Tobacco Leaves (tobacco + leaf)

Distribution by Scientific Domains


Selected Abstracts


Ubiquitination of TMV Coat Protein Aggregates in Infected Tobacco Leaves

JOURNAL OF PHYTOPATHOLOGY, Issue 11-12 2003
J. Hamacher
Abstract Protein extracts from Tobacco mosaic virus (TMV) infected young tobacco leaves exhibiting strong symptoms contain higher amounts of ubiquitin conjugates in comparison with non-infected control plants presumably due to stress reactions of host cells. Western blots with antibodies against TMV indicate as well an increase in coat protein or in ubiquitinated coat protein subunits. Immunogold labelling of infected leaf material revealed the accumulation of large amounts of coat protein in opaque inclusions which also reacted strongly with ubiquitin antibodies. These inclusions appear in close vicinity to chloroplasts in chlorotic areas of the leaf. It is concluded that most of the ubiquitin conjugates found in Western blots are due to ubiquitinated coat protein. As a consequence, a considerable amount of coat protein cannot be used for correct capsid construction as the stabilizing lysine residues are blocked by ubiquitination. [source]


The role of auxin-binding protein 1 in the expansion of tobacco leaf cells

THE PLANT JOURNAL, Issue 6 2001
Jin-Gui Chen
Summary Tobacco leaf was used to investigate the mechanism of action of auxin-binding protein 1 (ABP1). The distributions of free auxin, ABP1, percentage of leaf nuclei in G2 and the amount of auxin-inducible growth were each determined in control tobacco leaves and leaves over-expressing Arabidopsis ABP1. These parameters were compared with growth of tobacco leaves, measured both spatially and temporally throughout the entire expansion phase. Within a defined window of leaf development, juvenile leaf cells that inducibly expressed Arabidopsis ABP1 prematurely advanced nuclei to the G2 phase. The ABP1-induced increase in cell expansion occured before the advance to the G2 phase, indicating that the ABP1-induced G2 phase advance is an indirect effect of cell expansion. The level of ABP1 was highest at the position of maximum cell expansion, maximum auxin-inducible growth and where the free auxin level was the lowest. In contrast, the position of maximum cell division correlated with higher auxin levels and lower ABP1 levels. Consistent with the correlations observed in leaves, tobacco cells (BY-2) in culture displayed two dose-dependent responses to auxin. At a low auxin concentration, cells expanded, while at a relatively higher concentration, cells divided and incorporated [3H]-thymidine. Antisense suppression of ABP1 in these cells dramatically reduced cell expansion with negligible effect on cell division. Taken together, the data suggest that ABP1 acts at a relatively low level of auxin to mediate cell expansion, whereas high auxin levels stimulate cell division via an unidentified receptor. [source]


Disease stress-inducible genes of tobacco: expression profile of elicitor-responsive genes isolated by subtractive hybridization

PHYSIOLOGIA PLANTARUM, Issue 4 2003
Daigo Takemoto
In order to investigate the change in mRNA profile during tobacco disease response, a subtractive hybridization procedure was used to generate a cDNA library for genes induced in tobacco (Nicotiana tabacum cv. Samsun NN) treated with oomycete elicitor. Database searches with the randomly isolated genes revealed that this cDNA library was enriched for reported disease stress-responsive genes such as pathogenesis-related proteins and cell wall protein genes. The expressions of eight newly isolated genes were induced by inoculation with the non-pathogenic bacteria, Pseudomonas syringae pv. glycinea. The NtEIGs (N.tabacumelicitor- inducible genes) showed similarity to genes for stellacyanin-like protein (NtEIG-A1), glutathione peroxidase (NtEIG-C08), extensin-like protein (NtEIG-C29), WRKY transcription factor (NtEIG-D48), glycine rich protein (NtEIG-E17), , -1, 3-glucanase-like protein (NtEIG-E76), photoassimilate-responsive protein-1 (NtEIG-E80) and wound-induced protein (NtEIG-D10). The expression patterns of NtEIGs in tobacco leaf in response to P. syringae pv. glycinea, salicylic acid (SA), methyl jasmonate (MeJA) and wound stress were analysed. The individual expression patterns of NtEIGs indicate that the transcriptional activation of NtEIGs is regulated by various signals and the products of NtEIGs are involved in different processes at different stages of the plant defence responses. [source]


