F-box Gene TaFBA1 from Wheat Enhanced the Drought Tolerance in Transgenic Tobacco Plants without Impacting Growth and Development.

    Science.gov (United States)

    Kong, Xiangzhu; Zhou, Shumei; Yin, Suhong; Zhao, Zhongxian; Han, Yangyang; Wang, Wei

    2016-01-01

    E3 ligase plays an important role in the response to many environment stresses in plants. In our previous study, constitutive overexpression of an F-box protein gene TaFBA1 driven by 35S promoter improved the drought tolerance in transgenic tobacco plants, but the growth and development in transgenic plants was altered in normal conditions. In this study, we used stress-inducible promoter RD29A instead of 35S promoter, as a results, the stress-inducible transgenic tobacco plants exhibit a similar phenotype with wild type (WT) plants. However, the drought tolerance of the transgenic plants with stress-inducible expressed TaFBA1 was enhanced. The improved drought tolerance of transgenic plants was indicated by their higher seed germination rate and survival rate, greater biomass and photosynthesis than those of WT under water stress, which may be related to their greater water retention capability and osmotic adjustment. Moreover, the transgenic plants accumulated less reactive oxygen species, kept lower MDA content and membrane leakage under water stress, which may be related to their higher levels of antioxidant enzyme activity and upregulated gene expression of some antioxidant enzymes. These results suggest that stress induced expression of TaFBA1 confers drought tolerance via the improved water retention and antioxidative compete ability. Meanwhile, this stress-inducible expression strategy by RD29A promoter can minimize the unexpectable effects by 35S constitutive promoter on phenotypes of the transgenic plants.

  7. 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.

  8. Overexpression of a cytosolic abiotic stress responsive universal stress protein (SbUSP mitigates salt and osmotic stress in transgenic tobacco plants

    Directory of Open Access Journals (Sweden)

    Pushpika eUdawat

    2016-04-01

    Full Text Available The Universal Stress Protein (USP is a ubiquitous protein and plays an indispensable role in plant abiotic stress tolerance. The genome of Salicornia brachiata contains two homologues of intron less SbUSP gene which encodes for salt and osmotic responsive universal stress protein. In vivo localization reveals that SbUSP is a membrane bound cytosolic protein. The role of the gene was functionally validated by developing transgenic tobacco and compared with control (wild type and vector control plants under different abiotic stress condition. Transgenic lines (T1 exhibited higher chlorophyll, relative water, proline, total sugar, reducing sugar, free amino acids, polyphenol contents, osmotic potential, membrane stability and lower electrolyte leakage and lipid peroxidation (malondialdehyde content under stress treatments than control (WT and VC plants. Lower accumulation of H2O2 and O2- radicals was also detected in transgenic lines compared to control plants under stress conditions. Present study confers that overexpression of the SbUSP gene enhances plant growth, alleviates ROS buildup, maintains ion homeostasis and improves the physiological status of the plant under salt and osmotic stresses. Principal component analysis (PCA exhibited a statistical distinction of plant response to salinity stress, and a significant response was observed for transgenic lines under stress, which provides stress endurance to the plant. A possible signaling role is proposed that some downstream genes may get activated by abiotic stress responsive cytosolic SbUSP, which leads to the protection of cell from oxidative damages. The study unveils that ectopic expression of the gene mitigates salt or osmotic stress by scavenging ROS and modulating the physiological process of the plant.

  9. Overexpression of a Cytosolic Abiotic Stress Responsive Universal Stress Protein (SbUSP) Mitigates Salt and Osmotic Stress in Transgenic Tobacco Plants

    Science.gov (United States)

    Udawat, Pushpika; Jha, Rajesh K.; Sinha, Dinkar; Mishra, Avinash; Jha, Bhavanath

    2016-01-01

    The universal stress protein (USP) is a ubiquitous protein and plays an indispensable role in plant abiotic stress tolerance. The genome of Salicornia brachiata contains two homologs of intron less SbUSP gene which encodes for salt and osmotic responsive USP. In vivo localization reveals that SbUSP is a membrane bound cytosolic protein. The role of the gene was functionally validated by developing transgenic tobacco and compared with control [wild-type (WT) and vector control (VC)] plants under different abiotic stress condition. Transgenic lines (T1) exhibited higher chlorophyll, relative water, proline, total sugar, reducing sugar, free amino acids, polyphenol contents, osmotic potential, membrane stability, and lower electrolyte leakage and lipid peroxidation (malondialdehyde content) under stress treatments than control (WT and VC) plants. Lower accumulation of H2O2 and O2− radicals was also detected in transgenic lines compared to control plants under stress conditions. Present study confers that overexpression of the SbUSP gene enhances plant growth, alleviates ROS buildup, maintains ion homeostasis and improves the physiological status of the plant under salt and osmotic stresses. Principal component analysis exhibited a statistical distinction of plant response to salinity stress, and a significant response was observed for transgenic lines under stress, which provides stress endurance to the plant. A possible signaling role is proposed that some downstream genes may get activated by abiotic stress responsive cytosolic SbUSP, which leads to the protection of cell from oxidative damages. The study unveils that ectopic expression of the gene mitigates salt or osmotic stress by scavenging ROS and modulating the physiological process of the plant. PMID:27148338

  10. Involvement of ethylene in lesion development and systemic acquired resistance in tobacco during the hypersensitive reaction to tobacco mosaic virus

    NARCIS (Netherlands)

    Knoester, M.; Linthorst, H.J.M.; Bol, J.F.; Loon, L.C. van

    2001-01-01

    Different approaches were taken to investigate the significance of ethylene in lesion development and systemic acquired resistance (SAR) in tobacco (Nicotiana tabacum) reacting hypersensitively to tobacco mosaic virus (TMV). Gaseous ethylene, the ethylene precursor 1-aminocyclopropane-1-carboxylic

  11. Tissue-specific and pathogen-induced regulation of a Nicotiana plumbaginifolia beta-1,3-glucanase gene.

    Science.gov (United States)

    Castresana, C; de Carvalho, F; Gheysen, G; Habets, M; Inzé, D; Van Montagu, M

    1990-01-01

    The Nicotiana plumbaginifolia gn1 gene encoding a beta-1,3-glucanase isoform has been characterized. The gn1 product represents an isoform distinct from the previously identified tobacco beta-1,3-glucanases. By expressing gn1 in Escherichia coli, we have determined directly that the encoded protein does, indeed, correspond to a beta-1,3-glucanase. In N. plumbaginifolia, gn1 was found to be expressed in roots and older leaves. Transgenic tobacco plants containing the 5'-noncoding region of gn1 fused to the beta-glucuronidase (GUS) reporter gene also showed maximum levels of GUS activity in roots and older leaves. No detectable activity was present in the upper part of the transgenic plants with the exception of stem cells at the bases of emerging shoots. The expression conferred by the gn1 promoter was differentially induced in response to specific plant stress treatments. Studies of three plant-bacteria interactions showed high levels of GUS activity when infection resulted in a hypersensitive reaction. Increased gene expression was confined to cells surrounding the necrotic lesions. The observed expression pattern suggests that the characterized beta-1,3-glucanase plays a role both in plant development and in the defense response against pathogen infection. PMID:2152158

  12. Regulatory dephosphorylation of CDK at G₂/M in plants: yeast mitotic phosphatase cdc25 induces cytokinin-like effects in transgenic tobacco morphogenesis.

    Science.gov (United States)

    Lipavská, Helena; Masková, Petra; Vojvodová, Petra

    2011-05-01

    During the last three decades, the cell cycle and its control by cyclin-dependent kinases (CDKs) have been extensively studied in eukaryotes. This endeavour has produced an overall picture that basic mechanisms seem to be largely conserved among all eukaryotes. The intricate regulation of CDK activities includes, among others, CDK activation by CDC25 phosphatase at G₂/M. In plants, however, studies of this regulation have lagged behind as a plant Cdc25 homologue or other unrelated phosphatase active at G₂/M have not yet been identified. Failure to identify a plant mitotic CDK activatory phosphatase led to characterization of the effects of alien cdc25 gene expression in plants. Tobacco, expressing the Schizosaccharomyces pombe mitotic activator gene, Spcdc25, exhibited morphological, developmental and biochemical changes when compared with wild type (WT) and, importantly, increased CDK dephosphorylation at G₂/M. Besides changes in leaf shape, internode length and root development, in day-neutral tobacco there was dramatically earlier onset of flowering with a disturbed acropetal floral capacity gradient typical of WT. In vitro, de novo organ formation revealed substantially earlier and more abundant formation of shoot primordia on Spcdc25 tobacco stem segments grown on shoot-inducing media when compared with WT. Moreover, in contrast to WT, stem segments from transgenic plants formed shoots even without application of exogenous growth regulator. Spcdc25-expressing BY-2 cells exhibited a reduced mitotic cell size due to a shortening of the G₂ phase together with high activity of cyclin-dependent kinase, NtCDKB1, in early S-phase, S/G₂ and early M-phase. Spcdc25-expressing tobacco ('Samsun') cell suspension cultures showed a clustered, more circular, cell phenotype compared with chains of elongated WT cells, and increased content of starch and soluble sugars. Taken together, Spcdc25 expression had cytokinin-like effects on the characteristics studied

  13. Overexpression of a Medicago truncatula stress-associated protein gene (MtSAP1) leads to nitric oxide accumulation and confers osmotic and salt stress tolerance in transgenic tobacco.

    Science.gov (United States)

    Charrier, Aurélie; Planchet, Elisabeth; Cerveau, Delphine; Gimeno-Gilles, Christine; Verdu, Isabelle; Limami, Anis M; Lelièvre, Eric

    2012-08-01

    The impact of Medicago truncatula stress-associated protein gene (MtSAP1) overexpression has been investigated in Nicotiana tabacum transgenic seedlings. Under optimal conditions, transgenic lines overexpressing MtSAP1 revealed better plant development and higher chlorophyll content as compared to wild type seedlings. Interestingly, transgenic lines showed a stronger accumulation of nitric oxide (NO), a signaling molecule involved in growth and development processes. This NO production seemed to be partially nitrate reductase dependent. Due to the fact that NO has been also reported to play a role in tolerance acquisition of plants to abiotic stresses, the responses of MtSAP1 overexpressors to osmotic and salt stress have been studied. Compared to the wild type, transgenic lines were less affected in their growth and development. Moreover, NO content in MtSAP1 overexpressors was always higher than that detected in wild seedlings under stress conditions. It seems that this better tolerance induced by MtSAP1 overexpression could be associated with this higher NO production that would enable seedlings to reach a high protection level to prepare them to cope with abiotic stresses.

  14. A ThDREB gene from Tamarix hispida improved the salt and drought tolerance of transgenic tobacco and T. hispida.

    Science.gov (United States)

    Yang, Guiyan; Yu, Lili; Zhang, Kaimin; Zhao, Yulin; Guo, Yucong; Gao, Caiqiu

    2017-04-01

    Dehydration-responsive element-binding (DREB) transcription factors are important abiotic stress tolerance related genes, and some reports on the roles of DREB have primarily addressed herbal plants. To explore the abiotic stress tolerance role of DREB (ThDREB) from Tamarix hispida, a ThDREB gene with a complete ORF of 783 bp that encodes a 28.74 kDa protein with 260 amino acids, was isolated and functionally annotated. ThDREB expression was highly induced by NaCl, PEG, NaHCO 3 and CdCl 2 treatments, and the highest expression level (369.2-fold of control) was found for the roots that were under NaCl stress for 6 h. The tobacco plants that were transformed by ThDREB were conferred with higher germination rates, fresh weights and root lengths than the wild type (WT) tobacco plants under NaCl and mannitol treatments. The total chlorophyll content (tcc), superoxide dismutase (SOD) and peroxidase (POD) activities were also higher in the transgenic lines in comparison with the WT, and the malondialdehyde (MDA) and H 2 O 2 content, electrolyte leakage (EL) rate and ROS as tracked by staining were generated to a lesser degree in ThDREB transgenic plants than in the WT under NaCl and mannitol stress. Furthermore, the transient overexpression analysis of ThDREB in T. hispida also improved plant salt and drought tolerance in comparison with the empty vector-transformed lines. Our results indicated that ThDREB expression could effectively improve tolerance to salt and drought stress by enhancing the antioxidase activity that keeps the ROS at a low accumulation level and makes them easy to scavenge. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  15. A self-excising Cre recombinase allows efficient recombination of multiple ectopic heterospecific lox sites in transgenic tobacco

    NARCIS (Netherlands)

    Mlynarova, L.; Nap, J.P.H.

    2003-01-01

    To study the impact of different DNA configurations on the stability of transgene expression, a variant of the cre gene was developed. This variant allows for the highly efficient in planta removal of its own loxP-flanked coding sequence as well as other DNAs flanked by ectopic heterospecific lox

  16. Pronounced Phenotypic Changes in Transgenic Tobacco Plants Overexpressing Sucrose Synthase May Reveal a Novel Sugar Signaling Pathway

    Science.gov (United States)

    Nguyen, Quynh Anh; Luan, Sheng; Wi, Seung G.; Bae, Hanhong; Lee, Dae-Seok; Bae, Hyeun-Jong

    2016-01-01

    Soluble sugars not only serve as nutrients, but also act as signals for plant growth and development, but how sugar signals are perceived and translated into physiological responses in plants remains unclear. We manipulated sugar levels in transgenic plants by overexpressing sucrose synthase (SuSy), which is a key enzyme believed to have reversible sucrose synthesis and sucrose degradation functions. The ectopically expressed SuSy protein exhibited sucrose-degrading activity, which may change the flux of sucrose demand from photosynthetic to non-photosynthetic cells, and trigger an unknown sucrose signaling pathway that lead to increased sucrose content in the transgenic plants. An experiment on the transition from heterotrophic to autotrophic growth demonstrated the existence of a novel sucrose signaling pathway, which stimulated photosynthesis, and enhanced photosynthetic synthesis of sucrose, which was the direct cause or the sucrose increase. In addition, a light/dark time treatment experiment, using different day length ranges for photosynthesis/respiration showed the carbohydrate pattern within a 24-h day and consolidated the role of sucrose signaling pathway as a way to maintain sucrose demand, and indicated the relationships between increased sucrose and upregulation of genes controlling development of the shoot apical meristem (SAM). As a result, transgenic plants featured a higher biomass and a shorter time required to switch to reproduction compared to those of control plants, indicating altered phylotaxis and more rapid advancement of developmental stages in the transgenic plants. PMID:26793204

  17. Antioxidant protection during ageing and senescence in transgenic tobacco with enhanced activity of cytokinin oxidase/dehydrogenase

    Czech Academy of Sciences Publication Activity Database

    Procházková, Dagmar; Wilhelmová, Naděžda

    2009-01-01

    Roč. 53, č. 4 (2009), s. 691-696 ISSN 0006-3134 R&D Projects: GA ČR GP522/05/P558 Institutional research plan: CEZ:AV0Z50380511 Keywords : oxidative stress * reactive oxygen species * transgenic plants Subject RIV: ED - Physiology Impact factor: 1.656, year: 2009

  18. Overexpression of a Triticum aestivum Calreticulin gene (TaCRT1 Improves Salinity Tolerance in Tobacco.

    Directory of Open Access Journals (Sweden)

    Yang Xiang

    Full Text Available Calreticulin (CRT is a highly conserved and abundant multifunctional protein that is encoded by a small gene family and is often associated with abiotic/biotic stress responses in plants. However, the roles played by this protein in salt stress responses in wheat (Triticum aestivum remain obscure. In this study, three TaCRT genes were identified in wheat and named TaCRT1, TaCRT2 and TaCRT3-1 based on their sequence characteristics and their high homology to other known CRT genes. Quantitative real-time PCR expression data revealed that these three genes exhibit different expression patterns in different tissues and are strongly induced under salt stress in wheat. The calcium-binding properties of the purified recombinant TaCRT1 protein were determined using a PIPES/Arsenazo III analysis. TaCRT1 gene overexpression in Nicotiana tabacum decreased salt stress damage in transgenic tobacco plants. Physiological measurements indicated that transgenic tobacco plants showed higher activities of superoxide dismutase (SOD, peroxidase (POD and catalase (CAT than non-transgenic tobacco under normal growth conditions. Interestingly, overexpression of the entire TaCRT1 gene or of partial TaCRT1 segments resulted in significantly higher tolerance to salt stress in transgenic plants compared with their WT counterparts, thus revealing the essential role of the C-domain of TaCRT1 in countering salt stress in plants.

  19. Novel AroA from Pseudomonas putida Confers Tobacco Plant with High Tolerance to Glyphosate

    Science.gov (United States)

    Yan, Hai-Qin; Chang, Su-Hua; Tian, Zhe-Xian; Zhang, Le; Sun, Yi-Cheng; Li, Yan; Wang, Jing; Wang, Yi-Ping

    2011-01-01

    Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, also designated as AroA), a key enzyme in the aromatic amino acid biosynthesis pathway in microorganisms and plants. Previously, we reported that a novel AroA (PpAroA1) from Pseudomonas putida had high tolerance to glyphosate, with little homology to class I or class II glyphosate-tolerant AroA. In this study, the coding sequence of PpAroA1 was optimized for tobacco. For maturation of the enzyme in chloroplast, a chloroplast transit peptide coding sequence was fused in frame with the optimized aroA gene (PparoA1optimized) at the 5′ end. The PparoA1optimized gene was introduced into the tobacco (Nicotiana tabacum L. cv. W38) genome via Agrobacterium-mediated transformation. The transformed explants were first screened in shoot induction medium containing kanamycin. Then glyphosate tolerance was assayed in putative transgenic plants and its T1 progeny. Our results show that the PpAroA1 from Pseudomonas putida can efficiently confer tobacco plants with high glyphosate tolerance. Transgenic tobacco overexpressing the PparoA1optimized gene exhibit high tolerance to glyphosate, which suggest that the novel PpAroA1 is a new and good candidate applied in transgenic crops with glyphosate tolerance in future. PMID:21611121

  20. Phylogenetic fragrance patterns in Nicotiana sections Alatae and Suaveolentes.

    Science.gov (United States)

    Raguso, Robert A; Schlumpberger, Boris O; Kaczorowski, Rainee L; Holtsford, Timothy P

    2006-09-01

    We analyzed floral volatiles from eight tobacco species (Nicotiana; Solanaceae) including newly discovered Brazilian taxa (Nicotiana mutabilis and "Rastroensis") in section Alatae. Eighty-four compounds were found, including mono- and sesquiterpenoids, nitrogenous compounds, benzenoid and aliphatic alcohols, aldehydes and esters. Floral scent from recent accessions of Nicotiana alata, Nicotiana bonariensis and Nicotiana langsdorffii differed from previously published data, suggesting intraspecific variation in scent composition at the level of biosynthetic class. Newly discovered taxa in Alatae, like their relatives, emit large amounts of 1,8-cineole and smaller amounts of monoterpenes on a nocturnal rhythm, constituting a chemical synapomorphy for this lineage. Fragrance data from three species of Nicotiana sect. Suaveolentes, the sister group of Alatae, (two Australian species: N. cavicola, N. ingulba; one African species: N. africana), were compared to previously reported data from their close relative, N. suaveolens. Like N. suaveolens, N. cavicola and N. ingulba emit fragrances dominated by benzenoids and phenylpropanoids, whereas the flowers of N. africana lacked a distinct floral scent and instead emitted only small amounts of an aliphatic methyl ester from foliage. Interestingly, this ester also is emitted from foliage of N. longiflora and N. plumbaginifolia (both in section Alatae s.l.), which share a common ancestor with N. africana. This result, combined with the synapomorphic pattern of 1,8 cineole emission in Alatae s.s., suggests that phylogenetic signal explains a major component of fragrance composition among tobacco species in sections Alatae and Suaveolentes. At the intraspecific level, interpopulational scent variation is widespread in sect. Alatae, and may reflect edaphic specialization, introgression, local pollinator shifts, genetic drift or artificial selection in cultivation. Further studies with genetically and geographically well

  1. Targeted Modification of Homogalacturonan by Transgenic Expression of a Fungal Polygalacturonase Alters Plant Growth1

    Science.gov (United States)

    Capodicasa, Cristina; Vairo, Donatella; Zabotina, Olga; McCartney, Lesley; Caprari, Claudio; Mattei, Benedetta; Manfredini, Cinzia; Aracri, Benedetto; Benen, Jacques; Knox, J. Paul; De Lorenzo, Giulia; Cervone, Felice

    2004-01-01

    Pectins are a highly complex family of cell wall polysaccharides comprised of homogalacturonan (HGA), rhamnogalacturonan I and rhamnogalacturonan II. We have specifically modified HGA in both tobacco (Nicotiana tabacum) and Arabidopsis by expressing the endopolygalacturonase II of Aspergillus niger (AnPGII). Cell walls of transgenic tobacco plants showed a 25% reduction in GalUA content as compared with the wild type and a reduced content of deesterified HGA as detected by antibody labeling. Neutral sugars remained unchanged apart from a slight increase of Rha, Ara, and Gal. Both transgenic tobacco and Arabidopsis were dwarfed, indicating that unesterified HGA is a critical factor for plant cell growth. The dwarf phenotypes were associated with AnPGII activity as demonstrated by the observation that the mutant phenotype of tobacco was completely reverted by crossing the dwarfed plants with plants expressing PGIP2, a strong inhibitor of AnPGII. The mutant phenotype in Arabidopsis did not appear when transformation was performed with a gene encoding AnPGII inactivated by site directed mutagenesis. PMID:15247378

  2. Ectopic Expression of the Coleus R2R3 MYB-Type Proanthocyanidin Regulator Gene SsMYB3 Alters the Flower Color in Transgenic Tobacco.

    Directory of Open Access Journals (Sweden)

    Qinlong Zhu

    Full Text Available Proanthocyanidins (PAs play an important role in plant disease defense and have beneficial effects on human health. We isolated and characterized a novel R2R3 MYB-type PA-regulator SsMYB3 from a well-known ornamental plant, coleus (Solenostemon scutellarioides, to study the molecular regulation of PAs and to engineer PAs biosynthesis. The expression level of SsMYB3 was correlated with condensed tannins contents in various coleus tissues and was induced by wounding and light. A complementation test in the Arabidopsis tt2 mutant showed that SsMYB3 could restore the PA-deficient seed coat phenotype and activated expression of the PA-specific gene ANR and two related genes, DFR and ANS. In yeast two-hybrid assays, SsMYB3 interacted with the Arabidopsis AtTT8 and AtTTG1 to reform the ternary transcriptional complex, and also interacted with two tobacco bHLH proteins (NtAn1a and NtJAF13-1 and a WD40 protein, NtAn11-1. Ectopic overexpression of SsMYB3 in transgenic tobacco led to almost-white flowers by greatly reducing anthocyanin levels and enhancing accumulation of condensed tannins. This overexpression of SsMYB3 upregulated the key PA genes (NtLAR and NtANR and late anthocyanin structural genes (NtDFR and NtANS, but downregulated the expression of the final anthocyanin gene NtUFGT. The formative SsMYB3-complex represses anthocyanin accumulation by directly suppressing the expression of the final anthocyanin structural gene NtUFGT, through competitive inhibition or destabilization of the endogenous NtAn2-complex formation. These results suggested that SsMYB3 may form a transcription activation complex to regulate PA biosynthesis in the Arabidopsis tt2 mutant and transgenic tobacco. Our findings suggest that SsMYB3 is involved in the regulation of PA biosynthesis in coleus and has the potential as a molecular tool for manipulating biosynthesis of PAs in fruits and other crops using metabolic engineering.

  3. 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.

  4. Pea p68, a DEAD-box helicase, provides salinity stress tolerance in transgenic tobacco by reducing oxidative stress and improving photosynthesis machinery.

    Science.gov (United States)

    Tuteja, Narendra; Banu, Mst Sufara Akhter; Huda, Kazi Md Kamrul; Gill, Sarvajeet Singh; Jain, Parul; Pham, Xuan Hoi; Tuteja, Renu

    2014-01-01

    The DEAD-box helicases are required mostly in all aspects of RNA and DNA metabolism and they play a significant role in various abiotic stresses, including salinity. The p68 is an important member of the DEAD-box proteins family and, in animal system, it is involved in RNA metabolism including pre-RNA processing and splicing. In plant system, it has not been well characterized. Here we report the cloning and characterization of p68 from pea (Pisum sativum) and its novel function in salinity stress tolerance in plant. The pea p68 protein self-interacts and is localized in the cytosol as well as the surrounding of cell nucleus. The transcript of pea p68 is upregulated in response to high salinity stress in pea. Overexpression of p68 driven by constitutive cauliflower mosaic virus-35S promoter in tobacco transgenic plants confers enhanced tolerances to salinity stress by improving the growth, photosynthesis and antioxidant machinery. Under stress treatment, pea p68 overexpressing tobacco accumulated higher K+ and lower Na+ level than the wild-type plants. Reactive oxygen species (ROS) accumulation was remarkably regulated by the overexpression of pea p68 under salinity stress conditions, as shown from TBARS content, electrolyte leakage, hydrogen peroxide accumulation and 8-OHdG content and antioxidant enzyme activities. To the best of our knowledge this is the first direct report, which provides the novel function of pea p68 helicase in salinity stress tolerance. The results suggest that p68 can also be exploited for engineering abiotic stress tolerance in crop plants of economic importance.

  5. Pea p68, a DEAD-box helicase, provides salinity stress tolerance in transgenic tobacco by reducing oxidative stress and improving photosynthesis machinery.

    Directory of Open Access Journals (Sweden)

    Narendra Tuteja

    Full Text Available The DEAD-box helicases are required mostly in all aspects of RNA and DNA metabolism and they play a significant role in various abiotic stresses, including salinity. The p68 is an important member of the DEAD-box proteins family and, in animal system, it is involved in RNA metabolism including pre-RNA processing and splicing. In plant system, it has not been well characterized. Here we report the cloning and characterization of p68 from pea (Pisum sativum and its novel function in salinity stress tolerance in plant.The pea p68 protein self-interacts and is localized in the cytosol as well as the surrounding of cell nucleus. The transcript of pea p68 is upregulated in response to high salinity stress in pea. Overexpression of p68 driven by constitutive cauliflower mosaic virus-35S promoter in tobacco transgenic plants confers enhanced tolerances to salinity stress by improving the growth, photosynthesis and antioxidant machinery. Under stress treatment, pea p68 overexpressing tobacco accumulated higher K+ and lower Na+ level than the wild-type plants. Reactive oxygen species (ROS accumulation was remarkably regulated by the overexpression of pea p68 under salinity stress conditions, as shown from TBARS content, electrolyte leakage, hydrogen peroxide accumulation and 8-OHdG content and antioxidant enzyme activities.To the best of our knowledge this is the first direct report, which provides the novel function of pea p68 helicase in salinity stress tolerance. The results suggest that p68 can also be exploited for engineering abiotic stress tolerance in crop plants of economic importance.

  6. Enhanced production of resveratrol derivatives in tobacco plants by improving the metabolic flux of intermediates in the phenylpropanoid pathway.

    Science.gov (United States)

    Jeong, Yu Jeong; An, Chul Han; Woo, Su Gyeong; Park, Ji Hye; Lee, Ki-Won; Lee, Sang-Hoon; Rim, Yeonggil; Jeong, Hyung Jae; Ryu, Young Bae; Kim, Cha Young

    2016-09-01

    The biosynthesis of flavonoids such as anthocyanin and stilbenes has attracted increasing attention because of their potential health benefits. Anthocyanins and stilbenes share common phenylpropanoid precursor pathways. We previously reported that the overexpression of sweetpotato IbMYB1a induced anthocyanin pigmentation in transgenic tobacco (Nicotiana tabacum) plants. In the present study, transgenic tobacco (Nicotiana tabacum SR1) plants (STS-OX and ROST-OX) expressing the RpSTS gene encoding stilbene synthase from rhubarb (Rheum palmatum L. cv. Jangyeop) and the RpSTS and VrROMT genes encoding resveratrol O-methyltransferase from frost grape (Vitis riparia) were generated under the control of 35S promoter. Phenotypic alterations in floral organs, such as a reduction in floral pigments and male sterility, were observed in STS-OX transgenic tobacco plants. However, we failed to obtain STS-OX and ROST-OX plants with high levels of resveratrol compounds. Therefore, to improve the production of resveratrol derivatives in plants, we cross-pollinated flowers of STS-OX or ROST-OX and IbMYB1a-OX transgenic lines (SM and RSM). Phenotypic changes in vegetative and reproductive development of SM and RSM plants were observed. Furthermore, by HPLC and LC-MS analyses, we found enhanced production of resveratrol derivatives such as piceid, piceid methyl ether, resveratrol methyl ether O-hexoside, and 5-methyl resveratrol-3,4'-O-β-D-diglucopyranoside in SM and RSM cross-pollinated lines. Here, total contents of trans- and cis-piceids ranged from approximately 104-240 µg/g fresh weight in SM (F2). Collectively, we suggest that coexpression of RpSTS and IbMYB1a via cross-pollination can induce enhanced production of resveratrol compounds in plants by increasing metabolic flux into stilbenoid biosynthesis.

  7. Regulation of galactolipid biosynthesis by overexpression of the rice MGD gene contributes to enhanced aluminum tolerance in tobacco

    Directory of Open Access Journals (Sweden)

    Meijuan eZhang

    2016-03-01

    Full Text Available Membrane lipid alterations affect Al tolerance in plants, but little is known about the regulation of membrane lipid metabolism in response to Al stress. Transgenic tobacco (Nicotiana tabacum overexpressing rice monogalactosyldiacylglycerol (MGDG synthase (OsMGD gene and wild-type tobacco plants were exposed to AlCl3, and the impact of Al toxicity on root growth, Al accumulation, plasma membrane integrity, lipid peroxidation and membrane lipid composition were investigated. Compared with the wild type, the transgenic plants exhibited rapid regrowth of roots after removal of Al and less damage to membrane integrity and lipid peroxidation under Al stress, meanwhile, the Al accumulation showed no difference between wild-type and transgenic plants. Lipid analysis showed that Al treatment dramatically decreased the content of MGDG and the ratio of MGDG to digalactosyldiacylglycerol (DGDG in wild-type plants, while it was unchanged in transgenic plants. The stable of MGDG level and the ratio of MGDG/DGDG contribute to maintain the membrane stability and permeability. Moreover, Al caused a significant increase in phospholipids in wild-type plants, resulting in a high proportion of phospholipids and low proportion of galactolipids, but these proportions were unaffected in transgenic plants. The high proportion of phospholipids could contribute to a higher rate of Al3+ binding in the membrane and thereby leads to more membrane perturbation and damage. These results show that the regulation of galactolipid biosynthesis could play an important role in maintaining membrane structure and function under Al stress.

  8. Isolation and characterization of a Vitis vinifera transcription factor, VvWRKY1, and its effect on responses to fungal pathogens in transgenic tobacco plants.

    Science.gov (United States)

    Marchive, Chloé; Mzid, Rim; Deluc, Laurent; Barrieu, François; Pirrello, Julien; Gauthier, Adrien; Corio-Costet, Marie-France; Regad, Farid; Cailleteau, Bernard; Hamdi, Saïd; Lauvergeat, Virginie

    2007-01-01

    Pathogen attack represents a major problem for viticulture and for agriculture in general. At present, the use of phytochemicals is more and more restrictive, and therefore it is becoming essential to control disease by having a thorough knowledge of resistance mechanisms. The present work focused on the trans-regulatory proteins potentially involved in the control of the plant defence response, the WRKY proteins. A full-length cDNA, designated VvWRKY1, was isolated from a grape berry library (Vitis vinifera L. cv. Cabernet Sauvignon). It encodes a polypeptide of 151 amino acids whose structure is characteristic of group IIc WRKY proteins. VvWRKY1 gene expression in grape is regulated in a developmental manner in berries and leaves and by various signal molecules involved in defence such as salicylic acid, ethylene, and hydrogen peroxide. Biochemical analysis indicates that VvWRKY1 specifically interacts with the W-box in various nucleotidic contexts. Functional analysis of VvWRKY1 was performed by overexpression in tobacco, and transgenic plants exhibited reduced susceptibility to various fungi but not to viruses. These results are consistent with a possible role for VvWRKY1 in grapevine defence against fungal pathogens.

  9. Ntdin, a tobacco senescence-associated gene, is involved in molybdenum cofactor biosynthesis.

    Science.gov (United States)

    Yang, Seung Hwan; Berberich, Thomas; Miyazaki, Atsushi; Sano, Hiroshi; Kusano, Tomonobu

    2003-10-01

    To date, dozens of genes have been reported to be up-regulated with senescence in higher plants. Radish din1 and its ortholog sen1 of Arabidopsis are known as such, but their function is not clear yet. Here we have isolated their counterpart cDNA from tobacco and designated it as NTDIN: Its product, Ntdin, a 185 amino acid polypeptide with 56.8% and 54.2% identity to Atsen1 and Rsdin1, respectively, is localized in chloroplasts. Transcripts of Ntdin are induced by sulfate or nitrate but not by phosphate, suggesting its involvement in sulfur and nitrogen metabolism. A database search revealed that Ntdin shows similarity with the C-terminal region of Nicotiana plumbaginifolia Cnx5, which functions in molybdenum cofactor (Moco) biosynthesis. Transgenic tobacco plants with suppressed Ntdin are more tolerant to chlorate, a substrate analog of nitrate reductase, than controls, implying low nitrate reductase activity in the transgenic plants due to a deficiency of Moco. Indeed, enzymatic activities of two molybdoenzymes, nitrate reductase and xanthine dehydrogenase, in transgenic plants are found to be significantly lower than in control plants. Direct measurement of Moco contents reveals that those transgenic plants contain about 5% Moco of those of the control plants. Abscisic acid and indole-3-acidic acid, whose biosynthetic pathways require Moco, up-regulated Ntdin expression. Taken together, it is concluded that Ntdin functions in a certain step in Moco biosynthesis.

  10. Genomes and virulence difference between two physiological races of Phytophthora nicotianae.

    Science.gov (United States)

    Liu, Hui; Ma, Xiao; Yu, Haiqin; Fang, Dunhuang; Li, Yongping; Wang, Xiao; Wang, Wen; Dong, Yang; Xiao, Bingguang

    2016-01-01

    Black shank is a severe plant disease caused by the soil-borne pathogen Phytophthora nicotianae. Two physiological races of P. nicotianae, races 0 and 1, are predominantly observed in cultivated tobacco fields around the world. Race 0 has been reported to be more aggressive, having a shorter incubation period, and causing worse root rot symptoms, while race 1 causes more severe necrosis. The molecular mechanisms underlying the difference in virulence between race 0 and 1 remain elusive. We assembled and annotated the genomes of P. nicotianae races 0 and 1, which were obtained by a combination of PacBio single-molecular real-time sequencing and second-generation sequencing (both HiSeq and MiSeq platforms). Gene family analysis revealed a highly expanded ATP-binding cassette transporter gene family in P. nicotianae. Specifically, more RxLR effector genes were found in the genome of race 0 than in that of race 1. In addition, RxLR effector genes were found to be mainly distributed in gene-sparse, repeat-rich regions of the P. nicotianae genome. These results provide not only high quality reference genomes of P. nicotianae, but also insights into the infection mechanisms of P. nicotianae and its co-evolution with the host plant. They also reveal insights into the difference in virulence between the two physiological races.

  11. Silencing Nicotiana attenuata LHY and ZTL alters circadian rhythms in flowers.

    Science.gov (United States)

    Yon, Felipe; Joo, Youngsung; Cortés Llorca, Lucas; Rothe, Eva; Baldwin, Ian T; Kim, Sang-Gyu

    2016-02-01

    The rhythmic opening/closing and volatile emissions of flowers are known to attract pollinators at specific times. That these rhythms are maintained under constant light or dark conditions suggests a circadian clock involvement. Although a forward and reverse genetic approach has led to the identification of core circadian clock components in Arabidopsis thaliana, the involvement of these clock components in floral rhythms has remained untested, probably because of the weak diurnal rhythms in A. thaliana flowers. Here, we addressed the role of these core clock components in the flowers of the wild tobacco Nicotiana attenuata, whose flowers open at night, emit benzyl acetone (BA) scents and move vertically through a 140° arc. We first measured N. attenuata floral rhythms under constant light conditions. The results suggest that the circadian clock controls flower opening, BA emission and pedicel movement, but not flower closing. We generated transgenic N. attenuata lines silenced in the homologous genes of Arabidopsis LATE ELONGATED HYPOCOTYL (LHY) and ZEITLUPE (ZTL), which are known to be core clock components. Silencing NaLHY and NaZTL strongly altered floral rhythms in different ways, indicating that conserved clock components in N. attenuata coordinate these floral rhythms. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  12. 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.

  13. Overexpression of a flower-specific aerolysin-like protein from the dioecious plant Rumex acetosa alters flower development and induces male sterility in transgenic tobacco.

    Science.gov (United States)

    Manzano, Susana; Megías, Zoraida; Martínez, Cecilia; García, Alicia; Aguado, Encarnación; Chileh, Tarik; López-Alonso, Diego; García-Maroto, Federico; Kejnovský, Eduard; Široký, Jiří; Kubát, Zdeněk; Králová, Tereza; Vyskot, Boris; Jamilena, Manuel

    2017-01-01

    Sex determination in Rumex acetosa, a dioecious plant with a complex XY 1 Y 2 sex chromosome system (females are XX and males are XY 1 Y 2 ), is not controlled by an active Y chromosome but depends on the ratio between the number of X chromosomes and autosomes. To gain insight into the molecular mechanisms of sex determination, we generated a subtracted cDNA library enriched in genes specifically or predominantly expressed in female floral buds in early stages of development, when sex determination mechanisms come into play. In the present paper, we report the molecular and functional characterization of FEM32, a gene encoding a protein that shares a common architecture with proteins in different plants, animals, bacteria and fungi of the aerolysin superfamily; many of these function as β pore-forming toxins. The expression analysis, assessed by northern blot, RT-PCR and in situ hybridization, demonstrates that this gene is specifically expressed in flowers in both early and late stages of development, although its transcripts accumulate much more in female flowers than in male flowers. The ectopic expression of FEM32 under both the constitutive promoter 35S and the flower-specific promoter AP3 in transgenic tobacco showed no obvious alteration in vegetative development but was able to alter floral organ growth and pollen fertility. The 35S::FEM32 and AP3::FEM32 transgenic lines showed a reduction in stamen development and pollen viability, as well as a diminution in fruit set, fruit development and seed production. Compared with other floral organs, pistil development was, however, enhanced in plants overexpressing FEM32. According to these effects, it is likely that FEM32 functions in Rumex by arresting stamen and pollen development during female flower development. The aerolysin-like pore-forming proteins of eukaryotes are mainly involved in defence mechanisms against bacteria, fungi and insects and are also involved in apoptosis and programmed cell death (PCD

  14. High Levels of Bioplastic Are Produced in Fertile Transplastomic Tobacco Plants Engineered with a Synthetic Operon for the Production of Polyhydroxybutyrate1[C][OA

    Science.gov (United States)

    Bohmert-Tatarev, Karen; McAvoy, Susan; Daughtry, Sean; Peoples, Oliver P.; Snell, Kristi D.

