Phytohormone-induced GABA production in transformed root cultures of Datura stramonium: an in vivo 15N NMR study

Primary nitrogen metabolism in transformed root cultures ofDatura stramonium was observed by in vivo ¹⁵N NMR. Treatmentof the root cultures with the plant growth regulators -naphthaleneaceticacid (NAA) and kinetin caused a de-differentiation of the roottissue, together with perturbation of primary and secondarynitrogen metabolism. The levels of newly-synthesized glutamineand glutamate during ammonium assimilation were depleted relativeto control cultures, whereas GABA biosynthesis was enhanced.Although GABA production could be stimulated by a decrease incytoplasmic pH (whether imposed artificially or induced by hypoxia),observation of the roots during phytohormone treatment by ³¹PNMR showed that the cytoplasmic pH remained stable, indicatingthat the perturbation of nitrogen metabolism in the de-differentiatedroots must be due to other causes. Key words: Datura, -aminobutyric acid, nitrogen metabolism, NMR, root cultures     

The purification and immunocharacterisation of N-methylputrescine oxidase from transformed root cultures of Nicotiana tabacum L. cv SC58

The enzyme N-methylputrescine oxidase which catalyses the conversion of N-methylputrescine to N-methylpyrrolinium salt has been purified to homogeneity from transformed roots of Nicotiana tabacum L. cv SC58. The enzyme has an apparent sub-unit molecular weight of 53 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with gel-filtration studies, indicating that the native form is a dimer. The K _m of the enzyme for N-methylputrescine has been estimated to be 0.1 mM. Polyclonal antibodies raised to the purified protein recognise one product in an immunoblot of a crude extract of transformed root tissue and will immunoprecipitate N-methylputrescine oxidase activity from such an extract. The antibodies also show a high degree of specificity in immunoblots of crude extracts of transformed root cultures from a range of other solanaceous and non-solanaceous species but do not cross-react with a partially purified preparation of pea-seedling diamine oxidase.Abbreviations MPO N-methylputrescine oxidase - PVDF polyvinylidene difluoride - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We would like to thank members of the Plant Cell Biotechnology Group, Institute of Food Research, Norwich Laboratory, for their helpful discussions during the preparation of this paper.     

Product enhancement and recovery from transformed root cultures of Nicotiana glauca

Transformed roots of Nicotiana glauce synthesize the alkaloids nicotine and anabasine at levels reflecting the parent plants. Media composition, strength, and pH were evaluated with respect to biomass yield and productivity. Full-strength Gamborg's B5 medium proved the best for biomass yield while half-strength, or low-salt, medium enhanced alkaloid accumulation. A detailed investigation of media nitrate levels demonstrated how these may be manipulated to promote growth and intracellular or extracellular alkaloid levels. High nitrate concentrations were found to significantly enhance media alkaloid levels at the end of the growth phase. Media pH is also important, although transformed roots will grow in Gamborg's B5 medium between pH 3 and 9, root biomass is favored by an increase in medium alkalinity, while alkaloid release is encouraged by mildly acidic pH.Transformed roots release a proportion of their secondary metabolites into the growth medium. By continually removing root products, any feedback inhibition on enzymatic reactions is reduced, as are the toxic effects resulting from product accumulation. In this article we describe the use of Amberlite resins (XAD-2 and XAD-4) to enhance alkaloid levels (nicotine and anabasine) of hairy root cultures of Nicotiana glauca by a factor of 10 with no adverse effect on root growth. The performance of the Amberlite columns was subsequently investigated with respect to alkaloid adsorption and desorption, including an evaluation of the effects of pH and loading capacity. The resins also adsorb media constituents which are identified and quantified as part of this work. Resulting nutritional stresses are thought to be partly responsible for enhancing secondary metabolism at the expense of biomass yield. However, the net effects of using Amberlite resins as a means of product removal significantly increases the overall product yield and the extent to which products are released into the growth medium.     