SIMPEROTA 1/3 , a decision support system for blue mould disease of tobacco,

EPPO BULLETIN, Issue 2 2007
P. Racca
Blue mould (Peronospora tabacina) is the most serious threat to German tobacco crops. In order to efficiently control the disease whilst minimizing the risk of nontolerable fungicide residue levels on tobacco leaves, a decision support system has been developed which optimizes the timing of fungicide treatments. The DSS consists of two models, SIMPEROTA 1, which forecasts the dates of blue mould first appearance and SIMPEROTA 3 which forecasts the dates of fungicide applications. Crucial biological processes are included in the models (infection, mycelium growth, sporulation and spore release). Input parameters are temperature, relative humidity and leaf wetness recorded on an hourly basis. Validation with data from 2003 and 2006 showed that SIMPEROTA 1 gave satisfying results. The model is suitable for practical use and can be employed for steering monitoring efforts of extension services and for the timing of the first fungicide treatment. SIMPEROTA 3 gives advice on follow-up treatments and the length of spraying intervals, but this model needs to be validated before being introduced into practice. [source]


Alternaria alternata AT Toxin Induces Programmed Cell Death in Tobacco

JOURNAL OF PHYTOPATHOLOGY, Issue 10 2009
Elena T. Yakimova
Abstract Detached tobacco leaves were infiltrated with an AT toxin preparation from the foliar pathogen Alternaria alternata tobacco pathotype. The AT toxin preparation caused formation of necrotic lesions within 5 days post-infiltration in a concentration-dependent manner. Cell death was accompanied by increased levels of the stress metabolites hydrogen peroxide, malondialdehyde, free proline and by enhanced total protease activity. Lesion development and the production of stress metabolites were suppressed if the infiltration site was pre-infiltrated with caspase-specific peptide inhibitors (irreversible caspase-1 inhibitor acyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-CMK) and the broad range caspase inhibitor benzyoxycarbonyl-Asp-2,6-dichlorobenzoyloxymethylketone (Z-Asp-CH2-DCB)), the serine protease inhibitor N,-p-tosyl- l -lysine chloromethylketone and the polyamine spermine. Extensive accumulation of reactive oxygen species (ROS), as determined by staining with 3-3,-diaminobenzidine and 2,,7,-dichlorofluorescein diacetate, was found in the AT toxin-challenged lesions. The data show that AT toxin-induced cell death in tobacco is a type of programmed cell death in which caspase-like proteases and ROS signalling play a prominent role. [source]


Tobacco blue mould disease caused by Peronospora hyoscyami f. sp. tabacina

MOLECULAR PLANT PATHOLOGY, Issue 1 2010
ORLANDO BORRÁS-HIDALGO
SUMMARY Blue mould [Peronospora hyoscyami f. sp. tabacina (Adam) Skalicky 1964] is one of the most important foliar diseases of tobacco that causes significant losses in the Americas, south-eastern Europe and the Middle East. This review summarizes the current knowledge of the mechanisms employed by this oomycete pathogen to colonize its host, with emphasis on molecular aspects of pathogenicity. In addition, key biochemical and molecular mechanisms involved in tobacco resistance to blue mould are discussed. Taxonomy: Kingdom: Chromista (Straminipila); Phylum: Heterokontophyta; Class: Oomycete; Order: Peronosporales; Family: Peronosporaceae; Genus: Peronospora; Species: Peronospora hyoscyami f. sp. tabacina. Disease symptoms: The pathogen typically causes localized lesions on tobacco leaves that appear as single, or groups of, yellow spots that often coalesce to form light-brown necrotic areas. Some of the leaves exhibit grey to bluish downy mould on their lower surfaces. Diseased leaves can become twisted, such that the lower surfaces turn upwards. In such cases, the bluish colour of the diseased plants becomes quite conspicuous, especially under moist conditions when sporulation is abundant. Hence the name of the disease: tobacco blue mould. Infection process: The pathogen develops haustoria within plant cells that are thought to establish the transfer of nutrients from the host cell, and may also act in the delivery of effector proteins during infection. Resistance: Several defence responses have been reported to occur in the Nicotiana tabacum,P. hyoscyami f. sp. tabacina interaction. These include the induction of pathogenesis-related genes, and a correlated increase in the activities of typical pathogenesis-related proteins, such as peroxidases, chitinases, ,-1,3-glucanases and lipoxygenases. Systemic acquired resistance is one of the best characterized tobacco defence responses activated on pathogen infection. [source]