    2011-01-01

    An optimized genetic construct for plastid transformation of tobacco (Nicotiana tabacum) for the production of the renewable, biodegradable plastic polyhydroxybutyrate (PHB) was designed using an operon extension strategy. Bacterial genes encoding the PHB pathway enzymes were selected for use in this construct based on their similarity to the codon usage and GC content of the tobacco plastome. Regulatory elements with limited homology to the host plastome yet known to yield high levels of plastidial recombinant protein production were used to enhance the expression of the transgenes. A partial transcriptional unit, containing genes of the PHB pathway and a selectable marker gene encoding spectinomycin resistance, was flanked at the 5′ end by the host plant’s psbA coding sequence and at the 3′ end by the host plant’s 3′ psbA untranslated region. This design allowed insertion of the transgenes into the plastome as an extension of the psbA operon, rendering the addition of a promoter to drive the expression of the transgenes unnecessary. Transformation of the optimized construct into tobacco and subsequent spectinomycin selection of transgenic plants yielded T0 plants that were capable of producing up to 18.8% dry weight PHB in samples of leaf tissue. These plants were fertile and produced viable seed. T1 plants producing up to 17.3% dry weight PHB in samples of leaf tissue and 8.8% dry weight PHB in the total biomass of the plant were also isolated. PMID:21325565

  15. High levels of bioplastic are produced in fertile transplastomic tobacco plants engineered with a synthetic operon for the production of polyhydroxybutyrate.

    Science.gov (United States)

    Bohmert-Tatarev, Karen; McAvoy, Susan; Daughtry, Sean; Peoples, Oliver P; Snell, Kristi D

    2011-04-01

    An optimized genetic construct for plastid transformation of tobacco (Nicotiana tabacum) for the production of the renewable, biodegradable plastic polyhydroxybutyrate (PHB) was designed using an operon extension strategy. Bacterial genes encoding the PHB pathway enzymes were selected for use in this construct based on their similarity to the codon usage and GC content of the tobacco plastome. Regulatory elements with limited homology to the host plastome yet known to yield high levels of plastidial recombinant protein production were used to enhance the expression of the transgenes. A partial transcriptional unit, containing genes of the PHB pathway and a selectable marker gene encoding spectinomycin resistance, was flanked at the 5' end by the host plant's psbA coding sequence and at the 3' end by the host plant's 3' psbA untranslated region. This design allowed insertion of the transgenes into the plastome as an extension of the psbA operon, rendering the addition of a promoter to drive the expression of the transgenes unnecessary. Transformation of the optimized construct into tobacco and subsequent spectinomycin selection of transgenic plants yielded T0 plants that were capable of producing up to 18.8% dry weight PHB in samples of leaf tissue. These plants were fertile and produced viable seed. T1 plants producing up to 17.3% dry weight PHB in samples of leaf tissue and 8.8% dry weight PHB in the total biomass of the plant were also isolated.

  16. Screening and characterization a RAPD marker of tobacco brown ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-08-04

    Aug 4, 2008 ... Random amplified DNA polymorphism of Nicotiana tabacum L. cultivars. Biologia Plantarum. 49: 605-607. Zhang HY, Liu XZ, Li TS, Yang YM (2006). Genetic diversity among flue- cured tobacco (Nicotiana tabacum L.) revealed by amplified fragment length polymorphism. Botanical Studies. 47: 223-229.

  17. Assays of dioxins and dioxin-like compounds in actually contaminated soils using transgenic tobacco plants carrying a recombinant mouse aryl hydrocarbon receptor-mediated β-glucuronidase reporter gene expression system.

    Science.gov (United States)

    Inui, Hideyuki; Gion, Keiko; Utani, Yasushi; Wakai, Taketo; Kodama, Susumu; Eun, Heesoo; Kim, Yun-Seok; Ohkawa, Hideo

    2012-01-01

    The transgenic tobacco plant XD4V-26 carrying the recombinant mouse aryl hydrocarbon receptor XD4V-mediated β-glucuronidase (GUS) reporter gene expression system was used for assay of dioxins and dioxin-like compounds consisting of polychlorinated dibenzeno-p-dioxins, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls (Co-PCBs) in actually contaminated soils. The transgenic tobacco plant XD4V-26 showed a significant dose-dependent induced GUS activity when cultured on MS medium containing PCB126 [toxic equivalency factor (TEF) = 0.1]. In contrast, PCB169 and PCB180, which have 0.03 of TEF and unassigned TEF values, respectively, did not significantly induce GUS activity under the same conditions as with PCB126. When the tobacco plants were cultivated for up to 5 weeks on actually contaminated soils with dioxins and dioxin-like compounds collected from the periphery of an incinerator used for disposal of residential and industrial wastes, GUS activity in the leaves was dose-dependently increased. The plants clearly detected 360 pg-TEQ g(-1) of dioxins and dioxin-like compounds in this assay. There was a positive correlation between GUS activity and TEQ value of dioxins and dioxin-like compounds in the plants. This assay does not require any extraction and purification processes for the actually contaminated soil samples.

  18. Ecological costs and benefits correlated with trypsin protease inhibitor production in Nicotiana attenuata

    NARCIS (Netherlands)

    Glawe, G.A.; Zavala, J.A.; Kessler, A.; Van Dam, N.M.; Baldwin, I.T.

    2003-01-01

    Genotypes of the wild tobacco Nicotiana attenuata from different geographic regions in North America vary considerably in the level of constitutive and inducible trypsin protease inhibitors (TrypPIs), a potent direct defense, as well as in the production of herbivore-induced volatiles that function

  19. Potential of marker-assisted selection for Tobacco mosaic ...

    African Journals Online (AJOL)

    Tobacco mosaic tobamovirus (TMV) is one of the most destructive virus threatening worldwide tobacco production. Use of host resistance is the best method of control. The N-gene was introgressed into tobacco from Nicotiana glutinosa to confer hypersensitive resistance to TMV. Phenotypic selection of TMV resistant ...

  20. Up-regulation of phosphoinositide metabolism in tobacco cells constitutively expressing the human type I inositol polyphosphate 5-phosphatase

    Science.gov (United States)

    Perera, Imara Y.; Love, John; Heilmann, Ingo; Thompson, William F.; Boss, Wendy F.; Brown, C. S. (Principal Investigator)

    2002-01-01

    To evaluate the impact of suppressing inositol 1,4,5-trisphosphate (InsP(3)) in plants, tobacco (Nicotiana tabacum) cells were transformed with the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme which specifically hydrolyzes InsP(3). The transgenic cell lines showed a 12- to 25-fold increase in InsP 5-ptase activity in vitro and a 60% to 80% reduction in basal InsP(3) compared with wild-type cells. Stimulation with Mas-7, a synthetic analog of the wasp venom peptide mastoparan, resulted in an approximately 2-fold increase in InsP(3) in both wild-type and transgenic cells. However, even with stimulation, InsP(3) levels in the transgenic cells did not reach wild-type basal values, suggesting that InsP(3) signaling is compromised. Analysis of whole-cell lipids indicated that phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)), the lipid precursor of InsP(3), was greatly reduced in the transgenic cells. In vitro assays of enzymes involved in PtdInsP(2) metabolism showed that the activity of the PtdInsP(2)-hydrolyzing enzyme phospholipase C was not significantly altered in the transgenic cells. In contrast, the activity of the plasma membrane PtdInsP 5 kinase was increased by approximately 3-fold in the transgenic cells. In vivo labeling studies revealed a greater incorporation of (32)P into PtdInsP(2) in the transgenic cells compared with the wild type, indicating that the rate of PtdInsP(2) synthesis was increased. These studies show that the constitutive expression of the human type I InsP 5-ptase in tobacco cells leads to an up-regulation of the phosphoinositide pathway and highlight the importance of PtdInsP(2) synthesis as a regulatory step in this system.

  1. Oral Vaccination Against Anthrax Using a Transgenic Plant Expressing Protective Antigen.

    Science.gov (United States)

    1996-09-01

    Nicotiana plumbaginifolia )" Science 223:496-498. 15. Jefferson, R.A. (1987), "Assaying chimeric genes in plants: The GUS gene fusion system" Plant Mol.Biol...interest. Tobacco ( Nicotiana tabacum cv BY-2) cells were grown in Murashige and Skoog (MS; 1962) media containing 0.2 [tg/ml 2,4-D with shaking at 8

  2. Salicylic acid-mediated and RNA-silencing defense mechanisms cooperate in the restriction of systemic spread of plum pox virus in tobacco.

    Science.gov (United States)

    Alamillo, Josefa M; Saénz, Pilar; García, Juan Antonio

    2006-10-01

    Plum pox virus (PPV) is able to replicate in inoculated leaves of Nicotiana tabacum, but is defective in systemic movement in this host. However, PPV produces a systemic infection in transgenic tobacco expressing the silencing suppressor P1/HC-Pro from tobacco etch virus (TEV). In this work we show that PPV is able to move to upper non-inoculated leaves of tobacco plants expressing bacterial salicylate hydroxylase (NahG) that degrades salicylic acid (SA). Replication and accumulation of PPV is higher in the locally infected leaves of plants deficient in SA or expressing TEV P1/HC-Pro silencing suppressor. Accumulation of viral derived small RNAs was reduced in the NahG transgenic plants, suggesting that SA might act as an enhancer of the RNA-silencing antiviral defense in tobacco. Besides, expression of SA-mediated defense transcripts, such as those of pathogenesis-related (PR) proteins PR-1 and PR-2 or alternative oxidase-1, as well as that of the putative RNA-dependent RNA polymerase NtRDR1, is induced in response to PPV infection, and the expression patterns of these defense transcripts are altered in the TEV P1/HC-Pro transgenic plants. Long-distance movement of PPV is highly enhanced in NahG x P1/HC-Pro double-transgenic plants and systemic symptoms in these plants reveal that the expression of an RNA-silencing suppressor and the lack of SA produce additive but distinct effects. Our results suggest that SA might act as an enhancer of the RNA-silencing antiviral defense in tobacco, and that silencing suppressors, such as P1/HC-Pro, also alter the SA-mediated defense. Both an RNA-silencing and an SA-mediated defense mechanism could act together to limit PPV infection.

  3. DECOMPOSTION OF GENETICALLY ENGINEERED TOBACCO UNDER FIELD CONDITIONS: PERSISTENCE OF THE PROTEINASE INHIBITOR I PRODUCT AND EFFECTS OF SOIL MICROBIAL RESPIRATION AND PROTOZOA, NEMATODE AND MICROARTHR

    Science.gov (United States)

    1. To evaluate the potential effects of genetically engineered (transgenic) plants on soil ecosystems, litterbags containing leaves of non-engineered (parental) and transgenic tobacco plants were buried in field plots. The transgenic tobacco plants were genetically engineered to ...

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

    Czech Academy of Sciences Publication Activity Database

    Nováková, Martina; Macková, M.; Antošová, Z.; Viktorová, J.; Szekeres, M.; Demnerová, K.; Macek, Tomáš

    2010-01-01

    Roč. 1, č. 6 (2010), s. 419-423 ISSN 1949-1018 R&D Projects: GA MŠk 1M06030 Grant - others:GA MŠk(CZ) ME09024 Institutional research plan: CEZ:AV0Z40550506 Keywords : phytoremediation * transgenic plant * Nicotiana tabacum * bphC Subject RIV: EI - Biotechnology ; Bionics

  5. An Intergenic Region Shared by At4g35985 and At4g35987 in Arabidopsis thaliana Is a Tissue Specific and Stress Inducible Bidirectional Promoter Analyzed in Transgenic Arabidopsis and Tobacco Plants

    Science.gov (United States)

    Banerjee, Joydeep; Sahoo, Dipak Kumar; Dey, Nrisingha; Houtz, Robert L.; Maiti, Indu Bhushan

    2013-01-01

    On chromosome 4 in the Arabidopsis genome, two neighboring genes (calmodulin methyl transferase At4g35987 and senescence associated gene At4g35985) are located in a head-to-head divergent orientation sharing a putative bidirectional promoter. This 1258 bp intergenic region contains a number of environmental stress responsive and tissue specific cis-regulatory elements. Transcript analysis of At4g35985 and At4g35987 genes by quantitative real time PCR showed tissue specific and stress inducible expression profiles. We tested the bidirectional promoter-function of the intergenic region shared by the divergent genes At4g35985 and At4g35987 using two reporter genes (GFP and GUS) in both orientations in transient tobacco protoplast and Agro-infiltration assays, as well as in stably transformed transgenic Arabidopsis and tobacco plants. In transient assays with GFP and GUS reporter genes the At4g35985 promoter (P85) showed stronger expression (about 3.5 fold) compared to the At4g35987 promoter (P87). The tissue specific as well as stress responsive functional nature of the bidirectional promoter was evaluated in independent transgenic Arabidopsis and tobacco lines. Expression of P85 activity was detected in the midrib of leaves, leaf trichomes, apical meristemic regions, throughout the root, lateral roots and flowers. The expression of P87 was observed in leaf-tip, hydathodes, apical meristem, root tips, emerging lateral root tips, root stele region and in floral tissues. The bidirectional promoter in both orientations shows differential up-regulation (2.5 to 3 fold) under salt stress. Use of such regulatory elements of bidirectional promoters showing spatial and stress inducible promoter-functions in heterologous system might be an important tool for plant biotechnology and gene stacking applications. PMID:24260266

  6. An intergenic region shared by At4g35985 and At4g35987 in Arabidopsis thaliana is a tissue specific and stress inducible bidirectional promoter analyzed in transgenic arabidopsis and tobacco plants.

    Directory of Open Access Journals (Sweden)

    Joydeep Banerjee

    Full Text Available On chromosome 4 in the Arabidopsis genome, two neighboring genes (calmodulin methyl transferase At4g35987 and senescence associated gene At4g35985 are located in a head-to-head divergent orientation sharing a putative bidirectional promoter. This 1258 bp intergenic region contains a number of environmental stress responsive and tissue specific cis-regulatory elements. Transcript analysis of At4g35985 and At4g35987 genes by quantitative real time PCR showed tissue specific and stress inducible expression profiles. We tested the bidirectional promoter-function of the intergenic region shared by the divergent genes At4g35985 and At4g35987 using two reporter genes (GFP and GUS in both orientations in transient tobacco protoplast and Agro-infiltration assays, as well as in stably transformed transgenic Arabidopsis and tobacco plants. In transient assays with GFP and GUS reporter genes the At4g35985 promoter (P85 showed stronger expression (about 3.5 fold compared to the At4g35987 promoter (P87. The tissue specific as well as stress responsive functional nature of the bidirectional promoter was evaluated in independent transgenic Arabidopsis and tobacco lines. Expression of P85 activity was detected in the midrib of leaves, leaf trichomes, apical meristemic regions, throughout the root, lateral roots and flowers. The expression of P87 was observed in leaf-tip, hydathodes, apical meristem, root tips, emerging lateral root tips, root stele region and in floral tissues. The bidirectional promoter in both orientations shows differential up-regulation (2.5 to 3 fold under salt stress. Use of such regulatory elements of bidirectional promoters showing spatial and stress inducible promoter-functions in heterologous system might be an important tool for plant biotechnology and gene stacking applications.

  7. Nitrite reductase expression is regulated at the post-transcriptional level by the nitrogen source in Nicotiana plumbaginifolia and Arabidopsis thaliana.

    Science.gov (United States)

    Crété, P; Caboche, M; Meyer, C

    1997-04-01

    Higher plant nitrite reductase (NiR) is a monomeric chloroplastic protein catalysing the reduction of nitrite, the product of nitrate reduction, to ammonium. The expression of this enzyme is controlled at the transcriptional level by light and by the nitrogen source. In order to study the post-transcriptional regulation of NiR, Nicotiana plumbaginifolia and Arabidopsis thaliana were transformed with a chimaeric NiR construct containing the tobacco leaf NiR1 coding sequence driven by the CaMV 35S RNA promoter. Transformed plants did not show any phenotypic difference when compared with the wild-type, although they overexpressed NiR activity in the leaves. When these plants were grown in vitro on media containing either nitrate or ammonium as sole nitrogen source, NiR mRNA derived from transgene expression was constitutively expressed, whereas NiR activity and protein level were strongly reduced on ammonium-containing medium. These results suggest that, together with transcriptional control, post-transcriptional regulation by the nitrogen source is operating on NiR expression. This post-transcriptional regulation of tobacco leaf NiR1 expression was observed not only in the closely related species N. plumbaginifolia but also in the more distant species A. thaliana.

  8. Targeting of a Nicotiana plumbaginifolia H+ -ATPase to the plasma membrane is not by default and requires cytosolic structural determinants.

    Science.gov (United States)

    Lefebvre, Benoit; Batoko, Henri; Duby, Geoffrey; Boutry, Marc

    2004-07-01

    The structural determinants involved in the targeting of multitransmembrane-span proteins to the plasma membrane (PM) remain poorly understood. The plasma membrane H+ -ATPase (PMA) from Nicotiana plumbaginifolia, a well-characterized 10 transmembrane-span enzyme, was used as a model to identify structural elements essential for targeting to the PM. When PMA2 and PMA4, representatives of the two main PMA subfamilies, were fused to green fluorescent protein (GFP), the chimeras were shown to be still functional and to be correctly and rapidly targeted to the PM in transgenic tobacco. By contrast, chimeric proteins containing various combinations of PMA transmembrane spanning domains accumulated in the Golgi apparatus and not in the PM and displayed slow traffic properties through the secretory pathway. Individual deletion of three of the four cytosolic domains did not prevent PM targeting, but deletion of the large loop or of its nucleotide binding domain resulted in GFP fluorescence accumulating exclusively in the endoplasmic reticulum. The results show that, at least for this polytopic protein, the PM is not the default pathway and that, in contrast with single-pass membrane proteins, cytosolic structural determinants are required for correct targeting.

  9. Overexpression of cotton RAV1 gene in Arabidopsis confers transgenic plants high salinity and drought sensitivity.

    Science.gov (United States)

    Li, Xiao-Jie; Li, Mo; Zhou, Ying; Hu, Shan; Hu, Rong; Chen, Yun; Li, Xue-Bao

    2015-01-01

    RAV (related to ABI3/VP1) protein containing an AP2 domain in the N-terminal region and a B3 domain in the C-terminal region, which belongs to AP2 transcription factor family, is unique in higher plants. In this study, a gene (GhRAV1) encoding a RAV protein of 357 amino acids was identified in cotton (Gossypium hirsutum). Transient expression analysis of the eGFP:GhRAV1 fusion genes in tobacco (Nicotiana tabacum) epidermal cells revealed that GhRAV1 protein was localized in the cell nucleus. Quantitative RT-PCR analysis indicated that expression of GhRAV1 in cotton is induced by abscisic acid (ABA), NaCl and polyethylene glycol (PEG). Overexpression of GhRAV1 in Arabidopsis resulted in plant sensitive to ABA, NaCl and PEG. With abscisic acid (ABA) treatment, seed germination and green seedling rates of the GhRAV1 transgenic plants were remarkably lower than those of wild type. In the presence of NaCl, the seed germination and seedling growth of the GhRAV1 transgenic lines were inhibited greater than those of wild type. And chlorophyll content and maximum photochemical efficiency of the transgenic plants were significantly lower than those of wild type. Under drought stress, the GhRAV1 transgenic plants displayed more severe wilting than wild type. Furthermore, expressions of the stress-related genes were altered in the GhRAV1 transgenic Arabidopsis plants under high salinity and drought stresses. Collectively, our data suggested that GhRAV1 may be involved in response to high salinity and drought stresses through regulating expressions of the stress-related genes during cotton development.

  10. The use of phosphomannose isomerase selection system for Agrobacterium-mediated transformation of tobacco and flax aimed for phytoremediation.

    Science.gov (United States)

    Hilgert, Jitka; Sura-De Jong, Martina; Fišer, Jiří; Tupá, Kateřina; Vrbová, Miroslava; Griga, Miroslav; Macek, Tomáš; Žiarovská, Jana

    2017-05-04

    A plant selection system based on the phosphomannose isomerase gene (pmi) as a selectable marker is often used to avoid selection using antibiotic resistance. Nevertheless, pmi gene is endogenous in several plant species and therefore difficult to use in such cases. Here we evaluated and compared Agrobacterium-mediated transformation of Linum usitatissimum breeding line AGT-952 (without endogenous pmi gene) and Nicotiana tabacum var. WSC-38 (with endogenous pmi gene). Transformation was evaluated for vectors bearing transgenes that have the potential to be involved in improved phytoremediation of contaminated environment. Tobacco regenerants selection resulted in 6.8% transformation efficiency when using a medium supplemented with 30 g/L mannose with stepwise decrease of the sucrose concentration. Similar transformation efficiency (5.3%) was achieved in transformation of flax. Relatively low selection efficiency was achieved (12.5% and 34.8%, respectively). The final detection of efficient pmi selection was conducted using PCR and the non-endogenous genes; pmi transgene for flax and todC2 transgene for tobacco plants.

  11. 210Pb and 210Po concentrations determined in Nicotiana tabacum L., Burley variety, cultivated in Brazil

    International Nuclear Information System (INIS)

    Damatto, Sandra R.; Rocha, Rique J.; Da Silva, Carolina F.; Frujuele, Jonatan V.

    2013-01-01

    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 illnesses. 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 (compression, filter and paper) and the temperature variation resulting from the incomplete combustion of tobacco. 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 cultivate in Brazil of Nicotiana tabacum L.; this paper presents the preliminary results of 210 Pb and 210 Po concentration for the Burley variety. Plants from this variety cultivated in open air, both in pots with special soil and fertilizer; and in small farms in natural conditions. The whole plant was analyzed; root, steam, leaves and flowers. The results obtained presented higher values for 210 Pb in leaves when compared with the other parts of the plant. (author)

  12. RNAi-mediated transgenic tospovirus resistance broken by intraspecies NSs complementation

    NARCIS (Netherlands)

    Hassani-Mehraban, A.; Brenkman, A.B.; Broek, N.F.J.; Goldbach, R.W.; Kormelink, R.J.M.

    2009-01-01

    Extension of an inverted repeat transgene cassette, containing partial nucleoprotein (N) gene sequences from four different tomato-infecting Tospovirus spp. with a partial N gene sequence from the tomato strain of Tomato yellow ring virus (TYRV-t), renders transgenic Nicotiana benthamiana plants

  13. Both positive and negative regulatory elements mediate expression of a photoregulated CAB gene from Nicotiana plumbaginifolia.

    Science.gov (United States)

    Castresana, C; Garcia-Luque, I; Alonso, E; Malik, V S; Cashmore, A R

    1988-01-01

    We have analyzed promoter regulatory elements from a photoregulated CAB gene (Cab-E) isolated from Nicotiana plumbaginifolia. These studies have been performed by introducing chimeric gene constructs into tobacco cells via Agrobacterium tumefaciens-mediated transformation. Expression studies on the regenerated transgenic plants have allowed us to characterize three positive and one negative cis-acting elements that influence photoregulated expression of the Cab-E gene. Within the upstream sequences we have identified two positive regulatory elements (PRE1 and PRE2) which confer maximum levels of photoregulated expression. These sequences contain multiple repeated elements related to the sequence-ACCGGCCCACTT-. We have also identified within the upstream region a negative regulatory element (NRE) extremely rich in AT sequences, which reduces the level of gene expression in the light. We have defined a light regulatory element (LRE) within the promoter region extending from -396 to -186 bp which confers photoregulated expression when fused to a constitutive nopaline synthase ('nos') promoter. Within this region there is a 132-bp element, extending from -368 to -234 bp, which on deletion from the Cab-E promoter reduces gene expression from high levels to undetectable levels. Finally, we have demonstrated for a full length Cab-E promoter conferring high levels of photoregulated expression, that sequences proximal to the Cab-E TATA box are not replaceable by corresponding sequences from a 'nos' promoter. This contrasts with the apparent equivalence of these Cab-E and 'nos' TATA box-proximal sequences in truncated promoters conferring low levels of photoregulated expression. Images PMID:2901343

  14. Involvement of the putative Ca²⁺-permeable mechanosensitive channels, NtMCA1 and NtMCA2, in Ca²⁺ uptake, Ca²⁺-dependent cell proliferation and mechanical stress-induced gene expression in tobacco (Nicotiana tabacum) BY-2 cells.

    Science.gov (United States)

    Kurusu, Takamitsu; Yamanaka, Takuya; Nakano, Masataka; Takiguchi, Akiko; Ogasawara, Yoko; Hayashi, Teruyuki; Iida, Kazuko; Hanamata, Shigeru; Shinozaki, Kazuo; Iida, Hidetoshi; Kuchitsu, Kazuyuki

    2012-07-01

    To gain insight into the cellular functions of the mid1-complementing activity (MCA) family proteins, encoding putative Ca²⁺-permeable mechanosensitive channels, we isolated two MCA homologs of tobacco (Nicotiana tabacum) BY-2 cells, named NtMCA1 and NtMCA2. NtMCA1 and NtMCA2 partially complemented the lethality and Ca²⁺ uptake defects of yeast mutants lacking mechanosensitive Ca²⁺ channel components. Furthermore, in yeast cells overexpressing NtMCA1 and NtMCA2, the hypo-osmotic shock-induced Ca²⁺ influx was enhanced. Overexpression of NtMCA1 or NtMCA2 in BY-2 cells enhanced Ca²⁺ uptake, and significantly alleviated growth inhibition under Ca²⁺ limitation. NtMCA1-overexpressing BY-2 cells showed higher sensitivity to hypo-osmotic shock than control cells, and induced the expression of the touch-inducible gene, NtERF4. We found that both NtMCA1-GFP and NtMCA2-GFP were localized at the plasma membrane and its interface with the cell wall, Hechtian strands, and at the cell plate and perinuclear vesicles of dividing cells. NtMCA2 transcript levels fluctuated during the cell cycle and were highest at the G1 phase. These results suggest that NtMCA1 and NtMCA2 play roles in Ca²⁺-dependent cell proliferation and mechanical stress-induced gene expression in BY-2 cells, by regulating the Ca²⁺ influx through the plasma membrane.

  15. GUS gene expression driven by a citrus promoter in transgenic tobacco and 'Valencia' sweet orange Expressão do gene GUS controlado por promotor de citros em plantas transgênicas de tabaco e laranja 'Valência'

    Directory of Open Access Journals (Sweden)

    Fernando Alves de Azevedo

    2006-11-01

    Full Text Available The objective of this work was the transformation of tobacco and 'Valencia' sweet orange with the GUS gene driven by the citrus phenylalanine ammonia-lyase (PAL gene promoter (CsPP. Transformation was accomplished by co-cultivation of tobacco and 'Valência' sweet orange explants with Agrobacterium tumefaciens containing the binary vector CsPP-GUS/2201. After plant transformation and regeneration, histochemical analyses using GUS staining revealed that CsPP promoter preferentially, but not exclusively, conferred gene expression in xylem tissues of tobacco. Weaker GUS staining was also detected throughout the petiole region in tobacco and citrus CsPP transgenic plants.O objetivo deste trabalho foi realizar a transformação de plantas de tabaco e laranja 'Valência' com o gene GUS controlado pelo promotor do gene da fenilalanina amônia-liase (PAL de citros (CsPP. Foi realizada transformação genética por meio do co-cultivo de explantes de tabaco e laranja 'Valência' com Agrobacterium tumefaciens que continha o vetor binário CsPP-GUS/2201. Após a transformação e a regeneração, a detecção da atividade de GUS por ensaios histoquímicos revelou que o promotor CsPP, preferencialmente, mas não exclusivamente, confere expressão gênica em tecidos do xilema de tabaco. Expressão mais baixa de GUS também foi detectada na região de tecido de pecíolo, em plantas transgênicas (CsPP de tabaco e laranja 'Valência'.

  16. De invloed van auxine, tryptofaan en enige anorganische zouten op de infectie van Nicotiana glutinosa met tabaksmozaiekvirus

    NARCIS (Netherlands)

    Liem, A.S.N.

    1963-01-01

    The number of necrotic spots arising on leaves of Nicotiana glutinosa after inoculation with tobacco mosaic virus was less than in controls without additives, if the water in which the leaves floated hadβ-indoleacetic acid (IAA),α-naphthylacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D)

  17. Pengaruh Pemberian Fungi Mikoriza Arbuskular (Fma) Terhadap Pertumbuhan Dan Produksi Beberapa Varietas Tembakau (Nicotiana Tabaccum L.) Di Lapangan

    OpenAIRE

    Sinaga, Parulian; Purba, Edison; Ginting, Jonis

    2014-01-01

    The growth and yield of a selected tobacco varieties (Nicotiana tabaccum L) treated withmycorhiza fungi arbuskular were evaluated in a field experiment. The aimed of the research was todetermine the effect of mycorhiza fungi on the growth and yield of several varieties of tobacco. Theresearch was conducted outdoor in the field at Balai Benih Penelitian Tembakau Deli Medan withaltitude of about 25 meters above sea level at the beginning of February until May 2012, with twotreatment factors. Th...

  18. Proteomic analysis of cold stress responses in tobacco seedlings ...

    African Journals Online (AJOL)

    Cold stress is one of the major abiotic stresses limiting the productivity and the geographical distribution of many important crops. To gain a better understanding of cold stress responses in tobacco (Nicotiana tabacum), we carried out a comparative proteomic analysis. Five-week-old tobacco seedlings were treated at 4°C ...

  19. Chickpea WRKY70 Regulates the Expression of a Homeodomain-Leucine Zipper (HD-Zip) I Transcription Factor CaHDZ12, which Confers Abiotic Stress Tolerance in Transgenic Tobacco and Chickpea.

    Science.gov (United States)

    Sen, Senjuti; Chakraborty, Joydeep; Ghosh, Prithwi; Basu, Debabrata; Das, Sampa

    2017-11-01

    Drought and salinity are the two major environmental constraints that severely affect global agricultural productivity. Plant-specific HD-Zip transcription factors are involved in plant growth, development and stress responses. In the present study, we explored the functional characteristics and regulation of a novel HD-Zip (I) gene from chickpea, CaHDZ12, in response to water-deficit and salt-stress conditions. Transgenic tobacco lines over-expressing CaHDZ12 exhibited improved tolerance to osmotic stresses and increased sensitivity to abscisic acid (ABA). Physiological compatibility of transgenic lines was found to be more robust compared to the wild-type plants under drought and salinity stress. Additionally, expression of several stress-responsive genes was significantly induced in CaHDZ12 transgenic plants. On the other hand, silencing of CaHDZ12 in chickpea resulted in increased sensitivity to salt and drought stresses. Analysis of different promoter deletion mutants identified CaWRKY70 transcription factor as a transcriptional regulator of CaHDZ12 expression. In vivo and in vitro interaction studies detected an association between CaWRKY70 and CaHDZ12 promoter during stress responses. Epigenetic modifications underlying histone acetylation at the CaHDZ12 promoter region play a significant role in stress-induced activation of this gene. Collectively, our study describes a crucial and unique mechanistic link between two distinct transcription factors in regulating plant adaptive stress response. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  20. Tissue-specific expression of telomerase reverse transcriptase gene variants in Nicotiana tabacum

    Czech Academy of Sciences Publication Activity Database

    Jurečková, J.; Sýkorová, Eva; Hafidh, Said; Honys, David; Fajkus, Jiří; Fojtová, M.

    2017-01-01

    Roč. 245, č. 3 (2017), s. 549-561 ISSN 0032-0935 R&D Projects: GA ČR GA13-06943S; GA MŠk(CZ) LQ1601 Institutional support: RVO:68081707 ; RVO:61389030 Keywords : male gametophyte development * tobacco male gametophyte * allotetraploid nicotiana Subject RIV: EF - Botanics; EF - Botanics (UEB-Q) OBOR OECD: Plant sciences, botany; Plant sciences, botany (UEB-Q) Impact factor: 3.361, year: 2016

  1. An investigation of gene action on different traits of tobacco under ...

    African Journals Online (AJOL)

    enoh

    2012-03-13

    Nicotiana tabacum). Information Bulletin Coresta Congress japan. 183. SHoaei DM, Honarnejad R (2003). Gene effects and Combining ability of quantitative and qualitative characteristics of Burley Tobacco. Information Bulletin ...

  2. Photosynthesis and protective mechanisms during ageing in transgenic tobacco leaves with over-expressed cytokinin oxidase/dehydrogenase and thus lowered cytokinin content

    Czech Academy of Sciences Publication Activity Database

    Mýtinová, Zuzana; Haisel, Daniel; Wilhelmová, Naděžda

    2006-01-01

    Roč. 44, č. 4 (2006), s. 599-605 ISSN 0300-3604 R&D Projects: GA ČR GA522/03/0312 Institutional research plan: CEZ:AV0Z50380511 Keywords : senescence * cytokinins * carotenoids * chlorophyll * xanthophylls cycle * photosynthetic efficiency * chlorophyll fluorescence * tobacco Subject RIV: CE - Biochemistry Impact factor: 0.782, year: 2006

  3. Effect of ATP sulfurylase overexpression in bright yellow 2 tobacco cells: regulation of ATP sulfurylase and SO4(-2) transport activities

    International Nuclear Information System (INIS)

    Hatzfeld, Y.; Cathala, N.; Grignon, C.; Davidian, J.C.

    1998-01-01

    To determine if the ATP sulfurylase reaction is a regulatory step for the SO4(2-)-assimilation pathway in plants, an Arabidopsis thaliana ATP sulfurylase cDNA, APS2, was fused to the 355 promoter of the cauliflower mosaic virus and introduced by Agrobacterium tumefaciens-mediated transformation into isolated Bright Yellow 2 tobacco (Nicotiana tabacum) cells. The ATP sulfurylase activity in transgenic cells was 8-fold that in control cells, and was correlated with the expression of a specific polypeptide revealed by western analysis using an anti-ATP sulfurylase antibody. The molecular mass of this polypeptide agreed with that for the overexpressed mature protein. ATP sulfurylase overexpression had no effect on [35S]SO4(2-) influx or ATP sulfurylase activity regulation by S availability, except that ATP sulfurylase activity variations in response to S starvation in transgenic cells were 8 times higher than in the wild type. There were also no differences in cell growth or sensitivity to SeO4(2-) (a toxic SO4(2-) analog) between transgenic and wild-type cells. We propose that in Bright Yellow 2 tobacco cells, the ATP sulfurylase derepression by S deficiency may involve a posttranscriptional mechanism, and that the ATP sulfurylase abundance is not limiting for cell metabolism

  4. Distinct Calcium Signaling Pathways Regulate Calmodulin Gene Expression in Tobacco1

    Science.gov (United States)

    van der Luit, Arnold H.; Olivari, Claudio; Haley, Ann; Knight, Marc R.; Trewavas, Anthony J.

    1999-01-01

    Cold shock and wind stimuli initiate Ca2+ transients in transgenic tobacco (Nicotiana plumbaginifolia) seedlings (named MAQ 2.4) containing cytoplasmic aequorin. To investigate whether these stimuli initiate Ca2+ pathways that are spatially distinct, stress-induced nuclear and cytoplasmic Ca2+ transients and the expression of a stress-induced calmodulin gene were compared. Tobacco seedlings were transformed with a construct that encodes a fusion protein between nucleoplasmin (a major oocyte nuclear protein) and aequorin. Immunocytochemical evidence indicated targeting of the fusion protein to the nucleus in these plants, which were named MAQ 7.11. Comparison between MAQ 7.11 and MAQ 2.4 seedlings confirmed that wind stimuli and cold shock invoke separate Ca2+ signaling pathways. Partial cDNAs encoding two tobacco calmodulin genes, NpCaM-1 and NpCaM-2, were identified and shown to have distinct nucleotide sequences that encode identical polypeptides. Expression of NpCaM-1, but not NpCaM-2, responded to wind and cold shock stimulation. Comparison of the Ca2+ dynamics with NpCaM-1 expression after stimulation suggested that wind-induced NpCaM-1 expression is regulated by a Ca2+ signaling pathway operational predominantly in the nucleus. In contrast, expression of NpCaM-1 in response to cold shock is regulated by a pathway operational predominantly in the cytoplasm. PMID:10557218

  5. Lactoferrin derived resistance against plant pathogen in transgenic plants

    Science.gov (United States)

    Lactoferrin (LF) is a ubiquitous cationic iron-binding milk glycoprotein and it is known to exert a broad-spectrum primary defense activity against bacteria, fungi, protozoa and viruses in mammals. The Bovine lactoferrin gene was introduced to tobacco (Nicotiana tabacum var Xanthi), Arabidopsis (A. ...

  6. Identification and expression of three new Nicotiana plumbaginifolia genes which encode isoforms of a plasma-membrane H(+)-ATPase, and one of which is induced by mechanical stress.

    Science.gov (United States)

    Oufattole, M; Arango, M; Boutry, M

    2000-04-01

    To analyze in detail the multigene family encoding the plasma-membrane H(+)-ATPase (pma) in Nicotiana plumbaginifolia Viv., five new pma genes (pma 5-9) were isolated. Three of these (pma 6, 8, 9) were fully characterized and classified into new and independent subfamilies. Their cell-type expression was followed by the beta-glucuronidase (gusA) reporter-gene method. While the pma8-gusA transgene was not expressed in transgenic tobacco, expression of the two other transgenes (pma6- and pma9-gusA) was found to be restricted to particular cell types. In the vegetative tissues, pma6-gusA expression was limited to the head cells of the leaf short trichomes, involved in secretion, and to the cortical parenchyma of the young nodes where the developing leaves and axillary flowering stalks join the stem. In the latter tissues, gene expression was enhanced by mechanical stress, suggesting that H(+)-ATPase might be involved in the strength of the tissues and their resistance to mechanical trauma. The pma9-gusA transgene was mainly expressed in the apical meristem of adventitious roots and axillary buds as well as in the phloem tissues of the stem, in which expression depended on the developmental stage. In flowers, pma9-gusA expression was limited to the mature pollen grains and the young fertilized ovules, while that of pma6-gusA was identified in most of the organs. Reverse transcription-polymerase chain reaction of leaf and stem RNA confirmed the expression of pma 6 and 9, while pma8 was found to be expressed in both organs at a lower level. In conclusion, although pma 6 and 9 had a more restricted expression pattern than the previously characterized pma genes, they were nevertheless expressed in cell types in which H(+)-ATPase had not been previously detected.

  7. 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.

