Nicotiana Tobacum Research Articles

Phytotoxic effect of selected crude plant extracts on soil-borne fungi of common bean

Common bean (Phaseolus vulgaris L.) is an important food and cash crop particularly in Eastern, Southern and Great lake region. The efficacy of water based crude plant extracts of Neem (Azadirachta indica), Mexican marigold (Tagetes minuta), Tobacco (Nicotiana tobacum) and Peri-winkle (Vinca rosea) in controlling soil-borne fungi (Fusarium oxysporum Schl. f. sp. phaseoli) of common bean (Phaseolus vulgaris L.) was studied in the western province of Kenya. Thirty farms were used in the study carried out in August-September 2005 and March-June 2006. Broth microdilution method was used to determine minimum inhibitory concentration (MIC). All crude plant extracts controlled Fusarium yellows with varying efficacy. Neem extract was the most effective, while peri-winkle was the least. Wilt incidence was not affected by dosage regime. Neem is inhibitory to Fusarium growth at lower dosage than tobacco, Mexican marigold and peri-winkle with MIC ranging between 2.5 and 10.0 mg ml-1

Grain Discoloration of Rice Caused by Pantoea ananatis (synonym Erwinia uredovora) in China.

In the autumn of 2008, a new bacterial disease of rice was noted in paddy fields near Hangzhou, Zhejiang Province, China. The disease caused severe discoloration of rice grains on cv. Zhong-zhe-you 1 (Oryza sativa L.). It often occurred at early flowering of hybrid rice. Initially, light, rusty, water-soaked lesions appeared on the lemma or palea and then turned brown. More immature and lighter grains were observed on panicles at harvest. No bacterial ooze was observed. Ten bacterial isolates were recovered from eight samples of discolored rice grains (1). Six isolates were selected for identification. They were similar to those of the reference strain of Pantoea ananatis (Serrano, synonym Erwinia uredovora) LMG 2665T (ATCC 33244) from Belgium in phenotypic tests based on the Biolog Microbial Identification System, version 4.2 (Biolog Inc., Hayward, CA), pathogenicity tests, gas chromatographic analysis of fatty acid methyl esters (FAME) using the Microbial Identification System (MIDI Inc, Newark, DE) with the aerobic bacterial library (TAB 5.0), and electron microscopy (TEM,KYKY-1000B, Japan). All isolates were facultatively anaerobic, gram-negative rods that measured 1.6 to 2.5 × 0.5 to 0.7 μm and had three to six peritrichous flagella. Colonies on nutrient agar were yellow and raised with smooth margins. A hypersensitive reaction was observed on tobacco (Nicotiana tobacum cv. Benshi) 24 h after inoculation. All isolates were identified as P. ananatis with Biolog similarity indices of 0.716 to 0.852 and FAME similarity indices of 0.783 to 0.903. Further identification as P. ananatis was done by 16S rDNA sequence analysis. Amplicons were produced from three strains using the universal primers (3) fD2: 5'-AGA GTT TGA TCA TGG CTC AG-3' forward primer and rP1: 5'-ACG GTT ACC TTG TTA CGA CTT-3' reverse primer and then sequenced (GenBank Accession Nos. GU324769, GU324770, and GU338399). A BlastN search of GenBank revealed that they had 97 to 98% nt identity with P. ananatis strain 3Pe76 (GenBank Accession No. EF178449). Koch's postulates were completed by spray inoculating panicles of rice cv. Zhong-zhe-you 1 at booting stage, grown in pots, with cell suspensions containing 108 CFU/ml of the six strains at 25 to 29°C. Three plants were inoculated with each strain, controls were sprayed with water, and the experiment was repeated once. Three weeks after inoculation, all strains produced symptoms on panicles similar to those observed in the field. Yellow pigmented bacteria were reisolated from symptomatic panicles and their identity was confirmed by FAMEs. These results indicate that the pathogen is P. ananatis (2), which also causes leaf blight and bulb decay of onion. To our knowledge, this is the first report of rice grain discoloration caused by P. ananatis in China. The disease cycle on rice and the control strategies in the regions are being further studied.

Fungitoxic properties of four crude plant extacts on fusarium oxysporum schl. F. sp phaseoli.

