Studies on the Chemical Regulation of Alkaloid Biosynthesis in Tobacco Plants

Effects of helminthosporol and helminthosporic acid (H-acid) on nicotine biosynthesis in Nicotiana were examined. By the addition of these compounds into nutrient solution, nicotine formation in decapitated and intact plants of Nicotiana tabacum was decreased. In sterile culture of excised roots of Nicotiana rustica, H-acid reduced the yield of nicotine exerting no effect on growth rate. Incorporation of l-glutamic acid-U-¹⁴C and dl-ornithine-2-¹⁴C into nicotine in the roots was decreased by the addition of H-acid, and that of nicotinic acid-³H(G) was not influenced. Since H-acid did not enhance destruction of nicotine-³H exogenously added, it is probable that the decrease of nicotine yield by the acid in the root culture is due to reduction in the production rate of the alkaloid.     

The effect of varying hormonal and precursor supplementations on levels of nicotine and related alkaloids in cell cultures ofNicotiana tabacum

Cell cultures ofNicotiana tabacum were grown on M & S medium containing 0–2.0 ppm of two auxins. Cellular nicotine was estimated for given auxin concentrations and highest levels determined. Feeding suspensions with amino acids either alone, or in combination with nicotinic acid and nicotinamide produced decresed nicotine levels. The presence of anatabine, anabasine, myosmine, anatalline and nicotelline in the cultures was established and it was noted that precursor feeding which decreased nicotine concentration caused elevated levels of the other alkaloids, notably anatabine.     

Variation in nicotine content of tobacco callus cultures

Plants ofNicotiana tabacum L. cv. Burley 21 which showed no difference in nicotine content were used to establish callus cultures. Cultures were initiated from different plants and from different leaves within each plant. The nicotine content of the calli was determined, and the results subjected to an analysis of variance. Differences between plants and differences within plants significantly affected the nicotine content of the cultures. The differences between plants were transmitted sexually and asexually, providing evidence that they are genetically determined. No such differences in nicotine content were found between the plants from which the cultures were established, indicating that nicotine production in vitro involves additional genes to those which are needed for nicotine production in the plant. The differences within plants were further investigated by establishing callus cultures from pith explants taken from different parts of the stem. Explants from apical pith tissue gave calli having far more nicotine and more roots than cultures derived from basal pith explants. This results may reflect the proximity of the apical pith explants to the site of auxin synthesis in the stem apex. Callus cultures derived from pith explants showed greater growth and nicotine production than those derived from leaf explants when the calli were induced on Murashige-Skoog medium containing -naphthalene acetic acid. This result is in conflict with the widely held belief that explants from different parts of the plant give cultures with similar yields of species-specific compounds.Abbreviations HN High nicotine - LN low nicotine     

Analysis of Nicotiana tabacum PIN genes identifies NtPIN4 as a key regulator of axillary bud growth

The plant‐specific PIN‐FORMED (PIN) auxin efflux proteins have been well characterized in many plant species, where they are crucial in the regulation of auxin transport in various aspects of plant development. However, little is known about the exact roles of the PIN genes during plant development in Nicotiana species. This study investigated the PIN genes in tobacco (Nicotiana tabacum) and in two ancestral species (Nicotiana sylvestris and Nicotiana tomentosiformis). Genome‐wide analysis of the N. tabacum genome identified 20 genes of the PIN family. An in‐depth phylogenetic analysis of the PIN genes of N. tabacum, N. sylvestris and N. tomentosiformis was conducted. NtPIN4 expression was strongly induced by the application of exogenous indole‐3‐acetic acid (IAA), but was downregulated by the application of ABA, a strigolactone analogue, and cytokinin, as well as by decapitation treatments, suggesting that the NtPIN4 expression level is likely positively regulated by auxin. Expression analysis indicated that NtPIN4 was highly expressed in tobacco stems and shoots, which was further validated through analysis of the activity of the NtPIN4 promoter. We used CRISPR‐Cas9 technology to generate mutants for NtPIN4 and observed that both T₀ and T₁ plants had a significantly increased axillary bud growth phenotype, as compared with the wild‐type plants. Therefore, NtPIN4 offers an opportunity for studying auxin‐dependent branching processes.     