  8. Overexpression of a Grapevine Sucrose Transporter (VvSUC27 in Tobacco Improves Plant Growth Rate in the Presence of Sucrose In vitro

    Directory of Open Access Journals (Sweden)

    Yumeng Cai

    2017-06-01

    Full Text Available The import of sugar from source leaves and it further accumulation in grape berries are considerably high during ripening, and this process is mediated via sucrose transporters. In this study, a grape sucrose transporter (SUT gene, VvSUC27, located at the plasma membrane, was transferred to tobacco (Nicotiana tabacum. The transformants were more sensitive to sucrose and showed more rapid development, especially roots, when cultured on MS agar medium containing sucrose, considering that the shoot/root dry weight ratio was only half that of the control. Moreover, all transformed plants exhibited light-colored leaves throughout their development, which indicated chlorosis and an associated reduction in photosynthesis. The total sugar content in the roots and stems of transformants was higher than that in control plants. No significant difference was observed in the leaves between the transformants and control plants. The levels of growth-promoting hormones were increased, and those of stress-mediating hormones were reduced in transgenic tobacco plants. The qRT-PCR analysis revealed that the expression of VvSUC27 was 1,000 times higher than that of the autologous tobacco sucrose transporter, which suggested that the markedly increased growth rate of transformants was because of the heterogeneously expressed gene. The transgenic tobacco plants showed resistance to abiotic stresses. Strikingly, the overexpression of VvSUC27 leaded to the up regulation of most reactive oxygen species scavengers and abscisic acid-related genes that might enable transgenic plants to overcome abiotic stress. Taken together, these results revealed an important role of VvSUC27 in plant growth and response to abiotic stresses, especially in the presence of sucrose in vitro.

  9. Inter Simple Sequence Repeat DNA (ISSR) Polymorphism Utility in Haploid Nicotiana Alata Irradiated Plants for Finding Markers Associated with Gamma Irradiation and Salinity

    International Nuclear Information System (INIS)

    El-Fiki, A.; Adly, M.; El-Metabteb, G.

    2017-01-01

    Nicotiana alata is an ornamental plant. It is a member of family Solanasea. Tobacco (Nicotiana spp.) is one of the most important commercial crops in the world. Wild Nicotiana species, as a store house of genes for several diseases and pests, in addition to genes for several important phytochemicals and quality traits which are not present in cultivated varieties. Inter simple sequence repeat DNA (ISSR) analysis was used to determine the degree of genetic variation in treated haploid Nicotiana alata plants. Total genomic DNAs from different treated haploid plant lets were amplified using five specific primers. All primers were polymorphic. A total of 209 bands were amplified of which 135 (59.47%) polymorphic across the radiation treatments. Whilst, the level of polymorphism among the salinity treatments were 181 (85.6 %). Whereas, the polymorphism among the combined effects between gamma radiation doses and salinity concentrations were 283 ( 73.95% ). Treatments relationships were estimated through cluster analysis (UPGMA) based on ISSR data

  10. Enhanced heterologous expression of biologically active human granulocyte colony stimulating factor in transgenic tobacco BY-2 cells by localization to endoplasmic reticulum.

    Science.gov (United States)

    Nair, Nisha R; Chidambareswaren, M; Manjula, S

    2014-09-01

    Tobacco Bright Yellow-2 (BY-2) cells, one of the best characterized cell lines is an attractive expression system for heterologous protein expression. However, the expression of foreign proteins is currently hampered by their low yield, which is partially the result of proteolytic degradation. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine. Recombinant hG-CSF is successfully being used for the treatment of chemotherapy-induced neutropenia in cancer patients. Here, we describe a simple strategy for producing biologically active hG-CSF in tobacco BY-2 cells, localized in the apoplast of BY-2 cells, as well as targeted to the endoplasmic reticulum (ER). ER targeting significantly enhanced recombinant production which scaled to 17.89 mg/l from 4.19 mg/l when expressed in the apoplasts. Southern blotting confirmed the stable integration of hG-CSF in the BY-2 nuclear genome, and the expression of hG-CSF was analysed by Western blotting. Total soluble protein containing hG-CSF isolated from positive calli showed proliferative potential when tested on HL-60 cell lines by MTT assay. We also report the potential of a Fluorescence-activated cell sorting approach for an efficient sorting of the hG-CSF-expressing cell lines, which will enable the generation of homogenous high-producing cell lines.

  11. The aux1 gene of the Ri plasmid is sufficient to confer auxin autotrophy in tobacco BY-2 cells.

    Science.gov (United States)

    Nemoto, Keiichirou; Hara, Masamitsu; Goto, Shingo; Kasai, Kouji; Seki, Hikaru; Suzuki, Masashi; Oka, Atsuhiro; Muranaka, Toshiya; Mano, Yoshihiro

    2009-05-01

    Tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells are rapidly proliferating meristematic cells that require auxin for culture in vitro. We have established several transgenic BY-2 cell lines that carry the T-DNA of Agrobacterium rhizogenes 15834, which harbors an agropine-type root-inducing (Ri) plasmid. Two of these lines, BYHR-3 and BYHR-7, were used to test the role of auxin in the proliferation of plant cells. The lines grew rapidly in Linsmaier-Skoog (LS) medium lacking auxin and other phytohormones. The TR-DNA, containing the aux1 (tryptophan monooxygenase) and aux2 (indoleacetamide hydrolase) genes, was present in the genomes of both transgenic lines, whereas the TL-DNA, containing the rolA, B, C and D genes, was present in the genome of BYHR-7 but not BYHR-3. Since the introduction of the rolABCD genes alone did not affect the auxin requirement of BY-2 cells, the aux1 and aux2 genes, but not the rolABCD genes, appear to be relevant to the auxin autotrophy of these transgenic lines. Furthermore, the overexpression of aux1 allowed BY-2 cells to grow rapidly in the absence of auxin, suggesting the existence in plant cells of an unidentified gene whose product is functionally equivalent or similar to that of aux2 of the Ri plasmid.

  12. Nicotiana rustica L. var. Souffi

    African Journals Online (AJOL)

    edoja

    2013-03-20

    Mar 20, 2013 ... Key words: Assimilation, growth, salt stress, nitrogen, mineral nutrition, tobacco ... to its ability to compete with K+ for binding site essential .... salinity leads to a constant root to shoot DW ratio (R/S ..... the interaction between relative K+ and Na+ concentration .... Cadmium toxicity induced changes in nitrogen.

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

    International Nuclear Information System (INIS)

    Rocha, Rique J.F.X.; Silva, Carolina F.; Frujuele, Jonatan V.; Bovolini, Raquel R.; Damatto, Sandra R.

    2013-01-01

    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 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 210 Pb in leaves when compared with the other parts of the plant. Comparing the three study areas the highest results of 210 Pb concentration were obtained in the plants cultivated in the urban area probably due to its atmospheric deposition. (author)

  14. 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)

  15. Impact of ubiquitous inhibitors on the GUS gene reporter system: evidence from the model plants Arabidopsis, tobacco and rice and correction methods for quantitative assays of transgenic and endogenous GUS

    Directory of Open Access Journals (Sweden)

    Gerola Paolo D

    2009-12-01

    Full Text Available Abstract Background The β-glucuronidase (GUS gene reporter system is one of the most effective and employed techniques in the study of gene regulation in plant molecular biology. Improving protocols for GUS assays have rendered the original method described by Jefferson amenable to various requirements and conditions, but the serious limitation caused by inhibitors of the enzyme activity in plant tissues has thus far been underestimated. Results We report that inhibitors of GUS activity are ubiquitous in organ tissues of Arabidopsis, tobacco and rice, and significantly bias quantitative assessment of GUS activity in plant transformation experiments. Combined with previous literature reports on non-model species, our findings suggest that inhibitors may be common components of plant cells, with variable affinity towards the E. coli enzyme. The reduced inhibitory capacity towards the plant endogenous GUS discredits the hypothesis of a regulatory role of these compounds in plant cells, and their effect on the bacterial enzyme is better interpreted as a side effect due to their interaction with GUS during the assay. This is likely to have a bearing also on histochemical analyses, leading to inaccurate evaluations of GUS expression. Conclusions In order to achieve reliable results, inhibitor activity should be routinely tested during quantitative GUS assays. Two separate methods to correct the measured activity of the transgenic and endogenous GUS are presented.

  16. Overexpression of a 9-cis-Epoxycarotenoid Dioxygenase Gene in Nicotiana plumbaginifolia Increases Abscisic Acid and Phaseic Acid Levels and Enhances Drought Tolerance1

    Science.gov (United States)

    Qin, Xiaoqiong; Zeevaart, Jan A.D.

    2002-01-01

    The plant hormone abscisic acid (ABA) plays important roles in seed maturation and dormancy and in adaptation to a variety of environmental stresses. An effort to engineer plants with elevated ABA levels and subsequent stress tolerance is focused on the genetic manipulation of the cleavage reaction. It has been shown in bean (Phaseolus vulgaris) that the gene encoding the cleavage enzyme (PvNCED1) is up-regulated by water stress, preceding accumulation of ABA. Transgenic wild tobacco (Nicotiana plumbaginifolia Viv.) plants were produced that overexpress the PvNCED1 gene either constitutively or in an inducible manner. The constitutive expression of PvNCED1 resulted in an increase in ABA and its catabolite, phaseic acid (PA). When the PvNCED1 gene was driven by the dexamethasone (DEX)-inducible promoter, a transient induction of PvNCED1 message and accumulation of ABA and PA were observed in different lines after application of DEX. Accumulation of ABA started to level off after 6 h, whereas the PA level continued to increase. In the presence of DEX, seeds from homozygous transgenic line TN1 showed a 4-d delay in germination. After spraying with DEX, the detached leaves from line TN1 had a drastic decrease in their water loss relative to control leaves. These plants also showed a marked increase in their tolerance to drought stress. These results indicate that it is possible to manipulate ABA levels in plants by overexpressing the key regulatory gene in ABA biosynthesis and that stress tolerance can be improved by increasing ABA levels. PMID:11842158

  17. Overexpression of a 9-cis-epoxycarotenoid dioxygenase gene in Nicotiana plumbaginifolia increases abscisic acid and phaseic acid levels and enhances drought tolerance.

    Science.gov (United States)

    Qin, Xiaoqiong; Zeevaart, Jan A D

    2002-02-01

    The plant hormone abscisic acid (ABA) plays important roles in seed maturation and dormancy and in adaptation to a variety of environmental stresses. An effort to engineer plants with elevated ABA levels and subsequent stress tolerance is focused on the genetic manipulation of the cleavage reaction. It has been shown in bean (Phaseolus vulgaris) that the gene encoding the cleavage enzyme (PvNCED1) is up-regulated by water stress, preceding accumulation of ABA. Transgenic wild tobacco (Nicotiana plumbaginifolia Viv.) plants were produced that overexpress the PvNCED1 gene either constitutively or in an inducible manner. The constitutive expression of PvNCED1 resulted in an increase in ABA and its catabolite, phaseic acid (PA). When the PvNCED1 gene was driven by the dexamethasone (DEX)-inducible promoter, a transient induction of PvNCED1 message and accumulation of ABA and PA were observed in different lines after application of DEX. Accumulation of ABA started to level off after 6 h, whereas the PA level continued to increase. In the presence of DEX, seeds from homozygous transgenic line TN1 showed a 4-d delay in germination. After spraying with DEX, the detached leaves from line TN1 had a drastic decrease in their water loss relative to control leaves. These plants also showed a marked increase in their tolerance to drought stress. These results indicate that it is possible to manipulate ABA levels in plants by overexpressing the key regulatory gene in ABA biosynthesis and that stress tolerance can be improved by increasing ABA levels.

  18. 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.

  19. Ecological Roles and Biological Activities of Specialized Metabolites from the Genus Nicotiana.

    Science.gov (United States)

    Jassbi, Amir Reza; Zare, Somayeh; Asadollahi, Mojtaba; Schuman, Meredith C

    2017-10-11

    Species of Nicotiana grow naturally in different parts of the world and have long been used both medicinally and recreationally by human societies. More recently in our history, Nicotiana tabacum has attracted interest as one of the most economically important industrial crops. Nicotiana species are frequently investigated for their bioactive natural products, and the ecological role of their specialized metabolites in responses to abiotic stress or biotic stress factors like pathogens and herbivores. The interest of tobacco companies in genetic information as well as the success of a few wild tobacco species as experimental model organisms have resulted in growing knowledge about the molecular biology and ecology of these plants and functional studies of the plant's natural products. Although a large number of reviews and books on biologically active natural products already exists, mostly from N. tabacum, we focus our attention on the ecological roles and biological activity of natural products, versus products from cured and processed material, in this Review. The studied compounds include alkaloids, aromatic compounds, flavonoids, volatiles, sesquiterpenoids, diterpenes alcohols, and sugar esters from trichomes of the plants, and recently characterized acyclic hydroxygeranyllinalool diterpene glycosides (HGL-DTGs). In this Review (1800s-2017), we describe the above-mentioned classes of natural products, emphasizing their biological activities and functions as they have been determined either in bioassay-guided purification approaches or in bioassays with plants in which the expression of specific biosynthetic genes has been genetically manipulated. Additionally, a review on the history, taxonomy, ecology, and medicinal application of different Nicotiana species growing around the globe presented in this Review may be of interest for pharmacognosists, natural products, and ecological chemists.

  20. Pest and disease resistance enhanced by heterologous suppression of a Nicotiana plumbaginifolia cytochrome P450 gene CYP72A2.

    Science.gov (United States)

    Smigocki, Ann C; Wilson, Dennis

    2004-12-01

    The functional role of the Nicotiana plumbaginifolia cytochrome P450 gene CYP72A2 was investigated in transgenic plants. N. tabacum plants transformed with a sense or antisense CYP72A2 construct exhibited diminished heights, branched stems, smaller leaves and deformed flowers. Western blot analysis revealed reduced levels of a 58 kDa protein corresponding to CYP72A2, suggesting that the CYP72A2 homolog was suppressed in the sense and antisense plants. Transgenic plants had increased resistance to Manduca sexta larvae that consumed about 35 to 90 less of transgenic versus control leaves. A virulent strain of Pseudomonas syringae pv. tabaci induced a disease-limiting response followed by a delayed and decreased development of disease symptoms in the transgenics. CYP72A2 gene mediated resistance suggests that the plant-pest or -pathogen interactions may have been modified by changes in bioactive metabolite pools.

  1. The Role of Heterologous Chloroplast Sequence Elements in Transgene Integration and Expression1[W][OA

    Science.gov (United States)

    Ruhlman, Tracey; Verma, Dheeraj; Samson, Nalapalli; Daniell, Henry

    2010-01-01

    Heterologous regulatory elements and flanking sequences have been used in chloroplast transformation of several crop species, but their roles and mechanisms have not yet been investigated. Nucleotide sequence identity in the photosystem II protein D1 (psbA) upstream region is 59% across all taxa; similar variation was consistent across all genes and taxa examined. Secondary structure and predicted Gibbs free energy values of the psbA 5′ untranslated region (UTR) among different families reflected this variation. Therefore, chloroplast transformation vectors were made for tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa), with endogenous (Nt-Nt, Ls-Ls) or heterologous (Nt-Ls, Ls-Nt) psbA promoter, 5′ UTR and 3′ UTR, regulating expression of the anthrax protective antigen (PA) or human proinsulin (Pins) fused with the cholera toxin B-subunit (CTB). Unique lettuce flanking sequences were completely eliminated during homologous recombination in the transplastomic tobacco genomes but not unique tobacco sequences. Nt-Ls or Ls-Nt transplastomic lines showed reduction of 80% PA and 97% CTB-Pins expression when compared with endogenous psbA regulatory elements, which accumulated up to 29.6% total soluble protein PA and 72.0% total leaf protein CTB-Pins, 2-fold higher than Rubisco. Transgene transcripts were reduced by 84% in Ls-Nt-CTB-Pins and by 72% in Nt-Ls-PA lines. Transcripts containing endogenous 5′ UTR were stabilized in nonpolysomal fractions. Stromal RNA-binding proteins were preferentially associated with endogenous psbA 5′ UTR. A rapid and reproducible regeneration system was developed for lettuce commercial cultivars by optimizing plant growth regulators. These findings underscore the need for sequencing complete crop chloroplast genomes, utilization of endogenous regulatory elements and flanking sequences, as well as optimization of plant growth regulators for efficient chloroplast transformation. PMID:20130101

  2. The role of heterologous chloroplast sequence elements in transgene integration and expression.

    Science.gov (United States)

    Ruhlman, Tracey; Verma, Dheeraj; Samson, Nalapalli; Daniell, Henry

    2010-04-01

    Heterologous regulatory elements and flanking sequences have been used in chloroplast transformation of several crop species, but their roles and mechanisms have not yet been investigated. Nucleotide sequence identity in the photosystem II protein D1 (psbA) upstream region is 59% across all taxa; similar variation was consistent across all genes and taxa examined. Secondary structure and predicted Gibbs free energy values of the psbA 5' untranslated region (UTR) among different families reflected this variation. Therefore, chloroplast transformation vectors were made for tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa), with endogenous (Nt-Nt, Ls-Ls) or heterologous (Nt-Ls, Ls-Nt) psbA promoter, 5' UTR and 3' UTR, regulating expression of the anthrax protective antigen (PA) or human proinsulin (Pins) fused with the cholera toxin B-subunit (CTB). Unique lettuce flanking sequences were completely eliminated during homologous recombination in the transplastomic tobacco genomes but not unique tobacco sequences. Nt-Ls or Ls-Nt transplastomic lines showed reduction of 80% PA and 97% CTB-Pins expression when compared with endogenous psbA regulatory elements, which accumulated up to 29.6% total soluble protein PA and 72.0% total leaf protein CTB-Pins, 2-fold higher than Rubisco. Transgene transcripts were reduced by 84% in Ls-Nt-CTB-Pins and by 72% in Nt-Ls-PA lines. Transcripts containing endogenous 5' UTR were stabilized in nonpolysomal fractions. Stromal RNA-binding proteins were preferentially associated with endogenous psbA 5' UTR. A rapid and reproducible regeneration system was developed for lettuce commercial cultivars by optimizing plant growth regulators. These findings underscore the need for sequencing complete crop chloroplast genomes, utilization of endogenous regulatory elements and flanking sequences, as well as optimization of plant growth regulators for efficient chloroplast transformation.

  3. Growth modulation effects of CBM2a under the control of AtEXP4 and CaMV35S promoters in Arabidopsis thaliana, Nicotiana tabacum and Eucalyptus camaldulensis.

    Science.gov (United States)

    Keadtidumrongkul, Pornthep; Suttangkakul, Anongpat; Pinmanee, Phitsanu; Pattana, Kanokwan; Kittiwongwattana, Chokchai; Apisitwanich, Somsak; Vuttipongchaikij, Supachai

    2017-08-01

    The expression of cell-wall-targeted Carbohydrate Binding Modules (CBMs) can alter cell wall properties and modulate growth and development in plants such as tobacco and potato. CBM2a identified in xylanase 10A from Cellulomonas fimi is of particular interest for its ability to bind crystalline cellulose. However, its potential for promoting plant growth has not been explored. In this work, we tested the ability of CBM2a to promote growth when expressed using both CaMV35S and a vascular tissue-specific promoter derived from Arabidopsis expansin4 (AtEXP4) in three plant species: Arabidopsis, Nicotiana tabacum and Eucalyptus camaldulensis. In Arabidopsis, the expression of AtEXP4pro:CBM2a showed trends for growth promoting effects including the increase of root and hypocotyl lengths and the enlargements of the vascular xylem area, fiber cells and vessel cells. However, in N. tabacum, the expression of CBM2a under the control of either CaMV35S or AtEXP4 promoter resulted in subtle changes in the plant growth, and the thickness of secondary xylem and vessel and fiber cell sizes were generally reduced in the transgenic lines with AtEXP4pro:CBM2a. In Eucalyptus, while transgenics expressing CaMV35S:CBM2a showed very subtle changes compared to wild type, those transgenics with AtEXP4pro:CBM2a showed increases in plant height, enlargement of xylem areas and xylem fiber and vessel cells. These data provide comparative effects of expressing CBM2a protein in different plant species, and this finding can be applied for plant biomass improvement.

  4. Increased and altered fragrance of tobacco plants after metabolic engineering using three monoterpene synthases from lemon

    NARCIS (Netherlands)

    Lücker, J.; Schwab, W.; Hautum, van B.; Blaas, J.; Plas, van der L.H.W.; Bouwmeester, H.J.; Verhoeven, H.A.

    2004-01-01

    Wild-type tobacco (Nicotiana tabacum) plants emit low levels of terpenoids, particularly from the flowers. By genetic modification of tobacco cv Petit Havana SR1 using three different monoterpene synthases from lemon (Citrus limon L. Burm. f.) and the subsequent combination of these three into one

  5. Analyzing diversification possibilities on specialized tobacco farms in Argentina using a bio-economic farm model

    NARCIS (Netherlands)

    Chavez, M.D.; Berentsen, P.B.M.; Oude Lansink, A.G.J.M.

    2014-01-01

    Tobacco (Nicotiana tabacum L.) is the non-food crop with the largest acreage in the world. Tobacco is criticized because it causes health problems to its consumers and because production causes environmental damage such as soil degradation, deforestation and water pollution. Diversification has been

  6. Mutation of a Nicotiana tabacum L. eukaryotic translation-initiation factor gene reduces susceptibility to a resistance-breaking strain of Potato Virus Y.

    Science.gov (United States)

    Takakura, Yoshimitsu; Udagawa, Hisashi; Shinjo, Akira; Koga, Kazuharu

    2018-04-06

    Eukaryotic translation-initiation factors eIF4E and eIF(iso)4E in plants play key roles in infection by potyviruses and other plant RNA viruses. Mutations in the genes encoding these factors reduce susceptibility to the viruses, and are the basis of several recessive virus-resistance genes widely used in plant breeding. Because virus variants occasionally break such resistance, the molecular basis for this process must be elucidated. Although deletion mutants of eIF4E1-S of tobacco (Nicotiana tabacum L.) resist Potato virus Y (PVY; the type member of the genus Potyvirus), resistance-breaking strains of PVY threaten tobacco production worldwide. Here, we used RNA interference technology to knock down tobacco eIF4E2-S and eIF4E2-T genes or eIF(iso)4E-S and eIF(iso)4E-T genes. Transgenic plants with reduced transcript levels of both eIF(iso)4E-S and eIF(iso)4E-T showed reduced susceptibility to a resistance-breaking PVY strain with a K105E mutation in the viral genome-associated protein (VPg). By screening a population of chemically-induced mutants of eIF(iso)4E-S and eIF(iso)4E-T, we showed that plants with a nonsense mutation in eIF(iso)4E-T, but not eIF(iso)4E-S, showed reduced susceptibility to the resistance-breaking PVY strain. In a yeast two-hybrid assay, VPg of the resistance-breaking strain, but not wild-type PVY, physically interacted with the eIF(iso)4E-T protein. Thus, eIF4E1-S is required for infection by PVY, but eIF(iso)4E-T is required for infection by the resistance-breaking strain. Our study provides the first evidence for the involvement of a host eukaryotic translation-initiation factor in the infection cycle of a resistance-breaking virus strain. The eIF(iso)4E-T mutants will be useful in tobacco breeding to introduce resistance against resistance-breaking PVY strains. This article is protected by copyright. All rights reserved. © 2018 BSPP and John Wiley & Sons Ltd.

  7. Tobacco Products

    Science.gov (United States)

    ... Exposure is High in Multiunit Housing Smokeless Products Electronic Cigarettes Youth Tobacco Prevention Tobacco Products Tobacco Ingredient ... Tweet Share Compartir Find Fact Sheets on Products (Cigars, Bidis and Betel Quid with Tobacco (Gutka) and ...

  8. Salivary cotinine levels as a biomarker for green tobacco sickness in dry tobacco production among Thai traditional tobacco farmers.

    Science.gov (United States)

    Saleeon, Thanusin; Siriwong, Wattasit; Maldonado-Pérez, Héctor Luis; Robson, Mark Gregory

    2016-01-01

    Dry Thai traditional tobacco (Nicotiana Tabacum L.) production involves a unique process: (a) picking tobacco leaves, (b) curing tobacco leaves, (c) removing stems of tobacco leaves, cutting leaves and putting on a bamboo rack, (d) drying in the sun, reversing a rack, spraying a tobacco extract to adjust the tobacco's color, storing dried tobacco and packaging. These processes may lead to adverse health effects caused by dermal absorption of nicotine such as Green Tobacco Sickness (GTS). The aim of this study was to determine the correlation between GTS resulting from dry Thai traditional tobacco production and salivary cotinine levels among Thai traditional tobacco farmers in Nan Province, Thailand. A prospective cohort study was conducted with 20 tobacco farmers and 20 non-tobacco farmers in Praputtabath Sub-District and Phatow Sub-District. The participants were randomly selected and interviewed using in person questionnaires with bi-weekly follow-up for 14 weeks. During each contact, the cotinine concentration was measured by NicAlert(TM) Saliva strip tests (NCTS). Descriptive statistics and Spearman's correlation (Spearman's rho) was used to examine the relationship between the variables at both 0.01 and 0.05 significant probability levels. This study indicated that GTS from dry tobacco production has the potential to be considered a common occupational disease. This study demonstrated the usefulness of salivary cotinine level measurements by NCTS. The levels were well correlated with farmers who were employed in the dry Thai tobacco production industry. Salivary cotinine levels were also significantly correlated with the prevalence of GTS in the group of tobacco farmers at any given time within a crop season. However, the production process of dry Thai traditional tobacco is different from that evaluated in our previous studies where GTS and salivary cotinine level were correlated in workers working in humid conditions. The long-term effects of such exposure

  9. Overexpression of Nictaba-Like Lectin Genes from Glycine max Confers Tolerance towards Pseudomonas syringae Infection, Aphid Infestation and Salt Stress in Transgenic Arabidopsis Plants

    Directory of Open Access Journals (Sweden)

    Sofie Van Holle

    2016-10-01

    Full Text Available Plants have evolved a sophisticated immune system that allows them to recognize invading pathogens by specialized receptors. Carbohydrate-binding proteins or lectins are part of this immune system and especially the lectins that reside in the nucleocytoplasmic compartment are known to be implicated in biotic and abiotic stress responses. The class of Nictaba-like lectins (NLL groups all proteins with homology to the tobacco (Nicotiana tabacum lectin, known as a stress-inducible lectin. Here we focus on two Nictaba homologs from soybean (Glycine max, referred to as GmNLL1 and GmNLL2. Confocal laser scanning microscopy of fusion constructs with the green fluorescent protein either transiently expressed in Nicotiana benthamiana leaves or stably transformed in tobacco BY-2 suspension cells revealed a nucleocytoplasmic localization for the GmNLLs under study. RT-qPCR analysis of the transcript levels for the Nictaba-like lectins in soybean demonstrated that the genes are expressed in several tissues throughout the development of the plant. Furthermore, it was shown that salt treatment, Phytophthora sojae infection and Aphis glycines infestation trigger the expression of particular NLL genes. Stress experiments with Arabidopsis lines overexpressing the NLLs from soybean yielded an enhanced tolerance of the plant towards bacterial infection (Pseudomonas syringae, insect infestation (Myzus persicae and salinity. Our data showed a better performance of the transgenic lines compared to wild type plants, indicating that the NLLs from soybean are implicated in the stress response. These data can help to further elucidate the physiological importance of the Nictaba-like lectins from soybean, which can ultimately lead to the design of crop plants with a better tolerance to changing environmental conditions.

  10. Changes in cytokinins are sufficient to alter developmental patterns of defense metabolites in Nicotiana attenuata

    Czech Academy of Sciences Publication Activity Database

    Bruetting, C.; Schaefer, N.; Vaňková, Radomíra; Gase, K.; Baldwin, I.T.; Meldau, S.

    2017-01-01

    Roč. 89, č. 1 (2017), s. 15-30 ISSN 0960-7412 R&D Projects: GA MŠk LD14120 Institutional support: RVO:61389030 Keywords : proteinase-inhibitor production * plant defense * arabidopsis-thaliana * leaf senescence * insect interactions * tobacco plants * jasmonic acid * manduca-sexta * cis-zeatin * responses * cytokinins * optimal defense * herbivores * inducible defense * Nicotiana attenuata * Manduca sexta * plant development * immunosenescence * phytohormones Subject RIV: ED - Physiology OBOR OECD: Plant sciences, botany Impact factor: 5.901, year: 2016

  11. Reversible Heat-Induced Inactivation of Chimeric β-Glucuronidase in Transgenic Plants1

    Science.gov (United States)

    Almoguera, Concepción; Rojas, Anabel; Jordano, Juan

    2002-01-01

    We compared the expression patterns in transgenic tobacco (Nicotiana tabacum) of two chimeric genes: a translational fusion to β-glucuronidase (GUS) and a transcriptional fusion, both with the same promoter and 5′-flanking sequences of Ha hsp17.7 G4, a small heat shock protein (sHSP) gene from sunflower (Helianthus annuus). We found that immediately after heat shock, the induced expression from the two fusions in seedlings was similar, considering chimeric mRNA or GUS protein accumulation. Surprisingly, we discovered that the chimeric GUS protein encoded by the translational fusion was mostly inactive in such conditions. We also found that this inactivation was fully reversible. Thus, after returning to control temperature, the GUS activity was fully recovered without substantial changes in GUS protein accumulation. In contrast, we did not find differences in the in vitro heat inactivation of the respective GUS proteins. Insolubilization of the chimeric GUS protein correlated with its inactivation, as indicated by immunoprecipitation analyses. The inclusion in another chimeric gene of the 21 amino-terminal amino acids from a different sHSP lead to a comparable reversible inactivation. That effect not only illustrates unexpected post-translational problems, but may also point to sequences involved in interactions specific to sHSPs and in vivo heat stress conditions. PMID:12011363

  12. Advance of molecular marker application in the tobacco research ...

    African Journals Online (AJOL)

    Tobacco (Nicotiana spp.) is one of the most important commercial crops in the world. During the last two decades, molecular markers have entered the scene of genetic improvement in different fields of agricultural research. The principles and characteristics of several molecular markers such as RFLP, RAPD, AFLP, ...

  13. In Vitro Assay of Ethanolic Heat Reflux Extract of Nicotiana tabacum L. var Virginia Against Nosocomial Bacteria Pathogen

    Science.gov (United States)

    Pramono, Andri; Fauzantoro, Ahmad; Rizki Hidayati, Irma; Hygea, Arina; Puspita, Oktaviani Sandra; Muktamiroh, Hikmah; Simanjuntak, Kristina; Gozan, Misri

    2018-03-01

    Tobacco plays an important role in international trade as one of the export commodities. Indonesia is one of the good quality export contributors of tobacco leaves in the world. Nevertheless, tobacco is used only as a raw material for the cigarette industries, and the rise on anti-cigarette regulations prompted the exploration of alternative product from tobacco plants. The content of alkaloids, flavonoids, terpenoids and steroids in tobacco leaves were reported in literatures as antibacterial. Therefore, this study proposed in vitro assay of the ethanolic heat reflux extract (EHRE) of Nicotiana tabacum var. Virginia against nosocomial bacteria pathogen ((Pseudomonas aeruginosa (ATCC 27853), Eschericia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212)). Kirby-bauer diffusion method was used for this assay. The concentration of the EHRE for Kirby-bauer assay were 20; 40; 60; 80; and 100%. The presence of clear zones on Kirby-bauer test, against the growth of each nosocomial bacteria pathogen show that tobacco extract has antibacterial effect. Statistical analysis result showed that each extract concentration had significant difference value (p steroids) of tobacco leaf extracts (N. tabacum) has potential as antibacterial against nosocomial bacteria pathogen. Nevertheless, optimization of tobacco leaf extract to obtain maximum active ingredient still needs to be done. This study is important for further development of the tobacco leaf extract as antibacterial

  14. Evaluating the use of renewable fuel sources to heat flue-cured tobacco barns

    OpenAIRE

    Brown, Robert T

    2018-01-01

    Evaluating the use of renewable fuel sources to heat flue-cured tobacco barns Robert Taylor Brown ABSTRACT The curing of flue-cured tobacco (Nicotiana tabacum L.) is an energy intensive process and represents a significant portion of the overall cost of production. Given the goal of the industry to reduce the environmental footprint of tobacco production and the energy demand of curing, attention has been directed to explore options for the use of renewable fuels for heating to...

  15. Identifying Growth Conditions for Nicotiana benthimiana Resulting in Predictable Gene Expression of Promoter-Gus Fusion

    Science.gov (United States)

    Sandoval, V.; Barton, K.; Longhurst, A.

    2012-12-01

    Revoluta (Rev) is a transcription factor that establishes leaf polarity inArabidopsis thaliana. Through previous work in Dr. Barton's Lab, it is known that Revoluta binds to the ZPR3 promoter, thus activating the ZPR3 gene product inArabidopsis thaliana. Using this knowledge, two separate DNA constructs were made, one carrying revgene and in the other, the ZPR3 promoter fussed with the GUS gene. When inoculated in Nicotiana benthimiana (tobacco), the pMDC32 plasmid produces the Rev protein. Rev binds to the ZPR3 promoter thereby activating the transcription of the GUS gene, which can only be expressed in the presence of Rev. When GUS protein comes in contact with X-Gluc it produce the blue stain seen (See Figure 1). In the past, variability has been seen of GUS expression on tobacco therefore we hypothesized that changing the growing conditions and leaf age might improve how well it's expressed.

  16. Inhibition of tobacco mosaic virus replication in lateral roots is dependent on an activated meristem-derived signal.

    Science.gov (United States)

    Valentine, T A; Roberts, I M; Oparka, K J

    2002-05-01

    Viral invasion of the root system of Nicotiana benthamiana was studied noninvasively with a tobacco mosaic virus (TMV) vector expressing the green-fluorescent protein (GFP). Lateral root primordia, which developed from the pericycle of primary roots, became heavily infected as they emerged from the root cortex. However, following emergence, a progressive wave of viral inhibition occurred that originated in the lateral-root meristem and progressed towards its base. Excision of source and sink tissues suggested that the inhibition of virus replication was brought about by the basipetal movement of a root meristem signal. When infected plants were inoculated with tobacco rattle virus (TRV) expressing the red-fluorescent protein, DsRed, TRV entered the lateral roots and suppressed the host response, leading to a reestablishment of TMV infection in lateral roots. By infecting GFP-expressing transgenic plants with TMV carrying the complementary GFP sequence it was possible to silence the host GFP, leading to the complete loss of fluorescence in lateral roots. The data suggest that viral inhibition in lateral roots occurs by a gene-silencing-like mechanism that is dependent on the activation of a lateral-root meristem.

  17. Manduca sexta recognition and resistance among allopolyploid Nicotiana host plants

    OpenAIRE

    Lou, Yonggen; Baldwin, Ian T.

    2003-01-01

    Allopolyploid speciation occurs instantly when the genomes of different species combine to produce self-fertile offspring and has played a central role in the evolution of higher plants, but its consequences for adaptive responses are unknown. We compare herbivore-recognition and -resistance responses of the diploid species and putative ancestral parent Nicotiana attenuata with those of the two derived allopolyploid species Nicotiana clevelandii and Nicotiana bigelovii. Manduca sexta larvae a...

  18. DNA methylation polymorphism in flue-cured tobacco and candidate markers for tobacco mosaic virus resistance*

    Science.gov (United States)

    Zhao, Jie-hong; Zhang, Ji-shun; Wang, Yi; Wang, Ren-gang; Wu, Chun; Fan, Long-jiang; Ren, Xue-liang

    2011-01-01

    DNA methylation plays an important role in the epigenetic regulation of gene expression during plant growth, development, and polyploidization. However, there is still no distinct evidence in tobacco regarding the distribution of the methylation pattern and whether it contributes to qualitative characteristics. We studied the levels and patterns of methylation polymorphism at CCGG sites in 48 accessions of allotetraploid flue-cured tobacco, Nicotiana tabacum, using a methylation-sensitive amplified polymorphism (MSAP) technique. The results showed that methylation existed at a high level among tobacco accessions, among which 49.3% sites were methylated and 69.9% allelic sites were polymorphic. A cluster analysis revealed distinct patterns of geography-specific groups. In addition, three polymorphic sites significantly related to tobacco mosaic virus (TMV) resistance were explored. This suggests that tobacco breeders should pay more attention to epigenetic traits. PMID:22042659

  19. DNA methylation polymorphism in flue-cured tobacco and candidate markers for tobacco mosaic virus resistance.

    Science.gov (United States)

    Zhao, Jie-hong; Zhang, Ji-shun; Wang, Yi; Wang, Ren-gang; Wu, Chun; Fan, Long-jiang; Ren, Xue-liang

    2011-11-01

    DNA methylation plays an important role in the epigenetic regulation of gene expression during plant growth, development, and polyploidization. However, there is still no distinct evidence in tobacco regarding the distribution of the methylation pattern and whether it contributes to qualitative characteristics. We studied the levels and patterns of methylation polymorphism at CCGG sites in 48 accessions of allotetraploid flue-cured tobacco, Nicotiana tabacum, using a methylation-sensitive amplified polymorphism (MSAP) technique. The results showed that methylation existed at a high level among tobacco accessions, among which 49.3% sites were methylated and 69.9% allelic sites were polymorphic. A cluster analysis revealed distinct patterns of geography-specific groups. In addition, three polymorphic sites significantly related to tobacco mosaic virus (TMV) resistance were explored. This suggests that tobacco breeders should pay more attention to epigenetic traits.

  20. OBSERVATIONS ON THE SUSCEPTIBILITY OF SOME WILD SPECIES OF THE GENUS NICOTINA AND OF SOME VARIETIES OF NICOTIANA TABACUM L. AND N. RUSTICA L. TO BLUE MOULD (PERONOSPORA TABACINA ADAM) -- PULAWY 1962

    Science.gov (United States)

    Nicotiana , no manifestations of the mould were observed on the species of N. debneyi and N. exigua. Very weak symptoms appeared in N. paniculata and N... plumbaginifolia . In the group of cigarette-tobacco varieties only the Hicks Resistant and Hicks fixed A2 (varieties of Australian origin, obtained by

  1. Nectar Sugar Modulation and Cell Wall Invertases in the Nectaries of Day- and Night- Flowering Nicotiana.

    Science.gov (United States)

    Tiedge, Kira; Lohaus, Gertrud

    2018-01-01

    Nectar composition varies between species, depending on flowering time and pollinator type, among others. Various models of the biochemical and molecular mechanisms underlying nectar production and secretion have been proposed. To gain insights into these mechanisms, day- and night-flowering tobacco ( Nicotiana ) species with high or low proportions of hexoses in the nectar were analyzed. Nectar and nectaries were simultaneously collected, throughout the day and night. Soluble sugars and starch were determined and the activity and expression level of cell wall invertase (CW-INVs) were measured in nectaries. Nectaries and nectar of the five Nicotiana species contained different amounts of sucrose, glucose, and fructose. CW-INV activity was detected in the nectaries of all Nicotiana species and is probably involved in the hydrolysis of sucrose in the nectary tissue and during nectar secretion. The larger differences in the sucrose-to-hexose-ratio between nectaries and nectar in diurnal species compared to nocturnal species can be explained by higher sucrose cleavage within the nectaries in night-flowering species, and during secretion in day-flowering species. However, cell wall invertase alone cannot be responsible for the differences in sugar concentrations. Within the nectaries of the Nicotiana species, a portion of the sugars is transiently stored as starch. In general, night-flowering species showed higher starch contents in the nectaries compared to day-flowering species. Moreover, in night flowering species, the starch content decreased during the first half of the dark period, when nectar production peaks. The sucrose concentrations in the cytoplasm of nectarial cells were extrapolated from nectary sucrose contents. In day-flowering species, the sucrose concentration in the nectary cytoplasm was about twice as high as in nectar, whereas in night-flowering species the situation was the opposite, which implies different secretion mechanisms. The secreted nectar

  2. Overexpression of snapdragon Delila (Del) gene in tobacco enhances anthocyanin accumulation and abiotic stress tolerance.