Fusarium yellows is a disease of common beans (Phaseolus vulgaris, L.) caused by Fusarium oxysporum Schl. F. sp. phaseoli, it has been found to be important in Busia district of Western province, Kenya. The study on fungitoxic properties of four locally available crude plant extracts was aimed at evaluating their efficacy in controlling Fusarium yellows infestation under the field conditions. Crude plant extracts from Azadirachta indica, Tagetes minuta, Nicotiana tobacum and Vinca rosea were tested against Fusarium oxysporum Schl. F. sp. phaseoli. Participatory On-Farm Trials (POFT) in six (6) divisions were carried out in August-September 2005 and March- June 2006; a total of thirty (30) farms were randomly surveyed. Minimum Inhibitory Concentration (MIC) of crude plant extracts against Fusarium was determined by broth microdilution method. Analysis of variance (ANOVA) was performed on the data, using Genstat 8th edition statistical program (Release 8.11, Lawes Agricultural Trust, Rothamsted Experimental Station, Harpenden, UK). Means were separated using LSD. Crude plant extracts exhibited fungitoxic activity against Fusarium oxysporum Schl. F. sp. phaseoli, with varying degree of efficacy. Nicotiana tobacum and Vinca rosea were not effective, Azadirachta indica and Tagetes minuta exhibited significant control over Fusarium. Azadirachta indica performed better amongst all the plant extracts. Common bean treatment with Benomyl 1 significantly reduced (P≤0.05) wilt incidence and increased growth in comparison to negative (–ve) control. Azadirachta indica formulation gave a significant reduction in wilt incidence compared to the other three crude plant extracts formulations. It reduced the wilt incidence by 17.24% in comparison to Tagetes minuta, Nicotiana tobacum and Vinca rosea whose wilt incidence reduction ranged from 5.84-9.8%. Azadirachta indica inhibited Fusarium growth at lower dosage than Tagetes minuta, Nicotiana tobacum and Vinca rosea. Extracts from Azadirachta indica and Tagetes minuta are effective, cheap and ecofriendly promising methods for protecting common bean against Fusarium oxysporum Schl. F. sp. phaseoli.

ISOLATE AND PURIFY THE DIAGNOSIS AND COMPLEX STUDY OF NICOTINE AND ITS IMPACT AND WATER EXTRACTS OF THE TOBACCO PLANT TO SOME SPECIES OF BACTERIAL PATHOGENICITY

Extraction and purification were Nicotine alkaloid from leaves of tobacco plant Nicotiana tobacum medical use of some of the ways in diagnostic spectral IR, UV , TLC, Melting point and examined some of the chemical study of the impact of their effectiveness have been against some bacteria, the specter of disease and measure the inhibition of the effectiveness of this compound in addition to the abstract knowledge of the influence of the tobacco plant to the water, the results showed the superiority of Nicotine alkaloid extracted water to plant tobacco, and all concentrated on all types of bacteria . Gave the highest composite zone to discourage the amount of 25 mm to focus 50 mg / ml of Pseudomonas bacteria, while the less zone to discourage the concentration 1 mg / ml and 2 mm of the bacteria Staphylococcus and E. coli

Analysis of the tip-to-base gradient of CaM in pollen tube pulsant growth using in vivo CaM–GFP system

Ca(2+)-CaM signaling is involved in pollen tube development. However, the distribution and function of CaM and the downstream components of Ca(2+)-CaM signal in pollen tube development still need more exploration. Here we obtained the CaM-GFP fusion protein transgenic line of Nicotiana tobacum SRI, which allowed us to monitor CaM distribution pattern in vivo and provided a useful tool to observe CaM response to various exogenous stimulations and afforded solid evidences of the essential functions of CaM in pollen tube growth. CaM-GFP fusion gene was constructed under the control of Lat52-7 pollen-specific promoter and transformed into Nicotiana tobacum SRI. High level of CaM-GFP fluorescence was detected at the germinal pores and the tip-to-base gradient of fluorescence was observed in developing pollen tubes. The distribution of CaM at apical dome had close relationship with the pulsant growth mode of pollen tubes: when CaM aggregated at the apical dome, pollen tubes stepped into growth state; When CaM showed non-polarized distribution, pollen tubes stopped growing. In addition, after affording exogenous Ca(2+), calmidazolium (antagonism of CaM) or Brefeldin A (an inhibitor of membrane trafficking), CaM turned to a uniform distribution at the apical dome and pollen tube growth was held back. Taken together, our results showed that CaM played a vital role in pollen tube elongation and growth rate, and the oscillation of tip-to-base gradient of CaM was required for the normal pulsant growth of pollen tube.

