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At least 19 recordsLinked to original sources
PubMed2006

The chloroplast genome of Nicotiana sylvestris and Nicotiana tomentosiformis: complete sequencing confirms that the Nicotiana sylvestris progenitor is the maternal genome donor of Nicotiana tabacum.

The tobacco cultivar Nicotiana tabacum is a natural amphidiploid that is thought to be derived from ancestors of Nicotiana sylvestris and Nicotiana tomentosiformis. To compare these chloroplast genomes, DNA was prepared from isolated chloroplasts from green leaves of N. sylvestris and N. tomentosiformis, and subjected to whole-genome shotgun sequencing. The N. sylvestris chloroplast genome comprises of 155,941 bp and shows identical gene organization with that of N. tabacum, except one ORF. Detailed comparison revealed only seven different sites between N. tabacum and N. sylvestris; three in introns, two in spacer regions and two in coding regions. The chloroplast DNA of N. tomentosiformis is 155,745 bp long and possesses also identical gene organization with that of N. tabacum, except four ORFs and one pseudogene. However, 1,194 sites differ between these two species. Compared with N. tabacum, the nucleotide substitution in the inverted repeat was much lower than that in the single-copy region. The present work confirms that the chloroplast genome from N. tabacum was derived from an ancestor of N. sylvestris, and suggests that the rate of nucleotide substitution of the chloroplast genomes from N. tabacum and N. sylvestris is very low.

Chloroplasts↗
PubMed2004

Consistency of Nicotiana attenuata's herbivore- and jasmonate-induced transcriptional responses in the allotetraploid species Nicotiana quadrivalvis and Nicotiana clevelandii.

We examined the consistency of the native diploid Nicotiana attenuata (Na)'s herbivore-induced transcriptional changes in the two allotetraploid natives, Nicotiana clevelandii (Nc) and Nicotiana quadrivalvis (Nq), which are thought to be derived from hybridizations with an ancestral Na. An analysis of nuclear-encoded chloroplast-expressed Gln synthetase gene (ncpGS) sequences found strong similarity between Nc and Na and between N. trigonophylla and the two allopolyploids. All species were elicited with methyl jasmonate (MeJA), or were wounded and treated with either water, Manduca sexta oral secretions and regurgitant (R), or the two most abundant fatty acid amino acid conjugates (F) in R to simulate herbivory. The induced transcriptional responses in all three species were compared with a cDNA microarray enriched in Na genes. Na had the fastest transcriptional responses followed by Nc and then Nq. Na's R- and F-elicited responses were more similar to those from Nq, while the MeJA- or wound-elicited responses were more consistent in Nc. Treatment of wounds with the full cocktail of elicitors found in R elicits more complex responses than does treatment with F. The species differ in their elicited JA responses, and these differences are mirrored in the expression of oxylipin genes (LOX, HPL, AOS, and alpha-DOX) and downstream JA-elicited genes (TD). Elicitation decreases the expression of growth-related genes in all three species. We propose that this is a valuable system to examine the modification of complex, polygenic, adaptive responses during allopolyploid speciation.

Acetates↗
PubMed2006

Nicotiana (Nicotiana tobaccum, Nicotiana benthamiana).

Agrobacterium-mediated transformation of Nicotiana species, namely, Nicotiana tobaccum and Nicotiana benthamiana, using leaf disks as the target explant has provided the plant community with a valuable tool for rapid evaluation of transgenes in higher plants. This protocol has a number of desirable attributes: readily available explant material, high efficiency, and a relatively quick turnaround time. The in vitro regeneration scheme of the leaf disks is prolific and follows an indirect organogenic differentiation. N. tobaccum and N. benthamiana are highly susceptible to a variety of disarmed A. tumefaciens strains, and the transformation system is amenable to a number of selection agents. This protocol has proved to be easily transferable to a point at which a single investigator with minimal training can generate thousand of events per year. Moreover, seed size and quantity per event permit monitoring of segregation in Petri plates, and sufficient biomass can be accrued from an individual plant, which can be either clonally propagated or allowed to self-pollinate or easily outcrossed.