    Science.gov (United States)

    Naing, Aung Htay; Park, Kyeung Il; Ai, Trinh Ngoc; Chung, Mi Young; Han, Jeung Sul; Kang, Young-Wha; Lim, Ki Byung; Kim, Chang Kil

    2017-03-23

    Rosea1 (Ros1) and Delila (Del) co-expression controls anthocyanin accumulation in snapdragon flowers, while their overexpression in tomato strongly induces anthocyanin accumulation. However, little data exist on how Del expression alone influences anthocyanin accumulation. In tobacco (Nicotiana tabacum 'Xanthi'), Del expression enhanced leaf and flower anthocyanin production through regulating NtCHS, NtCHI, NtF3H, NtDFR, and NtANS transcript levels. Transgenic lines displayed different anthocyanin colors (e.g., pale red: T 0 -P, red: T 0 -R, and strong red: T 0 -S), resulting from varying levels of biosynthetic gene transcripts. Under salt stress, the T 2 generation had higher total polyphenol content, radical (DPPH, ABTS) scavenging activities, antioxidant-related gene expression, as well as overall greater salt and drought tolerance than wild type (WT). We propose that Del overexpression elevates transcript levels of anthocyanin biosynthetic and antioxidant-related genes, leading to enhanced anthocyanin production and antioxidant activity. The resultant increase of anthocyanin and antioxidant activity improves abiotic stress tolerance.

  3. Syringyl lignin is unaltered by severe sinapyl alcohol dehydrogenase suppression in tobacco.

    Science.gov (United States)

    Barakate, Abdellah; Stephens, Jennifer; Goldie, Alison; Hunter, William N; Marshall, David; Hancock, Robert D; Lapierre, Catherine; Morreel, Kris; Boerjan, Wout; Halpin, Claire

    2011-12-01

    The manipulation of lignin could, in principle, facilitate efficient biofuel production from plant biomass. Despite intensive study of the lignin pathway, uncertainty exists about the enzyme catalyzing the last step in syringyl (S) monolignol biosynthesis, the reduction of sinapaldehyde to sinapyl alcohol. Traditional schemes of the pathway suggested that both guaiacyl (G) and S monolignols are produced by a single substrate-versatile enzyme, cinnamyl alcohol dehydrogenase (CAD). This was challenged by the discovery of a novel sinapyl alcohol dehydrogenase (SAD) that preferentially uses sinapaldehyde as a substrate and that was claimed to regulate S lignin biosynthesis in angiosperms. Consequently, most pathway schemes now show SAD (or SAD and CAD) at the sinapaldehyde reduction step, although functional evidence is lacking. We cloned SAD from tobacco (Nicotiana tabacum) and suppressed it in transgenic plants using RNA interference-inducing vectors. Characterization of lignin in the woody stems shows no change to content, composition, or structure, and S lignin is normal. By contrast, plants additionally suppressed in CAD have changes to lignin structure and S:G ratio and have increased sinapaldehyde in lignin, similar to plants suppressed in CAD alone. These data demonstrate that CAD, not SAD, is the enzyme responsible for S lignin biosynthesis in woody angiosperm xylem.

  4. Syringyl Lignin Is Unaltered by Severe Sinapyl Alcohol Dehydrogenase Suppression in Tobacco[W

    Science.gov (United States)

    Barakate, Abdellah; Stephens, Jennifer; Goldie, Alison; Hunter, William N.; Marshall, David; Hancock, Robert D.; Lapierre, Catherine; Morreel, Kris; Boerjan, Wout; Halpin, Claire

    2011-01-01

    The manipulation of lignin could, in principle, facilitate efficient biofuel production from plant biomass. Despite intensive study of the lignin pathway, uncertainty exists about the enzyme catalyzing the last step in syringyl (S) monolignol biosynthesis, the reduction of sinapaldehyde to sinapyl alcohol. Traditional schemes of the pathway suggested that both guaiacyl (G) and S monolignols are produced by a single substrate-versatile enzyme, cinnamyl alcohol dehydrogenase (CAD). This was challenged by the discovery of a novel sinapyl alcohol dehydrogenase (SAD) that preferentially uses sinapaldehyde as a substrate and that was claimed to regulate S lignin biosynthesis in angiosperms. Consequently, most pathway schemes now show SAD (or SAD and CAD) at the sinapaldehyde reduction step, although functional evidence is lacking. We cloned SAD from tobacco (Nicotiana tabacum) and suppressed it in transgenic plants using RNA interference–inducing vectors. Characterization of lignin in the woody stems shows no change to content, composition, or structure, and S lignin is normal. By contrast, plants additionally suppressed in CAD have changes to lignin structure and S:G ratio and have increased sinapaldehyde in lignin, similar to plants suppressed in CAD alone. These data demonstrate that CAD, not SAD, is the enzyme responsible for S lignin biosynthesis in woody angiosperm xylem. PMID:22158465

  5. SolCyc: a database hub at the Sol Genomics Network (SGN) for the manual curation of metabolic networks in Solanum and Nicotiana specific databases

    Science.gov (United States)

    Foerster, Hartmut; Bombarely, Aureliano; Battey, James N D; Sierro, Nicolas; Ivanov, Nikolai V; Mueller, Lukas A

    2018-01-01

    Abstract SolCyc is the entry portal to pathway/genome databases (PGDBs) for major species of the Solanaceae family hosted at the Sol Genomics Network. Currently, SolCyc comprises six organism-specific PGDBs for tomato, potato, pepper, petunia, tobacco and one Rubiaceae, coffee. The metabolic networks of those PGDBs have been computationally predicted by the pathologic component of the pathway tools software using the manually curated multi-domain database MetaCyc (http://www.metacyc.org/) as reference. SolCyc has been recently extended by taxon-specific databases, i.e. the family-specific SolanaCyc database, containing only curated data pertinent to species of the nightshade family, and NicotianaCyc, a genus-specific database that stores all relevant metabolic data of the Nicotiana genus. Through manual curation of the published literature, new metabolic pathways have been created in those databases, which are complemented by the continuously updated, relevant species-specific pathways from MetaCyc. At present, SolanaCyc comprises 199 pathways and 29 superpathways and NicotianaCyc accounts for 72 pathways and 13 superpathways. Curator-maintained, taxon-specific databases such as SolanaCyc and NicotianaCyc are characterized by an enrichment of data specific to these taxa and free of falsely predicted pathways. Both databases have been used to update recently created Nicotiana-specific databases for Nicotiana tabacum, Nicotiana benthamiana, Nicotiana sylvestris and Nicotiana tomentosiformis by propagating verifiable data into those PGDBs. In addition, in-depth curation of the pathways in N.tabacum has been carried out which resulted in the elimination of 156 pathways from the 569 pathways predicted by pathway tools. Together, in-depth curation of the predicted pathway network and the supplementation with curated data from taxon-specific databases has substantially improved the curation status of the species–specific N.tabacum PGDB. The implementation of this

  6. The Ca(2+) status of the endoplasmic reticulum is altered by induction of calreticulin expression in transgenic plants

    Science.gov (United States)

    Persson, S.; Wyatt, S. E.; Love, J.; Thompson, W. F.; Robertson, D.; Boss, W. F.; Brown, C. S. (Principal Investigator)

    2001-01-01

    To investigate the endoplasmic reticulum (ER) Ca(2+) stores in plant cells, we generated tobacco (Nicotiana tabacum; NT1) suspension cells and Arabidopsis plants with altered levels of calreticulin (CRT), an ER-localized Ca(2+)-binding protein. NT1 cells and Arabidopsis plants were transformed with a maize (Zea mays) CRT gene in both sense and antisense orientations under the control of an Arabidopsis heat shock promoter. ER-enriched membrane fractions from NT1 cells were used to examine how altered expression of CRT affects Ca(2+) uptake and release. We found that a 2.5-fold increase in CRT led to a 2-fold increase in ATP-dependent (45)Ca(2+) accumulation in the ER-enriched fraction compared with heat-shocked wild-type controls. Furthermore, after treatment with the Ca(2+) ionophore ionomycin, ER microsomes from NT1 cells overproducing CRT showed a 2-fold increase in the amount of (45)Ca(2+) released, and a 2- to 3-fold increase in the amount of (45)Ca(2+) retained compared with wild type. These data indicate that altering the production of CRT affects the ER Ca(2+) pool. In addition, CRT transgenic Arabidopsis plants were used to determine if altered CRT levels had any physiological effects. We found that the level of CRT in heat shock-induced CRT transgenic plants correlated positively with the retention of chlorophyll when the plants were transferred from Ca(2+)-containing medium to Ca(2+)-depleted medium. Together these data are consistent with the hypothesis that increasing CRT in the ER increases the ER Ca(2+) stores and thereby enhances the survival of plants grown in low Ca(2+) medium.

  7. The Ca2+ Status of the Endoplasmic Reticulum Is Altered by Induction of Calreticulin Expression in Transgenic Plants1

    Science.gov (United States)

    Persson, Staffan; Wyatt, Sarah E.; Love, John; Thompson, William F.; Robertson, Dominique; Boss, Wendy F.

    2001-01-01

    To investigate the endoplasmic reticulum (ER) Ca2+ stores in plant cells, we generated tobacco (Nicotiana tabacum; NT1) suspension cells and Arabidopsis plants with altered levels of calreticulin (CRT), an ER-localized Ca2+-binding protein. NT1 cells and Arabidopsis plants were transformed with a maize (Zea mays) CRT gene in both sense and antisense orientations under the control of an Arabidopsis heat shock promoter. ER-enriched membrane fractions from NT1 cells were used to examine how altered expression of CRT affects Ca2+ uptake and release. We found that a 2.5-fold increase in CRT led to a 2-fold increase in ATP-dependent 45Ca2+ accumulation in the ER-enriched fraction compared with heat-shocked wild-type controls. Furthermore, after treatment with the Ca2+ ionophore ionomycin, ER microsomes from NT1 cells overproducing CRT showed a 2-fold increase in the amount of 45Ca2+ released, and a 2- to 3-fold increase in the amount of 45Ca2+ retained compared with wild type. These data indicate that altering the production of CRT affects the ER Ca2+ pool. In addition, CRT transgenic Arabidopsis plants were used to determine if altered CRT levels had any physiological effects. We found that the level of CRT in heat shock-induced CRT transgenic plants correlated positively with the retention of chlorophyll when the plants were transferred from Ca2+-containing medium to Ca2+-depleted medium. Together these data are consistent with the hypothesis that increasing CRT in the ER increases the ER Ca2+ stores and thereby enhances the survival of plants grown in low Ca2+ medium. PMID:11457960

  8. Transposition of a Ds element from a plasmid into the plant genome in Nicotiana plumbaginifolia protoplast-derived cells.

    Science.gov (United States)

    Houba-Hérin, N; Domin, M; Pédron, J

    1994-07-01

    Nicotiana plumbaginifolia haploid protoplasts were co-transformed with two plasmids, one with a NPT-II/Ds element and one with a gene encoding an amino-terminal truncated Ac transposase. It is shown that Ds can efficiently transpose from extrachromosomal DNA to N. plumbaginifolia chromosomes when the Ac transposase gene is present in trans. Ds has been shown to have transposed into the plant genome in a limited number of copies (1.9 copies per genome), for 21/32 transgenic lines tested. The flanking sequences present in the original plasmid are missing in these 21 plants. In only two of 21 plants was part of the transposase construct integrated. By segregation analysis of transgenic progeny, Ds was shown to be present in the heterozygous state in 10 lines even though haploid protoplasts had been originally transformed. This observation could indicate that integration occurred after or during DNA replication that leads to protoplast diploidization.

  9. Methylation-sensitive amplified polymorphism-based genome-wide analysis of cytosine methylation profiles in Nicotiana tabacum cultivars.

    Science.gov (United States)

    Jiao, J; Wu, J; Lv, Z; Sun, C; Gao, L; Yan, X; Cui, L; Tang, Z; Yan, B; Jia, Y

    2015-11-26

    This study aimed to investigate cytosine methylation profiles in different tobacco (Nicotiana tabacum) cultivars grown in China. Methylation-sensitive amplified polymorphism was used to analyze genome-wide global methylation profiles in four tobacco cultivars (Yunyan 85, NC89, K326, and Yunyan 87). Amplicons with methylated C motifs were cloned by reamplified polymerase chain reaction, sequenced, and analyzed. The results show that geographical location had a greater effect on methylation patterns in the tobacco genome than did sampling time. Analysis of the CG dinucleotide distribution in methylation-sensitive polymorphic restriction fragments suggested that a CpG dinucleotide cluster-enriched area is a possible site of cytosine methylation in the tobacco genome. The sequence alignments of the Nia1 gene (that encodes nitrate reductase) in Yunyan 87 in different regions indicate that a C-T transition might be responsible for the tobacco phenotype. T-C nucleotide replacement might also be responsible for the tobacco phenotype and may be influenced by geographical location.

  10. The stylar 120 kDa glycoprotein is required for S-specific pollen rejection in Nicotiana.

    Science.gov (United States)

    Hancock, C Nathan; Kent, Lia; McClure, Bruce A

    2005-09-01

    S-RNase participates in at least three mechanisms of pollen rejection. It functions in S-specific pollen rejection (self-incompatibility) and in at least two distinct interspecific mechanisms of pollen rejection in Nicotiana. S-specific pollen rejection and rejection of pollen from Nicotiana plumbaginifolia also require additional stylar proteins. Transmitting-tract-specific (TTS) protein, 120 kDa glycoprotein (120K) and pistil extensin-like protein III (PELP III) are stylar glycoproteins that bind S-RNase in vitro and are also known to interact with pollen. Here we tested whether these glycoproteins have a direct role in pollen rejection. 120K shows the most polymorphism in size between Nicotiana species. Larger 120K-like proteins are often correlated with S-specific pollen rejection. Sequencing results suggest that the polymorphism primarily reflects differences in glycosylation, although indels also occur in the predicted polypeptides. Using RNA interference (RNAi), we suppressed expression of 120K to determine if it is required for S-specific pollen rejection. Transgenic SC N. plumbaginifolia x SI Nicotiana alata (S105S105 or SC10SC10) hybrids with no detectable 120K were unable to perform S-specific pollen rejection. Thus, 120K has a direct role in S-specific pollen rejection. However, suppression of 120K had no effect on rejection of N. plumbaginifolia pollen. In contrast, suppression of HT-B, a factor previously implicated in S-specific pollen rejection, disrupts rejection of N. plumbaginifolia pollen. Thus, S-specific pollen rejection and rejection of N. plumbaginifolia pollen are mechanistically distinct, because they require different non-S-RNase factors.

  11. 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

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

    Directory of Open Access Journals (Sweden)

    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

  13. Tobacco Addiction

    Science.gov (United States)

    ... and lighters—anything that you connect with your smoking habit. Get rid of all old chewing tobacco containers ... nicotine addiction and more to do with the habit of smoking or using chewing tobacco. Some people gain weight ...

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Small heat shock proteins can release light dependence of tobacco seed during germination.

    Science.gov (United States)

    Koo, Hyun Jo; Park, Soo Min; Kim, Keun Pill; Suh, Mi Chung; Lee, Mi Ok; Lee, Seong-Kon; Xinli, Xia; Hong, Choo Bong

    2015-03-01

    Small heat shock proteins (sHSPs) function as ATP-independent molecular chaperones, and although the production and function of sHSPs have often been described under heat stress, the expression and function of sHSPs in fundamental developmental processes, such as pollen and seed development, have also been confirmed. Seed germination involves the breaking of dormancy and the resumption of embryo growth that accompany global changes in transcription, translation, and metabolism. In many plants, germination is triggered simply by imbibition of water; however, different seeds require different conditions in addition to water. For small-seeded plants, like Arabidopsis (Arabidopsis thaliana), lettuce (Lactuca sativa), tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum), light is an important regulator of seed germination. The facts that sHSPs accumulate during seed development, sHSPs interact with various client proteins, and seed germination accompanies synthesis and/or activation of diverse proteins led us to investigate the role of sHSPs in seed germination, especially in the context of light dependence. In this study, we have built transgenic tobacco plants that ectopically express sHSP, and the effect was germination of the seeds in the dark. Administering heat shock to the seeds also resulted in the alleviation of light dependence during seed germination. Subcellular localization of ectopically expressed sHSP was mainly observed in the cytoplasm, whereas heat shock-induced sHSPs were transported to the nucleus. We hypothesize that ectopically expressed sHSPs in the cytoplasm led the status of cytoplasmic proteins involved in seed germination to function during germination without additional stimulus and that heat shock can be another signal that induces seed germination. © 2015 American Society of Plant Biologists. All Rights Reserved.

  16. Small Heat Shock Proteins Can Release Light Dependence of Tobacco Seed during Germination1[OPEN

    Science.gov (United States)

    Koo, Hyun Jo; Park, Soo Min; Kim, Keun Pill; Suh, Mi Chung; Lee, Mi Ok; Lee, Seong-Kon; Xinli, Xia

    2015-01-01

    Small heat shock proteins (sHSPs) function as ATP-independent molecular chaperones, and although the production and function of sHSPs have often been described under heat stress, the expression and function of sHSPs in fundamental developmental processes, such as pollen and seed development, have also been confirmed. Seed germination involves the breaking of dormancy and the resumption of embryo growth that accompany global changes in transcription, translation, and metabolism. In many plants, germination is triggered simply by imbibition of water; however, different seeds require different conditions in addition to water. For small-seeded plants, like Arabidopsis (Arabidopsis thaliana), lettuce (Lactuca sativa), tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum), light is an important regulator of seed germination. The facts that sHSPs accumulate during seed development, sHSPs interact with various client proteins, and seed germination accompanies synthesis and/or activation of diverse proteins led us to investigate the role of sHSPs in seed germination, especially in the context of light dependence. In this study, we have built transgenic tobacco plants that ectopically express sHSP, and the effect was germination of the seeds in the dark. Administering heat shock to the seeds also resulted in the alleviation of light dependence during seed germination. Subcellular localization of ectopically expressed sHSP was mainly observed in the cytoplasm, whereas heat shock-induced sHSPs were transported to the nucleus. We hypothesize that ectopically expressed sHSPs in the cytoplasm led the status of cytoplasmic proteins involved in seed germination to function during germination without additional stimulus and that heat shock can be another signal that induces seed germination. PMID:25604531

  17. Determination of gold and arsenic in Indian tobacco leaves

    International Nuclear Information System (INIS)

    Purkayastha, B.C.; Bhattacharyya, D.K.

    1975-01-01

    Two varieties of Indian Tobacco leaves have been analysed for gold and arsenic by neutron activation ( 76 As, 198 Au). Nicotiana rustica variety from North Bengal was found to contain 3.7x10 -1 ppm of gold and 4.0x10 -3 ppm of arsenic and the nicotiana tabaccum variety from Andhra Pradesh contains 1.26x10 -1 ppm of gold and 5.1x10 -3 ppm of arsenic, respectively. Unlike those in other countries Indian tobacco leaves seem to be enriched in the gold content and depleted in the arsenic content. The soil of North Bengal is richer in gold than the soil of Andhra Pradesh which requires further investigation, and the amount of arsenic in both soils is physiologically insignificant. Irradiation of leaf samples was done in a CIRUS reactor at a neutron flux of 10 13 n cm -2 s -1 for seven days. (F.G.)

  18. Large-scale development of PIP and SSR markers and their complementary applied in Nicotiana.

    Science.gov (United States)

    Huang, L; Cao, H; Yang, L; Yu, Yu; Wang, Yu

    2013-08-01

    PIP (Potential Intron Polymorphism) and SSR (Simple Sequence Repeats) were used in many species, but large-scale development and combined use of these two markers have not been reported in tobacco. In this study, a total of 12,388 PIP and 76,848 SSR markers were designed and uploaded to a web-accessible database (http://yancao.sdau.edu.cn/tgb/). E-PCR analysis showed that PIP and SSR rarely overlapped and were strongly complementary in the tobacco genome. The density was 3.07 PIP and 1.72 SSR markers per 10 kb of the known sequences. A total of 153 and 166 alleles were detectedby 22 PIP and 22 SSR markers in 64 Nicotiana accessions. SSR produced higher PIC (polymorphism information content) values and identified more alleles than PIP, whereas PIP could identify larger numbers of rare alleles. Mantel testing demonstrated a high correlation coefficient (r = 0.949, P SSR. The UPGMA dendrogram created from the combined PIP and SSR markers was clearer and more reliable than the individual PIP or SSR dendrograms. It suggested that PIP and SSR can make up the deficiency of molecular markers not only in tobacco but other plant.

  19. Effect of virus infection on symplastic transport of fluorescent tracers in Nicotiana clevelandii leaf epidermis.

    Science.gov (United States)

    Derrick, P M; Barker, H; Oparka, K J

    1990-07-01

    The molecular weight exclusion limit of plasmodesmata in subveinal epidermal cells of Nicotiana clevelandii (Gray) leaves was estimated by microinjection and fluorescence microscopy using fluorescein isothiocyanate-peptide conjugates, carboxyfluorescein and Lucifer Yellow CH. The largest fluorochrome which moved symplastically between cells had a molecular weight of 749, although movement did not appear to depend purely on molecular weight parameters. Systemic infection of plants by tobacco rattle tobravirus, tomato black ring nepovirus or potato Y potyvirus did not alter the limits of plasmodesmatal conductance of the fluorochromes. However, carrot mottle umbravirus and groundnut rosette umbravirus diminished the symplastic mobility of some fluorescent tracers. These results imply that intercellular movement of these viruses does not involve a long-lasting increase in the plasmodesmatal molecular size exclusion limit.

  20. Growth and nitrogen metabolism changes in NaCl-stressed tobacco ...

    African Journals Online (AJOL)

    Growth and nitrogen metabolism changes in NaCl-stressed tobacco (Nicotiana rustica L. var. Souffi) seedlings. Chokri Zaghdoud, Houda Maâroufi-Dguimi, Youssef Ouni, Mokhtar Guerfel, Houda Gouia, Kamel-Eddine Negaz, Ali Ferchichi, Mohamed Debouba ...

  1. Response of Green Peach Aphids and Other Arthropods to Garlic Intercropped with Tobacco

    NARCIS (Netherlands)

    Lai, R.; You, M.; Lotz, L.A.P.; Vasseur, L.

    2011-01-01

    The green peach aphid, Myzus persicae (Sulzer), is an insect pest that causes extensive damage to tobacco (Nicotiana tabacum L.) in China. Field trials were conducted in 2008 and 2009 at Longyan in the Fujian Province (China) to evaluate the effects of garlic (Allium sativum L.) as a deterrent to

  2. Expression of a wolf spider toxin in tobacco inhibits the growth of microbes and insects

    Science.gov (United States)

    Abstract Lycotoxin I, from the wolf spider (Lycosa carolinensis), is an amphipathic pore-forming peptide that has antimicrobial and anti-insect activity. Constitutive expression of a lycotoxin I odified for oral toxicity to insects in tobacco (Nicotiana abacum) conferred significantly enhanced resis...

  3. Isolation and expression analysis of a tobacco AINTEGUMENTA ortholog (NtANTL).

    Science.gov (United States)

    Rieu, Ivo; Bots, Marc; Mariani, Celestina; Weterings, Koen A P

    2005-05-01

    The Arabidopsis AINTEGUMENTA (ANT) protein is essential for proper ovule development, but functions in cell proliferation and organ growth throughout the plant. Here we report the isolation of a full-length cDNA clone from tobacco (Nicotiana tabacum L.) that encodes a protein with high similarity to ANT and is preferentially expressed in the pistil. In situ hybridization analysis on the tobacco ovary shows that the expression pattern of the corresponding gene is different from that of ANT in Arabidopsis.

  4. Metabolite profiling reveals a role for atypical cinnamyl alcohol dehydrogenase CAD1 in the synthesis of coniferyl alcohol in tobacco xylem.

    Science.gov (United States)

    Damiani, Isabelle; Morreel, Kris; Danoun, Saïda; Goeminne, Geert; Yahiaoui, Nabila; Marque, Christiane; Kopka, Joachim; Messens, Eric; Goffner, Deborah; Boerjan, Wout; Boudet, Alain-Michel; Rochange, Soizic

    2005-11-01

    In angiosperms, lignin is built from two main monomers, coniferyl and sinapyl alcohol, which are incorporated respectively as G and S units in the polymer. The last step of their synthesis has so far been considered to be performed by a family of dimeric cinnamyl alcohol dehydrogenases (CAD2). However, previous studies on Eucalyptus gunnii xylem showed the presence of an additional, structurally unrelated, monomeric CAD form named CAD1. This form reduces coniferaldehyde to coniferyl alcohol, but is inactive on sinapaldehyde. In this paper, we report the functional characterization of CAD1 in tobacco (Nicotiana tabacum L.). Transgenic tobacco plants with reduced CAD1 expression were obtained through an RNAi strategy. These plants displayed normal growth and development, and detailed biochemical studies were needed to reveal a role for CAD1. Lignin analyses showed that CAD1 down-regulation does not affect Klason lignin content, and has a moderate impact on G unit content of the non-condensed lignin fraction. However, comparative metabolic profiling of the methanol-soluble phenolic fraction from basal xylem revealed significant differences between CAD1 down-regulated and wild-type plants. Eight compounds were less abundant in CAD1 down-regulated lines, five of which were identified as dimers or trimers of monolignols, each containing at least one moiety derived from coniferyl alcohol. In addition, 3-trans-caffeoyl quinic acid accumulated in the transgenic plants. Together, our results support a significant contribution of CAD1 to the synthesis of coniferyl alcohol in planta, along with the previously characterized CAD2 enzymes.

  5. Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in high nitrite excretion and NO emission from leaf and root tissue.

    Science.gov (United States)

    Lea, Unni S; Ten Hoopen, Floor; Provan, Fiona; Kaiser, Werner M; Meyer, Christian; Lillo, Cathrine

    2004-05-01

    In wild-type Nicotiana plumbaginifolia Viv. and other higher plants, nitrate reductase (NR) is regulated at the post-translational level and is rapidly inactivated in response to, for example, a light-to-dark transition. This inactivation is caused by phosphorylation of a conserved regulatory serine residue, Ser 521 in tobacco, and interaction with divalent cations or polyamines, and 14-3-3 proteins. The physiological importance of the post-translational NR modulation is presently under investigation using a transgenic N. plumbaginifolia line. This line expresses a mutated tobacco NR where Ser 521 has been changed into aspartic acid (Asp) by site-directed mutagenesis, resulting in a permanently active NR enzyme. When cut leaves or roots of this line (S(521)) were placed in darkness in a buffer containing 50 mM KNO(3), nitrite was excreted from the tissue at rates of 0.08-0.2 micromol (g FW)(-1) h(-1) for at least 5 h. For the control transgenic plant (C1), which had the regulatory serine of NR intact, nitrite excretion was low and halted completely after 1-3 h. Without nitrate in the buffer in which the tissue was immersed, nitrite excretion was also low for S(521), although 20-40 micromol (g FW)(-1) nitrate was present inside the tissue. Apparently, stored nitrate was not readily available for reduction in darkness. Leaf tissue and root segments of S(521) also emitted much more nitric oxide (NO) than the control. Importantly, NO emission from leaf tissue of S(521) was higher in the dark than in the light, opposite to what was usually observed when post-translational NR modulation was operating.

  6. Neuroanatomy and transgenic technologies

    Science.gov (United States)

    This is a short review that introduces recent advances of neuroanatomy and transgenic technologies. The anatomical complexity of the nervous system remains a subject of tremendous fascination among neuroscientists. In order to tackle this extraordinary complexity, powerful transgenic technologies a...

  7. Wild tobacco genomes reveal the evolution of nicotine biosynthesis.

    Science.gov (United States)

    Xu, Shuqing; Brockmöller, Thomas; Navarro-Quezada, Aura; Kuhl, Heiner; Gase, Klaus; Ling, Zhihao; Zhou, Wenwu; Kreitzer, Christoph; Stanke, Mario; Tang, Haibao; Lyons, Eric; Pandey, Priyanka; Pandey, Shree P; Timmermann, Bernd; Gaquerel, Emmanuel; Baldwin, Ian T

    2017-06-06

    Nicotine, the signature alkaloid of Nicotiana species responsible for the addictive properties of human tobacco smoking, functions as a defensive neurotoxin against attacking herbivores. However, the evolution of the genetic features that contributed to the assembly of the nicotine biosynthetic pathway remains unknown. We sequenced and assembled genomes of two wild tobaccos, Nicotiana attenuata (2.5 Gb) and Nicotiana obtusifolia (1.5 Gb), two ecological models for investigating adaptive traits in nature. We show that after the Solanaceae whole-genome triplication event, a repertoire of rapidly expanding transposable elements (TEs) bloated these Nicotiana genomes, promoted expression divergences among duplicated genes, and contributed to the evolution of herbivory-induced signaling and defenses, including nicotine biosynthesis. The biosynthetic machinery that allows for nicotine synthesis in the roots evolved from the stepwise duplications of two ancient primary metabolic pathways: the polyamine and nicotinamide adenine dinucleotide (NAD) pathways. In contrast to the duplication of the polyamine pathway that is shared among several solanaceous genera producing polyamine-derived tropane alkaloids, we found that lineage-specific duplications within the NAD pathway and the evolution of root-specific expression of the duplicated Solanaceae-specific ethylene response factor that activates the expression of all nicotine biosynthetic genes resulted in the innovative and efficient production of nicotine in the genus Nicotiana Transcription factor binding motifs derived from TEs may have contributed to the coexpression of nicotine biosynthetic pathway genes and coordinated the metabolic flux. Together, these results provide evidence that TEs and gene duplications facilitated the emergence of a key metabolic innovation relevant to plant fitness.

  8. Reduced Position Effect in Mature Transgenic Plants Conferred by the Chicken Lysozyme Matrix-Associated Region

    NARCIS (Netherlands)

    Mlynárová, Ľudmila; Loonen, Annelies; Heldens, Jos; Jansen, Ritsert C.; Keizer, Paul; Stiekema, Willem J.; Nap, Jan-Peter

    1994-01-01

    Matrix-associated regions may be useful for studying the role of chromatin architecture in transgene activity of transformed plants. The chicken lysozyme A element was shown to have specific affinity for tobacco nuclear matrices, and its influence on the variability of transgene expression in

  9. 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.

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

    Science.gov (United States)

    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

  11. Tobacco Rotated with Rapeseed for Soil-Borne Phytophthora Pathogen Biocontrol: Mediated by Rapeseed Root Exudates

    Directory of Open Access Journals (Sweden)

    Yuting Fang

    2016-06-01

    Full Text Available Black shank, caused by Phytophthora parasitica var. nicotianae, is a widespread and destructive disease of tobacco. Crop rotation is essential in controlling black shank. Here, we confirmed that rotating black shank-infested fields with rapeseed (Brassica napus suppressed the incidence this disease. Further study demonstrated that rapeseed roots have a strong ability to attract zoospores and subsequently stop the swimming of zoospores into cystospores. Then, rapeseed roots secrete a series of antimicrobial compounds, including 2-butenoic acid, benzothiazole, 2-(methylthiobenzothiazole, 1-(4-ethylphenyl-ethanone, and 4-methoxyindole, to inhibit the cystospore germination and mycelial growth of P. parasitica var. nicotianae. Thus, rapeseed rotated with tobacco suppresses tobacco black shank disease through the chemical weapons secreted by rapeseed roots.

  12. Youth and Tobacco Use

    Science.gov (United States)

    ... past 30 days. † Any tobacco product includes cigarettes, cigars, smokeless tobacco (including chewing tobacco, snuff, dip, snus, and dissolvable tobacco), tobacco pipes, bidis, hookah, and electronic cigarettes. § Where percentages are missing, sample sizes were ...

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

    Directory of Open Access Journals (Sweden)

    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.

  14. Larval Helicoverpa zea Transcriptional, Growth and Behavioral Responses to Nicotine and Nicotiana tabacum

    Directory of Open Access Journals (Sweden)

    Linus Gog

    2014-09-01

    Full Text Available The polyphagous feeding habits of the corn earworm, Helicoverpa zea (Boddie, underscore its status as a major agricultural pest with a wide geographic distribution and host plant repertoire. To study the transcriptomic response to toxins in diet, we conducted a microarray analysis of H. zea caterpillars feeding on artificial diet, diet laced with nicotine and Nicotiana tabacum (L. plants. We supplemented our analysis with growth and aversion bioassays. The transcriptome reflects an abundant expression of proteases, chitin, cytochrome P450 and immune-related genes, many of which are shared between the two experimental treatments. However, the tobacco treatment tended to elicit stronger transcriptional responses than nicotine-laced diet. The salivary factor glucose oxidase, known to suppress nicotine induction in the plant, was upregulated by H. zea in response to tobacco but not to nicotine-laced diet. Reduced caterpillar growth rates accompanied the broad regulation of genes associated with growth, such as juvenile hormone epoxide hydrolase. The differential expression of chemosensory proteins, such as odorant binding-protein-2 precursor, as well as the neurotransmitter nicotinic-acetylcholine-receptor subunit 9, highlights candidate genes regulating aversive behavior towards nicotine. We suggest that an observed coincidental rise in cannibalistic behavior and regulation of proteases and protease inhibitors in H. zea larvae signify a compensatory response to induced plant defenses.

  15. Properties of purified cytosolic isoenzyme I of Cu,Zn-superoxide dismutase from Nicotiana plumbaginifolia leaves.

    Science.gov (United States)

    Ragusa, S; Cambria, M T; Scarpa, M; Di Paolo, M L; Falconi, M; Rigo, A; Cambria, A

    2001-11-01

    The isoenzyme I of cytosolic Cu,Zn-superoxide dismutase (SOD) from Nicotiana plumbaginifolia (tobacco) leaves has been purified to apparent homogeneity. The relative molecular mass of the native isoenzyme, determined by gel filtration chromatography, is about 33.2 kDa. SDS-polyacrylamide gel electrophoresis shows that the enzyme is composed of two equal subunits of 16.6 kDa The isolectric point, assayed by isoelectric focusing, in the pH range of 3.5-6.5, is 4.3. The enzyme stability was tested at different temperatures, pH, and concentration of inhibitors (KCN and H(2)O(2)). The catalytic constant (k(cat)) was 1.17 +/- 0.14 x 10(9) M(-1) s(-1) at pH 9.9 and 0.1 M ionic strength. The activation energy of the thermal denaturation process is 263 kJ mol(-1). The electrostatic surface potential of the modeled tobacco Cu,Zn-SOD I was calculated showing that the functional spatial network of charges on the protein surface has been maintained, independently of the amino acid substitution around the active sites. Copyright 2001 Academic Press.

  16. Growth of nicotiana in response to atmospheric CO sub 2 enrichment and various light regimes

    Energy Technology Data Exchange (ETDEWEB)

    Pope, S.; Thomas, J.F. (North Carolina State Univ., Raleigh (USA))

    1989-04-01

    Nicotiana tabacum NCTG-22, N. tabacum Petite Havana and N. plumbaginifolia were grown in chambers (24 C, 12-h light) under daytime atmospheric CO{sub 2} levels of 340 ppm (ambient) or 1000 ppm (enriched). All 3 types of tobacco grew faster and had open flowers sooner under CO2 enrichment, but patterns of dry weight distribution varied with type of tobacco. In N. plumbaginifolia significant proportions of dry weight were allocated to stems and branches, while in tabacum types, less was allocated to stems and more to leaves and roots. Increases in dry weight due to CO2 enrichment were accompanied by increases in leaf area and thickness. Plants given a far-red low intensity night break exhibited few differences from controls except having thinner leaves under ambient CO2; but under enriched CO2, had greater total dry weight and thicker leaves containing a higher proportion of spongy mesophyll than controls. A 50% reduction in light intensity led to a comparable reduction in dry weight and leaf area across treatments.

  17. 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.

  18. Shine-dalgarno sequences play an essential role in the translation of plastid mRNAs in tobacco

    DEFF Research Database (Denmark)

    Scharff, Lars; Ehrnthaler, Miriam; Janowski, Marcin

    2017-01-01

    SD]). Although many chloroplast mRNAs harbor putative SDs in their 5' untranslated regions and the aSD displays strong conservation, the functional relevance of SD-aSD interactions in plastid translation is unclear. Here, by generating transplastomic tobacco (Nicotiana tabacum) mutants with point mutations...

  19. Optimization and utilization of Agrobacterium-mediated transient protein production in Nicotiana.

    Science.gov (United States)

    Shamloul, Moneim; Trusa, Jason; Mett, Vadim; Yusibov, Vidadi

    2014-04-19

    Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana × N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

  20. 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.

  1. SPECIFICITY OF THE PRECIPITIN REACTION IN TOBACCO MOSAIC DISEASE.

    Science.gov (United States)

    Beale, H P

    1931-09-30

    1. Leaf extracts of Sudan grass, Hippeastrum equestre Herb., lily, and Abutilon striatum Dicks. (A. Thompsoni hort.), each affected with its respective mosaic disease, and peach affected with yellows disease, were tested for their ability to precipitate antiserum for virus extract of tobacco mosaic disease. No precipitate occurred. 2. Nicotiana glutinosa L., N. rustica L., and Martynia louisiana Mill. were added to the list of hosts of tobacco mosaic virus which have been tested with antiserum for the same virus in N. tabacum L. var. Turkish. The object was to determine the presence or absence of material reacting with the specific precipitins such as that already demonstrated in extracts of tomato, pepper, and petunia affected with the same virus. The presence of specific substances was demonstrated in every case. 3. The viruses of ringspot and cucumber mosaic diseases were multiplied in Turkish tobacco and leaf extracts of the affected plants were used in turn as antigens in precipitin tests with antiserum for tobacco mosaic virus extract of Turkish tobacco. A slight precipitation resulted in the tubes containing undiluted antiserum and virus extract such as occurs when juice from normal tobacco is used with undiluted antiserum. No precipitate was demonstrable that was specific for virus extracts of tobacco affected with either ringspot or cucumber mosaic disease. 4. The results favor the interpretation that the specific antigenic substance in virus extract of tobacco mosaic disease is foreign antigenic material, possibly virus itself, not altered host protein.

