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.     

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.     

Absolute stereochemistry of leucinopine,a crown gall opine

Crown gall tumors incited by Agrobacterium tumefaciens strain Bo542 have been reported to synthesize a tumor-specific substance identified as N-(1,3-dicarboxypropyl)-leucine (leucinopine), a compound with two centers of asymmetry. We report here evidence that leucinopine is indeed a crown gall opine, in that it is specifically catabolized by A. tumefaciens strains carrying the tumor-inducing plasmid pTi Bo542, as well as strains carrying closely related plasmids pTi AT1 and pTi AT4. We further report catabolism of leucinopine by the succinamopine-type strains A518, A519 and A532, carrying pTi EU6, pTi AT181 and pTi T10/73, respectively. Strains lacking any virulence plasmid, as well as those carrying octopine or nopaline type Ti plasmids or mannopine type Ri plasmids, did not catabolize leucinopine. On the basis of specificity of catabolism by bacteria carrying pTi Bo542, we conclude that the stereochemistry of natural leucinopine is l-threo, i.e. l^glu,l^leu. Such stereochemistry is novel in the opines known thus far: octopine, nopaline and succinamopine have d,l-stereochemistry: d^ala,l^arg (octopine), d^glu,l^arg (nopaline) and d^glu,l^asn (succinamopine).     

Octopine as a marker for the induction of tumorous growth by agrobacterium tumefaciens strain B6.

Octopine [N²-(D-1-carboxyethyl)-L-arginine] was detected in all tobacco and sunflower crown gall tumors incited by $\text{Agrobacterium tumefaciens}$ (E. F. Sm. and Town.) Conn strain B₆ at levels between 1 and 2.5 μmoles/20 g fresh weight. Most tissue cultures derived from plant tumors contained octopine at levels between 0.3 and 1 μmole/20 g fresh wt. Normal plant tissues and tissue cultures derived from normal tissues contained no detectable octopine when assayed by a [³H] arginine incorporation technique designed to detect low levels of octopine (less than 0.5 nmole/20 g fresh wt).     

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.     

Octopine Accumulation Early in Crown Gall Development is Progressive

A purification of octopine from crown gall tissue was developed to quantitate conversion of precursor [³H]arginine into [³H]octopine. Plant wound tissue which was sterile or infected with an avirulent strain of Agrobacterium tumefaciens did not accumulate detectable quantities of octopine, consistent with opine synthesis not being induced by wounding or infection. Octopine was only recovered from tissue infected with virulent tumor-inducing strains of A. tumefaciens. In every case tested, the morphological appearance of tumors preceded the accumulation of octopine by at least 1 week, and in some instances 3 weeks. Thus, what was necessary and sufficient for the expression of plant hormones (auxin and cytokinin) required for tumor growth was not sufficient for the accumulation of octopine. The possible nature of the temporal difference in the expression of hormone autotrophy and octopine synthesis is discussed.     

Fingerprints of Agrobacterium Ti plasmids

Many crown gall-inducing Agrobacterium tumefaciens strains have multiple plasmids, only one of which, the tumor-inducing (Ti) plasmid, is essential for oncogenicity. For comparison of Ti plasmids, single-plasmid-containing transconjugant or transformant derivatives were used as sources of pure Ti-plasmid DNA. Fingerprinting was undertaken using the restriction endonuclease SmaI because it produced a relatively simple cleavage pattern. Three groups of Ti plasmids are discernible based upon both their genetic characteristics and their SmaI fingerprints: (1) Octopine-type Ti plasmids, which confer oncogenicity and octopine utilization on the bacterium. Tumors incited produce octopine. This group of plasmids is highly conservative; fingerprints of all members were identical except for two minor variations. (2) Nopaline-type Ti plasmids, which confer oncogenicity and nopaline utilization on the bacterium. Tumors incited may or may not produce nopaline; these plasmids have fingerprints that suggest variable degrees of relationship, including one that appears unrelated to the rest (3) Null-type plasmids, which confer oncogenicity but neither utilization trait on the bacterium. Tumors incited by this class of strains produce neither octopine nor nopaline. Only one member of this group has been examined thus far. Fingerprints of plasmids from several nononcogenic strains examined bore no resemblance to fingerprints of any of the Ti plasmids.     