Agrobacterium tumefaciens↗
PubMed1992

The function of vacuolar beta-1,3-glucanase investigated by antisense transformation. Susceptibility of transgenic Nicotiana sylvestris plants to Cercospora nicotianae infection.

Vacuolar class I beta-1,3-glucanases (EC 3.2.1.39) are believed to be important in the induced defense reaction of plants to fungal infection. We used antisense transformation to test this hypothesis and to identify other possible physiological functions of this enzyme. Nicotiana sylvestris plants were transformed with antisense constructions containing the region from position 27 to 608 of the coding sequence of the basic, vacuolar beta-1,3-glucanase gene GLA of tobacco regulated by cauliflower mosaic virus 35S RNA expression signals. Plants homozygous for this transgene showed a marked, ca. 20-fold reduction in the constitutive expression of class I beta-1,3-glucanase antigen in their leaves. RNA blot analysis indicated that the antisense plants expressed low levels of the sense transcript of the host beta-1,3-glucanase gene and the antisense transcript of the transgene. Immune blot analysis of plant extracts indicated that only expression of the N. sylvestris homologue of class I tobacco beta-1,3-glucanase and not the acidic, class II isoforms of the enzyme was blocked in the antisense plants. Class I isoforms of beta-1,3-glucanase and chitinase were coordinately induced in leaves of untransformed and empty-vector-transformed N. sylvestris plants treated with ethylene or infected with the fungal leaf pathogen Cercospora nicotianae. In antisense plants, chitinase but not beta-1,3-glucanase was induced under these conditions indicating that antisense transformation effectively blocks constitutive as well as induced expression of class I beta-1,3-glucanase. Under greenhouse conditions, antisense plants developed normally and were fertile. The plants did not exhibit increased susceptibility to C. nicotianae infection. These results suggest that expression of the beta-1,3-glucanase isoform blocked by antisense transformation is not necessary for 'housekeeping' functions of N. sylvestris nor defense against the fungal pathogen tested.

Blotting, Northern↗
PubMed1991

High-level expression of a tobacco chitinase gene in Nicotiana sylvestris. Susceptibility of transgenic plants to Cercospora nicotianae infection.

Endochitinases (E.C.3.2.14, chitinase) are believed to be important in the biochemical defense of plants against chitin-containing fungal pathogens. We introduced a gene for class I (basic) tobacco chitinase regulated by Cauliflower Mosaic Virus 35S-RNA expression signals into Nicotiana sylvestris. The gene was expressed to give mature, enzymatically active chitinase targeted to the intracellular compartment of leaves. Most transformants accumulated extremely high levels of chitinase-up to 120-fold that of non-transformed plants in comparable tissues. Unexpectedly, some transformants exhibited chitinase levels lower than in non-transformed plants suggesting that the transgene inhibited expression of the homologous host gene. Progeny tests indicate this effect is not permanent. High levels of chitinase in transformants did not substantially increase resistance to the chitin-containing fungus Cercospora nicotiana, which causes Frog Eye disease. Therefore class I chitinase does not appear to be the limiting factor in the defense reaction to this pathogen.

Base Sequence↗
PubMed1995

An S-RNase promoter from Nicotiana alata functions in transgenic N. alata plants but not Nicotiana tabacum.

Nicotiana tabacum and Nicotiana alata plants were transformed with genomic clones of two S-RNase alleles from N. alata. Neither the S2 clone, with 1.6 kb of 5' sequence, nor the S6 clone, with 2.8 kb of 5' sequence, were expressed at detectable levels in transgenic N. tabacum plants. In N. alata, expression of the S2 clone was not detected, however the S6 clone was expressed (at low levels) in three out of four transgenic plants. An S6-promoter-GUS fusion gene was also expressed in transgenic N. alata but not N. tabacum. Although endogenous S-RNase genes are expressed exclusively in floral pistils, the GUS fusion was expressed in both styles and leaves.