T-DNA analysis of plants regenerated from hairy root tumors

Summary Plants regenerated from hairy root tumors induced on Nicotiana glauca and Nicotiana tabacum by Agrobacterium rhizogenes strain A4 were examined for the presence of T-DNA. Regenerated N. tabacum plants contained intact copies of both TL-DNA and TR-DNA. However, plants regenerated from N. glauca tumors did not contain the TR-DNA region corresponding to the tms (auxin synthesis) genes. Some of the regenerants exhibited an abnormal phenotype which is characterized by severe leaf wrinkling. This phenotype is correlated with the presence of TL-DNA, but not TR-DNA.     

Correlation between the nicotine content of tobacco plants and callus cultures

Callus cultures of two low-alkaloid lines of Nicotiana tabacum L. had considerably lower nicotine contents than cultures from the respective highalkaloid cultivars which were isogenic except for the two loci for alkaloid accumulation. Thus, there was a strong correlation between the nicotine content of callus cultures and the plants from which they were derived.     

Inhibitors of the carrier-mediated influx of auxin in suspension-cultured tobacco cells

 Active auxin transport in plant cells is catalyzed by two carriers working in opposite directions at the plasma membrane, the influx and efflux carriers. A role for the efflux carrier in polar auxin transport (PAT) in plants has been shown from studies using phytotropins. Phytotropins have been invaluable in demonstrating that PAT is essential to ensure polarized and coordinated growth and to provide plants with the capacity to respond to environmental stimuli. However, the function of the influx carrier at the whole-plant level is unknown. Our work aims to identify new auxin-transport inhibitors which could be employed to investigate its function. Thirty-five aryl and aryloxyalkylcarboxylic acids were assayed for their ability to perturb the accumulation of 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (1-NAA) in suspension-cultured tobacco (Nicotiana tabacum L.) cells. As 2,4-D and 1-NAA are preferentially transported by the influx and efflux carriers, respectively, accumulation experiments utilizing synthetic auxins provide independant information on the activities of both carriers. The majority (60%) of compounds half-inhibited the carrier-mediated influx of [¹⁴C]2,4-D at concentrations of less than 10 μM. Most failed to interfere with [³H]NAA efflux, at least in the short term. Even though they increasingly perturbed auxin efflux when given a prolonged treatment, several compounds were much better at discriminating between influx and efflux carrier activities than naphthalene-2-acetic acid which is commonly employed to investigate influx-carrier properties. Structure-activity relationships and factors influencing ligand specificity with regard to auxin carriers are discussed. Received: 28 June 1999 / Accepted: 28 August 1999     

Comparison of mechanisms controlling uptake and accumulation of 2,4-dichlorophenoxy acetic acid,naphthalene-1-acetic acid,and indole-3-acetic acid in suspension-cultured tobacco cells

Accumulation of radiolabelled naphthalene-1-acetic acid (1-NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and indole-3-acetic acid (IAA) has been measured in suspension-cultured tobacco (Nicotiana tabacum) cells. In this paper is presented a simple methodology allowing activities of the auxin influx and efflux carriers to be monitored independently by measuring the cellular accumulation of [³H]NAA and [¹⁴C]2,4-D. We have shown that 1-NAA enters cells by passive diffusion and has its accumulation level controlled by the efflux carrier. By contrast, 2,4-D uptake is mostly ensured by the influx carrier and this auxin is not secreted by the efflux carrier. Both auxin carriers contribute to IAA accumulation. The kinetic parameters and specificity of each carrier have been determined and new information concerning interactions with naphthylphthalamic acid, pyrenoylbenzoic acid, and naphthalene-2-acetic acid are provided. The relative contributions of diffusion and carrier-mediated influx and efflux to the membrane transport of 2,4-D, 1-NAA, and IAA have been quantified, and the data indicate that plant cells are able to modulate over a large range their auxin content by modifying the activity of each carrier.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 1-NAA naphthalene-1-acetic acid - 2-NAA naphthalene-2-acetic acid - NPA N-1-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid - V_m maximum transport capacity of the carrier In honour of Professor Dieter Klämbt's 65th birthdayThe authors thank Drs. A.E. Geissler and G.F. Katekar (CSIRO, Canberra City, Australia) for providing auxin efflux carrier inhibitors CPD, CPP, and PBA, and Dr. H. Barbier-Brygoo (Institut des Sciences Végétales, CNRS, Gif-sur-Yvette, France) for helpful discussions. This work was supported by funds from the Centre National de la Recherche Scientifique (UPR0040).     

