Studies on the Essential Oils of Tobacco Leaves

An investigation was conducted on the neutral fraction in the essential oil of Virginia tobacco leaves. α-Pyrryl methyl ketone, ethyl alcohol, ethyl acetate and furfuryl alcohol were isolated and identified. An alcohol, C₄, and an ester of benzoic acid were also separated. Hydrocarbons were separated from the total neutral fraction by liquid chromatography, prior to any further procedure; and a straight chain paraffin near C₃₀ and an unsaturated hydrocarbon resembling myrcene were also isolated and their contents were determined. A technique of chromatostrip was found to be very useful for detecting liquid chromatographic separation.     

Fungi Isolated from Damaged Flue-cured Tobacco

Species of Aspergillus were the most prevalent fungi isolated from 51 samples of damaged flue-cured tobacco of the 1966 U.S. crop, comprising 57% of the total isolates. Other prevalent fungi were Penicillium (16%), Alternaria (8%), Cladosporium (4%), and Chaetomium (4%). Members of the Aspergillus glaucus group were isolated most frequently from samples with moisture contents ranging from 18 to 28%, whereas Alternaria, Cladosporium, and Penicillium were isolated consistently from samples containing 24 to 32% moisture. Aspergillus niger was prevalent in tobacco ranging in moisture content from 18 to 30%.     

Phytochemical Studies on the Tobacco Alkaloids. XII. Identification of gamma-Methylaminobutyraldehyde and its Precursor Role in Nicotine Biosynthesis

γ-Methylaminobutyraldehyde (N-methylpyrroline) labeled with ¹⁴C was isolated from tobacco roots which had metabolized ornithine-2-¹⁴C. It was labeled most strongly 4 hours after adding ornithine-2-¹⁴C to the root, also labeled by putrescine-1,4-¹⁴C and methionine-¹⁴CH₃, and observed in the root but not in the aerial portions of tobacco plants. γ-Methyl-aminobutyraldehyde when added back to the root was an efficient precursor of nicotine. Identity of γ-methylaminobutyraldehyde from tobacco roots was confirmed by comparison with the authentic compound.     

Effect of Tobacco Mosaic Virus Infection on Amino Acid Incorporation into Isolated Mitochondria from Tobacco Leaves

Mitochondria isolated from tobacco leaves incorporated ¹⁴C-leucine into the protein and the rate was enhanced by tobacco mosaic virus (TMV) infection as compared with noninfected level. In vitro amino acid incorporation by mitochondria required adenosine triphosphate (ATP), Mg²⁺, and KC1 and the energy sources from oxidative phosphorylation as well as from ATP-generating system. This incorporation was inhibited by ribonuclease (RNase), deoxyribonuclease (DNase), actinomycin D, mitomycin C, puromycin, and chloramphenicol added in the reaction medium. The pretreatment of the mitochondria with DNase and actinomycin D reduced the rate of incorporation. The mitochondria incorporated ³H-guanosine triphosphate (GTP) and this activity was blocked by actinomycin D. The presence in this system of 15,000 g supernatant cell sap fraction or bacterial contamination was carefully checked obtaining a negative result. The reaction product into which ^l4C-amino acids incorporated was solubilized by trypsin. The nature of the amino acid incorporating activity of isolated mitochondria obtained from TMV-infected tobacco leaves is discussed.     

Fungi Isolated from Flue-cured Tobacco at Time of Sale and After Storage

The fungi isolated from 100 samples of flue-cured tobacco from 12 markets in 2 tobacco belts comprised 11 genera, including 10 species of Aspergillus. The mean percentage per sample isolated from 62 samples of tobacco from Middle Belt markets was Alternaria, 40.6%; Aspergillus niger, 47.8%; Aspergillus repens, 38.0%; and Penicillium, 25.8%. The mean percentage per sample isolated from 38 samples of tobacco from Old Belt markets was Alternaria, 74.0%; Penicillium, 52.5%; Aspergillus repens, 38.0%; and Aspergillus ruber, 36.2%. Damaged (74 samples) and nondamaged (26 samples) stored tobacco yielded species of six genera of fungi, including eight species of Aspergillus. Species of Aspergillus and Penicillium were commonly isolated from both damaged and nondamaged tobacco, whereas species of Alternaria, Cladosporium, Fusarium, and Rhizopus were isoalted more frequently from nondamaged tobacco. The fungi that occurred in the highest population in damaged tobacco were Aspergillus repens, A. niger, A. ruber, and Penicillium species.     