Analysis of residues of imidacloprid in tobacco by high-performance liquid chromatography with liquid,liquid partition cleanup

PEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 5 2005
Hongxia Liu
Abstract A practical method for analysis of residues of imidacloprid in baked tobacco leaves has been developed using HPLC with liquid,liquid partition clean-up. Imidacloprid was extracted with ethyl acetate under ultrasound, and cleaned up by liquid,liquid partition with 50 g litre,1 aqueous sodium chloride followed by dichloromethane. The water-soluble and fat-soluble components in tobacco, which interfere with imidacloprid in HPLC, were removed. The separation was performed on a Supelco LC-18 column (250 mm × 4.6 mm ID) with a mobile phase of acetonitrile + 5 mM ammonium acetate (20 + 80 by volume) at a flow-rate of 1 ml min,1. The baseline separation between imidacloprid and the tobacco blank was achieved within 10 min. With a detection wavelength at 270 nm, the limit of quantitation was 0.04 mg kg,1. The recovery ranged from 89.8 to 95.4% and the RSDs were less than 2.3%. The proposed method was successfully employed for the determination of imidacloprid residues in 300 samples of flue-cured tobacco leaves. Copyright © 2004 Society of Chemical Industry [source]


Overexpression of the apple alcohol acyltransferase gene alters the profile of volatile blends in transgenic tobacco leaves

PHYSIOLOGIA PLANTARUM, Issue 3 2008
Dapeng Li
Alcohol acyltransferases (AATs) are key enzymes in ester biosynthesis. Previous studies have found that AAT may be a stress-related gene. To investigate further the function of the apple alcohol acyltransferase gene (MdAAT2), transgenic tobacco plants overexpressing MdAAT2 were generated. Gas chromatography,mass spectroscopy analysis showed that the volatile blends were altered in these transgenic tobacco leaves. Although no apple-fruity volatile esters were detected in transgenic tobacco leaves, methyl caprylate, methyl caprate, and methyl dodecanoate were newly generated, and the concentrations of methyl benzoate and methyl tetradecanoate were significantly increased, suggesting that MdAAT2 may use medium-chain fatty acyl CoA and benzoyl-CoA as acyl donors together with methanol acceptors as substrates. Surprisingly, the concentrations of linalool were significantly increased in transgenic tobacco leaves, which may mediate the repellent effect on Myzus persicae (Sulzer) aphids. Using methyl jasmonate (MeJA) and wounding treatments, we found that MdAAT2 may substitute for the partial ability of MeJA to induce the production of linalool in transgenic plants. These data suggest that MdAAT2 may be involved in the response to the MeJA signal and may play a role in the response to biotic and abiotic stress. [source]


A new catalytic activity from tobacco converting 2-coumaric acid to salicylic aldehyde

PHYSIOLOGIA PLANTARUM, Issue 3 2007
Jacek Malinowski
Salicylic acid (SA) mediates plant response to pathogen invasion, resulting in hypersensitive response and in the formation of systemic acquired resistance. It is well known that Nicotiana tabacum and other plants respond to Tobacco Mosaic Virus (TMV) infection by increasing the content of SA but the details of SA biosynthesis are still not fully understood. Generally, SA may originate directly from isochorismate (Arabidopsis thaliana), or its C6,C1 skeleton could be synthesized via the phenylpropanoid pathway by ,-oxidation of trans -cinnamic acid (N. tabacum), 2-coumaric acid (OCA) (Gaulteria procumbens, Lycopersicum esculentum) or by retro-aldol reaction of trans -cinnamoyl-CoA (Hypericum androsaemum). We report here a novel putative enzyme activity from tobacco, salicylic aldehyde synthase (SAS), catalysing non-oxidative formation of salicylic aldehyde (SALD) directly from OCA. This chain-shortening activity is similar to that of 4-hydroxybenzaldehyde synthase from Vanilla planifolia, Lithospermum erythrorhizon, Daucus carota, Solanum tuberosum and Polyporus hispidus but the enzyme differs in the kinetics of the reaction, substrate specificity and requirements for reducing cofactors. SAS activity is constitutively expressed in healthy tobacco leaves and doubles as a result of infection with TMV. Moreover, the product of SAS activity,SALD, applied exogenously on tobacco leaves, stimulates peroxidase activity and enhances resistance to consecutive infection with TMV. These observations could suggest a contribution of SAS and SALD to the response of tobacco to TMV infection. [source]