Anther cultures of Nicotiana tabacum L. mutants

Summary The theoretically expected and experimentally observed phenotypic ratios have been compared in populations of haploids derived from chlorophyll mutants of Nicotiana tabacum L. with a known genotypic constitution. The frequencies of mutant genotypes were significantly lower than the expected values, proving the existence of selection in a system of haploid embryoids developing in the anther.The anthers from M₁ plants of a diploidized Nicotiana tabacum haploid cv. Samsun, treated with various concentrations of N-nitroso-N-methylurea and n-butylmethane sulphonate, were cultivated in vitro. The number of anthers which gave rise to haploids (embryogenic anthers) was stimulated by lower concentrations of both the mutagens. The stimulation at the level of M₁ sporophyte is explained by internal genetic heterogeneity induced by adequate mutagen concentration. The average number of haploids per embryogenic anther decreased in all the treatments. The frequency of haploid plants of the mutant phenotypes increased with increasing mutagen concentration.     

Floral morphogenesis in thin-layer tissue cultures of Nicotiana tabacum

The morphological changes in thin-layer tissues of Nicotiana labacum L. cv. Samsun, cultured on Murashige and Skoog medium with 1 μ M each of naphthalene acetic acid (NAA) and benzyladenine (BA), were studied during the first 8 days of culture with light and scanning electron microscopy. The first three days of culture arc characterized by enlargement of all cells and cell divisions starling in the cortical parenchyma cells adjacent to the medium. Between days 3 and 6, epidermal and/or subepidermal cells start to divide, resulting in division centers, which lead to flower bud formation. The hormones NAA and BA in different concentrations affect the formation and distribution of flower buds, bud morphology and callus formation. BA influences particularly bud formation and bud morphology, while NAA affects callus formation in particular. In addition, polarity may occur in the formation of both callus and flower buds, the degree of which depends upon the hormone concentrations.     

Development of auxin autotrophy in Nicotiana tabacum callus cultures

Changes of auxin and ethylene metabolism of Nicotiana tabacum var. Xanthi callus were investigated in relation to auxin-independent growth. During the habituation process, changes occur progressively in hormone metabolism in auxin-heterotrophic tissues: the potential for the destruction of indoleacetic acid (IAA) increases, the IAA level in the cultures rises slightly, and the auxin sensitivity of the callus becomes modified. Preceding the onset of habituation, ethylene production is enhanced although the tissues retain their ability to undergo regeneration.
Gradual changes in auxin metabolism and ethylene production confirm the epigenetic character of the habituation process.     

De-novo synthesis and release of peroxidases in cell suspension cultures of Nicotiana tabacum L.

De-novo synthesis of acid and basic peroxidases has been studied in cell suspension cultures of tobacco by incorporation of ³H- and ¹⁴C-amino acids. Incorporation rates were found to be high for acid peroxidases and low for basic peroxidases. Synthesis of all peroxidases was inhibited by cycloheximide and actinomycin D. Subculturing of the cells increased the rates of radioactive amino-acid incorporation into all peroxidases within the first 24 h. This rise in peroxidase synthesis was correlated with the age of the transferred cells. The older the cells were the more pronounced was the effect. During the culture cycle the high rates of peroxidase synthesis at the second day dropped back to initial values. Peroxidase synthesis was thus inversely related to peroxidase accumulation which was very low at the beginning and increased continuously. By pulse-chase experiments it has been shown that newly synthesized acid peroxidases accumulated in the medium. This process was inhibited by monensin. Only the acid peroxidases were secreted into the cell wall and from there released. The basic peroxidases were not detectable in the medium.Abbreviations AA^* radioactive amino-acid mixture - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Biosynthesis of calystegines: 15N NMR and kinetics of formation in root cultures of Calystegia sepium

Calystegines are nortropane alkaloids bearing between three and five hydroxyl groups in various positions. [15N]Tropinone was administered to root cultures of Calystegia sepium and the incorporation into calystegines was followed. Increase of label in calystegines was measured by one-dimensional 15N NMR and inverse-detected 2D NMR techniques. The results show that tropinone and pseudotropine are metabolites in the biosynthetic pathway of calystegines. The velocity of calystegine accumulation was followed kinetically by transfer of root cultures from 15N-enriched medium to 14N-medium and analysis by GC-MS. A constant calystegine formation with no interference by excretion or degradation was observed. A biosynthetic rate for individual calystegines at each time point was calculated, the maximum was 0.4 mg/day/g of biomass. This allowed the velocity of individual biosynthetic steps to be estimated.     