Phytochemical Studies on Tobacco Alkaloids

From the findings of the feeding experiments of d- and l-nicotine-¹⁴CH₃ and d-nicotine to the excised tobacco leaves, it was demonstrated that demethylation of nicotine was stereospecific for the d-form in tobacco leaves. Such preferential demethylation to the enantiomer was also observed with N-methylanabasine and 1-(3′-pyridyl)-1-methylaminoethane which are analogous compounds to nicotine.     

Phytochemical Studies on the Tobacco Alkaloids

Duplicate feeding experiments of dl-ornithine-2-¹⁴C to the excised tobacco root culture were made, and the radioactive nornicotine was isolated. Approximately two thirds of the radioactivity was located in the 2-position of the pyrrolidine of the nornicotine in these experiments. This fact indicates that there are two modes in nornicotine biosynthesis: exclusive incorporation to the C-2 and equal incorporation to C-2 and C-5 from C-2 of ornithine.

On the basis of this finding, biosynthetic route was discussed.

dl-Ornithine-2-¹⁴C, dl-methionine-¹⁴CH₃ and partially racemized l-nornicotine-2,5-¹⁴C were administered to aseptically grown excised roots (N. rustica var. Brasilia). Incorporation of their radioactivity to nicotine was compared. The extent of their radioactive incorporation to nicotine was high in the order of ornithine, methionine and nornicotine; incorporation of radioactivity of nornicotine to nicotine was extraordinarily low. ¹⁵N-Labeled nornicotine was also fed to the same materials and ¹⁵N distribution was examined. Most of ¹⁵N still remained in the nornicotine reisolated. Marked amounts of ¹⁵N were located in the ethanol-insoluble fraction, the amino acid fraction and the substances having chromatographic R_F value close to that of nicotine. Only small amount of ¹⁵N was incorporated to the isolated nicotine.

Nornicotine is generally accepted to be a direct precursor of nicotine in tobacco plants. From these findings, however, it can be said that the biosynthesis of nicotine can occur through other routes without going through nornicotine.     

Expression and Biological Properties of a Novel Methionine Sulfoxide Reductase A in Tobacco (Nicotiana tabacum)

Methionine (Met) residues in proteins/peptides are extremely susceptible to oxidation mediated by reactive oxygen species, resulting in the formation of methionine sulfoxide, which could be inversely reduced back to Met by methionine sulfoxide reductase (MSR). In the present study, an A-type MSR gene, termed NtMSRA4, was isolated from tobacco (Nicotiana tabacum). Sequence analysis of NtMSRA4 amino acid sequence indicated that the gene, encoded a polypeptide with a molecular weight of 21 kDa, possessed the highly conserved motif, ‘GCFWG’ in the N-terminus and ‘KGCNDPIRCY’ motif in the C-terminus respectively. Substrate specific analysis revealed that recombinant NtMSRA4 protein could reduce specifically S-isomer of Dabsyl-MetSO to Dabsyl-Met in vitro using dithiothreitol as an electron donor. Enzymatic properties analysis showed that the temperature of 42 °C and pH 9.0 were optimum for NtMSRA4 activity. The K _m and K _cat values of NtMSRA4 were determined to be 40.04 μM and 0.048 S^?1 in the thioredoxin dependent reduction system. Overexpression of NtMSRA4 in E. coli cells enhanced resistance to H₂O₂ toxicity. Subcellular localization result showed that NtMSRA4 was located in the chloroplast. The expression level of NtMSRA4 was affected differently after exposure to various abiotic stresses.     

Transgenic Tobacco Plants Constitutively Overexpressing a Rice Thaumatin-like Protein (PR-5) Show Enhanced Resistance to Alternaria alternata

Overexpression of antifungal pathogenesis-related (PR) proteins in crop plants has the potential for enhancing resistance against fungal pathogens. Thaumatin-like proteins (TLPs) are one group (PR-5, permatins) of antifungal PR-proteins isolated from various plants. In the present study, a plasmid containing a cDNA of rice tlp (D34) under the control of the CaMV-35S promoter was introduced into tobacco plants through Agrobacterium-mediated transformation system. A considerable overproduction of TLP was observed in transformed tobacco plants by Western blot analysis. There was a large accumulation of tlp mRNA in transgenic plants as revealed by Northern blot analysis. Southern blot analysis of the DNA from transgenic tobacco plants confirmed the presence of the rice tlp gene in the genomic DNA of transgenic tobacco plants. Immunoblot analysis of intracellular and extracellular proteins of transgenic tobacco leaves using a Pinto bean TLP antibody demonstrated that the 23-kDa TLP was secreted into the extracellular matrix. T₂ progeny of regenerated plants transformed with TLP gene were tested for their disease reaction to Alternaria alternata, the brown spot pathogen. Transgenic tobacco plants expressing TLP at high levels showed enhanced tolerance to necrotization caused by the pathogen. This revised version was published online in July 2006 with corrections to the Cover Date.     