Isolation in vitro of naphthaleneacetic acid-tolerant mutants of Nicotiana tabacum, which are impaired in root morphogenesis

Summary Mutants resistant to the toxic effect of the auxin analogue naphthaleneacetic acid have been selected in vitro in mutagenized populations of haploid mesophyll protoplasts of Nicotiana tabacum. Among the regenerated clones obtained, two clones impaired in root morphogenesis were further characterized. The parental mutant clones isolated were found to be heterozygotes for the mutations conferring the inability to root. These mutations were transmissible to the progeny as single nuclear dominant traits and cosegregated with resistance to naphthaleneacetic acid at the cellular level. Apart from the inability to root and a slightly modified leaf morphology, no obvious modification of stem structure, apical dominance, and flower fertility was detected in the development of homozygotes obtained in the progeny of one of the parental mutant clones when they were grafted onto normal plants.Abbreviations NAA 1-Naphthaleneacetic acid - PE plating efficiency - p-cells protoplast-derived dividing cells - MNNG N-methyl-N'-nitro-N-nitrosoguanidine - UV Ultraviolet light - 2,4-D 2,4-dichlorophenoxyacetic acid Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Acclimation of tobacco plantlets to ex vitro conditions as affected by application of abscisic acid

Plantlets of Nicotiana tabacum L. cv. Petit Havana SR1 were grown in vitro on Murashige and Skoog medium containing 2% saccharose, and then transplanted ex vitro into pots with coarse sand and Hewitt nutrient solution. In the first day after transplantation, the anti-transpirant abscisic acid (ABA; 0.01, 0.05 or 0.10 mM) was added to the substrate. Leaf stomatal conductance (gs), which was high in plants during the first days after transplantation similarly as in plantlets grown in vitro, was considerably decreased by ABA-treatment. However, in the further days gs decreased more quickly in control than in ABA-treated plants, and after 2 or 3 weeks gs was significantly lower than that of plantlets grown in vitro but similar in control and ABA-treated plants. Two weeks after transplantation, net photosynthetic rate, chlorophyll a + b content, maximal photochemical efficiency, and actual quantum yield of photosystem II in plant leaves were higher in comparison with those in plantlets grown in vitro. ABA-treatment had slight positive or insignificant effect on photosynthetic parameters and enhanced plant growth. Thus ABA application can alleviate 'transplant shock' and speed up acclimation of plantlets to ex vitro conditions.     

Amyloplast formation in cultured tobacco cells; effects of plant hormones on multiplication, size, and starch content

In BY-2 cultured tobacco cells (Nicotiana tabacum L.), depletion of 2,4-dichlorophenoxyacetic acid (2,4-D) and addition of benzyladenine (BA) caused amyloplast formation, a decrease in cell multiplication, and an increase in cell size. These changes were primarily triggered by the depletion of 2,4-D, and facilitated by the addition of BA. An increase in the starch content of BY-2 cells was always accompanied by a reduction in cell multiplication. However, when hormonal conditions were unsuitable for amyloplast formation, the starch content of the cells did not increase, even if cell multiplication was forcibly terminated by the addition of aphidicolin. This result indicates that the hormonal conditions themselves, and not the decrease in cell multiplication, induce amyloplast formation in BY-2 cultured tobacco cells.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 4,6-diamidino-2-phenylindole - DMSO dimethyl sulfoxide - NAA 1-naphthylacetic acid - PMSF phenyl methyl sulfonyl fluoride     

Short-Lived and Phosphorylated Proteins Contribute to Carrier-Mediated Efflux, but Not to Influx, of Auxin in Suspension-Cultured Tobacco Cells

