Kinetin incorporated into tobacco callus ribosomal RNA and transfer RNA preparations

Kinetin, N⁶-furfuryladenine, was incorporated into tobacco (nicotiana tabacum L., var. Wis. No. 38) callus RNA isolated from rapidly growing tissue cultured in the presence of N⁶-furfuryladenine-8-¹⁴C or unlabeled kinetin. Approximately 0.7% of the radioactivity in the labeled kinetin added to the medium was recovered as N⁶-furfuryladenosine (fr⁶A) in the rRNA and tRNA preparations from the tobacco callus. The rRNA contained over 90% of these fr⁶ A moieties. The extent of kinetin incorporation was four times greater than that observed for N⁶-benzyladenine. The radiochemical purity of the recovered fr⁶ A was confirmed by three successive chromatographic purifications on Sephadex columns (LH-20 eluted with 35% ethanol, G-10 eluted with 20% ethanol, and LH-20 eluted with water). A cytokinin-active ribonucleoside with elution volumes corresponding to fr⁶ A was isolated from the tobacco callus rRNA preparation. This compound was analyzed by gas-liquid chromatography and rigorously characterized as N⁶-furfuryladenosine by gas-liquid chromatography-mass spectrometry of the trimethylsilyl derivative.     

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.     

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.     

Stabilization of cortical microtubules by the cell wall in cultured tobacco cells

Cortical microtubules (MTs) in protoplasts prepared from tobacco (Nicotiana tabacum L.) BY-2 cells were found to be sensitive to cold. However, as the protoplasts regenerated cell walls they became resistant to cold, indicating that the cell wall stabilizes cortical MTs against the effects of cold. Since poly-l-lysine was found to stabilize MTs in protoplasts, we examined extensin, an important polycationic component of the cell wall, and found it also to be effective in stabilizing the MTs of protoplasts. Both extensin isolated from culture filtrates of tobacco BY-2 cells and extensin isolated in a similar way from cultures of tobacco XD-6S cells rendered the cortical MTs in protoplasts resistant to cold. Extensin at 0.1 mg·ml⁻¹ was as effective as the cell wall in this respect. It is probable that extensin in the cell wall plays an important role in stabilizing cortical MTs in tobacco BY-2 cells.     

N′-isopropylnornicotine: Its formation from nicotine in aged leaves of Nicotiana tabacum

An aqueous solution of nicotine-[2′-¹⁴C] was painted on the leaves of 4-month-old tobacco plants (Nicotiana tabacum) which were harvested 3 weeks later. This tracer was similarly applied to excised tobacco leaves which were allowed to dry in air for 4 weeks. The alkaloids, were extracted with the addition of N′-isopropylnornicotine, a compound which has been previously isolated from air-cured tobacco. Radioactive nicotine and nornicotine were isolated from the intact plants with only minute activity in the N′-isopropylnornicotine. All three of these alkaloids were radioactive from the air-cured leaves, and degradation of the labelled N'-isopropylnornicotine indicated that all the activity was located at the C-2′ position. A higher level of activity was found in N′-isopropylnornicotine which was obtained from excised leaves which were fed the nicotine- [2′- ¹⁴C] in aqueous acetone, and were treated on subsequent days with aqueous acetone. These results are consistent with the hypothesis that N′-isopropylnornicotine is produced in the curing of tobacco leaves by reaction of nornicotine (formed by the demethylation of nicotine) with acetoacetate, followed by decarboxylation and reduction. The ¹³C NMR chemical shifts of the methyl groups of N′-isopropylnornicotine and related 1-isopropylpyrrolidines which have chirality at the α-position of the pyrrolidine ring, are significantly different (up to 7.5 ppm).     

Absorption of Iron by Enzymically Isolated Leaf Cells

Iron uptake was studied using cells enzymically isolated from green tobacco leaves. Absorption was increased both by light and succinate as probable energy sources. Bicarbonate in the incubation mixture was inhibitory, and citrate also reduced absorption presumably by chelation with the metal. Absorption of iron was temperature sensitive and optimal at 25°C. Temperature coefficients and activation energies suggested that absorption was energy mediated. NaN₃ and DNP inhibited uptake at concentrations of 10-³M and 10^?4M, respectively. The inhibition caused by DNP was not negated by an external supply of ATP. The results suggest that iron absorption is an active metabolic process in cells enzymically isolated from green tobacco leaves. Cells from Fe-chlorotic leaves of PI 54619–5–1 soybean absorbed less iron than those derived from healthy leaves of the same variety, while leaf cells from the variety Hawkeye showed no such differences.     

