High production of beta-thujaplicin glycosides by immobilized plant cells of Nicotiana tabacum

beta-Thujaplicin (hinokitiol) is a tropolone derivative present in the heartwood of cupressaceous plants and is used as a medicine, a food additive, and a preservative, and in cosmetics as hair tonic. The cultured plant cells of Nicotiana tabacum glycosylated beta-thujaplicin to two glucosides, 4-isopropyltropolone 2-O-beta-D-glucoside (6%) and 6-isopropyltropolone 2-O-beta-D-glucoside (12%), and two gentiobiosides, 4-isopropyltropolone 2-O-beta-D-gentiobioside (2%) and 6-isopropyltropolone 2-O-beta-D-gentiobioside (5%) after 48 h incubation. The use of immobilized cells of N. tabacum in sodium alginate gel much improved the yield of the products; the glycosylation of beta-thujaplicin with immobilized N. tabacum gave the glycoside products, 4-isopropyltropolone 2-O-beta-D-glucoside (11%), 4-isopropyltropolone 2-O-beta-D-gentiobioside (6%), 6-isopropyltropolone 2-O-beta-D-glucoside (20%), and 6-isopropyltropolone 2-O-beta-D-gentiobioside (10%). On the other hand, 4-isopropyltropolone 2-O-beta-D-glucoside (14%), 4-isopropyltropolone 2-O-beta-D-gentiobioside (7%), 6-isopropyltropolone 2-O-beta-D-glucoside (33%), and 6-isopropyltropolone 2-O-beta-D-gentiobioside (13%) were obtained through the biotransformation with immobilized cells in the medium without iron ions. In comparison with the case of bioconversion in the normal medium containing iron ions, removal of iron ions improved the yields of products.     

Phenolics production by encapsulated Nicotiana tabacum cells

Plant cells of tobacco (Nicotiana tabacum L.) were grown for several generations in suspension cultures. Cells were immobilized in continuous bioreactors in calcium alginate (Ca Alg) beads or in poly-L-lysine (PLL) encapsulated calcium alginatehydrogels. In each case, the cells were fed continuously a modified Linsmaier-Skoog plant cell culture medium. The bioreactor effluent was analyzed for total phenolic compounds. The net specific productivity of phenolics was calculated on a daily basis for several test runs. For comparison, productivity in suspension cultures was monitored. Productivity of suspended cells declined to zero within 9 d; both immobilized and encapsulated cells remained productive for 16 d following inoculation. Specific productivity by encapsulated cells was higher than that by immobilized cells; in both types similar rates of decline in productivity occurred.     

PEG- and electroporation-induced transformation in Nicotiana tabacum: influence of genotype on transformation frequencies

Summary Experimental parameters for direct gene transfer with recombinant DNA encoding neomycin phosphotransferase II (NPTII) under control of eukaryotic expression signals were established. The introduced gene was shown by the growth of transformants on media containing kanamycin, by genomic blotting and by assaying NPTII activity. Leaf protoplasts from three green genotypes of varieties xanthii and petit havanna, and from four plastome-encoded albino genotypes of Nicotiana tabacum were analyzed with respect to cell division kinetics and yield of kanamycin-tolerant colonies after direct gene transfer. No clear correlation was found between the time of onset of cell division and transformation frequency.     