Auxin is transported across the plasma membrane of plant cells by diffusion and by two carriers operating in opposite directions, the influx and efflux carriers. Both carriers most likely play an important role in controlling auxin concentration and distribution in plants but little is known regarding their regulation. We describe the influence of modifications of the transmembrane pH gradient and the effect of agents interfering with protein synthesis, protein traffic, and protein phosphorylation on the activity of the auxin carriers in suspension-cultured tobacco (Nicotiana tabacum L.) cells. Carrier-mediated influx and efflux were monitored independently by measuring the accumulation of [¹⁴C]2,4-dichlorophenoxyacetic acid and [³H]naphthylacetic acid, respectively. The activity of the influx carrier decreased on increasing external pH and on decreasing internal pH, whereas that of the efflux carrier was only impaired on internal acidification. The efflux carrier activity was inhibited by cycloheximide, brefeldin A, and the protein kinase inhibitors staurosporine and K252a, as shown by the increased capability of treated cells to accumulate [³H]naphthylacetic acid. Kinetics and reversibility of the effect of brefeldin A were consistent with one or several components of the efflux system being turned over at the plasma membrane with a half-time of less than 10 min. Inhibition of efflux by protein kinase inhibitors suggested that protein phosphorylation was essential to sustain the activity of the efflux carrier. On the contrary, the pharmacological agents used in this study failed to inhibit [¹⁴C]2,4-dichlorophenoxyacetic acid accumulation, suggesting that rapidly turned-over proteins or proteins activated by phosphorylation are not essential to carrier-mediated auxin influx. Our data support the idea that the efflux carrier in plants constitutes a complex system regulated at multiple levels, in marked contrast with the influx carrier. Physiological implications of the kinetic features of this regulation are discussed.     

Influence of accumulated phosphate on culture growth and formation of cinnamoyl putrescines in medium-induced cell suspension cultures of Nicotiana tabacum

The inorganic phosphate of the liquid nutrient medium was completely taken up by freshly inoculated cells of Nicotiana tabacum L. within the first 2 d of culture. Thus intracellular ortho-phosphate concentrations of approx. 0.06 M were accumulated, which upon growth of the cultures were diluted by cell division and subsequent cell growth. Cells from different stages of the growth cycle containing progressively decreasing levels of phosphate were transferred to a phosphate-free medium which normally stimulates the formation of cinnamoyl putrescines. The resulting accumulation of these compounds was inversely correlated with the intracellular phosphate level, whereas a direct linear relationship in the phosphate concentration was found with further growth in the phosphate-free medium.Abbreviations 2,4D 2,4-dichlorophenoxyacetic acid - FW fresh weight - DW dry weight - MS-medium Murashige-Skoog-medium (Murashige and Skoog 1962)     

Effect of abscisic acid on photosynthetic parameters during <Emphasis Type="Italic">ex vitro</Emphasis> transfer of micropropagated tobacco plantlets

The aim of this research was to determine whether exogenous abscisic acid (ABA) applied immediately after ex vitro transfer of in vitro grown plants can improve their acclimatization. Tobacco (Nicotiana tabacum L.) plantlets were transferred into pots with Perlite initially moistened either by water or 50 μM ABA solution and they were grown under low (LI) or high (HI) irradiance of 150 and 700 μmol m⁻² s⁻¹, respectively. Endogenous content of ABA in tobacco leaves increased considerably after ABA application and even more in plants grown under HI. Stomatal conductance, transpiration rate and net photosynthetic rate decreased considerably 1 d after ex vitro transfer and increased thereafter. The gas exchange parameters were further decreased by ABA application and so wilting of these plants was limited. Chlorophyll (a+b) and β-carotene contents were higher in ABA-treated plants, but the content of xanthophyll cycle pigments was not increased. However, the degree of xanthophyll cycle pigments deepoxidation was decreased what also suggested less stress in ABA-treated plants. No dramatic changes in most chlorophyll a fluorescence parameters after ex vitro transfer suggested that the plants did not suffer from restriction of electron transport or photosystem damage.     