Grafting tomato plant on tobacco plant and its effect on tomato plant yield and nicotine content

AbstractBACKGROUND:Two different tomato scions, cv. Elazig and cv. Sweet (cherry) (Solanum lycopersicumL.) were self‐grafted and grafted onto tobacco root stock (Nicotiana tobacumL.). Then, grafted tomato plants were evaluated in terms of flower and fruit yield. Tobacco‐grafted tomato plant fruits were also evaluated for nicotine content.RESULTS:Tobacco grafting had a positive effect on the tomato plant cultivation performance; the onset of flowering was almost 15 days earlier and the tomato flower and fruit yields increased in both tomato cultivars. Tobacco grafting resulted in 5.0% and 30.1% increase in total fruit weight for cv. Sweet and cv. Elazig, respectively. Because the level of nicotine was within acceptable ranges, tobacco‐grafted tomato fruits were considered to be safe for consumption. Self‐grafted tomato cultivars also had flowering time onsets almost 11 days earlier. However, self‐grafting caused 6.0% and 7.6% less total fruit yield per cv. Sweet and cv. Elazig, respectively.CONCLUSION:In conclusion, our results show that tomato–tobacco grafting is a novel and promising technique for improvement of not only tomato plant performance and yield, but also that it can be employed to various tomato varieties. Copyright © 2009 Society of Chemical Industry

Nitrogen Use Efficiency of Base Fertilizer and Top Dressing in Flue-Cured Tobacco in Rainy Regions

A field experiment was conducted using tobacco (Nicotiana tobacum L.) cultivar K326 grown in random block design with three replications in three treatments by 15N isotopes tracer in Liuyang institute of Hunan in 2007. The traits associated with nitrogen use efficiency were investigated during different growth stages of flue-cured tobacco. The results showed that tobacco plant mainly absorbed nitrogen from fertilizers in root elongation and vigorous growth stages and from soil in maturing stage. Nitrogen uptake reduced from base fertilizer, but increased from top dressing with the increase of top dressing under the same fertilizer application amount. At the same time, nitrogen use efficiency rose for base fertilizer, but declined for top dressing. The utilization of top dressing was significantly higher than that of base fertilizer in different treatments at different growth stages. After harvesting of tobacco leaves, nitrogen residue in base fertilizer was less than that in top dressing with the increase of top dressing ratio, but the total nitrogen residue and loss decreased. So enhancing properly the ratio of top dressing can improve ni-trogen use efficiency of flue-cured tobacco in rainy regions.

Biology and life table studies of the oriental tobacco budworm, Helicoverpa assulta (Lepidoptera: Noctuidae), influenced by different larval diets

AbstractDevelopment, survivorship, pupal weight, oviposition, and life table parameters of the oriental tobacco budworm, Helicoverpa assulta Guenée, were evaluated in the laboratory on an artificial diet, pepper (Capsicum frutescens L.), and tobacco (Nicotiana tobacum L.). We found that the average developmental time of immature stages was longest on tobacco (36.2 d), intermediate on pepper (34.4 d), and shortest on artificial diet (33.5 d). Immature survival from egg to pupa varied from 31% on tobacco, 43% on pepper, and 74% on artificial diet. Pupal weight ranged from 197.4 mg/pupa on tobacco, 233.1 mg/pupa on pepper and 253.4 mg/pupa on artificial diet. The average numbers of eggs laid by adults reared as larvae on the artificial diet, pepper, or tobacco were 614, 421 and 334 eggs/female, respectively. Numbers of remaining eggs in ovaries of the adult females reared as larvae on the artificial diet, pepper, or tobacco were 16, 26, and 42 eggs/female, respectively. The longevity of adult females developed from larvae reared on the three diets was not significantly different, whereas the longevity of male adults from the larvae reared on artificial diet was longer (16.8 d) than that for males reared on tobacco (13.8 d) and pepper (13.3 d). The intrinsic, finite, gross, and net rates of increase were highest for females reared as larvae on artificial diet, lowest for females emerging from larvae reared on tobacco, and intermediate for females emerging from larvae reared on pepper. Generation times and doubling time of H. assulta were shortest for larvae fed artificial diet, intermediate from larvae reared on pepper, and longest from larvae reared on tobacco. We concluded that the artificial diet was the most suitable larval diet of H. assulta followed by pepper, and tobacco.