Recombinant human GAD65 accumulates to high levels in transgenic tobacco plants when expressed as an enzymatically inactive mutant

PLANT BIOTECHNOLOGY JOURNAL, Issue 8 2010
Linda Avesani
Summary The 65-kDa isoform of glutamic acid decarboxylase (GAD65) is the major autoantigen implicated in the development of type 1 diabetes mellitus (T1DM). The bulk manufacture of GAD65 is a potential issue in the fight against T1DM but current production platforms are expensive. We show that a catalytically inactive form of GAD65 (GAD65mut) accumulates at up to 2.2% total soluble protein in transgenic tobacco leaves, which is more than 10-fold the levels achieved with active GAD65, yet the protein retains the immunogenic properties required to treat T1DM. This higher yield was found to be a result of a higher rate of protein synthesis and not transcript availability or protein stability. We found that targeting GAD65 to the endoplasmic reticulum, a strategy that increases the accumulation of many recombinant proteins expressed in plants, did not improve production of GAD65mut. The production of a catalytically inactive autoantigen that retains its immunogenic properties could be a useful strategy to provide high-quality therapeutic protein for treatment of autoimmune T1DM. [source]


Chloroplast-derived vaccine antigens confer dual immunity against cholera and malaria by oral or injectable delivery

PLANT BIOTECHNOLOGY JOURNAL, Issue 2 2010
Abdoreza Davoodi-Semiromi
Summary Cholera and malaria are major diseases causing high mortality. The only licensed cholera vaccine is expensive; immunity is lost in children within 3 years and adults are not fully protected. No vaccine is yet available for malaria. Therefore, in this study, the cholera toxin-B subunit (CTB) of Vibrio cholerae fused to malarial vaccine antigens apical membrane antigen-1 (AMA1) and merozoite surface protein-1 (MSP1) was expressed in lettuce and tobacco chloroplasts. Southern blot analysis confirmed homoplasmy and stable integration of transgenes. CTB-AMA1 and CTB-MSP1 fusion proteins accumulated up to 13.17% and 10.11% (total soluble protein, TSP) in tobacco and up to 7.3% and 6.1% (TSP) in lettuce, respectively. Nine groups of mice (n = 10/group) were immunized subcutaneously (SQV) or orally (ORV) with purified antigens or transplastomic tobacco leaves. Significant levels of antigen-specific antibody titres of immunized mice completely inhibited proliferation of the malarial parasite and cross-reacted with the native parasite proteins in immunoblots and immunofluorescence studies. Protection against cholera toxin challenge in both ORV (100%) and SQV (89%) mice correlated with CTB-specific titres of intestinal, serum IgA and IgG1 in ORV and only IgG1 in SQV mice, but no other immunoglobulin. Increasing numbers of interleukin-10+ T cell but not Foxp3+ regulatory T cells, suppression of interferon-, and absence of interleukin-17 were observed in protected mice, suggesting that immunity is conferred via the Tr1/Th2 immune response. Dual immunity against two major infectious diseases provided by chloroplast-derived vaccine antigens for long-term (>300 days, 50% of mouse life span) offers a realistic platform for low cost vaccines and insight into mucosal and systemic immunity. [source]


Effects of growth and measurement light intensities on temperature dependence of CO2 assimilation rate in tobacco leaves

PLANT CELL & ENVIRONMENT, Issue 3 2010
WATARU YAMORI
ABSTRACT Effects of growth light intensity on the temperature dependence of CO2 assimilation rate were studied in tobacco (Nicotiana tabacum) because growth light intensity alters nitrogen allocation between photosynthetic components. Leaf nitrogen, ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) and cytochrome f (cyt f) contents increased with increasing growth light intensity, but the cyt f/Rubisco ratio was unaltered. Mesophyll conductance to CO2 diffusion (gm) measured with carbon isotope discrimination increased with growth light intensity but not with measuring light intensity. The responses of CO2 assimilation rate to chloroplast CO2 concentration (Cc) at different light intensities and temperatures were used to estimate the maximum carboxylation rate of Rubisco (Vcmax) and the chloroplast electron transport rate (J). Maximum electron transport rates were linearly related to cyt f content at any given temperature (e.g. 115 and 179 µmol electrons mol,1 cyt f s,1 at 25 and 40 °C, respectively). The chloroplast CO2 concentration (Ctrans) at which the transition from RuBP carboxylation to RuBP regeneration limitation occurred increased with leaf temperature and was independent of growth light intensity, consistent with the constant ratio of cyt f/Rubisco. In tobacco, CO2 assimilation rate at 380 µmol mol,1 CO2 concentration and high light was limited by RuBP carboxylation above 32 °C and by RuBP regeneration below 32 °C. [source]