  2. Mechanisms of nitric-oxide-induced increase of free cytosolic Ca2+ concentration in Nicotiana plumbaginifolia cells.

    Science.gov (United States)

    Lamotte, Olivier; Courtois, Cécile; Dobrowolska, Grazyna; Besson, Angélique; Pugin, Alain; Wendehenne, David

    2006-04-15

    In this study, we investigated a role for nitric oxide (NO) in mediating the elevation of the free cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in plants using Nicotiana plumbaginifolia cells expressing the Ca(2+) reporter apoaequorin. Hyperosmotic stress induced a fast increase of [Ca(2+)](cyt) which was strongly reduced by pretreating cell suspensions with the NO scavenger carboxy PTIO, indicating that NO mediates [Ca(2+)](cyt) changes in plant cells challenged by abiotic stress. Accordingly, treatment of transgenic N. plumbaginifolia cells with the NO donor diethylamine NONOate was followed by a transient increase of [Ca(2+)](cyt) sensitive to plasma membrane Ca(2+) channel inhibitors and antagonist of cyclic ADP ribose. We provided evidence that NO might activate plasma membrane Ca(2+) channels by inducing a rapid and transient plasma membrane depolarization. Furthermore, NO-induced elevation of [Ca(2+)](cyt) was suppressed by the kinase inhibitor staurosporine, suggesting that NO enhances [Ca(2+)](cyt) by promoting phosphorylation-dependent events. This result was further supported by the demonstration that the NO donor induced the activation of a 42-kDa protein kinase which belongs to SnRK2 families and corresponds to Nicotiana tabacum osmotic-stress-activated protein kinase (NtOSAK). Interestingly, NtOSAK was activated in response to hyperosmotic stress through a NO-dependent process, supporting the hypothesis that NO also promotes protein kinase activation during physiological processes.

  3. Constitutive expression of a putative high-affinity nitrate transporter in Nicotiana plumbaginifolia: evidence for post-transcriptional regulation by a reduced nitrogen source.

    Science.gov (United States)

    Fraisier, V; Gojon, A; Tillard, P; Daniel-Vedele, F

    2000-08-01

    The NpNRT2.1 gene encodes a putative inducible component of the high-affinity nitrate (NO3-) uptake system in Nicotiana plumbaginifolia. Here we report functional and physiological analyses of transgenic plants expressing the NpNRT2.1 coding sequence fused to the CaMV 35S or rolD promoters. Irrespective of the level of NO3- supplied, NO3- contents were found to be remarkably similar in wild-type and transgenic plants. Under specific conditions (growth on 10 mM NO3-), the steady-state NpNRT2. 1 mRNA level resulting from the deregulated transgene expression was accompanied by an increase in 15NO3- influx measured in the low concentration range. This demonstrates for the first time that the NRT2.1 sequence codes a limiting element of the inducible high-affinity transport system. Both 15NO3- influx and mRNA levels decreased in the wild type after exposure to ammonium, in agreement with previous results from many species. Surprisingly, however, influx was also markedly decreased in transgenic plants, despite stable levels of transgene expression in independent transformants after ammonium addition. We conclude that the conditions associated with the supply of a reduced nitrogen source such as ammonium, or with the generation of a further downstream metabolite, probably exert a repressive effect on NO3- influx at both transcriptional and post-transcriptional levels.

  4. Immunity to potato mop-top virus in Nicotiana benthamiana plants expressing the coat protein gene is effective against fungal inoculation of the virus.

    Science.gov (United States)

    Reavy, B; Arif, M; Kashiwazaki, S; Webster, K D; Barker, H

    1995-01-01

    Nicotiana benthamiana stem tissue was transformed with Agrobacterium tumefaciens harboring a binary vector containing the potato mop-top virus (PMTV) coat protein (CP) gene. PMTV CP was expressed in large amounts in some of the primary transformants. The five transgenic lines which produced the most CP were selected for resistance testing. Flowers on transformed plants were allowed to self-fertilize. Transgenic seedlings selected from the T1 seed were mechanically inoculated with two strains of PMTV. Virus multiplication, assayed by infectivity, was detected in only one transgenic plant of 98 inoculated. T1 plants were also highly resistant to graft inoculation; PMTV multiplied in only one plant of 45 inoculated. Transgenic T1 seedlings were challenged in a bait test in which they were grown in soil containing viruliferous spores of the vector fungus Spongospora subterranea. In these tests only two plants out of 99 became infected. Of the five transgenic lines tested, plants of three lines were immune to infection following manual, graft, or fungal inoculation.

  5. Vesicular transport route of horseradish C1a peroxidase is regulated by N- and C-terminal propeptides in tobacco cells.

    Science.gov (United States)

    Matsui, T; Nakayama, H; Yoshida, K; Shinmyo, A

    2003-10-01

    Peroxidases (PRX, EC 1.11.1.7) are widely distributed across microorganisms, plants, and animals; and, in plants, they have been implicated in a variety of secondary metabolic reactions. In particular, horseradish (Armoracia rusticana) root represents the main source of commercial PRX production. The prxC1a gene, which encodes horseradish PRX (HRP) C, is expressed mainly in the roots and stems of the horseradish plant. HRP C1a protein is shown to be synthesized as a preprotein with both a N-terminal (NTPP) and a C-terminal propeptide (CTPP). These propeptides, which might be responsible for intracellular localization or secretion, are removed before or concomitant with production of the mature protein. We investigated the functional role of HRP C1a NTPP and CTPP in the determination of the vesicular transport route, using an analytical system of transgenically cultured tobacco cells (Nicotiana tabacum, BY2). Here, we report that NTPP and CTPP are necessary and sufficient for accurate localization of mature HRP C1a protein to vacuoles of the vesicular transport system. We also demonstrate that HRP C1a derived from a preprotein lacking CTPP is shunted into the secretory pathway.

  6. Omzettingen van koolhydraten in het blad van Nicotiana tabacum L.

    NARCIS (Netherlands)

    Tollenaar, D.

    1925-01-01

    Nicotiana tabacum L. was chosen as an experimental plant for several practical reasons. The plants were grown in large pots in a glasshouse at 22 °C and great humidity in February-March and September-October until 4 normal leaves were present. Each day at 16.00 h the plants were brought into

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

    Directory of Open Access Journals (Sweden)

    C. Poggialini

    2014-01-01

    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.

  8. Selectable tolerance to herbicides by mutated acetolactate synthase genes integrated into the chloroplast genome of tobacco.

    Science.gov (United States)

    Shimizu, Masanori; Goto, Maki; Hanai, Moeko; Shimizu, Tsutomu; Izawa, Norihiko; Kanamoto, Hirosuke; Tomizawa, Ken-Ichi; Yokota, Akiho; Kobayashi, Hirokazu

    2008-08-01

    Strategies employed for the production of genetically modified (GM) crops are premised on (1) the avoidance of gene transfer in the field; (2) the use of genes derived from edible organisms such as plants; (3) preventing the appearance of herbicide-resistant weeds; and (4) maintaining transgenes without obstructing plant cell propagation. To this end, we developed a novel vector system for chloroplast transformation with acetolactate synthase (ALS). ALS catalyzes the first step in the biosynthesis of the branched amino acids, and its enzymatic activity is inhibited by certain classes of herbicides. We generated a series of Arabidopsis (Arabidopsis thaliana) mutated ALS (mALS) genes and introduced constructs with mALS and the aminoglycoside 3'-adenyltransferase gene (aadA) into the tobacco (Nicotiana tabacum) chloroplast genome by particle bombardment. Transplastomic plants were selected using their resistance to spectinomycin. The effects of herbicides on transplastomic mALS activity were examined by a colorimetric assay using the leaves of transplastomic plants. We found that transplastomic G121A, A122V, and P197S plants were specifically tolerant to pyrimidinylcarboxylate, imidazolinon, and sulfonylurea/pyrimidinylcarboxylate herbicides, respectively. Transplastomic plants possessing mALSs were able to grow in the presence of various herbicides, thus affirming the relationship between mALSs and the associated resistance to herbicides. Our results show that mALS genes integrated into the chloroplast genome are useful sustainable markers that function to exclude plants other than those that are GM while maintaining transplastomic crops. This investigation suggests that the resistance management of weeds in the field amid growing GM crops is possible using (1) a series of mALSs that confer specific resistance to herbicides and (2) a strategy that employs herbicide rotation.

  9. 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.

  10. Purification of nitrate reductase from Nicotiana plumbaginifolia by affinity chromatography using 5'AMP-sepharose and monoclonal antibodies.

    Science.gov (United States)

    Moureaux, T; Leydecker, M T; Meyer, C

    1989-02-15

    Nitrate reductase was purified from leaves of Nicotiana plumbaginifolia using either 5'AMP-Sepharose chromatography or two steps of immunoaffinity chromatography involving monoclonal antibodies directed against nitrate reductase from maize and against ribulose-1,5-bisphosphate carboxylase from N. plumbaginifolia. Nitrate reductase obtained by the first method was purified 1000-fold to a specific activity of 9 units/mg protein. The second method produced an homogenous enzyme, purified 21,000-fold to a specific activity of 80 units/mg protein. SDS/PAGE of nitrate reductase always resulted in two bands of 107 and 99.5 kDa. The 107-kDa band was the nitrate reductase subunit of N. plumbaginifolia; the smaller one of 99.5 kDa is thought, as commonly reported, to result from proteolysis of the larger protein. The molecular mass of 107 kDa is close to the values calculated from the coding sequences of the two nitrate reductase genes recently cloned from tobacco (Nicotiana tabacum cv Xanthi).

  11. Gene Inactivation by CRISPR-Cas9 in Nicotiana tabacum BY-2 Suspension Cells.

    Science.gov (United States)

    Mercx, Sébastien; Tollet, Jérémie; Magy, Bertrand; Navarre, Catherine; Boutry, Marc

    2016-01-01

    Plant suspension cells are interesting hosts for the heterologous production of pharmacological proteins such as antibodies. They have the advantage to facilitate the containment and the application of good manufacturing practices. Furthermore, antibodies can be secreted to the extracellular medium, which makes the purification steps much simpler. However, improvements are still to be made regarding the quality and the production yield. For instance, the inactivation of proteases and the humanization of glycosylation are both important targets which require either gene silencing or gene inactivation. To this purpose, CRISPR-Cas9 is a very promising technique which has been used recently in a series of plant species, but not yet in plant suspension cells. Here, we sought to use the CRISPR-Cas9 system for gene inactivation in Nicotiana tabacum BY-2 suspension cells. We transformed a transgenic line expressing a red fluorescent protein (mCherry) with a binary vector containing genes coding for Cas9 and three guide RNAs targeting mCherry restriction sites, as well as a bialaphos-resistant (bar) gene for selection. To demonstrate gene inactivation in the transgenic lines, the mCherry gene was PCR-amplified and analyzed by electrophoresis. Seven out of 20 transformants displayed a shortened fragment, indicating that a deletion occurred between two target sites. We also analyzed the transformants by restriction fragment length polymorphism and observed that the three targeted restriction sites were hit. DNA sequencing of the PCR fragments confirmed either deletion between two target sites or single nucleotide deletion. We therefore conclude that CRISPR-Cas9 can be used in N. tabacum BY2 cells.

  12. You(th) & Tobacco

    Science.gov (United States)

    ... Exposure is High in Multiunit Housing Smokeless Products Electronic Cigarettes Youth Tobacco Prevention Tobacco Products Tobacco Ingredient ... Performance Don’t get trapped. Nicotine in cigarettes, cigars, and spit tobacco is addictive. Nicotine narrows your ...

  13. Regulation of carotenoid and ABA accumulation during the development and germination of Nicotiana plumbaginifolia seeds.

    Science.gov (United States)

    Frey, Anne; Boutin, Jean-Pierre; Sotta, Bruno; Mercier, Raphaël; Marion-Poll, Annie

    2006-08-01

    Abscisic acid (ABA) is derived from epoxycarotenoid cleavage and regulates seed development and maturation. A detailed carotenoid analysis was undertaken to study the contribution of epoxycarotenoid synthesis to the regulation of ABA accumulation in Nicotiana plumbaginifolia developing seeds. Maximal accumulation of xanthophylls occurred at mid-development in wild type seeds, when total ABA levels also peaked. In contrast, in ABA-deficient mutants xanthophyll synthesis was delayed, in agreement with the retardation in seed maturation. Seed dormancy was restored in mutants impaired in the conversion of zeaxanthin into violaxanthin by zeaxanthin epoxidase (ZEP), by the introduction of the Arabidopsis AtZEP gene under the control of promoters inducing expression during later stages of seed development compared to wild type NpZEP, and in dry and imbibed seeds. Alterations in the timing and level of ZEP expression did not highly affect the temporal regulation of ABA accumulation in transgenic seeds, despite notable perturbations in xanthophyll accumulation. Therefore, major regulatory control of ABA accumulation might occur downstream of epoxycarotenoid synthesis.

  14. Congenital skeletal malformations and cleft palate induced in goats by ingestion of Lupinus, Conium and Nicotiana species.

    Science.gov (United States)

    Panter, K E; Keeler, R F; Bunch, T D; Callan, R J

    1990-01-01

    Three piperidine alkaloid containing plants, Conium maculatum (poison-hemlock), Nicotiana glauca (tree tobacco) and Lupinus formosus (lunara lupine), induced multiple congenital contractures (MCC) and palatoschisis in goat kids when their dams were gavaged with the plant during gestation days 30-60. The skeletal abnormalities included fixed extension or flexure of the carpal, tarsal, and fetlock joints, scoliosis, lordosis, torticollis and rib cage abnormalities. Clinical signs of toxicity included those reported in sheep, cattle and pigs--ataxia, incoordination, muscular weakness, prostration and death. One quinolizidine alkaloid containing plant, Lupinus caudatus (tailcup lupine), on the other hand, which is also known to cause MCC in cows, caused only slight signs of toxicity in pregnant goats and no teratogenic effects in their offspring.

  15. 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.

  16. The rise of the photosynthetic rate when light intensity increases is delayed in ndh gene-defective tobacco at high but not at low CO2 concentrations

    Directory of Open Access Journals (Sweden)

    Mercedes eMartin

    2015-02-01

    Full Text Available The 11 plastid ndh genes have hovered frequently on the edge of dispensability, being absent in the plastid DNA of many algae and certain higher plants. We have compared the photosynthetic activity of tobacco (Nicotiana tabacum, cv. Petit Havana with five transgenic lines (ndhF, pr-ndhF, T181D, T181A and ndhF FC and found that photosynthetic performance is impaired in transgenic ndhF-defective tobacco plants at rapidly fluctuating light intensities and higher than ambient CO2 concentrations. In contrast to wild type and ndhF FC, which reach the maximum photosynthetic rate in less than one min when light intensity suddenly increases, ndh defective plants (ndhF and T181A show up to a 5 min delay in reaching the maximum photosynthetic rate at CO2 concentrations higher than the ambient 360 ppm. Net photosynthesis was determined at different CO2 concentrations when sequences of 130, 870, 61, 870 and 130 μmol m−2 s−1 PAR sudden light changes were applied to leaves and photosynthetic efficiency and entropy production were determined as indicators of photosynthesis performance. The two ndh-defective plants, ndhF and T181A, had lower photosynthetic efficiency and higher entropy production than wt, ndhF FC and T181D tobacco plants, containing full functional ndh genes, at CO2 concentrations above 400 ppm. We propose that the Ndh complex improves cyclic electron transport by adjusting the redox level of transporters during the low light intensity stage. In ndhF-defective strains, the supply of electrons through the Ndh complex fails, transporters remain over-oxidized (specially at high CO2 concentrations and the rate of cyclic electron transport is low, impairing the ATP level required to rapidly reach high CO2 fixation rates in the following high light phase. Hence, ndh genes could be dispensable at low but not at high atmospheric concentrations of CO2.

  17. A jasmonate ZIM-domain protein NaJAZd regulates floral jasmonic acid levels and counteracts flower abscission in Nicotiana attenuata plants.

    Directory of Open Access Journals (Sweden)

    Youngjoo Oh

    Full Text Available Jasmonic acid is an important regulator of plant growth, development and defense. The jasmonate-ZIM domain (JAZ proteins are key regulators in jasmonate signaling ubiquitously present in flowering plants but their functional annotation remains largely incomplete. Recently, we identified 12 putative JAZ proteins in native tobacco, Nicotiana attenuata, and initiated systematic functional characterization of these proteins by reverse genetic approaches. In this report, Nicotiana attenuata plants silenced in the expression of NaJAZd (irJAZd by RNA interference were used to characterize NaJAZd function. Although NaJAZd transcripts were strongly and transiently up-regulated in the rosette leaves by simulated herbivory treatment, we did not observe strong defense-related phenotypes, such as altered herbivore performance or the constitutive accumulation of defense-related secondary metabolites in irJAZd plants compared to wild type plants, both in the glasshouse and the native habitat of Nicotiana attenuata in the Great Basin Desert, Utah, USA. Interestingly, irJAZd plants produced fewer seed capsules than did wild type plants as a result of increased flower abscission in later stages of flower development. The early- and mid-developmental stages of irJAZd flowers had reduced levels of jasmonic acid and jasmonoyl-L-isoleucine, while fully open flowers had normal levels, but these were impaired in NaMYB305 transcript accumulations. Previously, NaMYB305-silenced plants were shown to have strong flower abscission phenotypes and contained lower NECTARIN 1 transcript levels, phenotypes which are copied in irJAZd plants. We propose that the NaJAZd protein is required to counteract flower abscission, possibly by regulating jasmonic acid and jasmonoyl-L-isoleucine levels and/or expression of NaMYB305 gene in Nicotiana attenuata flowers. This novel insight into the function of JAZ proteins in flower and seed development highlights the diversity of functions

  18. in transgenic cucumber

    African Journals Online (AJOL)

    Jane

    2011-07-18

    Jul 18, 2011 ... College of Horticulture, South China Agriculture University, Guangzhou 510642, Guangdong ... The pattern of expression vector pBI-PacPAP. ..... Disease scale ... These transgenic T0 plants were self-pollinated and the.

  19. Arabidopsis Vacuolar Pyrophosphatase gene (AVP1) induces drought and salt tolerance in Nicotiana tabacum plants (abstract)

    International Nuclear Information System (INIS)

    Arif, A.; Mohsin, A.M.; Shafiq, S.; Zafar, Y.; Hameed, S.M.; Arif, M.; Javed, M.; Gaxiola, R.A.

    2005-01-01

    Drought and salinity are global problems. In Pakistan these problems are increasing to an alarming situation due to low rain-fall and bad agricultural practices. Salt and drought stress shows a high degree of similarity with respect to physiological, biochemical, molecular and genetic effects. This is due to the fact that sub-lethal salt-stress condition is ultimately an osmotic effect which is apparently similar to that brought in by water deficit. Genetic engineering allows the re-introduction of plant genes into their genomes by increasing their expression level. Plant vacuoles play a central role in cellular mechanisms of adaptation to salinity and drought stresses. In principle, increased vacuolar solute accumulation should have a positive impact in the adaptation of plants to salinity and drought. The active transport of the solutes depends on the proton gradients established by proton pumps. We have over expressed Arabidopsis gene AVP1 (Arabidopsis thaliana vacuolar pyro phosphatase H/sup +/ pump) to increase drought/salt tolerance in tobacco. The AVP1 ORF with a tandem repeat of 358 promoter was cloned in pPZP212 vector and Agrobacterium-mediated transformation was performed. Transgenic plants were selected on plant nutrient agar medium supplemented with 50 mg/liter kanamycin. Transgenic plants were confirmed for transfer of genes by AVP1 and nptll gene specific PCR and Southern hybridization. AVP1 transgenic plants were screened for salt tolerance by providing NaCl solution in addition to nutrient solution. AVP1 transgenic plants showed tolerance up to 300 mM NaCl as compared to control which died ten days after 200 mM NaCl. Sodium and potassium were measured in salt treated and control plants. Results showed that sodium ion uptake in the salt treated transgenic plants was four times more as compared to wild type. This remarkable increase in Na/sup +/ ion uptake indicates that AVP1 vacuole proton pumps are actively involved in the transport of Na

  20. Transgene mus som sygdomsmodeller

    DEFF Research Database (Denmark)

    Schuster, Mikkel Bruhn; Porse, Bo Torben

    2003-01-01

    Transgenic animal models have proven to be useful tools in understanding both basic biology and the events associated with disease. Recent technical advances in the area of genomic manipulation in combination with the availability of the human and murine genomic sequences now allow the precise...... tailoring of the mouse genome. In this review we describe a few systems in which transgenic animal models have been employed for the purpose of studying the etiology of human diseases. Udgivelsesdato: 2003-Feb-17...

  1. Gene conversion of ribosomal DNA in Nicotiana tabacum is associated with undermethylated, decondensed and probably active gene units.

    Science.gov (United States)

    Lim, K Y; Kovarik, A; Matýăsek, R; Bezdĕk, M; Lichtenstein, C P; Leitch, A R

    2000-06-01

    We examined the structure, intranuclear distribution and activity of ribosomal DNA (rDNA) in Nicotiana sylvestris (2n = 2x = 24) and N. tomentosiformis (2n = 2x = 24) and compared these with patterns in N. tabacum (tobacco, 2n = 4x = 48). We also examined a long-established N. tabacum culture, TBY-2. Nicotiana tabacum is an allotetraploid thought to be derived from ancestors of N. sylvestris (S-genome donor) and N. tomentosiformis (T-genome donor). Nicotiana sylvestris has three rDNA loci, one locus each on chromosomes 10, 11, and 12. In root-tip meristematic interphase cells, the site on chromosome 12 remains condensed and inactive, while the sites on chromosomes 10 and 11 show activity at the proximal end of the locus only. Nicotiana tomentosiformis has one major locus on chromosome 3 showing activity and a minor, inactive locus on chromosome 11. In N. tabacum cv. 095-55, there are four rDNA loci on T3, S10, S11/t and S12 (S11/t carries a small T-genome translocation). The locus on S12 remains condensed and inactive in root-tip meristematic cells while the others show activity, including decondensation at interphase and secondary constrictions at metaphase. Nicotiana tabacum DNA digested with methylcytosine-sensitive enzymes revealed a hybridisation pattern for rDNA that resembled that of N. tomentosiformis and not N. sylvestris. The data indicate that active, undermethylated genes are of the N. tomentosiformis type. Since S-genome chromosomes of N. tabacum show rDNA expression, the result indicates rDNA gene conversion of the active rDNA units on these chromosomes. Gene conversion in N. tabacum is consistent with the results of previous work. However, using primers specific for the S-genome rDNA intergenic sequences (IGS) in the polymerase chain reaction (PCR) show that rDNA gene conversion has not gone to completion in N. tabacum. Furthermore, using methylation-insensitive restriction enzymes we demonstrate that about 8% of the rDNA units remain of the N

  2. Response of antioxidant enzymes in Nicotiana tabacum clones during phytoextraction of heavy metals.

    Science.gov (United States)

    Lyubenova, Lyudmila; Nehnevajova, Erika; Herzig, Rolf; Schröder, Peter

    2009-07-01

    Tobacco, Nicotiana tabacum, is a widely used model plant for growth on heavy-metal-contaminated sites. Its high biomass and deep rooting system make it interesting for phytoextraction. In the present study, we investigated the antioxidative activities and glutathione-dependent enzymes of different tobacco clones optimized for better Cd and Zn accumulation in order to characterize their performance in the field. The improved heavy metal resistance also makes the investigated tobacco clones interesting for understanding the plant defense enzyme system in general. Freshly harvested plant material (N. tabacum leaves) was used to investigate the antioxidative cascade in plants grown on heavy metal contaminated sites with and without amendments of different ammonium nitrate and ammonium sulfate fertilizers. Plants were grown on heavily polluted soils in north-east Switzerland. Leaves were harvested at the field site and directly deep frozen in liquid N(2). Studies were concentrated on the antioxidative enzymes of the Halliwell-Asada cycle, and spectrophotometric measurements of catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), superoxide dismutase (SOD, EC 1.15.1.1), glutathione peroxidase (GPX, EC 1.11.1.9), glutathione reductase (GR, EC 1.6.4.2), glutathione S-transferase (GST, EC 2.5.1.18) were performed. We tried to explain the relationship between fertilizer amendments and the activity of the enzymatic defense systems. When tobacco (N. tabacum) plants originating from different mutants were grown under field conditions with varying fertilizer application, the uptake of cadmium and zinc from soil increased with increasing biomass. Depending on Cd and Zn uptake, several antioxidant enzymes showed significantly different activities. Whereas SOD and CAT were usually elevated, several other enzymes, and isoforms of GST were strongly inhibited. Heavy metal uptake represents severe stress to plants, and specific antioxidative enzymes are induced at the

  3. TOBACCO CONTROL

    International Development Research Centre (IDRC) Digital Library (Canada)

    Tobacco is farmed in more than 125 countries and the problems associated with this ... Canada's International Development Research Centre (IDRC) is one of the world's leading institutions in the generation and application of new ... assumptions about the relative safety ... In Kenya, researchers at Maseno University work.

  4. 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.

  5. Transient Expression and Purification of Horseradish Peroxidase C in Nicotiana benthamiana.

    Science.gov (United States)

    Huddy, Suzanne M; Hitzeroth, Inga I; Meyers, Ann E; Weber, Brandon; Rybicki, Edward P

    2018-01-01

    Horseradish peroxidase (HRP) is a commercially important reagent enzyme used in molecular biology and in the diagnostic product industry. It is typically purified from the roots of the horseradish ( Armoracia rusticana ); however, this crop is only available seasonally, yields are variable and often low, and the product is a mixture of isoenzymes. Engineering high-level expression in transiently transformed tobacco may offer a solution to these problems. In this study, a synthetic Nicotiana benthamiana codon-adapted full-length HRP isoenzyme gene as well as C-terminally truncated and both N- and C-terminally truncated versions of the HRP C gene were synthesized, and their expression in N. benthamiana was evaluated using an Agrobacterium tumefaciens -mediated transient expression system. The influence on HRP C expression levels of co-infiltration with a silencing suppressor (NSs) construct was also evaluated. Highest HRP C levels were consistently obtained using either the full length or C-terminally truncated HRP C constructs. HRP C purification by ion exchange chromatography gave an overall yield of 54% with a Reinheitszahl value of >3 and a specific activity of 458 U/mg. The high level of HRP C production in N. benthamiana in just five days offers an alternative, viable, and scalable system for production of this commercially significant enzyme.

  6. Introgression of a Tombusvirus Resistance Locus from Nicotiana edwardsonii var. Columbia to N. clevelandii.

    Science.gov (United States)

    Schoelz, James E; Wiggins, B Elizabeth; Wintermantel, William M; Ross, Kathleen

    2006-05-01

    ABSTRACT A new variety of Nicotiana, N. edwardsonii var. Columbia, was evaluated for its capacity to serve as a new source for virus resistance genes. Columbia was developed from a hybridization between N. glutinosa and N. clevelandii, the same parents used for the formation of the original N. edwardsonii. However, in contrast to the original N. edwardsonii, crosses between Columbia and either of its parents are fertile. Thus, the inheritance of virus resistance genes present in N. glutinosa could be characterized by using Columbia as a bridge plant in crosses with the susceptible parent, N. clevelandii. To determine how virus resistance genes would segregate in interspecific crosses between Columbia and N. clevelandii, we followed the fate of the N gene, a single dominant gene that specifies resistance to Tobacco mosaic virus (TMV). Our genetic evidence indicated that the entire chromosome containing the N gene was introgressed into N. clevelandii to create an addition line, designated N. clevelandii line 19. Although line 19 was homozygous for resistance to TMV, it remained susceptible to Tomato bushy stunt virus (TBSV) and Cauliflower mosaic virus (CaMV) strain W260, indicating that resistance to these viruses must reside on other N. glutinosa chromosomes. We also developed a second addition line, N. clevelandii line 36, which was homozygous for resistance to TBSV. Line 36 was susceptible to TMV and CaMV strain W260, but was resistant to other tombusviruses, including Cucumber necrosis virus, Cymbidium ringspot virus, Lettuce necrotic stunt virus, and Carnation Italian ringspot virus.

  7. Natural variation in floral nectar proteins of two Nicotiana attenuata accessions.

    Science.gov (United States)

    Seo, Pil Joon; Wielsch, Natalie; Kessler, Danny; Svatos, Ales; Park, Chung-Mo; Baldwin, Ian T; Kim, Sang-Gyu

    2013-07-13

    Floral nectar (FN) contains not only energy-rich compounds to attract pollinators, but also defense chemicals and several proteins. However, proteomic analysis of FN has been hampered by the lack of publically available sequence information from nectar-producing plants. Here we used next-generation sequencing and advanced proteomics to profile FN proteins in the opportunistic outcrossing wild tobacco, Nicotiana attenuata. We constructed a transcriptome database of N. attenuata and characterized its nectar proteome using LC-MS/MS. The FN proteins of N. attenuata included nectarins, sugar-cleaving enzymes (glucosidase, galactosidase, and xylosidase), RNases, pathogen-related proteins, and lipid transfer proteins. Natural variation in FN proteins of eleven N. attenuata accessions revealed a negative relationship between the accumulation of two abundant proteins, nectarin1b and nectarin5. In addition, microarray analysis of nectary tissues revealed that protein accumulation in FN is not simply correlated with the accumulation of transcripts encoding FN proteins and identified a group of genes that were specifically expressed in the nectary. Natural variation of identified FN proteins in the ecological model plant N. attenuata suggests that nectar chemistry may have a complex function in plant-pollinator-microbe interactions.

  8. A fundamental research of growth, metabolism and product formation of tobacco suspension cells at different scales

    OpenAIRE

    Ullisch, David

    2012-01-01

    For over two decades, plant cell cultures have been promising hosts for the expression of recombinant proteins such as hormones, growth factors, full-size antibodies and antigens. So far, over 700 different plant cell cultures are stored in the German Collection of Microorganisms and Cell Cultures (DSMZ) in Braunschweig. Among these plant cell cultures, the tobacco cell line Nicotiana tabacum Bright Yellow 2 (BY-2) was chosen as a good host cell line for the production of recombinant proteins...

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

    OpenAIRE

    Md. Shafiullah Shajib; Bidyut Kanti Datta; Md. Hossain Sohrab; Mohammad Abdur Rashid; Lutfun Nahar; Satyajit Dey Sarker

    2017-01-01

    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...

  10. Host-Pathogen Interactions : XXXII. A Fungal Glucan Preparation Protects Nicotianae against Infection by Viruses.

    Science.gov (United States)

    Kopp, M; Rouster, J; Fritig, B; Darvill, A; Albersheim, P

    1989-05-01

    A glucan preparation obtained from the mycelial walls of the fungus Phytophthora megasperma f.sp. glycinea and known as an elicitor of phytoalexins in soybean was shown to be a very efficient inducer of resistance against viruses in tobacco. The glucan preparation protected against mechanically transmitted viral infections on the upper and lower leaf surfaces. Whether the glucan preparation was applied by injection, inoculation, or spraying, it protected the plants if applied before, at the same time as, or not later than 8 hours after virus inoculation. At concentrations ranging from 0.1 to 10 micrograms per milliliter, the glucan preparation induced protection ranging from 50 to 100% against both symptom production (necrotic local lesions, necrotic rings, or systemic mosaic) and virus accumulation in all Nicotiana-virus combinations examined. However, no significant protection against some of the same viruses was observed in bean or turnip. The host plants successfully protected included N. tabacum (9 different cultivars), N. sylvestris, N. glutinosa, and N. clevelandii. The viruses belonged to several taxonomic groups including tobacco mosaic virus, alfalfa mosaic virus, and tomato black ring virus. The glucan preparation did not act directly on the virus and did not interfere with virus disassembly; rather, it appeared to induce changes in the host plant that prevented infections from being initiated or recently established infections from enlarging. The induced resistance does not depend on induction of pathogenesis-related proteins, the phenylpropanoid pathway, lignin-like substances, or callose-like materials. We believe the induced resistance results from a mechanism that has yet to be described.

  11. Retrotransposons of the Tnt1B family are mobile in Nicotiana plumbaginifolia and can induce alternative splicing of the host gene upon insertion.

    Science.gov (United States)

    Leprinc, A S; Grandbastien, M A; Christian, M

    2001-11-01

    Active retrotransposons have been identified in Nicotiana plumbaginifolia by their ability to disrupt the nitrate reductase gene in chlorate-resistant mutants selected from protoplast-derived cultures. In mutants E23 and F97, two independent insertions of Tnp2, a new retrotransposon closely related to the tobacco Tnt1 elements, were detected in the nitrate reductase gene. These two Tnp2 elements are members of the Tnt1B subfamily which shows that Tnt1B elements can be active and mutagenic in the N. plumbaginifolia genome. Furthermore, these results suggest that Tnt1B is the most active family of Tntl elements in N. plumbaginifolia, whereas in tobacco only members of the Tnt1A subfamily were found inserted in the nitrate reductase gene. The transcriptional regulations of Tnp2 and Tnt1A elements are most probably different due to non-conserved U3 regions. Our results thus support the hypothesis that different Nicotiana species contain different active Tntl subfamilies and that only one active Tntl subfamily might be maintained in each of these species. The Tnp2 insertion found in the F97 mutant was found to be spliced out of the nitrate reductase mRNA by activation of cryptic donor and acceptor sites in the nitrate reductase and the Tnp2 sequences respectively.

  12. Secondhand Tobacco Smoke (Environmental Tobacco Smoke)

    Science.gov (United States)

    Learn about secondhand tobacco smoke, which can raise your risk of lung cancer. Secondhand tobacco smoke is the combination of the smoke given off by a burning tobacco product and the smoke exhaled by a smoker. Also called environmental tobacco smoke, involuntary smoke, and passive smoke.

  13. Large-scale development of SSR markers in tobacco and construction of a linkage map in flue-cured tobacco.

    Science.gov (United States)

    Tong, Zhijun; Xiao, Bingguang; Jiao, Fangchan; Fang, Dunhuang; Zeng, Jianmin; Wu, Xingfu; Chen, Xuejun; Yang, Jiankang; Li, Yongping

    2016-06-01

    Tobacco (Nicotiana tabacum L.), particularly flue-cured tobacco, is one of the most economically important nonfood crops and is also an important model system in plant biotechnology. Despite its importance, only limited molecular marker resources are available for genome analysis, genetic mapping, and breeding. Simple sequence repeats (SSR) are one of the most widely-used molecular markers, having significant advantages including that they are generally co-dominant, easy to use, abundant in eukaryotic organisms, and produce highly reproducible results. In this study, based on the genome sequence data of flue-cured tobacco (K326), we developed a total of 13,645 mostly novel SSR markers, which were working in a set of eighteen tobacco varieties of four different types. A mapping population of 213 backcross (BC1) individuals, which were derived from an intra-type cross between two flue-cured tobacco varieties, Y3 and K326, was selected for mapping. Based on the newly developed SSR markers as well as published SSR markers, we constructed a genetic map consisting of 626 SSR loci distributed across 24 linkage groups and covering a total length of 1120.45 cM with an average distance of 1.79 cM between adjacent markers, which is the highest density map of flue-cured tobacco till date.

  14. Molecular characterization of tocopherol biosynthetic genes in sweetpotato that respond to stress and activate the tocopherol production in tobacco.

    Science.gov (United States)

    Ji, Chang Yoon; Kim, Yun-Hee; Kim, Ho Soo; Ke, Qingbo; Kim, Gun-Woo; Park, Sung-Chul; Lee, Haeng-Soon; Jeong, Jae Cheol; Kwak, Sang-Soo

    2016-09-01

    Tocopherol (vitamin E) is a chloroplast lipid that is presumed to be involved in the plant response to oxidative stress. In this study, we isolated and characterized five tocopherol biosynthetic genes from sweetpotato (Ipomoea batatas [L.] Lam) plants, including genes encoding 4-hydroxyphenylpyruvate dioxygenase (IbHPPD), homogentisate phytyltransferase (IbHPT), 2-methyl-6-phytylbenzoquinol methyltransferase (IbMPBQ MT), tocopherol cyclase (IbTC) and γ-tocopherol methyltransferase (IbTMT). Fluorescence microscope analysis indicated that four proteins localized into the chloroplast, whereas IbHPPD observed in the nuclear. Quantitative RT-PCR analysis revealed that the expression patterns of the five tocopherol biosynthetic genes varied in different plant tissues and under different stress conditions. All five genes were highly expressed in leaf tissues, whereas IbHPPD and IbHPT were highly expressed in the thick roots. The expression patterns of these five genes significantly differed in response to PEG, NaCl and H2O2-mediated oxidative stress. IbHPPD was strongly induced following PEG and H2O2 treatment and IbHPT was strongly induced following PEG treatment, whereas IbMPBQ MT and IbTC were highly expressed following NaCl treatment. Upon infection of the bacterial pathogen Pectobacterium chrysanthemi, the expression of IbHPPD increased sharply in sweetpotato leaves, whereas the expression of the other genes was reduced or unchanged. Additionally, transient expression of the five tocopherol biosynthetic genes in tobacco (Nicotiana bentamiana) leaves resulted in increased transcript levels of the transgenes expressions and tocopherol production. Therefore, our results suggested that the five tocopherol biosynthetic genes of sweetpotato play roles in the stress defense response as transcriptional regulators of the tocopherol production. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  15. Bean Yellow Dwarf Virus replicons for high-level transgene expression in transgenic plants and cell cultures.

    Science.gov (United States)

    Zhang, Xiuren; Mason, Hugh

    2006-02-05

    A novel stable transgenic plant expression system was developed using elements of the replication machinery of Bean Yellow Dwarf Virus (BeYDV). The system contains two transgenes: 1) The BeYDV replicon vector with an expression cassette flanked by cis-acting DNA elements of BeYDV, and 2) The viral replication initiator protein (Rep) controlled by an alcohol-inducible promoter. When Rep expression was triggered by treatment with ethanol, it induced release of the BeYDV replicon from stably integrated T-DNA and episomal replication to high copy number. Replicon amplification resulted in substantially increased transgene mRNA levels (up to 80-fold) and translation products (up to 10-fold) after induction of Rep expression by ethanol treatment in tobacco NT1 cells and leaves of whole potato plants. Thus, the BeYDV stable transformant replicon system is a powerful tool for plant-based production of recombinant proteins. (c) 2005 Wiley Periodicals, Inc.

  16. Effects of arbuscular mycorrhizal inoculation on cadmium accumulation by different tobacco (Nicotiana tabacum L.) types

    Czech Academy of Sciences Publication Activity Database

    Janoušková, Martina; Vosátka, Miroslav; Rossi, L.; Lugon-Moulin, M.

