Hormonal control of tobacco crown gall tumor morphology

The endogenous levels of auxin and cytokinin in teratoma and unorganized tobacco (Nicotiana tabacum L. var Wisconsin #38) crown gall tumor tissues were determined. Teratoma tissues contain levels of auxin and cytokinin favorable for shoot formation, whereas unorganized tumors contain levels of auxin that suppress shoot formation. This conclusion is based upon the observation that when levels of auxin and cytokinin similar to those found in a teratoma were added to the growth medium of nontumorous tobacco tissue, shoot formation resulted; when levels similar to those found in unorganized tumors were added, the normal tissue grew as unorganized callus.     

Physiology of Hormone Autonomous Tissue Lines Derived From Radiation-Induced Tumors of Arabidopsis thaliana

γ-Radiation-induced tumors of Arabidopsis thaliana L. have been produced as a novel approach to isolation of genes that regulate plant development. Tumors excised from irradiated plants are hormone autonomous in culture and have been maintained on hormone-free medium for up to 4 years. Five tumor tissue lines having different morphologies and growth rates were analyzed for auxin, cytokinin, and 1-aminocyclopropane-1-carboxylic acid (ACC) content, ethylene production, and response to exogenous growth regulators. Normal tissues and two crown gall tissue lines were analyzed for comparison. Rosettes and whole seedlings each contained approximately 30 nanograms· (gram fresh weight)⁻¹ free indoleacetic acid (IAA), 150 nanograms· (gram fresh weight)⁻¹ ester-conjugated IAA, and 10 to 20 micrograms· (gram fresh weight)⁻¹ amide-conjugated IAA. The crown gall lines contained similar amounts of free and ester-conjugated IAA but less amide conjugates. Whereas three of the radiation-induced tumor lines had IAA profiles similar to normal tissues, one line had 10- to 100-fold more free IAA and three- to 10-fold less amide-conjugated IAA. The fifth line had normal free IAA levels but more conjugated IAA than control tissues. Whole seedlings contained approximately 2 nanograms· (gram fresh weight)⁻¹ of both zeatin riboside and isopentenyladenosine. The crown gall lines had 100- to 1000-fold higher levels of each cytokinin. In contrast, the three radiation-induced tumor lines analyzed contained cytokinin levels similar to the control tissue. The radiation-induced tumor tissues produced very little ethylene, although each contained relatively high levels of ACC. Normal callus contained similar amounts of ACC but produced several times more ethylene than the radiation-induced tumor lines. Each of the radiation-induced tumor tissues displayed a unique set of responses to exogenously supplied growth regulators. Only one tumor line showed the same response as normal callus to both auxin and cytokinin feeding. In some cases, one or more tumor lines showed increased sensitivity to certain growth substances. In other cases, growth regulator feeding had no significant effect on tumor tissue growth. Morphology of the radiation-induced tumor tissues generally did not correlate with auxin to cytokinin ratio in the expected manner. The results suggest that a different primary genetic event led to the formation of each tumor and that growth and differentiation in the tumor tissue lines are uncoupled from the normal hormonal controls.     

INDOLE ACETIC ACID OXIDASE AND PEROXIDASE ACTIVITIES IN NORMAL AND CROWN GALL TISSUE CULTURES OF PARTHENOCISSUS TRICUSPIDATA

Lipetz , Jacques , and Arthur W. Galston . (Yale U., New Haven.) Indole acetic acid oxidase and peroxidase activities in normal and crown gall tissue cultures of Parthenocissus tricuspidata. Amer. Jour. Bot. 46(3) : 193-196. Illus. 1959.—Normal and crown gall cells of P. tricuspidata grown in pure culture were examined for IAA oxidase and peroxidase activities. No IAA oxidase activity could be demonstrated in dialyzed or undialyzed homogenates of either tissue; however, crown gall tissue, but not normal tissue, was found to produce an extracellular IAA oxidase which required Mn⁺⁺ and DCP as co-factors. Normal tissue, but not crown gall tissue was found to contain high levels of substances which spared IAA from destruction by a pea IAA oxidase preparation. Peroxidase activity was found to be higher in normal than in crown gall homogenates, but crown gall tissue released considerably more peroxidase into the external medium. The differences in the auxin requirements and growth rate between normal and crown gall cells appear not to be easily explicable in terms of differential auxin destruction.     

