Ribosylzeatin and zeatin in tobacco crown gall tissue

Cytokinins were extracted from two cultures of tobacco crown gall tumor tissue: an unorganized tissue and a teratoma which produced leafy shoots. On Sephadex LH-20 column chromatography, extracts of both types of tissue yielded two peaks of cytokinin activity with elution volumes similar to ribosylzeatin and zeatin. Ribosylzeatin and zeatin were detected and quantified by coupled gas chromatography — mass spectrometry selected ion monitoring (GC/MS SIM), comparable quantities being found in the two extracts. Full mass spectral evidence for the presence of ribosylzeatin in both tissues was obtained. No evidence was found for the presence of N⁶-(²-isopentenyl)adenosine (i⁶Ade) or N⁶-(²-isopentenyl)adenine (i⁶Ade) although these compounds have been reported to occur in cytokinin-habituated tobacco callus tissues.Abbreviations BAP 6-benzylaminopurine - GC gas chromatography - GC/MS coupled gas chromatography-mass spectrometry - i⁶ Ade N⁶-(²-isopentenyl)adenine - i⁶ Ado N⁶-(²-isopentenyl)adenosine - RFE rotary film evaporation - SIM selected ion monitoring - TLC thin-layer chromatography - TMS trimethylsilyl     

Acid invertase in Nicotiana tabacum crown-gall cells: Molecular properties of the cell-wall isoform

Cell-wall invertase (CWI; EC 3.2.1.26) was salt-eluted from non-disrupted Agrobacterium tumefaciens-transformed Nicotiana tabacum L. cells and purified to homogeneity. More than 90% of total cellular invertase activity (measured at pH 4.8) was recovered in the NaCl-eluted fraction whereas for the cytoplasmic marker glucose-6-phosphate dehydrogenase 96% of total activity could be extracted from the tissue after salt-elution, indicating absence of appreciable stress-induced cell disruption. Likewise, appreciable contamination of CWI with vacuolar acid invertase could be excluded. Tobacco CWI cross-reacted with an antiserum directed against deglycosylated carrot CWI; however, during some purification steps CWI enzyme activity did not correlate with CWI immunosignal. In fractions of low CWI activity and strong immunosignal, a putative inhibitor peptide with an apparent M_r of 17 kDa was detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining (Weil et al. 1994, Planta 193, 438–445). The CWI of transformed tobacco cells has an apparent M_r of 69 kDa (SDS-PAGE) and is a basic (pI 9.5) glycoprotein. Gel-permeation chromatography indicated that enzymatically active CWI is a monomer. Deglycosylation of the denatured CWI by treatment with endo--N-acetylglucosaminidase, peptide-N-glycosidase F and trifluoromethanesulfonic acid indicated the presence of two high-mannose and two complex glycans. In partially purified CWI fractions the carrot CWI antiserum cross-reacted with one other tobacco cell-wall peptide (M_r 28 kDa). To address the possibility of a second invertase isoenzyme cross-reacting with the carrot antiserum, intact CWI and the 28-kDa peptide were digested in vitro with Staphylococcus aureus V8 protease and cyanogen bromide. A comparison of the resulting peptide patterns identified the 28-kDa polypeptide as a cleavage product of CWI. Running electroeluted CWI (69 kDa) on a second SDS-polyacrylamide gel led to substantial formation of the 28-kDa peptide. This suggests that the intrinsic 28-kDa cleavage product is the result of an intrinsic lability of tobacco CWI, rather than being a proteolytic degradation product.Abbreviations Con A concanavalin A - CWI cell-wall invertase - Endo H endo--N-acetylglucosaminidase - Glc-6-P-DH glucose-6-phosphate dehydrogenase - 1-OMG methyl -d-glucopyranoside - p17 17 kDa peptide - pI isoelectric point - PNGase F peptide-N-glycosidase F - TFMS trifluoromethanesulfonic acid This work was supported by grants from the Deutsche Forschungsgemeinschaft. The gift of an antiserum directed against carrot cellwall invertase from Dr. Arnd Sturm (Friedrich-Miescher-Institut, Basel, Switzerland) is kindly acknowledged. Furthermore, the authors thank Sigrid Ranostaj for excellent technical assistence.     