Reciprocal diurnal changes of phosphoenolpyruvate carboxylase expression and cytosolic pyruvate kinase, citrate synthase and NADP-isocitrate dehydrogenase expression regulate organic acid metabolism during nitrate assimilation in tobacco leaves

PLANT CELL & ENVIRONMENT, Issue 11 2000
W.-R. Scheible
ABSTRACT Diurnal changes of transcript levels for key enzymes in nitrate and organic acid metabolism and the accompanying changes of enzyme activities and metabolite levels were investigated in nitrogen-sufficient wild-type tobacco, in transfomants with decreased expression of nitrate reductase, and in nitrate-deficient wild-type tobacco. (i) In nitrogen-sufficient wild-type plants, transcript levels for nitrate reductase (NR, EC 1.6.6.1), nitrite reductase (NIR, EC 1.7.7.1) and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) were high at the end of the night and decreased markedly during the light period. The levels of these three transcripts were increased and the diurnal changes were damped in genotypes with decreased expression of nitrate reductase. The levels of these transcripts were very low in nitrate-limited wild-type plants, except for a small rise after irrigation with 0·2 mM nitrate. (ii) The levels of the transcripts for cytosolic pyruvate kinase (PK, EC 2.7.1.40), mitochondrial citrate synthase (CS, EC 4.1.3.7) and NADP-isocitrate dehydrogenase (NADP-ICDH, EC 1.1.1.42) were highest at the end of the light period and beginning of the night. These three transcripts increase and the diurnal changes were damped in genotypes with decreased expression of NR. (iii) The diurnal changes of transcript levels were accompanied by changes in the activities of the encoded enzymes. The activities of NR and PEPC were highest in the early part of the light period, whereas the activities of PK and NADP-ICDH were highest later in the light period and during the first part of the night and CS activity was highest at the end of the night. Activity of PEPC, PK, CS and NADP-ICDH increased and the diurnal changes were damped in genotypes with low expression of NR. Activity of all four enzymes decreased in nitrate-limited wild-type plants. (iv) In the light, malate accumulated, citrate decreased, and about 30% of the assimilated nitrate accumulated temporarily as glutamine, ammonium, glycine and serine. These changes were reversed during the night. (v) It is proposed that the diurnal changes of expression facilitate preferential synthesis of malate to act as a counter-anion for pH regulation during the first part of the light period when NR activity is high, and preferential synthesis of 2-oxoglutarate to act as a nitrogen acceptor later in the day when large amounts of nitrogen have accumulated in ammonium, glutamine and other amino acids including glycine in the photorespiration pathway, and NR activity has been decreased. [source]


Hydroxyproline-rich glycoprotein accumulation in tobacco leaves protected against Erysiphe cichoracearum by potato virus Y infection

PLANT PATHOLOGY, Issue 2 2000
V. Raggi
Tobacco cv. Havana 425 acquired resistance to a compatible isolate of Erysiphe cichoracearum after infection by a strain of potato virus Y (PVYN) that causes veinal necrosis; another common strain (PVYO) that does not cause necrosis gave less protection. Hydroxyproline-rich glycoproteins (HRGPs), believed to be involved in resistance, were determined by analysing hydroxyproline (Hyp) in purified cell walls. Hyp content increased significantly in PVYN -protected leaves, compared with untreated controls, 2,4 days after necrotic lesion symptoms developed. No further increase in Hyp was noted in PVYN -protected leaves after E. cichoracearum challenge. Hyp increases were significantly higher in protected leaves of plants showing symptoms on day 7 than on day 10. Infection with the PVYO strain caused significant decrease in Hyp content, compared with uninoculated controls. Inoculation of virus-free plants with E. cichoracearum induced moderate and transitory Hyp increases on day 2 or 3, followed by a quick decrease associated with a weak response by the compatible host. It is suggested that HRGP accumulation induced by PVYN (but not by PVYO) causes changes in the host cell wall that result in resistance to E. cichoracearum. [source]