Differential accumulation of proteinase inhibitor I in normal and crown gall tissue of tobacco, tomato, and potato

A proteinase inhibitor (inhibitor I) is induced in crown gall tumors of tobacco (Nicotiana tabacum) initiated through infection with the tumorinducing bacterium, Agrobacterium tumefaciens, strains B6 or CG-14. Uninfected tissues do not contain immunologically detectable quantities of inhibitor I. Inhibitor I synthesis in tobacco crown gall tumors paralleled tumor growth at the average rate of about 4.5 μg of inhibitor I per 200 mg of fresh tissue per day. Infection of variegated tobacco mutant Dp-I with A. tumefaciens strain CG-14 produced tumors with 25% more inhibitor than tumors induced with strain B6. Unlike tobacco, tumors induced by either bacterial strain on potato (Solanum tuberosum) and on tomato (Lycopersicum esculentum) did not accumulate inhibitor I. Consequently, inhibitor I accumulation is modulated by the type of plant host used in spite of familial relatedness (Solanaceae) and the strain of A. tumefaciens used for infection.     

Crown Gall Tumor Disc Bioassay: A POSSIBLE AID IN THE DETECTION OF COMPOUNDS WITH ANTITUMOR ACTIVITY

Seventeen samples consisting of purified compounds and various ethanol extracts from plant sources were tested for activity on the initiation of crown gall tumors on potato discs. The results demonstrated definite correlation between the ability of these samples to inhibit the formation of crown gall tumors and their activity on the P388 leukemia system in mice. Samples showing only cytotoxic effects in KB cell cultures did not affect tumor initiation in our system. The active materials had no effects on bacterial viability or on the ability of the bacteria to attach to a tumorbinding site.     

Characterization of the Opine-Utilizing Microflora Associated with Samples of Soil and Plants

Microorganisms utilizing an opine as the sole carbon source were recovered from crown gall tumors, soil, and surface-disinfected potato tubers. The effect of the opines octopine, nopaline, succinamopine, and mannopine as selective substrates was compared with that of the auxin indoleacetic acid. Selection on octopine and indoleacetic acid favored the fluorescent pseudomonads, whereas mannopine allowed the frequent recovery of agrobacteria. Coryneforms which utilized succinamopine or mannopine were detected in soil, but not in tumors. Fungi growing on succinamopine or mannopine and a mannopine-utilizing Pseudomonas putida were isolated from tumor and soil, respectively.     

T-DNA organization in homogeneous and heterogeneous octopine-type crown gall tissues of nicotiana tabacum

Octopine-type tumor tissue was obtained both by infection of plants or isolated protoplasts with Agrobacterium tumefaciens and by somatic hybridization of normal and crown gall tobacco cells. Analysis of T-DNA by Southern blotting of clones and uncloned tissue reveals that, whereas tumors induced on plants are heterogeneous mixtures of cells differing in T-DNA organization, each tissue derived from transformed protoplasts or from somatic hybridization is homogeneous. Detailed analysis of T-DNA organization showed that T_L- or “core” T-DNA was always present at one or two copies per diploid genome. However, sometimes it was present in a modified form, either deleted, extended, tandemly duplicated or probably methylated. T_R-DNA was not detected. The observed variation in the organization of T-DNA in octopine crown gall tissue did not appear to be a characteristic of the way the tissue was derived.     

Bioassay and attributes of a growth factor associated with crown gall tumors

An improved bioassay is described for a factor that promotes tumor growth which was first obtained from extracts of pinto bean leaves with crown gall tumors. Sixteen primary pinto bean leaves per sample are inoculated with sufficient Agrobacterium tumefaciens to initiate about 5 to 10 tumors per leaf and treated with tumor growth factor at day 3 after inoculation. The diameters of 30 to 48 round tumors (no more than 3 randomly selected per leaf) are measured per test sample at day 6. Mean tumor diameter increased linearly with the logarithm of the concentration of tumor growth factor applied. The tumor growth factor was separated by column chromatography from an ultraviolet light-absorbing compound previously reported to be associated with fractions having maximal tumor growth factor activity. Partly purified tumor growth factor showed no activity in a cytokinin bioassay or an auxin bioassay, and negligible activity in gibberellin bioassays. Representatives of these three classes of growth factors did not promote tumor growth. Extracts from crown gall tumors on primary pinto bean leaves, primary castor bean leaves, Bryophyllum leaves, carrot root slices, and tobacco stems showed tumor growth factor activity, whereas extracts from healthy control tissues did not. Extracts from actively growing parts of healthy pinto beans, Bryophyllum, and tobacco, however, showed tumor growth factor activity. Tumor growth factor is proposed to be a normal plant growth factor associated with rapidly growing tissues. Its synthesis may be activated in nongrowing tissues by infection with Agrobacterium sp.     