    2007-01-01

    Roč. 35, č. 3 (2007), s. 502-510 ISSN 0929-1393 R&D Projects: GA AV ČR KSK6005114 Institutional research plan: CEZ:AV0Z60050516 Keywords : agriculture * Glomus * heavy metals Subject RIV: EF - Botanics Impact factor: 1.810, year: 2007

  17. Histological study of smoke extract of Tobacco nicotiana on the heart ...

    African Journals Online (AJOL)

    The heart, liver, lungs, kidney, and testes were carefully excised, blotted dry, and fixed in formol saline for histological analysis using Hematoxylin and Eosin stain. Results: Using the light microscope, it was observed that the histoarchitectural profiles of the studied organs in the sections obtained from the control animals ...

  18. Photosynthesis in leaves of Nicotiana tabacum L. infected with tobacco mosaic virus

    Czech Academy of Sciences Publication Activity Database

    Wilhelmová, Naděžda; Procházková, Dagmar; Šindelářová, Milada; Šindelář, Luděk

    2005-01-01

    Roč. 43, č. 4 (2005), s. 597-602 ISSN 0300-3604 R&D Projects: GA ČR GA522/02/0708 Institutional research plan: CEZ:AV0Z50380511 Keywords : carotenoids * chlorophyll * chlorophyll fluorescence Subject RIV: CE - Biochemistry Impact factor: 0.810, year: 2005

  19. Characterization of natural leaf senescence in tobacco (Nicotiana tabacum) plants grown in vitro

    Czech Academy of Sciences Publication Activity Database

    Uzelac, B.; Janošević, D.; Simonović, A.; Motyka, Václav; Dobrev, Petre; Budimir, S.

    2016-01-01

    Roč. 253, č. 2 (2016), s. 259-275 ISSN 0033-183X R&D Projects: GA ČR(CZ) GAP506/11/0774 Institutional support: RVO:61389030 Keywords : Leaf senescence * Mesophyll ultrastructure * Phytohormones Subject RIV: EF - Botanics Impact factor: 2.870, year: 2016

  20. Youth access to tobacco.

    Science.gov (United States)

    Rigotti, N A

    1999-01-01

    To start smoking, young people need a supply of tobacco products. Reducing youth access to tobacco is a new approach to preventing tobacco use that has been a focus of federal, state, and local tobacco control efforts over the past decade. All 50 states ban tobacco sales to minors, but compliance is poor because laws are not enforced. Consequently, young people have little trouble obtaining tobacco products. Commercial sources of tobacco (stores and vending machines) are important for underage smokers, who often purchase their own cigarettes. Underage youths also obtain tobacco from noncommercial sources such as friends, relatives, older adolescents, and adults. Educating retailers about tobacco sales laws has not produced long-term improvement in their compliance. Active enforcement of tobacco sales laws changes retailer behavior, but whether this reduces young people's access to tobacco or their tobacco use is not clear. The effectiveness of new local, state, and federal actions that aim to reduce youth access to tobacco remains to be determined. Can enforcing tobacco sales laws reduce young people's access to tobacco? If so, will this prevent or delay the onset of their tobacco use? How will youths' sources of tobacco change as commercial sources are restricted? What are the social (noncommercial) sources of tobacco for minors and how can youths' access to tobacco from these sources be reduced? What is the impact of the new federal policies aimed at reducing youth access to tobacco? Do new state and local laws that ban youth possession or use of tobacco have a net positive or negative impact on youth attitudes, access to tobacco, or tobacco use? What is the relative effectiveness and cost-effectiveness of efforts to reduce the supply of tobacco compared to those that aim to reduce demand for tobacco? Will either work alone or are both necessary to achieve reductions in youth smoking?

  1. Expression of a constitutively activated plasma membrane H+-ATPase in Nicotiana tabacum BY-2 cells results in cell expansion.

    Science.gov (United States)

    Niczyj, Marta; Champagne, Antoine; Alam, Iftekhar; Nader, Joseph; Boutry, Marc

    2016-11-01

    Increased acidification of the external medium by an activated H + -ATPase results in cell expansion, in the absence of upstream activating signaling. The plasma membrane H + -ATPase couples ATP hydrolysis with proton transport outside the cell, and thus creates an electrochemical gradient, which energizes secondary transporters. According to the acid growth theory, this enzyme is also proposed to play a major role in cell expansion, by acidifying the external medium and so activating enzymes that are involved in cell wall-loosening. However, this theory is still debated. To challenge it, we made use of a plasma membrane H + -ATPase isoform from Nicotiana plumbaginifolia truncated from its C-terminal auto-inhibitory domain (ΔCPMA4), and thus constitutively activated. This protein was expressed in Nicotiana tabacum BY-2 suspension cells using a heat shock inducible promoter. The characterization of several independent transgenic lines showed that the expression of activated ΔCPMA4 resulted in a reduced external pH by 0.3-1.2 units, as well as in an increased H + -ATPase activity by 77-155 % (ATP hydrolysis), or 70-306 % (proton pumping) of isolated plasma membranes. In addition, ΔCPMA4-expressing cells were 17-57 % larger than the wild-type cells and displayed abnormal shapes. A proteomic comparison of plasma membranes isolated from ΔCPMA4-expressing and wild-type cells revealed the altered abundance of several proteins involved in cell wall synthesis, transport, and signal transduction. In conclusion, the data obtained in this work showed that H + -ATPase activation is sufficient to induce cell expansion and identified possible actors which intervene in this process.

  2. Tobacco-Related Mortality

    Science.gov (United States)

    ... Exposure is High in Multiunit Housing Smokeless Products Electronic Cigarettes Youth Tobacco Prevention Tobacco Products Tobacco Ingredient ... 2004 [accessed 2015 Aug 17]. National Cancer Institute. Cigars: Health Effects and Trends [ PDF –2.93 MB] . ...

  3. Risks of tobacco

    Science.gov (United States)

    Secondhand smoke - risks; Cigarette smoking - risks; Smoking and smokeless tobacco - risks; Nicotine - risks ... tobacco that are known to cause cancer. HEALTH RISKS OF SMOKING OR USING SMOKELESS TOBACCO Knowing the ...

  4. 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...

  5. A hyper-thermostable α-amylase from Pyrococcus furiosus accumulates in Nicotiana tabacum as functional aggregates.

    Science.gov (United States)

    Zhu, Hong; Reynolds, L Bruce; Menassa, Rima

    2017-06-19

    Alpha amylase hydrolyzes α-bonds of polysaccharides such as starch and produces malto-oligosaccharides. Its starch saccharification applications make it an essential enzyme in the textile, food and brewing industries. Commercially available α-amylase is mostly produced from Bacillus or Aspergillus. A hyper-thermostable and Ca 2++ independent α-amylase from Pyrococcus furiosus (PFA) expressed in E.coli forms insoluble inclusion bodies and thus is not feasible for industrial applications. We expressed PFA in Nicotiana tabacum and found that plant-produced PFA forms functional aggregates with an accumulation level up to 3.4 g/kg FW (fresh weight) in field conditions. The aggregates are functional without requiring refolding and therefore have potential to be applied as homogenized plant tissue without extraction or purification. PFA can also be extracted from plant tissue upon dissolution in a mild reducing buffer containing SDS. Like the enzyme produced in P. furiosus and in E. coli, plant produced PFA preserves hyper-thermophilicity and hyper-thermostability and has a long shelf life when stored in lyophilized leaf tissue. With tobacco's large biomass and high yield, hyper-thermostable α-amylase was produced at a scale of 42 kg per hectare. Tobacco may be a suitable bioreactor for industrial production of active hyperthermostable alpha amylase.

  6. Manduca sexta recognition and resistance among allopolyploid Nicotiana host plants

    Science.gov (United States)

    Lou, Yonggen; Baldwin, Ian T.

    2003-01-01

    Allopolyploid speciation occurs instantly when the genomes of different species combine to produce self-fertile offspring and has played a central role in the evolution of higher plants, but its consequences for adaptive responses are unknown. We compare herbivore-recognition and -resistance responses of the diploid species and putative ancestral parent Nicotiana attenuata with those of the two derived allopolyploid species Nicotiana clevelandii and Nicotiana bigelovii. Manduca sexta larvae attack all three species, and in N. attenuata attack is recognized when larval oral secretions are introduced to wounds during feeding, resulting in a jasmonate burst, a systemic amplification of trypsin inhibitor accumulation, and a release of volatile organic compounds, which function as a coordinated defense response that slows caterpillar growth and increases the probability of their being attacked. Most aspects of this recognition response are retained with modifications in one allotetraploid (N. bigelovii) but lost in the other (N. clevelandii). Differences between diploid and tetraploid species were apparent in delays (maximum 1 and 0.5 h, respectively) in the jasmonate burst, the elicitation of trypsin inhibitors and release of volatile organic compounds, and the constitutive levels of nicotine, trypsin inhibitors, diterpene glycosides, rutin, and caffeoylputrescine in the leaves. Resistance to M. sexta larvae attack was most strongly associated with diterpene glycosides, which were higher in the diploid than in the two allotetraploid species. Because M. sexta elicitors differentially regulate a large proportion of the N. attenuata transcriptome, we propose that these species are suited for the study of the evolution of adaptive responses requiring trans-activation mechanisms. PMID:14530394

  7. Transgenics in Agriculture

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 6; Issue 2. Transgenics in Agriculture. D Rex Arunraj B Gajendra Babu. Classroom Volume 6 Issue 2 February 2001 pp 83-92. Fulltext. Click here to view fulltext PDF. Permanent link: https://www.ias.ac.in/article/fulltext/reso/006/02/0083-0092 ...

  8. Chlorogenic acid in a Nicotiana plumbaginifolia cell suspension.

    Science.gov (United States)

    Gillet; Mesnard; Fliniaux; Monti; Fliniaux

    1999-11-01

    A phenylpropanoid compound has been characterized in a Nicotiana plumbaginifolia cell suspension. This compound has been isolated and purified by semi-preparative reverse phase-high performance liquid chromatography. Its structure has been identified by NMR spectroscopy as 5-O-caffeoylquinic acid, which is chlorogenic acid (CA). The influence of culture conditions on the accumulation of this metabolite by N. plumbaginifolia cell suspensions has been studied. Darkness strongly inhibits the CA accumulation. Moreover, it has been shown that feeding experiments with caffeic acid had a deleterious effect upon the CA content. This one was not influenced by a supplementation with quinic acid.

  9. Interactions among tobacco sieve element occlusion (SEO) proteins.

    Science.gov (United States)

    Jekat, Stephan B; Ernst, Antonia M; Zielonka, Sascia; Noll, Gundula A; Prüfer, Dirk

    2012-12-01

    Angiosperms transport their photoassimilates through sieve tubes, which comprise longitudinally-connected sieve elements. In dicots and also some monocots, the sieve elements contain parietal structural proteins known as phloem proteins or P-proteins. Following injury, P proteins disperse and accumulate as viscous plugs at the sieve plates to prevent the loss of valuable transport sugars. Tobacco (Nicotiana tabacum) P-proteins are multimeric complexes comprising subunits encoded by members of the SEO (sieve element occlusion) gene family. The existence of multiple subunits suggests that P-protein assembly involves interactions between SEO proteins, but this process is largely uncharacterized and it is unclear whether the different subunits perform unique roles or are redundant. We therefore extended our analysis of the tobacco P-proteins NtSEO1 and NtSEO2 to investigate potential interactions between them, and found that both proteins can form homomeric and heteromeric complexes in planta.

  10. Genotoxicity of Nicotiana tabacum leaves on Helix aspersa.

    Science.gov (United States)

    da Silva, Fernanda R; Erdtmann, Bernardo; Dalpiaz, Tiago; Nunes, Emilene; Ferraz, Alexandre; Martins, Tales L C; Dias, Johny F; da Rosa, Darlan P; Porawskie, Marilene; Bona, Silvia; da Silva, Juliana

    2013-07-01

    Tobacco farmers are routinely exposed to complex mixtures of inorganic and organic chemicals present in tobacco leaves. In this study, we examined the genotoxicity of tobacco leaves in the snail Helix aspersa as a measure of the risk to human health. DNA damage was evaluated using the micronucleus test and the Comet assay and the concentration of cytochrome P450 enzymes was estimated. Two groups of snails were studied: one fed on tobacco leaves and one fed on lettuce (Lactuca sativa L) leaves (control group). All of the snails received leaves (tobacco and lettuce leaves were the only food provided) and water ad libitum. Hemolymph cells were collected after 0, 24, 48 and 72 h. The Comet assay and micronucleus test showed that exposure to tobacco leaves for different periods of time caused significant DNA damage. Inhibition of cytochrome P450 enzymes occurred only in the tobacco group. Chemical analysis indicated the presence of the alkaloid nicotine, coumarins, saponins, flavonoids and various metals. These results show that tobacco leaves are genotoxic in H. aspersa and inhibit cytochrome P450 activity, probably through the action of the complex chemical mixture present in the plant.

  11. Genotoxicity of Nicotiana tabacum leaves on Helix aspersa

    Directory of Open Access Journals (Sweden)

    Fernanda R. da Silva

    2013-01-01

    Full Text Available Tobacco farmers are routinely exposed to complex mixtures of inorganic and organic chemicals present in tobacco leaves. In this study, we examined the genotoxicity of tobacco leaves in the snail Helix aspersa as a measure of the risk to human health. DNA damage was evaluated using the micronucleus test and the Comet assay and the concentration of cytochrome P450 enzymes was estimated. Two groups of snails were studied: one fed on tobacco leaves and one fed on lettuce (Lactuca sativa L leaves (control group. All of the snails received leaves (tobacco and lettuce leaves were the only food provided and water ad libitum. Hemolymph cells were collected after 0, 24, 48 and 72 h. The Comet assay and micronucleus test showed that exposure to tobacco leaves for different periods of time caused significant DNA damage. Inhibition of cytochrome P450 enzymes occurred only in the tobacco group. Chemical analysis indicated the presence of the alkaloid nicotine, coumarins, saponins, flavonoids and various metals. These results show that tobacco leaves are genotoxic in H. aspersa and inhibit cytochrome P450 activity, probably through the action of the complex chemical mixture present in the plant.

  12. Phosphorus acquisition by citrate- and phytase-exuding Nicotiana tabacum plant mixtures depends on soil phosphorus availability and root intermingling.

    Science.gov (United States)

    Giles, Courtney D; Richardson, Alan E; Cade-Menun, Barbara J; Mezeli, Malika M; Brown, Lawrie K; Menezes-Blackburn, Daniel; Darch, Tegan; Blackwell, Martin Sa; Shand, Charles A; Stutter, Marc I; Wendler, Renate; Cooper, Patricia; Lumsdon, David G; Wearing, Catherine; Zhang, Hao; Haygarth, Philip M; George, Timothy S

    2018-03-02

    Citrate and phytase root exudates contribute to improved phosphorus (P) acquisition efficiency in Nicotiana tabacum (tobacco) when both exudates are produced in a P deficient soil. To test the importance of root intermingling in the interaction of citrate and phytase exudates, Nicotiana tabacum plant-lines with constitutive expression of heterologous citrate (Cit) or fungal phytase (Phy) exudation traits were grown under two root treatments (roots separated or intermingled) and in two soils with contrasting soil P availability. Complementarity of plant mixtures varying in citrate efflux rate and mobility of the expressed phytase in soil was determined based on plant biomass and P accumulation. Soil P composition was evaluated using solution 31 P NMR spectroscopy. In the soil with limited available P, positive complementarity occurred in Cit+Phy mixtures with roots intermingled. Root separation eliminated positive interactions in mixtures expressing the less mobile phytase (Aspergillus niger PhyA) whereas positive complementarity persisted in mixtures that expressed the more mobile phytase (Peniophora lycii PhyA). Soils from Cit+Phy mixtures contained less inorganic P and more organic P compared to monocultures. Exudate-specific strategies for the acquisition of soil P were most effective in P-limited soil and depended on citrate efflux rate and the relative mobility of the expressed phytase in soil. Plant growth and soil P utilization in plant systems with complementary exudation strategies are expected to be greatest where exudates persist in soil and are expressed synchronously in space and time. This article is protected by copyright. All rights reserved.

  13. 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.

  14. Zoospore exudates from Phytophthora nicotianae affect immune responses in Arabidopsis.

    Science.gov (United States)

    Kong, Ping; McDowell, John M; Hong, Chuanxue

    2017-01-01

    Zoospore exudates play important roles in promoting zoospore communication, homing and germination during plant infection by Phytophthora. However, it is not clear whether exudates affect plant immunity. Zoospore-free fluid (ZFF) and zoospores of P. nicotianae were investigated comparatively for effects on resistance of Arabidopsis thaliana Col-0 and mutants that affect signaling mediated by salicylic acid (SA) and jasmonic acid (JA): eds16 (enhanced disease susceptibility16), pad4 (phytoalexin deficient4), and npr1 (nonexpressor of pathogenesis-related genes1). Col-0 attracted more zoospores and had severe tissue damage when flooded with a zoospore suspension in ZFF. Mutants treated with ZFF alone developed disease symptoms similar to those inoculated with zoospores and requirements of EDS16 and PAD4 for plant responses to zoospores and the exudates was apparent. Zoospore and ZFFs also induced expression of the PR1 and PDF1.2 marker genes for defense regulated by SA and JA, respectively. However, ZFF affected more JA defense signaling, down regulating PR1 when SA signaling or synthesis is deficient, which may be responsible for Arabidopsis mutant plants more susceptible to infection by high concentration of P. nicotianae zoospores. These results suggest that zoospore exudates can function as virulence factors and inducers of plant immune responses during plant infection by Phytophthora.

  15. Zoospore exudates from Phytophthora nicotianae affect immune responses in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Ping Kong

    Full Text Available Zoospore exudates play important roles in promoting zoospore communication, homing and germination during plant infection by Phytophthora. However, it is not clear whether exudates affect plant immunity. Zoospore-free fluid (ZFF and zoospores of P. nicotianae were investigated comparatively for effects on resistance of Arabidopsis thaliana Col-0 and mutants that affect signaling mediated by salicylic acid (SA and jasmonic acid (JA: eds16 (enhanced disease susceptibility16, pad4 (phytoalexin deficient4, and npr1 (nonexpressor of pathogenesis-related genes1. Col-0 attracted more zoospores and had severe tissue damage when flooded with a zoospore suspension in ZFF. Mutants treated with ZFF alone developed disease symptoms similar to those inoculated with zoospores and requirements of EDS16 and PAD4 for plant responses to zoospores and the exudates was apparent. Zoospore and ZFFs also induced expression of the PR1 and PDF1.2 marker genes for defense regulated by SA and JA, respectively. However, ZFF affected more JA defense signaling, down regulating PR1 when SA signaling or synthesis is deficient, which may be responsible for Arabidopsis mutant plants more susceptible to infection by high concentration of P. nicotianae zoospores. These results suggest that zoospore exudates can function as virulence factors and inducers of plant immune responses during plant infection by Phytophthora.

  16. Cloning and functional analysis in transgenic tobacco of a tapetum ...

    African Journals Online (AJOL)

    Jane

    2010-10-11

    Oct 11, 2010 ... “anther-box”, have been already used for genetic engineering of ... tabacum var. NC89) was used for transformation in Murashige and ..... 265-272. Koltunow AM, Truettner T, Cox KH, Wallroth M, Goldberg RB (1990). Different ...

  17. The effect of synchrotron radiation on nicotiana tabacum-roots in oxygen atmosphere

    International Nuclear Information System (INIS)

    Avakyan, Ts.M.; Karagezyan, A.S.; Danielyan, A.Kh.

    1977-01-01

    The question of mutual action of sVnchrotron radiation (SR) and living objects and the influence of powerful radiations on the peculiarities of their functioning is a major problem in all fields where SR in applied, as well as in medicobiological aspects of space flights. The present report summarizes new experimental findings concerning the action of magnetic-inhibiting radiation on Nicotiana tabacum - roots in oxygen and helium atmosphere. Comparative studies have been carried out on ''oxygen effect'' of SR and X-ray radiation by traditional radiobiological equipment. The experiments have been performed on the 2 synchrotron channel of Yerevan Physical Institute Electron Accelerator. The circular current of the accelerator equals 1 ma at a maximal energy of electrons in the ring 4.5 GeV. Nonmonochromatized SR coming out from the beryllium window of the current conductor entered a special maylar chamber which was filled with oxygen and helium. 4-day old roots of tobacco seeds were radiated in the chamber. The radiation dose in X-ray, as well as in SR equals 500 rad/min. X-ray radiation was carried out with the use of a RUP-200/20 equipment at a regime of J=15 ma, E=183 kV. In applying 500, 00 and 2500 rad in oxygen atmosphere a marked maximum of chromosome aberration frequency was noted at 2500 rad. Comparative investigations have shown that in radiating the roots by X-ray in oxygen atmosphere, the percentage of chromosome aberrations constitutes 4.5 at 2500 rad, while in SR it equals 24. The ''oxygen effect'' has been demonstrated, and the protective effect in helium atmosphere. The question of dosimetry is discussed, and basing on modern views a working hypothesis is presented which explains the marked damaging effect of SR action in oxygen atmosphere

  18. 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

  19. 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

  20. 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.

    Directory of Open Access Journals (Sweden)

    Kira Tiedge

    Full Text Available 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

  1. Recovery of Nicotiana benthamiana plants from a necrotic response induced by a nepovirus is associated with RNA silencing but not with reduced virus titer.

    Science.gov (United States)

    Jovel, Juan; Walker, Melanie; Sanfaçon, Hélène

    2007-11-01

    Recovery of plants from virus-induced symptoms is often described as a consequence of RNA silencing, an antiviral defense mechanism. For example, recovery of Nicotiana clevelandii from a nepovirus (tomato black ring virus) is associated with a decreased viral RNA concentration and sequence-specific resistance to further virus infection. In this study, we have characterized the interaction of another nepovirus, tomato ringspot virus (ToRSV), with host defense responses during symptom induction and subsequent recovery. Early in infection, ToRSV induced a necrotic phenotype in Nicotiana benthamiana that showed characteristics typical of a hypersensitive response. RNA silencing was also activated during ToRSV infection, as evidenced by the presence of ToRSV-derived small interfering RNAs (siRNAs) that could direct degradation of ToRSV sequences introduced into sensor constructs. Surprisingly, disappearance of symptoms was not accompanied by a commensurate reduction in viral RNA levels. The stability of ToRSV RNA after recovery was also observed in N. clevelandii and Cucumis sativus and in N. benthamiana plants carrying a functional RNA-dependent RNA polymerase 1 ortholog from Medicago truncatula. In experiments with a reporter transgene (green fluorescent protein), ToRSV did not suppress the initiation or maintenance of transgene silencing, although the movement of the silencing signal was partially hindered. Our results demonstrate that although RNA silencing is active during recovery, reduction of virus titer is not required for the initiation of this phenotype. This scenario adds an unforeseen layer of complexity to the interaction of nepoviruses with the host RNA silencing machinery. The possibility that viral proteins, viral RNAs, and/or virus-derived siRNAs inactivate host defense responses is discussed.

  2. Dynamic behavior of tobacco waste in the coal-fired fluidized-bed boiler

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Kai; Chang, Jian; Chen, Honggang; Yang, Yongping [North China Electric Power Univ., Beijing (China). National Eng Lab for Biomass Power Generation Equipment; Yu, Bangting [China Univ. of Petroleum, Beijing (China). State Key Lab. of Heavy Oil Processing

    2013-07-01

    Circulating fluidized bed (CFB) technology is an advanced method for utilizing coal and other solid fuels in an environmentally acceptable manner. During the processing procedure in the nicotiana tabacum plants, lots of tobacco stem wastes are produced, which are normally being dumped to the landfill field. If this kind of waste can be used as a part of the fuel to be added into the coal in a CFB combustor, it will reduce the use of coal and then cut the net carbon emissions. To understand the complicated fluid dynamics of nicotiana tabacum wastes in the coal-fired CFB boiler, the mixing and segregation behavior of tobacco stalk are preliminary measured in a cylindrical fluidized bed. Obvious segregation behavior is found due to distinct differences in density and shape between tobacco stem and coal, which results in poor fluidization quality and bad combustion efficiency. To overcome this disadvantage, a jet with high gas velocity is introduced through the air distributor and a detailed experimental study is conducted in a fluidized bed made up of stem-sand mixture with different solid components at various jet velocities, which greatly improve the mixing performance of stem in the fluidized bed. The above findings are helpful for the technological upgrading of small- or middle-sized CFB boiler with adding tobacco stem into coal.

  3. NaStEP: A Proteinase Inhibitor Essential to Self-Incompatibility and a Positive Regulator of HT-B Stability in Nicotiana alata Pollen Tubes1[W][OA

    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. PMID:23150644

  4. Root-specific expression of opine genes and opine accumulation in some cultivars of the naturally occurring genetically modified organism Nicotiana tabacum.

    Science.gov (United States)

    Chen, Ke; de Borne, François Dorlhac; Julio, Emilie; Obszynski, Julie; Pale, Patrick; Otten, Léon

    2016-08-01

    Previous studies have shown that Nicotiana tabacum contains three Agrobacterium-derived T-DNA sequences inherited from its paternal ancestor Nicotiana tomentosiformis. Among these, the TB locus carries an intact mannopine synthase 2' gene (TB-mas2'). This gene is similar to the Agrobacterium rhizogenes A4-mas2' gene that encodes the synthesis of the Amadori compound deoxyfructosyl-glutamine (DFG or santhopine). In this study we show that TB-mas2' is expressed at very low levels in N. tomentosiformis and in most N. tabacum cultivars; however, some cultivars show high TB-mas2' expression levels. The TB-mas2' promoter sequences of low- and high-expressing cultivars are identical. The low/high level of expression segregates as a single Mendelian factor in a cross between a low- and a high-expression cultivar. pTB-mas2'-GUS and pA4-mas2'-GUS reporter genes were stably introduced in N. benthamiana. Both were mainly expressed in the root expansion zone and leaf vasculature. Roots of tobacco cultivars with high TB-mas2' expression contain detectable levels of DFG. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  5. Repression of the DCL2 and DCL4 genes in Nicotiana benthamiana plants for the transient expression of recombinant proteins.

    Science.gov (United States)

    Matsuo, Kouki; Matsumura, Takeshi

    2017-08-01

    The production of recombinant proteins in plants has many advantages, including safety and reduced costs. However, this technology still faces several issues, including low levels of production. The repression of RNA silencing seems to be particularly important for improving recombinant protein production because RNA silencing effectively degrades transgene-derived mRNAs in plant cells. Therefore, to overcome this, we used RNA interference technology to develop DCL2- and DCL4-repressed transgenic Nicotiana benthamiana plants (ΔD2, ΔD4, and ΔD2ΔD4 plants), which had much lower levels of NbDCL2 and/or NbDCL4 mRNAs than wild-type plants. A transient gene expression assay showed that the ΔD2ΔD4 plants accumulated larger amounts of green fluorescent protein (GFP) and human acidic fibroblast growth factor (aFGF) than ΔD2, ΔD4, and wild-type plants. Furthermore, the levels of GFP and aFGF mRNAs were also higher in ΔD2ΔD4 plants than in ΔD2, ΔD4, and wild-type plants. These findings demonstrate that ΔD2ΔD4 plants express larger amounts of recombinant proteins than wild-type plants, and so would be useful for recombinant protein production. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  6. Expression of ACC oxidase promoter-GUS fusions in tomato and Nicotiana plumbaginifolia regulated by developmental and environmental stimuli.

    Science.gov (United States)

    Blume, B; Grierson, D

    1997-10-01

    The enzyme ACC oxidase, catalysing the last step in the biosynthesis of the plant hormone ethylene, is encoded by a small multigene family in tomato, comprising three members, LEACO1, LEACO2 and LEACO3. LEACO1 is the major gene expressed during ripening, leaf senescence, and wounding (Barry et al., 1996). To investigate the transcriptional regulation of ACC oxidase gene expression, chimeric fusions between the beta-glucuronidase reporter gene and 97 bp of 5' UTR plus 124, 396 and 1825 bp, respectively, of 5' untranscribed LEACO1 sequence were constructed and introduced into Lycopersicon esculentum (Mill cv. Ailsa Craig) and Nicotiana plumbaginifolia. Analysis of transgenic tomatoes indicated that the region containing nucleotides -124 to +97 of the LEACO1 gene is sufficient to confer a marked increase in GUS activity during fruit ripening, albeit at very low levels. Fusion of 396 and 1825 bp of LEACO1 upstream sequence resulted in strong and specific induction of GUS expression in situations known to be accompanied by enhanced ethylene production. Reporter gene expression was similar to that of the endogenous LEACO1 gene, with major increases especially during fruit ripening, senescence and abscission of leaves and, to a lesser extent, of flowers. Analysis of transgenic N. plumbaginifolia plants confirmed the pattern of LEACO1 promoter activity detected in tomato leaves and flowers. Reporter gene expression was also induced following wounding, treatment with ethylene, and pathogen infection. Histochemical analysis illustrated localized GUS activity in the pericarp of ripening fruit, abscission zones of senescent petioles and unfertilized flowers, and at wound sites. These results demonstrate that ACC oxidase is regulated at the transcriptional level in a wide range of cell types at different developmental stages and in response to several external stimuli.

  7. Transgenic algae engineered for higher performance

    Science.gov (United States)

    Unkefer, Pat J; Anderson, Penelope S; Knight, Thomas J

    2014-10-21

    The present disclosure relates to transgenic algae having increased growth characteristics, and methods of increasing growth characteristics of algae. In particular, the disclosure relates to transgenic algae comprising a glutamine phenylpyruvate transaminase transgene and to transgenic algae comprising a glutamine phenylpyruvate transaminase transgene and a glutamine synthetase.

  8. Differential expression of catalase genes in Nicotiana plumbaginifolia (L.).

    Science.gov (United States)

    Willekens, H; Langebartels, C; Tiré, C; Van Montagu, M; Inzé, D; Van Camp, W

    1994-10-25

    We have analyzed the expression of three catalase (Cat; EC 1.11.1.6) genes from Nicotiana plumbaginifolia by means of RNA blot and in situ hybridizations. Our data demonstrate that the expression of each catalase is associated with a particular H2O2-producing process. Cat1 appears to be specifically involved in the scavenging of photorespiratory H2O2 and is under control of a circadian rhythm, Cat2 is uniformly expressed in different organs with a cellular preference for vascular tissues, and the expression profile of Cat3 points to a role in glyoxysomal processes. Differential expression of these catalases is also manifested in response to temperature changes. DNA sequence comparison with other dicotyledonous catalases led to the identification of at least three distinct classes, which indicates that the functional organization of catalases is generally conserved in dicotyledonous plants.

  9. Molecular identification of catalases from Nicotiana plumbaginifolia (L.).

    Science.gov (United States)

    Willekens, H; Villarroel, R; Van Montagu, M; Inzé, D; Van Camp, W

    1994-09-19

    We have isolated three different catalase cDNAs from Nicotiana plumbaginifolia (cat1, cat2, and cat3) and a partial sequence of a fourth catalase gene (cat4) that shows no discernible expression based on Northern analysis. The catalase sequences were used to determine the similarity with other plant catalases and to study the transcriptional response to paraquat, 3-aminotriazole, and salicylic acid. 3-Aminotriazole induces mRNA levels of cat1, cat2 and cat3, indicating that a reduction in catalase activity positively affects catalase mRNA abundance. Salicylic acid that binds catalase in vitro, had no effect on catalase transcript levels at physiological concentrations. Paraquat resulted in the induction of cat1.

  10. Cysteine proteinases regulate chloroplast protein content and composition in tobacco leaves: a model for dynamic interactions with ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) vesicular bodies.

    Science.gov (United States)

    Prins, Anneke; van Heerden, Philippus D R; Olmos, Enrique; Kunert, Karl J; Foyer, Christine H

    2008-01-01

    The roles of cysteine proteinases (CP) in leaf protein accumulation and composition were investigated in transgenic tobacco (Nicotiana tabacum L.) plants expressing the rice cystatin, OC-1. The OC-1 protein was present in the cytosol, chloroplasts, and vacuole of the leaves of OC-1 expressing (OCE) plants. Changes in leaf protein composition and turnover caused by OC-1-dependent inhibition of CP activity were assessed in 8-week-old plants using proteomic analysis. Seven hundred and sixty-five soluble proteins were detected in the controls compared to 860 proteins in the OCE leaves. A cyclophilin, a histone, a peptidyl-prolyl cis-trans isomerase, and two ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase isoforms were markedly altered in abundance in the OCE leaves. The senescence-related decline in photosynthesis and Rubisco activity was delayed in the OCE leaves. Similarly, OCE leaves maintained higher leaf Rubisco activities and protein than controls following dark chilling. Immunogold labelling studies with specific antibodies showed that Rubisco was present in Rubisco vesicular bodies (RVB) as well as in the chloroplasts of leaves from 8-week-old control and OCE plants. Western blot analysis of plants at 14 weeks after both genotypes had flowered revealed large increases in the amount of Rubisco protein in the OCE leaves compared to controls. These results demonstrate that CPs are involved in Rubisco turnover in leaves under optimal and stress conditions and that extra-plastidic RVB bodies are present even in young source leaves. Furthermore, these data form the basis for a new model of Rubisco protein turnover involving CPs and RVBs.

  11. Ascorbate oxidase-dependent changes in the redox state of the apoplast modulate gene transcript accumulation leading to modified hormone signaling and orchestration of defense processes in tobacco.

    Science.gov (United States)

    Pignocchi, Cristina; Kiddle, Guy; Hernández, Iker; Foster, Simon J; Asensi, Amparo; Taybi, Tahar; Barnes, Jeremy; Foyer, Christine H

    2006-06-01

    The role of the redox state of the apoplast in hormone responses, signaling cascades, and gene expression was studied in transgenic tobacco (Nicotiana tabacum) plants with modified cell wall-localized ascorbate oxidase (AO). High AO activity specifically decreased the ascorbic acid (AA) content of the apoplast and altered plant growth responses triggered by hormones. Auxin stimulated shoot growth only when the apoplastic AA pool was reduced in wild-type or AO antisense lines. Oxidation of apoplastic AA in AO sense lines was associated with loss of the auxin response, higher mitogen-activated protein kinase activities, and susceptibility to a virulent strain of the pathogen Pseudomonas syringae. The total leaf glutathione pool, the ratio of reduced glutathione to glutathione disulfide, and glutathione reductase activities were similar in the leaves of all lines. However, AO sense leaves exhibited significantly lower dehydroascorbate reductase and ascorbate peroxidase activities than wild-type and antisense leaves. The abundance of mRNAs encoding antioxidant enzymes was similar in all lines. However, the day/night rhythms in the abundance of transcripts encoding the three catalase isoforms were changed in response to the AA content of the apoplast. Other transcripts influenced by AO included photorespiratory genes and a plasma membrane Ca(2+) channel-associated gene. We conclude that the redox state of the apoplast modulates plant growth and defense responses by regulating signal transduction cascades and gene expression patterns. Hence, AO activity, which modulates the redox state of the apoplastic AA pool, strongly influences the responses of plant cells to external and internal stimuli.

  12. Ectopic Expression of the Grape Hyacinth (Muscari armeniacum R2R3-MYB Transcription Factor Gene, MaAN2, Induces Anthocyanin Accumulation in Tobacco

    Directory of Open Access Journals (Sweden)

    Kaili Chen

    2017-06-01

    Full Text Available Anthocyanins are responsible for the different colors of ornamental plants. Grape hyacinth (Muscari armeniacum, a monocot plant with bulbous flowers, is popular for its fascinating blue color. In the present study, we functionally characterized an R2R3-MYB transcription factor gene MaAN2 from M. armeniacum. Our results indicated that MaAN2 participates in controlling anthocyanin biosynthesis. Sequence alignment and phylogenetic analysis suggested that MaAN2 belonged to the R2R3-MYB family AN2 subgroup. The anthocyanin accumulation of grape hyacinth flowers was positively correlated with the expression of MaAN2. And the transcriptional expression of MaAN2 was also consistent with that of M. armeniacum dihydroflavonol 4-reductase (MaDFR and M. armeniacum anthocyanidin synthase (MaANS in flowers. A dual luciferase transient expression assay indicated that when MaAN2 was co-inflitrated with Arabidopsis thaliana TRANSPARENT TESTA8 (AtTT8, it strongly activated the promoters of MaDFR and MaANS, but not the promoters of M. armeniacum chalcone synthase (MaCHS, M. armeniacum chalcone isomerase (MaCHI, and M. armeniacum flavanone 3-hydroxylase (MaF3H. Bimolecular fluorescence complementation assay confirmed that MaAN2 interacted with AtTT8 in vivo. The ectopic expression of MaAN2 in Nicotiana tabacum resulted in obvious red coloration of the leaves and much redder flowers. Almost all anthocyanin biosynthetic genes were remarkably upregulated in the leaves and flowers of the transgenic tobacco, and NtAn1a and NtAn1b (two basic helix–loop–helix anthocyanin regulatory genes were highly expressed in the transformed leaves, compared to the empty vector transformants. Collectively, our results suggest that MaAN2 plays a role in anthocyanin biosynthesis.

  13. RNaseI from Escherichia coli cannot substitute for S-RNase in rejection of Nicotiana plumbaginifolia pollen.

    Science.gov (United States)

    Beecher, B; Murfett, J; McClure, B A

    1998-03-01

    Unilateral incompatibility often occurs between self-incompatible (SI) species and their self-compatible (SC) relatives. For example, SI Nicotiana alata rejects pollen from SC N. plumbaginifolia, but the reciprocal pollination is compatible. This interspecific pollen rejection system closely resembles intraspecific S-allele-specific pollen rejection. However, the two systems differ in degree of specificity. In SI, rejection is S-allele-specific, meaning that only a single S-RNase causes rejection of pollen with a specific S genotype. Rejection of N. plumbaginifolia pollen is less specific, occurring in response to almost any S-RNase. Here, we have tested whether a non-S-RNase can cause rejection of N. plumbaginifolia pollen. The Escherichia coli rna gene encoding RNAseI was engineered for expression in transgenic (N. plumbaginifolia x SC N. alata) hybrids. Expression levels and pollination behavior of hybrids expressing E. coli RNaseI were compared to controls expressing SA2-RNase from N. alata. Immunoblot analysis and RNase activity assays showed that RNaseI and SA2-RNase were expressed at comparable levels. However, expression of SA2-RNase caused rejection of N. plumbaginifolia pollen, whereas expression of RNaseI did not. Thus, in this system, RNase activity alone is not sufficient for rejection of N. plumbaginifolia pollen. The results suggest that S-RNases may be specially adapted to function in pollen rejection.

  14. Root jasmonic acid synthesis and perception regulate folivore-induced shoot metabolites and increase Nicotiana attenuata resistance.

