Studies on the structure of cointegrates between octopine and nopaline Ti-plasmids and their tumour-inducing properties

Stable cointegrates between incRh-1 octopine (Ach5) and nopaline (C58) Ti-plasmids, present in ten independently isolated Agrobacterium tumefaciens strains, showed identical restriction endonuclease patterns. Each cointegration event had taken place in the common sequence between the T-regions of both Ti-plasmids. This illustrates a high preference for this region when used in the formation of cointegrates. Four crown gall tissues, obtained after transformation of Nicotiana tabacum cells by one of the mutants, were analysed by using Southern blot analysis for their T-DNA structure. The borders of T-DNA frequently appeared to differ from T-DNA borders previously detected in tumour tissues that had been induced by Agrobacterium strain C58 or Ach5. Therefore, it was concluded that possibly a less stringent mechanism exists for the integration into plant DNA of T-DNA, derived from a composite (octopine/nopaline) T-region than for integration of T-DNA from a normal (octopine or nopaline) T-region.Abbreviations Agr sensitivity to agrocin 84 - Ape phage Apl exclusion - Cb resistance to carbenicillin - Occ octopine catabolism - Ocs octopine synthesis - Noc nopaline catabolism - Nos nopaline synthesis - Rec recombination - Tra transfer - Vir virulence     

Anatomical and chemical studies of vitrified shoots of chestnut regenerated in vitro

The growth of crown-gall cells cultured in vitro ( Nicotiana tabacum L. cv. White Burley and Parthenocissus tricuspidata cv. Veitchii) is inhibited by alstonine (BG-8), a plant alkaloid, the anti-cancer effect of which has previously been demonstrated on animals and plants. The growth of normal cells is only slightly affected. The inhibitory effect of BG-8 on crown-gall cells is antagonized by indole-3-acetic acid (IAA) added to the culture medium. Kinetin associated with IAA does not prevent this inhibitory effect. BG-8 present in the culture medium containing the two types of hormones seems to modify the later hormonal requirement of Parthenocissus crown-gall tissues.
BG-8 exhibits high binding affinity for crown-gall DNA and, therefore, strongly inhibits its in vitro synthesis. The alkaloid has practically no effect on DNA from healthy cells. The inhibition by BG-8 is dependent on the level of DNA strand separation and on the origin and nature of the tissues (crown-gall DNA is more destabilized than healthy DNA; DNA from habituated tissues is intermediate). IAA and kinetin have opposite effects on the in vitro strand separation of the DNAs from crown-gall cells and, consequently, antagonistic effects on DNA replication (IAA stimulates and kinetin inhibits). It is possible to establish a close relationship between in situ development of crown-gall tissues of the two species studied (in the presence or absence of BG-8 or cell-growth factors), in vitro DNA synthesis and DNA strand separation.     

Diversity of Opines and Opine-Catabolizing Bacteria Isolated from Naturally Occurring Crown Gall Tumors

The diversity of opines from 43 naturally occurring crown gall tumors on several plant species was analyzed for the presence of agropine, chrysopine, iminodiacid, an unidentified leucinopine-like iminodiacid (IDA-B), mannopine, octopine, nopaline, DL- and LL-succinamopine, leucinopine and heliopine. Opine utilization patterns of agrobacteria and fluorescent pseudomonads resident in a tumor were then analyzed and compared for agreement with the opine isolated from that tumor. Nopaline was the most common opine found and was detected in tumors from cherry, blackberry, grape, and plum. Octopine was not found, although octopine-catabolizing bacteria were isolated from several tumors. A new, previously undescribed iminodiacid of the succinamopine-leucinopine type (provisionally designated IDA-B) was isolated from tumors of wild blackberry. Field tumors from apple, blueberry and grape yielded no detectable opines, even though opine-utilizing bacteria were present. Bacterial isolates from plum and cherry showed the best correspondence between the opine in tumors (nopaline) and the presence of bacteria that catabolized that opine. However, several unusual opine catabolic combinations were identified, including isolates that catabolized a variety of opines but were nonpathogenic. More variability was observed among isolates from field tumors on the remaining plant species. We isolated novel mannopine-nopaline type agrobacteria from field tumors of cherry, plum and blackberry that induced tumors containing either mannopine (plus agropine) or nopaline, but not both. Epidemiologically, the galled plants from an area were not of clonal origin (same Ti plasmid), indicating that the field tumors from a small area were incited by more than one type of Ti plasmid.     

Ornithine cyclodeaminase from octopine Ti plasmid Ach5: identification, DNA sequence, enzyme properties, and comparison with gene and enzyme from nopaline Ti plasmid C58.

