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.     

Studies on the production of indole-3-acetic acid with auxin-heterotrophic mutants derived from cultured crown gall cells

Mutants in the indole-3-acetic acid metabolism derived fromcultured crown gall cells were tested to see whether they couldutilize any one of eight indolic compounds in place of indole-3-aceticacid. Two auxin-heterotrophic mutant cell lines could not utilizeindolepyruvic acid, but growth recovered when there was a supplementof indole-3-acetic acid. Indoleacetonitril and indoleacetaldoximeinhibited the growth of mutant cell lines and their parentalcrown gall cells. Cultured crown gall cells may have synthesizedindole-3-acetic acid from tryptophan via indolepyruvic acidand indole-acetaldehyde, and also may be able to produce indole-3-aceticacid from tryptophan via tryptamine (Received May 6, 1980; )     

Studies on the production of indole-3-acetic acid with auxin-heterotrophic mutants derived from cultured crown gall cells

Mutants in the indole-3-acetic acid metabolism derived fromcultured crown gall cells were tested to see whether they couldutilize any one of eight indolic compounds in place of indole-3-aceticacid. Two auxin-heterotrophic mutant cell lines could not utilizeindolepyruvic acid, but growth recovered when there was a supplementof indole-3-acetic acid. Indoleacetonitril and indoleacetaldoximeinhibited the growth of mutant cell lines and their parentalcrown gall cells. Cultured crown gall cells may have synthesizedindole-3-acetic acid from tryptophan via indolepyruvic acidand indole-acetaldehyde, and also may be able to produce indole-3-aceticacid from tryptophan via tryptamine (Received May 6, 1980; )     

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.     

Analysis of a range of crown gall and normal plant tissues for Ti plasmid-determined compounds

Summary Twenty-five plant tissues from several species, including thirteen crown gall tissues, were analysed for the full range of unusual compounds (the opines) whose synthesis in crown gall cells and utilization by Agrobacterium tumefaciens are genetically determined by the Ti plasmids found in this bacterial species. A technique for the analysis of the non-guanidino opines by GC and GC/MS is described. None of the opines were detected in any of the various normal tissues analysed. In the crown gall tissues, on the other hand, these compounds were often present at very high levels. The type of opines found in the crown gall tissues was dependent on the strain of initiating bacterium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GC gas chromatography - GC/MS coupled gas chromatography-mass spectrometry - HFB heptafluorobutyryl - SIM selected ion monitoring - TLC thin layer chromatography     

Internal organization,boundaries and integration of Ti-plasmid DNA in nopaline crown gall tumours

Eight lines of nopaline crown gall tumours were analysed by Southern (1975) blot hybridization to determine the size, internal organization, boundaries, possible plant DNA integration and accuracy of transfer of the Ti-plasmid DNA segment (T-DNA) transferred from Agrobacterium tumefaciens to crown gall plant cells. The conservation of this T-DNA in tumour tissues and tissues derived from plants regenerated from crown gall teratomas was also studied.A defined plasmid segment (the T-region) of about 15 × 10⁶M_r is accurately transferred and integrated into nuclear plant DNA without any major internal rearrangements. Furthermore, common composite fragments covalently linking the left and the right boundary of the T-region were observed, thus indicating either tandem duplications of integrated T-DNA segments or polymeric circles of T-DNA segments. The length of the transferred segment is not determined by size, since insertions in the T-region were found to be co-transferred with the T-DNA. The results indicate that sequences at the boundaries of the region may play a role in the transfer mechanism, although the right boundary could be replaced by a Tn1 insertion. Cells from plants regenerated from crown gall teratomas were shown to contain T-DNA without internal rearrangements but with minor modifications of the boundary fragments. In plants obtained from meiotic products of teratomaderived regenerated plants no T-DNA was observed.     

The fine structure of plant tumors

Summary Crown gall cells of several plant species contain considerably more endoplasmic reticulum than their normal or hyperplastic counterparts. This characteristic appears to be stable even in crown gall tissues grownin vitro for many years. No evidence for the presence of an etiological agent was found in any of the crown gall cells, nor was there any evidence of any structure peculiar to these cells. It is not possible at this time to determine if this increase in endoplasmic reticulum is responsible for the increased biosynthetic capacities of tumor cells.     

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.     