    Science.gov (United States)

    Fragoso, Variluska; Rothe, Eva; Baldwin, Ian T; Kim, Sang-Gyu

    2014-06-01

    While jasmonic acid (JA) signaling is widely accepted as mediating plant resistance to herbivores, and the importance of the roots in plant defenses is recently being recognized, the role of root JA in the defense of above-ground parts remains unstudied. To restrict JA impairment to the roots, we micrografted wildtype Nicotiana attenuata shoots to the roots of transgenic plants impaired in JA signaling and evaluated ecologically relevant traits in the glasshouse and in nature. Root JA synthesis and perception are involved in regulating nicotine production in roots. Strikingly, systemic root JA regulated local leaf JA and abscisic acid (ABA) concentrations, which were associated with differences in nicotine transport from roots to leaves via the transpiration stream. Root JA signaling also regulated the accumulation of other shoot metabolites; together these account for differences in resistance against a generalist, Spodoptera littoralis, and a specialist herbivore, Manduca sexta. In N. attenuata's native habitat, silencing root JA synthesis increased the shoot damage inflicted by Empoasca leafhoppers, which are able to select natural jasmonate mutants. Silencing JA perception in roots also increased damage by Tupiocoris notatus. We conclude that attack from above-ground herbivores recruits root JA signaling to launch the full complement of plant defense responses. © 2014 Max Planck Society. New Phytologist © 2014 New Phytologist Trust.

  15. Transgenic plants expressing the AaIT/GNA fusion protein show increased resistance and toxicity to both chewing and sucking pests.

    Science.gov (United States)

    Liu, Shu-Min; Li, Jie; Zhu, Jin-Qi; Wang, Xiao-Wei; Wang, Cheng-Shu; Liu, Shu-Sheng; Chen, Xue-Xin; Li, Sheng

    2016-04-01

    The adoption of pest-resistant transgenic plants to reduce yield losses and decrease pesticide use has been successful. To achieve the goal of controlling both chewing and sucking pests in a given transgenic plant, we generated transgenic tobacco, Arabidopsis, and rice plants expressing the fusion protein, AaIT/GNA, in which an insecticidal scorpion venom neurotoxin (Androctonus australis toxin, AaIT) is fused to snowdrop lectin (Galanthus nivalis agglutinin, GNA). Compared with transgenic tobacco and Arabidopsis plants expressing AaIT or GNA, transgenic plants expressing AaIT/GNA exhibited increased resistance and toxicity to one chewing pest, the cotton bollworm, Helicoverpa armigera. Transgenic tobacco and rice plants expressing AaIT/GNA showed increased resistance and toxicity to two sucking pests, the whitefly, Bemisia tabaci, and the rice brown planthopper, Nilaparvata lugens, respectively. Moreover, in the field, transgenic rice plants expressing AaIT/GNA exhibited a significant improvement in grain yield when infested with N. lugens. This study shows that expressing the AaIT/GNA fusion protein in transgenic plants can be a useful approach for controlling pests, particularly sucking pests which are not susceptible to the toxin in Bt crops. © 2015 Institute of Zoology, Chinese Academy of Sciences.

  16. Plant biotechnology: transgenic crops.

    Science.gov (United States)

    Shewry, Peter R; Jones, Huw D; Halford, Nigel G

    2008-01-01

    Transgenesis is an important adjunct to classical plant breeding, in that it allows the targeted manipulation of specific characters using genes from a range of sources. The current status of crop transformation is reviewed, including methods of gene transfer, the selection of transformed plants and control of transgene expression. The application of genetic modification technology to specific traits is then discussed, including input traits relating to crop production (herbicide tolerance and resistance to insects, pathogens and abiotic stresses) and output traits relating to the composition and quality of the harvested organs. The latter include improving the nutritional quality for consumers as well as the improvement of functional properties for food processing.

  17. Smokeless Tobacco - An Overview

    Directory of Open Access Journals (Sweden)

    Klus H

    2014-12-01

    Full Text Available Smoking, especially cigarette smoking, is the most common form of tobacco consumption world-wide. It is generally accepted that smoking carries health risks for smokers. The combustion and pyrolysis products of tobacco generated during smoking are considered to be responsible for the harmful effects. Smokeless tobacco, another wide-spread form of tobacco use, is not subjected to burning and produces no combustion or pyrolysis products. Therefore, there is an increasingly intense debate about the potential role of smokeless tobacco in reducing the harm of tobacco use.

  18. Effect of Radiation Dosage on Efficiency of Chloroplast Transfer by Protoplast Fusion in Nicotiana

    OpenAIRE

    Menczel, László; Galiba, Gábor; Nagy, Ferenc; Maliga, Pál

    1982-01-01

    Chloroplasts of Nicotiana tabacum SR1 were transferred into Nicotiana plumbaginifolia by protoplast fusion. The protoplasts of the organelle donor were irradiated with different lethal doses using a 60Co source, to facilitate the elimination of their nuclei from the fusion products. After fusion induction, clones derived from fusion products and containing streptomycin-resistant N. tabacum SR1 chloroplasts were selected by their ability to green on a selective medium. When N. tabacum protopla...

  19. [TSA improve transgenic porcine cloned embryo development and transgene expression].

    Science.gov (United States)

    Kong, Qing-Ran; Zhu, Jiang; Huang, Bo; Huan, Yan-Jun; Wang, Feng; Shi, Yong-Qian; Liu, Zhong-Feng; Wu, Mei-Ling; Liu, Zhong-Hua

    2011-07-01

    Uncompleted epigenetic reprogramming is attributed to the low efficiency of producing transgenic cloned animals. Histone modification associated with epigenetics can directly influence the embryo development and transgene expression. Trichostatin A (TSA), as an inhibitor of histone deacetylase, can change the status of histone acetylation, improve somatic cell reprogramming, and enhance cloning efficiency. TSA prevents the chromatin structure from being condensed, so that transcription factor could binds to DNA sequence easily and enhance transgene expression. Our study established the optimal TSA treatment on porcine donor cells and cloned embryos, 250 nmol/L, 24 h and 40 nmol/L, 24 h, respectively. Furthermore, we found that both the cloned embryo and the donor cell treated by TSA resulted in the highest development efficiency. Meanwhile, TSA can improve transgene expression in donor cell and cloned embryo. In summary, TSA can significantly improve porcine reconstructed embryo development and transgene expression.

  20. Radiation-induced organogenesis: effects of irradiated medium and its components on tobacco tissue culture

    International Nuclear Information System (INIS)

    Degani, N.

    1975-01-01

    Gamma irradiated medium induces the formation of buds in non-irradiated dark growth tobacco callus (Nicotiana tabacum Var. Wisconsin No.38). Experiments were conducted to determine the component(s) of the medium that is effective in this radiation-induced organogenesis. Fraction of medium were irradiated singly and in combination, then combined with non-irradiated fractions to form the complete growth medium. The results showed that irradiated indoleacetic acid (IAA) was not the effective component in the induction of organogensis. Omission of IAA from the medium resulted in the formation of buds, as expected. Irradiated myo-inositol induced organogenesis more consistently than the other irradiated components. The age of the inoculum tissue and its passage number from the tobacco stem affected the potency of the tobacco callus to organise. (author)

  1. Introduction of transformed chloroplasts from tobacco into petunia by asymmetric cell fusion.

    Science.gov (United States)

    Sigeno, Asako; Hayashi, Sugane; Terachi, Toru; Yamagishi, Hiroshi

    2009-11-01

    Plastid engineering technique has been established only in Nicotiana tabacum, and the widespread application is severely limited so far. In order to exploit a method to transfer the genetically transformed plastomes already obtained in tobacco into other plant species, somatic cell fusion was conducted between a plastome transformant of tobacco and a cultivar of petunia (Petunia hybrida). A tobacco strain whose plastids had been transformed with aadA (a streptomycin/spectinomycin adenylyltransferase gene) and mdar [a gene for monodehydroascorbate reductase (MDAR)] and a petunia variety, 'Telstar', were used as cell fusion partners. An efficient regeneration system from the protoplasts of both the parents, and effectiveness of selection for the aadA gene with spectinomycin were established before the cell fusion. In addition, the influence of UV irradiation on the callus development from the protoplasts and shoot regeneration of tobacco was investigated. Protoplasts were cultured after cell fusion treatment with polyethylene glycol, and asymmetric somatic cybrids were selected using the aadA gene as a marker. Although many shoots of tobacco that had escaped the UV irradiation regenerated, several shoots possessing the morphology of petunia and the resistance to spectinomycin were obtained. Molecular analyses of the petunia type regenerants demonstrated that they had the nuclear and mitochondrial genomes derived from petunia besides the chloroplasts of tobacco transformed with aadA and mdar. Furthermore, it was ascertained that mdar was transcribed in the somatic cybrids. The results indicate the success in intergeneric transfer of transformed plastids of tobacco into petunia.

  2. High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture

    DEFF Research Database (Denmark)

    Shin, Yun-Ji; Hong, Shin-Young; Kwon, Tae-Ho

    2003-01-01

    Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) has been previously produced in tobacco cell suspension cultures. However, the amount of hGM-CSF accumulated in the culture medium dropped quickly from its maximum of 150 microg/L at 5 d after incubation. To overcome...... of recombinant hGM-CSF in transgenic rice cell suspension culture and protease activity of this culture medium was low compared to that of tobacco culture system....

  3. Smokeless Tobacco: Health Effects

    Science.gov (United States)

    ... t start. If you do use them, quit. Addiction to Smokeless Tobacco Smokeless tobacco contains nicotine, which ... Smoking and Health E-mail: tobaccoinfo@cdc.gov Phone: 1-800-CDC-INFO Media Inquiries: Contact CDC’s ...

  4. Allegheny County Tobacco Vendors

    Data.gov (United States)

    Allegheny County / City of Pittsburgh / Western PA Regional Data Center — The tobacco vendor information provides the location of all tobacco vendors in Allegheny County in 2015. Data was compiled from administrative records managed by...

  5. Smokeless Tobacco and Cancer

    Science.gov (United States)

    ... in smokeless tobacco include polonium–210 (a radioactive element found in tobacco fertilizer) and polynuclear aromatic hydrocarbons ( ... study of the 40 most widely used popular brands of moist snuff showed that the amount of ...

  6. Cadmium resistance in tobacco plants expressing the MuSI gene.

    Science.gov (United States)

    Kim, Young-Nam; Kim, Ji-Seoung; Seo, Sang-Gyu; Lee, Youngwoo; Baek, Seung-Woo; Kim, Il-Sup; Yoon, Ho-Sung; Kim, Kwon-Rae; Kim, Sun-Hyung; Kim, Kye-Hoon

    2011-10-01

    MuSI, a gene that corresponds to a domain that contains the rubber elongation factor (REF), is highly homologous to many stress-related proteins in plants. Since MuSI is up-regulated in the roots of plants treated with cadmium or copper, the involvement of MuSI in cadmium tolerance was investigated in this study. Escherichia coli cells overexpressing MuSI were more resistant to Cd than wild-type cells transfected with vector alone. MuSI transgenic plants were also more resistant to Cd. MuSI transgenic tobacco plants absorbed less Cd than wild-type plants. Cd translocation from roots to shoots was reduced in the transgenic plants, thereby avoiding Cd toxicity. The number of short trichomes in the leaves of wild-type tobacco plants was increased by Cd treatment, while this was unchanged in MuSI transgenic tobacco. These results suggest that MuSI transgenic tobacco plants have enhanced tolerance to Cd via reduced Cd uptake and/or increased Cd immobilization in the roots, resulting in less Cd translocation to the shoots.

  7. [Tobacco--a highly efficient producer of vaccines].

    Science.gov (United States)

    Budzianowski, Jaromir

    2010-01-01

    Along with the depreciation of tobacco as a source of nicotine-containing commercial products, the increase of its appreciation as a potential producer of recombinant therapeutical proteins can be observed. Two species of tobacco--Nicotiana tabacum L. and N. benthamiana are easily grown by well established methods of field or green-house cultivation or cell culture, yield high biomass and soluble protein content, can be easily transformed by several methods and are not food for humans or feed for animals. Expression of foreign proteins, including vaccines, can be achieved in those plants either through stable transformation of nuclear or plastid (chloroplast) genomes or by transient transformation using infection with plant virus or bacteria--Agrobacterium tumefaciens (agroinfiltration). The most advanced mode of agrofiltration termed magnifection, which combines benefits of virus and Agrobacterium and depends on using Agrobacterium with viral pro-vectors, enables high-yield and rapid expression of therapeutical proteins, even in a few days, and can be employed on an industrial scale. Expression of many antigenic proteins, which may serve as antiviral, antibacterial, antiprotozoan and anticancer vaccines, and additionally a few autoantigens designed for the treatment of autoimunogenic diseases, like diabetes, have been achieved in tobacco. To date, a vaccine against Newcastle virus disease in poultry produced by tobacco cell culture has been approved for commercial application and several other vaccines are in advanced stage of development. The possibility of a high-level production of vaccines in tobacco against pandemic influenza or anthrax and plague due to a bioterroristic attack, as well as of individualised anticancer vaccines against non-Hodgkin's lymphoma (NHL) in a much shorter period of time than by traditional methods became realistic and hence caused increased interest in tobacco as a high-efficient producer of vaccines not only of specialistic

  8. Pea early-browning virus -mediated genome editing via the CRISPR/Cas9 system in Nicotiana benthamiana and Arabidopsis

    KAUST Repository

    Ali, Zahir; Eid, Ayman; Ali, Shawkat; Mahfouz, Magdy M.

    2017-01-01

    The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system has enabled efficient genome engineering in diverse plant species. However, delivery of genome engineering reagents, such as the single guide RNA (sgRNA), into plant cells remains challenging. Here, we report the engineering of Tobacco rattle virus (TRV) and Pea early browning virus (PEBV) to deliver one or multiple sgRNAs into Nicotiana benthamiana and Arabidopsis thaliana (Col-0) plants that overexpress a nuclear localization signal containing Cas9. Our data showed that TRV and PEBV can deliver sgRNAs into inoculated and systemic leaves, and this resulted in mutagenesis of the targeted genomic loci. Moreover, in N. benthamiana, PEBV-based sgRNA delivery resulted in more targeted mutations than TRV-based delivery. Our data indicate that TRV and PEBV can facilitate plant genome engineering and can be used to produce targeted mutations for functional analysis and other biotechnological applications across diverse plant species.Key message: Delivery of genome engineering reagents into plant cells is challenging and inefficient and this limit the applications of this technology in many plant species. RNA viruses such as TRV and PEBV provide an efficient tool to systemically deliver sgRNAs for targeted genome modification.

  9. Pea early-browning virus -mediated genome editing via the CRISPR/Cas9 system in Nicotiana benthamiana and Arabidopsis

    KAUST Repository

    Ali, Zahir

    2017-10-17

    The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system has enabled efficient genome engineering in diverse plant species. However, delivery of genome engineering reagents, such as the single guide RNA (sgRNA), into plant cells remains challenging. Here, we report the engineering of Tobacco rattle virus (TRV) and Pea early browning virus (PEBV) to deliver one or multiple sgRNAs into Nicotiana benthamiana and Arabidopsis thaliana (Col-0) plants that overexpress a nuclear localization signal containing Cas9. Our data showed that TRV and PEBV can deliver sgRNAs into inoculated and systemic leaves, and this resulted in mutagenesis of the targeted genomic loci. Moreover, in N. benthamiana, PEBV-based sgRNA delivery resulted in more targeted mutations than TRV-based delivery. Our data indicate that TRV and PEBV can facilitate plant genome engineering and can be used to produce targeted mutations for functional analysis and other biotechnological applications across diverse plant species.Key message: Delivery of genome engineering reagents into plant cells is challenging and inefficient and this limit the applications of this technology in many plant species. RNA viruses such as TRV and PEBV provide an efficient tool to systemically deliver sgRNAs for targeted genome modification.

  10. Microspores irradiation in anther culture: testing a new technique to obtain mutations immediatly detected and fixed (Application to Nicotiana tabacum)

    International Nuclear Information System (INIS)

    Mondeil, Fanja

    1974-01-01

    In order to consider the effects of microspores irradiation on embryo development, and in order to observe the morphological responses of haploid plantlets derived from androgenetic anthers to ionizing irradiation, 1000, 1500 and 2000r of gamma rays were delivered on anthers of Nicotiana tabacum (DL 50 range calculated: 1500r). The cytological studies of embryo development revealed an apparent increase in irradiated microspores: cell division is stimulated but followed by an early mortality. A sharp rise in lethality effects was observed when gamma rays were applied beyond the seventh day of culture, when the proembryo contains an average of 4 cells. Morphological aberrations and colour changes in the Mo progeny derived from irradiated microspores are diverse. But after chromosome doubling and mutation checking out, all the plants were not recorded to have transmitted their aberrant characters. Thus, heritable character 'mutations) and not heritable character (variations) were obtained. The variations characters include dwarfing, excessive branching, fasciation and dichotomy of the stems, altered flower form, especially of petals. As to the leaves, they usually show induced changes in their colour (chlorotic areas, mosaic-colour changes, or an over-all colour changes), in their form (irregularity in outline) and in their texture (thickening, hairless leaf). Among the mutants, a monster tobacco, with excrescences on the leaves and the flowers is certainly the most conspicuous. But mutants also include altered leaf colour (over-all pale green) and altered flower colour, (dark red, clear pink, white) [fr

  11. TL transgenic mouse strains

    International Nuclear Information System (INIS)

    Obata, Y.; Matsudaira, Y.; Hasegawa, H.; Tamaki, H.; Takahashi, T.; Morita, A.; Kasai, K.

    1993-01-01

    As a result of abnormal development of the thymus of these mice, TCR αβ lineage of the T cell differentiation is disturbed and cells belonging to the TCR γδ CD4 - CD8 - double negative (DN) lineage become preponderant. The γδ DN cells migrate into peripheral lymphoid organs and constitute nearly 50% of peripheral T cells. Immune function of the transgenic mice is severely impaired, indicating that the γδ cells are incapable of participating in these reactions. Molecular and serological analyses of T-cell lymphomas reveal that they belong to the γδ lineage. Tg.Tla a -3-1 mice should be useful in defining the role of TL in normal and abnormal T cell differentiation as well as in the development of T-cell lymphomas, and further they should facilitate studies on the differentiation and function of γδ T cells. We isolated T3 b -TL gene from B6 mice and constructed a chimeric gene in which T3 b -TL is driven by the promoter of H-2K b . With the chimeric gene, two transgenic mouse strains, Tg. Con.3-1 and -2 have been derived in C3H background. Both strains express TL antigen in various tissues including skin. The skin graft of transgenic mice on C3H and (B6 X C3H)F 1 mice were rejected. In the mice which rejected the grafts, CD8 + TCRαβ cytotoxic T cells (CTL) against TL antigens were recognized. The recognition of TL by CTL did not require the antigen presentation by H-2 molecules. The results indicated that TL antigen in the skin becomes a transplantation antigen and behaves like a typical allogeneic MHC class I antigen. The facts that (B6 X C3H)F 1 mice rejected the skin expressing T3 b -TL antigen and induced CTL that killed TL + lymphomas of B6 origin revealed that TL antigen encoded by T3 b -TL is recognized as non-self in B6 mice. Experiments are now extended to analyze immune responses to TL antigen expressed on autochthonous T cell lymphomas. (J.P.N.)

  12. Tobacco and Pregnancy

    Science.gov (United States)

    This paper will review the epidemiology of the impact of cigarette smoking and other forms of tobacco exposure on human development. Sources of exposure described include cigarettes and other forms of smoked tobacco, secondhand (environmental) tobacco smoke, several forms of smok...

  13. Untranslatable tospoviral NSs fragment coupled with L conserved region enhances transgenic resistance against the homologous virus and a serologically unrelated tospovirus.

    Science.gov (United States)

    Yazhisai, Uthaman; Rajagopalan, Prem Anand; Raja, Joseph A J; Chen, Tsung-Chi; Yeh, Shyi-Dong

    2015-08-01

    Tospoviruses cause severe damages to important crops worldwide. In this study, Nicotiana benthamiana transgenic lines carrying individual untranslatable constructs comprised of the conserved region of the L gene (denoted as L), the 5' half of NSs coding sequence (NSs) or the antisense fragment of whole N coding sequence (N) of Watermelon silver mottle virus (WSMoV), individually or in combination, were generated. A total of 15-17 transgenic N. benthamiana lines carrying individual transgenes were evaluated against WSMoV and the serologically unrelated Tomato spotted wilt virus (TSWV). Among lines carrying single or chimeric transgenes, the level of resistance ranged from susceptible to completely resistant against WSMoV. From the lines carrying individual transgenes and highly resistant to WSMoV (56-63% of lines assayed), 30% of the L lines (3/10 lines assayed) and 11% of NSs lines (1/9 lines assayed) were highly resistant against TSWV. The chimeric transgenes provided higher degrees of resistance against WSMoV (80-88%), and the NSs fragment showed an additive effect to enhance the resistance to TSWV. Particularly, the chimeric transgenes with the triple combination of fragments, namely L/NSs/N or HpL/NSs/N (a hairpin construct), provided a higher degree of resistance (both 50%, with 7/14 lines assayed) against TSWV. Our results indicate that the untranslatable NSs fragment is able to enhance the transgenic resistance conferred by the L conserved region. The better performance of L/NSs/N and HpL/NSs/N in transgenic N. benthamiana lines suggests their potential usefulness in generating high levels of enhanced transgenic resistance against serologically unrelated tospoviruses in agronomic crops.

  14. Production of complex multiantennary N-glycans in Nicotiana benthamiana plants.

    Science.gov (United States)

    Nagels, Bieke; Van Damme, Els J M; Pabst, Martin; Callewaert, Nico; Weterings, Koen

    2011-03-01

    In recent years, plants have been developed as an alternative expression system to mammalian hosts for the production of therapeutic proteins. Many modifications to the plant glycosylation machinery have been made to render it more human because of the importance of glycosylation for functionality, serum half-life, and the safety profile of the expressed proteins. These modifications include removal of plant-specific β1,2-xylose and core α1,3-fucose, and addition of bisecting N-acetylglucosamine, β1,4-galactoses, and sialic acid residues. Another glycosylation step that is essential for the production of complex human-type glycans is the synthesis of multiantennary structures, which are frequently found on human N-glycans but are not generated by wild-type plants. Here, we report both the magnICON-based transient as well as stable introduction of the α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnT-IV isozymes a and b) and α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase (GnT-V) in Nicotiana benthamiana plants. The enzymes were targeted to the Golgi apparatus by fusing their catalytic domains to the plant-specific localization signals of xylosyltransferase and fucosyltransferase. The GnT-IV and -V modifications were tested in the wild-type background, but were also combined with the RNA interference-mediated knockdown of β1,2-xylosyltransferase and α1,3-fucosyltransferase. Results showed that triantennary Gn[GnGn] and [GnGn]Gn N-glycans could be produced according to the expected activities of the respective enzymes. Combination of the two enzymes by crossing stably transformed GnT-IV and GnT-V plants showed that up to 10% tetraantennary [GnGn][GnGn], 25% triantennary, and 35% biantennary N-glycans were synthesized. All transgenic plants were viable and showed no aberrant phenotype under standard growth conditions.

  15. Production of Complex Multiantennary N-Glycans in Nicotiana benthamiana Plants1[W][OA

    Science.gov (United States)

    Nagels, Bieke; Van Damme, Els J.M.; Pabst, Martin; Callewaert, Nico; Weterings, Koen

    2011-01-01

    In recent years, plants have been developed as an alternative expression system to mammalian hosts for the production of therapeutic proteins. Many modifications to the plant glycosylation machinery have been made to render it more human because of the importance of glycosylation for functionality, serum half-life, and the safety profile of the expressed proteins. These modifications include removal of plant-specific β1,2-xylose and core α1,3-fucose, and addition of bisecting N-acetylglucosamine, β1,4-galactoses, and sialic acid residues. Another glycosylation step that is essential for the production of complex human-type glycans is the synthesis of multiantennary structures, which are frequently found on human N-glycans but are not generated by wild-type plants. Here, we report both the magnICON-based transient as well as stable introduction of the α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnT-IV isozymes a and b) and α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase (GnT-V) in Nicotiana benthamiana plants. The enzymes were targeted to the Golgi apparatus by fusing their catalytic domains to the plant-specific localization signals of xylosyltransferase and fucosyltransferase. The GnT-IV and -V modifications were tested in the wild-type background, but were also combined with the RNA interference-mediated knockdown of β1,2-xylosyltransferase and α1,3-fucosyltransferase. Results showed that triantennary Gn[GnGn] and [GnGn]Gn N-glycans could be produced according to the expected activities of the respective enzymes. Combination of the two enzymes by crossing stably transformed GnT-IV and GnT-V plants showed that up to 10% tetraantennary [GnGn][GnGn], 25% triantennary, and 35% biantennary N-glycans were synthesized. All transgenic plants were viable and showed no aberrant phenotype under standard growth conditions. PMID:21233332

  16. North Carolina Tobacco Farmers' Changing Perceptions of Tobacco Control and Tobacco Manufacturers

    Science.gov (United States)

    Crankshaw, Erik C.; Beach, Robert H.; Austin, W. David; Altman, David G.; Jones, Alison Snow

    2009-01-01

    Purpose: To examine tobacco farmers' attitudes toward tobacco control, public health, and tobacco manufacturers in order to determine the extent to which rapidly changing economic conditions have influenced North Carolina tobacco farmer attitudes in ways that may provide tobacco control advocates with new opportunities to promote tobacco control…

  17. 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

    The plant respiratory chain contains a complex setup of non-energy conserving NAD(P)H dehydrogenases, the physiological consequences of which are highly unclear. An expression construct for the potato (Solanum tuberosum L., cv. Desiree) ndb1 gene, a homologue of bacterial and fungal type II NAD...

  18. Transgenics, agroindustry and food sovereignty

    Directory of Open Access Journals (Sweden)

    Xavier Alejandro León Vega

    2014-10-01

    Full Text Available Food sovereignty has been implemented constitutionally in Ecuador; however, many of the actions and policies are designed to benefit the dominant model of food production, based in agroindustry, intensive monocultures, agrochemicals and transgenics. This article reflects upon the role of family farming as a generator of food sovereignty, and secondly the threat to them by agroindustry agriculture based in transgenic. The role played by food aid in the introduction of transgenic in Latin America and other regions of the world is also analyzed.

  19. 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.

  20. Two tandemly repeated telomere-associated sequences in Nicotiana plumbaginifolia.

    Science.gov (United States)

    Chen, C M; Wang, C T; Wang, C J; Ho, C H; Kao, Y Y; Chen, C C

    1997-12-01

    Two tandemly repeated telomere-associated sequences, NP3R and NP4R, have been isolated from Nicotiana plumbaginifolia. The length of a repeating unit for NP3R and NP4R is 165 and 180 nucleotides respectively. The abundance of NP3R, NP4R and telomeric repeats is, respectively, 8.4 x 10(4), 6 x 10(3) and 1.5 x 10(6) copies per haploid genome of N. plumbaginifolia. Fluorescence in situ hybridization revealed that NP3R is located at the ends and/or in interstitial regions of all 10 chromosomes and NP4R on the terminal regions of three chromosomes in the haploid genome of N. plumbaginifolia. Sequence homology search revealed that not only are NP3R and NP4R homologous to HRS60 and GRS, respectively, two tandem repeats isolated from N. tabacum, but that NP3R and NP4R are also related to each other, suggesting that they originated from a common ancestral sequence. The role of these repeated sequences in chromosome healing is discussed based on the observation that two to three copies of a telomere-similar sequence were present in each repeating unit of NP3R and NP4R.

  1. Expression studies of the zeaxanthin epoxidase gene in nicotiana plumbaginifolia

    Science.gov (United States)

    Audran; Borel; Frey; Sotta; Meyer; Simonneau; Marion-Poll

    1998-11-01

    Abscisic acid (ABA) is a plant hormone involved in the control of a wide range of physiological processes, including adaptation to environmental stress and seed development. In higher plants ABA is a breakdown product of xanthophyll carotenoids (C40) via the C15 intermediate xanthoxin. The ABA2 gene of Nicotiana plumbaginifolia encodes zeaxanthin epoxidase, which catalyzes the conversion of zeaxanthin to violaxanthin. In this study we analyzed steady-state levels of ABA2 mRNA in N. plumbaginifolia. The ABA2 mRNA accumulated in all plant organs, but transcript levels were found to be higher in aerial parts (stems and leaves) than in roots and seeds. In leaves ABA2 mRNA accumulation displayed a day/night cycle; however, the ABA2 protein level remained constant. In roots no diurnal fluctuation in mRNA levels was observed. In seeds the ABA2 mRNA level peaked around the middle of development, when ABA content has been shown to increase in many species. In conditions of drought stress, ABA levels increased in both leaves and roots. A concomitant accumulation of ABA2 mRNA was observed in roots but not in leaves. These results are discussed in relation to the role of zeaxanthin epoxidase both in the xanthophyll cycle and in the synthesis of ABA precursors.

  2. Effects of different potting growing media for Petunia grandiflora and Nicotiana alata Link & Otto on photosynthetic capacity, leaf area, and flowering potential

    Directory of Open Access Journals (Sweden)

    Gheorghe Cristian Popescu

    2015-03-01

    Full Text Available Petunia grandiflora Juss. and Nicotiana alata Link & Otto are two of the most widely spread plants on the market for annual potted ornamental plants. In order to identify the most adequate substrate formula we analyzed the effects of different potting growing media used for P. hybrida grandiflora 'Bravo' and N. alata 'Dinamo' on their photosynthetic capacity, leaf area, and flowering potential. Optimization of growing media formula for petunia and ornamental tobacco was performed by preparing four growing media mixing fallow soil (FS, Biolan peat (BP, acid peat (AP, leaf compost (C, and perlite (P in different proportions. The physiological potential of petunia and ornamental tobacco was investigated by photosynthesis and respiration rate and chlorophyll pigments in leaves, while the vegetative and flowering phenological stages were evaluated by number of leaves per plant, leaf area, number of flowers per plant and leaf area/flowers ratio. These measurements were significantly influenced by the different potting growing media used in this study. In the flowering stage, the highest photosynthesis rates (8.612 μmol CO2 m-2 s-1 as well as leaf area (1.766 dm² of petunias were obtained on growing media with 60% biolan peat, 30% acid peat and 10% perlite (BP60-AP30-P10. Flowering responses to growing conditions vary greatly among plants and the biggest number of ornamental tobacco flowers (22 flowers plant-1 was registered as an effect of BP60-AP30-P10 media. Growing media with the BP60-AP30-P10 formula seem to be the most adequate growth substrate to develop profitable crops for petunias and ornamental tobacco with high decorative value.

  3. Engineering and expression of a human rotavirus candidate vaccine in Nicotiana benthamiana.

    Science.gov (United States)

    Pêra, Francisco F P G; Mutepfa, David L R; Khan, Ayesha M; Els, Johann H; Mbewana, Sandiswa; van Dijk, Alberdina A A; Rybicki, Edward P; Hitzeroth, Inga I

    2015-12-02

    Human rotaviruses are the main cause of severe gastroenteritis in children and are responsible for over 500 000 deaths annually. There are two live rotavirus vaccines currently available, one based on human rotavirus serotype G1P[8], and the other a G1-G4 P[8] pentavalent vaccine. However, the recent emergence of the G9 and other novel rotavirus serotypes in Africa and Asia has prompted fears that current vaccines might not be fully effective against these new varieties. We report an effort to develop an affordable candidate rotavirus vaccine against the new emerging G9P[6] (RVA/Human-wt/ZAF/GR10924/1999/G9P[6]) strain. The vaccine is based on virus-like particles which are both highly immunogenic and safe. The vaccine candidate was produced in Nicotiana benthamiana by transient expression, as plants allow rapid production of antigens at lower costs, without the risk of contamination by animal pathogens. Western blot analysis of plant extracts confirmed the successful expression of two rotavirus capsid proteins, VP2 and VP6. These proteins assembled into VLPs resembling native rotavirus particles when analysed by transmission electron microscopy (TEM). Expression of the rotavirus glycoprotein VP7 and the spike protein VP4 was also tried. However, VP7 expression caused plant wilting during the course of the time trial and expression could never be detected for either protein. We therefore created three fusion proteins adding the antigenic part of VP4 (VP8*) to VP6 in an attempt to produce more appropriately immunogenic particles. Fusion protein expression in tobacco plants was detected by western blot using anti-VP6 and anti-VP4 antibodies, but no regular particles were observed by TEM, even when co-expressed with VP2. Our results suggest that the rotavirus proteins produced in N. benthamiana are candidates for a subunit vaccine specifically for the G9P[6] rotavirus strain. This could be more effective in developing countries, thereby possibly providing a higher

  4. Assessment of natural radionuclides concentration from 238U and 232Th series in Virginia and Burley varieties of Nicotiana tabacum L

    International Nuclear Information System (INIS)

    Silva, Carolina Fernanda da

    2015-01-01

    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 238 U, 234 U, 230 Th, 22 '6Ra, 210 Pb and 210 Po, members from the 238 U decay series, and the radionuclides 232 Th and 228 Ra members of the 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 210 Po determination, and gross alpha and beta counting, 228 Ra, 226 Ra and 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 radionuclides 226 Ra, 228 Ra, 210 Pb, and 210 Po, were determined in most

  5. Rapid expression of transgenes driven by seed-specific constructs in leaf tissue: DHA production

    Directory of Open Access Journals (Sweden)

    Zhou Xue-Rong

    2010-03-01

    Full Text Available Abstract Background Metabolic engineering of seed biosynthetic pathways to diversify and improve crop product quality is a highly active research area. The validation of genes driven by seed-specific promoters is time-consuming since the transformed plants must be grown to maturity before the gene function can be analysed. Results In this study we demonstrate that genes driven by seed-specific promoters contained within complex constructs can be transiently-expressed in the Nicotiana benthamiana leaf-assay system by co-infiltrating the Arabidopsis thaliana LEAFY COTYLEDON2 (LEC2 gene. A real-world case study is described in which we first assembled an efficient transgenic DHA synthesis pathway using a traditional N. benthamiana Cauliflower Mosaic Virus (CaMV 35S-driven leaf assay before using the LEC2-extended assay to rapidly validate a complex seed-specific construct containing the same genes before stable transformation in Arabidopsis. Conclusions The LEC2-extended N. benthamiana assay allows the transient activation of seed-specific promoters in leaf tissue. In this study we have used the assay as a rapid preliminary screen of a complex seed-specific transgenic construct prior to stable transformation, a feature that will become increasingly useful as genetic engineering moves from the manipulation of single genes to the engineering of complex pathways. We propose that the assay will prove useful for other applications wherein rapid expression of transgenes driven by seed-specific constructs in leaf tissue are sought.

  6. Multinational Tobacco Companies and Tobacco Consumption (China)

    International Development Research Centre (IDRC) Digital Library (Canada)

    Until recently, the Chinese tobacco industry has been run as a state-owned monopoly. It is reported ... New funding opportunity for gender equality and climate change ... IDRC invests in research and knowledge to empower women in India.

  7. Transgene teknikker erstatter problematisk avl

    DEFF Research Database (Denmark)

    Alstrup, Aage Kristian Olsen; Hansen, Axel Kornerup

    2016-01-01

    Dyremodeller har ofte været baseret på avl, der ud fra et alment velfærdsmæssigt synspunkt var problematisk. Transgene teknikker kan ofte forbedre dyrevelfærden ved at erstatte disse traditionelle avlsmetoder.......Dyremodeller har ofte været baseret på avl, der ud fra et alment velfærdsmæssigt synspunkt var problematisk. Transgene teknikker kan ofte forbedre dyrevelfærden ved at erstatte disse traditionelle avlsmetoder....

  8. [The tobacco in the light of history and medicine].

    Science.gov (United States)

    de Micheli, Alfredo

    2015-01-01

    Super trajectory is reported of tobacco from his first meeting with the European man October 15, 1492. This plant was known in Europe by the publications of the Sevillan physician Nicolas Monardes (1574), the relations of friar Andrés Thevet (1575) and the famous botanical treatise of Charles de l'Écluse (1605). The Swedish botanist Karl Linnaeus inclused tobacco plant in the family Solanaceae and deleted from this group other plants that were intermixed with it. Its botanical name (Nicotiana tabacum) derived from the surname of the French ambassador to Portugal, Jean Nicot of Villemain, who in 1560 sent it to the Queen Mother of France Cathérine de Medicis. The use of snuff quickly spread throughout Europe, were it became common in the seventeenth century. By the late eighteenth century in New Spain, in addition to cigars, cigarettes and due in packs of different content the tobacco is concocted and price. The preparation of the different presentations of snuff, tobacco made in factories in the capital and several provincial cities, originated in 1796 the creation of the first kindergartens for the children of those working in them. This thanks to the successful initiative of then viceroy Marquis of Branciforte. But contrary to the forecasts of Father F. J. Clavijero and Mrs. F. Calderón de la Barca, wife of the first Spanish diplomatic representative to the government of Mexico, the use of tobacco, with the passage of time, far from waning has been increasing in every social class. And now, more than men, women are smokers. Copyright © 2014 Instituto Nacional de Cardiología Ignacio Chávez. Published by Masson Doyma México S.A. All rights reserved.

  9. Two widely expressed plasma membrane H(+)-ATPase isoforms of Nicotiana tabacum are differentially regulated by phosphorylation of their penultimate threonine.

    Science.gov (United States)

    Bobik, Krzysztof; Duby, Geoffrey; Nizet, Yannick; Vandermeeren, Caroline; Stiernet, Patrick; Kanczewska, Justyna; Boutry, Marc

    2010-04-01

    The plasma membrane H(+)-ATPases PMA2 and PMA4 are the most widely expressed in Nicotiana plumbaginifolia, and belong to two different subfamilies. Both are activated by phosphorylation of a Thr at the penultimate position and the subsequent binding of 14-3-3 proteins. Their expression in Saccharomyces cerevisiae revealed functional and regulatory differences. To determine whether different regulatory properties between PMA2 and PMA4 exist in plants, we generated two monoclonal antibodies able to detect phosphorylation of the penultimate Thr of either PMA2 or PMA4 in a total protein extract. We also raised Nicotiana tabacum transgenic plants expressing 6-His-tagged PMA2 or PMA4, enabling their individual purification. Using these tools we showed that phosphorylation of the penultimate Thr of both PMAs was high during the early exponential growth phase of an N. tabacum cell culture, and then progressively declined. This decline correlated with decreased 14-3-3 binding and decreased plasma membrane ATPase activity. However, the rate and extent of the decrease differed between the two isoforms. Cold stress of culture cells or leaf tissues reduced the Thr phosphorylation of PMA2, whereas no significant changes in Thr phosphorylation of PMA4 were seen. These results strongly suggest that PMA2 and PMA4 are differentially regulated by phosphorylation. Analysis of the H(+)-ATPase phosphorylation status in leaf tissues indicated that no more than 44% (PMA2) or 32% (PMA4) was in the activated state under normal growth conditions. Purification of either isoform showed that, when activated, the two isoforms did not form hetero-oligomers, which is further support for these two H(+)-ATPase subfamilies having different properties.