Cloning, Molecular↗
PubMed2007

Analysis of an introgressed Nicotiana tomentosa genomic region affecting leaf number and correlated traits in Nicotiana tabacum.

Germplasm from closely related diploid relatives of tobacco (Nicotiana tabacum L.) could be of value for continued genetic modification of this species and for mapping quantitative trait loci (QTLs). We examined near isogenic tobacco lines and hybrids differing for an introgressed genomic region from N. tomentosa Ruiz and Pavon designated as Many Leaves that exhibits a large influence on leaf number and correlated traits. Within a 'Red Russian' genetic background, the region acted in an additive to partially dominant fashion to delay flowering time, and increase leaf number, plant height, and green leaf yield. Evidence of epistasis was observed as the region affected these traits to varying degrees in diverse near isogenic hybrids. Fifteen amplified fragment length polymorphism (AFLP) markers of N. tomentosa origin were mapped within a single linkage group of 34.5 cM using a population of 207 BC(1)F(1) individuals segregating for Many Leaves. Composite interval mapping produced 2-LOD confidence intervals for likely QTL positions influencing leaf number (3.1 cM region), plant height (2.9 cM region), and days to flowering (3.3 cM region). These intervals were overlapping. Results demonstrate that genomic regions with large genetic effects can be transferred to tobacco from closely related diploid relatives, and that sufficient recombination within these regions may permit mapping of genes controlling quantitative traits. Materials and results described here may be useful in future research to gain insight on the genetic control of the transition from vegetative to reproductive development in Nicotiana.

Amplified Fragment Length Polymorphism Analysis↗
PubMed2005

Characterization of genes encoding metal tolerance proteins isolated from Nicotiana glauca and Nicotiana tabacum.

We have isolated a metal tolerance protein (MTP) gene, NgMTP1, from Nicotiana glauca (a potential phytoremediator plant) and two MTP genes, NtMTP1a and NtMTP1b, from Nicotiana tabacum. These three genes shared approximately 95% homology at the amino acid level. Heterologous expression of any of these three genes complemented Zn and Co tolerance in yeast mutants to a similar extent. In yeast, these proteins were shown to be located to vacuole membrane. These results suggest that the three MTPs operate by sequestering Zn and Co into vacuoles, thereby reducing the toxicity of these metals.

Amino Acid Sequence↗
PubMed2006

Cocksfoot mottle virus P1 suppresses RNA silencing in Nicotiana benthamiana and Nicotiana tabacum.

The Sobemovirus genome consists of positive sense, single-stranded polycistronic RNA. The 5'-terminal ORF, encoding the protein P1, is its most variable region. Sobemoviral P1 has been described as dispensable for replication but indispensable for systemic infection. The P1 of Rice yellow mottle virus-Nigerian isolate (RYMV-N) is the only RNA silencing suppressor reported for sobemoviruses until now. Using an agrobacterium-mediated transient assay, we demonstrate here that P1 of Cocksfoot mottle virus-Norwegian isolate (CfMV-NO) suppresses RNA silencing in Nicotiana benthamiana and Nicotiana tabacum, two non-host plants. CfMV-NO P1 was able to suppress the initiation and maintenance of silencing. The suppression of systemic silencing was weaker with CfMV-NO P1 than in the case of RYMV-N P1. In the case of suppression at the local level, the reduction in the amount of 25-nucleotide small interfering RNAs (siRNAs) was less pronounced for CfMV-NO P1 than it was when RYMV-N P1 was used. At the same time, we show that CfMV-NO P1 did not bind siRNAs.

Down-Regulation↗
PubMed2007

Solar ultraviolet-B radiation and insect herbivory trigger partially overlapping phenolic responses in Nicotiana attenuata and Nicotiana longiflora.