Auxin autonomy in cultured tobacco teratoma tissues transformed by an auxin-mutant strain ofAgrobacterium tumefaciens

We have studied the mechanism of auxin autonomy in tobacco (Nicotiana tabacum L.) crowngall tissues transformed by the auxin-mutant (tms ^–) A66 strain of Agrobacterium tumefaciens. Normally, tms ^– tobacco tumor tissues require the formation of shoots to exhibit auxin-independent growth in culture. We have isolated from tms ^– tobacco cells several stable variants that are fully hormone-independent and grow rapidly as friable, unorganized tissues, thus mimicking the growth and morphology of tms ⁺ tobacco cells that produce high levels of auxin. However, none of the variants contained the high levels of auxin found in tms ⁺ tumor cells. The variants could be divided into two classes with respect to their response to applied auxin. The first class was highly sensitive to applied auxin: low concentrations (1 M) of -naphthaleneacetic acid (NAA) severely inhibited growth and markedly stimulated the accumulation of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). The second class of variants showed a low sensitivity to applied auxin: growth was promoted by concentrations of NAA up to 10 M, and growth inhibition and high ACC levels were observed only at high NAA concentrations (100 M). Unorganized variants with low auxin sensitivity were also isolated from a variant line with high auxin sensitivity. The isolation of tumor cells that exhibited the growth phenotype of tms ⁺ cells while retaining the low auxin content and low auxin sensitivity of tms ^– cells indicates that full hormone autonomy, characteristic of wild-type crown-gall tumors, can be achieved by a mechanism that is independent of changes in the auxin physiology of the cells.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - IAA indole-3-acetic acid - MACC N-malonyl ACC - NAA naphthaleneacetic acid - tms tumor morphology shooty, the auxin biosynthesis locus of Agrobacterium Ti plasmids The authors thank Dr. Andrew Binns (University of Pennsylvania, Philadelphia, USA) for providing cell lines TA6-5 and TA66C3-78, and Mr. James Dacey for preparation of the composite photograph used in Fig. 1. Support for this work by the National Science Foundation (DMB84-17087) and the U.S. Department of Agriculture (86-CRCR-1-2150) is gratefully acknowledged.     

Cytokinins in transfer RNA of normal and crown-gall tissue of Vinca rosea

cis-Zeatin riboside was identified in transfer-RNA hydrolysates from both normal and crown-gall tissue of Vinca rosea L. The trans-isomer was associated exclusively with the crowngall transfer-RNA. The importance of these observations is discussed in relation to biosynthesis of free cytokinins.Abbreviations GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - TLC thin-layer chromatography - TMSi trimethylsilyl - tRNA transfer RNA - ZR zeatin riboside     

Mass-spectrometric quantification of cytokinin nucleotides and glycosides in tobacco crown-gall tissue

the cytokinins of tobacco crown-gall tissue have been analysed by quantitative mass spectrometry using ²H₂-labelled cytokinin riboside 5-monophosphates and ¹⁵N₄-labelled cytokinin glycosides as internal standards. The principal endogenous cytokinin of this tissue is zeatin riboside 5-monophosphate. The biologically inactive 7-glucoside of zeatin is the most abundant basic cytokinin in the tissue. These findings expose the limitations of previously reported analyses of similar tissues, which were restricted to biologically active basic cytokinins. The present study demonstrates that the endogenous cytokinins of tobacco crowngall tissue show a clear correspondence to the range of metabolites formed when exogenous cytokinins are supplied to nontumorous tobacco cells.Abbreviations DHZ dihydrozeatin - DHZ7G dihydrozeatin 7-glucoside - DHZMP dihydrozeatin 9-riboside 5-monophosphate - DHZR dihydrozeatin 9-riboside - GC-MS coupled gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - Z7G zeatin 7-glucoside - Z9G zeatin 9-glucoside - ZOG zeatin O-glucoside - ZMP zeatin 9-riboside 5-monophosphate - ZR zeatin 9-riboside - ZROG zeatin 9-riboside O-glucoside     