Temperature-sensitive loss of tumor characteristics in a tobacco crown gall strain

Summary Three independently isolated tobacco crown gall strains incited byAgrobacterium tumefaciens C58 required phytohormone (auxin and cytokinin) supplements in the basal medium to grow, at 37°C. Six other tobacco crown gall strains incited, respectively, byA. tumefaciens IIBV7, B6, CGIC, A6NC, 27 and AT4 expressed, at 37°C, the tumor characteristic of ability to grow in vitro on medium lacking phytohormones. Nopaline was not detectable in C58 tumors cultured at 37°C, but octopine was produced by B6 tumor tissues incibated at the elevated temperature. C58 tumor strains kept at 37°C for 1 week or more lost the ability to express tumor characteristics at 27°C such as tissue morphology, growth on basal medium lacking phytohormones and nopaline production. Heat-treated C58 tissues also differed from the original tumor strain in regeneration ability and phytohormone requirements of explants; i.e. explants from regenerated, heart-treated C58 tumors required both auxin and cytokinin for growth in vitro.     

Functional Expression of Agrobacterium tumefaciens T-DNA onc-genes in Asparagus Crown Gall Tissues

We analyzed Asparagus crown gall tissues transformed with A.tumefaciens C58 and C58C1 (pTiB6S3) and selected for hormoneautotrophic growth. No increased IAA levels were observed inthe Asparagus tumor lines notwithstanding the presence of allthree T-DNA onc genes. The endogenous cytokinin levels indicatethat Asparagus crown gall is dependent on enhanced zeatin ribosideequivalent levels for its growth. We conclude that phytohormone autotrophic growth of Asparaguscrown gall tissue seems only to be dependent upon an activegene 4, inducing enhanced cytokinin levels. Moreover, the presenceof an active gene 1 seems to be lethal as was indicated by theabsence of tryptophan-2-mono-oxygenase activity in transformedtissues and the toxicity of exogenously supplied indole-3-acetamide(IAM) or naphthalene-1-acetamide (NAM) as a substitute for anactive gene 1. (Received August 7, 1989; Accepted October 31, 1989)     

Differential tolerance ofArabidopsis thaliana crown gall tumors and calli to 5-bromo-2′-deoxyuridine

The degree of tolerance of two crown gall tumors and leaf calli ofArabidopsis thaliana to BUdR was compared. The nopaline producing teratoma tumor tolerated BUdR in concentration as high as 2.10^?4 M. The tolerance of octopine producing unorganized crown gall tumor to BUdR was lower, but both exceeded significantly the degree of tolerance to BUdR of untransformedA. thaliana calli, where 10^?5 M BUdR already show some inhibitory effect on the growth rate.     

l-Tryptophan metabolism in wound-activated and Agrobacterium tumefaciens-transformed potato tuber cells

The in vivo metabolism of L-tryptophan in wound-activated and Agrobacterium tumefaciens , strain C 58, transformed tissues of white potato tubers ( Solanum tuberosum L. cv. Saskia) was investigated. The following metabolites of L-tryptophan were identified in both tissues by co-chromatography with authentic standards in several thinlayer chromotography (TLC) and high pressure liquid chromatographic (HPLC) systems: indole-3-acetic acid (IAA), indole-3-acetaldehyde, indole-3-ethanol, indole-3-acetamide and tryptamine. Labelled indole-3-acetaldoxime was only found in transformed tissue. Crown gall tissue generally incorporated [¹⁴C]-L-tryptophan into precursors of IAA at a distinctly higher rate than did wound tissue. Tryptamine and indole-3-ethanol accumulated about ten-fold more label in crown gall cells than in cells from wounded tissue. The incorporation of radioactivity into indole-3-acetamide as determined by 2 consecutive TLC systems followed by HPLC analysis was rather low, though consistently observed in both tissues. An indole-3-acetamide hydrolyzing enzyme, the putative product of gene 2 on the T-DNA, could be extracted from the transformed tissue only. The indole-3-ethanol level was 4.3 nmol (g dry weight)⁻¹ and 41 nmol (g dry weight)⁻¹ for wounded tissue and primary crown gall tissue, respectively, as determined by HPLC with a [¹⁴C]-labelled internal standard. The experiments are critically discussed in relation to recent reports on a T-DNA encoded enzyme of IAA biosynthesis in crown gall tumors.     