Octopine and nopaline are two arginine-derived opines synthesized in plant cells transformed with octopine or nopaline plasmids. Utilization in Agrobacterium tumefaciens is mediated by Ti plasmid regions called occ or noc (octopine or nopaline catabolism), and recent experiments showed that noc in pTiC58 codes for a pathway from nopaline to L-proline. The last enzyme is ornithine cyclodeaminase (OCD), an unusual protein converting L-ornithine directly into L-proline. We investigated whether octopine plasmid pTiAch5 also harbors a gene for OCD. The results revealed an ocd gene which is induced by octopine and maps in the occ region. DNA sequence analysis and comparison with the gene from pTiC58 showed that the two genes are related (69% homology in DNA and deduced amino acid sequence), and antiserum against OCD(C58) also reacted with OCD(Ach5). The enzyme activity was characterized, and a comparison with OCD(C58) showed that the properties are similar, but not identical. Differences were detected in the regulation of enzyme activity by L-arginine and L-proline and in the response to varying ratios of NAD+/NADH. It is proposed that this reflects different mechanisms for integration of opine catabolism into general metabolism.     

DNA from Agrobacterium rhizogenes in transferred to and expressed in axenic hairy root plant tissues

Summary Axenic root tissue cultures were established from primary hairy roots induced on carrot and potato by Agrobacterium rhizogenes strain 15834. cDNA made towards poly-A⁺ RNA isolated from these tissues, hybridized with a limited number of well-defined fragments of the plasmid DNA present in the inciting A. rhizogenes strain. These data therefore demonstrate that at least part of the rootinducing (Ri) plasmid of Agrobacterium rhizogenes is transferred, stably maintained and expressed in hairy-root plant tissues and confirm that hairy roots are a special type of crown gall. The T-DNA in hairy-root cells appears to have several regions which are related in terms of sequence homology and probably also function to the T-DNA in octopine and nopaline crown gall tumours.     

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.     

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.     

Arginine catabolism: a new function of both octopine and nopaline Ti-plasmids of Agrobacterium.

Summary The oncogenic plasmids of Agrobacterium, the Ti-plasmids, carry genes that enable their bacterial host to catabolize opines. Opines are unusual amino acid derivatives that are only produced in crown gall tumours incited by oncogenic strains of Agrobacterium. The 2 opines, octopine and nopaline, are degraded by Agrobacterium strains carrying the octopine or the nopoline Ti-plasmid, respectively, to arginine and pyruvic acid, and to arginine and -ketoglutaric acid. In this paper it is shown that the Ti-plasmids carry gene(s) involved in the utilisation of arginine as a carbon source. Strains harbouring wild type octopine or nopaline Ti-plasmids in the chromosomal context of strain C58C1 do not grow on arginine as a carbon source. However, they are able to grow on arginine provided that they are induced, or constitutive for opine catabolism. The features of ornithine utilisation are identical. The gene(s) involved in arginine and ornithine utilization in C58C1 (pTi-oct) or C58C1 (pTi-nop) are under the control of the regulator gene that controls octopine or nopaline catabolism. A tentative pathway of octopine utilization is proposed, in which at least two steps are Ti-plasmid coded, and probably belong to the same operon: 1-scission of octopine into arginine and pyruvic acid 2-transformation of an arginine derivative (GSA?) to glutamic acid.Arginine utilization as a carbon source is therefore a new function of the Ti-plasmid. As this function is not inducible by arginine but by opines, it provides a method for selecting regulatory mutants of opine catabolism in the genetic background of strain C58.     

In Vivo Synthesis of Crown Gall-specific Agrobacterium tumefaciens-directed Derivatives of Basic Amino Acids

Several kinds of primary sunflower (Helianthus annuus) crown gall tissues were established in tissue culture and then labeled in vivo with either [¹⁴C]arginine, [¹⁴C]histidine, [³H]lysine, or [³H]ornithine. Crown gall tissues incited by Agrobacterium tumefaciens strains that utilize octopine as a sole source of carbon or nitrogen for growth synthesized the four members of the N²-(1-carboxyethyl)-amino acid family: octopine, histopine, lysopine, and octopinic acid. Those tissues incited by A. tumefaciens strains that utilize nopaline synthesized nopaline and two new compounds, a lysine and an ornithine derivative (ornaline). A normal tissue culture, a habituated tissue culture, and a crown gall culture from a strain of the bacteria unable to utilize either octopine or nopaline did not synthesize any of the amino acid derivatives. We could not detect any other crown gall-specific derivatives of the four basic amino acids.     

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.     

Octopine and nopaline synthesis and breakdown genetically controlled by a plasmid of Agrobacterium tumefaciens

Summary Several nopaline degrading strains and one octopine degrading strain are shown to loose oncogenicity as well as the ability to utilize these guanidine compounds when they are cured of their TI plasmid. To investigate whether the specific genes involved in the utilization of one or the other compound are located on the plasmid, plasmid-transfer experiments have been performed.The plasmid from a nopaline degrading strain has been transferred to a naturally non oncogenic Agrobacterium namely A. radiobacter. Furthermore, the plasmid from an octopine degrading strain has been transferred to a plasmid-cured strain which originally had the capacity to utilize nopaline. Both kinds of experiments prove that the TI plasmid determines the strain specificity with regard to the utilization of either octopine or nopaline.They also demonstrate that the synthesis of either octopine or nopaline in crown gall cells is also determined by genes located on the TI plasmid harboured by the transforming A. tumefaciens strains.     