Mass-spectrometric quantitation of cytokinins in tobacco crown-gall tumours induced by mutated octopine Ti plasmids of Agrobacterium tumefaciens

The levels of the major cytokinins, zeatin, zeatin riboside, zeatin riboside-5-monophosphate and zeatin-7-glucoside were measured in tobacco (Nicotiana tabacum L.) crown-gall tissues carrying insertion and deletion mutations in the T-DNA. Measurements were made by combined gas chromatography-mass spectrometry using selected ion monitoring with ¹⁵N- and ²H-labelled internal standards. The results demonstrate that, relative to wild-type tumour tissue, cytokinin levels are considerably elevated in tissues lacking functional T-DNA auxin-biosynthetic genes. From a detailed analysis of the major cytokinin metabolites it is concluded that a reduction in the extent of cytokinin degradation via N⁶-side-chain cleavage is an important factor leading to increased cytokinin levels in these tissues.Abbreviations IAA indole-3-acetic acid - SIM selected ion monitoring - Z zeatin - [7G]Z zeatin-7-glucoside - [9R]Z zeatin-9-riboside - [9R-5P]Z zeatin riboside-5-monophosphate     

Neoplastic progression in crown gall in tobacco without elevated auxin levels

We have isolated two stable variants from a crown-gall teratoma tissue of tobacco (Nicotiana tabacum L.) transformed by Agrobacterium tumefaciens strain A66, a mutant of the virulent A6 strain containing an insertion sequence in the tumor-inducing (Ti) plasmid at the locus coding for auxin biosynthesis. Normally tobacco cells transformed by strain A66 spontaneously form shoots in culture and will not grow on hormone-free medium unless shoots develop. The variant tissue lines, isolated from the teratoma tissue after prolonged culture in the dark, grew as friable and unorganized tissues on hormone-free growth medium. Growth of the variants was more sensitive to auxin feeding than growth of the parental teratoma line, and the auxin dose-response curves of the variant lines were similar to those obtained with A6-transformed tobacco cells. Southern blot analysis of DNA from the parental teratoma line and one of the variants showed no differences in copy number or organization of the oncogenic DNA sequence (T-DNA) transferred from the bacterium, indicating that the variant phenotype did not result from reversion of the A66 mutation. Radio-immunoassay analysis showed similar levels of indole-3-acetic acid (IAA) in the variants and parental teratoma line (3–50 and 38–42 pmol·(gFW)^-1, respectively), whereas an A6-transformed cell line contained much higher IAA levels (150–1200 pmol·(g FW)^-1). Low levels of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid in the variants and the parental teratoma line (<5 nmol·(g FW)^-1) as compared with that found in the A6-transformed line (>100 nmol· (g FW)^-1) provided additional, indirect evidence for low auxin levels in the variant lines. These results indicate that crown-gall teratoma tissues of tobacco may switch to the unorganized, auxin-sensitive phenotype without an increase in auxin content.Abbreviations IAA indole-3-acetic acid - kb kilobase - NAA -naphthalene acetic acid - NAM -naphthaleneacetamide - T-DNA DNA transferred from the Ti plasmid to the plant - T_L-DNA the left transferred region of pTiA6 containing the T-DNA oncogenes     

Studies on the use of toxic precursor analogs of opines to select transformed plant cells

Summary S-(2-aminoethyl-)L-cysteine and L-canavanine were less toxic for octopine-type crown gall tissues that contained lysopine dehydrogenase than for other crown gall or habituated tissues. These analogs are substrates for lysopine dehydrogenase in vitro and in vivo. Thus toxic analogs of amino acid precursors of opines may be useful in selecting for cells that contain an opine dehydrogenase.     