BACKGROUND AND AIMS: Plants exposed to solar ultraviolet-B radiation (UV-B, 280-315 nm) frequently suffer less insect herbivory than do plants that receive attenuated levels of UV-B. This anti-herbivore effect of solar UV-B exposure, which has been documented in several ecosystems, is in part mediated by changes in plant tissue quality. Exposure to UV-B can modify the abundance of a number of secondary metabolites, including phenolic compounds with potential impacts on insect herbivores. The aim of this study is to assess the potential anti-herbivore role of UV-B-induced phenolic compounds by comparing the phenolic profiles induced by UV-B and simulated insect herbivory in two wild species of the genus Nicotiana. METHODS: Plants grown under field and glasshouse conditions were exposed to contrasting levels of UV-B. Half of the plants of the attenuated UV-B treatment were given a simulated herbivory treatment, where leaves were mechanically damaged and immediately treated with oral secretions of Manduca sexta caterpillars. This treatment is known to mimic the impact of real herbivory on the expression of plant defences in Nicotiana. Phenolic profiles induced by UV-B and simulated herbivory were characterized using high-performance liquid chromatography-mass spectrometry (HPLC-MS). KEY RESULTS: UV-B induced the accumulation of several UV-absorbing phenolic compounds that are known to play a significant role in UV-B screening. Interestingly, there was a significant convergence in the phenolic profiles induced by UV-B and simulated herbivory: chlorogenic acid and dicaffeoylspermidine isomers, in particular, displayed a similar pattern of response to these stimuli. In contrast, rutin, the only flavonoid that accumulated in significant quantities in the experiments, was only induced by UV-B. CONCLUSIONS: The results suggest that the anti-herbivory effect induced by UV-B may be mediated at least in part by the accumulation of phenylpropanoid derivatives that are similar to those induced by the plant in response to insect herbivory.

Animals↗
PubMed2003

Virus-induced silencing of sterol biosynthetic genes: identification of a Nicotiana tabacum L. obtusifoliol-14alpha-demethylase (CYP51) by genetic manipulation of the sterol biosynthetic pathway in Nicotiana benthamiana L.

Obtusifoliol-14alpha-demethylase (CYP51) is implicated in plant sterol biosynthesis. An Arabidopsis expressed sequence tag encoding a CYP51 was used as a probe to isolate Nicotiana tabacum L. cDNAs. Two types of cDNA clones were identified. Nt CYP51-1 and Nt CYP51-2 shared 97% identity together and around 75% with other plant CYP51s. The function of the encoded enzyme has been demonstrated in planta by manipulating the sterol biosynthetic pathway at the gene level. The endogenous CYP51 of Nicotiana benthamiana was silenced upon inoculation of the plantlets with POTATO VIRUS X::Nt CYP51-1 transcripts. This resulted in the accumulation of obtusifoliol, the substrate of CYP51, and other 14alpha-methyl sterols, with a concomitant growth reduction phenotype. Virus-induced gene silencing was also applied to another steroidogenic enzyme, the Delta7-sterol-C5(6)-desaturase, and this resulted in the accumulation of Delta7-sterols in infected plants instead of the pathway end-products Delta5-sterols.

Cloning, Molecular↗
PubMed2002

Cell death processes during expression of hybrid lethality in interspecific F1 hybrid between Nicotiana gossei Domin and Nicotiana tabacum.