Studies on Auxin Protectors X. Protector Levels and Lignification in Sunflower Crown Gall Tissue

The lignification and differentiation of phloem fibers in sunflower stems is inhibited by growing crown gall tumors. Crown gall tumor tissue has previously been shown to contain large quantities of auxin protectors. Since auxin protectors are antioxidants which inhibit peroxidase-catalyzed reactions, and since the formation of lignin is known to involve a peroxidase-catalyzed reaction, an investigation was undertaken to examine the relationship between auxin protectors and lignification in sunflower crown gall tissue. Sunflower crown gall tissue placed into media low in mineral content, rapidly lignifies. In the low mineral media, protectors appear in the medium within an hour or two, implying that endogenously-synthesized protectors rapidly leak out of the tissue. In control media, the tissue neither lignified appreciably, nor did it exhibit an excessive amount of protector release. The addition of Ca²⁺ to the low mineral medium markedly slowed, but did not entirely prevent lignification; similarly Ca²⁺ markedly slowed the release of protector into the low mineral medium. Auxin protectors added to the low mineral medium did not inhibit lignification apparently because, in the medium, the protectors are rapidly oxidized to quinones. The addition of catechol, a substance which mimics protector, also failed to inhibit lignification and also formed a colored compound in the medium suggesting o-qui none formation. In contrast, dithiothreitol, a strong anti-oxidant which upon oxidation does not form a strong oxidant (such as o-quinone), when added to the low mineral medium does inhibit lignification. It is suggested that in the in vitro situation lignification and senescence occurs in low mineral media because the protectors leak out rapidly causing the cell's metabolism to favor peroxidase-catalyzed oxidations including those leading to lignification, while in the in vivo situation the excess protectors produced by crown gall tumor tissue diffuse into surrounding tissue, maintaining a reduced state in such tissues and thereby inhibiting differentiation and lignification. The synthesis of large quantities of protectors by the tumor tissue therefore could account for the anaplasia of the bundle caps observed in sunflower internodes in the vicinity of growing crown gall tumors.     

The in vitro effects of opines and other compounds on DNAs originating from bacteria, and from healthy and tumorous plant tissues

Purified total DNAs were isolated from oncogenic or nononcogenic Agrobacterium tumefaciens cells as well as from normal and crown gall tissues. Opines (octopine, nopaline, lysopine), plant hormone (auxin IAA) and some carcinogenic compounds were used in order to correlate their effects on in vitro strand separation and synthesis of DNAs with in vivo tumorous cell multiplication. Octopine (or nopaline) induced chain opening of DNAs originating from octopine (or nopaline)-metabolizing bacteria and from same bacteria strain-induced tumorous cells. This phenomenon was measured by the increase in DNA hyperchromicity which is concentration dependent. The tested compounds stimulated the in vitro synthesis of the same DNAs. Under the same conditions, in vitro strand separation and synthesis of healthy plant DNA was not (or only slightly) enhanced, except in the case of particular hormone-connected healthy cell DNA. IAA and carcinogens stimulated in vitro synthesis and induced in vitro strand separation (dose-dependent effect) of DNAs isolated from crown gall cells and inducing bacteria. Compared to healthy cell DNAs, these DNAs were thus susceptible to structurally very diversified molecules and in this way behave as do mammalian tissue DNAs. The opine and IAA actions observed here were specific for plant tissue DNA; cancerous human or animal tissue DNAs were insensitive. By their presence in the crown gall cells, opines possibly maintain destabilized areas (required for rapid growth and division) on tumor cell DNA. The cooperative actions of IAA and opines as well as small RNA and RNA fragments on gene activation, might explain the autonomy of plant tumor cells.     

Über die Physiologischen Eigenschaften von Gewebekulturen aus Tumor- und normalem Gewebe vonPicea glauca

Summary In contrast to other plant tumor tissues (crown gall and genetic tumors), that fromPicea glauca can be cultivated in vitro only in the presence of IAA. The auxin requirement for maximal growth of the tumor is appreciably higher (10^–5 g/ml IAA) than that of normal Picea cultures (10^–8 g/ml). A similar marked difference is also found in the ascorbic acid requirement, which is 10^–5 g/ml for neoplastic, but 10^–7 g/ml for normal tissue. The significance of these results for the characterisation and the in vitro growth of plant tumors is discussed.