,Senescence-associated vacuoles' are involved in the degradation of chloroplast proteins in tobacco leaves

THE PLANT JOURNAL, Issue 2 2008
Dana E. Martínez
Summary Massive degradation of photosynthetic proteins is the hallmark of leaf senescence; however the mechanism involved in chloroplast protein breakdown is not completely understood. As small ,senescence-associated vacuoles' (SAVs) with intense proteolytic activity accumulate in senescing leaves of soybean and Arabidopsis, the main goal of this work was to determine whether SAVs are involved in the degradation of chloroplastic components. SAVs with protease activity were readily detected through confocal microscopy of naturally senescing leaves of tobacco (Nicotiana tabacum L.). In detached leaves incubated in darkness, acceleration of the chloroplast degradation rate by ethylene treatment correlated with a twofold increase in the number of SAVs per cell, compared to untreated leaves. In a tobacco line expressing GFP targeted to plastids, GFP was re-located to SAVs in senescing leaves. SAVs were isolated by sucrose density gradient centrifugation. Isolated SAVs contained chloroplast-targeted GFP and the chloroplast stromal proteins Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) and glutamine synthetase, but lacked the thylakoid proteins D1 and light-harvesting complex II of the photosystem II reaction center and photosystem II antenna, respectively. In SAVs incubated at 30°C, there was a steady decrease in Rubisco levels, which was completely abolished by addition of protease inhibitors. These results indicate that SAVs are involved in degradation of the soluble photosynthetic proteins of the chloroplast stroma during senescence of leaves. [source]


The Arabidopsis thaliana TIR-NB-LRR R-protein, RPP1A; protein localization and constitutive activation of defence by truncated alleles in tobacco and Arabidopsis

THE PLANT JOURNAL, Issue 6 2006
L. Michael Weaver
Summary Specific recognition of Hyaloperonospora parasitica isolate Cala2 by Arabidopsis thaliana Ws-0 is mediated by the resistance gene RPP1A. Transient expression of different truncations of RPP1A in tobacco leaves revealed that its TIR-NB-ARC portion is sufficient to induce an elicitor-independent cell death. In stable transgenic lines of Arabidopsis, overexpression of the RPP1A TIR-NB-ARC domains (E12) using the 35S promoter leads to broad-spectrum resistance to virulent strains of H. parasitica and Pseudomonas syringae DC3000. The TIR-NB-ARC-mediated constitutive immunity is due to activation of the salicylic acid-dependent resistance pathway and is relieved by either a mutation in EDS1 or the presence of the salicylate hydroxylase gene, NahG. Growth of 35S::E12 plants is reduced, a phenotype observed in many constitutively resistant mutants. RPP1A carries a hydrophobic peptide at its N-terminus that directs the RPP1A protein into membranes, though it may not be the sole determinant mediating membrane association of RPP1A. Two-phase partitioning and sucrose density gradient sedimentation established that RPP1A resides in the endoplasmic reticulum and/or Golgi apparatus. [source]


Continuous expression in tobacco leaves of a Brassica napus PEND homologue blocks differentiation of plastids and development of palisade cells

THE PLANT JOURNAL, Issue 1 2005
Paul Wycliffe
Summary Brassica napus complementary deoxyribonucleic acid (cDNA) clones encoding a DNA-binding protein, BnPEND, were isolated by Southwestern screening. A distinctive feature of the protein was a bZIP-like sequence in the amino-terminal portion, which, after expression in Escherichia coli, bound DNA. BnPEND transcripts were present in B. napus roots and flower buds, and to a lesser extent in stems, flowers and young leaves. Treatment in the dark for 72 h markedly increased the amount of BnPEND transcript in leaves of all ages. Sequence comparison showed that BnPEND was similar to a presumed transcription factor from B. napus, GSBF1, a protein deduced from an Arabidopsis thaliana cDNA (BX825084) and the PEND protein from Pisum sativum, believed to anchor the plastid DNA to the envelope early during plastid development. Homology to expressed sequence tag (EST) sequences from additional species suggested that BnPEND homologues are widespread among the angiosperms. Transient expression of BnPEND fused with green fluorescent protein (GFP) in Nicotiana benthamiana epidermal cells showed that BnPEND is a plastid protein, and that the 15 amino acids at the amino-terminal contain information about plastid targeting. Expression of BnPEND in Nicotiana tabacum from the Cauliflower Mosaic Virus 35S promoter gave stable transformants with different extents of white to light-green areas in the leaves, and even albino plants. In the white areas, but not in adjacent green tissue, the development of palisade cells and chloroplasts was disrupted. Our data demonstrate that the BnPEND protein, when over-expressed at an inappropriate stage, functionally blocks the development of plastids and leads to altered leaf anatomy, possibly by preventing the release of plastid DNA from the envelope. [source]