Ethylene production by shoot-forming and unorganized crown-gall tumor tissues of Nicotiana and Lycopersicon cultured in vitro

We have studied ethylene biosynthesis in cloned crown-gall cell lines of Nicotiana tabacum L., N. glutinosa L., and Lycopersicon esculentum (L.) Mill. transformed by the A6 strain of Agrobacterium tumefaciens (Smith and Townsend) Conn. or a tms (shooty) mutant strain, A66. Both the synthesis of the ethylene precursor 1-aminocyclo-propane-1-carboxylic acid (ACC) and the conversion of ACC to ethylene were affected by crown-gall transformation. All A6-transformed cell lines contained about 50 times more ACC than the A66-transformed cell lines, indicating that the tms genes stimulate ACC synthesis. On the other hand, A6-transformed N. tabacum and L. esculentum cell lines showed a very low capacity to convert ACC to ethylene when compared with A66-transformed cells of the same species. These differences in ACC-dependent ethylene formation were stable and could not be modified by supplying auxin to the culture medium. In contrast, both the A6- and A66-transformed N. glutinosa cell lines showed a low capacity for ACC-dependent ethylene production. Thus, the low-ethylene-forming phenotype did not seem to be under direct control of the tms genes and appeared to be part of the host response to crown-gall transformation. All cell lines exhibiting the low-ethylene-forming phenotype grew as unorganized tissues in culture, whereas cell lines showing a high capacity to convert ACC to ethylene formed shoots. Thus, ACC-dependent ethylene formation may be useful for studying host factors important in determining tumor phenotype.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - NAA -naphthalencacetic acid     

Regulation of developmental pathways in cultured microspores of tobacco and snapdragon by medium pH

The regulation of developmental pathways in cultured microspores of tobacco (Nicotiana tabacum L) and snapdragon (Antirrhinum majus L) by medium pH is described for the first time. Unicellular tobacco and snapdragon microspores developed into normal, fertile pollen when cultured in media T1 and AT3 at pH 7.0 and 25°C for 6 and 8 days, respectively. First, pollen mitosis was asymmetric and mature pollen grains were filled with starch granules and germinated upon transfer to a germination medium. However, when tobacco and snapdragon microspores were cultured in media T1 and AT3, respectively, at pH 8.0–8.5 for 4–6 days at 25 °C, the frequency of symmetric division increased significantly with the formation two nuclei of equal size, and the gametophytic pathway was blocked, as seen by the lack of starch accumulation and the inhibition of pollen germination. The transfer of these microspores to embryogenesis medium AT3 at pH 6.5 resulted in the formation of multicellular structures in both species and, in tobacco, in the formation of embryos and plants. In order to understand the possible mechanisms of the action of high pH, sucrose metabolism was analysed in isolated microspores of tobacco cultured at various pH values. Invertase (EC 3.2.1.26) activity in microspores was maximal at pH 5.0 and strongly decreased at higher pH, leading to a slow-down of sucrose cleavage. At the same time the incorporation of ¹⁴C-labelled sucrose from the medium into microspores was drastically reduced at high pH. These data suggest that isolated microspores are not able to metabolise carbohydrates at high pH and thus undergo starvation stress, which was shown earlier to block the gametophytic pathway and trigger sporophytic development.     

Phenotypic and genetic variations in crown-gall tumour cells of tobacco

Summary Phenotypic and genetic variations of tumour cells were analysed both quantitatively and qualitatively in clones and subclones of a crown-gall strain. Thus, growth rates, grafting tests, octopine synthesis, estimations of the T-DNA contents, modifications in the numbers, and structures of chromosomes were examined. Phenotypic variations are closely associated with genetic changes, including variation in chromosome number (which is shown to be non-specific to the tumoral state) and, above all, variation in the copy-number of T-DNA sequences per cell, and structural rearrangements of chromosomes. Such rearrangements are characterized by specific marker chromosomes in the tumour cells and they correlate with the degree of oncogenicity of the cells.     