Naphthylphthalamic acid-binding sites in cultured cells from Nicotiana tabacum

Naphthylphthalamic acid (NPA), an inhibitor of polar auxin transport, binds with high affinity to membrane preparations from callus and cell suspension cultures derived from Nicotiana tabacum (K _d approx. 2·10^–9 M). The concentration of membrane-bound binding sites is higher in cell suspension than in callus cultures. The binding of NPA to these sites seems to be a simple process, in contrast to the binding of the synthetic auxin naphthylacetic acid (1-NAA) to membrane preparations from callus cultures, which is more complex (A.C. Maan et al., 1983, Planta 158, 10–15). Naphthylacetic acid, a number of structurally related compounds and the auxin-transport inhibitor triiodobenzoic acid were all able to compete with NPA for the same binding site with K _d values ranging from 10^–6 to 10^–4 M. On the other hand, NPA was not able to displace detectable amounts of NAA from the NAA-binding site. A possible explantation is the existence of two different membrane-bound binding sites, one exclusively for auxins and one for NPA as well as auxins, that differ in concentration. The NPA-binding site is probably an auxin carrier.Abbreviations 1-NAA 1-Naphthylacetic acid - 2-NAA 2-Naphthylacetic acid - NPA N-1-Naphthylphthalamic acid     

Immobilization of cells of Nicotiana tabacum L. on polyphenyleneoxide coated with poly-L-lysine

Cells of Nicotiana tabacum L. cv. Wisconsin 38 were immobilized on poly (2,6-dimethyl)-p-phenyleneoxide in powder form (Sorfix) coated with poly-L-lysine (molecular weight 40 000 daltons). The dependence of cell immobilization on the amount of bound polyL-lysine was estimated.Abbreviations MW molecular weight - dwt dry weight - fwt fresh weight     

The jasmonate-induced expression of the Nicotiana tabacum leaf lectin

Previous experiments with tobacco (Nicotiana tabacum L. cv Samsun NN) plants revealed that jasmonic acid methyl ester (JAME) induces the expression of a cytoplasmic/nuclear lectin in leaf cells and provided the first evidence that jasmonates affect the expression of carbohydrate-binding proteins in plant cells. To corroborate the induced accumulation of relatively large amounts of a cytoplasmic/nuclear lectin, a detailed study was performed on the induction of the lectin in both intact tobacco plants and excised leaves. Experiments with different stress factors demonstrated that the lectin is exclusively induced by exogeneously applied jasmonic acid and JAME, and to a lesser extent by insect herbivory. The lectin concentration depends on leaf age and the position of the tissue in the leaf. JAME acts systemically in intact plants but very locally in excised leaves. Kinetic analyses indicated that the lectin is synthesized within 12 h exposure time to JAME, reaching a maximum after 60 h. After removal of JAME, the lectin progressively disappears from the leaf tissue. The JAME-induced accumulation of an abundant nuclear/cytoplasmic lectin is discussed in view of the possible role of this lectin in the plant.     

High efficiency transient and stable transformation by optimized DNA microinjection into Nicotiana tabacum protoplasts

An efficient system has been established that allows well controlledDNA microinjection into tobacco (Nicotiana tabacum) mesophyllprotoplasts with partially regenerated cell walls and subsequentanalysis of transient as well as stable expression of injectedreporter genes in particular targeted cells or derived clones.The system represents an effective tool to study parametersimportant for the successful transformation of plant cells bymicroinjection and other techniques. Protoplasts were immobilizedin a very thin layer of medium solidified with agarose or alginate.DNA microinjection was routinely monitored by coinjecting FITC-dextranand aimed at the cytoplasm of target cells. The injection procedurewas optimized for efficient delivery of injection solution intothis compartment. Cells were found to be at the optimal stagefor microinjection about 24 h after immobilization in solidmedium. Embedded cells could be kept at this stage for up to4 d by incubating them at 4 C in the dark. Within 1 h successfuldelivery of injection, solution was routinely possible into20–40 cells. Following cytoplasmic coinjection of FITC-dextran and pSHI913,a plasmid containing the neo (neomycin phosphotransferase II)gene, stably transformed, paromomycin-resistant clones couldbe recovered through selection. Transgenic tobacco lines havebeen established from such clones. Injection solutions containingpSHI913 at a concentration of either 50 µg ml^–1or 1 mg ml^–1 have been tested. With 1 mg ml^–1 plasmidDNA the percentage of resistant clones per successfully injectedcell was determined to be about 3.5 times higher. Incubationof embedded protoplasts at 4C before microinjection was foundto reduce the percentage of resistant clones obtained per injectedcell Protoplasts were immobilized above a grid pattern and the locationof injected cells was recorded by Polaroid photography. Thefate of particular targeted cells could be observed. Isolationand individual culture of clones derived from injected cellswas possible. Following cytoplasmic coinjection of FITC-dextranand 1 mg ml^–1 plasmid DNA on average about 20% of thetargeted cells developed into microcalli and roughly 50% ofthese calli were stably transformed. Transient expression ofthe firefly luciferase gene (Luc) was nondestructively analysed24 h after injection of pAMLuc. Approximately 50% of the injectedcells that were alive at this time point expressed the Luc genetransiently. Apparently, stable integration of the injectedgenes occurred in essentially all transiently expressing cellsthat developed into clones. Key words: DNA microinjection, firefly luciferase, FITCdextran, Nicotiana tabacum, protoplast transformation     