Spatio-temporal expression of patatin-like lipid acyl hydrolases and accumulation of jasmonates in elicitor-treated tobacco leaves are not affected by endogenous levels of salicylic acid

THE PLANT JOURNAL, Issue 5 2002
Sandrine Dhondt
Summary We have previously isolated three tobacco genes (NtPat) encoding patatin-like proteins, getting rapidly induced during the hypersensitive response (HR) to tobacco mosaic virus, in advance to jasmonate accumulation. NtPAT enzymes are lipid acyl hydrolases that display high phospholipase A2 (PLA2) activity and may mobilize fatty acid precursors of oxylipins. Here, we performed a detailed study of NtPat gene regulation under various biotic and abiotic stresses. PLA2 activity was poorly induced in response to drought, wounding, reactive oxygen intermediates, salicylic acid (SA) or methyl-jasmonate (MJ) whereas the ethylene (ET) precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), provoked a moderate induction. In contrast, PLA2 activity was strongly induced when ACC was combined with MJ, and in response to the bacterium Erwinia carotovora or to the fungus Botrytis cinerea, as well as to treatment with ,-megaspermin, a cell death-inducing protein elicitor. A simplified system based on the infiltration of ,-megaspermin into leaves was used to dissect the spatio-temporal activation of PLA2 activity with regards to the accumulation of jasmonates and to the influence of endogenous SA. NtPat -encoded PLA2 activity was rapidly induced in the infiltrated zone before the appearance of cell death and with some delay in the surrounding living cells. A massive accumulation of 12-oxo-phytodienoic and jasmonic acids occurred in the elicitor-infiltrated zone, but only low levels were detectable outside this area. A similar picture was found in SA-deficient plants, showing that in tobacco, accumulation of jasmonates is not affected by the concomitant HR-induced build-up of endogenous SA. Finally, ET-insensitive plants showed a weakened induction of PLA2 activity outside the elicitor-infiltrated tissue. [source]


The role of auxin-binding protein 1 in the expansion of tobacco leaf cells

THE PLANT JOURNAL, Issue 6 2001
Jin-Gui Chen
Summary Tobacco leaf was used to investigate the mechanism of action of auxin-binding protein 1 (ABP1). The distributions of free auxin, ABP1, percentage of leaf nuclei in G2 and the amount of auxin-inducible growth were each determined in control tobacco leaves and leaves over-expressing Arabidopsis ABP1. These parameters were compared with growth of tobacco leaves, measured both spatially and temporally throughout the entire expansion phase. Within a defined window of leaf development, juvenile leaf cells that inducibly expressed Arabidopsis ABP1 prematurely advanced nuclei to the G2 phase. The ABP1-induced increase in cell expansion occured before the advance to the G2 phase, indicating that the ABP1-induced G2 phase advance is an indirect effect of cell expansion. The level of ABP1 was highest at the position of maximum cell expansion, maximum auxin-inducible growth and where the free auxin level was the lowest. In contrast, the position of maximum cell division correlated with higher auxin levels and lower ABP1 levels. Consistent with the correlations observed in leaves, tobacco cells (BY-2) in culture displayed two dose-dependent responses to auxin. At a low auxin concentration, cells expanded, while at a relatively higher concentration, cells divided and incorporated [3H]-thymidine. Antisense suppression of ABP1 in these cells dramatically reduced cell expansion with negligible effect on cell division. Taken together, the data suggest that ABP1 acts at a relatively low level of auxin to mediate cell expansion, whereas high auxin levels stimulate cell division via an unidentified receptor. [source]


The effect of plant cytokinin hormones on the production of ethylene, nitric oxide, and protein nitrotyrosine in ageing tobacco leaves