Response of Respiration of Tobacco Leaves in Light and Darkness and the CO(2) Compensation Concentration to Prior Illumination and Oxygen

The course of respiration in control leaves of tobacco (Nicotiana tabacum L.) that were illuminated 4 to 5 hours and then darkened 0.25 to 10 hours and in tobacco leaves starved of carbohydrate by 14 hours or more of darkness was measured as CO₂ efflux in light and darkness into CO₂-free atmospheres containing 0.04, 2.23, 21, 40, and 100% O₂.     

Degradation of Metolachlor in Tobacco Field Soil

The extensive use of metolachlor to control weeds in tobacco fields in China has aroused concern about its environmental fate. The aim of this study was to investigate the degradation and residue fate of metolachlor in tobacco field soil (silt loam) under laboratory and field conditions. In laboratory experiments, metolachlor in bulk soil exhibited fast degradation in a temperature range from 10 to 35°C and a soil moisture level of 20–80%, with half-lives (T_1/2) from 66.7 to 28.8 days. The degradation rate of metolachlor decreased as the application dose increased. Owing to higher microbial populations and enzymatic activities, metolachlor rapidly dissipated in rhizosphere soil as compared to bulk soil. Field persistence of metolachlor was evaluated in the same soil during the tobacco (Nicotiana tabacum K326) growing season in 2012 and 2013. The dissipation of metolachlor followed the first-order kinetics and its T_1/2 values were 11.7–13.5 days in soil and 9.0–9.6 days in green tobacco leaves, respectively. At harvest time, the residual levels of metolachlor in soil and green tobacco leaves were in the range of 0.626–1.623 and 0.083-0.481 mg kg^?1, respectively. These findings might have practical implications for the fate of metolachlor residue in tobacco fields. Environmental factors, especially temperature and moisture, should be considered in combination with the appropriate application dose of metolachlor for achieving satisfactory weed-control efficacy, reducing runoff, and minimizing effects on environmental quality.     

Effects of Hydroxybenzoic Acids on Oxidation of Reduced Nicotinamide Adenine Dinucleotide by Enzymes from Tobacco Leaves

Hydroxyhenzoic acids were tested for their effects on oxidation of the reduced nicotinamide adenine dinucleotide (NADH) in the absence of added H₂O₂ and Mn²* by an enzyme preparation from tobacco leaves (Nicotiana tabacum, var. White Gold). For comparison, a commercial horseradish peroxidase was also used. The rate of NADH oxidation was followed spectruphotometrically at 340 nm. Mono- and dihydroxybenzoic acids exerted significant effect on the rate of NADU oxidation, yet their effectiveness was determined by the number and position of the hydroxyl group on the ring. 4-Hydroxybenzoic acid was very effective in stimulating the reaction. Shifting the hydroxyl from the 4- to the 3-position and from the 3- to the 2-position decreased activity. 2,4- And 2,5-dihydroxybenzoic aeids were more active than the other dihydroxy-iscuners in stinulating oxidation of NADH. the dihydroxybenzoic acids with the hydroxyls in adjacent positions were less effective, and their activity was affected by other phenolic activators. In the presence of 4-hydroxybenzoic acid which enhanced oxidation of NADH, 2,4- and 2,5-dihydroxybenzoic acids further stimulated the reaction, but 3,4-, 2,3- and 2,6-dibydoxybenzoic acids were inhibitory. The inhibition by 3,4- and 2,3-dihydroxybenzoic aciils was non-competitive. The enzymes extracted by a L-cysteine-containing buffer showed lower NADH-oxidase activity. The enzyme preparation possessed peroxidase activity. The activity of NADH-oxidase inereased when H₂O₂ and Mi²* were present in addition to 4-hydroxy-benzoic acid. The effect of the position and number of hydroxyl substitution on the rate of NADH oxidation by borseradish peroxidase was also significant. This suggests the involvement of peroxidase in the NADH-oxidase system of tobacco leaves. However, a combination of the inactivated enzyme solution and active horseradish peroxidase with peroxidase activity equivalent to that of the enzyme preparation from tobacco leaves did not reconstitute the NADH-oxidase activity of tobacco leaves. This and other evidence suggests that the soluble NADH-oxidizing zyme system of tobacco leaves is more complicated than peroxidase.     