Biodegradation of nicotine by a newly isolated Agrobacterium sp. strain S33

Aims: To isolate and characterize bacteria capable of degrading nicotine from the rhizospheric soil of a tobacco plant and to use them to degrade the nicotine in tobacco solid waste. Methods and Results: A bacterium, strain S33, was newly isolated from the rhizospheric soil of a tobacco plant, and identified as Agrobacterium sp. based on morphology, physiological tests, Biolog MicroLog3 4·20 system and 16S rRNA gene sequence. Using nicotine as the sole source of carbon and nitrogen in the medium, it grew optimally with 1·0 g l^?1 of nicotine at 30°C and pH 7·0, and nicotine was completely degraded within 6 h. The resting cells prepared from the glucose‐ammonium medium or LB medium could not degrade nicotine within 10 h, while those prepared from the nicotine medium could completely degrade 3 g l^?1 of nicotine in 1·5 h at a maximal rate of 1·23 g nicotine h^?1 g^?1 dry cell. Using the medium containing nicotine, glucose and ammonium simultaneously to cultivate strain S33, the resting cells could degrade 98·87% of nicotine in tobacco solid waste with the concentration as 30 mg nicotine g^?1 dry weight tobacco solid waste within 7 h at a maximal rate of 0·46 g nicotine h^?1 g^?1 dry cell. Conclusions: This is the first report that Agrobacterium sp. has the ability to degrade nicotine. Agrobacterium sp. S33 could use nicotine as the sole source of carbon and nitrogen. The use of resting cells of the strain S33 prepared from the nicotine–glucose–ammonium medium was an effective method to degrade nicotine and detoxify tobacco solid waste. Significance and Impact of the Study: Nicotine in tobacco wastes is both toxic and harmful to human health and the environment. This study showed that Agrobacterium sp. S33 may be suitable for the disposal of tobacco wastes and reducing the nicotine content in tobacco leaves.     

Growth cycle stage-dependent NaCl induction of plasma membrane H+-ATPase mRNA accumulation in de-adapted tobacco cells

A cDNA clone encoding an isoform of the plasma membrane H⁺-ATPase was isolated from Nicotiana tabacum. The steady-state plasma membrane H⁺-ATPase message levels were the same in unadapted tobacco cells and tobacco cells adapted to 428 mol m⁻³ NaCl. When cells adapted to 428 mol m⁻³ NaCl maintained in the absence of NaCl (deadapted) for an excess of 100 passages were exposed to 400 mol m⁻³ NaCl for 24 h, there was an increased accumulation of plasma membrane H⁺-ATPase message. The NaCl responsiveness of the deadapted cells was dependent upon the growth cycle stage. Alterations in the levels of plasma membrane FT-ATPase message during the growth cycle support a role for the H⁺-ATPase in cell growth. These results document the induction by NaCl of plasma membrane FT-ATPase message accumulation in tobacco cells, and suggest that enhanced expression of the plasma membrane FT-ATPase has a role in the short term response of cells of NaCl, but is not necessarily involved in long-term adaptation.     

Auxin-binding by particulate fractions from tobacco leaf protoplasts

In vitro binding of 1-naphthaleneacetic acid (NAA) to particulate fractions from tobacco leaf protoplasts was studied. In freshly isolated protoplasts no specific binding could be detected, whereas it was present in particulate fractions from tobacco leaves. It is concluded that the NAA-binding-sites are probably located at the external face of the plasma membrane; they are destroyed during protoplast isolation by proteolytic enzymes in the cellulase and macerozyme preparations. After culturing the protoplasts for 3–4 d, the first cell divisions were observed and at the same time specific NAA-binding became detectable. The affinity constant for NAA was approx. 2·10⁶ mol^-1 and the number of binding sites increased during further culture.Abbreviations MES 4-morpholinoethanesulfonic acid - NAA 1-naphthaleneacetic acid     

Enhanced H Transport Capacity and ATP Hydrolysis Activity of the Tonoplast H-ATPase after NaCl Adaptation

Tonoplast enriched membrane vesicle fractions were isolated from unadapted and NaCl (428 millimolar) adapted tobacco cells (Nicotiana tabacum L. var Wisconsin 38). Polypeptides from the tonoplast enriched vesicle fractions were separated by SDS-PAGE and analyzed by Western blots using polyclonal antibodies to the 70 kilodalton subunit of the red beet tonoplast H⁺-ATPase. These antibodies cross-reacted exclusively to a tobacco polypeptide of an apparent molecular weight of 69 kilodaltons. The antibodies inhibited ATP-dependent, NO₃⁻ sensitive H⁺ transport into vesicles in tonoplast enriched membrane fractions from both unadapted and NaCl adapted cells. The relative H⁺ transport capacity per unit of 69 kilodalton subunit of the tonoplast ATPase of vesicles from NaCl adapted cells was fourfold greater than that observed for vesicles from unadapted cells. The increase in specific H⁺ transport capacity after adaptation was also observed for ATP hydrolysis.     