Integration of Agrobacterium T-DNA into a tobacco chromosome: Possible involvement of DNA homology between T-DNA and plant DNA

Summary We established tobacco tumour cell lines from crown galls induced by Agrobacterium. Restriction fragments containing T-DNA/plant DNA junctions were cloned from one of the cell lines, which has a single copy of the T-DNA in a unique region of its genome. We also isolated a DNA fragment that contained the integration target site from nontransformed tobacco cells. Nucleotide sequence analyses showed that the right and left breakpoints of the T-DNA mapped ca. 7.3 kb internal to the right 25 by border and ca. 350 by internal to the left border respectively. When the nucleotide sequences around these breakpoints were compared with the sequence of the target, significant homology was seen between the region adjacent to the integration target site and both external regions of the T-DNA breakpoints. In addition, a short stretch of plant DNA in the vicinity of the integration site was deleted. This deletion seems to have been promoted by homologous recombination between short repeated sequences that were present on both sides of the deleted stretch. Minor rearrangements, which included base substitutions, insertions and deletions, also took place around the integration site in the plant DNA. These results, together with previously reported results showing that in some cases sequences homologous to those in T-DNA are present in plant DNA regions adjacent to left recombinational junctions, indicate that sequence homology between the incoming T-DNA and the plant chromosomal DNA has an important function in T-DNA integration. The homology may promote close association of both termini of a T-DNA molecule on a target sequence; then TDNA may in some cases be integrated by a mechanism at least in part analogous to homologous recombination.Shogo Matsumoto is on leave from Biochemical Research Institute, Nippon Menard Cosmetic Co., Ltd, Ogaki, Gifu-ken 503, Japan     

DNA-dependent RNA polymerase II from nuclei of suspension-cultured tobacco cells

From isolated nuclei of suspension cultured cells of Nicotiana tabacum. DNA-dependent RNA polymerase II (E.C. 2.7.76) has been purified to homogeneity as evidenced by polyacrylamidegel electrophoresis under non-denaturing conditions. The purified enzyme had a specific activity of more than 15 nmol min^-1·mg^-1 with denatured calf thymus DNA as template. Sodium-dodecyl-sulfate gel electrophoresis and protein highperformance liquid chromatography revealed a subunit composition of four proteins with molecular weights of 165 000, 135 000, 35 000 and 25 000 and with a stoichiometry of 1:1:2:2. The RNA polymerase did not exhibit any detectable proteinkinase activity. The 25 000 subunit binds ADP in a molar ratio of 1:1; it could not be decided whether this subunit has an ATPase activity or is merely an acceptor of ADP.Abbreviations HPLC high-performance liquid chromatography - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate This contribution is dedicated to Professor Fritz Cramer on the occasion of his 60th birthday     

Cytoplasmic acidification as an early phosphorylation-dependent response of tobacco cells to elicitors

Elicitor-induced cytoplasmic pH changes of tobacco (Nicotiana tabacum L. cv. Xanthi) cells grown in suspension cultures were explored under a variety of conditions by using a flexible technique based on the distribution of [¹⁴C] benzoic acid between the intracellular and extracellular compartments. Comparison of data obtained by this technique and by ³¹P-nuclear magnetic resonance spectrometry qualifies the benzoic acid distribution method as a convenient and reliable way to probe cytoplasmic pH variations. Various elicitors shown to induce several defense-related responses in tobacco cells, namely oligogalacturonides of degree of polymerization 7–20, pectolyase from Aspergillus japonicus, Phytophthora megasperma crude elicitors and purified cryptogein, triggered cytoplasmic acidifications differing in intensity and kinetics according to the signal molecule. In contrast, no changes in cytoplasmic protons and external pH were observed in cells treated with short galacturonide oligomers, or with soybean-specific hepta -glucoside from P. megasperma, which are devoid of elicitor activity in tobacco cells. The oligogalacturonide-induced cytoplasmic acidification was inhibited by two structurally unrelated protein kinase inhibitors, staurosporine and 6-dimethylaminopurine, which both reduced the external alkalinization response to the elicitor. The protein phosphatase inhibitor calyculin A alone behaved as an elicitormimicking molecule in triggering cytoplasmic acidification, again associated with extracellular alkalinization. These results indicate that the increase in the cytoplasmic concentration of protons may be considered as a common early intracellular response of tobacco cells to elicitors, associated with the extracellular alkalinization response and controlled by protein phosphorylation.Abbreviations BA(H) benzoic acid (protonated form) - 6-DMAP 6-dimethylaminopurine - DP degree of polymerization - Mes 2-(N-morpholino)ethanesulfonic acid - OG oligogalacturonide - pHc cytoplasmic pH - ³¹P-NMR nuclear magnetic resonance spectroscopy of ³¹P atoms The authors thank P. Albersheim (CCRC, Athens, Georgia, USA) for providing the purified oligogalacturonides and the hepta -glucoside and P. Ricci (INRA, Antibes, France) for providing the purified cryptogein.     