Agrocinopine A, a phosphorylated opine is secreted from crown gall cells

We showed that phosphorus-containing metabolites of crown gall tissues were all taken up by appropriate pTi+ agrobacteria. All but one were also taken up by pTi- bacteria. This one compound, produced by nopaline-, but not by octopine-type tumours, was the only phosphorylated organic compound actively secreted by healthy crown gall cells, and it appears to be agrocinopine A. Testing crown gall cell exudates may be a general procedure for the identification of opines by transformed plant cells.     

Transport of nonmetabolizable opines by Agrobacterium tumefaciens.

We have examined the uptake of [14C]octopine and [14C]nopaline by Agrobacterium tumefaciens strains containing the C58 chromosomal background in medium suitable for the induction of vir genes. All strains tested could transport both of these opines, regardless of the presence or type of Ti plasmid (octopine or nopaline) present in the bacterium. The transport of these opines required active cellular metabolism. Nonradioactive octopine, nopaline, and arginine competed effectively with [14C]octopine and [14C]nopaline for transport into A. tumefaciens A136, suggesting that the transport of these opines occurs via an arginine transport pathway not encoded by the Ti plasmid.     

The opine synthase genes carried by Ti plasmids contain all signals necessary for expression in plants

Signals necessary for in vivo expression of Ti plasmid T-DNA-encoded octopine and nopaline synthase genes were studied in crown gall tumors by constructing mutated genes carrying various lengths of sequences upstream of the 5' initiation site of their mRNAs. Deletions upstream of position -294 did not interfere with expression of the octopine synthase gene while those extending upstream of position -170 greatly reduced the gene expression. The estimated size of the octopine synthase promoter is therefore 295 bp. The maximal length of 5' upstream sequences involved in the in vivo expression of the nopaline synthase gene is 261 bp. Our results also demonstrated that Ti plasmid-derived sequences contain all signals essential for expression of opine synthase genes in plants. Expression of these genes, therefore, is independent of the direct vicinity of the plant DNA sequences and is not activated by formation of plant DNA and T-DNA border junction.     

Differential Expression of Oncogenicity and Nopaline Synthesis in Intact Leaves Derived from Crown Gall Teratomas of Tobacco

The results reported in the present study demonstrate that oncogenicity and nopaline synthesis are differentially expressed under conditions in which the neoplastic state is persistently suppressed in crown gall teratoma tissues of tobacco. While an active and continued synthesis of nopaline occurs in cells present in intact teratoma-derived leaves, the reexpression of oncogenicity depends on the development of new cell divisions. It is suggested that these new cell divisions are required either (a) to reestablish positive feedback control mechanisms upon which the development of a capacity for autonomous growth of these plant tumour tissues appears to depend, or (b) to establish the pattern of metabolism concerned with cell growth and division which is then maintained in the plant tumour cells by positive feedback control mechanisms.     

Agrocinopine A, a tumor-inducing plasmid-coded enzyme product, is a phosphodiester of sucrose and L-arabinose

Opines are unusual compounds found specifically in plant crown gall tumors. Genes for their synthesis and catabolism reside in agrobacteria as tumor-inducing (Ti) plasmid DNA. Only a small Ti-plasmid segment (24 kilobase pairs), the T-DNA, is transferred to the plant cell where it commonly codes for enzymes involved in the biosynthesis of nitrogenous opines such as nopaline (N2-(1,3-D-dicarboxypropyl)-L-arginine) as well as the tumor phenotype. Ellis and Murphy, (Ellis, J.G., and Murphy, P.J. (1981) Mol. Gen. Genet. 181, 36-43) reported the existence of the phosphorylated opines, agrocinopines A and B in tumors containing nopaline. Pure agrocinopine A has now been isolated in a yield of 0.05-0.06 g/100 g, fresh weight, from such tumors. Physical, chemical, and biological data establish the structure of agrocinopine A as an unusual non-nitrogenous opine of sucrose and L-arabinose with a phosphodiester linkage from the 2-hydroxyl of the arabinose to the 4-hydroxyl of the fructose moiety in sucrose. Agrocinopine B is the corresponding phosphodiester, in which the glucose has been hydrolyzed from the sucrose portion of agrocinopine A. Borohydride reduction of the free L-arabinose anomeric carbon of agrocinopine A, to the corresponding arabinitol derivative eliminates the characteristic inhibition zone enhancement produced by both agrocinopines A and B in the agrocin 84 (a fraudulent adenine nucleotide) bioassay. Because of the limited number of genes in the T-DNA, a generalization is proposed, whereby all opines will be found to comprise two common plant cell constituents linked in an uncommon manner by the minimum number of enzymes.     

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