  10. Cell cycle-dependent O-GlcNAc modification of tobacco histones and their interaction with the tobacco lectin.

    Science.gov (United States)

    Delporte, Annelies; De Zaeytijd, Jeroen; De Storme, Nico; Azmi, Abdelkrim; Geelen, Danny; Smagghe, Guy; Guisez, Yves; Van Damme, Els J M

    2014-10-01

    The Nicotiana tabacum agglutinin or Nictaba is a nucleocytoplasmic lectin that is expressed in tobacco after the plants have been exposed to jasmonate treatment or insect herbivory. Nictaba specifically recognizes GlcNAc residues. Recently, it was shown that Nictaba is interacting in vitro with the core histone proteins from calf thymus. Assuming that plant histones - similar to their animal counterparts - undergo O-GlcNAcylation, this interaction presumably occurs through binding of the lectin to the O-GlcNAc modification present on the histones. Hereupon, the question was raised whether this modification also occurs in plants and if it is cell cycle dependent. To this end, histones were purified from tobacco BY-2 suspension cells and the presence of O-GlcNAc modifications was checked. Concomitantly, O-GlcNAcylation of histone proteins was studied. Our data show that similar to animal histones plant histones are modified by O-GlcNAc in a cell cycle-dependent fashion. In addition, the interaction between Nictaba and tobacco histones was confirmed using lectin chromatography and far Western blot analysis. Collectively these findings suggest that Nictaba can act as a modulator of gene transcription through its interaction with core histones. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  11. Transcriptome profiling of male gametophyte development in Nicotiana tabacum

    Czech Academy of Sciences Publication Activity Database

    Bokvaj, Pavel; Hafidh, Said; Honys, David

    2015-01-01

    Roč. 3, MAR (2015), s. 106-111 ISSN 2213-5960 R&D Projects: GA ČR(CZ) GAP501/11/1462; GA ČR(CZ) GAP305/12/2611; GA MŠk(CZ) LD14109; GA ČR(CZ) GA13-06943S; GA MŠk(CZ) LD13049 Institutional support: RVO:61389030 Keywords : Pollen development transcriptome * Tobacco * Reproduction Subject RIV: EB - Genetics ; Molecular Biology

  12. Tobacco-control policies in tobacco-growing states: where tobacco was king.

    Science.gov (United States)

    Fallin, Amanda; Glantz, Stanton A

    2015-06-01

    POLICY POINTS: The tobacco companies prioritized blocking tobacco-control policies in tobacco-growing states and partnered with tobacco farmers to oppose tobacco-control policies. The 1998 Master Settlement Agreement, which settled state litigation against the cigarette companies, the 2004 tobacco-quota buyout, and the companies' increasing use of foreign tobacco led to a rift between the companies and tobacco farmers. In 2003, the first comprehensive smoke-free local law was passed in a major tobacco-growing state, and there has been steady progress in the region since then. Health advocates should educate the public and policymakers on the changing reality in tobacco-growing states, notably the major reduction in the volume of tobacco produced. The 5 major tobacco-growing states (Kentucky, North Carolina, South Carolina, Tennessee, and Virginia) are disproportionately affected by the tobacco epidemic, with higher rates of smoking and smoking-induced disease. These states also have fewer smoke-free laws and lower tobacco taxes, 2 evidence-based policies that reduce tobacco use. Historically, the tobacco farmers and hospitality associations allied with the tobacco companies to oppose these policies. This research is based on 5 detailed case studies of these states, which included key informant interviews, previously secret tobacco industry documents (available at http://legacy.library.ucsf.edu), and media articles. This was supplemented with additional tobacco document and media searches specifically for this article. The tobacco companies were particularly concerned about blocking tobacco-control policies in the tobacco-growing states by promoting a pro-tobacco culture, beginning in the late 1960s. Nevertheless, since 2003, there has been rapid progress in the tobacco-growing states' passage of smoke-free laws. This progress came after the alliance between the tobacco companies and the tobacco farmers fractured and hospitality organizations stopped opposing smoke

  13. 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

  14. Radioactivity of tobacco

    International Nuclear Information System (INIS)

    Nashawati, A.; Al-Dalal, Z.; Al-Akel, B.; Al-Masri, M. S.

    2002-04-01

    This report shows the results of studies related to radioactivity in tobacco and its pathways to human being. Tobacco contains high concentrations of natural radioactive materials especially polonium 210 and lead 210, which may reach a value of 27 mBq/g. The amount of polonium 210 in tobacco is related to the concentration of radon (the main source of polonium 210 in the agricultural areas) in addition to the over use of phosphate fertilizers for tobacco plantation. Radioactive materials present in tobacco enter the human body through smoking where 210 Po concentrates in the Alveolar lung; this may cause health risks including lung cancer. In addition, radiation doses due to smoking have been reported and some results of the studies carried out for radioactivity in tobacco at the Syrian Atomic Energy Commission. (author)

  15. Nicotiana plumbaginifolia: A Rich Antimicrobial and Antioxidant Source

    International Nuclear Information System (INIS)

    Ajaib, M.; Perveen, S.

    2016-01-01

    Antimicrobial and antioxidant activities of plant Nicotiana plumbaginifolia Viv. Were carried out using various techniques. The petroleum ether, chloroform, methanol and aqueous extracts of the N. plumbaginifolia were obtained by maceration technique. The maximum antibacterial potential was exhibited by chloroform leaves extract (76.3 ± 0.3 mm), methanolic root extract (69 ± 0.8 mm) and petroleum ether root extract (67 ± 1.7 mm) against P. aureginosa. Methanolic root extract possessed 64 ± 2.3 mm zone of inhibition against E. coli, whereas chloroform root extract displayed 49 ± 0.8 mm against B. subtilis. Chloroform root extract showed 48 ±1.2 against S. aureus. The maximum zone of inhibition of antifungal potential was displayed by methanolic extracts of leaves against A. niger (43 ± 0.8 mm) and F. solani (43 ± 1.6 mm). The MIC assay was determine for further analysis which showed the MIC value of methanolic root extract (0.04 ± 0.1 mg/mL) against E. coli and the MIC value was noticed (0.108 ± 0.04 mg/mL) against A. niger by methanolic root extract. Antioxidant potential was determined using four methods i.e. (1,1-diphenyl-2-picrylhydrazyl radical (DPPH) scavenging activity, total antioxidant activity (TAA), total phenolic contents (TPC) and metal chelating activity. The highest value of percent DPPH was observed 90.56 at 1000 microL concentration in petroleum ether extract. The maximum values of TAA, TPC, FRAP and FTC were 1.352 ± 0.01, 1.683 ± 0.09 and 80.66 ± 0.08, respectively. (author)

  16. Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana.

    Science.gov (United States)

    Phoolcharoen, Waranyoo; Bhoo, Seong H; Lai, Huafang; Ma, Julian; Arntzen, Charles J; Chen, Qiang; Mason, Hugh S

    2011-09-01

    Filoviruses (Ebola and Marburg viruses) cause severe and often fatal haemorrhagic fever in humans and non-human primates. The US Centers for Disease Control identifies Ebola and Marburg viruses as 'category A' pathogens (defined as posing a risk to national security as bioterrorism agents), which has lead to a search for vaccines that could prevent the disease. Because the use of such vaccines would be in the service of public health, the cost of production is an important component of their development. The use of plant biotechnology is one possible way to cost-effectively produce subunit vaccines. In this work, a geminiviral replicon system was used to produce an Ebola immune complex (EIC) in Nicotiana benthamiana. Ebola glycoprotein (GP1) was fused at the C-terminus of the heavy chain of humanized 6D8 IgG monoclonal antibody, which specifically binds to a linear epitope on GP1. Co-expression of the GP1-heavy chain fusion and the 6D8 light chain using a geminiviral vector in leaves of N. benthamiana produced assembled immunoglobulin, which was purified by ammonium sulphate precipitation and protein G affinity chromatography. Immune complex formation was confirmed by assays to show that the recombinant protein bound the complement factor C1q. Size measurements of purified recombinant protein by dynamic light scattering and size-exclusion chromatography also indicated complex formation. Subcutaneous immunization of BALB/C mice with purified EIC resulted in anti-Ebola virus antibody production at levels comparable to those obtained with a GP1 virus-like particle. These results show excellent potential for a plant-expressed EIC as a human vaccine. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  17. Antimicrobial Activity of Bacteriophage Endolysin Produced in Nicotiana benthamiana Plants.

    Science.gov (United States)

    Kovalskaya, Natalia; Foster-Frey, Juli; Donovan, David M; Bauchan, Gary; Hammond, Rosemarie W

    2016-01-01

    The increasing spread of antibiotic-resistant pathogens has raised the interest in alternative antimicrobial treatments. In our study, the functionally active gram-negative bacterium bacteriophage CP933 endolysin was produced in Nicotiana benthamiana plants by a combination of transient expression and vacuole targeting strategies, and its antimicrobial activity was investigated. Expression of the cp933 gene in E. coli led to growth inhibition and lysis of the host cells or production of trace amounts of CP933. Cytoplasmic expression of the cp933 gene in plants using Potato virus X-based transient expression vectors (pP2C2S and pGR107) resulted in death of the apical portion of experimental plants. To protect plants against the toxic effects of the CP933 protein, the cp933 coding region was fused at its Nterminus to an N-terminal signal peptide from the potato proteinase inhibitor I to direct CP933 to the delta-type vacuoles. Plants producing the CP933 fusion protein did not exhibit the severe toxic effects seen with the unfused protein and the level of expression was 0.16 mg/g of plant tissue. Antimicrobial assays revealed that, in contrast to gram-negative bacterium E. coli (BL21(DE3)), the gram-positive plant pathogenic bacterium Clavibacter michiganensis was more susceptible to the plant-produced CP933, showing 18% growth inhibition. The results of our experiments demonstrate that the combination of transient expression and protein targeting to the delta vacuoles is a promising approach to produce functionally active proteins that exhibit toxicity when expressed in plant cells.

  18. A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants.

    Science.gov (United States)

    Chen, Longzheng; Li, Wei; Katin-Grazzini, Lorenzo; Ding, Jing; Gu, Xianbin; Li, Yanjun; Gu, Tingting; Wang, Ren; Lin, Xinchun; Deng, Ziniu; McAvoy, Richard J; Gmitter, Frederick G; Deng, Zhanao; Zhao, Yunde; Li, Yi

    2018-01-01

    Developing CRISPR/Cas9-mediated non-transgenic mutants in asexually propagated perennial crop plants is challenging but highly desirable. Here, we report a highly useful method using an Agrobacterium -mediated transient CRISPR/Cas9 gene expression system to create non-transgenic mutant plants without the need for sexual segregation. We have also developed a rapid, cost-effective, and high-throughput mutant screening protocol based on Illumina sequencing followed by high-resolution melting (HRM) analysis. Using tetraploid tobacco as a model species and the phytoene desaturase ( PDS ) gene as a target, we successfully created and expediently identified mutant plants, which were verified as tetra-allelic mutants. We produced pds mutant shoots at a rate of 47.5% from tobacco leaf explants, without the use of antibiotic selection. Among these pds plants, 17.2% were confirmed to be non-transgenic, for an overall non-transgenic mutation rate of 8.2%. Our method is reliable and effective in creating non-transgenic mutant plants without the need to segregate out transgenes through sexual reproduction. This method should be applicable to many economically important, heterozygous, perennial crop species that are more difficult to regenerate.

  19. Construction and Quality Analysis of Transgenic Rehmannia glutinosa Containing TMV and CMV Coat Protein

    Directory of Open Access Journals (Sweden)

    Zhongqiu Teng

    2016-08-01

    Full Text Available Plant viruses, especially tobacco mosaic virus (TMV and cucumber mosaic virus (CMV are serious threats to Rehmannia glutinosa which is a “top grade” herb in China. In the present study, TMV- and CMV-resistant Rehmannia glutinosa Libosch. plants were constructed by transforming the protein (CP genes of TMV and CMV into Rehmannia glutinosa via a modified procedure of Agrobacterium tumefaciens-mediated transformation. Integration and expression of TMV CP and CMV CP transgenes in 2 lines, LBA-1 and LBA-2, were confirmed by PCR, Southern blot and RT-PCR. Both LBA-1 and LBA-2 were resistant to infection of homologous TMV and CMV strains. The quality of transgenic Rehmanniae Radix was evaluated based on fingerprint analysis and components quantitative analysis comparing with control root tubes. These results showed that chemical composition of transgenic Rehmanniae Radix were similar to non-transgenic ones, which demonstrated that the medical quality and biosafety of transgenic Rehmanniae Radix were equivalent to non-transgenic material when consumed as traditional Chinese medicinal (TCM.

  20. A wheat calreticulin gene (TaCRT1) contributes to drought tolerance in transgenic arabidopsis

    International Nuclear Information System (INIS)

    Xiang, V.; Du, C.; Jia, H.; Song, M.; Wang, Y.; Ma, Z.

    2018-01-01

    The TaCRT1 gene is a member of calreticulin (CRT) family in wheat. In our previous study, we showed that transgenic tobacco lines over expressing wheat TaCRT1 showed enhanced tolerance to salt stress. This study aimed to determine whether TaCRT1 over expression would increase drought tolerance in transgenic Arabidopsis. Over expression of TaCRT1 in Arabidopsis plants enhances tolerance to drought stress. However, the transgenic line was found to retard the growth. Moreover, the transgenic line showed decreased water loss but higher sensitivity to exogenous abscisic acid (ABA) compared with the wild type (Col-0). Meanwhile, the transgenic line had the elevated endogenous ABA level. The semi-quantitative RT-PCR (sqRT-PCR) analysis showed that transcription levels of ABA-biosynthesizing gene (NCED3) and ABA-responsive gene (ABF3) were higher in the transgenic line than that in the Col-0 under normal condition. The above results implied that the TaCRT1 might be able to used as a potential target to improve the drought tolerance in crops. (author)

  1. Production of Active Bacillus licheniformis Alpha-Amylase in Tobacco and its Application in Starch Liquefaction

    NARCIS (Netherlands)

    Pen, J; MOLENDIJK, L; Quax, Wim J.; SIJMONS, PC; VANOOYEN, AJJ; VANDENELZEN, PJM; RIETVELD, K; HOEKEMA, A

    As a first example of the feasibility of producing industrial bulk enzymes in plants, we have expressed Bacillus licheniformis alpha-amylase in transgenic tobacco, and applied the seeds directly in starch liquification. The enzyme was properly secreted into the intercellular space, and maximum

  2. Online Tobacco Marketing and Subsequent Tobacco Use.

    Science.gov (United States)

    Soneji, Samir; Yang, JaeWon; Knutzen, Kristin E; Moran, Meghan Bridgid; Tan, Andy S L; Sargent, James; Choi, Kelvin

    2018-02-01

    Nearly 2.9 million US adolescents engaged with online tobacco marketing in 2013 to 2014. We assess whether engagement is a risk factor for tobacco use initiation, increased frequency of use, progression to poly-product use, and cessation. We analyzed data from 11 996 adolescents sampled in the nationally representative, longitudinal Population Assessment for Tobacco and Health study. At baseline (2013-2014), we ascertained respondents' engagement with online tobacco marketing. At follow-up (2014-2015), we determined if respondents had initiated tobacco use, increased frequency of use, progressed to poly-product use, or quit. Accounting for known risk factors, we fit a multivariable logistic regression model among never-users who engaged at baseline to predict initiation at follow-up. We fit similar models to predict increased frequency of use, progression to poly-product use, and cessation. Compared with adolescents who did not engage, those who engaged reported higher incidences of initiation (19.5% vs 11.9%), increased frequency of use (10.3% vs 4.4%), and progression to poly-product use (5.8% vs 2.4%), and lower incidence of cessation at follow-up (16.1% vs 21.5%). Accounting for other risk factors, engagement was positively associated with initiation (adjusted odds ratio [aOR] = 1.26; 95% confidence interval [CI]: 1.01-1.57), increased frequency of use (aOR = 1.58; 95% CI: 1.24-2.00), progression to poly-product use (aOR = 1.70; 95% CI: 1.20-2.43), and negatively associated with cessation (aOR = 0.71; 95% CI: 0.50-1.00). Engagement with online tobacco marketing represents a risk factor for adolescent tobacco use. FDA marketing regulation and cooperation of social-networking sites could limit engagement. Copyright © 2018 by the American Academy of Pediatrics.

  3. The pepper Bs4C proteins are localized to the endoplasmic reticulum (ER) membrane and confer disease resistance to bacterial blight in transgenic rice.

    Science.gov (United States)

    Wang, Jun; Zeng, Xuan; Tian, Dongsheng; Yang, Xiaobei; Wang, Lanlan; Yin, Zhongchao

    2018-03-30

    Transcription activator-like effector (TALE)-dependent dominant disease resistance (R) genes in plants, also referred to as executor R genes, are induced on infection by phytopathogenic bacteria of the genus Xanthomonas harbouring the corresponding TALE genes. Unlike the traditional R proteins, the executor R proteins do not determine the resistance specificity and may function broadly in different plant species. The executor R gene Bs4C-R in the resistant genotype PI 235047 of the pepper species Capsicum pubescens (CpBs4C-R) confers disease resistance to Xanthomonas campestris pv. vesicatoria (Xcv) harbouring the TALE genes avrBsP/avrBs4. In this study, the synthetic genes of CpBs4C-R and two other Bs4C-like genes, the susceptible allele in the genotype PI585270 of C. pubescens (CpBs4C-S) and the CaBs4C-R homologue gene in the cultivar 'CM334' of Capsicum annum (CaBs4C), were characterized in tobacco (Nicotiana benthamiana) and rice (Oryza sativa). The Bs4C genes induced cell death in N. benthamiana. The functional Bs4C-eCFP fusion proteins were localized to the endoplasmic reticulum (ER) membrane in the leaf epidermal cells of N. benthamiana. The Xa10 promoter-Bs4C fusion genes in transgenic rice conferred strain-specific disease resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight in rice, and were specifically induced by the Xa10-incompatible Xoo strain PXO99 A (pHM1avrXa10). The results indicate that the Bs4C proteins from pepper species function broadly in rice and the Bs4C protein-mediated cell death from the ER is conserved between dicotyledonous and monocotyledonous plants, which can be utilized to engineer novel and enhanced disease resistance in heterologous plants. © 2018 TEMASEK LIFE SCIENCES LABORATORY. MOLECULAR PLANT PATHOLOGY © 2018 JOHN WILEY & SONS LTD.

  4. Sincronización de Células de Tabaco (Nicotiana tabacum) NT-1

    OpenAIRE

    Ruiz, León F; Higareda, Ana E; Pardo, Marco A

    2010-01-01

    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. L...

  5. Tobacco as a production platform for biofuel: overexpression of Arabidopsis DGAT and LEC2 genes increases accumulation and shifts the composition of lipids in green biomass.

    Science.gov (United States)

    Andrianov, Vyacheslav; Borisjuk, Nikolai; Pogrebnyak, Natalia; Brinker, Anita; Dixon, Joseph; Spitsin, Sergei; Flynn, John; Matyszczuk, Paulina; Andryszak, Karolina; Laurelli, Marilyn; Golovkin, Maxim; Koprowski, Hilary

    2010-04-01

    When grown for energy production instead for smoking, tobacco can generate a large amount of inexpensive biomass more efficiently than almost any other agricultural crop. Tobacco possesses potent oil biosynthesis machinery and can accumulate up to 40% of seed weight in oil. In this work, we explored two metabolic engineering approaches to enhance the oil content in tobacco green tissues for potential biofuel production. First, an Arabidopsis thaliana gene diacylglycerol acyltransferase (DGAT) coding for a key enzyme in triacylglycerol (TAG) biosynthesis, was expressed in tobacco under the control of a strong ribulose-biphosphate carboxylase small subunit promoter. This modification led to up to a 20-fold increase in TAG accumulation in tobacco leaves and translated into an overall of about a twofold increase in extracted fatty acids (FA) up to 5.8% of dry biomass in Nicotiana tabacum cv Wisconsin, and up to 6% in high-sugar tobacco variety NC-55. Modified tobacco plants also contained elevated amounts of phospholipids. This increase in lipids was accompanied by a shift in the FA composition favourable for their utilization as biodiesel. Second, we expressed in tobacco Arabidopsis gene LEAFY COTYLEDON 2 (LEC2), a master regulator of seed maturation and seed oil storage under the control of an inducible Alc promoter. Stimulation of LEC2 expression in mature tobacco plants by acetaldehyde led to the accumulation of up to 6.8% per dry weight of total extracted FA. The obtained data reveal the potential of metabolically modified plant biomass for the production of biofuel.

  6. National Adult Tobacco Survey (NATS)

    Data.gov (United States)

    U.S. Department of Health & Human Services — 2013-2014. The National Adult Tobacco Survey (NATS) was created to assess the prevalence of tobacco use, as well as the factors promoting and impeding tobacco use...

  7. Promoting scopolamine biosynthesis in transgenic Atropa belladonna plants with pmt and h6h overexpression under field conditions.

    Science.gov (United States)

    Xia, Ke; Liu, Xiaoqiang; Zhang, Qiaozhuo; Qiang, Wei; Guo, Jianjun; Lan, Xiaozhong; Chen, Min; Liao, Zhihua

    2016-09-01

    Atropa belladonna is one of the most important plant sources for producing pharmaceutical tropane alkaloids (TAs). T1 progeny of transgenic A. belladonna, in which putrescine N-methyltransferase (EC. 2.1.1.53) from Nicotiana tabacum (NtPMT) and hyoscyamine 6β-hydroxylase (EC. 1.14.11.14) from Hyoscyamus niger (HnH6H) were overexpressed, were established to investigate TA biosynthesis and distribution in ripe fruits, leaves, stems, primary roots and secondary roots under field conditions. Both NtPMT and HnH6H were detected at the transcriptional level in transgenic plants, whereas they were not detected in wild-type plants. The transgenes did not influence the root-specific expression patterns of endogenous TA biosynthetic genes in A. belladonna. All four endogenous TA biosynthetic genes (AbPMT, AbTRI, AbCYP80F1 and AbH6H) had the highest/exclusive expression levels in secondary roots, suggesting that TAs were mainly synthesized in secondary roots. T1 progeny of transgenic A. belladonna showed an impressive scopolamine-rich chemotype that greatly improved the pharmaceutical value of A. belladonna. The higher efficiency of hyoscyamine conversion was found in aerial than in underground parts. In aerial parts of transgenic plants, hyoscyamine was totally converted to downstream alkaloids, especially scopolamine. Hyoscyamine, anisodamine and scopolamine were detected in underground parts, but scopolamine and anisodamine were more abundant than hyoscyamine. The exclusively higher levels of anisodamine in roots suggested that it might be difficult for its translocation from root to aerial organs. T1 progeny of transgenic A. belladonna, which produces scopolamine at very high levels (2.94-5.13 mg g(-1)) in field conditions, can provide more valuable plant materials for scopolamine production. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  8. Very bright orange fluorescent plants: endoplasmic reticulum targeting of orange fluorescent proteins as visual reporters in transgenic plants

    Directory of Open Access Journals (Sweden)

    Mann David GJ

    2012-05-01

    Full Text Available Abstract Background The expression of fluorescent protein (FP genes as real-time visual markers, both transiently and stably, has revolutionized plant biotechnology. A palette of colors of FPs is now available for use, but the diversity has generally been underutilized in plant biotechnology. Because of the green and far-red autofluorescent properties of many plant tissues and the FPs themselves, red and orange FPs (RFPs, and OFPs, respectfully appear to be the colors with maximum utility in plant biotechnology. Within the color palette OFPs have emerged as the brightest FP markers in the visible spectra. This study compares several native, near-native and modified OFPs for their “brightness” and fluorescence, therefore, their usability as marker genes in transgenic plant tissues. Results The OFPs DsRed2, tdTomato, mOrange and pporRFP were all expressed under the control of the CaMV 35S promoter in agroinfiltration-mediated transient assays in Nicotiana benthamiana. Each of these, as well as endoplasmic reticulum (ER-targeted versions, were stably expressed in transgenic Nicotiana tabacum and Arabidopsis thaliana. Congruent results were observed between transient and stable assays. Our results demonstrated that there are several adequate OFP genes available for plant transformation, including the new pporRFP, an unaltered tetramer from the hard coral Porites porites. When the tandem dimer tdTomato and the monomeric mOrange were targeted to the ER, dramatic, ca. 3-fold, increase in plant fluorescence was observed. Conclusions From our empirical data, and a search of the literature, it appears that tdTomato-ER and mOrange-ER are the two highest fluorescing FPs available as reporters for transgenic plants. The pporRFP is a brightly fluorescing tetramer, but all tetramer FPs are far less bright than the ER-targeted monomers we report here.

  9. Aquaporins of the PIP2 class are required for efficient anther dehiscence in tobacco.

    Science.gov (United States)

    Bots, Marc; Vergeldt, Frank; Wolters-Arts, Mieke; Weterings, Koen; van As, Henk; Mariani, Celestina

    2005-03-01

    Several processes during sexual reproduction in higher plants involve the movement of water between cells or tissues. Before flower anthesis, anther and pollen dehydration takes place before the release of mature pollen at dehiscence. Aquaporins represent a class of proteins that mediates the movement of water over cellular membranes. Aquaporins of the plasmamembrane PIP2 family are expressed in tobacco (Nicotiana tabacum) anthers and may therefore be involved in the movement of water in this organ. To gain more insight into the role these proteins may play in this process, we have analyzed their localization using immunolocalizations and generated plants displaying RNA interference of PIP2 aquaporins. Our results indicate that PIP2 protein expression is modulated during anther development. Furthermore, in tobacco PIP2 RNA interference plants, anther dehydration was slower, and dehiscence occurred later when compared with control plants. Together, our results suggest that aquaporins of the PIP2 class are required for efficient anther dehydration prior to dehiscence.

  10. 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

  11. Ionizing radiation from tobacco

    International Nuclear Information System (INIS)

    Westin, J.B.

    1987-01-01

    Accidents at nuclear power facilities seem inevitably to bring in their wake a great deal of concern on the part of both the lay and medical communities. Relatively little attention, however, is given to what may be the largest single worldwide source of effectively carcinogenic ionizing radiation: tobacco. The risk of cancer deaths from the Chernobyl disaster are tobacco smoke is discussed

  12. Stomatal Closure and SA-, JA/ET-Signaling Pathways Are Essential for Bacillus amyloliquefaciens FZB42 to Restrict Leaf Disease Caused by Phytophthora nicotianae in Nicotiana benthamiana

    Directory of Open Access Journals (Sweden)

    Liming Wu

    2018-04-01

    Full Text Available Bacillus amyloliquefaciens FZB42 is a plant growth-promoting rhizobacterium that induces resistance to a broad spectrum of pathogens. This study analyzed the mechanism by which FZB42 restricts leaf disease caused by Phytophthora nicotianae in Nicotiana benthamiana. The oomycete foliar pathogen P. nicotianae is able to reopen stomata which had been closed by the plant innate immune response to initiate penetration and infection. Here, we showed that root colonization by B. amyloliquefaciens FZB42 restricted pathogen-mediated stomatal reopening in N. benthamiana. Abscisic acid (ABA and salicylic acid (SA-regulated pathways mediated FZB42-induced stomatal closure after pathogen infection. Moreover, the defense-related genes PR-1a, LOX, and ERF1, involved in the SA and jasmonic acid (JA/ethylene (ET signaling pathways, respectively, were overexpressed, and levels of the hormones SA, JA, and ET increased in the leaves of B. amyloliquefaciens FZB42-treated wild type plants. Disruption of one of these three pathways in N. benthamiana plants increased susceptibility to the pathogen. These suggest that SA- and JA/ET-dependent signaling pathways were important in plant defenses against the pathogen. Our data thus explain a biocontrol mechanism of soil rhizobacteria in a plant.

  13. Why does anatabine, but not nicotine, accumulate in jasmonate-elicited cultured tobacco BY-2 cells?

    Science.gov (United States)

    Shoji, Tsubasa; Hashimoto, Takashi

    2008-08-01

    Suspension-cultured cells of Nicotiana tabacum cv. Bright Yellow-2 (BY-2) grow rapidly in a highly homogenous population and still exhibit the general behavior of plant cells, and thus are often used as model systems in several areas of plant molecular and cellular biology, including secondary metabolism. While the parental tobacco variety synthesizes nicotine as a major alkaloid, the cultured tobacco cells mainly produce a related alkaloid anatabine, instead of nicotine, when elicited with jasmonates. We report here that cultured BY-2 cells scarcely express N-methylputrescine oxidase (MPO) genes even after jasmonate elicitation. MPO is the second enzyme in the biosynthetic pathway that supplies the pyrrolidine moiety of nicotine and nornicotine, but is predicted to be dispensable for the biosynthesis of anatabine, anabasine and anatalline, which do not contain the pyrrolidine moiety. When MPO was overexpressed in tobacco BY-2 cells, nicotine synthesis was dramatically enhanced while anatabine formation was effectively suppressed. As a complementary approach, we suppressed MPO expression by RNA interference in tobacco hairy roots that normally accumulate nicotine. In the MPO-suppressed roots, the contents of anatabine, anabasine and anatalline, as well as N-methylputrescine and putrescine, markedly increased to compensate for suppressed formation of nicotine and nornicotine. These results identify the transcriptional regulation of MPO as a critical rate-limiting step that restricts nicotine formation in cultured tobacco BY-2 cells.

  14. Evaluation of DNA damage and mutagenicity induced by lead in tobacco plants.

    Science.gov (United States)

    Gichner, Tomás; Znidar, Irena; Száková, Jirina

    2008-04-30

    Tobacco (Nicotiana tabacum L. var. xanthi) seedlings were treated with aqueous solutions of lead nitrate (Pb2+) at concentrations ranging from 0.4 mM to 2.4 mM for 24 h and from 25 microM to 200 microM for 7 days. The DNA damage measured by the comet assay was high in the root nuclei, but in the leaf nuclei a slight but significant increase in DNA damage could be demonstrated only after a 7-day treatment with 200 microM Pb2+. In tobacco plants growing for 6 weeks in soil polluted with Pb2+ severe toxic effects, expressed by the decrease in leaf area, and a slight but significant increase in DNA damage were observed. The tobacco plants with increased levels of DNA damage were severely injured and showed stunted growth, distorted leaves and brown root tips. The frequency of somatic mutations in tobacco plants growing in the Pb2+-polluted soil did not significantly increase. Analytical studies by inductively coupled plasma optical emission spectrometry demonstrate that after a 24-h treatment of tobacco with 2.4 mM Pb2+, the accumulation of the heavy metal is 40-fold higher in the roots than in the above-ground biomass. Low Pb2+ accumulation in the above-ground parts may explain the lower levels or the absence of Pb2+-induced DNA damage in leaves.

  15. Regulation of Expression of the prb-1b / ACC Deaminase gene by UV-B in Transgenic tomatoes

    International Nuclear Information System (INIS)

    Tamot, B.K.; Pauls, K.P.; Glick, R.

    2003-01-01

    Transgenic tomato plants with 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase gene from Enterobacter cloacae UWA4 under the control of a pathogenesis-related promoter (prb-1b) from tobacco were challenged by abiotic stresses to determine the expression patterns of the transgene. No ACC deaminase RNA or protein was detected bu RT-PCR and in western blots prepared from leaf proteins of transgenic plants after wounding or treatment with alpha-amino butyric acid, xylanase, ethephon, salicylic acid, jasmonic acid , ethylene, or ethylene plus jasmonic acid. However, expression of the ACC deaminase transgene was observed in leaves and roots of transformed tomato lines exposed to UV light. The UV response required a minimum of 48 h of exposure and was specific to UV-B light

  16. Mice orally immunized with a transgenic plant expressing the glycoprotein of Crimean-Congo hemorrhagic fever virus

    DEFF Research Database (Denmark)

    Ghiasi, Seyed Mojtaba; Salmanian, A H; Chinikar, S

    2011-01-01

    in their serum and feces, respectively. The mice in the fed/boosted group showed a significant rise in specific IgG antibodies after a single boost. Our results imply that oral immunization of animals with edible materials from transgenic plants is feasible, and further assessments are under way. In addition......While Crimean-Congo hemorrhagic fever (CCHF) has a high mortality rate in humans, the associated virus (CCHFV) does not induce clinical symptoms in animals, but animals play an important role in disease transmission to humans. Our aim in this study was to examine the immunogenicity of the CCHFV...... glycoprotein when expressed in the root and leaf of transgenic plants via hairy roots and stable transformation of tobacco plants, respectively. After confirmatory analyses of transgenic plant lines and quantification of the expressed glycoprotein, mice were either fed with the transgenic leaves or roots, fed...

  17. Anxiety and Tobacco

    Directory of Open Access Journals (Sweden)

    Cristina Mae Wood

    2010-02-01

    Full Text Available Tobacco use is the first preventable cause of death. This is associated not only with physical illness and a shorter life expectancy, but also with different mental disorders such as anxiety disorders. Given the low risk perception of use, this paper reports a systematic review of the scientific literature on the relationship between anxiety and tobacco from an emotional perspective, including data on smoking prevalence, factors associated with the onset and maintenance of tobacco use, as well as those factors that hamper smoking cessation and increase relapse rates. The high rates of comorbidity between tobacco use and anxiety disorders make necessary the development of new and better tobacco cessation treatments, especially designed for those smokers with high state anxiety or anxiety sensitivity, with the aim of maximizing the efficacy.

  18. Tobacco packaging design for reducing tobacco use.

    Science.gov (United States)

    McNeill, Ann; Gravely, Shannon; Hitchman, Sara C; Bauld, Linda; Hammond, David; Hartmann-Boyce, Jamie

    2017-04-27

    Tobacco use is the largest single preventable cause of death and disease worldwide. Standardised tobacco packaging is an intervention intended to reduce the promotional appeal of packs and can be defined as packaging with a uniform colour (and in some cases shape and size) with no logos or branding, apart from health warnings and other government-mandated information, and the brand name in a prescribed uniform font, colour and size. Australia was the first country to implement standardised tobacco packaging between October and December 2012, France implemented standardised tobacco packaging on 1 January 2017 and several other countries are implementing, or intending to implement, standardised tobacco packaging. To assess the effect of standardised tobacco packaging on tobacco use uptake, cessation and reduction. We searched MEDLINE, Embase, PsycINFO and six other databases from 1980 to January 2016. We checked bibliographies and contacted study authors to identify additional peer-reviewed studies. Primary outcomes included changes in tobacco use prevalence incorporating tobacco use uptake, cessation, consumption and relapse prevention. Secondary outcomes covered intermediate outcomes that can be measured and are relevant to tobacco use uptake, cessation or reduction. We considered multiple study designs: randomised controlled trials, quasi-experimental and experimental studies, observational cross-sectional and cohort studies. The review focused on all populations and people of any age; to be included, studies had to be published in peer-reviewed journals. We examined studies that assessed the impact of changes in tobacco packaging such as colour, design, size and type of health warnings on the packs in relation to branded packaging. In experiments, the control condition was branded tobacco packaging but could include variations of standardised packaging. Screening and data extraction followed standard Cochrane methods. We used different 'Risk of bias' domains for

  19. Biochemical properties of the matrix metalloproteinase NtMMP1 from Nicotiana tabacum cv. BY-2 suspension cells.

    Science.gov (United States)

    Mandal, Manoj K; Fischer, Rainer; Schillberg, Stefan; Schiermeyer, Andreas

    2010-09-01

    A zinc-dependent matrix metalloproteinase (NtMMP1) found in the plasma membrane of Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells is thought to be responsible for the degradation of recombinant proteins secreted into the culture supernatant. We have characterized the proteolytic activity of NtMMP1 by expressing a recombinant derivative lacking the C-terminal transmembrane domain in yeast. After purifying the protein by affinity chromatography, its autocatalytic activity was analyzed using monoclonal antibodies raised against its N-terminal and C-terminal portions. Both the unprocessed and processed forms of NtMMP1 displayed caseinolytic activity and N-terminal sequencing identified an autocatalytic cleavage site within the sequence motif HFSFFP, which is similar to the corresponding sequences of the human matrix metalloproteinases stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10). Unlike all other matrix metalloproteinases investigated so far, NtMMP1 contains a disulfide bond within its propeptide thus rendering the proenzyme catalytically active. Kinetic analysis of NtMMP1 with a synthetic substrate revealed a K(m) of 10.55 +/- 0.9 microM, a k(cat) of 0.6 +/- 0.01 s(-1) and maximum activity at pH 7.5. We found that NtMMP1 degrades Desmodus rotundus salivary plasminogen activator alpha 1 (DSPAalpha1), a biopharmaceutical protein, that has proven difficult to produce in tobacco BY-2 cells. This provides a likely explanation for the frequent instability of secreted recombinant biopharmaceuticals produced in plant suspension cell cultures. Our data suggest new avenues that can be explored to improve the production of pharmaceutical proteins in plants and plant cells.

  20. Nitrogen Supply Influences Herbivore-Induced Direct and Indirect Defenses and Transcriptional Responses in Nicotiana attenuata[w

    Science.gov (United States)

    Lou, Yonggen; Baldwin, Ian T.

    2004-01-01

    Although nitrogen (N) availability is known to alter constitutive resistance against herbivores, its influence on herbivore-induced responses, including signaling pathways, transcriptional signatures, and the subsequently elicited chemical defenses is poorly understood. We used the native tobacco, Nicotiana attenuata, which germinates in the postfire environment and copes with large changes in soil N during postfire succession, to compare a suite of Manduca sexta- and elicitor-induced responses in plants grown under high- and low-N (LN) supply rates. LN supply decreased relative growth rates and biomass by 35% at 40 d compared to high-N plants; furthermore, it also attenuated (by 39 and 60%) the elicitor-induced jasmonate and salicylate bursts, two N-intensive direct defenses (nicotine and trypsin proteinase inhibitors, albeit by different mechanisms), and carbon-containing nonvolatile defenses (rutin, chlorogenic acid, and diterpene glycosides), but did not affect the induced release of volatiles (cis-α-bergamotene and germacrene A), which function as indirect defenses. M. sexta and methyl jasmonate-induced transcriptional responses measured with a microarray enriched in herbivore-induced genes were also substantially reduced in plants grown under LN supply rates. In M. sexta-attacked LN plants, only 36 (45%) up-regulated and 46 (58%) down-regulated genes showed the same regulation as those in attacked high-N plants. However, transcriptional responses frequently directly countered the observed metabolic changes. Changes in a leaf's sensitivity to elicitation, an attacked leaf's waning ability to export oxylipin wound signals, and/or resource limitations in LN plants can account for the observed results, underscoring the conclusion that defense activation is a resource-intensive response. PMID:15133153

  1. Silencing of a Germin-Like Gene in Nicotiana attenuata Improves Performance of Native Herbivores1[W

    Science.gov (United States)

    Lou, Yonggen; Baldwin, Ian T.

    2006-01-01

    Germins and germin-like proteins (GLPs) are known to function in pathogen resistance, but their involvement in defense against insect herbivores is poorly understood. In the native tobacco Nicotiana attenuata, attack from the specialist herbivore Manduca sexta or elicitation by adding larval ora