BIOFACTORS, Issue 1-4 2006
N. Wilhelmová
Abstract Transgenic plants with genetically increased or decreased levels of cytokinins were used to investigate the effect of cytokinin level on the production of ethylene, a plant hormone with suggested role in senescence, and the production of nitric oxide, potentially important signalling and regulatory molecule. The production of these gases was followed during the course of leaf development and senescence. The production of ethylene and nitric oxide is under genetic control of genes other than those involved in regulation of senescence. The difference in basic ethylene and NO levels in different tobacco cultivars was higher than their changes in senescence. The results of this study did not indicate a direct link between ethylene production and cytokinin levels. However, there was a decreased production of NO in senescent leaves. Low cytokinins level was associated with increased NO production during leaf development. Protein nitrotyrosine proved to be a better indicator of the reactive nitrogen species than measuring of the NO production. Higher nitrotyrosine concentrations were found in insoluble proteins than in the soluble ones, pointing to membrane proteins as the primary targets of the reactive nitrogen species. In plants with elevated cytokinin levels the content of nitrated proteins decreased both in soluble and insoluble fractions. This finding indicates an antioxidative function of cytokinins against reactive nitrogen species. [source]


High-level bacterial cellulase accumulation in chloroplast-transformed tobacco mediated by downstream box fusions

BIOTECHNOLOGY & BIOENGINEERING, Issue 4 2009
Benjamin N. Gray
Abstract The Thermobifida fusca cel6A gene encoding an endoglucanase was fused to three different downstream box (DB) regions to generate cel6A genes with 14 amino acid fusions. The DB-Cel6A fusions were inserted into the tobacco (Nicotiana tabacum cv. Samsun) chloroplast genome for protein expression. Accumulation of Cel6A protein in transformed tobacco leaves varied over approximately two orders of magnitude, dependent on the identity of the DB region fused to the cel6A open reading frame (ORF). Additionally, the DB region fused to the cel6A ORF affected the accumulation of Cel6A protein in aging leaves, with the most effective DB regions allowing for high level accumulation of Cel6A protein in young, mature, and old leaves, while Cel6A protein accumulation decreased with leaf age when less effective DB regions were fused to the cel6A ORF. In the most highly expressed DB-Cel6A construct, enzymatically active Cel6A protein accumulated at up to 10.7% of total soluble leaf protein (%TSP). The strategy used for high-level endoglucanase expression may be useful for expression of other cellulolytic enzymes in chloroplasts, ultimately leading to cost-effective heterologous enzyme production for cellulosic ethanol using transplastomic plants. Biotechnol. Bioeng. 2009;102: 1045,1054. © 2008 Wiley Periodicals, Inc. [source]


Production of native and modified recombinant Der p 1 molecules in tobacco plants

CLINICAL & EXPERIMENTAL ALLERGY, Issue 5 2009
D. Burtin
Summary Background As a complex molecule requiring post-translational processing, it has been difficult to produce the Der p 1 major allergen from the Dermatophagoides pteronyssinus house dust mite in a recombinant form. Objective Here, we tested whether transgenic tobacco plants are suitable to express Der p 1, either as a wild-type molecule or as variants lacking N -glycosylation sites (Gly,) and/or cysteine protease activity (Enz,). Methods Using Agrobacterium tumefaciens -based transformation, pro Der p 1 molecules bearing mutations within either the N -glycosylation sites (N34Q, N150Q) and/or the cysteine protease-active site (C132V) were expressed in tobacco plants. After purification by ion exchange chromatography, allergens were characterized using immunoblotting, circular dichroism (CD), as well as basophil and T lymphocyte stimulation assays. Results Four forms of recombinant Der p 1 (i.e. wild-type Gly+/Enz+, as well as Gly,/Enz+, Gly+/Enz, or Gly,/Enz, variants) were successfully expressed in tobacco leaves as pro Der p 1 molecules. Spontaneous cleavage of the pro-peptide was observed in tobacco leaf extracts for all forms of recombinant Der p 1 (r Der p 1). CD confirmed that all r Der p 1 molecules, with the exception of the Gly,/Enz, variant, exhibited secondary structures comparable to the natural protein. A cysteine protease activity was associated only with the Gly+/Enz+ form. All these molecules exhibit a profile similar to natural Der p 1 with respect to IgE immunoreactivity, basophil activation and T cell recognition. Conclusion A tobacco plant expression system allows the production of various forms of mature Der p 1, which could be used for diagnostic or immunotherapeutic purposes. [source]