Comparative study of in vivo stigmasterol biosynthesis in Nicotiana tabacum and Hordeum vulgare

Six-day-old tobacco (Nicotiana tabacum) and barley (Hordeum vulgare) seedlings rapidly incorporated and metabolized exogenously supplied [4-¹⁴C]sitosterol but neither plant was able to convert it into stigmasterol. However, a sterol metabolite was isolated from both species and the acetate derivative was slightly more polar, on AgNO₃—silica gel TLC, than stigmasteryl acetate. A similar metabolite was also obtained with [4-¹⁴C]cholesterol, indicating a general metabolic reaction of plants to exogenous sterols. Both species incorporated [2-¹⁴C]mevalonic acid into sitosterol and stigmasterol. We suggest that in vascular plants, whether monocotyledons or dicotyledons, the pathway of stigmasterol biosynthesis is not via sitosterol but through a common precursor which is derived from mevalonic acid.     

Sesquiterpenoid phytoalexins from suspended callus cultures of Nicotiana tabacum

Treatment of suspended callus cultures of Nicotiana tabacum with commercial cellulase elicited four principal stress metabolites including the phytoalexin capsidiol and a second eremophilane-type diol, shown on the basis of chemical and spectroscopic evidence to be 4-epieremophil-9-ene-11 /gx, 12-diol (without assignment of absolute configuration). This diol appears to be structurally identical with debneyol isolated from N. debneyi (see accompanying paper). Among minor metabolites were an isomer and a dehydro-analogue of the diol. GC/MS of cyclic derivatives (boronates and di-t-butylsilylene derivatives) of vicinal diols was useful for their detection and characterisation. The remaining two major metabolites appeared to be phytuberol and phytuberin.     

Determination of the sites of synthesis of chlorophyll synthesizing enzymes in cell cultures of Nicotiana tabacum

The activity of a sequence of enzymes involved in chlorophyll biosynthesis (δ-aminolevulinic acid synthetase (ALAS), δ-aminolevulinic acid dehydratase, porphobilinogenase and chlorophyllase) was followed during greening of tobacco cell cultures under the influence of chloramphenicol (CAP). The photosynthetic enzymes ribulose diphosphate carboxylase (RuDPCO) and NADP linked glyceraldehyde dehydrogenase (NADP-GDH) were used as markers for penetration and action of the inhibitor. RuCPCO was inhibited at concentrations of CAP which still allowed good chlorophyll accumulation. The enzymes of chlorophyll biosynthesis, the activity of which increased during illumination and CAP treatment, behaved like NADP-GDH which is known to be synthesized in the cytoplasm. The results suggest that synthesis of enzymes of chlorophyll biosynthesis takes place in the cytoplasm. Decreasing light induced increment of ALAS activity caused by CAP may possibly be taken as an indication that things are more complicated with this enzyme.     

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 S ₂ clone, with 1.6 kb of 5 sequence, nor the S ₆ clone, with 2.8 kb of 5 sequence, were expressed at detectable levels in transgenic N. tabacum plants. In N. alata, expression of the S ₂ clone was not detected, however the S ₆ clone was expressed (at low levels) in three out of four transgenic plants. An S ₆-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.     

Transfer of transformed chloroplasts from Nicotiana tabacum to the Lycium barbarum plants

Plastid transformation is an attractive technology for obtaining crop plants with new useful characteristics and for fundamental researches of plastid functioning and nuclear-plastid interaction. The aim of our experiments was to obtain plants with Lycium barbarum nucleus and transformed Nicotiana tabacum plastids. Plastome of previously engineered transplastomic tobacco plants contains reporter uidA gene and selective aadA gene that confers resistance to antibiotics spectinomycin and streptomycin. Asymmetric somatic hybridization was performed for transferring transformed tobacco plastids from transplastomic tobacco plants into recipient L. barbarum wild type plants. Hybrid L. barbarum plants containing transformed tobacco plastome with active aadA and uidA genes were obtained as a result of the experiments. The work shows the possibility of obtaining transplastomic plants by transferring the transformed plastids to remote species by using somatic hybridization technology. The developed technique is especially effective for obtaining transplastomic plants that have low regeneration and transformation ability.     