Production and Analysis of Tobacco Transgenic Plants Expressing the Agrobacterial Gene for Tryptophan Monooxygenase

The expression of the agrobacterial iaaM gene for tryptophan monooxygenase, the enzyme catalyzing the first step in the auxin biosynthesis, induced substantial physiological and biochemical changes in transgenic tobacco (Nicotiana tabacum L.) plants. All lines of transgenic plants grown in vitro manifested abnormal phenotypes: enhanced root formation, adventitious roots on stems, and curled leaves. When grown in vivo, plants manifested abnormal, normal, or intermediate phenotype. Under conditions of a greenhouse, the abnormal plants contained the highest amount of auxins in their leaves and manifested an increased number of adventitious roots, poor reproductivity, and the loss in seed germination. Transgenic plants with the normal phenotype did not substantially differ from the wild-type plants in their morphology, and their auxin content was lower than in the abnormal plants. The intermediate-phenotype plants were devoid of some morphological properties characteristic of the abnormal plants. Only the seeds of normal- and intermediate-phenotype transgenic plants germinated at a high rate.     

Auxin Conjugation by Tobacco Mesophyll Protoplasts : Correlations between Auxin Cytotoxicity under Low Density Growth Conditions and Induction of Conjugation Processes at High Density

Auxin induction of the proliferation of Nicotiana tabacum (cv Xanthi) mesophyll protoplasts and of protoplast-derived cells was studied. The growth-promoting properties and cytotoxicities at high concentrations of IAA and naphthaleneacetic acid were strongly affected by cell density. The induction of growth by 2,4-dichlorophenoxyacetic acid and picloram was not affected by cell density. The comparison of catabolism of these [¹⁴C]-labeled auxins by protoplasts showed that IAA and naphthalene-acetic acid were rapidly accumulated and conjugated unlike 2,4-dichlorophenoxyacetic acid and picloram. The major catabolite derived from naphthaleneacetic acid was identified as naphthaleneacetyl-l-aspartate. The biosynthesis of this conjugate in protoplasts was inducible by naphthaleneacetic acid concentrations found to be cytotoxic under low density growth conditions. However, although it was taken up by cells, the conjugate was not cytotoxic at concentrations as high as 0.2 mm under low density growth conditions. The relationship between conjugation processes and auxin cytotoxicity is discussed.     

Influence of Auxin-Like Herbicides on Tobacco Mosaic Virus Multiplication

Tobacco (Nicotiana tabacum L., cv. Samsun) leaf discs inoculated with tobacco mosaic virus (TMV) were treated with auxin-like herbicides 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid (MCPA), 3-amino-1,2,4-triazol (Amitrol) and 6-chloro-2-ethylamino-4-isopropylamino-1,3,5-triazine (Atrazin). All herbicides in the concentration of 10^–7 M enhanced the virus content (MCPA to 227.4 %, Amitrol to 218.1 % and Atrazin to 257.3 % of values found in TMV-infected, herbicide untreated discs). The 2,4-D alone did not affect the activity of the glucose-6-phosphate dehydrogenase and ribonucleases, but the 2,4-D treatment together with TMV infection raised their activities twice as high as in the untreated control discs. Polyacrylamide gel electrophoresis of acidic extracellular proteins washed from leaf discs treated with 2,4-D did not prove the induction of PR-proteins.     

The effect of auxin starvation on the growth of auxin-dependent tobacco cell culture: dynamics of auxin-binding activity and endogenous free IAA content

The growth of a cell strain derived from the stem pith of tobacco(Nicotiana tabacum L., cv. Virginia Bright Italia) was investigatedin subcultures grown at various levels of synthetic auxins.Both partial and complete auxin starvation resulted in a decreaseof the frequency of cell division. For these treatments theendogenous free indole-3-acetic acid content increased substantiallyat the commencement of the exponential growth phase. The possibilitythat the receptivity of the cells to auxin changed during thegrowth cycle was examined by measuring the activity of a membrane-boundauxin-binding site. In subcultures grown in a medium with anoptimal auxin concentration the maximum auxin-binding activitywas restricted to the end of the exponential growth phase. Inthe cells cultivated in partially or completely auxin deprivedmedia the auxin-binding activity increased to varying extents.These results probably reflect mechanisms controlling both theintracellular content of free auxin and the sensitivity of thecells to exogenous auxin supply (including auxin binding) withrespect to the cell division and/or growth Key words: Nicotiana tabacum L., plant cell culture, IAA, auxin-binding site, cell division