Targeting and glycosylation of patatin the major potato tuber protein in leaves of transgenic tobacco

Patatin, the most abundant protein in the storage parenchyma cells of potato (Solanum tuberosum L.) tubers, is a vacuolar glycoprotein that consists of a number of closely related polypeptides and is encoded by a large gene family. To analyse the glycosylation pattern and the nature of the glycans on a single patatin polypeptide in a heterologous tissue we introduced a single chimaeric patatin gene into tobacco (Nicotiana tabacum L.) and studied its product in leaves. Patatin isolated from the leaves of transgenic tobacco plants is glycosylated at asparagine (Asn)⁶⁰, and Asn⁹⁰, but the third glycosylation site (Asn²⁰²) has no glycan. The two glycans are typical small complex glycans with xylose, fucose, mannose and N-acetylglucosamine in a ratio 1:1:3:2, the same ratio as found on patatin isolated from potato tubers. Expression of patatin in tobacco leaves was accompanied by the correct processing of the signal peptide, and the proper targeting of the glyco-protein to the vacuoles of mesophyll cells.Abbreviations Asn asparagine - ConA concanavalin A - EndoH endoglycosidase H - Fuc fucose - GlcNAc N-acetylglucosamine - HPLC high-performance liquid chromatography - Man mannose - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl-sulfate - Ser serine - TFMS trifluoromethanesulfonic acid - Thr threonine - Xyl xylose     

Flow cytometric analysis of tobacco and cowpea protoplasts infected in vivo and in vitro with alfalfa mosaic virus

Immunofluorescence flow cytometry was used to study the distribution of viral antigen in protoplast populations. Protoplasts were isolated from healthy and alfalfa mosaic virus (AMV) infected tobacco leaves (designated in vivo infected). Furthermore isolated tobacco and cowpea protoplasts were infected in vitro with AMV. The FITC-conjugated antibodies could penetrate formaldehyde fixed protoplasts. The flow cytometric measurements were rapid and reproducible. Comparable immunofluorescence patterns were found for all infected samples (per sample 10⁴ protoplasts were measured). Infectious virus could only be detected in in vivo infected tobacco protoplasts and in in vitro infected cowpea protoplasts.     

An acidic xylan from extracellular polysaccharides of suspension-cultured cells of Nicotiana tabacum

An acidic xylan was isolated from the extracellular polysaccharides of suspension-cultured tobacco cells. Its structure was investigated by methylation analysis and ¹³C NMR spectroscopy and was shown to consist of a main chain of β-(1→4)-linked D-xylopyranosyl residues to which were attached as side-chains, α-D-glucuronic acid residues at O-2.     

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.     

Biosynthesis of azetidine-2-carboxylic acid from methionine in Nicotiana tabacum

The administration of dl-methionine-[1¹⁴C] to Nicotia tabacum resulted in the formation of radioactive azetidine-2-carboxylic acid (isolated by dilution) which was specifically labelled on its carboxyl group. This result and other evidence strongly indicates that this imino acid is a normal component of tobacco.     

Binding of Tobacco Mosaic Virus to Membrane Material Isolated from Tobacco Leaves

Binding of tobacco mosaic virus (TMV) to disrupted tobacco leaf membrane was studied. Membrane isolated from tobacco leaves was treated successively with (NH₄)₂SO₄, Li-diiodosalicylate and then pronase. TMV-binding substance was thus isolated in a soluble form. From enzymatic digestion experiments, it was suggested that the binding substance was composed of lipid and carbohydrate.     

Characterization of sub-genomic double-stranded RNAs from virus-infected plants

Double-stranded RNAs (sub-RFs) smaller than the double-stranded RNAs (RFs) corresponding to genomic RNAs of tobacco mosaic (TMV) and cowpea chlorotic mottle (CCMV) viruses were isolated from infected plants and characterized. Seven of the 12 sub-RFs of TMV that were found ranging in size from 3.00 – 0.42 × 10⁶ daltons corresponded to twice the size of the 7 sub-genomic mRNAs reported by Goelet and Karn (8). Six sub-RFs of CCMV were found ranging from 0.98 – 0.39 × 10⁶ daltons with the most abundant species corresponding to twice the size of RNA 4. The kinetics of incorporation of ³H-uridine into sub-RFs were different from that into RFs. Incorporation into sub-RFs was slow and linear whereas that into RF turned over rapidly.     

Functional Similarities of Recombinant OLP and Cytokinin-Binding Protein 2

CBP1 and CBP2 are cytokinin-binding proteins isolated from tobacco callus. In particularly, CBP2 is a 26-kDa protein with high affinity (Kd=1.08×10^-6M) for cytokinin[Kobayashi et al. Plant Cell Physiol. 41(2): 148-157 (2000)] and the N-terminal amino acid analysis of CBP2 showed high sequence homology (92.9%) to tobacco osmotin-like protein (OLP). To compare the properties of OLP and CBP2, recombinant OLP was purified, and binding to benzyladenine (BA) was examined. The inclusion bodies of recombinant OLP were solubilized in 8 M urea and purified on an SP-Sepharose column. SDS-PAGE analysis of the purified recombinant OLP revealed a single band of 26 kDa. The Kd of solublized recombinant OLP to BA obtained from a Scachard plot was 1.10×10^-6M, which was similar to the Kd of CBP2 to BA (1.08×10^-6M).