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Studies on superoxide dismutase activities in virulent and avirulent strains of Agrobacterium tumefaciens and also in normal and crown gall tumor cells of Bryophyllum calycinum

Superoxide dismutase activity in virulent strains of Agrobacterium tumefaciens was found to be higher than that in avirulent strains. Polyacrylamide gel electrophoresis revealed two isoenzymes in both these strains. These isoenzymes are suggested to be iron and manganese containing superoxide dismutases. Crown gall tumor cells of the plant Bryophyllum calycinum were found to have higher superoxide dismutase activity than the normal plant cells. Polyacrylamide gel electrophoresis revealed two isoenzymes in both normal and crown gall tumor cells. Advantages of the higher superoxide dismutase activities in respect of the survival of virulent strains of A. tumefaciens and crown gall tumor growth have been discussed.     

Regeneration of tobacco plants from crown gall tumors

Summary Tissue culture methods have been developed for regeneration of normal appearing tobacco plants from bacteria-free crown gall strains incited byAgrobacterium tumefaciens C58, IIBV7, B6, CGIC, A6NC, 27, and AT4. Regenerants fall into two categories depending on the properties of tissues from these plants. The first type of regenerant was obtained from tumors incited byA. tumefaciens C58 and it retained the potential for expression of tumor characteristics such as a nonrequirement for phytohormones (auxin and cytokinin) by explants in vitro and the presence of detectable concentrations of nopaline. Normal appearing plants obtained from C58 tumors had much lower concentrations of nopaline than the corresponding tumor tissue (130 versus 1700 μg per g dry wt) indicating a parallel repression of abnormal growth and nopaline concentrations in regenerants. The second type of regenerant was obtained from tumors incited by the otherA. tumefaciens strains and was characterized by requirements for phytohormones by explants in vitro and the apparent lack of octopine or nopaline in regenerant tissues.     

Phytohormones in the formation of crown gall tumors

Crown gall tumors were initiated in a variety of plant species by infection with Agrobacterium tumefaciens strain B6 and the concomitant changes in the tissue levels of phytohormones, mainly indole-3-acetic acid (IAA) and cytokinins, were analyzed. A comparison was made of these hormones with those produced by virulent and avirulent strains of the bacterium in liquid culture and with those of bacteria-free crown gall callus cultures. Specific radioimmunoassays were employed for hormone determinations. An assay for the quantitation of femto-mol amounts of isopentenyladenosine and related cytokinins was newly developed and is described in detail. The results can be summarized as follows: Virulence in strain B 6 is associated with the ability to release trans-zeatin and increased amounts of IAA into the surrounding environment. In many, but not all plants analyzed, the development of crown gall tumors is also associated with a sharp rise in the levels of trans-zeatin-type zytokinins and IAA (e.g., Euphorbia lathyris, Catharanthus roseus). Crown gall calli growing on hormone-free media varied greatly in their cytokinin levels. In a culture of Nicotiana tabacum, both trans-zeatin and isopentenyladenine or related cytokinins were not detected. Thus, tumor growth cannot be explained on the basis of elevated levels of IAA and/or cytokinins alone.Abbreviations ABA abscisic acid - GC-MS gas chromatography-mass spectroscopy - HPLC high pressure liquid chromatography - IAA indole-3-acetic acid - RIA radioimmunoassay - TLC thin layer chromatography Part 19 in the series Use of immunoassay in plant science     

Primary Action of Indole-3-acetic Acid in Crown Gall Tumors: Increase of Solute Uptake

Exogenously added indole-3-acetic acid at a concentration of 100 micromolars stimulates d-glucose uptake (or 3-O-methyl-d-glucose uptake) by 25% in crown gall tumors induced on potato tuber tissue by Agrobacterium tumefaciens strain C 58. The titration of the endogenous IAA with the auxin antagonist 2-naphthaleneacetic acid at 100 micromolars reduces d-glucose uptake by about 80%. The apparent inhibition constant K_i is 21 micromolars. Other auxin antagonists like 1-naphthoxyacetic acid and 2-(p-chlorophenoxy)-2-methylpropionic acid show similar effects. The uptake of the amino acids leucine, methionine, tryptophan, lysine, and aspartic acid is also inhibited by 2-naphthaleneacetic acid to similar degrees. The auxins 1-naphthaleneacetic acid and 2-naphthoxyacetic acid at concentrations between 10 and 100 micromolars inhibit solute uptake only slightly (inhibition less than 20%). The impact of the results on the postulated role of indole-3-acetic acid as a modifier of the electrochemical proton gradient across the plasmalemma in crown gall tumor tissue is discussed.