The reversible inhibition of pollen germination of Nicotiana tabacum L.: an entry into a transformation system

Summary The hydrodynamics of mature pollen rehydration in Nicotiana tabacum was used to study reversible inhibition of pollen germination in vitro. Tobacco pollen was incubated for various times in media containing calcium, potassium and magnesium salts, boric acid, and exhibiting different osmotic pressures as a function of sucrose concentration. Total inhibition of germination with complete viability preservation was achieved for 56 h by keeping the grains in medium with 80% sucrose, since typical percentages of germination and pollen tube lengths were recovered after this treatment. These effects were considered as consequences of natural osmoregulation of rehydration/germination in mature pollen. The possibility of applying these findings to the incubation of pollen with Agrobacterium tumefaciens to develop a pollen transformation method is discussed.     

Evidence for O-acetylserine in Nicotiana tabacum

O-Acetylserine, a precursor of cysteine in plants, was isolated from cells of Nicotiana tabacum cultured in liquid medium.     

A damascone derivative from Nicotiana tabacum

A new member of the damascone series, 4-hydroxy-β-damascone, was identified in the steam distillable oil from Virginia tobacco.     

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.     

The biotransformation of carvoxime and dihydrocarvoxime with cell suspension cultures of Nicotiana tabacum

In newly initiated cell suspension cultures of Nicotiana tabacum, (4R)-(?) and (4S)(+)-carvoximes and (1S,4R)(+)-dihydrocarvoxime were hydrolysed to the corresponding ketones and then the resultant ketones were reduced to the corresponding alcohols.     

Subcellular localization and topology of homogalacturonan methyltransferase in suspension-cultured Nicotiana tabacum cells

Pectin is a complex polysaccharide in the primary walls of all plant cells that is thought to be synthesized in the cellular endomembrane system and inserted into the wall via exocytosis. The most abundant pectic polysaccharide, homogalacturonan, is partially methylesterified within the cell by the pectin methyltransferase homogalacturonan methyltransferase (HGA-MT). The subcellular location of HGA-MT activity was determined in tobacco (Nicotiana tabacum L. cv. Samsun) cell membranes separated on linear sucrose gradients. The activity of HGA-MT and two enzymatic markers of the Golgi apparatus, IDPase and UDPase, were found to be located in the same membrane fraction. No NADH cytochrome c reductase activity, a marker for the endoplasmic reticulum, was detected in the Golgi fraction. Homogalacturonan methyltransferase activity was not reduced by protease treatment of intact membranes or membranes treated with 0.01% Triton X-100. In contrast, HGA-MT activity was reduced by protease treatment of membranes permeabilized with 0.02% Triton X-100. The sensitivity of HGA-MT in detergent-permeabilized membranes, and the lack of inhibition of HGA-MT activity by protease-treatment of intact membranes, provides evidence that the catalytic site of HGA-MT is located on the lumenal side of the Golgi. Received: 2 December 1998 / Accepted: 9 February 1999