Endogenous Levels of Gibberellins, IAA and Cytokinins in Tobacco Crown Gall Tissues of Different Morphologies

Endogenous levels of gibberellin (GA) as well as IAA and cytokininsin teratomas and unorganized crown gall tissues of tobacco wereexamined by GC-SIM (for GA and cytokinin) or HPLC with a fluorescencedetector (for IAA). Two different types of crown gall inducedby octopine-type and nopaline-type Ti-plasmids were used. Inboth types, GA contents were higher in shoot-forming teratomasthan in unorganized calluses, while IAA contents were higherin unorganized calluses. But cytokinin contents in octopine-typecells were higher in unorganized calluses than in teratomas,whereas the contents in nopaline-type cells were higher in teratomas.Our results suggest that there is not always a relationshipbetween the cytokinin/IAA balance and tobacco crown gall morphology,but GA production in tobacco tissues is closely related to itsdifferentiation. ⁴ Present address: Agency for the Assessment and Applicationof Technology (BPPT), Jakarta Pusat, Indonesia. (Received September 1, 1986; Accepted February 16, 1987)     

A new amino acid derivative present in crown gall tumor tissue

A new amino acid derivative has been found in primary and secondary sunflower crown gall tissue cultures and in fresh crown gall tumors from sunflower plants wound-inoculated with Agrobacterium tumefaciens B₆. Normal plant tissue does not contain detectable levels of the compound. Radioactive labeling and cochromatography experiments strongly suggest that the natural derivative is identical to synthetic N²-(1-carboxyethyl)-L-histidine (histopine). Crown gall tissue cultures contain 1 μmole of histopine/20 g fresh weight. A. tumefaciens strain B₆, but not strain C₅₈, can utilize natural histopine and incorporate the products into macromolecules.     

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.     

Characterization of the enzyme responsible for nopaline and ornaline synthesis in sunflower crown gall tissues

Extracts prepared from sunflower (Helianthus annuus L.) crown gall tissues induced by Agrobacterium tumefaciens strains C58 and T37 (nopaline utilizers) catalyze the synthesis of nopaline and ornaline. These compounds are not synthesized in extracts of crown gall tissues induced by strains B6, 15955 (octopine utilizers), and AT1 (utilizes neither octopine nor nopaline) or in extracts of habituated sunflower callus. Both synthetic activities require NADPH, α-ketoglutarate, and either arginine or ornithine; histidine and lysine will not substitute. Incorporation of arginine or ornithine into product is inhibited by the other substrate but not by histidine or lysine. On the basis of inhibition and K_m data, both activities appear to be catalyzed by one enzyme and the same enzyme is apparently present in crown gall tissues induced by strains C58 and T37.     

Seed Transmission of Pea Seed-borne Mosaic Virus in Peas: Early and Late Expression of the Virus in the Progeny

Abstract Field trials at Summerland and Creston in 1984 and 1985 on loam soils naturally intested with Agrobadonum nonfactures showed that A radiobacter Strain 84 K-S4, did not control crown gall on Antonovka apple seedlingy when applied as a root-dip. K-84 controiled crown gall at Sidney on sandy loam soil m 1984 and 1985, but tailed in 1986. At Creston, higher than standard concentrations of K-84 did not control crown gall of seedlings, whether the seedlings were grown in unsterililzed soil 1986) or in sterile soil, 1987 before transplanting them into A nonfactures naturally intested soil. It a field trial in 1986 at Summerland, root-dip treatment with K-84 alone or in combination with broadeast treatments of melam-sodium of formalin tailed to control crown gall on Antonovka seedlings. However, crown gall intection was significantly reduced with metam-sodium or K-84 treatment alone when seedlings were raised in sterile soil before transplanting in naturally instead safe at Summerland in 1987.     

Enhanced 3-methoxytyramine levels in crown gall tumours and other undifferentiated plant tissues.

An amine, after dansylation, has been isolated from Nicotiana tabacum crown gall tumours for the first time and characterized as 4-hydroxy-3-methoxy-beta-phenylethylamine (3-methoxytyramine). The compound cannot be detected in differentiated N. tabacum tissues but appears in the corresponding callus controls. Its concentration is further increased 5-42-fold when N. tabacum is transformed with all strains of Agrobacterium tumefaciens so far tested.     

丹参的冠瘿组织培养和丹参酮的产生

用根癌农杆菌感染丹参无菌苗获得冠瘿组织,除菌后的冠瘿组织在无激素的Ms培养基上生长良好。经高压纸电泳检查,冠瘿组织中含有冠痿碱,证实根癌农杆菌的Ti质粒转化成功。冠瘿组织的生长和丹参酮的积累与基本培养基有关,B5和Ms培养基有利于生长．月增殖倍数分别达到102倍和90倍,而67-V和WP培养基则有利于丹参酮的合成,在培养过程中丹参酮能分泌到培养液中。研究表明用冠瘿组织作为培养系统,生产药用植物有效成分具有良好的开发前景。     

The host range of crown gall

Crown gall is a plant tumor disease caused by the specific action of the bacteriumAgrobacterium tumefaciens. In the current literature its host range is not clearly defined or is thought to be restricted to the dicotyledonous class of the angiosperms. We reviewed the susceptibility of 1193 species belonging to 588 genera and 138 families; 643 are host plants belonging to 331 genera and 93 families. Our list seems to be so far the most extensive source of information on crown gall susceptibility of plants. We attempted to correlate the susceptibility of plants to crown gall with known and/or presumed taxonomic relationships (according to the taxonomic systems of Engler and Takhtajan). No lower plant is known to be a host for crown gall. About 60% of the gymnosperms and the dicotyledonous angiosperms examined were sensitive for crown gall. In the latter class, there is no significant relationship between the taxonomic position of a plant family and its susceptibility. According to the literature, the susceptible monocots are limited to theLiliales andArales. The common opinion that the host range of crown gall is restricted to the dicotyledonous plants, is thus incorrect.     