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.     

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.     

Biosynthesis of N2-(1,3-dicarboxypropyl) ornithine [nopalinic acid] in crown gall tumour tissue

Protein extract from crown gall tumour tissue, induced on Nicotiana tabacum by Agrobacterium tumefaciens strain T37, synthesized nopalinic acid [N²-(1,3-dicarboxypropyl)ornithine] from l-ornithine and α-ketoglutarate in the presence of NADPH. Label was incorporated into nopalinic acid from both l-ornithine-[¹⁴C] and α-ketoglutarate-[¹⁴C] in vivo. Nopaline [N²-(1,3-dicarboxypropyl)arginine] did not appear to be metabolized to nopalinic acid in vivo.     

Expression of nopaline synthesis in tumorous and regenerant tobacco crown galls

Summary Tissues formed in liquid cultures of tobacco (Nicotiana tabacum cv. Wisconsin 38) crown galls incited byAgrobacterium tumefaciens C58 were of three types: unorganized callus, organized teratoma, and organized normal appearing. These tissues contained 400±12, 410±17, and 614±53 μg nopaline/g fresh weight, respectively. Using [¹⁴C]arginine, methods were developed for measuring in vivo nopaline biosynthetic rates. Tissues were incubated in a low concentration (i.e., 3 μM) of [¹⁴C]arginine to minimize disruption of the internal pool (approximately 140 μM free arginine). Radioactivity in the tissue was assayed and the specific radioactivity of free arginine, the precursor of nopaline, was determined. The linear rate of incorporation of radioactivity into nopaline was used to calculate the following biosynthetic rates (expressed as microgram nopaline per gram fresh weight per 24 h): callus, 14; teratoma, 21; normal appearing, 24. These results show conclusively that normal appearing tissues obtained from crown gall tumors can synthesize nopaline. Abnormal growth and opine biosynthesis, therfore, can be expressed independently.     

Comparison of octopine, histopine, lysopine, and octopinic acid synthesizing activities in sunflower crown gall tissues.

Extracts of sunflower crown gall tissues induced by $\text{Agrobacterium tumefaciens}$ strain B₆ catalyze the synthesis of octopine, histopine, lysopine and octopinic acid. These compounds are not synthesized either in extracts of crown gall tissues induced by strains AT1 and C58 or in extracts of habituated sunflower callus. All four synthetic activities require NADPH or NADH, pyruvate, and the appropriate basic amino acid. Incorporation of radioactivity from any one of the four labeled, basic amino acids into its product is inhibited by the other three basic amino acids. All the reactions are inhibited by ε-aminocaproic acid but none are inhibited by the neutral amino acids alanine and phenylalanine.     

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     

Broad-Host-Range Agrocin of Agrobacterium tumefaciens

Eighteen strains of Agrobacterium tumefaciens isolated from crown galls were tested for agrocin production. Of six agrocin-producing strains, one (D286) produced a broad-host-range agrocin active against strains carrying nopaline, octopine, and agropine type Ti plasmids. Sensitivity to agrocin D286 was found to map in the 11- to 18-megadalton region of the nopaline Ti plasmid pTiC58. The agrocin was partially purified, and its physical characteristics were consistent with its being a nucleotide, as is agrocin 84. Agrocin D286 was shown to inhibit DNA, RNA, and protein syntheses. Strain D286 spontaneously lost its pathogenicity, and its potential for use in the biological control of crown gall is discussed.     

