Detection of nopaline dehydrogenase and lysopine dehydrogenase activities in crown gall tumors of different plant species

The LpDH and NpDH activities in crown gall tumors incited byAgrobacterium tumefaciens strain C58 were followed in 7 plant species and 100 tumors were assayed in each experimental variant. The NpDH activity was found in 790 out of 800 crown galls observed. The LpDH activity was tested, after induction of tumors withA. tumefaciens strain B6-806 and 37400, in three experimental variants. The LpDH activity was found in 290 out of 300 crown galls. In the small fraction of the LpDH and NpDH negative tumors, the activity was possibly actually present, but it was below the limits of the sensitivity of the detection technique used.     

Unusual plasmid DNA organization in an octopine crown gall tumor.

A cloned tobacco crown gall tumor, 1595501, incited by A. tumefaciens strain 15955 was studied. Molecular analysis of the organization of the T-DNA by means of Southern transfer and hybridization techniques indicated that the 1595501 tumor has about 10 copies of TL DNA, five of which are complete TL DNA, whereas most octopine tumors have only one to two copies of complete TL DNA. Hybridization studies and genomic cloning indicated that some segments of the T-DNA have undergone deletions. One of the clones contained two copies of T-DNA which were inverted in orientation with respect to each other. Two left ends of TL DNA from the 1595501 tumor line and the corresponding region of the octopine plasmid were sequenced. Comparison of the various cloned T-DNA sequences with Ti-plasmid sequence indicated that while there is an association with a 25 base pair direct repeat, there is no specific set of base pairs in the T-DNA at which divergence from Ti-plasmid sequences occurred.     

Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism.

Mutants of Agrobacterium tumefaciens which affect virulence or the ability to catabolize octopine were isolated after Tn5-induced mutagenesis. Of 8,900 colonies tested, 7 mutants with Tn5 insertions in a specific region of other Ti plasmid unable to catabolize octopine were isolated. Thirty-seven mutants affected in tumorigenesis resulted from insertions in the Ti plasmid and the Agrobacterium chromosome. Of these mutations, 12 were chromosomal and 25 mapped on the plasmid. Twenty-three mapped within a 20-megadalton region, which is distinct from the Ti plasmid sequences found stably integrated into the plant cell genome T-deoxyribonucleic acid). Included in these were mutants that were either a virulent or produced tumors with unusual morphologies. Three mutants contained insertions in the T-deoxyribonucleic acid. These three mutants incited tumors which synthesized octopine but had an altered morphology due to either extensive proliferation of shoots or roots from the tumor callus. Three additional mutants not caused by Tn5 contained mutations in the Ti plasmid.     

Structure and synthesis of histopine, a histidine derivative produced by crown gall tumors

Histopine, an unusual amino acid derivative of histidine isolated from crown gall tumors of sunflowers (Helianthus annus) inoculated with Agrobacterium tumefaciens strain B6, was previously assigned the gross structure N-(1-carboxyethyl) histidine (2). A diastereomeric mixture containing histopine (2a and 2b) was readily prepared by reductive alkylation of (S)-histidine (1) with pyruvic acid and sodium cyanoborohydride. The individual diastereomers were prepared by reaction of (S)-histidine with (R)- and (S)-2-bromopropionic acid. (R)-N-(1-Carboxyethyl)-(S)-histidine (2a) supports the growth of A. tumefaciens whereas (S)-N-(1-carboxyethyl)-(S)-histidine (2b) is inactive. Therefore, we assign structure 2a to histopine.     

Further insight on the transferred-DNA of octopine crown gall

Summary Six octopine tumour lines incited by pTiB6S3, pTiAch5 and pTiA6 on tobacco, Arabidopsis and Petunia were studied by the Southern blotting hybridisation technique in order to define accurately the dimensions of the segments of plasmid origin transferred to the tumourous cell and their organisation in the plant genome. Emphasis has been put on the comparison between octopine and nopaline T-DNAs and on the lines presented here compared with those studied previously (Thomashow et al. 1980).The lenght of the transferred DNA segment does not depend on the plasmids used, nor on the host plants. The octopine T-DNA organisation in the cell nucleus is significantly different from that of nopaline T-DNAs: tandem arrangements of T-DNA segments could not be detected and the T-DNA itself is much shorter.The tumour lines described here can be compared to some extent with those studied by another group (Thomashow et al. 1980) by the same technique. However, some differences were observed. The transferred DNA was seen as a unique stretch of about 11 kb present only once per cell. No amplification of any part was noticed in any of these six lines.Examination of the restriction patterns presented by the boundary fragments of the T-DNA in these lines suggested that some of them were of common origin.     

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.     

An electron microscope study of sunflower crown gall tumor

Summary The inception and developmental phases of sunflower (Helianthus annuus) crown gall tumor growth induced byAgrobacterium tumefaciens were studied submicroscopically. Bacteria were found only in the inception phase. Chloroplasts from tumor cells of both phases showed several kinds of structural variation. Other cytoplasmic organelles remained more or less normal.This investigation was supported in part by an American Cancer Society Institutional Grant.     