Expression of Protein Elicitor-Encoding Gene pemG1 in Tobacco ( Nicotiana tobacum cv. Samsun NN) Plants and Enhancement of Resistance to TMV

The pemG1 gene from Magnaporthe grisea was transferred into tobacco ( Nicotiana tobacum cv. Samsun NN) to test its functions. Plant expression vector pCAMBIA2300- Ubi-pemG1-Oc harboring elicitor-encoding gene pemG1 was constructed. The maize ubiquitin promoter/octopine synthase terminator system and kanamycin-resistant gene npt II were used for constitutive expression systems. The vector was then introduced into Agrobacterium tumefaciens strain AGL-1 with freeze-thaw method. Tobacco primary transformants were produced by leaf disc transformation. The kanamycin-resistant regenerated plants were confirmed to be electropositive by PCR. Integration and expression of the pemG1 gene were further confirmed by Southern blotting and Western blotting analyses. Transgenic tobacco plants of T 2 generation were successively inoculated with Tobacco Mosaic Virus (TMV) at virus concentrations of 0.2 and 0.5 mg per leaf. In comparison with TMV-infected wild-type plants, PemG1-expressed plants displayed reduced hypersensitive-response lesions in both inoculation treatments. Furthermore, accumulation level of pemG1 steady-state transcripts was examined at 24 h after inoculation. The results indicated that the reduction of lesions corresponded to the accumulation of pemG1 steady-state transcripts as monitored by Northern blotting analysis. All these indicated that the expression of pemG1 in tobacco plants improved the resistance to TMV.

Monogalactosyldiacylglycerol deficiency affects jasmonic acid biosynthesis and defense responses to insect herbivores in Nicotiana tobacum

Jasmonic acid (JA) plays critical roles in plant development and defense. Linolenic acid (18:3) and hexadecatrienoic acid (16:3) released from chloroplast lipids are now known to be precursors for JA biosynthesis, but the relationship between chloroplast lipids, especially galactolipids and JA biosynthesis still remains unclear. Here, this question was addressed by characterizing the transgenic tobacco plants, which had reduction in monogalactosyldiacylglycerol (MGDG) and trienoic fatty acids owing to the fact that MGDG synthase activity was down-regulated by using RNA interference technology. In response to wounding, the transgenic plants produced lower levels of JA than wild-type plants. Moreover, the expression of genes encoding lipoxygenase ( LOX1), allene oxide cyclase ( AOC), hydroperoxide lyase ( HPL) and proteinase inhibitor ( PI-I and PI-II) was strongly activated by mechanical wounding in wild-type plants but was diminished in transgenic plants. In addition, the transgenic plants were shown to be more susceptible to attack by Helicoverpa armigera larvae. Treatment of transgenic plants with methyl jasmonate restored resistance to H. armigera and expression of HPL, PI-I and PI-II. Our results suggest that MGDG plays important roles as source of 18:3 and 16:3 in JA biosynthesis and JA-mediated defense responses to insect herbivores in tobacco.

Continuous counter current extraction, isolation and determination of solanesol in Nicotiana tobacum L. by non-aqueous reversed phase high performance liquid chromatography

A method of continuous counter current extraction in a large-scale production of solanesol from tobacco leaves was developed. The crude extract containing 15–20% solanesol was subjected to a series of steps, viz., saponification, solvent recrystallization and column chromatography. The pure material was characterized by FT-IR, ESI-MS, 1H NMR and 13C NMR spectrometry. The analysis was carried out by a simple and rapid non-aqueous reversed-phase high performance liquid chromatographic method on a Hypersil BDS C 18 column (250 mm × 4.6 mm, particle size 5 μm) with isopropyl alcohol–methanol (60:40, v/v) as mobile phase and detection at 215 nm. The product purity was between 95 and 98% (w/w) as determined by HPLC.