Cold-sensitive expression of cytokinin habituation by tobacco pith cells in culture

Cloned, cytokinin-habituated tissues of Nicotiana tobacum L. cv. Havana 425 are able to grow in culture at 25° C without added cytokinin. These tissues vary in their expression of the habituated phenotype at 16° C. When cytokinin-requiring pith tissues are converted to the habituated state by 35° C treatment, all of the habituated cells are cold sensitive. After several transfers in culture, some of these habituated cells give rise to stable, cold resistant variants. Both phenotypes are inherited by individual cells. Cold sensitive clones at 16° C and non-habituated clones at 16° C as well as 25° C show the same dose response to the cytokinin, kinetin. This suggests that at the physiological level, cold sensitivity results from a decreased production of cell division factors rather than from a decreased affinity of cellular receptors for these factors.Abbreviations CDF Cell division factor(s) - NAA -naphthalencacetic acid     

Biosynthesis of nicotianamine in the suspension-cultured cells of tobacco (Nicotiana megalosiphon)

Summary Seven kinds of suspension cell cultures from five species ofNicotiana were screened for the occurrence of nicotianamine. Nicotianamine was detected in the cultured cells ofN. megalosiphon andN. plumbaginifolia.l-[l-¹⁴C]Methionine, which is the precursor of the mugineic-acid-family phytosiderophores and nicotianamine in barley plants, was incorporated into nicotianamine by the cultured cells ofN. megalosiphon both in vivo and in vitro. The advantage of the cultured tobacco cells for the study of the biosynthesis of nicotianamine and the mugineic-acid-family phytosiderophores is discussed.     

Wild species of Nicotiana as a new source of tobacco resistance to the tobacco hornworm Manduca sexta

Sixteen species of Nicotiana were examined for resistance to second instar larvae of the tobacco hornworm, Manduca sexta (L.). The tests were designed so as to discriminate between antibiosis and nonpreference. High levels of antibiosis resistance were observed in several wild species such as N. gossei and species in the Repandae section. Several other species, such as N. excelsior, showed antibiosis that appeared to be due to a different mechanism than the alkaloid-trichome exudate based resistance of the above species. The data indicate that these species of Nicotiana may be used as a new source of resistance to larvae of M. sexta.

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D'espèces sauvages de Nicotiania comme nouvelle source de résistance du tabac à Manduca sexta

Résumé La résistance aux chenilles de second stade de Manduca sexta L. a été examinée chez seize espèces de Nicotiana. Les essais ont été conçus pour distinguer absence d'appétance et antibiose. Plusieurs espèces sauvages comme N. gossei et les espèces de la section Repandae ont présenté un niveau élevé d'antibiose. D'autres espèces, comme N. excelsior ont présenté une antibiose vraisemblablement due à un mécanisme différent de la résistance des espèces précédentes provoquée par un exsudat alcaloïde des trichomes. Les résultats indiquent que ces espèces de Nicotiana peuvent être utilisées comme nouvelle source de résistance aux chenilles de M. sexta.