Induction of Proteinase Inhibitors in Tobacco Cell Suspension Culture by Elicitors of Phytophthora parasitica var. nicotianae

An elicitor preparation obtained from Phytophthora parasitica var. nicotianae, a pathogen of tobacco, induced an accumulation of proteinase inhibitors and a stimulation of ethylene synthesis in a tobacco (Nicotiana tabacum) cell suspension culture. About 30 micrograms per milliliter of elicitor were necessary for maximal induction of proteinase inhibitor accumulation, and the response was detectable after 12 hours of incubation with elicitor. Accumulation of proteinase inhibitors required de novo protein synthesis, since cycloheximide completely inhibited its elicitation, and actinomycin D inhibited it partially. One of the inhibitors was purified by a procedure that included heating, (NH₄)₂SO₄ precipitation, ion-exchange chromatography, and affinity chromatography. The purified inhibitor was shown to be a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of about 10,500. It inhibited trypsin but not chymotrypsin.     

First Record of Fusarium Wilt of Tobacco in Greece Imported as Seedborne Inoculum

Leaf yellowing and brown discoloration was observed in tobacco plants cv. Burley TN97 in tobacco fields of central Greece in 2002. Fusarium oxysporum f. sp. nicotianae was isolated from symptomatic plants and Koch's postulates were fulfilled. The pathogenicity of the isolated fungus was examined on five tobacco cultivars (Burley TN97, BurleyB21, VirginiaBE9, Virginia Niki and Anatolika KE26/2). The pathogen was present in tobacco seed batches imported in 2000 and 2001, which indicates that the infected seed is most probably the primary source of the disease in Greece. As Fusarium oxysporum f. sp. vasinfectum can also cause vascular wilt in tobacco, the hypothesis that the isolated F. oxysporum strain belongs to f. sp. vasinfectum was excluded by a pathogenicity test to cotton cv. Acala SJ‐2. This is the first report of F. oxysporum f. sp. nicotianae in Greece and the second in the European Union, although the seedborne nature of the pathogen has not been previously reported in Europe.     

Disassembly of Tobacco Mosaic Virus by Membrane Lipid Isolated from Tobacco Leaves and Polyornithine

In vitro disassembly of tobacco mosaic virus (TMV) virions occurred in the presence of both polyornithine and a lipid fraction isolated from tobacco leaf membrane. The latter could be replaced by lecithine. Disassembly of 10 μg of TMV virions was attained in the presence of a 500-mg leaf equivalent of membrane lipid and 20 μg of polyornithine in 1 ml of 0.01 M Tris-HCl buffer, pH 7.4 at 30 C. Similarity and dissimilarity between the in vitro disassembly and the in vivo uncoating mechanisms are discussed.     

Alteration of Hormone Levels in Transgenic Tobacco Plants Overexpressing the Rice Homeobox Gene OSH1

The rice (Oryza sativa L.) homeobox gene OSH1 causes morphological alterations when ectopically expressed in transgenic rice, Arabidopsis thaliana, and tobacco (Nicotiana tabacum L.) and is therefore believed to function as a morphological regulator gene. To determine the relationship between OSH1 expression and morphological alterations, we analyzed the changes in hormone levels in transgenic tobacco plants exhibiting abnormal morphology. Levels of the plant hormones indole-3-acetic acid, abscisic acid, gibberellin (GA), and cytokinin (zeatin and trans-zeatin [Z]) were measured in leaves of OSH1-transformed and wild-type tobacco. Altered plant morphology was found to correlate with changes in hormone levels. The more severe the alteration in phenotype of transgenic tobacco, the greater were the changes in endogenous hormone levels. Overall, GA₁ and GA₄ levels decreased and abscisic acid levels increased compared with wild-type plants. Moreover, in the transformants, Z (active form of cytokinin) levels were higher and the ratio of Z to Z riboside (inactive form) also increased. When GA₃ was supplied to the shoot apex of transformants, internode extension was restored and normal leaf morphology was also partially restored. However, such GA₃-treated plants still exhibited some morphological abnormalities compared with wild-type plants. Based on these data, we propose the hypothesis that OSH1 affects plant hormone metabolism either directly or indirectly and thereby causes changes in plant development.     