Increased production of cadaverine and anabasine in hairy root cultures of Nicotiana tabacum expressing a bacterial lysine decarboxylase gene

Several hairy root cultures of Nicotiana tabacum varieties, carrying two direct repeats of a bacterial lysine decarboxylase (ldc) gene controlled by the cauliflower mosaic virus (CaMV) 35S promoter expressed LDC activity up to 1 pkat/mg protein. Such activity was, for example, sufficient to increase cadaverine levels of the best line SR3/1-K1,2 from ca. 50 g (control cultures) to about 700 g/g dry mass. Some of the overproduced cadaverine of this line was used for the formation of anabasine, as shown by a 3-fold increase of this alkaloid. In transgenic lines with lower LDC activity the changes of cadaverine and anabasine levels were correspondingly lower and sometimes hardly distinguishable from controls. Feeding of lysine to root cultures, even to those with low LDC activity, greatly enhanced cadaverine and anabasine livels, while the amino acid had no or very little effect on controls and LDC-negative lines.     

PSTV infection in tobacco (Nicotiana tabacum L.) transformed with PSTV cDNA

Tobacco cv. White Burley was transformed with disarmed expression vector pCB1314 containing dimeric cDNA of potato spindle tuber viroid (PSTV, severe strain) in plus orientation regulated from the mannopine promoter. Amount of PSTV specific (?) and (+) sequences and PSTV circular forms was measured in transformed tobacco stock and compared with PSTV content in untransformed tomato and tobacco grafts. It follows from the results that lower rate of accumulation of PSTV in tobacco as compared with tomato is due to less intensive viroid transportation through the cytoplasm and/or cell to cell moverment, whereas both, viroid replication and processing showed comparable characteristics in tomato and tobacco with respect to accumulation of minus and plus strands and circular forms in infected tissues. Despite of accumulation of viroid in comparable amount in both transformed tobacco and infected tomato, no expression of any morphological symptom of disease was observed in transgenic tobacco.     

The biosynthesis of sesquiterpenoid phytoalexins in suspended callus cultures of Nicotiana tabacum

Suspended callus cultures of Nicotiana tabacum treated with commercial cellulase (ex Trichoderma viride) incorporated sodium [1,2-¹³C₂]acetate to give a¹³C-enrichment of ca 1.6% of the carbon content of debneyol.¹³C NMR data indicated that the angular methyl group in debneyol arises by migration from the C-10 position of a eudesmane-type intermediate.¹³C enrichments of 0.8% and 1.3% respectively were observed in phytuberol and phytuberin isolated from the cultures.     

Dynamics of root growth stimulation in Nicotiana tabacum in increasing light intensity

Light intensity is crucial for plant growth. In this study, the hypothesis was tested whether a sudden increase in light intensity leads to an immediate increase of root growth. Seedlings of Nicotiana tabacum grown in agar-filled Petri dishes were subjected to light intensities of 60 and 300 micromol m(-2) s(-1), respectively. Seedling biomass, sucrose, glucose and fructose concentration as well as primary root growth increased significantly with light intensity. The dynamics of the increase in root growth were analysed here in more detail. In transition experiments from low to high light intensities, root growth increased by a factor of four within 4 d, reaching the steady-state level measured in plants that were cultivated in high-light conditions. The distribution of relative elemental growth rates along the root growth zone retained a constant shape throughout this transition. During the first three hours after light increase, strong growth fluctuations were repeatedly observed with the velocity of the root tip cycling in a sinusoidal pattern between 120 and 180 microm h(-1). These dynamic patterns are discussed in the context of hydraulic and photosynthetic acclimation to the altered conditions. Experiments with externally applied sucrose and with transgenic plants having reduced capacities for sucrose synthesis indicated clearly that increasing light intensity rapidly enhanced root growth by elevating sucrose export from shoot to root.