Cloning of Salt Stress Responsive cDNA from Wheat and Resistant Analysis of Differential Fragment SR07 in Transgenic Tobacco

Analysis of the gene expression differentiation in leaves of wheat ( Triticum aestivum L.) cultivar Baofeng 7228, under salt stress, was carried out by Differential-Display Reverse Transcription-polymerase Chain Reaction (DDRT-PCR.) Twenty-seven differential cDNA fragments were obtained. The expression of the SR07 fragment was induced noticeably by salt treatment, and the nucleotide sequence homology of 87% between the SR07 fragment and PIPs (water channel proteins) was observed. Further research showed that a 561 bp open read frame was present in the SR07 fragment. Plant expression vector of pCAMBIA-SR07 was constructed and three transformants of tobacco ( Nicotiana tobacum) mediated by Agrobacterium tumefaciens plasmid were obtained. Resistance to salt, PEG, and mannitol stresses of the three transformants were examined. No significant difference ( P > 0.05) was observed between the control and the transformants in resistance to salt stress, but there was significant difference ( P < 0.05) between the control and the transformants in resistance to PEG and mannitol stresses. Therefore, the expression of the SR07 fragment may play an important role in the water regulation of the plant.

Analysis on genetic contribution of agronomic traits to total sugar in flue-cured tobacco ( Nicotiana tabacum L.)

To uncover the genetic contributions of agronomic traits to content of total sugar (TS) and find indicator traits for indirect selection on TS in the flue-cured tobacco ( Nicotiana tobacum L.), multivariable conditional analysis was conducted based on a genetic model containing additive–dominance and their interactions with environments. Fourteen cultivars (or breeding lines) and derived 41 F 1 crosses were grown at four locations in Yunnan province, China. Significant phenotypic contribution to TS was detected for six agronomic traits, plant height (PH), girth of stem (GS), internode length (INL), number of leaves (NL), length of middle leaves (LML) and width of middle leaves (WML). There was large contribution of additive effects due to each of the five agronomic traits (PH, GS, INL, LML and WML). The contribution ratio of dominance effect was high due to PH. By serving as high contributor of additive effects to TS and having high ratios of additive variance to phenotypic variance, INL and PH could be used as indicative agronomic traits for selecting breeding lines with suitable TS. Among the six agronomic traits, PH had the highest contribution to dominance effects of TS for most F 1 crosses, and could be used for selecting the crosses with suitable TS.

In Vitro Comparative Kinetic Analysis of the Chloroplast Toc GTPases

A unique aspect of protein transport into plastids is the coordinate involvement of two GTPases in the translocon of the outer chloroplast membrane (Toc). There are two subfamilies in Arabidopsis, the small GTPases (Toc33 and Toc34) and the large acidic GTPases (Toc90, Toc120, Toc132, and Toc159). In chloroplasts, Toc34 and Toc159 are implicated in precursor binding, yet mechanistic details are poorly understood. How the GTPase cycle is modulated by precursor binding is complex and in need of careful dissection. To this end, we have developed novel in vitro assays to quantitate nucleotide binding and hydrolysis of the Toc GTPases. Here we present the first systematic kinetic characterization of four Toc GTPases (cytosolic domains of atToc33, atToc34, psToc34, and the GTPase domain of atToc159) to permit their direct comparison. We report the KM, Vmax, and Ea values for GTP hydrolysis and the Kd value for nucleotide binding for each protein. We demonstrate that GTP hydrolysis by psToc34 is stimulated by chloroplast transit peptides; however, this activity is not stimulated by homodimerization and is abolished by the R133A mutation. Furthermore, we show peptide stimulation of hydrolytic rates are not because of accelerated nucleotide exchange, indicating that transit peptides function as GTPase-activating proteins and not guanine nucleotide exchange factors in modulating the activity of psToc34. Finally, by using the psToc34 structure, we have developed molecular models for atToc33, atToc34, and atToc159G. By combining these models with the measured enzymatic properties of the Toc GTPases, we provide new insights of how the chloroplast protein import cycle may be regulated.