Promotion of crown-gall tumor growth by lysopine, octopine, nopaline, and carnosine

The growth of crown-gall tumors on primary bean leaves (Phaseolus vulgaris L. cv. “Pinto”) was promoted by the addition of d-lysopine, d-octopine, l-carnosine, or nopaline. Assayed on tumors induced by Agrobacterium tumefaciens strain B6, the relative activity was octopine = carnosine > lysopine nopaline; assayed on tumors induced by A. tumefaciens strain T-37, which induces tumors which form nopaline, the relative activity was nopaline = octopine = carnosine > lysopine. From one to three applications of carnosine or octopine gave equal additive increments in tumor growth, showing that a continual supply of these substances is required to maintain an increased rate of growth. At concentrations above 0.1 mm, pairs of these growth-promoting substances were less active than when applied singly. Inhibition of octopine-induced growth was obtained by applying 0.01 mm carnosine with 1 mm octopine and partial inhibition was obtained when carnosine was added 10 hr after octopine. Equimolar mixtures of lysopine, octopine, and carnosine, however, were at least as active in promoting tumor growth as any of the compounds added singly at equivalent concentrations. The activity of 0.1 to 0.5 mm lysopine, octopine, and carnosine was inhibited, respectively, by 1 mml-lysine, l-arginine, and l-histidine and this inhibition was limited in each case to the basic amino acid corresponding to that of the growth factor. Arginine fully inhibited octopine-induced tumor growth when applied as much as 6 hr after octopine, indicating that this inhibition was not due to prevention of octopine uptake. Although four separate substances were found which promoted tumor growth, the molecular specificity required for activity of each compound was high. Evidence is presented which suggests that a tumor growth-promoting substance extracted from tumorous leaves is a carnosine-like derivative of l-histidine.     

Genetic studies on the role of octopine T-DNA border regions in crown gall tumor formation

Summary Crown gall tumors result from transfer and integration of the T-DNA from the Ti plasmid of Agrobacterium tumefaciens into plant nuclear DNA. In the present study, recombinant plasmids containing deletion and rearrangement deriviatives of the T-DNA region of the octopine Ti plasmid pTiA6 were tested in a binary tumorigenesis system (Hoekema et al. 1983) to determine the requirements for T-DNA border regions in tumor formation. Since two defined segments of the T-DNA region of octopine Ti plasmids can be detected in tumor DNA (the left (T_L-) and right (T_R-) DNA), four border regions exist in this Ti plasmid. Agrobacteria harboring plasmid constructs which contain a T-DNA gene capable of inciting tumors (gene 4, the tmr gene, which is involved in cytokinin biosynthesis) and various T-DNA border regions were tested for ability to cause tumors on Nicotiana glauca and other host plants. Such tmr constructs containing as their only border region the right border of either the T_L-DNA or the T_R-DNA are fully tumorigenic. Analogous tmr constructs containing only the T_L-DNa left border region are not tumorigenic. These results do not depend on the orientation or position of the single border with respect to the tmr gene; furthermore, the T_R-DNA right border can confer tumor-forming ability despite the presence of an intervening copy of the T_L-DNA left border.These results for relatively small plasmids are contrasted with previously determined requirements for border regions in tumorigenesis by intact Ti plasmids. A model previously proposed by Wang et al. (1984) for the role of border regions in DNA transfer to plant cells is extended in order to explain the tumor-forming ability of plasmid constructs containing a single border region. The results of this study interpreted according to the model suggest that the octopine T_L-DNA left border is defective in this DNA-transfer process.     

The lysopinedehydrogenase gene used as a marker for the selection of octopine crown gall cells

Plant cells transformed into octopine-synthesizing tumour cells by the bacterium Agrobacterium tumefaciens survive when cultured in the presence of homo-arginine (HA), whereas both normal plant cells and nopaline producing plant tumour cells do not. Survival of octopine crown gall cells is due to the activity of the enzyme lysopinedehydrogenase (LpDH) in these cells, which converts toxic homo-arginine into non-toxic homo-octopine. The selective toxicity of homo-arginine for normal cells can be applied for the enrichment of octopine Ti plasmid transformed plant cells vs normal plant cells in mixed cultures.     

Relation between auxin autotrophy and tryptophan content in sunflower crown gall cells in culture

The content of indoleacetic acid (IAA) in cultured sunflowercrown gall cells was high in the transitional stage from thelog to the stationary stage. In contrast, the content of tryptophanwas low in the log, transitional and stationary stages, butin the necrotic stage it showed an increase of ca. 80 timesthat of the log and stationary stages. Although tryptophan transaminaseactivity per gram of fresh weight of the cells was detectedthroughout the culture period, with a slight increase in thetransitional stage, its specific activity remained low in thelog and stationary stages, but increased in the necrotic stageto two or three times that of the log or stationary stages.The accumulation of IAA in the transitional stage was not accompaniedby an increase in tryptophan. It might be regulated by the actionof the IAA protector, and the accumulation of IAA in the necroticstage might be regulated by the amount of tryptophan and theIAA protector. (Received April 11, 1980; )     

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.     

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.