Hybrid lethality, a type of reproductive isolation, is a genetically controlled event appearing at the seedling stage in interspecific hybrids. We characterized the lethality of F(1) hybrid seedlings from Nicotiana gossei Domin and Nicotiana tabacum cv Bright-Yellow 4 using a number of traits including growth rate, microscopic features of tissues and cells, ion leakage, DNA degradation, reactive oxygen intermediates including superoxide radical (O(2)(-)) and hydrogen peroxide (H(2)O(2)), and expression of stress response marker genes. Lethal symptoms appeared at 4 d after germination in the basal hypocotyl and extended toward both the hypocotyl and root of the plants grown at 26 degrees C. Microscopic analysis revealed a prompt lysis of cell components during cell death. Membrane disruption and DNA degradation were found in the advanced stage of the lethality. The death of mesophyll cells in the cotyledon was initiated by the vascular bundle, suggesting that a putative factor inducing cell death diffused into surrounding cells from the vascular tissue. In contrast, these symptoms were not observed in the plants grown at 37 degrees C. Seedlings grown at 26 degrees C generated larger amounts of reactive oxygen intermediate in the hypocotyl than those grown at 37 degrees C. A number of stress response marker genes were expressed at 26 degrees C but not at 37 degrees C. We proposed that a putative death factor moving systemically through the vascular system induced a prompt and successive lysis of the cytoplasm of cells and that massive cell death eventually led to the loss of the hybrid plant.

Apoptosis↗
PubMed1993

Expression of the [beta]-Glucuronidase Gene in Pollen of Lily (Lilium longiflorum), Tobacco (Nicotiana tabacum), Nicotiana rustica, and Peony (Paeonia lactiflora) by Particle Bombardment.

A [beta]-glucuronidase (GUS) gene that is under the control of the anther-specific LAT52 promoter of tomato (Lycopersicon esculentum) and the nopaline synthetase polyadenylation terminator was successfully expressed in pollen of Lilium longiflorum, Nicotiana tabacum, Nicotiana rustica, and Paeonia lactiflora using a pneumatic particle gun. The GUS gene in plasmid pBI221 was also expressed, to a lesser extent, in pollen of all of these species. The presence of methanol in the substrate solution for histochemical GUS assay and the incubation time in this solution influenced successful detection of GUS expression in bombarded pollen. Cytological analysis of GUS-expressing pollen of lily showed that introduced gold particles were seen in intracellular compartments of pollen, including the vegetative cytoplasm, vegetative nucleus, and generative cytoplasm.

Journal Article↗
PubMed1975

Polypeptide composition of fraction I protein from Nicotiana glauca and from cultivars of Nicotiana tabacum, including a male sterile line.

The polypeptide compositions of fraction I protein isolated from six collections of Nicotiana glauca and from ten cultivars of N. tabacum, as well as a polyploid series and a male sterile line, have been analyzed by isoelectric focusing in polyacrylamide gel slabs containing 8 M urea. Apart from the male sterile line, none of the plants showed any variation from the species pattern of polypeptides. Fraction I protein from the male sterile Burley 21 cultivar of N. tabacum contained the two small subunit polypeptides of N. tabacum and the three large subunit polypeptides of an Australian species, probably N. megalosiphon. This indicates a changed chloroplast genome in the male sterile line in comparison to the normal fertile N. tabacum.

Genetic Variation↗
PubMed2005

Transfer of resistance to potato virus Y (PVY) from Nicotiana africana to Nicotiana tabacum: possible influence of tissue culture on the rate of introgression.

A disomic chromosome addition line of tobacco, Nicotiana tabacum L., was established previously that possesses a single chromosome pair from N. africana [Merxm. and Buttler]. This addition chromosome carries a gene that confers increased resistance to severe strains of potato virus Y (PVY). Methods to increase the probability of gene transfer from alien chromosomes to tobacco (2n=48) are desired. In the research described here, the PVY resistance gene was transferred to a tobacco chromosome from the N. africana addition chromosome in seven independent cases. One introgression event was obtained using conventional backcrossing of the disomic addition line to N. tabacum cv. Petite Havana, while the remaining six events were obtained using a scheme that involved exposure of explants of the addition line to tissue culture. Twenty-six derived 2n=48 individuals heterozygous for PVY resistance were found to exhibit 24 bivalents or 23 bivalents + 2 univalents at metaphase I. Ovular transmission rates for the PVY resistance factor ranged from 25% to 52%, while pollen transmission rates were much lower, ranging from 0 to 39%. Fifty-one random amplified polymorphic DNA (RAPD) markers specific for the intact addition chromosome were identified and used to characterize derived 2n=48/PVY-resistant genotypes. Variability was observed among these plants with respect to the total number of N. africana RAPD markers that were present, which is an indication that crossing over was occurring within each of the seven introgressed chromosome segments. A limited molecular marker-assisted backcrossing experiment allowed for selection of a 2n=48/PVY-resistant individual that possessed only 6 of the 51 original N. africana RAPD markers. In vitro culture is potentially a valuable system for increasing the rate of alien gene transfer in tobacco, and the successful transfer of PVY resistance from N. africana may allow for an increased level and range of resistance to this virus in tobacco.