Control of seed and root development by WIPK-activated transcription factor, NtWIF in tobacco plants

Mitogen-activated protein kinase (MAPK) is one of the critical mediators of signaling systems by constituting phosphorylation pathways together with upstream kinases. Despite the importance, few direct targets have so far been identified, making it difficult to understand the whole features of so called MAPK cascade. Notably, a novel transcription factor, which was recently identified from tobacco, is activated upon phosphorylation by wound-induced protein kinase (WIPK), a typical MAPK in tobacco plants, and designated as NtWIF (Nicotiana tobacum WIPK interacting factor). To identify genes that are controlled by NtWIF, a microarray screening was performed using NtWIF overexpressing transgenic tobacco, and a set of affected clones were isolated. Approximately 28% of them were related to defense, confirming the critical role of WIPK in stress response. In addition, 15% of the clones were related to differentiation, suggesting pleiotrophic effects of NtWIF. When NtWIF was overexpressed or silenced by RNAi in tobacco plants, abnormal seed development and root growth were observed. Seeds of RNAi lines exhibited immature endosperm, resulting in germination failure. Root growth was retarded in both transgenic seedlings due to abnormal cell numbers in meristem. These results implicated the participation of phytohormones, of which function might be controlled by NtWIF. Since NtWIF possesses a B3-DNA binding motif, which specifically recognizes auxin-responsive elements found in many auxin-responsive genes, the present finding suggested that WIPK/NtWIF functions not only in stress response but also in developmental process through hormonal control.

Stable transformation ofphaC2 gene in tobacco chloroplast genome

AbstractMedium-chain-length polyhydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters. The key enzyme for mcl-PHA biosynthesis is type II PHA synthase. The genephaC2 encoding type II PHA synthase was placed under the control ofpsbA-pro andpsbA-ter of rice (Oryza sativa) to construct aphaC2 cassette, which was ligated with the screening marker geneaadAcassette (prrn–aadA–TpsbA-ter). These recombined fragments were cloned between the plastidrbcLandaccDgenes for targeting to the large single copy region of the chloroplast genome. A chloroplast transformation vector, pTC2, was constructed and introduced into the tobacco (Nicotiana tobacum) chloroplast genome by particle bombardment. PCR and Southern blot analysis confirmed stable integration ofphaC2 into the chloroplast genomes of T0and T1transgenic plants, and T1transgenic plants exhibited homoplasmy. The expression ofphaC2 at transcription level was detected by reverse transcriptase–polymerase chain reaction (RT-PCR). Recombinant transgenes in the tobacco chloroplast genome were maternally inherited and were not transmitted via pollen when out-crossed with untransformed female plants. To our knowledge, this is the first report on the stable transformation ofphaC2 encoding type II PHA synthase in tobacco via chloroplast genetic engineering.

Trace element concentrations of soils, plants and waters caused by a copper smelting plant and other industries, Northeast Turkey

The study area is situated in a large agricultural field which produces tobacco, maize, and other yearly cultivated vegetables in Tekkekoy, Samsun (NE-Turkey). In addition, a significant part of this area to the north along the Black Sea coast is occupied by several industrial plants such as a copper smelting plant (KBI), a fertilizer plant (Tugsas) and industrial park facilities (IPF). In order to reveal their environmental impacts, heavy metal analyses were conducted on soil, plant leaves and water samples collected within an area of approximately 30 km2 around these plants. Soil samples within an area of 10 km2 around these facilities are found to be highly polluted with Cu, Zn, Pb, Fe and Mn. Pollution occurs at surface and sharply dies out at 20 cm downwards in soil profile. Since the region is polluted mostly with base metals, the copper smelting factory appears to be the main source of pollution as it processes the massive sulfidic ores of the Black Sea area. Plants show Cu, Pb, Zn and Fe pollution around KBI and Tugsas and Cu and Pb around IPF. Pollutants observed in tobacco (Nicotiana tobacum) are Cu, Pb, Zn, Fe and S; in maize (Zea mays) Cu, Zn and Fe; and in cabbage (Brassica oleracea) Cu, Pb, Fe and S. The analyses of water samples collected from the study area reveal that Pb and, to a lesser degree, Cu and Fe pollution stem from KBI; Cu, Fe and Mn pollution from Tugsas; Pb and minor amounts of Fe and Mn pollution from IPF. Factor analyses from analyzed metals and anionic complexes in water show three distinct groups: (a) an association of heavy metals with Na, K and Mg referring to pollution and acid leaching of soil, (b) an association of NH4, Fe, SO4, Cl and Br indicating agricultural pollution and sea-water invasion in land near the shore line, and (c) HCO3 behaving in a different manner in heavy metal precipitation.