Biological control of crown gall, Agrobacterium tumefaciens

Biological control of crown gall caused by Agrobacteriurn turnefaciens (Smith & Townsend) Conn, pioneered by Dr A. Kerr in South Australia, is effected through the establishment of a high population of the related non-pathogen A. radiobacter (Beijerinck & van Delden) Conn, strain 84 in the rhizosphere of susceptible plants. Strain 84 produces a bacteriocin to which many strains of the pathogen in Australasia, North America and Britain are sensitive. The disease is present in Britain on a variety of hosts including cherry. At East Malling cherry leaf scars, invaluable as an avenue of infection for bacterial canker infectivity titrations, have been used successfully in crown gall studies. Live cells of strain 84, but neither an avirulent strain of the pathogen nor a soil bacterium highly antagonistic to A. tumefaciens in vitro, inhibited gall formation in cherry leaf scars. Heat-killed cells had no effect. In a field experiment at East Malling hardwood cuttings of the new rootstock Colt have been dipped in strain 84 and total inhibition of crown gall is expected to ensue. The results of other experiments where the disease is already established on cherry-rootstock layer-beds and in blackberry plantations are less predictable. In time we hope to solve this problem. Only time will show whether this method of biological control is long lasting or will eventually break down.     

Evaluation of Biological and Chemical Treatments for Control of Crown Gall on Young Apple Trees in the Kootenay Valley of British Columbia

Field trials at Creston in 1988, 1989, and 1990 on silty loam soil naturally infested with Agrobacterium tumefaciens showed that Agrobacterium radiobacter strain K84 did not control crown gall on young Antonovka apple trees when apphed as a root-dip treatment. Copper oxychloride applied as a root-dip treatment at 2.5 and 5.0 g ai/1 and sewage sludge applied at 260 g per tree as broadcast were effective in reducing crown gall infection but these treatments were toxic to young apple trees in 1989. Lower rates of copper oxychloride, 0.3, 0.6 and 0.9 g ai/1, and sewage sludge at 130 g per tree, did not control crown gall in the 1990 field trial. The biological treatment with the strain AB8 of Bacillus subtilis applied as a root-dip effectively controlled crown gall and was not phytotoxic to young Antonovka apple trees. These results suggest that strain AB8 of B. subtilis has the potential to control crown gall on young apple trees under field conditions.     

Methylglyoxal destroys <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> crown gall tumours in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> without any adverse effect on the host plant

Methylglyoxal (MG) is a highly reactive α-oxoaldehyde, demonstrating anticancer effect on plant neoplastic tumours. In in vivo studies it was observed that MG destroyed crown gall tumours in Nicotiana tabacum produced by Agrobacterium tumefaciens, without any adverse effect on the host. The efficacy of MG in comparison to other anticancer drugs viz. cisplatin and ellagic acid in the treatment of crown gall was investigated. A slight degeneration of galls was noted in plants treated with cisplatin and ellagic acid but the plants died subsequently. With MG however, crown galls were completely cured and the plants completed their usual life cycle by flowering and producing seeds. MG inhibited the respiration of crown gall calluses suggesting that energy depletion resulted in tumour destruction.     

Mechanisms of crown gall formation: T-DNA transfer fromAgrobacterium tumefaciens to plant cells

Agrobacterium tumefaciens harbouring the Ti plasmid incites crown gall tumor on dicotyledonous species. Upon infection of these plants, T-DNA in the Ti plasmid is transferred by unknown mechanisms to plant cells to be integrated into nuclear DNA. WhenAgrobacterium is incubated with protoplasts or seedlings of dicotyledonous plants, circulation of T-DNA and expression ofvir (virulence) genes on the Ti plasmid are induced. The circularization event is efficiently induced by mesophyll protoplasts of tobacco which are highly competent for transformation by the T-DNA, and is also induced by diffusible phenolic compounds excreted from the protoplasts. The circularization and formation of crown gall both require the expression of thevirD locus, one of the induciblevir genes. These results suggest that the circularization of T-DNA reflects one of steps of the T-DNA transfer during formation of crown gall. In contrast to dicotyledonous plants, monocotyledonous plants are thought to be unresponsive to infection byAgrobacterium. We showed that monocotyledonous plants do not excrete diffusible inducers for the expression ofvir genes, while they contain a novel type of a signal substance(s). This inducer is not detected in the exudates of seedlings of monocotyledonous plants, but is found in the extracts from the seedlings, and also those from the seeds, bran and germ of wheat and oats. This finding suggests that T-DNA processing, and possibly its transfer, should take place whenAgrobacterium invades seedlings and seeds of monocotyledonous plants. Recipient of the Botanical Society Award for Young Scientists, 1987.