Chromosomes, Plant↗
PubMed1977

[Effect of gamma radiation on organs and cells of higher plants cultivated in vitro. Dose effect on survival of axillary buds of haploid and diploid Nicotiana sylvestris and Nicotiana tabacum].

The Dose/Action relationship between gamma irradiation and bud survival, plotted on log-linear axes, produces a plateau (no decrease in survival) followed by a straight line decrease. The length of the plateau and the slope of the line appear to depend on the degree of ploïdy and the structure of the genome. There could be redondant parts in the genome of Nicotiana sylvestris.

Cell Survival↗
PubMed1992

Expression of a Self-Incompatibility Glycoprotein (S2-Ribonuclease) from Nicotiana alata in Transgenic Nicotiana tabacum.

In Nicotiana alata, self-incompatibility is controlled by a single locus, designated the S-locus, with multiple alleles. Stylar products of these alleles are ribonucleases that are secreted mainly in the transmitting tract tissues. N. tabacum plants were transformed with constructs containing the S2-cDNA and genomic S2-sequences from N. alata that were linked to the cauliflower mosaic virus 35S promoter. Unlike other genes controlled by this promoter, the genes were expressed most highly in mature floral organs. This pattern of expression was observed at both the protein and RNA levels. The S2-glycoprotein was detected in the stylar transmitting tract tissues of the transgenic plants. The transgene product was secreted, had ribonuclease activity, and was glycosylated with the correct number of glycan chains. However, the maximum level of S2-glycoprotein in styles of the transgenic plants was approximately 100-fold lower than that found in N. alata styles carrying the S2-allele. Perhaps because of this lower protein level, the plants showed no changes in the incompatibility phenotype.

Journal Article↗
PubMed2005

Analysis of RNA silencing in agroinfiltrated leaves of Nicotiana benthamiana and Nicotiana tabacum.

In this study we analyse several aspects of cytoplasmic RNA silencing by agroinfiltration of DNA constructs encoding single- and double-stranded RNAs derived from a GFP transgene and from the endogenous Virp1 gene. Both types of inductors resulted after 2-4 days in much higher concentration of siRNAs in the agroinfiltrated zone than normally seen during systemic silencing. More specifically, infiltration of two transgene hairpin constructs resulted in elevated levels of siRNAs. However, differences between the two constructs were observed: the antisense-sense arrangement was more effective than the sense-antisense order. For both double-stranded forms, we observed a relative increase of the 24-mer size class of siRNAs. When a comparable hairpin construct of the endogenous Virp1 gene was assayed, the portion of the 24-mer siRNA class remained low as observed for all kinds of single-stranded inducers. The lack of increase of Virp1-derived 24-mers was independent of the expression level, as demonstrated by agroinfiltration into a transgenic plant that overexpressed Virp1 and showed the same pattern. Using transducer constructs, we could detect within a week transitive silencing from GFP to GUS sequences in the infiltrated zone and in either direction 5'-3' and 3'-5'. Conversely, for the endogenous Virp1 gene neither transitive silencing nor the induction of systemic silencing could be observed. These results are discussed in view of the current models of RNA silencing.

Agrobacterium tumefaciens↗