Cytokinin-controlled Indoleacetic Acid Oxidase Isoenzymes in Tobacco Callus Cultures

Indoleacetic acid oxidase in tobacco callus tissues (Nicotiana tabacum L., cultivar White Gold) was resolved into seven anionic isoenzymes by polyacrylamide gel disc electrophoresis. Different concentrations of kinetin and zeatin in the presence of indoleacetic acid affected the level of this enzyme, particularly two fast-moving isoenzymes, A₅ and A₆. The optimal concentration of kinetin was 0.2 μm; increasing concentrations above this level progressively lowered the total activity of indoleacetic acid oxidase and repressed the development of isoenzymes A₅ and A₆. Actinomycin D and cycloheximide inhibited the development of these two isoenzymes under the influence of 0.2 μm kinetin, suggesting a requirement for RNA and protein synthesis. The cytokinin-promoted indoleacetic acid oxidase isoenzymes A₅ and A₆ increased with time and paralleled the dry weight increase of tobacco callus tissues, but the total activity of indoleacetic acid oxidase per unit dry weight of tobacco callus varied with time depending on the stage of plant growth.     

Tobacco plants can use nitrogen taken up before mechanical wounding to synthesize nicotine afterwards

Mechanical wounding stimulates nicotine synthesis in tobacco plants. In the practice of tobacco production, most nitrogen (N) is taken up before removal of the shoot apex, while nicotine is mainly synthesized afterwards. Since N is required for nicotine synthesis, it is interesting to know whether plants can use N taken up before removal of the shoot apex to synthesize nicotine after wounding. To address this question, a hydroponics culture experiment was carried out, in which N was supplied as NH₄NO₃ at two levels (1 mM and 6 mM) in pre-culture, and N was either withdrawn or replaced by ¹⁵N after removing the shoot apex for the next seven days. Removal of the shoot apex caused a marked increase in nicotine concentration in various organs, also when plants grew under low-N conditions and showed symptoms of N deficiency. Increased nicotine accumulation even occurred when N was withdrawn from the growth medium before the apex was removed, indicating that tobacco plants can use N taken up previously to synthesize nicotine after mechanical wounding. The amount of N used for nicotine synthesis accounted for 5–6% of the total N, irrespective of treatment. Although most of the nicotine in intact plants and plants with the apex removed was synthesized de novo, as evidenced by the data when N was replaced by ¹⁵N-labeled NH₄NO₃, a large amount of the N absorbed before the N replacement was incorporated into the newly formed nicotine. The proportion of nicotine-¹⁵N to total nicotine-N was almost the same as that of ¹⁵N to total N in various organs. The results show the utilization of remobilized N taken up before excision of the shoot apex for nicotine synthesis afterwards, and highlight the importance of N cycling within plants, both when grown under N-sufficient and N-deficient conditions.Key words: ¹⁵N-isotope nitrogen, mechanical wounding, nicotine concentration, nicotine synthesis, nitrogen deficiency, removal of the shoot apex, tobacco (Nicotiana tabacum L.)     

Genetic and Pathogenic Variation Among Tobacco Black Shank Strains of Phytophthora parasitica var. nicotianae from the Main Tobacco Growing in China

Pathogenic and genetic variability among seven populations of Phytophthora parasitica var. nicotianae from individual tobacco fields (Yunnan, Shandong, Henan, Heilongjiang, Shanxi, Fujian and Sichuan provinces) were investigated using pathogenicity and randomly amplified polymorphic DNA (RAPD) analyses; 63 strains were isolated from different fields of seven tobacco growing regions, using tobacco cv. Hongda as a baiting host. Pathogenic variability was evaluated in greenhouse studies using five tobacco cultivars that have different levels of resistance to tobacco black shank; 75 and 73% of the strains were pathogenic on M3 and M4, 29 and 33% on M1 and M2, and 94% were pathogenic on M5, respectively. Disease severity incited by different strains varied significantly on individual tobacco cultivars. The percentage of strains pathogenic on different cultivars varied among locations. Genotypic variation among 63 strains was evaluated by RAPD analysis. Ten primers detected 89 polymorphic bands. Cluster and principal coordinates analysed cluster groups. the minor group contained 26 strains, and major group contained 37 strains. Estimates of genetic diversity based on RAPD analysis ranged from 0.24 to 0.34 within populations to 0.36 among all strains from all populations. Phytophthora parasitica var. nicotianae populations were genotypically and phenotypically variable, but no distinct genotypic differences were identified among populations from the seven locations.