Roles of Salicylic Acid-responsive &amp;lt;italic&amp;gt;Cis&amp;lt;/italic&amp;gt;-acting Elements and W-boxes in Salicylic Acid Induction of &amp;lt;italic&amp;gt;VCH3&amp;lt;/italic&amp;gt; Promoter in Transgenic Tobaccos

A salicylic acid (SA)-inducible VCH3 promoter was recently identified from grapevine (Vitis amurensis) that contains two inverse SA-responsive cis-acting elements and four W-boxes. To further demonstrate the roles of these elements, four fragments with lengths from -1187, -892, -589, -276 to +7 bp were fused with the b-glucuronidase (GUS) reporter gene and transferred to Nicotiana tobacum, together with another four VCH3 promoter fragments with mutation in the two inverse SA-responsive elements. The functions of each promoter fragment were examined by analysis of GUS activity in the transgenic tobacco root treated with SA. Enhanced GUS activity was shown in the roots of transgenic tobaccos with the VCH3 (-1187)-GUS construct containing two SA-responsive cis-acting elements and four W-boxes. However, GUS activity directed by the VCH3 (-892)-GUS construct, containing one SA cis-acting element and four W-boxes, was reduced by up to 35% compared with that in tobaccos transformed with the VCH3 (-1187)-GUS construct, indicating that the SA cis-acting element plays an important role in SA induction of the VCH3 promoter. Neither the m2VCH3 (-1187)-GUS nor the mVCH3 (-892)-GUS construct, with mutation on the SA-responsive elements, abolished the expression of GUS activity, demonstrating that the W-boxes in the VCH3 promoter are also involved in SA induction. Histochemical analysis of GUS activity directed by each of the eight VCH3 promoter fragments showed that GUS was expressed specifically in vascular tissue. It was concluded that both the SA-responsive cis-acting elements and the W-boxes are important for the SA induction of the VCH3 promoter. This promoter might have a potential use in plant genetic engineering.

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

Transgenic tobacco (Nicotiana tobacum) plants carrying the gene coding for potato virus A (PVA) non-structural P3 protein were prepared by inoculation with Agrobacterium tumefaciens. Seeds from self-pollinated flowers (T1 generation) were collected. To estimate the effectiveness of vertical transfer of the introduced gene and usefulness of respective plant lines for further experiments, the T1 generation was characterized by testing its ability to grow in the presence of kanamycin (Km) and by PCR of both neomycin phosphotransferase (nptII) and PVA P3 genes. Eight and ten of 29 lines showed Mendelian segregation of Km-resistant phenotype 3:1 and ≥15:1, respectively, the T1 of eleven lines showed low Km resistance. Selected PCR-positive lines were tested for the presence of P3 mRNA. In most cases the transgene transcription was dependent on the presence or absence of Km in the plant growth medium. Prepared transgenic plants were furthermore tested for sensitivity to PVA and potato virus Y (PVY) infection. All of them showed identical symptom development as the non-transgenic control plants.

A general procedure for the enantioselective synthesis of the minor tobacco alkaloids nornicotine, anabasine, and anatabine.

The minor tobacco alkaloids nornicotine, anabasine, and anatabine from Nicotiana tobacum are known to possess nicotinic receptor agonist activity, although they are relatively less potent than S-(-)-nicotine, the principal tobacco alkaloid. Previous pharmacological investigations and structure-activity studies have been limited owing to the lack of availability of the optically pure forms of these minor alkaloids. We now report a 2-step synthetic procedure for the enantioselective synthesis of the optical isomers of nornicotine and anabasine, and a modified procedure for the synthesis of anatabine enantiomers. These procedures involve initial formation of the chiral ketimine resulting from the condensation of either 1R, 2R, 5R-(+)- or 1S, 2S, 5S-(-)-2-hydroxy-3-pinanone with 3-(aminomethyl)pyridine followed by enantioselective C-alkylation with an appropriate halogenoalkane or halogenoalkene species, N-deprotection, and base-catalyzed intramolecular ring closure, to form the appropriate, chirally pure minor tobacco alkaloid. Using this approach, the R-(+)- and S-(-)-enantiomers of the above minor tobacco alkaloids were obtained in good overall chemical yield and excellent enantomeric excess.