Isolation of Agrobacterium Ti-plasmid regulatory mutants

Summary Crown gall tumors incited by Agrobacterium tumefaciens synthesize basic amino acid derivatives called opines. Opine production in tumours and opine catabolism by A. tumefaciens are coded by Ti-plasmids which confer oncogenicity on this bacterium. Catabolism of opines is inducible, and a method for isolation of regulatory mutants is described. From octopine-type bacteria, by plating on non-inducing substrates (noroctopine, noroctopine acid, D-histopine) we have isolated regulatory mutants of three types: constitutive, partially constitutive, and fully inducible by the analogue. From nopaline-type bacteria, by plating on octopine (a non inducing substrate) we have isolated analogous regulatory mutants.Synthetic opines, in which the amino acid moiety has been replaced by toxic arginine analogues, are toxic for these regulatory mutants. We isolated mutants resistant to such synthetic opines, and found that some had lost the capacity to utilize octopine. A survey of a large number of such mutants revealed that all of them still incited octopine synthetizing tumors.Mutants constitutive for octopine catabolism are in some instances also constitutive for Ti-plasmid transfer. A simple method for screening regulatory mutants for constitutive Ti-plasmid transfer is described.This work has been supported in part by grants from the Centre National de la Recherche Scientifique (contrats ATP 2814 and 3363).     

Growth requirements ofArabidopsis thaliana crown galls

Crown galls induced onArabidopsis thaliana plants by octopine or nopaline strains ofAgrobacterium tumefaciens were grownin vitro on different media. Dark growth of all tumor tissues was strictly hormone-dependent. In contrast, hormonal autonomy was observed in the light where crown gall calli readily differentiated into teratomas and (sometimes fertile) plants. Differentiating tissues always grew more vigorously than subtended calli. The growth of transformed calli was stimulated by vitamins and partly inhibited by growth regulators in concentrations used for the maintenance of untransformed calli. Crown gall calli, teratomas and sometimes regenerated plants were shown to express lysopine or nopaline dehydrogenase activities.     

Brassica grown gall tumourigenesis and in vitro of transformed tissue

A number of Brassica species and cultivars were tested and found to be highly susceptible to crown gall induction by both nopaline and octopine strains of Agrobacterium tumefaciens. Only B. napus did not form tumours when inoculated with octopine strains. Seedlings of very young plants were poor hosts but efficient infection occurred after 8–10 weeks of growth. Teratomas arising on tumours in planta were relatively frequent on induction with nopaline strains. Axenically cultured tumour calli of Brassicas were very active in opine synthase activity and stably maintained this transformed phenotype; however, transformed plants could not be regenerated. These results suggest that disarmed nopaline Ti plasmid vectors are well suited for the genetic engineering of this important crop family.     

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.     

N2-(1,3-dicarboxypropyl) ornithine in crown gall tumours

A novel acidic amino acid present in crown gall tumours induced on Nicotiana tabacum cv White Burley by Agrobacterium tumefaciens has been identified as N²-(1,3-dicarboxypropyl) ornithine.     

Specificity of Octopine Uptake by Rhizobium and Pseudomonas Strains

The octopine-utilizing strain Agrobacterium tumefaciens B6S3 and three nonagrobacteria which had the capacity to utilize this opine were compared for octopine uptake. The characteristics of uptake by Rhizobium meliloti A3 and strain B6S3 were similar. In both bacteria, uptake activity was inducible by octopine and by the related opine octopinic acid, and competition assays showed that these two opine substrates were accepted by the same uptake system with an equivalent affinity. Cells of Pseudomonas putida 203 accumulated octopine against a concentration gradient, and this activity was induced specifically by octopine. While strain 203 did not utilize octopinic acid, a spontaneous mutant with a combined capacity for octopine and octopinic acid utilization was obtained. Both opines induced octopine uptake by this mutant, but octopinic acid was not a substrate for the induced system. Thus, the Pseudomonas uptake system exhibited a different specificity for octopine than the corresponding Agrobacterium system. The nonfluorescent pseudomonad GU187j, which utilized the three related opines octopine, octopinic acid, and nopaline, was constitutive for octopine uptake. Strain GU187j possessed a system which accepted these three opines, but not arginine or ornithine, with a similar affinity.